Apoptotic and anti-proliferative activity of novel platinum complex [Pt((E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2- (pyridine-2-ylmethylene)hydrazine-1-carbothioamide)] against Ehrlich Ascites carcinoma (EAC) cells in vivo

Apoptotic and anti-proliferative activity of novel platinum complex [Pt((E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2- (pyridine-2-ylmethylene)hydrazine-1-carbothioamide)] against Ehrlich Ascites carcinoma (EAC) cells in vivo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Apoptotic and anti-proliferative activity of novel platinum complex [Pt((E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2- (pyridine-2-ylmethylene)hydrazine-1-carbothioamide)] against Ehrlich Ascites carcinoma (EAC) cells in vivo Tasnima Kamal, Azmin Akter, Asmaulhusna Biswas, Sharmin Akhter, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7229944/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Objectives Several platinum complexes have been used in clinical studies to address adverse effects and tumor resistance to cisplatin. Hence, the objective of the current study was to synthesize, characterize, and examine the anticancer activity of a novel platinum complex in EAC cells. Methods A synthetic compound was synthesized from Platinum and Schiff base ligands. The anticancer activity of the complex was tested against EAC cells in Swiss albino mice by monitoring several parameters, such as tumor cell growth, survival time, tumor mass, and hematological profile. Morphological observation and modulation of apoptotic regulatory genes’ expression were used to study its anticancer mechanisms. Results The IUPAC name of the ligand is ((E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2-(pyridine-2-ylmethylene) hydrazine-1-carbothioamide) [L]. The complex exhibited significant anticancer activity against EAC cells. It showed 45.01% (p < 0.01) and 62.57% (p < 0.001) cell growth inhibition at doses of 2.0 and 5.0 mg/kg/day, respectively, and significantly prolonged survival (30 versus 19 days; p < .01). Also, it reduced (37.1%) tumor weight at 5.0 mg/kg/day on EAC bearing Swiss albino mice. Moreover, EAC-bearing mice receiving the treatment restored blood parameters. It did not exhibit any long-term adverse effects on hematological, biochemical, or tissue parameters in mice. The compound-treated EAC cells showed increased expression of pro-apoptotic genes such as p53, Bax, Cas-3, 9 , and decreased expression of anti-apoptotic gene Bcl2 , indicating mitochondrial intrinsic pathway activation. Conclusions The compound showed potential anticancer activity by inducing apoptosis; however, further preclinical and clinical research is imperative before using animal and human models. Anticancer cell growth survival platinum complex and EAC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cancer is a complex and devastating disease characterized by the uncontrolled growth and spread of abnormal cells. It arises from a disruption of the delicate balance between cell proliferation and cell death [ 1 ], leading to the accumulation of cells that can invade the surrounding tissues and metastasize to distant organs. This intricate process is driven by a multitude of factors including genetic mutations [ 2 ], environmental exposures and lifestyle choices [ 3 ]. The global burden of cancer is substantial and continues to increase, posing a significant challenge to public health. According to the World Health Organization (WHO), cancer is one of the leading causes of death worldwide. It is estimated that there were 19.3 million new cases of cancer and approximately 10 million deaths from cancer in 2020 (Sung et al., 2021). This affects millions of individuals and families, transcending borders, cultures, and economics, posing challenges not only to the health system but also to socioeconomic development. Female breast cancer (2.26 million cases), lung (2.21 million), and prostate cancer (1.41 million) were the most frequently diagnosed malignancies globally. The most prevalent causes of cancer mortality were lung (1.79 million deaths), liver (830,000), and stomach cancers (769000) [ 4 ]. It is anticipated that there will be 28.4 million new cases of cancer worldwide in the year 2040, which represents a 47% increase from the 19.3 million cases reported in the year 2020. This is based on the assumption that the national rates calculated in 2020 will remain unchanged (5), and the incidence and mortality rates vary considerably across different cancer types and geographical regions, highlighting the need for targeted prevention and treatment strategies [ 5 ]. The development of effective anticancer drugs has been the central focus of biomedical research for decades. Chemotherapy, the cornerstone of cancer treatment, utilizes cytotoxic agents to eliminate rapidly dividing cancer cells [ 6 ]. However, the lack of specificity of these agents often leads to severe side effects that limit their therapeutic efficacy. In recent years, significant advances have been made in understanding the molecular mechanisms that drive cancer development and progression. This knowledge has paved the way for the development of targeted therapies that selectively target specific molecules or pathways involved in cancer growth [ 7 ]. Metal-based compounds have been used for therapeutic purposes since ancient times. During this time frame, the ancient Assyrians, Egyptians, and Chinese people understood the significance of employing metal-based compounds in the treatment of medical conditions [ 8 ]. Platinum-based chemotherapeutic agents, including cisplatin, carboplatin, and oxaliplatin, have been widely used as cornerstone treatments for various cancers, such as lung, ovarian, testicular, and colorectal cancers [ 9 ]. These drugs exert their anticancer effects primarily through the formation of DNA adducts, leading to the disruption of DNA replication and transcription and ultimately inducing apoptosis in cancer cells [ 10 ]. Despite their significant contribution to cancer treatment, these agents have several limitations that reduce their therapeutic efficacy and compromise patient outcomes. The primary challenge with platinum-based drugs is the development of drug resistance. Cancer cells often acquire resistance through multiple mechanisms, including reduced drug uptake, increased efflux, enhanced DNA repair, and detoxification by intracellular thiols such as glutathione [ 11 ]. This resistance not only limits the effectiveness of these agents but also necessitates the use of higher doses, increasing the risk of severe side effects. Another major drawback of cisplatin, carboplatin, and oxaliplatin is their notable toxic effects. For instance, cisplatin is well documented to cause nephrotoxicity, neurotoxicity, and ototoxicity, which can severely impact a patient’s quality of life and limit its clinical use [ 12 ]. While carboplatin is associated with reduced nephrotoxicity, it frequently induces myelosuppression, particularly thrombocytopenia [ 13 ]. Oxaliplatin, commonly used in the treatment of colorectal cancer, is known for its dose-limiting peripheral neuropathy, which can be either acute or cumulative, often necessitating dose reduction or treatment discontinuation [ 14 ]. The clinical success of the platinum-based drug cisplatin (cis-diamminedichloroplatinum (1r)) in anticancer chemotherapy has prompted an all-out search for analogs with reduced toxicity, better therapeutic indices, and enhanced activity [ 4 ]. The development of new platinum drugs with anticancer activity has been a topic of significant interest in the field of bioinorganic chemistry. The success of cisplatin as an anticancer drug has stimulated research in this area, leading to the approval of carboplatin and oxaliplatin for routine clinical use [ 15 ]. Other platinum drugs, such as nedaplatin, lobaplatin, and heptaplatin, have been approved in specific regions, highlighting the importance of exploring novel platinum complexes for their anticancer potential [ 15 ]. Efforts have been made to elucidate the biochemical mechanisms of cisplatin cytotoxicity to design new platinum-based drugs with improved pharmacological profiles [ 16 ]. The rational design of anticancer platinum complexes emphasizes the importance of understanding the structure-activity relationship to optimize the anticancer activities of these compounds [ 17 ]. This process involves consideration of the mechanisms of action, potential resistance mechanisms of cancer cells, and pharmacokinetic and toxicity properties to ensure the clinical usefulness of new platinum drugs [ 17 ]. Recent advances in platinum-based chemotherapeutics have focused on the development of drugs with inhibitory and targeted mechanisms of action [ 18 ]. The study of platinum (II) dithiocarbamate complexes as anticancer and DNA-damaging agents further underscores the importance of exploring new platinum compounds for their potential anticancer activities [ 19 ]. Additionally, the development of strategies for the design of platinum anticancer drugs has been highlighted as a crucial aspect in advancing the field [ 20 ]. Overall, the importance of new platinum drugs for anticancer activity lies in their potential to provide alternative treatment options with improved efficacy and fewer side effects. By understanding the structure-activity relationship and exploring novel platinum complexes, researchers aim to develop innovative anticancer drugs that can address the limitations of current platinum-based chemotherapeutics [ 17 ]. Therefore, the focus of the present study was to delve into the current landscape of the Platinum Schiff base ligand complex for the development of anticancer drugs, explore its activity against EAC (Ehrlich ascites carcinoma) cells, and investigate its underlying molecular mechanism. Materials and Methods Chemicals and reagents All chemicals and reagents used in this experiment were of analytical grade and purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA), Promega (Madison, Wisconsin, USA), Amresco (11 Speen Street Framingham, Massachusetts, USA), Life Technologies (5791 Van Allen Way Carlsbad, CA, USA), and Applied Biosystems (180 Oyster Point Blvd South San Francisco, CA, USA). Synthesis and characterization of Platinum complex The unbound ligand L[(E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2-(pyridine-2-ylmethylene) hydrazine-1-carbothioamide] and its platinum complex were synthesized according to a previously published protocol [ 21 ]. As shown in Fig. 1 , the Platinum complex was synthesized in three steps: (1) synthesis of ( E )-2-(pyridine-2-ylmethylene) hydrazine-carbothioamide, (2) Synthesis of Ligand [(E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2-(pyridine-2-ylmethylene) hydrazine-1-carbothioamide] (L), and (3) Synthesis of Pt (II) L complex. The final product was extracted with chloroform. The chloroform extract was removed by evaporation and dried over silica gel in a vacuum desiccator. The compound was characterized based on its physical properties (melting temperature, color, and solubility), IR spectra, and 1 H NMR. Animal, cell line, and ethical statement The University Ethics Committee (Institutional Animal, Medical Ethics, Biosafety, and Biosecurity Committee for Experiments on Animal, Human, Microbes, and Living Natural Sources (No. 293(13)/320-IAMEBBC/IBSc), Institute of Biological Sciences, Rajshahi University, Bangladesh) approved the use of experimental animals (mice). This study was carried out in strict accordance with the approved guidelines. Adult male Swiss albino mice weighing 20–25 g were used. The mice were meticulously raised in our laboratory according to the standards, protocols, and regulations established by the Institutional Animal, Medical Ethics, Biosafety, and Biosecurity Committee. In addition, the work described herein was carried out following the National Institutes of Health Office of Laboratory Animal Welfare policies and laws and complied with the ARRIVE guidelines. Mice were housed in standard polypropylene cages in well-ventilated rooms, under a 12 h light/12 h dark cycle at a temperature of 24 ± 2 ℃, and maintained under hygienic conditions. Standard food and drinking water were given ad libitum at a natural day-night cycle. All animal welfare considerations, including efforts to minimize the suffering and distress of the animals, were taken. The health and behavior of animals were monitored twice every day (morning and evening) during the experiments. At the end of the experiments, the mice were placed in a 30 cm × 10 cm anesthesia chamber for acclimation. The induction of anesthesia was carried out using 5% Isoflurane in oxygen at a flow rate of 1.2 L/minute for 2–4 minutes until the animal was fully anesthetized. The breathing pattern, anesthesia depth in mice were monitored using regular toe pinch tests to examine the anesthesia levels to ensure that the animals were adequately sedated without being overanesthetized. After that, the animals were placed on a surgical table, and 1–2% isoflurane in oxygen with a flow rate of 1.2 L/minute was used during all surgeries to maintain anesthesia, and the cervical dislocation method was used to sacrifice them to ensure the humane endpoints of the animals. All efforts were made to minimize the suffering of the animals. Ehrlich Ascites Carcinoma (EAC) cells were obtained from the Indian Institute for Chemical Biology (IICB) in Kolkata, India, with their kind assistance. Under controlled ambient conditions in our department laboratory, 10 5 cells per animal were administered intraperitoneally every two weeks to maintain the viability of these cells. No animals were dead before the completion (endpoints) of the experiment. Research students were trained in animal handling and maintenance for better management of the experimental animals. Inhibition of EAC cell growth In vivo inhibition of EAC cell growth was assessed according to a previously published protocol [ 22 ]. Four groups of mice, each consisting of six mice, were used in this experiment. Each of the mice was inoculated with 1.6 × 10⁶ EAC cells intraperitoneally on the first day. The treatment was initiated 24 hours after inoculation and was administered for five consecutive days. At concentrations of 2.0 mg/kg and 5.0 mg/kg body weight, the platinum (II) complex was administered intraperitoneally to Groups 2, 3, and 4, and the standard drug (Bleomycin 0.3 mg/kg), respectively. Each mouse received daily injections of 0.1 ml. Normal saline was administered intraperitoneally to Group 1, which served as the control. On the sixth day (24 hours after the last treatment), the mice were sacrificed, and peritoneal fluid was harvested using 0.98% saline. Viable tumor cells were quantified using a hemocytometer following trypan blue staining under an inverted microscope (XDS-1R; Optika, Bergamo, Italy), and tumor cell growth inhibition was calculated using the following formula: % Cell growth inhibition = (C- T / C) × 100 where T = Mean number of tumor cells in the treated group of mice and C = Mean number of tumor cells in the control group of mice. Average tumor weight and survival time The previously described method was employed to assess the tumor weight and survival time of EAC-bearing mice [ 23 ]. For this experiment, three groups (6 mice in each group) of Swiss albino mice were taken, and each of them was inoculated with 1.6×10 6 EAC cells. Group 1 was considered as control, and 24 hours after inoculation, groups 2 and 3 received 2.0 & 5.0 mg/kg/day body weight of the treatment intraperitoneally. The treatment was continued for 10 consecutive days. Daily variations in weight were recorded to track tumor progression. Survival data were analyzed using the Kaplan-Meier method to estimate the survival probability over time for each experimental group. Differences in survival distributions between groups were evaluated using the log-rank (Mantel-Cox) test [ 24 ]. The following formula was used to calculate the percentage increase in lifetime after host survival was recorded and expressed as the mean survival time in days: $$\:\text{M}\text{e}\text{a}\text{n}\:\text{s}\text{u}\text{r}\text{v}\text{i}\text{v}\text{a}\text{l}\:\text{t}\text{i}\text{m}\text{e}\:\left(\text{M}\text{S}\text{T}\right)\:=\sum\:\frac{\text{S}\text{u}\text{r}\text{v}\text{i}\text{v}\text{a}\text{l}\:\text{t}\text{i}\text{m}\text{e}\:\left(\text{d}\text{a}\text{y}\text{s}\right)\text{o}\text{f}\:\text{e}\text{a}\text{c}\text{h}\:\text{m}\text{o}\text{u}\text{s}\text{e}\:\text{i}\text{n}\:\text{a}\:\text{g}\text{r}\text{o}\text{u}\text{p}}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{m}\text{i}\text{c}\text{e}}$$ $$\:\text{P}\text{e}\text{r}\text{c}\text{e}\text{n}\text{t}\text{a}\text{g}\text{e}\:\text{i}\text{n}\text{c}\text{r}\text{e}\text{a}\text{s}\text{e}\:\text{o}\text{f}\:\text{l}\text{i}\text{f}\text{e}\:\text{s}\text{p}\text{a}\text{n}\:\left(\text{I}\text{L}\text{S}\text{%}\right)=\frac{\text{M}\text{S}\text{T}\:\text{o}\text{f}\:\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:\text{g}\text{r}\text{o}\text{u}\text{p}}{\text{M}\text{S}\text{T}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{g}\text{r}\text{o}\text{u}\text{p}}-1\times\:100$$ Evaluation of hematological parameters Hematological parameters were evaluated by observing the changes in hemoglobin (Hb), RBC, WBC, and platelet counts in comparison with normal mice. A hemocytometer and cell dilution fluids were used to assess the effects of the platinum (II) ligand complex. Four groups of mice were used, each comprising six mice [ 25 ]. Normal mice were assigned to group 1, whereas 0.1 ml of EAC (1.6×10 6 ) cells was inoculated intraperitoneally into the rest of the groups. Here, group 2 served as the control group, and groups 3 and 4 received a dose of 2.0 mg/kg and 5.0 mg/kg body weight of the Platinum (II) ligand complex per day, for 10 days consecutively. Blood samples were obtained through tail puncture on the 12th day after inoculation, and hematological parameters were subsequently assessed [ 25 ]. Morphological changes and nuclear damage The induction of apoptosis by the platinum (II) ligand complex was examined as previously reported [ 26 ]. Morphological observations of both treated and untreated EAC cells were studied by observing the changes in phase contrast images and staining them with Geimsa stain using an inverted microscope (XDS-1R, Optika, Bergamo, Italy). Further nuclear damage was confirmed by PI staining, using a fluorescence microscope (Olympus IX71, Japan). First, EAC cells were collected from the mice receiving platinum (II) ligand complex at a dose of 5.0 mg/kg body weight and saline (non-treated control) and stained with 10 µL (1 mg/ml) of Propidium Iodide (PI) at 37°C for 20 min in the dark. Subsequently, the cells were washed with phosphate-buffered saline (PBS), and morphological changes were visualized using a fluorescence microscope. In addition, a DNA fragmentation assay was performed to examine the apoptotic cleavages of the genomic materials of EAC cells receiving the treatment [ 22 ]. Effect of Pt (II) L complex on normal peritoneal cells The influence of the Pt (II) L complex on normal peritoneal cells was assessed by quantifying the total peritoneal cells and macrophages. Mice were divided into three groups (n = 6 per group), with two groups receiving the Pt (II) L complex (5.0 and 2.0 mg/kg, i.p.) for three consecutive days, while the third group served as an untreated control. Twenty-four hours after the final treatment, 5 mL 0.98% normal saline was injected into the peritoneal cavity before sacrificing the animals. The collected intraperitoneal exudate cells and macrophages were stained with 1% neutral red and counted using a hemocytometer. Extraction of mRNA Total RNA from the EAC cells was extracted using the manufacturer’s guidelines for the total RNA extraction kit (Favorgen Biotech Corp. Ping-Tung, Taiwan) from mice receiving 5.0 mg/kg/day and control EAC-bearing mice on day six of tumor implantation. RNA concentration and purity were precisely quantified using a NanoDrop spectrophotometer (NanoDrop One, Thermo Scientific, Waltham, MA, USA). Additionally, the mean absorption ratios, A260/280 and A260/230 were carefully evaluated to verify the purity of the sample. The structural integrity of the RNA was further confirmed by electrophoresis on a 1.8% agarose gel. The extracted RNA was stored at -80°C. Synthesis of cDNA cDNA was prepared for High-Capacity Reverse Transcription PCR (polymerase chain reaction) using the GoScript™ Reverse Transcription System (MA, Wisconsin, USA) according to the manufacturer’s instructions. For further analysis, the synthesized cDNA was stored at -20°C. Reverse transcriptase polymerase chain reaction (RT-PCR) The expression of five growth regulatory genes, namely, Bcl-2, Bax, p53, Caspase-3, and Caspase-9 , was examined using these cDNA as templates for RT-qPCR, with GAPDH as the control. The reaction mixture (20 µL) was prepared using the GoTaq® qPCR Master Mix Kit (MA, Wisconsin, USA) according to the manufacturer’s guidelines. The primer sequences and thermal cycling conditions are listed in Supplementary Information (SI 1 Table). Quantitative RT-PCR experiments were performed using three biological replicates for each experimental group, with each biological sample analyzed in two technical duplicates. A gradient thermal cycler was used for amplification (LightCycler® 96 Instrument, Roche Diagnostics, Forrenstrasse 2, 6343 Rotkreuz, Switzerland) was used to amplify the target genes. The comparative Ct (ΔΔCt) method was used to analyze the data. This method involved comparing the quantity of the target gene to that of the endogenous control GAPDH to determine the relative expression levels [ 27 ]. Toxicity studies in vivo Effect of test compound on biochemical and hematological parameters The toxicological effects of the compounds were evaluated by observing anomalies in the blood and biochemical parameters. For this evaluation, 40 mice, each weighing between 22 and 25 g, were divided into two groups: a control group consisting of 20 normal male mice, and a high-dose treatment group comprising 20 male mice. Baseline blood samples were collected from all mice prior to administration of the test compound to establish reference biochemical and hematological parameters. Additionally, the mice in the treatment group received the platinum (II) ligand complex intraperitoneally at a dosage of 5.0 mg/kg using a 1 ml syringe over 10 days. Following treatment, both groups of mice were anesthetized with ethyl acetate, and samples were collected on days 5, 10, and 25 post-treatment to evaluate sub-acute toxic effects of the compound using surgical blade no.22. Using a 3 ml syringe, fresh blood was collected from the heart in EDTA-free tubes. The blood samples were allowed to clot at room temperature for approximately 30 min. This allowed the blood cells to settle at the bottom of the tube. Following clotting, the blood samples were centrifuged at 7000 rpm for 12 min on a WIFUNG centrifuge (LABO-50M). Finally, clear straw-colored serum was collected in microcentrifuge tubes using a Pasteur pipette and refrigerated at -20°C. Finally, biochemical parameters of these sera were analyzed using a bioanalyzer (Humalyzer 3000, HUMAN Diagnostics Worldwide, Wiesbaden, Germany) following the previously mentioned method [ 28 ]. For the assessment of hematological parameters, fresh blood was extracted from the heart using a 3 ml syringe and collected in an Eppendorf tube containing ethylenediaminetetraacetic acid (EDTA) [ 29 ]. hematological parameters, including WBC count, RBC count, platelet count, and Hb level, were evaluated using a standardized procedure [ 30 ]. Furthermore, the effects of chemicals at the cellular level were assessed through histopathological analysis of the major organs in animals treated with the compound according to previously established protocols [ 31 ]. All tissues were immediately fixed using 10% neutral buffered formalin [ 32 ]. The tissues were subsequently dissected to accommodate cassettes. Fixed specimens of the liver, kidney, heart, and lungs were subjected to overnight processing for dehydration, clearing, and impregnation using an automatic tissue processor (automatic sample preparation system, Histo-line laboratories, Milan, Italy). The specimens were embedded in paraffin blocks using an embedding station (TEC2900 embedding center, Histo-line laboratories, Milan, Italy), and serial sections of 5 µm thickness were obtained using a microtome (Histo-line Laboratories, Milan, Italy). The sections were stained with Hematoxylin and Eosin [ 34 ]. The mounted specimens were examined under a light microscope. Statistical analysis The mean ± SEM (Standard Error of Mean (SEM) was used to express the data (which included the percentage of cell growth inhibition, increase in life span, body/tumor weight, and haematological profile). GraphPad Prism 8 software was used to conduct one-way analysis of variance (ANOVA) and Duncan's multiple range test. Statistical significance was defined as P < 0.05. Results Characterization of the compound The compound was synthesized as shown in the scheme (Fig. 1 ) and characterized by its physical constants, such as surface, color, solubility, and IR and NMR spectra (SI 2). The physical form was a red-orange powder with a melting temperature of 519°C. This compound was soluble in DMSO and partially soluble in water (H 2 O). [Insert Fig. 1 about here] FT-IR spectral analysis of ligand (L) The FT-IR spectrum of ligand L, recorded using a KBr disk, revealed key functional groups (SI 2 Table). A broad absorption at 3200–3500 cm⁻¹ suggests O–H or N–H stretching, while a sharp band at 1600–1700 cm⁻¹ indicates C = O stretching from the carbonyl-containing groups. The peaks at 1500–1600 cm⁻¹ correspond to C = C stretching, suggesting aromatic or conjugated systems. The fingerprint region (600–1500 cm⁻¹) shows characteristic bending and stretching vibrations. These spectral features confirm the structural composition of the ligand, which was further validated using complementary spectroscopic techniques (SI 2 Fig). ¹H-NMR spectral analysis of ligand (L) The ¹H-NMR spectrum of ligand L was recorded in DMSO-d₆, and the chemical shifts (δ) were reported in ppm (SI 3 Table). A downfield signal at δ ~ 11.63 ppm suggests an exchangeable proton (-OH or -NH). The aromatic region (δ 6.5–9.0 ppm) exhibited multiple peaks, confirming the presence of aromatic or heteroaromatic rings. Peaks in the aliphatic region (δ 1.0–4.0 ppm) correspond to -CH₂ or -CH₃ groups, with a singlet at δ 2.50 ppm from the solvent. The spectrum supports the expected structure of ligand L, indicating the presence of aromatic, aliphatic, and hydrogen-bonded functional groups. Further structural confirmation was achieved through ²D-NMR, ¹³C-NMR, and mass spectrometry (SI 3 Fig). Antineoplastic of Platinum Schiff base ligand complex Figure 2 (A & B) shows the effects of the complex on the proliferation of EAC cells on day 6 after tumor transplantation at doses of 2.0 mg and 5.0 mg/kg. At dosages of 2.0 and 5.0 mg/kg/day body weight of mice, the compound exhibited 45.01% and 62.52% suppression of cell proliferation, respectively, compared to the control. However, bleomycin reduced cell proliferation by 79.57% when administered intraperitoneally at a dose of 0.3 mg/kg. Thus, compared with untreated EAC-bearing mice, this outcome suggests that the test compound exhibited substantial inhibition of EAC cell growth (***p < 0.001). [Insert Fig. 2 about here] Survival time and tumor weight management Hematological profile During tumor progression, significant deviations in hematological parameters from baseline levels were observed. EAC-bearing mice showed significantly decreased RBC and Hb levels following EAC cell inoculation (Fig. 2 F and G). However, WBC and platelet levels were elevated (Fig. 2 H and I) compared to normal mice, showing immune dysfunction. Blood parameters were somewhat restored toward normal levels with the treatment of EAC-bearing mice with the Pt (II) L complex at doses of 5.0 and 2.0 mg/kg/day (Fig. 2 ). Morphological appearance and nuclear damage Morphological alterations of EAC cells collected from both control and treated mice were analyzed using PI and Giemsa staining. In Fig. 3 , the morphological changes in both control and treated cells, noted under the fluorescence and optical microscope, are indicated by arrows. Cells from control animals were observed to have round, regular, and normal-shaped nuclei under a microscope (Fig. 3 A and B). In contrast, treated cells exhibited apoptotic features, including condensed, fragmented, irregular nuclei and chromatin, as well as the formation of apoptotic bodies (Fig. 3 C and D). In addition, the generation of DNA fragmentation, a hallmark of apoptosis, results in smear-like DNA bands in agarose gel, indicating the induction of apoptosis of EAC cells, followed by the treatment of the compound (Fig. 3 E). [Insert Fig. 3 about here] Effect of Pt (II) L complex on normal peritoneal cells Pt (II) L complex shows a dose-dependent increase in macrophage and total peritoneal cell counts. In the control group (normal mice), the total number of peritoneal cell exudates was (3.17 ± 3.03) × 10 6 , of which (1.04 ± 2.19) × 10 6 (Fig. 4 ) were macrophages after five days of treatment. Mice treated with 5.0 mg/kg body weight of platinum complex showed an increase in both macrophage count and total peritoneal cells. At a lower dose (2.0 mg/kg), there is still an increase in macrophage and total peritoneal cell counts, however, the effect is less pronounced than with 5.0 mg/kg, suggesting that Pt (II) L complex may enhance immune cell recruitment or proliferation in the peritoneal cavity (Fig. 4 ). [Insert Fig. 4 about here] Analysis of gene expression Following the inhibition of cell growth and apoptotic body formation, we hypothesized that the Pt (II) L complex would demonstrate potential antiproliferative efficacy against EAC cells by inducing apoptosis. Consequently, we attempted to determine the expression levels of the apoptosis-regulatory genes. We assessed the effect of the Pt (II) L complex on the expression of pro-apoptotic genes, including p53, Bax, Caspase 3 , and Caspase 9 , along with the anti-apoptotic gene Bcl-2 , using RT-PCR. While the pro-apoptotic genes p53, Bax, Caspase 3 , and Caspase 9 were upregulated in the treatment group compared to the control, the reduced expression of the anti-apoptotic gene Bcl-2 indicated the mitochondrial apoptotic pathway (Fig. 5 ). [Insert Fig. 5 about here] Toxicological studies Blood parameters We investigated the hematological parameters in Swiss albino mice (without EAC cells) administered the test compound at a dose of 5.0 mg/kg/day for 10 consecutive days, and the blood parameters were measured on days 5, 10, and 25 to assess the detrimental effects of the Pt (II) L complex host (Table 1 ). RBC, WBC, platelets, and % Hb were found to vary somewhat under treatment; however, they reversed practically towards normal following treatment (p < 0.05). This provided evidence that the Pt (II) L complex did not have long-term toxic side effects on the host (Table 1 ). Table 1 Effects of Pt (II) L complex on blood parameters in normal mice on days, 0, 5, 10 and 25 at dose 5.0 mg/kg body weight Experiment Days RBC cells/ml WBC cells/ml Platelet cells/ml % of Hb Control (normal) 0 (7.28 ± 0.039) ×10 9 (9.1 ± 0.4) ×10 6 (32.7 ± 2.8) ×10 6 7.7 ± 0.26 Normal + test compound 5 (5.25 ± 11.57) ×10 6 *** (14.5 ± 0.8) ×10 6 *** (22.4 ± 0.6) ×10 6 *** 6.2 ± 0.28* 10 (5.72 ± 1.71) ×10 6 *** (11.5 ± 1.8) ×10 6** (18.1 ± 0.7) ×10 6 *** 5.67 ± 0.3** 25 (6.91 ± 2.45) ×10 6 * (9.89 ± 2.4) ×10 6* (25.8 ± 9.8) ×10 6 ** 6.8 ± 0.1 Number of mice in each group were 6; the results are shown as mean ± SEM and compared with control with normal mice (without treatment) where significant values are *p < 0.05 **p < 0.01 and***p < 0.001 Biochemical profile analysis Key biochemical parameters, such as triglycerides, serum cholesterol, creatinine, bilirubin, serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and alkaline phosphatase (ALP), showed notable alterations following a 10-day intraperitoneal administration of 5.0 mg/kg/day of the Pt (II) L complex in Swiss albino mice. Fluctuations in these serum parameters from the normal range were observed on day 5 day 5 and 10, but the parameters seemed to revert towards normal levels on day 25, indicating that the Pt (II) L complex did not have any long-term toxic side effects in the host (Table 2 ). Table 2 Effects of Pt (II) L complex on biochemical parameters in normal mice on days, 0, 5, 10 and 25 at dose 5.0 mg/kg body weight Test Unit Group Day 0 (Mean ± SEM) Day 5 (Mean ± SEM) Day 10 (Mean ± SEM) Day 25 (Mean ± SEM) Liver Function Tests SGPT (ALT) U/L Normal 18.75 ± 1.79*** 18.8 ± 1.29*** 18.85 ± 1.03*** 19.55 ± 1.04*** Treatment 18.72 ± 1.79** 31.02 ± 3.06 16.5 ± 1.08** 18.37 ± 1.4* SGOT (AST) U/L Normal 75 ± 6.58*** 74.2 ± 5.9*** 72.7 ± 7.13*** 74.5 ± 6.75*** Treatment 74.5 ± 6.75* 121.75 ± 6.07 64.5 ± 4.1** 79.5 ± 5.75 ALP U/L Normal 113.67 ± 1.21*** 112.7 ± 1.8*** 111.4 ± 1.2*** 113 ± 1.29*** Treatment 113.67 ± 12.1** 172.7 ± 8.2** 89.3 ± 5.6** 128.45 ± 1.75 Bilirubin mg/dL Normal 0.2625 ± 0.07*** 0.2625 ± 0.04*** 0.2375 ± 0.05*** 0.265 ± 0.05*** Treatment 0.3 ± 0.08* 0.4 ± 0.18 0.2 ± 0.08** 0.22 ± 0.05* Kidney Function Test Creatinine mg/dL Normal 1.12 ± 0.22*** 1.17 ± 0.3*** 1.2 ± 0.2*** 1.14 ± 0.24*** Treatment 0.37 ± 0.12* 2.17 ± 0.31 0.85 ± 0.22* 0.9 ± 0.26* Lipid Profile Tests Cholesterol mg/dL Normal 158 ± 1.5*** 160 ± 6.2*** 161.2 ± 1.05*** 157.5 ± 6.8*** Treatment 177.5 ± 1.07 163 ± 9.6 181.5 ± 1.61 147.1 ± 6.9** Triglycerides mg/dL Normal 139.2 ± 3.9*** 141.1 ± 3.4*** 139.9 ± 3.9*** 138.9 ± 3.96*** Treatment 131.7 ± 7.6** 166.6 ± 3.24 155.2 ± 1.06 150. ±7.34 Number of mice in each group were 6; the results are shown as mean ± SEM and compared with control with normal mice (without treatment) where significant values are *p < 0.05 **p < 0.01 and ***p < 0.001 Histopathological analysis The effects of the tested complexes at the cellular level were examined using histological analysis. Tissue sections were observed using an inverted microscope at 40X magnification (Fig. 6 ). It was noted that there was no damage (regeneration, degeneration, etc.) in the kidney, lung, heart, and liver tissues of treated mice, indicating that the tested compound had no adverse effects on tissue levels in mice (Fig. 6 ). [Insert Fig. 6 about here] Discussion Platinum-based chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin are effective oncological therapies, especially for solid neoplasms such as ovarian, testicular, bladder, and lung malignancies [ 35 ]. However, their clinical application is limited by adverse toxic side effects and the development of resistance to these therapeutics. Thus, new drugs are required to reduce or eliminate toxic side effects, enhance efficacy, improve target specificity, and combat aggressive and metastatic cancers [ 36 ]. In the present study, the antineoplastic effect of a novel Pt (II) ligand complex was demonstrated using EAC cells in a mouse model. Through the assessment of anti-proliferative activity against EAC cells, tumor weight reduction, growth inhibition, restoration of hematological parameters, and survival, followed by the investigation of the possible mechanisms of activating the intrinsic mitochondrial pathway, the potential of the chemical as an anticancer agent was evaluated. The newly synthesized compound was characterized by physicochemical properties, such as physical form, appearance, and melting point, along with spectrometric (IR spectroscopy, 1 H nuclear magnetic resonance, and 13 C nuclear magnetic resonance) analysis. The antitumor efficacy of synthetic platinum complexes against EAC in mice has been documented in scientific literature [ 37 ]. These studies demonstrated that such complexes can significantly inhibit tumor growth, reduce tumor volume, and enhance the lifespan of EAC-bearing mice [ 38 ]. Our survival analysis demonstrated that treatment with the platinum (II) ligand complex led to a significant increase in lifespan compared to controls. The Kaplan-Meier curves and median survival times clearly indicate a dose-dependent survival benefit [ 24 ]. In this investigation, we observed that the Pt (II) L complex inhibited cell growth by 62.52%, increased life span by 58.28%, and reduced tumor weight by 40.24% at 5 mg/kg compared to the control group. These data suggest that the platinum complex not only inhibits tumor growth, but also positively influences overall survival, an encouraging indication for its potential use in cancer therapy. Changes in blood parameters can reflect the tumor burden and progression, providing insights into the severity of the disease and the effectiveness of potential treatments. The significance of fluctuations in blood parameters in EAC-bearing mice is crucial for understanding the effects of cancer on hematological and overall physiological states of the animal. Tumor progression is associated with specific hematological changes, including a gradual decrease in hemoglobin content, red blood cells, and platelets, along with an increase in white blood cell counts; these changes were detected in EAC-bearing mice [ 28 ]. Deteriorated blood parameters, such as hemoglobin levels and red blood cell counts in the control group, can indicate anemia, which can lead to decreased oxygen transport, impacting the overall health of the mice [ 39 ]. In EAC-bearing mice, elevated WBCs often reflect immune dysregulation or the impact of the tumor on the immune system. Elevated levels of WBC can also be the result of tumor progression, which stimulates the bone marrow to produce more WBC via cytokines such as tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF). A declining platelet count can be explained by the release of inflammatory cytokines such as TNF-α and interleukins, which can suppress bone marrow activity. The suppression of bone marrow reduces platelet production (thrombopoiesis), leading to low platelet counts [ 40 ]. Cancer treatments, such as chemotherapy and radiation, can directly affect the bone marrow and the site of blood cell production. This may result in decreased red blood cells (anemia) and platelets (thrombocytopenia), and an increase in white blood cells (leukocytosis) [ 41 ]. However, it was reported that when EAC-bearing mice were treated with platinum complexes, these parameters seemed to be restored to normal levels compared to the control group [ 37 ]. It is intriguing that treatment with the Pt (II) L complex substantially increased the hemoglobin, RBC, and platelet counts and decreased the WBC count in the treatment group compared to the control. Consequently, it is hypothesized that this compound may exert a protective effect on the hematopoietic system. Hence, the potential chemotherapy drug derived from this compound may yield more tolerable medications with reduced side effects, thereby providing improved protective benefits for patients with cancer. The increased macrophage count observed in mice treated with a platinum complex compared to that in normal mice highlights the significant role of macrophages in the anti-tumor immune response. Platinum-based compounds, widely known for their cytotoxic effects on cancer cells, may also stimulate an immune response by inducing immunogenic cell death (ICD), leading to the recruitment and activation of macrophages in the tumor microenvironment. This macrophage accumulation could contribute to tumor suppression through enhanced phagocytosis, cytokine secretion, and antigen presentation, thereby strengthening anti-tumor immunity [ 42 ]. Platinum treatment may also affect macrophage polarization and therapeutic effectiveness. Proinflammatory (M1) macrophages may emit nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α), leading to tumor cell eradication [ 43 ]. In contrast, excessive M2 macrophage recruitment may suppress the immune system. Understanding macrophage behavior in response to platinum-based therapy is critical for enhancing its efficacy and potential immune-modulating drug combinations. Therefore, treatment with the Pt (II) L complex resulted in a dose-dependent increase in macrophage and total peritoneal cell count. The increase in both macrophages and total peritoneal cells suggests a potential immunostimulatory effect, which should be explored further in functional immune response studies. Apoptosis is a self-initiated cellular suicide mechanism and a defining hallmark of potential chemotherapeutic agents [ 44 ]. The evasion of apoptosis is a hallmark of cancer, enabling abnormal cell proliferation and contributing to tumor progression. The promotion of apoptosis in cancer cells while sparing healthy cells is a key mechanism for effective and safer anticancer therapies [ 45 ]. In vivo analysis of EAC cells demonstrated that the compound suppressed cell proliferation in a dose-dependent manner, subsequently triggering apoptosis. Synthetic pharmaceuticals used in clinical settings can trigger apoptosis in certain cancer cells. Giemsa staining is a cytological method that facilitates the examination of cellular morphology, enabling the identification of apoptotic features, such as membrane blebbing, cytoplasmic condensation, and apoptotic bodies. For instance, a study evaluating the apoptogenic effects of Averrhoa bilimbi extract on EAC-bearing mice employed Giemsa staining to detect these morphological changes, indicating apoptosis in the treated cells [ 26 ]. Propidium iodide is a fluorescent dye that intercalates into DNA. However, it is impermeable to live cells with intact membranes. It is commonly used, in conjunction with other stains, to assess cell viability and apoptosis. In a study investigating the sensitization of Ehrlich ascites tumor cells to methotrexate by inhibiting glutaminase, immunofluorescence staining with annexin V and propidium iodide was conducted to assess the number of apoptotic cells [ 46 ]. The cells treated with the tested compound showed remarkable apoptotic properties, including cell membrane blebbing, appearance of apoptotic bodies, chromosomal condensation, and fragmentation of the nucleus under a fluorescence and light microscope. Other platinum-based ligand complexes have been reported to cause apoptosis in EAC cells, which was confirmed by cytoplasmic shrinkage, ROS generation, mitochondrial membrane depolarization, and DNA damage [ 47 ]. Furthermore, Ca 2+ /Mg 2+ -dependent endonuclease activation, a key hallmark of apoptosis induction, produces oligonucleotide fragmentation followed by cleavages of inter-nucleosomes, resulting in the formation of smear-like DNA degradation [ 27 ]. EAC cells treated with the test compound in the current study caused DNA fragmentation, as previously reported for apoptosis-inducing substances [ 22 , 27 ]. Thus, the apoptotic death of cancer cells following treatment with our compound indicates the potential therapeutic implications of the assessed chemicals. We assessed the expression of apoptosis-regulating genes, such as p53, BAX, BCL-2, CAS-3, CAS-8, CAS-9, and PARP, using RT-qPCR to gain a better understanding of the activation of apoptotic pathways in MCF7 cells following drug treatment. Multiple growth-related genes govern the activation and inhibition of apoptosis, including pro-apoptotic (e.g., p53, Bax, Bid, and Bak) and anti-apoptotic factors (e.g., Bcl-2, Bcl-X, and Bcl-Wact). The expression of these genes in cancer cells changes in response to therapeutic intervention, resulting in the activation or deactivation of apoptosis [ 48 ]. We conducted a parallel analysis of the expression levels of apoptosis-related genes (p53, Bax, bcl-2, caspase-3, and caspase-9) in EAC cells after exposure to the tested drug. All intrinsic apoptotic events are predominantly controlled by Bcl-2 family proteins and the p53 tumor suppressor gene, which are majorly involved in the activation of Bcl-2 family proteins [ 49 ]. The groups treated with the Pt (II) L complex exhibited an upregulation of p53 and downregulation of the Bcl-2 gene, leading to apoptosis in EAC cells, indicating its potential as an anticancer drug. Bax and Bak, proapoptotic BH3-only proteins, can induce changes in the permeability of the mitochondrial membrane by creating pores [ 50 ]. The Bax gene was upregulated in the treated group, which implies that the compound triggers apoptosis in EAC cells. Cytochrome C interacts with Apaf-1 to form an apoptosome complex and activates pro-caspase-9, ultimately leading to the activation of caspase-3. Caspase-3 is triggered by both intrinsic and extrinsic apoptotic mechanisms [ 51 ]. Caspase-3 cleaves a variety of proteins, including kinases, DNA regulatory proteins, cytoskeletal proteins, and endonuclease inhibitors, in a shared mechanism for both intrinsic and extrinsic triggers [ 52 ]. In this gene expression analysis, the expression of both cas-9 and cas-3 was upregulated in groups treated with the Pt (II) L complex, representing apoptosis that occurred through the intrinsic pathway. Overall, at the mechanistic level, apoptosis assays conducted via RT-qPCR provided insights into how the Pt (II) L complex exerts its anticancer effects. This evidence suggests that the complex induces apoptosis as a primary mechanism for its anticancer activity, selectively promoting programmed cell death in tumor cells (Fig. 7 ). [Insert Fig. 7 about here] The toxicological profile of chemotherapeutic agents is crucial to determine their safety and therapeutic potential. In this study, the toxicological effects of a synthetic platinum complex were evaluated in mice, focusing on hematological, biochemical, and histopathological parameters to assess systemic toxicity and organ-specific damage. Hematological toxicity is a common side effect of platinum-based chemotherapeutics because of its effect on rapidly dividing bone marrow cells [ 53 ]. However, in this study, mice treated with the Pt (II) L complex at 5 mg/kg showed minimal alterations in red blood cell (RBC) count, hemoglobin levels, and white blood cell (WBC) count during treatment. For example, RBC and Hb levels were low on day 5; however, they returned to the normal range on day 25. WBC slightly increased on days 5 and 10 and returned to normal levels on day 25. Platelet levels showed a slight decrease within the normal range, indicating that the complex had a milder myelosuppressive effect than traditional platinum drugs such as cisplatin, which often cause severe thrombocytopenia and neutropenia. Biochemical markers, including liver enzymes (SGPT, SGOT, Bilirubin, ALP), kidney function indicators (creatinine), and lipid profiles (triglycerides and cholesterol), were assessed to evaluate the systemic toxicity of the compound. SGPT, SGOT, and bilirubin are crucial biomarkers for assessing liver and heart health [ 54 ]. Elevated levels of these enzymes in the bloodstream typically indicate damage to the liver or heart tissues as they are released during cellular injury. In this study, SGOT, SGPT, and bilirubin levels in treated mice remained close to those in normal mice throughout the treatment period on day 10 and returned to normal levels thereafter (day 25). This stability suggests that treatment with the Pt (II) L complex did not cause significant liver tissue damage, indicating that the compounds had fewer toxic side effects. Elevated ALP level may indicate biliary obstruction or liver damage. Cisplatin can elevate ALP levels, causing hepatocellular and bone damage [ 56 , 57 ]. This increase in ALP was correlated with higher levels of other liver enzymes, such as ALT and AST, which are common indicators of hepatocellular damage [ 58 ]. Mice treated with the Pt (II) L complex displayed normal ALP levels on day 10, suggesting that the compound does not induce hepatotoxicity, nephrotoxicity, or bone toxicity. Creatinine is a by-product of normal muscle metabolism and serves as a key indicator of renal function. Elevated serum creatinine levels can signal impaired kidney function owing to reduced clearance [ 59 ]. In the present study, slight changes in serum creatinine levels were observed in mice receiving the compound on (day 10), which reversed to normal levels after treatment (day 25). This finding implied that the experimental treatment had mild adverse effects on the kidneys. However, kidney function markers remained within normal limits, in contrast to conventional platinum drugs such as cisplatin, which are known to induce nephrotoxicity through oxidative stress and tubular necrosis [ 60 ]. These results suggest that the tested complex exhibits a safer toxicological profile with reduced organ damage. These lipid profiles serve as biomarkers for evaluating the therapeutic efficacy and side effects of treatments [ 61 ]. Cisplatin can decrease total cholesterol levels (hypocholesterolemia) [ 62 ]. Treatment of mice with the Pt (II) L complex resulted in triglyceride levels remaining within the normal range on days 10 and 25; however, total cholesterol levels were notably lower than those in the normal group, suggesting a disruption in cholesterol synthesis and transport. The levels of triglycerides and cholesterol are crucial for understanding the metabolic impact of platinum-based chemotherapeutic agents such as cisplatin in cancer patients. These lipid profiles can serve as biomarkers for evaluating the therapeutic efficacy and side effects of treatment. Histological examination of the major organs, including the liver, kidneys, heart, and lungs, revealed no significant pathological alterations at the tested doses of the Pt (II) L complex. Liver sections showed normal architecture, with very few hepatocellular vacuolizations observed in the high-dose group. Kidney sections of mice treated with the Pt (II) L complex displayed intact glomeruli and tubular structures with no evidence of necrosis or fibrosis. This contrasts sharply with cisplatin-induced histopathological changes, which cause acute kidney injury (AKI) characterized by proximal tubular injury, oxidative stress, inflammation, and vascular damage within the kidney [ 63 ]. The heart and lung tissue sections of the treated mice did not show any histological changes in contrast to the normal group, indicating that the compound caused no organ-specific toxicity. Toxicological evaluation of the synthetic Pt (II) L complex highlights its potential as a safer alternative to conventional platinum-based chemotherapeutics. Although mild renal stress and slightly low cholesterol levels were observed, the overall systemic and organ-specific toxicity was significantly lower than that of cisplatin. These findings warrant further investigation, including chronic toxicity studies and clinical trials, to validate its safety and efficacy. The study of the platinum Schiff base ligand complex presents promising results; however, several limitations must be considered. Testing was limited to a single cancer model (EAC-bearing mice), restricting the generalizability of the findings to other cancers. Pharmacokinetic and pharmacodynamic profiles have not been fully explored, leaving safety and efficacy in mammalian systems uncertain. Mechanistic studies have been limited to in vivo apoptosis analysis, and further in vitro testing could clarify the molecular pathways. While our study demonstrates promising therapeutic effects of the platinum (II) ligand complex in the Ehrlich Ascites Carcinoma (EAC) mouse model, it is important to acknowledge the inherent limitations of this preclinical model. EAC is a murine tumor derived from mammary adenocarcinoma cells and, although it shares some systemic and immunological features with human cancers, it does not fully recapitulate the complexity of human tumor biology, including tumor heterogeneity, microenvironment interactions, and metastatic behavior [ 64 ]. Moreover, mouse tumor models often differ from human tumors in terms of genetic, physiological, and immunological characteristics, which may limit the direct translatability of findings to clinical settings [ 65 ]. Therefore, while the EAC model provides valuable initial insights into drug efficacy and toxicity, further studies using more advanced and clinically relevant models—such as genetically engineered mouse models (GEMMs), patient-derived xenografts (PDXs), or organoid systems—are warranted to better mimic human tumor pathophysiology and improve predictive power [ 66 ]. The study's short duration and limited dosage variation restrict the understanding of long-term effects and dose-response relationships. Additionally, a comparative analysis with a broader range of standard treatments is required to validate the therapeutic advantages of the compound. This study might also have been affected by uncontrollable external factors, such as social, political, or environmental events, which might have affected the results. Limited funding restricted access to certain databases or tools that might have provided more robust data for the analysis. Constraints related to the tools, instruments, or technologies used for data collection and analysis may have affected the accuracy of the results. Conclusion Altogether, we can conclude that this study’s findings highlight the synthesized platinum Schiff base ligand complex as a potent candidate for drug screening, as the compound has shown promising results as an anticancer drug. Its anticancer efficacy, characterized by significant tumor growth inhibition, extended survival time, normalized hematological parameters, induced DNA fragmentation, activation of pro-apoptotic and inactivation of anti-apoptotic genes, and toxicological studies, warrants further investigation. Future studies should focus on optimizing the bioactivity of the compound, understanding its safety profile, and exploring potential synergies with existing therapeutic agents to unlock its full potential in the clinical setting. Additionally, integrating immune-competent models will be critical to evaluate the compound’s effects within the tumor microenvironment and immune system context. Further studies and advanced-level research using human cell lines and observing the accurate mechanism of action of the drug by detecting its molecular pathways are crucial before undergoing clinical trials. Declarations The authors have no conflict of interest. Funding This work was supported by grants from the Dean of Science, Rajshahi University, Rajshahi-6205, Bangladesh (Grant No 2002.5/52/RU). /Science-35/2023–2024). There was no additional external funding received for this study. Author Contribution Writing – original draft: TK; Methodology, Investigation, and Data curation: TK, AA, AB, SA, MI; Validation and visualization: ZAM, ARB, AA; Supervision: MTH, MAH; Conceptualization, review, and editing: MAH, TH, and FI. Data availability All the data reported in the manuscript are presented in figures and tables. Unprocessed and raw data will be available upon request to the corresponding author. References Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell. 2011;144(5):646–74. Stratton MR, Campbell PJ, Futreal PA. The cancer genome. Nature. 2009;458(7239):719–24. Anand P, Kunnumakara AB, Sundaram C, Harikumar KB, Tharakan ST, Lai OS. Cancer is a preventable disease that requires major lifestyle changes. Pharm Res. 2008;25(9):2097–1165. Pasini A, Zunino F. New Cisplatin Analogues—On the Way to Better Antitumor Agents. Angew Chem Int Ed Engl. 1987;26(7):615–24. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209–49. Chabner BA, Roberts TG. Chemotherapy and the war on cancer. Nat Rev Cancer. 2005;5(1):65–72. Woude GFV, Klein G. Advances in Cancer Research. Elsevier; 2004. p. 219. Norn S, Permin H, Kruse E, Kruse PR. Mercury-a major agent in the history of medicine and alchemy. Dan Med Arbog. 2008;36:21–40. Dasari S, Bernard P. Cisplatin in cancer therapy: Molecular mechanisms of action. Eur J Pharmacol. 2014;740:364–78. Wheate NJ, Walker S, Craig GE, Oun R. The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Trans. 2010;39(35):8113. Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, et al. Molecular mechanisms of cisplatin resistance. Oncogene. 2012;31(15):1869–83. Pabla N, Dong Z. Curtailing side effects in chemotherapy: a tale of PKCδ in cisplatin treatment. Oncotarget. 2012;3(1):107–11. McKeage MJ, et al. Comparative Adverse Effect Profiles of Platinum Drugs. Drug Saf. 1995;13(4):228–44. Gamelin E, Gamelin L, Bossi L, Quasthoff S. Clinical aspects and molecular basis of oxaliplatin neurotoxicity: Current management and development of preventive measures. Semin Oncol. 2002;29(5 Suppl 15):21–33. Galanski M, Jakupec MA, Keppler BK. Update of the preclinical situation of anticancer platinum complexes: novel design strategies and innovative analytical approaches. Curr Med Chem. 2005;12(18):2075–94. Cepeda V, Fuertes MA, Castilla J, Alonso C, Quevedo C, Perez JM. Biochemical Mechanisms of Cisplatin Cytotoxicity. Anti-Cancer Agents Med Chem- Anti-Cancer Agents. 2007;7(1):3–18. Montana AM, Batalla C. The Rational Design of Anticancer Platinum Complexes: The Importance of the Structure-Activity Relationship. Curr Med Chem. 2009;16(18):2235–60. Khoury A, Deo KM, Aldrich-Wright JR. Recent advances in platinum-based chemotherapeutics that exhibit inhibitory and targeted mechanisms of action. J Inorg Biochem. 2020;207:111070. Kashif Amir M, Hogarth G, Khan Z, Imran M. Zia-ur-Rehman. Platinum(II) dithiocarbamate complexes [Pt(S2CNR2)Cl(PAr3)] as anticancer and DNA-damaging agents. Inorganica Chim Acta. 2020;512:119853. Akitsu T, Tsvetkova D, Yamamoto Y, Nakane D, Kostova I. From Basics of Coordination Chemistry to Understanding Cisplatin-analogue Pt Drugs. Curr Pharm Des. 2023;29(22):1747–74. Wilson JJ, Lippard SJ. Synthetic methods for the preparation of platinum anticancer complexes. Chem Rev. 2014;114(8):4470–95. Islam F, Khatun H, Khatun M, Ali SMM, Khanam JA. Growth inhibition and apoptosis of Ehrlich ascites carcinoma cells by the methanol extract of Eucalyptus camaldulensis. Pharm Biol. 2014;52(3):281–90. Yesmin Y, Das R, Belal PK, Aktar H, Siddika S, Asha M. Anticancer potential of Michelia champaca Linn. bark against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice. Nat Prod J. 2021;11(1):85–96. Goel MK, Khanna P, Kishore J. Understanding survival analysis: Kaplan-Meier estimate. Int J Ayurveda Res. 2010;1(4):274–8. Dudley WN 1. Wickham,2 R, COOMBS,3 N. An Introduction to Survival Statistics: Kaplan-Meier Analysis. J Adv Pract Oncol. 2016;7(1):91–100. Perveen R, Islam F, Khanum J, Yeasmin T. Preventive effect of ethanol extract of Alpinia calcarata Rosc on Ehrlich’s ascitic carcinoma cell induced malignant ascites in mice. Asian Pac J Trop Med. 2012;5(2):121–5. Jyoti BC, Balaji KS, Jayarama S, Wethroe K. Evaluation of apoptogenic effects of Averrhoa bilambi extract on enrlich ascites carcinoma bearing mice. Asian J Pharm Clin Res. 2016;9:348–52. Islam F, Ali SMM, Khanam JA. Hepatoprotective effect of acetone semicarbazone on Ehrlich ascites carcinoma induced carcinogenesis in experimental mice. Asian Pac J Trop Biomed. 2013;3(2):105–10. Islam F, Khanam JA, Khatun M, Zuberi N, Khatun L, Kabir SR, et al. A p-Menth-1-ene-4,7-diol (EC-1) from Eucalyptus camaldulensis Dhnh. Triggers Apoptosis and Cell Cycle Changes in Ehrlich Ascites Carcinoma Cells. Phytother Res. 2015;29(4):573–81. Islam F, Ghosh S, Khanam JA. Antiproliferative and hepatoprotective activity of metabolites from Corynebacterium xerosis against Ehrlich Ascites Carcinoma cells. Asian Pac J Trop Biomed. 2014;4:S284–92. Auwal A, Al Banna MH, Pronoy TUH, Hossain MM, Rashel KM, Kabir SR et al. In vitro and In vivo Growth Inhibition and Apoptosis of Cancer Cells by Ethyl 4-[(4-methylbenzyl)oxy] Benzoate Complex. Anticancer Agents Med Chem. 2025. Cardiff RD, Munn RJ, Galvez JJ. The tumor pathology of genetically engineered mice: a new approach to molecular pathology. In The mouse in biomedical research 2007 Jan 1 (pp. 581–622). Academic. Knoblaugh SE, Hohl TM, La Perle KMD. Pathology Principles and Practices for Analysis of Animal Models. ILAR J. 2018;59(1):40–50. Ibrahim KE, Al-Mutary MG, Bakhiet AO, Khan HA. Histopathology of the Liver, Kidney, and Spleen of Mice Exposed to Gold Nanoparticles. Molecules. 2018;23(8):1848. Esteban-Fernández D, Verdaguer JM, Ramirez-Camacho R, Palacios MA, Gómez-Gómez MM. Accumulation, fractionation, and analysis of platinum in toxicologically affected tissues after cisplatin, oxaliplatin, and carboplatin administration. J Anal Toxicol. 2008;32(2):140–6. Johnstone TC, Park GY, Lippard SJ. Understanding and Improving Platinum Anticancer Drugs – Phenanthriplatin. Anticancer Res. 2014;34(1):471–6. Noureldeen AFH, Qusti SY, Al-shammari EM. Antitumor and Antioxidant Activities of a Novel Platinum(II)-PyrazineCarboxamide Complex Against Ehrlich Ascites Carcinoma Induced in Mice. Int J Pharm Phytopharm Res. 2017;7(6). Hossain MM, Soha K, Rahman A, Auwal A, Pronoy TUH, Islam F. Rhodium complex [RhLI2] I: a novel anticancer agent inducing tumor inhibition and apoptosis. Discover Oncol. 2024;15(1):782. Gaspar BL, Sharma P, Das R. Anemia in malignancies: Pathogenetic and diagnostic considerations. Hematology. 2015;20(1):18–25. Gilreath JA, Stenehjem DD, Rodgers GM. Diagnosis and treatment of cancer-related anemia. Am J Hematol. 2014;89(2):203–12. Jameus A, Kennedy AE, Thome C. Hematological changes following low dose radiation therapy and comparison to current standard of care cancer treatments. Dose-Response. 2021;19(4):15593258211056196. Galluzzi L, Buqué A, Kepp O, Zitvogel L, Kroemer G. Immunogenic cell death in cancer and infectious disease. Nat Rev Immunol. 2017;17(2):97–111. Mantovani A, Marchesi F, Malesci A, Laghi L, Allavena P. Tumour-associated macrophages as treatment targets in oncology. Nat Rev Clin Oncol. 2017;14(7):399–416. Lawen A. Apoptosis—an introduction. BioEssays. 2003;25(9):888–96. Pfeffer CM, Singh ATK. Apoptosis: A Target for Anticancer Therapy. Int J Mol Sci. 2018;19(2):448. Alonso FJ, Segura JA, Lora J, Lobo C, Fernández-Molina B, Márquez J et al. Sensitisation of Ehrlich Ascitic Tumour Cells to Methotrexate by Inhibiting Glutaminase. ANTICANCER Res. 2005. Icsel C, Yilmaz VT, Aygun M, Cevatemre B, Alper P, Ulukaya E. Palladium(ii) and platinum(ii) saccharinate complexes with bis(diphenylphosphino)methane/ethane: synthesis, S-phase arrest and ROS-mediated apoptosis in human colon cancer cells. Dalton Trans. 2018;47(33):11397–410. Reed JC. Mechanisms of Apoptosis. Am J Pathol. 2000;157(5):1415–30. Kiraz Y, Adan A, Kartal Yandim M, Baran Y. Major apoptotic mechanisms and genes involved in apoptosis. Tumor Biol. 2016;37(7):8471–86. Savitskaya MA, Onishchenko GE. Mechanisms of apoptosis. Biochem Mosc. 2015;80(11):1393–405. Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999;6(2):99–104. Thornberry NA, Lazebnik Y, Caspases. Enemies Within Sci. 1998;281(5381):1312–6. Oun R, Moussa YE, Wheate NJ. The side effects of platinum-based chemotherapy drugs: a review for chemists. Dalton Trans. 2018;47(19):6645–53. Herradón E, González C, Uranga JA, Abalo R, Martín MI, López-Miranda V. Characterization of cardiovascular alterations induced by different chronic cisplatin treatments. Front Pharmacol. 2017;8:196. Singh TD, Barbhuiya MA, Poojary S, Shrivastav BR, Tiwari PK. The liver function test enzymes and glucose level are positively correlated in gallbladder cancer: a cancer registry data analysis from north central India. Indian J Cancer. 2012;49(1):125–36. Ghadrdan E, Sadighi S, Ebrahimpour S, Abdollahi A, Hadjibabaei M, Gholami K, et al. The effect of melatonin on cisplatin-induced nephrotoxicity: A pilot, randomized, double-blinded, placebo-controlled clinical trial. Eur J Integr Med. 2020;34:101065. Young DR, Virolainen P, Inoue N, Frassica FJ, Chao EYS. The Short-Term Effects of Cisplatin Chemotherapy on Bone Turnover. J Bone Min Res. 1997;12(11):1874–82. Pezeshki Z, Khosravi A, Nekuei M, Khoshnood S, Zandi E, Eslamian M, et al. Time course of cisplatin-induced nephrotoxicity and hepatotoxicity. J Nephropathol. 2017;6(3):163–7. Zhang WR, Parikh CR. Biomarkers of Acute and Chronic Kidney Disease. Annu Rev Physiol. 2019;81:309–33. Pabla N, Dong Z. Cisplatin nephrotoxicity: Mechanisms and renoprotective strategies. Kidney Int. 2008;73(9):994–1007. Wang G, Su C, Yin T. Paclitaxel and platinum-based chemotherapy results in transient dyslipidemia in cancer patients. Mol Clin Oncol. 2017;6(2):261–5. Okafor IA, Nnamah US, Nnaka J. Lipid and cardiac profile assessment following the co-administration of Cisplatin and methanolic extract of Portulaca oleracea in adult Wistar rats: an experimental study. Bali Med J. 2020;9(3):799–805. Ozkok A, Edelstein CL. Pathophysiology of Cisplatin-Induced Acute Kidney Injury. BioMed Res Int. 2014;2014(1):967826. Feitosa IB, Mori B, Teles CBG, Costa AGD. What are the immune responses during the growth of Ehrlich’s tumor in ascitic and solid form? Life Sci. 2021;264:118578. De Jong M, Maina T. Of Mice and Humans: Are They the Same?—Implications in Cancer Translational Research: TABLE 1. J Nucl Med. 2010;51(4):501–4. Bhat N, Al-Mathkour M, Maacha S, Lu H, El-Rifai W, Ballout F. Esophageal adenocarcinoma models: a closer look. Front Mol Biosci. 2024;11:1440670. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7229944","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":521760522,"identity":"1b650f78-91c3-4f60-a42d-3857689b7f5b","order_by":0,"name":"Tasnima Kamal","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Tasnima","middleName":"","lastName":"Kamal","suffix":""},{"id":521760524,"identity":"2aa8fe9a-60d2-458e-80db-9f2ef813e8eb","order_by":1,"name":"Azmin Akter","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Azmin","middleName":"","lastName":"Akter","suffix":""},{"id":521760527,"identity":"48f31bcc-51af-4735-a059-aeb13089faa0","order_by":2,"name":"Asmaulhusna Biswas","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Asmaulhusna","middleName":"","lastName":"Biswas","suffix":""},{"id":521760528,"identity":"5f72bd89-5283-4699-b52a-ff0aba537fe4","order_by":3,"name":"Sharmin Akhter","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Sharmin","middleName":"","lastName":"Akhter","suffix":""},{"id":521760531,"identity":"df6eb634-284d-4ade-8323-1b67a6432245","order_by":4,"name":"Mahmud Ismail","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Mahmud","middleName":"","lastName":"Ismail","suffix":""},{"id":521760533,"identity":"33253307-4af3-4a9e-96cf-283567856c2c","order_by":5,"name":"Zinnat Ara Moni","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Zinnat","middleName":"Ara","lastName":"Moni","suffix":""},{"id":521760535,"identity":"509e3c47-e50f-456a-ab27-e8eb7025593d","order_by":6,"name":"Azadur Rahman Bhuiyan","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Azadur","middleName":"Rahman","lastName":"Bhuiyan","suffix":""},{"id":521760538,"identity":"4afb3737-7ce6-4f2c-882b-f786b4b06030","order_by":7,"name":"Abdul Auwal","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Abdul","middleName":"","lastName":"Auwal","suffix":""},{"id":521760539,"identity":"416901a7-18d5-4f94-9f86-10f2c867aebd","order_by":8,"name":"Md. 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1","display":"","copyAsset":false,"role":"figure","size":170727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the synthetic route of the target ligand and metal complex\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7229944/v1/ad5b1c425e603e2fbb7ee221.jpg"},{"id":92444030,"identity":"70683c35-fef2-4f71-9db3-7e6329e69585","added_by":"auto","created_at":"2025-09-29 19:43:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":399547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo antineoplastic activity of the compound and effect of the test complex on haematological parameters of EAC-bearing mice. \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003eA)\u003c/strong\u003e denotes the number of viable EAC cells in different doses. \u003cstrong\u003eB)\u003c/strong\u003e Shows the percentage of cell growth inhibition. \u003cstrong\u003eC) \u003cbr\u003e\n \u003c/strong\u003eKaplan–Meier survival curves of tumor-bearing mice treated with varying doses of a test compound. Mice were divided into three groups: untreated control (black), low dose (blue), and high dose (purple). Survival was monitored for up to 35 days. Both treatment groups showed improved survival compared to the control, with the high dose group demonstrating the most prolonged survival. Data are presented as mean ± SEM (Standard Error of the Mean).\u003cstrong\u003e D)\u003c/strong\u003e Increase in lifespan of treated and non-treated mice. \u003cstrong\u003eE)\u003c/strong\u003e Management of tumor weight. The number of mice in each group (n=6); the results were shown as mean ± SEM. Where significant values are *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001. \u003cstrong\u003eF) \u003c/strong\u003eRed Blood Cell (RBC) count (cells per mL): The control group shows a reduced RBC count compared to the normal group. However, the high-dose treatment significantly increases RBC count compared to the control group (***p \u0026lt; 0.001), suggesting a protective or restorative effect. \u003cstrong\u003eG) \u003c/strong\u003eWhite Blood Cell (WBC) count (cells per mL): The control group exhibits a markedly increased WBC count compared to the normal group, indicating a potential inflammatory response. Both low-dose and high-dose treatments appear to reduce WBC levels, suggesting a modulatory effect. \u003cstrong\u003eH)\u003c/strong\u003e Hemoglobin (Hb) levels (g/dL): The control group shows decreased hemoglobin levels relative to the normal group. Low-dose and high-dose treatments improve hemoglobin levels, with the high dose showing the most restoration. \u003cstrong\u003eI)\u003c/strong\u003e Platelet count (cells per mL): The control group has a reduced platelet count compared to the normal group. While the low-dose treatment shows partial recovery, the high-dose treatment fully restores platelet levels to near-normal values\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7229944/v1/6fb23ce0c54ae9686796712e.jpg"},{"id":92444027,"identity":"6045cc66-4437-4cd5-bed6-85a995e4a1c7","added_by":"auto","created_at":"2025-09-29 19:43:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":412035,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopic view of control and treated EAC cells\u003c/strong\u003e. \u003cstrong\u003eA).\u003c/strong\u003e Control EAC cells showed no apoptotic features under an optical microscope. \u003cstrong\u003eB).\u003c/strong\u003e EAC cells treated with the compound showed nuclear condensation, fragmentation, cell membrane blebbing, apoptotic bodies, etc., (indicated by arrows) under an optical microscope. \u003cstrong\u003eC)\u003c/strong\u003e Cells showed no apoptotic (round and smooth nuclear material, intact membrane, etc.) features under a fluorescence microscope. \u003cstrong\u003eD)\u003c/strong\u003e. EAC cells treated with the compound showed nuclear condensation, fragmentation, cell membrane blebbing, apoptotic bodies, etc. (indicated by arrows) under the fluorescence microscope. Scale bar: 40 µm. \u003cstrong\u003eE).\u003c/strong\u003eDNA fragmentation assays showed the smear-like DNA in agarose gel (1.5%) electrophoresis\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7229944/v1/f6261d85dfa2f31861ed88f0.jpg"},{"id":92445135,"identity":"d3ecf075-0a1b-4f45-8732-b1f2323928e3","added_by":"auto","created_at":"2025-09-29 19:59:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":353637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Pt (II) L complex on normal peritoneal cells.\u003c/strong\u003eStaining with Neutral Red to determine the number of macrophage cells in normal and treated mice. (A) Macrophage cells from normal mice treated with Pt (II) L complex, showing an increased number of stained macrophages. (B) Macrophage cells from untreated normal mice (control), displaying a comparatively lower number of stained macrophages. (C) Quantitative representation of macrophage cell numbers in both groups, indicating a significant increase in macrophage count in the Pt (II) L complex-treated group compared to the control. Scale bar: 40 µm\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7229944/v1/8fdeba1bece3b6d9b0bf81a9.jpg"},{"id":92444033,"identity":"05201bd4-a457-44d1-8820-f20ebf666780","added_by":"auto","created_at":"2025-09-29 19:43:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":298835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of the test compound on apoptotic gene expression\u003c/strong\u003e. \u003cstrong\u003eA)\u003c/strong\u003e Relative expression of \u003cem\u003ep53, Bax, Bcl2, Caspase-9, \u003c/em\u003eand\u003cem\u003e Caspase-3\u003c/em\u003e in EAC cells \u003cem\u003ein vivo\u003c/em\u003e treated and untreated with the Pt (II) L complex. Total RNA was extracted and reverse transcribed. Then, the generated cDNA was used as a template for a Rt-qPCR relative expression analysis with a qPCR Master Mix. Data were analyzed by ΔΔ CTs and normalized to the house-keeping gene (GAPDH). The experiments were carried out using three biological replicates, each with two technical duplicates. Significant values are indicated by * p \u0026lt; 0.05, ** p \u0026lt; 0.01, and *** p \u0026lt; 0.001 when compared to the control group. \u003cstrong\u003eB)\u003c/strong\u003e RT-PCR products of the tested genes in control and compound-treated EAC cells\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7229944/v1/da9d4dcb051ca8d8902c9729.jpg"},{"id":92443675,"identity":"7af9908c-80cc-4c15-9221-de3e74a9fdeb","added_by":"auto","created_at":"2025-09-29 19:35:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1173925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Platinum Schiff base ligand complex on mouse tissues.\u003c/strong\u003e Histopathological examination of key mouse organs. Mice were assessed on the 25th day of treatment. No abnormalities or damage were observed in the organs of the examined mice following the treatment\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7229944/v1/3f4d98181a6352181d83274f.jpg"},{"id":92444254,"identity":"1cb6ef0c-708d-44c8-8c24-a129ee3cf703","added_by":"auto","created_at":"2025-09-29 19:51:21","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":509857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePossible mechanism of anticancer activity of the compound\u003c/strong\u003e. Treatment of EAC cells with Pt (II) L complex induces upregulation of proapoptotic genes such as p53, Bax, Caspase-9, -3 and downregulates antiapoptotic Bcl-2. The Pt (II) L complex binds with a receptor and enters the nucleus, where it attaches itself to the DNA and induces damage, causing an upregulation of p53 (pro-apoptotic gene) followed by the disruption of Bax/ Bcl-2 equipoise between them, ultimately triggering caspase-mediated apoptosis of the EAC cells. This indicates that the Pt (II) L complex uses an intrinsic pathway for its anticancer activity\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7229944/v1/3ba60dd6b518c78e5edf31f4.jpg"},{"id":94988059,"identity":"afb8dd7c-7e6d-45fc-ad46-230385074b04","added_by":"auto","created_at":"2025-11-03 07:04:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4761689,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7229944/v1/e6c25a84-ab02-47db-a4ef-38a586753c47.pdf"},{"id":92444031,"identity":"8b8cb5ca-4095-4f47-b5e0-9623e5790600","added_by":"auto","created_at":"2025-09-29 19:43:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":910125,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7229944/v1/ff3b75f95527479b7674b068.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Apoptotic and anti-proliferative activity of novel platinum complex [Pt((E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2- (pyridine-2-ylmethylene)hydrazine-1-carbothioamide)] against Ehrlich Ascites carcinoma (EAC) cells in vivo","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer is a complex and devastating disease characterized by the uncontrolled growth and spread of abnormal cells. It arises from a disruption of the delicate balance between cell proliferation and cell death [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], leading to the accumulation of cells that can invade the surrounding tissues and metastasize to distant organs. This intricate process is driven by a multitude of factors including genetic mutations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], environmental exposures and lifestyle choices [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe global burden of cancer is substantial and continues to increase, posing a significant challenge to public health. According to the World Health Organization (WHO), cancer is one of the leading causes of death worldwide. It is estimated that there were 19.3\u0026nbsp;million new cases of cancer and approximately 10\u0026nbsp;million deaths from cancer in 2020 (Sung et al., 2021). This affects millions of individuals and families, transcending borders, cultures, and economics, posing challenges not only to the health system but also to socioeconomic development. Female breast cancer (2.26\u0026nbsp;million cases), lung (2.21\u0026nbsp;million), and prostate cancer (1.41\u0026nbsp;million) were the most frequently diagnosed malignancies globally. The most prevalent causes of cancer mortality were lung (1.79\u0026nbsp;million deaths), liver (830,000), and stomach cancers (769000) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It is anticipated that there will be 28.4\u0026nbsp;million new cases of cancer worldwide in the year 2040, which represents a 47% increase from the 19.3\u0026nbsp;million cases reported in the year 2020. This is based on the assumption that the national rates calculated in 2020 will remain unchanged (5), and the incidence and mortality rates vary considerably across different cancer types and geographical regions, highlighting the need for targeted prevention and treatment strategies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe development of effective anticancer drugs has been the central focus of biomedical research for decades. Chemotherapy, the cornerstone of cancer treatment, utilizes cytotoxic agents to eliminate rapidly dividing cancer cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the lack of specificity of these agents often leads to severe side effects that limit their therapeutic efficacy. In recent years, significant advances have been made in understanding the molecular mechanisms that drive cancer development and progression. This knowledge has paved the way for the development of targeted therapies that selectively target specific molecules or pathways involved in cancer growth [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Metal-based compounds have been used for therapeutic purposes since ancient times. During this time frame, the ancient Assyrians, Egyptians, and Chinese people understood the significance of employing metal-based compounds in the treatment of medical conditions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Platinum-based chemotherapeutic agents, including cisplatin, carboplatin, and oxaliplatin, have been widely used as cornerstone treatments for various cancers, such as lung, ovarian, testicular, and colorectal cancers [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These drugs exert their anticancer effects primarily through the formation of DNA adducts, leading to the disruption of DNA replication and transcription and ultimately inducing apoptosis in cancer cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite their significant contribution to cancer treatment, these agents have several limitations that reduce their therapeutic efficacy and compromise patient outcomes. The primary challenge with platinum-based drugs is the development of drug resistance. Cancer cells often acquire resistance through multiple mechanisms, including reduced drug uptake, increased efflux, enhanced DNA repair, and detoxification by intracellular thiols such as glutathione [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This resistance not only limits the effectiveness of these agents but also necessitates the use of higher doses, increasing the risk of severe side effects. Another major drawback of cisplatin, carboplatin, and oxaliplatin is their notable toxic effects. For instance, cisplatin is well documented to cause nephrotoxicity, neurotoxicity, and ototoxicity, which can severely impact a patient\u0026rsquo;s quality of life and limit its clinical use [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While carboplatin is associated with reduced nephrotoxicity, it frequently induces myelosuppression, particularly thrombocytopenia [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Oxaliplatin, commonly used in the treatment of colorectal cancer, is known for its dose-limiting peripheral neuropathy, which can be either acute or cumulative, often necessitating dose reduction or treatment discontinuation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe clinical success of the platinum-based drug cisplatin (cis-diamminedichloroplatinum (1r)) in anticancer chemotherapy has prompted an all-out search for analogs with reduced toxicity, better therapeutic indices, and enhanced activity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The development of new platinum drugs with anticancer activity has been a topic of significant interest in the field of bioinorganic chemistry. The success of cisplatin as an anticancer drug has stimulated research in this area, leading to the approval of carboplatin and oxaliplatin for routine clinical use [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Other platinum drugs, such as nedaplatin, lobaplatin, and heptaplatin, have been approved in specific regions, highlighting the importance of exploring novel platinum complexes for their anticancer potential [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Efforts have been made to elucidate the biochemical mechanisms of cisplatin cytotoxicity to design new platinum-based drugs with improved pharmacological profiles [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The rational design of anticancer platinum complexes emphasizes the importance of understanding the structure-activity relationship to optimize the anticancer activities of these compounds [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This process involves consideration of the mechanisms of action, potential resistance mechanisms of cancer cells, and pharmacokinetic and toxicity properties to ensure the clinical usefulness of new platinum drugs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Recent advances in platinum-based chemotherapeutics have focused on the development of drugs with inhibitory and targeted mechanisms of action [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The study of platinum (II) dithiocarbamate complexes as anticancer and DNA-damaging agents further underscores the importance of exploring new platinum compounds for their potential anticancer activities [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, the development of strategies for the design of platinum anticancer drugs has been highlighted as a crucial aspect in advancing the field [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Overall, the importance of new platinum drugs for anticancer activity lies in their potential to provide alternative treatment options with improved efficacy and fewer side effects. By understanding the structure-activity relationship and exploring novel platinum complexes, researchers aim to develop innovative anticancer drugs that can address the limitations of current platinum-based chemotherapeutics [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, the focus of the present study was to delve into the current landscape of the Platinum Schiff base ligand complex for the development of anticancer drugs, explore its activity against EAC (Ehrlich ascites carcinoma) cells, and investigate its underlying molecular mechanism.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eChemicals and reagents\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll chemicals and reagents used in this experiment were of analytical grade and purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA), Promega (Madison, Wisconsin, USA), Amresco (11 Speen Street Framingham, Massachusetts, USA), Life Technologies (5791 Van Allen Way Carlsbad, CA, USA), and Applied Biosystems (180 Oyster Point Blvd South San Francisco, CA, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynthesis and characterization of Platinum complex\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe unbound ligand L[(E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2-(pyridine-2-ylmethylene) hydrazine-1-carbothioamide] and its platinum complex were synthesized according to a previously published protocol [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the Platinum complex was synthesized in three steps: (1) synthesis of (\u003cem\u003eE\u003c/em\u003e)-2-(pyridine-2-ylmethylene) hydrazine-carbothioamide, (2) Synthesis of Ligand [(E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2-(pyridine-2-ylmethylene) hydrazine-1-carbothioamide] (L), and (3) Synthesis of Pt (II) L complex. The final product was extracted with chloroform. The chloroform extract was removed by evaporation and dried over silica gel in a vacuum desiccator. The compound was characterized based on its physical properties (melting temperature, color, and solubility), IR spectra, and \u003csup\u003e1\u003c/sup\u003eH NMR.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal, cell line, and ethical statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe University Ethics Committee (Institutional Animal, Medical Ethics, Biosafety, and Biosecurity Committee for Experiments on Animal, Human, Microbes, and Living Natural Sources (No. 293(13)/320-IAMEBBC/IBSc), Institute of Biological Sciences, Rajshahi University, Bangladesh) approved the use of experimental animals (mice). This study was carried out in strict accordance with the approved guidelines. Adult male Swiss albino mice weighing 20\u0026ndash;25 g were used. The mice were meticulously raised in our laboratory according to the standards, protocols, and regulations established by the Institutional Animal, Medical Ethics, Biosafety, and Biosecurity Committee. In addition, the work described herein was carried out following the National Institutes of Health Office of Laboratory Animal Welfare policies and laws and complied with the ARRIVE guidelines. Mice were housed in standard polypropylene cages in well-ventilated rooms, under a 12 h light/12 h dark cycle at a temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ℃, and maintained under hygienic conditions. Standard food and drinking water were given \u003cem\u003ead libitum\u003c/em\u003e at a natural day-night cycle. All animal welfare considerations, including efforts to minimize the suffering and distress of the animals, were taken. The health and behavior of animals were monitored twice every day (morning and evening) during the experiments. At the end of the experiments, the mice were placed in a 30 cm \u0026times; 10 cm anesthesia chamber for acclimation. The induction of anesthesia was carried out using 5% Isoflurane in oxygen at a flow rate of 1.2 L/minute for 2\u0026ndash;4 minutes until the animal was fully anesthetized. The breathing pattern, anesthesia depth in mice were monitored using regular toe pinch tests to examine the anesthesia levels to ensure that the animals were adequately sedated without being overanesthetized. After that, the animals were placed on a surgical table, and 1\u0026ndash;2% isoflurane in oxygen with a flow rate of 1.2 L/minute was used during all surgeries to maintain anesthesia, and the cervical dislocation method was used to sacrifice them to ensure the humane endpoints of the animals. All efforts were made to minimize the suffering of the animals.\u003c/p\u003e\u003cp\u003eEhrlich Ascites Carcinoma (EAC) cells were obtained from the Indian Institute for Chemical Biology (IICB) in Kolkata, India, with their kind assistance. Under controlled ambient conditions in our department laboratory, 10\u003csup\u003e5\u003c/sup\u003e cells per animal were administered intraperitoneally every two weeks to maintain the viability of these cells. No animals were dead before the completion (endpoints) of the experiment. Research students were trained in animal handling and maintenance for better management of the experimental animals.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInhibition of EAC cell growth\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e inhibition of EAC cell growth was assessed according to a previously published protocol [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Four groups of mice, each consisting of six mice, were used in this experiment. Each of the mice was inoculated with 1.6 \u0026times; 10⁶ EAC cells intraperitoneally on the first day. The treatment was initiated 24 hours after inoculation and was administered for five consecutive days. At concentrations of 2.0 mg/kg and 5.0 mg/kg body weight, the platinum (II) complex was administered intraperitoneally to Groups 2, 3, and 4, and the standard drug (Bleomycin 0.3 mg/kg), respectively. Each mouse received daily injections of 0.1 ml. Normal saline was administered intraperitoneally to Group 1, which served as the control. On the sixth day (24 hours after the last treatment), the mice were sacrificed, and peritoneal fluid was harvested using 0.98% saline. Viable tumor cells were quantified using a hemocytometer following trypan blue staining under an inverted microscope (XDS-1R; Optika, Bergamo, Italy), and tumor cell growth inhibition was calculated using the following formula:\u003c/p\u003e\u003cp\u003e% Cell growth inhibition = (C- T / C) \u0026times; 100\u003c/p\u003e\u003cp\u003ewhere T\u0026thinsp;=\u0026thinsp;Mean number of tumor cells in the treated group of mice and C\u0026thinsp;=\u0026thinsp;Mean number of tumor cells in the control group of mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAverage tumor weight and survival time\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe previously described method was employed to assess the tumor weight and survival time of EAC-bearing mice [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For this experiment, three groups (6 mice in each group) of Swiss albino mice were taken, and each of them was inoculated with 1.6\u0026times;10\u003csup\u003e6\u003c/sup\u003e EAC cells. Group 1 was considered as control, and 24 hours after inoculation, groups 2 and 3 received 2.0 \u0026amp; 5.0 mg/kg/day body weight of the treatment intraperitoneally. The treatment was continued for 10 consecutive days. Daily variations in weight were recorded to track tumor progression. Survival data were analyzed using the Kaplan-Meier method to estimate the survival probability over time for each experimental group. Differences in survival distributions between groups were evaluated using the log-rank (Mantel-Cox) test [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The following formula was used to calculate the percentage increase in lifetime after host survival was recorded and expressed as the mean survival time in days:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{M}\\text{e}\\text{a}\\text{n}\\:\\text{s}\\text{u}\\text{r}\\text{v}\\text{i}\\text{v}\\text{a}\\text{l}\\:\\text{t}\\text{i}\\text{m}\\text{e}\\:\\left(\\text{M}\\text{S}\\text{T}\\right)\\:=\\sum\\:\\frac{\\text{S}\\text{u}\\text{r}\\text{v}\\text{i}\\text{v}\\text{a}\\text{l}\\:\\text{t}\\text{i}\\text{m}\\text{e}\\:\\left(\\text{d}\\text{a}\\text{y}\\text{s}\\right)\\text{o}\\text{f}\\:\\text{e}\\text{a}\\text{c}\\text{h}\\:\\text{m}\\text{o}\\text{u}\\text{s}\\text{e}\\:\\text{i}\\text{n}\\:\\text{a}\\:\\text{g}\\text{r}\\text{o}\\text{u}\\text{p}}{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}\\:\\text{o}\\text{f}\\:\\text{m}\\text{i}\\text{c}\\text{e}}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{P}\\text{e}\\text{r}\\text{c}\\text{e}\\text{n}\\text{t}\\text{a}\\text{g}\\text{e}\\:\\text{i}\\text{n}\\text{c}\\text{r}\\text{e}\\text{a}\\text{s}\\text{e}\\:\\text{o}\\text{f}\\:\\text{l}\\text{i}\\text{f}\\text{e}\\:\\text{s}\\text{p}\\text{a}\\text{n}\\:\\left(\\text{I}\\text{L}\\text{S}\\text{%}\\right)=\\frac{\\text{M}\\text{S}\\text{T}\\:\\text{o}\\text{f}\\:\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d}\\:\\text{g}\\text{r}\\text{o}\\text{u}\\text{p}}{\\text{M}\\text{S}\\text{T}\\:\\text{o}\\text{f}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:\\text{g}\\text{r}\\text{o}\\text{u}\\text{p}}-1\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of hematological parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHematological parameters were evaluated by observing the changes in hemoglobin (Hb), RBC, WBC, and platelet counts in comparison with normal mice. A hemocytometer and cell dilution fluids were used to assess the effects of the platinum (II) ligand complex. Four groups of mice were used, each comprising six mice [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Normal mice were assigned to group 1, whereas 0.1 ml of EAC (1.6\u0026times;10\u003csup\u003e6\u003c/sup\u003e) cells was inoculated intraperitoneally into the rest of the groups. Here, group 2 served as the control group, and groups 3 and 4 received a dose of 2.0 mg/kg and 5.0 mg/kg body weight of the Platinum (II) ligand complex per day, for 10 days consecutively. Blood samples were obtained through tail puncture on the 12th day after inoculation, and hematological parameters were subsequently assessed [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eMorphological changes and nuclear damage\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe induction of apoptosis by the platinum (II) ligand complex was examined as previously reported [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Morphological observations of both treated and untreated EAC cells were studied by observing the changes in phase contrast images and staining them with Geimsa stain using an inverted microscope (XDS-1R, Optika, Bergamo, Italy). Further nuclear damage was confirmed by PI staining, using a fluorescence microscope (Olympus IX71, Japan). First, EAC cells were collected from the mice receiving platinum (II) ligand complex at a dose of 5.0 mg/kg body weight and saline (non-treated control) and stained with 10 \u0026micro;L (1 mg/ml) of Propidium Iodide (PI) at 37\u0026deg;C for 20 min in the dark. Subsequently, the cells were washed with phosphate-buffered saline (PBS), and morphological changes were visualized using a fluorescence microscope. In addition, a DNA fragmentation assay was performed to examine the apoptotic cleavages of the genomic materials of EAC cells receiving the treatment [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of Pt (II) L complex on normal peritoneal cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe influence of the Pt (II) L complex on normal peritoneal cells was assessed by quantifying the total peritoneal cells and macrophages. Mice were divided into three groups (n\u0026thinsp;=\u0026thinsp;6 per group), with two groups receiving the Pt (II) L complex (5.0 and 2.0 mg/kg, i.p.) for three consecutive days, while the third group served as an untreated control. Twenty-four hours after the final treatment, 5 mL 0.98% normal saline was injected into the peritoneal cavity before sacrificing the animals. The collected intraperitoneal exudate cells and macrophages were stained with 1% neutral red and counted using a hemocytometer.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction of mRNA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA from the EAC cells was extracted using the manufacturer\u0026rsquo;s guidelines for the total RNA extraction kit (Favorgen Biotech Corp. Ping-Tung, Taiwan) from mice receiving 5.0 mg/kg/day and control EAC-bearing mice on day six of tumor implantation. RNA concentration and purity were precisely quantified using a NanoDrop spectrophotometer (NanoDrop One, Thermo Scientific, Waltham, MA, USA). Additionally, the mean absorption ratios, A260/280 and A260/230 were carefully evaluated to verify the purity of the sample. The structural integrity of the RNA was further confirmed by electrophoresis on a 1.8% agarose gel. The extracted RNA was stored at -80\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynthesis of cDNA\u003c/b\u003e\u003c/p\u003e\u003cp\u003ecDNA was prepared for High-Capacity Reverse Transcription PCR (polymerase chain reaction) using the GoScript\u0026trade; Reverse Transcription System (MA, Wisconsin, USA) according to the manufacturer\u0026rsquo;s instructions. For further analysis, the synthesized cDNA was stored at -20\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReverse transcriptase polymerase chain reaction (RT-PCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression of five growth regulatory genes, namely, \u003cem\u003eBcl-2, Bax, p53, Caspase-3, and Caspase-9\u003c/em\u003e, was examined using these cDNA as templates for RT-qPCR, with GAPDH as the control. The reaction mixture (20 \u0026micro;L) was prepared using the GoTaq\u0026reg; qPCR Master Mix Kit (MA, Wisconsin, USA) according to the manufacturer\u0026rsquo;s guidelines. The primer sequences and thermal cycling conditions are listed in Supplementary Information (SI 1 Table). Quantitative RT-PCR experiments were performed using three biological replicates for each experimental group, with each biological sample analyzed in two technical duplicates. A gradient thermal cycler was used for amplification (LightCycler\u0026reg; 96 Instrument, Roche Diagnostics, Forrenstrasse 2, 6343 Rotkreuz, Switzerland) was used to amplify the target genes. The comparative Ct (ΔΔCt) method was used to analyze the data. This method involved comparing the quantity of the target gene to that of the endogenous control GAPDH to determine the relative expression levels [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eToxicity studies\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of test compound on biochemical and hematological parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe toxicological effects of the compounds were evaluated by observing anomalies in the blood and biochemical parameters. For this evaluation, 40 mice, each weighing between 22 and 25 g, were divided into two groups: a control group consisting of 20 normal male mice, and a high-dose treatment group comprising 20 male mice. Baseline blood samples were collected from all mice prior to administration of the test compound to establish reference biochemical and hematological parameters. Additionally, the mice in the treatment group received the platinum (II) ligand complex intraperitoneally at a dosage of 5.0 mg/kg using a 1 ml syringe over 10 days. Following treatment, both groups of mice were anesthetized with ethyl acetate, and samples were collected on days 5, 10, and 25 post-treatment to evaluate sub-acute toxic effects of the compound using surgical blade no.22. Using a 3 ml syringe, fresh blood was collected from the heart in EDTA-free tubes. The blood samples were allowed to clot at room temperature for approximately 30 min. This allowed the blood cells to settle at the bottom of the tube. Following clotting, the blood samples were centrifuged at 7000 rpm for 12 min on a WIFUNG centrifuge (LABO-50M). Finally, clear straw-colored serum was collected in microcentrifuge tubes using a Pasteur pipette and refrigerated at -20\u0026deg;C. Finally, biochemical parameters of these sera were analyzed using a bioanalyzer (Humalyzer 3000, HUMAN Diagnostics Worldwide, Wiesbaden, Germany) following the previously mentioned method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor the assessment of hematological parameters, fresh blood was extracted from the heart using a 3 ml syringe and collected in an Eppendorf tube containing ethylenediaminetetraacetic acid (EDTA) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. hematological parameters, including WBC count, RBC count, platelet count, and Hb level, were evaluated using a standardized procedure [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, the effects of chemicals at the cellular level were assessed through histopathological analysis of the major organs in animals treated with the compound according to previously established protocols [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. All tissues were immediately fixed using 10% neutral buffered formalin [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The tissues were subsequently dissected to accommodate cassettes. Fixed specimens of the liver, kidney, heart, and lungs were subjected to overnight processing for dehydration, clearing, and impregnation using an automatic tissue processor (automatic sample preparation system, Histo-line laboratories, Milan, Italy). The specimens were embedded in paraffin blocks using an embedding station (TEC2900 embedding center, Histo-line laboratories, Milan, Italy), and serial sections of 5 \u0026micro;m thickness were obtained using a microtome (Histo-line Laboratories, Milan, Italy). The sections were stained with Hematoxylin and Eosin [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The mounted specimens were examined under a light microscope.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (Standard Error of Mean (SEM) was used to express the data (which included the percentage of cell growth inhibition, increase in life span, body/tumor weight, and haematological profile). GraphPad Prism 8 software was used to conduct one-way analysis of variance (ANOVA) and Duncan's multiple range test. Statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of the compound\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe compound was synthesized as shown in the scheme (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and characterized by its physical constants, such as surface, color, solubility, and IR and NMR spectra (SI 2). The physical form was a red-orange powder with a melting temperature of 519\u0026deg;C. This compound was soluble in DMSO and partially soluble in water (H\u003csub\u003e2\u003c/sub\u003eO).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[Insert\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cstrong\u003eabout here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFT-IR spectral analysis of ligand (L)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FT-IR spectrum of ligand L, recorded using a KBr disk, revealed key functional groups (SI 2 Table). A broad absorption at 3200\u0026ndash;3500 cm⁻\u0026sup1; suggests O\u0026ndash;H or N\u0026ndash;H stretching, while a sharp band at 1600\u0026ndash;1700 cm⁻\u0026sup1; indicates C\u0026thinsp;=\u0026thinsp;O stretching from the carbonyl-containing groups. The peaks at 1500\u0026ndash;1600 cm⁻\u0026sup1; correspond to C\u0026thinsp;=\u0026thinsp;C stretching, suggesting aromatic or conjugated systems. The fingerprint region (600\u0026ndash;1500 cm⁻\u0026sup1;) shows characteristic bending and stretching vibrations. These spectral features confirm the structural composition of the ligand, which was further validated using complementary spectroscopic techniques (SI 2 Fig).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026sup1;H-NMR spectral analysis of ligand (L)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u0026sup1;H-NMR spectrum of ligand L was recorded in DMSO-d₆, and the chemical shifts (\u0026delta;) were reported in ppm (SI 3 Table). A downfield signal at \u0026delta;\u0026thinsp;~\u0026thinsp;11.63 ppm suggests an exchangeable proton (-OH or -NH). The aromatic region (\u0026delta; 6.5\u0026ndash;9.0 ppm) exhibited multiple peaks, confirming the presence of aromatic or heteroaromatic rings. Peaks in the aliphatic region (\u0026delta; 1.0\u0026ndash;4.0 ppm) correspond to -CH₂ or -CH₃ groups, with a singlet at \u0026delta; 2.50 ppm from the solvent. The spectrum supports the expected structure of ligand L, indicating the presence of aromatic, aliphatic, and hydrogen-bonded functional groups. Further structural confirmation was achieved through \u0026sup2;D-NMR, \u0026sup1;\u0026sup3;C-NMR, and mass spectrometry (SI 3 Fig).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntineoplastic of Platinum Schiff base ligand complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (A \u0026amp; B) shows the effects of the complex on the proliferation of EAC cells on day 6 after tumor transplantation at doses of 2.0 mg and 5.0 mg/kg. At dosages of 2.0 and 5.0 mg/kg/day body weight of mice, the compound exhibited 45.01% and 62.52% suppression of cell proliferation, respectively, compared to the control. However, bleomycin reduced cell proliferation by 79.57% when administered intraperitoneally at a dose of 0.3 mg/kg. Thus, compared with untreated EAC-bearing mice, this outcome suggests that the test compound exhibited substantial inhibition of EAC cell growth (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[Insert\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cstrong\u003eabout here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival time and tumor weight management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematological profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring tumor progression, significant deviations in hematological parameters from baseline levels were observed. EAC-bearing mice showed significantly decreased RBC and Hb levels following EAC cell inoculation (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF and G). However, WBC and platelet levels were elevated (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH and I) compared to normal mice, showing immune dysfunction. Blood parameters were somewhat restored toward normal levels with the treatment of EAC-bearing mice with the Pt (II) L complex at doses of 5.0 and 2.0 mg/kg/day (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological appearance and nuclear damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphological alterations of EAC cells collected from both control and treated mice were analyzed using PI and Giemsa staining. In Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the morphological changes in both control and treated cells, noted under the fluorescence and optical microscope, are indicated by arrows. Cells from control animals were observed to have round, regular, and normal-shaped nuclei under a microscope (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). In contrast, treated cells exhibited apoptotic features, including condensed, fragmented, irregular nuclei and chromatin, as well as the formation of apoptotic bodies (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). In addition, the generation of DNA fragmentation, a hallmark of apoptosis, results in smear-like DNA bands in agarose gel, indicating the induction of apoptosis of EAC cells, followed by the treatment of the compound (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[Insert\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cstrong\u003eabout here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of Pt (II) L complex on normal peritoneal cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePt (II) L complex shows a dose-dependent increase in macrophage and total peritoneal cell counts. In the control group (normal mice), the total number of peritoneal cell exudates was (3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.03) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, of which (1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) were macrophages after five days of treatment. Mice treated with 5.0 mg/kg body weight of platinum complex showed an increase in both macrophage count and total peritoneal cells. At a lower dose (2.0 mg/kg), there is still an increase in macrophage and total peritoneal cell counts, however, the effect is less pronounced than with 5.0 mg/kg, suggesting that Pt (II) L complex may enhance immune cell recruitment or proliferation in the peritoneal cavity (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[Insert\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cstrong\u003eabout here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of gene expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the inhibition of cell growth and apoptotic body formation, we hypothesized that the Pt (II) L complex would demonstrate potential antiproliferative efficacy against EAC cells by inducing apoptosis. Consequently, we attempted to determine the expression levels of the apoptosis-regulatory genes. We assessed the effect of the Pt (II) L complex on the expression of pro-apoptotic genes, including \u003cem\u003ep53, Bax, Caspase 3\u003c/em\u003e, and \u003cem\u003eCaspase 9\u003c/em\u003e, along with the anti-apoptotic gene \u003cem\u003eBcl-2\u003c/em\u003e, using RT-PCR. While the pro-apoptotic genes \u003cem\u003ep53, Bax, Caspase 3\u003c/em\u003e, and \u003cem\u003eCaspase 9\u003c/em\u003e were upregulated in the treatment group compared to the control, the reduced expression of the anti-apoptotic gene \u003cem\u003eBcl-2\u003c/em\u003e indicated the mitochondrial apoptotic pathway (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[Insert\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cstrong\u003eabout here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eToxicological studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the hematological parameters in Swiss albino mice (without EAC cells) administered the test compound at a dose of 5.0 mg/kg/day for 10 consecutive days, and the blood parameters were measured on days 5, 10, and 25 to assess the detrimental effects of the Pt (II) L complex host (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). RBC, WBC, platelets, and % Hb were found to vary somewhat under treatment; however, they reversed practically towards normal following treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This provided evidence that the Pt (II) L complex did not have long-term toxic side effects on the host (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of Pt (II) L complex on blood parameters in normal mice on days, 0, 5, 10 and 25 at dose 5.0 mg/kg body weight\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExperiment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDays\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRBC cells/ml\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWBC cells/ml\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlatelet cells/ml\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% of Hb\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl (normal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(7.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039) \u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(9.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4) \u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8) \u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eNormal\u0026thinsp;+\u0026thinsp;test compound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;11.57) \u0026times;10\u003csup\u003e6 ***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8) \u0026times;10\u003csup\u003e6 ***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(22.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6) \u0026times;10\u003csup\u003e6 ***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(5.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71) \u0026times;10\u003csup\u003e6 ***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8) \u0026times;10\u003csup\u003e6**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7) \u0026times;10\u003csup\u003e6 ***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(6.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45) \u0026times;10\u003csup\u003e6 *\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(9.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4) \u0026times;10\u003csup\u003e6*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8) \u0026times;10\u003csup\u003e6 **\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eNumber of mice in each group were 6; the results are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and compared with control with normal mice (without treatment) where significant values are *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical profile analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKey biochemical parameters, such as triglycerides, serum cholesterol, creatinine, bilirubin, serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and alkaline phosphatase (ALP), showed notable alterations following a 10-day intraperitoneal administration of 5.0 mg/kg/day of the Pt (II) L complex in Swiss albino mice. Fluctuations in these serum parameters from the normal range were observed on day 5 day 5 and 10, but the parameters seemed to revert towards normal levels on day 25, indicating that the Pt (II) L complex did not have any long-term toxic side effects in the host (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of Pt (II) L complex on biochemical parameters in normal mice on days, 0, 5, 10 and 25 at dose 5.0 mg/kg body weight\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTest\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUnit\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 0 (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 5 (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 10 (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 25 (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eLiver Function Tests\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSGPT (ALT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.02\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSGOT (AST)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u0026thinsp;\u0026plusmn;\u0026thinsp;6.58***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.13***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.75***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.75*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e121.75\u0026thinsp;\u0026plusmn;\u0026thinsp;6.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eALP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e112.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113.67\u0026thinsp;\u0026plusmn;\u0026thinsp;12.1**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e172.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBilirubin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emg/dL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2625\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2625\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2375\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.265\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eKidney Function Test\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCreatinine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emg/dL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eLipid Profile Tests\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCholesterol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emg/dL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e158\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e160\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e161.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e157.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e177.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e163\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e181.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e147.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTriglycerides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emg/dL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e141.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e138.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.96***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e166.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e155.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150. \u0026plusmn;7.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNumber of mice in each group were 6; the results are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and compared with control with normal mice (without treatment) where significant values are *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of the tested complexes at the cellular level were examined using histological analysis. Tissue sections were observed using an inverted microscope at 40X magnification (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). It was noted that there was no damage (regeneration, degeneration, etc.) in the kidney, lung, heart, and liver tissues of treated mice, indicating that the tested compound had no adverse effects on tissue levels in mice (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[Insert\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e \u003cstrong\u003eabout here]\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlatinum-based chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin are effective oncological therapies, especially for solid neoplasms such as ovarian, testicular, bladder, and lung malignancies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, their clinical application is limited by adverse toxic side effects and the development of resistance to these therapeutics. Thus, new drugs are required to reduce or eliminate toxic side effects, enhance efficacy, improve target specificity, and combat aggressive and metastatic cancers [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the present study, the antineoplastic effect of a novel Pt (II) ligand complex was demonstrated using EAC cells in a mouse model. Through the assessment of anti-proliferative activity against EAC cells, tumor weight reduction, growth inhibition, restoration of hematological parameters, and survival, followed by the investigation of the possible mechanisms of activating the intrinsic mitochondrial pathway, the potential of the chemical as an anticancer agent was evaluated.\u003c/p\u003e\u003cp\u003eThe newly synthesized compound was characterized by physicochemical properties, such as physical form, appearance, and melting point, along with spectrometric (IR spectroscopy, \u003csup\u003e1\u003c/sup\u003eH nuclear magnetic resonance, and \u003csup\u003e13\u003c/sup\u003eC nuclear magnetic resonance) analysis. The antitumor efficacy of synthetic platinum complexes against EAC in mice has been documented in scientific literature [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese studies demonstrated that such complexes can significantly inhibit tumor growth, reduce tumor volume, and enhance the lifespan of EAC-bearing mice [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our survival analysis demonstrated that treatment with the platinum (II) ligand complex led to a significant increase in lifespan compared to controls. The Kaplan-Meier curves and median survival times clearly indicate a dose-dependent survival benefit [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this investigation, we observed that the Pt (II) L complex inhibited cell growth by 62.52%, increased life span by 58.28%, and reduced tumor weight by 40.24% at 5 mg/kg compared to the control group. These data suggest that the platinum complex not only inhibits tumor growth, but also positively influences overall survival, an encouraging indication for its potential use in cancer therapy.\u003c/p\u003e\u003cp\u003eChanges in blood parameters can reflect the tumor burden and progression, providing insights into the severity of the disease and the effectiveness of potential treatments. The significance of fluctuations in blood parameters in EAC-bearing mice is crucial for understanding the effects of cancer on hematological and overall physiological states of the animal. Tumor progression is associated with specific hematological changes, including a gradual decrease in hemoglobin content, red blood cells, and platelets, along with an increase in white blood cell counts; these changes were detected in EAC-bearing mice [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Deteriorated blood parameters, such as hemoglobin levels and red blood cell counts in the control group, can indicate anemia, which can lead to decreased oxygen transport, impacting the overall health of the mice [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In EAC-bearing mice, elevated WBCs often reflect immune dysregulation or the impact of the tumor on the immune system. Elevated levels of WBC can also be the result of tumor progression, which stimulates the bone marrow to produce more WBC via cytokines such as tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF). A declining platelet count can be explained by the release of inflammatory cytokines such as TNF-α and interleukins, which can suppress bone marrow activity. The suppression of bone marrow reduces platelet production (thrombopoiesis), leading to low platelet counts [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Cancer treatments, such as chemotherapy and radiation, can directly affect the bone marrow and the site of blood cell production. This may result in decreased red blood cells (anemia) and platelets (thrombocytopenia), and an increase in white blood cells (leukocytosis) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, it was reported that when EAC-bearing mice were treated with platinum complexes, these parameters seemed to be restored to normal levels compared to the control group [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It is intriguing that treatment with the Pt (II) L complex substantially increased the hemoglobin, RBC, and platelet counts and decreased the WBC count in the treatment group compared to the control. Consequently, it is hypothesized that this compound may exert a protective effect on the hematopoietic system. Hence, the potential chemotherapy drug derived from this compound may yield more tolerable medications with reduced side effects, thereby providing improved protective benefits for patients with cancer.\u003c/p\u003e\u003cp\u003eThe increased macrophage count observed in mice treated with a platinum complex compared to that in normal mice highlights the significant role of macrophages in the anti-tumor immune response. Platinum-based compounds, widely known for their cytotoxic effects on cancer cells, may also stimulate an immune response by inducing immunogenic cell death (ICD), leading to the recruitment and activation of macrophages in the tumor microenvironment. This macrophage accumulation could contribute to tumor suppression through enhanced phagocytosis, cytokine secretion, and antigen presentation, thereby strengthening anti-tumor immunity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Platinum treatment may also affect macrophage polarization and therapeutic effectiveness. Proinflammatory (M1) macrophages may emit nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α), leading to tumor cell eradication [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In contrast, excessive M2 macrophage recruitment may suppress the immune system. Understanding macrophage behavior in response to platinum-based therapy is critical for enhancing its efficacy and potential immune-modulating drug combinations. Therefore, treatment with the Pt (II) L complex resulted in a dose-dependent increase in macrophage and total peritoneal cell count. The increase in both macrophages and total peritoneal cells suggests a potential immunostimulatory effect, which should be explored further in functional immune response studies.\u003c/p\u003e\u003cp\u003eApoptosis is a self-initiated cellular suicide mechanism and a defining hallmark of potential chemotherapeutic agents [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The evasion of apoptosis is a hallmark of cancer, enabling abnormal cell proliferation and contributing to tumor progression. The promotion of apoptosis in cancer cells while sparing healthy cells is a key mechanism for effective and safer anticancer therapies [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In vivo analysis of EAC cells demonstrated that the compound suppressed cell proliferation in a dose-dependent manner, subsequently triggering apoptosis. Synthetic pharmaceuticals used in clinical settings can trigger apoptosis in certain cancer cells. Giemsa staining is a cytological method that facilitates the examination of cellular morphology, enabling the identification of apoptotic features, such as membrane blebbing, cytoplasmic condensation, and apoptotic bodies. For instance, a study evaluating the apoptogenic effects of \u003cem\u003eAverrhoa bilimbi\u003c/em\u003e extract on EAC-bearing mice employed Giemsa staining to detect these morphological changes, indicating apoptosis in the treated cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Propidium iodide is a fluorescent dye that intercalates into DNA. However, it is impermeable to live cells with intact membranes. It is commonly used, in conjunction with other stains, to assess cell viability and apoptosis. In a study investigating the sensitization of Ehrlich ascites tumor cells to methotrexate by inhibiting glutaminase, immunofluorescence staining with annexin V and propidium iodide was conducted to assess the number of apoptotic cells [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The cells treated with the tested compound showed remarkable apoptotic properties, including cell membrane blebbing, appearance of apoptotic bodies, chromosomal condensation, and fragmentation of the nucleus under a fluorescence and light microscope. Other platinum-based ligand complexes have been reported to cause apoptosis in EAC cells, which was confirmed by cytoplasmic shrinkage, ROS generation, mitochondrial membrane depolarization, and DNA damage [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Furthermore, Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e-dependent endonuclease activation, a key hallmark of apoptosis induction, produces oligonucleotide fragmentation followed by cleavages of inter-nucleosomes, resulting in the formation of smear-like DNA degradation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. EAC cells treated with the test compound in the current study caused DNA fragmentation, as previously reported for apoptosis-inducing substances [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Thus, the apoptotic death of cancer cells following treatment with our compound indicates the potential therapeutic implications of the assessed chemicals.\u003c/p\u003e\u003cp\u003eWe assessed the expression of apoptosis-regulating genes, such as p53, BAX, BCL-2, CAS-3, CAS-8, CAS-9, and PARP, using RT-qPCR to gain a better understanding of the activation of apoptotic pathways in MCF7 cells following drug treatment. Multiple growth-related genes govern the activation and inhibition of apoptosis, including pro-apoptotic (e.g., p53, Bax, Bid, and Bak) and anti-apoptotic factors (e.g., Bcl-2, Bcl-X, and Bcl-Wact). The expression of these genes in cancer cells changes in response to therapeutic intervention, resulting in the activation or deactivation of apoptosis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We conducted a parallel analysis of the expression levels of apoptosis-related genes (p53, Bax, bcl-2, caspase-3, and caspase-9) in EAC cells after exposure to the tested drug. All intrinsic apoptotic events are predominantly controlled by Bcl-2 family proteins and the p53 tumor suppressor gene, which are majorly involved in the activation of Bcl-2 family proteins [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The groups treated with the Pt (II) L complex exhibited an upregulation of p53 and downregulation of the Bcl-2 gene, leading to apoptosis in EAC cells, indicating its potential as an anticancer drug. Bax and Bak, proapoptotic BH3-only proteins, can induce changes in the permeability of the mitochondrial membrane by creating pores [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The Bax gene was upregulated in the treated group, which implies that the compound triggers apoptosis in EAC cells. Cytochrome C interacts with Apaf-1 to form an apoptosome complex and activates pro-caspase-9, ultimately leading to the activation of caspase-3. Caspase-3 is triggered by both intrinsic and extrinsic apoptotic mechanisms [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Caspase-3 cleaves a variety of proteins, including kinases, DNA regulatory proteins, cytoskeletal proteins, and endonuclease inhibitors, in a shared mechanism for both intrinsic and extrinsic triggers [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In this gene expression analysis, the expression of both cas-9 and cas-3 was upregulated in groups treated with the Pt (II) L complex, representing apoptosis that occurred through the intrinsic pathway. Overall, at the mechanistic level, apoptosis assays conducted via RT-qPCR provided insights into how the Pt (II) L complex exerts its anticancer effects. This evidence suggests that the complex induces apoptosis as a primary mechanism for its anticancer activity, selectively promoting programmed cell death in tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e \u003cb\u003eabout here]\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe toxicological profile of chemotherapeutic agents is crucial to determine their safety and therapeutic potential. In this study, the toxicological effects of a synthetic platinum complex were evaluated in mice, focusing on hematological, biochemical, and histopathological parameters to assess systemic toxicity and organ-specific damage. Hematological toxicity is a common side effect of platinum-based chemotherapeutics because of its effect on rapidly dividing bone marrow cells [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, in this study, mice treated with the Pt (II) L complex at 5 mg/kg showed minimal alterations in red blood cell (RBC) count, hemoglobin levels, and white blood cell (WBC) count during treatment. For example, RBC and Hb levels were low on day 5; however, they returned to the normal range on day 25. WBC slightly increased on days 5 and 10 and returned to normal levels on day 25. Platelet levels showed a slight decrease within the normal range, indicating that the complex had a milder myelosuppressive effect than traditional platinum drugs such as cisplatin, which often cause severe thrombocytopenia and neutropenia.\u003c/p\u003e\u003cp\u003eBiochemical markers, including liver enzymes (SGPT, SGOT, Bilirubin, ALP), kidney function indicators (creatinine), and lipid profiles (triglycerides and cholesterol), were assessed to evaluate the systemic toxicity of the compound. SGPT, SGOT, and bilirubin are crucial biomarkers for assessing liver and heart health [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Elevated levels of these enzymes in the bloodstream typically indicate damage to the liver or heart tissues as they are released during cellular injury. In this study, SGOT, SGPT, and bilirubin levels in treated mice remained close to those in normal mice throughout the treatment period on day 10 and returned to normal levels thereafter (day 25). This stability suggests that treatment with the Pt (II) L complex did not cause significant liver tissue damage, indicating that the compounds had fewer toxic side effects. Elevated ALP level may indicate biliary obstruction or liver damage. Cisplatin can elevate ALP levels, causing hepatocellular and bone damage [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. This increase in ALP was correlated with higher levels of other liver enzymes, such as ALT and AST, which are common indicators of hepatocellular damage [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Mice treated with the Pt (II) L complex displayed normal ALP levels on day 10, suggesting that the compound does not induce hepatotoxicity, nephrotoxicity, or bone toxicity. Creatinine is a by-product of normal muscle metabolism and serves as a key indicator of renal function. Elevated serum creatinine levels can signal impaired kidney function owing to reduced clearance [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In the present study, slight changes in serum creatinine levels were observed in mice receiving the compound on (day 10), which reversed to normal levels after treatment (day 25). This finding implied that the experimental treatment had mild adverse effects on the kidneys. However, kidney function markers remained within normal limits, in contrast to conventional platinum drugs such as cisplatin, which are known to induce nephrotoxicity through oxidative stress and tubular necrosis [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. These results suggest that the tested complex exhibits a safer toxicological profile with reduced organ damage.\u003c/p\u003e\u003cp\u003eThese lipid profiles serve as biomarkers for evaluating the therapeutic efficacy and side effects of treatments [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Cisplatin can decrease total cholesterol levels (hypocholesterolemia) [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Treatment of mice with the Pt (II) L complex resulted in triglyceride levels remaining within the normal range on days 10 and 25; however, total cholesterol levels were notably lower than those in the normal group, suggesting a disruption in cholesterol synthesis and transport. The levels of triglycerides and cholesterol are crucial for understanding the metabolic impact of platinum-based chemotherapeutic agents such as cisplatin in cancer patients. These lipid profiles can serve as biomarkers for evaluating the therapeutic efficacy and side effects of treatment.\u003c/p\u003e\u003cp\u003eHistological examination of the major organs, including the liver, kidneys, heart, and lungs, revealed no significant pathological alterations at the tested doses of the Pt (II) L complex. Liver sections showed normal architecture, with very few hepatocellular vacuolizations observed in the high-dose group. Kidney sections of mice treated with the Pt (II) L complex displayed intact glomeruli and tubular structures with no evidence of necrosis or fibrosis. This contrasts sharply with cisplatin-induced histopathological changes, which cause acute kidney injury (AKI) characterized by proximal tubular injury, oxidative stress, inflammation, and vascular damage within the kidney [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The heart and lung tissue sections of the treated mice did not show any histological changes in contrast to the normal group, indicating that the compound caused no organ-specific toxicity. Toxicological evaluation of the synthetic Pt (II) L complex highlights its potential as a safer alternative to conventional platinum-based chemotherapeutics. Although mild renal stress and slightly low cholesterol levels were observed, the overall systemic and organ-specific toxicity was significantly lower than that of cisplatin. These findings warrant further investigation, including chronic toxicity studies and clinical trials, to validate its safety and efficacy.\u003c/p\u003e\u003cp\u003eThe study of the platinum Schiff base ligand complex presents promising results; however, several limitations must be considered. Testing was limited to a single cancer model (EAC-bearing mice), restricting the generalizability of the findings to other cancers. Pharmacokinetic and pharmacodynamic profiles have not been fully explored, leaving safety and efficacy in mammalian systems uncertain. Mechanistic studies have been limited to in vivo apoptosis analysis, and further in vitro testing could clarify the molecular pathways. While our study demonstrates promising therapeutic effects of the platinum (II) ligand complex in the Ehrlich Ascites Carcinoma (EAC) mouse model, it is important to acknowledge the inherent limitations of this preclinical model. EAC is a murine tumor derived from mammary adenocarcinoma cells and, although it shares some systemic and immunological features with human cancers, it does not fully recapitulate the complexity of human tumor biology, including tumor heterogeneity, microenvironment interactions, and metastatic behavior [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Moreover, mouse tumor models often differ from human tumors in terms of genetic, physiological, and immunological characteristics, which may limit the direct translatability of findings to clinical settings [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Therefore, while the EAC model provides valuable initial insights into drug efficacy and toxicity, further studies using more advanced and clinically relevant models\u0026mdash;such as genetically engineered mouse models (GEMMs), patient-derived xenografts (PDXs), or organoid systems\u0026mdash;are warranted to better mimic human tumor pathophysiology and improve predictive power [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The study's short duration and limited dosage variation restrict the understanding of long-term effects and dose-response relationships. Additionally, a comparative analysis with a broader range of standard treatments is required to validate the therapeutic advantages of the compound. This study might also have been affected by uncontrollable external factors, such as social, political, or environmental events, which might have affected the results. Limited funding restricted access to certain databases or tools that might have provided more robust data for the analysis. Constraints related to the tools, instruments, or technologies used for data collection and analysis may have affected the accuracy of the results.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAltogether, we can conclude that this study\u0026rsquo;s findings highlight the synthesized platinum Schiff base ligand complex as a potent candidate for drug screening, as the compound has shown promising results as an anticancer drug. Its anticancer efficacy, characterized by significant tumor growth inhibition, extended survival time, normalized hematological parameters, induced DNA fragmentation, activation of pro-apoptotic and inactivation of anti-apoptotic genes, and toxicological studies, warrants further investigation. Future studies should focus on optimizing the bioactivity of the compound, understanding its safety profile, and exploring potential synergies with existing therapeutic agents to unlock its full potential in the clinical setting. Additionally, integrating immune-competent models will be critical to evaluate the compound\u0026rsquo;s effects within the tumor microenvironment and immune system context. Further studies and advanced-level research using human cell lines and observing the accurate mechanism of action of the drug by detecting its molecular pathways are crucial before undergoing clinical trials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from the Dean of Science, Rajshahi University, Rajshahi-6205, Bangladesh (Grant No 2002.5/52/RU). /Science-35/2023\u0026ndash;2024). There was no additional external funding received for this study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWriting \u0026ndash; original draft: TK; Methodology, Investigation, and Data curation: TK, AA, AB, SA, MI; Validation and visualization: ZAM, ARB, AA; Supervision: MTH, MAH; Conceptualization, review, and editing: MAH, TH, and FI.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eAll the data reported in the manuscript are presented in figures and tables. Unprocessed and raw data will be available upon request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell. 2011;144(5):646\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStratton MR, Campbell PJ, Futreal PA. The cancer genome. Nature. 2009;458(7239):719\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnand P, Kunnumakara AB, Sundaram C, Harikumar KB, Tharakan ST, Lai OS. Cancer is a preventable disease that requires major lifestyle changes. Pharm Res. 2008;25(9):2097\u0026ndash;1165.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePasini A, Zunino F. New Cisplatin Analogues\u0026mdash;On the Way to Better Antitumor Agents. Angew Chem Int Ed Engl. 1987;26(7):615\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChabner BA, Roberts TG. Chemotherapy and the war on cancer. Nat Rev Cancer. 2005;5(1):65\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWoude GFV, Klein G. Advances in Cancer Research. Elsevier; 2004. p. 219.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNorn S, Permin H, Kruse E, Kruse PR. Mercury-a major agent in the history of medicine and alchemy. Dan Med Arbog. 2008;36:21\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDasari S, Bernard P. Cisplatin in cancer therapy: Molecular mechanisms of action. Eur J Pharmacol. 2014;740:364\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWheate NJ, Walker S, Craig GE, Oun R. The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Trans. 2010;39(35):8113.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGalluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, et al. Molecular mechanisms of cisplatin resistance. Oncogene. 2012;31(15):1869\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePabla N, Dong Z. Curtailing side effects in chemotherapy: a tale of PKCδ in cisplatin treatment. Oncotarget. 2012;3(1):107\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcKeage MJ, et al. Comparative Adverse Effect Profiles of Platinum Drugs. Drug Saf. 1995;13(4):228\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGamelin E, Gamelin L, Bossi L, Quasthoff S. Clinical aspects and molecular basis of oxaliplatin neurotoxicity: Current management and development of preventive measures. Semin Oncol. 2002;29(5 Suppl 15):21\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGalanski M, Jakupec MA, Keppler BK. Update of the preclinical situation of anticancer platinum complexes: novel design strategies and innovative analytical approaches. Curr Med Chem. 2005;12(18):2075\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCepeda V, Fuertes MA, Castilla J, Alonso C, Quevedo C, Perez JM. Biochemical Mechanisms of Cisplatin Cytotoxicity. Anti-Cancer Agents Med Chem- Anti-Cancer Agents. 2007;7(1):3\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMontana AM, Batalla C. The Rational Design of Anticancer Platinum Complexes: The Importance of the Structure-Activity Relationship. Curr Med Chem. 2009;16(18):2235\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhoury A, Deo KM, Aldrich-Wright JR. Recent advances in platinum-based chemotherapeutics that exhibit inhibitory and targeted mechanisms of action. J Inorg Biochem. 2020;207:111070.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKashif Amir M, Hogarth G, Khan Z, Imran M. Zia-ur-Rehman. Platinum(II) dithiocarbamate complexes [Pt(S2CNR2)Cl(PAr3)] as anticancer and DNA-damaging agents. Inorganica Chim Acta. 2020;512:119853.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkitsu T, Tsvetkova D, Yamamoto Y, Nakane D, Kostova I. From Basics of Coordination Chemistry to Understanding Cisplatin-analogue Pt Drugs. Curr Pharm Des. 2023;29(22):1747\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWilson JJ, Lippard SJ. Synthetic methods for the preparation of platinum anticancer complexes. Chem Rev. 2014;114(8):4470\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIslam F, Khatun H, Khatun M, Ali SMM, Khanam JA. Growth inhibition and apoptosis of Ehrlich ascites carcinoma cells by the methanol extract of Eucalyptus camaldulensis. Pharm Biol. 2014;52(3):281\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYesmin Y, Das R, Belal PK, Aktar H, Siddika S, Asha M. Anticancer potential of Michelia champaca Linn. bark against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice. Nat Prod J. 2021;11(1):85\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoel MK, Khanna P, Kishore J. Understanding survival analysis: Kaplan-Meier estimate. Int J Ayurveda Res. 2010;1(4):274\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDudley WN 1. Wickham,2 R, COOMBS,3 N. An Introduction to Survival Statistics: Kaplan-Meier Analysis. J Adv Pract Oncol. 2016;7(1):91\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerveen R, Islam F, Khanum J, Yeasmin T. Preventive effect of ethanol extract of Alpinia calcarata Rosc on Ehrlich\u0026rsquo;s ascitic carcinoma cell induced malignant ascites in mice. Asian Pac J Trop Med. 2012;5(2):121\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJyoti BC, Balaji KS, Jayarama S, Wethroe K. Evaluation of apoptogenic effects of Averrhoa bilambi extract on enrlich ascites carcinoma bearing mice. Asian J Pharm Clin Res. 2016;9:348\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIslam F, Ali SMM, Khanam JA. Hepatoprotective effect of acetone semicarbazone on Ehrlich ascites carcinoma induced carcinogenesis in experimental mice. Asian Pac J Trop Biomed. 2013;3(2):105\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIslam F, Khanam JA, Khatun M, Zuberi N, Khatun L, Kabir SR, et al. A p-Menth-1-ene-4,7-diol (EC-1) from Eucalyptus camaldulensis Dhnh. Triggers Apoptosis and Cell Cycle Changes in Ehrlich Ascites Carcinoma Cells. Phytother Res. 2015;29(4):573\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIslam F, Ghosh S, Khanam JA. Antiproliferative and hepatoprotective activity of metabolites from Corynebacterium xerosis against Ehrlich Ascites Carcinoma cells. Asian Pac J Trop Biomed. 2014;4:S284\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAuwal A, Al Banna MH, Pronoy TUH, Hossain MM, Rashel KM, Kabir SR et al. In vitro and In vivo Growth Inhibition and Apoptosis of Cancer Cells by Ethyl 4-[(4-methylbenzyl)oxy] Benzoate Complex. Anticancer Agents Med Chem. 2025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCardiff RD, Munn RJ, Galvez JJ. The tumor pathology of genetically engineered mice: a new approach to molecular pathology. In The mouse in biomedical research 2007 Jan 1 (pp. 581\u0026ndash;622). Academic.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKnoblaugh SE, Hohl TM, La Perle KMD. Pathology Principles and Practices for Analysis of Animal Models. ILAR J. 2018;59(1):40\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIbrahim KE, Al-Mutary MG, Bakhiet AO, Khan HA. Histopathology of the Liver, Kidney, and Spleen of Mice Exposed to Gold Nanoparticles. Molecules. 2018;23(8):1848.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEsteban-Fern\u0026aacute;ndez D, Verdaguer JM, Ramirez-Camacho R, Palacios MA, G\u0026oacute;mez-G\u0026oacute;mez MM. Accumulation, fractionation, and analysis of platinum in toxicologically affected tissues after cisplatin, oxaliplatin, and carboplatin administration. J Anal Toxicol. 2008;32(2):140\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJohnstone TC, Park GY, Lippard SJ. Understanding and Improving Platinum Anticancer Drugs \u0026ndash; Phenanthriplatin. Anticancer Res. 2014;34(1):471\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNoureldeen AFH, Qusti SY, Al-shammari EM. Antitumor and Antioxidant Activities of a Novel Platinum(II)-PyrazineCarboxamide Complex Against Ehrlich Ascites Carcinoma Induced in Mice. Int J Pharm Phytopharm Res. 2017;7(6).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHossain MM, Soha K, Rahman A, Auwal A, Pronoy TUH, Islam F. Rhodium complex [RhLI2] I: a novel anticancer agent inducing tumor inhibition and apoptosis. Discover Oncol. 2024;15(1):782.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGaspar BL, Sharma P, Das R. Anemia in malignancies: Pathogenetic and diagnostic considerations. Hematology. 2015;20(1):18\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGilreath JA, Stenehjem DD, Rodgers GM. Diagnosis and treatment of cancer-related anemia. Am J Hematol. 2014;89(2):203\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJameus A, Kennedy AE, Thome C. Hematological changes following low dose radiation therapy and comparison to current standard of care cancer treatments. Dose-Response. 2021;19(4):15593258211056196.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGalluzzi L, Buqu\u0026eacute; A, Kepp O, Zitvogel L, Kroemer G. Immunogenic cell death in cancer and infectious disease. Nat Rev Immunol. 2017;17(2):97\u0026ndash;111.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMantovani A, Marchesi F, Malesci A, Laghi L, Allavena P. Tumour-associated macrophages as treatment targets in oncology. Nat Rev Clin Oncol. 2017;14(7):399\u0026ndash;416.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLawen A. Apoptosis\u0026mdash;an introduction. BioEssays. 2003;25(9):888\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePfeffer CM, Singh ATK. Apoptosis: A Target for Anticancer Therapy. Int J Mol Sci. 2018;19(2):448.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlonso FJ, Segura JA, Lora J, Lobo C, Fern\u0026aacute;ndez-Molina B, M\u0026aacute;rquez J et al. Sensitisation of Ehrlich Ascitic Tumour Cells to Methotrexate by Inhibiting Glutaminase. ANTICANCER Res. 2005.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIcsel C, Yilmaz VT, Aygun M, Cevatemre B, Alper P, Ulukaya E. Palladium(ii) and platinum(ii) saccharinate complexes with bis(diphenylphosphino)methane/ethane: synthesis, S-phase arrest and ROS-mediated apoptosis in human colon cancer cells. Dalton Trans. 2018;47(33):11397\u0026ndash;410.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReed JC. Mechanisms of Apoptosis. Am J Pathol. 2000;157(5):1415\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKiraz Y, Adan A, Kartal Yandim M, Baran Y. Major apoptotic mechanisms and genes involved in apoptosis. Tumor Biol. 2016;37(7):8471\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSavitskaya MA, Onishchenko GE. Mechanisms of apoptosis. Biochem Mosc. 2015;80(11):1393\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePorter AG, J\u0026auml;nicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999;6(2):99\u0026ndash;104.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThornberry NA, Lazebnik Y, Caspases. Enemies Within Sci. 1998;281(5381):1312\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOun R, Moussa YE, Wheate NJ. The side effects of platinum-based chemotherapy drugs: a review for chemists. Dalton Trans. 2018;47(19):6645\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHerrad\u0026oacute;n E, Gonz\u0026aacute;lez C, Uranga JA, Abalo R, Mart\u0026iacute;n MI, L\u0026oacute;pez-Miranda V. Characterization of cardiovascular alterations induced by different chronic cisplatin treatments. Front Pharmacol. 2017;8:196.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh TD, Barbhuiya MA, Poojary S, Shrivastav BR, Tiwari PK. The liver function test enzymes and glucose level are positively correlated in gallbladder cancer: a cancer registry data analysis from north central India. Indian J Cancer. 2012;49(1):125\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhadrdan E, Sadighi S, Ebrahimpour S, Abdollahi A, Hadjibabaei M, Gholami K, et al. The effect of melatonin on cisplatin-induced nephrotoxicity: A pilot, randomized, double-blinded, placebo-controlled clinical trial. Eur J Integr Med. 2020;34:101065.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoung DR, Virolainen P, Inoue N, Frassica FJ, Chao EYS. The Short-Term Effects of Cisplatin Chemotherapy on Bone Turnover. J Bone Min Res. 1997;12(11):1874\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePezeshki Z, Khosravi A, Nekuei M, Khoshnood S, Zandi E, Eslamian M, et al. Time course of cisplatin-induced nephrotoxicity and hepatotoxicity. J Nephropathol. 2017;6(3):163\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang WR, Parikh CR. Biomarkers of Acute and Chronic Kidney Disease. Annu Rev Physiol. 2019;81:309\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePabla N, Dong Z. Cisplatin nephrotoxicity: Mechanisms and renoprotective strategies. Kidney Int. 2008;73(9):994\u0026ndash;1007.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G, Su C, Yin T. Paclitaxel and platinum-based chemotherapy results in transient dyslipidemia in cancer patients. Mol Clin Oncol. 2017;6(2):261\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOkafor IA, Nnamah US, Nnaka J. Lipid and cardiac profile assessment following the co-administration of Cisplatin and methanolic extract of Portulaca oleracea in adult Wistar rats: an experimental study. Bali Med J. 2020;9(3):799\u0026ndash;805.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOzkok A, Edelstein CL. Pathophysiology of Cisplatin-Induced Acute Kidney Injury. BioMed Res Int. 2014;2014(1):967826.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeitosa IB, Mori B, Teles CBG, Costa AGD. What are the immune responses during the growth of Ehrlich\u0026rsquo;s tumor in ascitic and solid form? Life Sci. 2021;264:118578.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDe Jong M, Maina T. Of Mice and Humans: Are They the Same?\u0026mdash;Implications in Cancer Translational Research: TABLE 1. J Nucl Med. 2010;51(4):501\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhat N, Al-Mathkour M, Maacha S, Lu H, El-Rifai W, Ballout F. Esophageal adenocarcinoma models: a closer look. Front Mol Biosci. 2024;11:1440670.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anticancer, cell growth, survival, platinum complex, and EAC","lastPublishedDoi":"10.21203/rs.3.rs-7229944/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7229944/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and Objectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral platinum complexes have been used in clinical studies to address adverse effects and tumor resistance to cisplatin. Hence, the objective of the current study was to synthesize, characterize, and examine the anticancer activity of a novel platinum complex in EAC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA synthetic compound was synthesized from Platinum and Schiff base ligands. The anticancer activity of the complex was tested against EAC cells in Swiss albino mice by monitoring several parameters, such as tumor cell growth, survival time, tumor mass, and hematological profile. Morphological observation and modulation of apoptotic regulatory genes’ expression were used to study its anticancer mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe IUPAC name of the ligand is ((E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2-(pyridine-2-ylmethylene) hydrazine-1-carbothioamide) [L]. The complex exhibited significant anticancer activity against EAC cells. It showed 45.01% (p \u0026lt; 0.01) and 62.57% (p \u0026lt; 0.001) cell growth inhibition at doses of 2.0 and 5.0 mg/kg/day, respectively, and significantly prolonged survival (30 versus 19 days; p \u0026lt; .01). Also, it reduced (37.1%) tumor weight at 5.0 mg/kg/day on EAC bearing Swiss albino mice. Moreover, EAC-bearing mice receiving the treatment restored blood parameters. It did not exhibit any long-term adverse effects on hematological, biochemical, or tissue parameters in mice. The compound-treated EAC cells showed increased expression of pro-apoptotic genes such as \u003cem\u003ep53, Bax, Cas-3, 9\u003c/em\u003e, and decreased expression of anti-apoptotic gene \u003cem\u003eBcl2\u003c/em\u003e, indicating mitochondrial intrinsic pathway activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe compound showed potential anticancer activity by inducing apoptosis; however, further preclinical and clinical research is imperative before using animal and human models.\u003c/p\u003e","manuscriptTitle":"Apoptotic and anti-proliferative activity of novel platinum complex [Pt((E)-N-((E)-4-hydroxy-3-methoxybenzylidene)-2- (pyridine-2-ylmethylene)hydrazine-1-carbothioamide)] against Ehrlich Ascites carcinoma (EAC) cells in vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 19:35:16","doi":"10.21203/rs.3.rs-7229944/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5c2e5e73-097d-464b-b391-7cc75d4ca704","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-02T00:23:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 19:35:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7229944","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7229944","identity":"rs-7229944","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}