Dispiro-indanedione hybrid of parthenin induces p53-independent apoptosis and suppresses tumor progression in colorectal cancer models

Dispiro-indanedione hybrid of parthenin induces p53-independent apoptosis and suppresses tumor progression in colorectal cancer models | 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 Dispiro-indanedione hybrid of parthenin induces p53-independent apoptosis and suppresses tumor progression in colorectal cancer models Manzoor Ahmed, Diljeet Kumar, Chetan Paul Singh, Samriti Dogra, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8268375/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 Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide, with mutation of the p53 gene found in up to 80% of advanced CRC cases, resulting in apoptotic halt and significant drug resistance. The development of new drugs targeting the mutated p53 gene in CRC patients is limited. This study explored the anticancer potential of a dispiro-indanedione hybrid of parthenin (DIHP) in a p53-independent manner using HCT 116 (human colorectal carcinoma cell line). DIHP induced apoptosis by blocking JAK2-STAT3-dependent survival protein like Bcl-xL, in cooperation with TRAIL (Apo2L), leading to dose-dependent cell death and cell cycle arrest. Acute toxicity and pharmacokinetic (PK) studies revealed DIHP is significantly less toxic than its parent parthenin and exhibits a favorable PK profile respectively. Efficacy assessments in both HCT 116 xenograft and AOM (Azoxymethane)/DSS (Dextran Sulfate Sodium) induced colitis-associated colorectal cancer (CAC) mouse models demonstrated significant tumor growth suppression and improved survival. Mechanistically, DIHP downregulated key oncogenic survival pathways including PI3K/AKT, NF-κB, STAT3, and ERK. Additionally, DIHP attenuated pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in the CAC model. These findings highlight the potential of DIHP in combating CRC, providing a new opportunity for the development of anticancer treatment with improved efficacy and possible therapeutic benefits. HCT 116 DIHP Apoptosis p53 Colorectal cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. INTRODUCTION Cancer greatly impacts the life expectancy of every country, which is one of the significant causes of mortality. In 2024, colorectal cancer (CRC) ranked as the second leading cause of cancer-related deaths, with an estimated 903,859 deaths, and the third most commonly diagnosed cancer worldwide 1 . While advances in therapy have improved the median overall survival for patients with metastatic colorectal cancer, current research is focused on more effective strategies that overcome drug resistance and reduce adverse side effects 2 . During the development of CRC, many genetic defects occur that confer treatment resistance and reduce cellular vulnerability to apoptosis 3 . The p53 tumor suppressor gene, necessary for inducing apoptosis in response to chemotherapy, is either absent or inactive in most of the colorectal malignancies, 4,5 limiting the efficacy of both targeted and conventional treatment. Consequently, as alternative treatment approaches, p53-independent apoptotic mechanisms such as caspase activation and mitochondrial outer membrane permeabilization (MOMP) attract more attention 6 . Researchers have also made efforts with other death receptors, such as Fas and TRAIL, which have shown good activity in preclinical studies 7 , 8 . Notably, TRAIL induces apoptosis through DR4/DR5 receptors, leading to caspase-8 activation, which further cleaves BID, a pro-apoptotic BH3-only Bcl-2 family member into, its truncated form tBID. This induces permeability of the mitochondrial membrane by activating BAX 9 , 10 , through which cytochrome c is released into the cytosol for the induction of apoptosis. However, clinical translation faces challenges, including chemoresistance and side effects. Proteins like Bcl-xL and upregulated STAT3 can inhibit TRAIL-mediated, p53-independent apoptosis, emphasizing the need for new drugs that activate alternative apoptotic pathways in CRC with minimal toxicity. Cancer research uses animal models to assess pharmacological treatments, especially human tumor xenografts using HCT 116 cells in nude mice. Before proceeding on to human clinical trials, these models provide an accurate preclinical evaluation by considering the tumor microenvironment and non-neoplastic cells, which gives insight into drug efficacy, toxicity, and safety 11 , 12 . CAC is a form of CRC, initiated by chronic inflammation, which ultimately progresses to carcinoma. This chronic inflammation leads to the activation of PI3K/AKT, NF-κB, STAT3, and ERK pathways, which are responsible for cellular proliferation and apoptotic halt 13 , 14 . It also induces the release of pro-inflammatory cytokines like IL-1, IL-6, and TNF-α, providing a favourable tumor microenvironment that supports the progression and development of CRC 15 . Currently, the drugs such as 5-aminosalicyclic acid (5-ASA), immunosuppressant etc, used for the treatment, increases the susceptibility of the patients to infection and cause adverse side effects 16 . Total colectomy is recommended after the detection of a malignant tumor, but this does not always eliminate the risk of developing cancer 17 . Due to the limitations of current therapies, alternative drugs with the potential to prevent tumor progression and minimal side effects should be developed to inhibit the development of CAC. Parthenin, a sesquiterpene lactone, has been recognized for its anti-inflammatory and anti-cancer activity. However, its toxicity remains a significant concern. Parthenin is a potent allergen causing contact dermatitis, allergic reactions, and skin inflammation. It induces oxidative stress, inflammatory responses, chromosomal aberrations, and nuclear alterations, indicating genotoxic potential. These combined effects highlight its dermatological and systemic toxicity 18 . To address this issue, we have synthesized a series of novel derivatives of parthenin to reduce toxicity while enhancing therapeutic benefits. Among the series of derivatives, DIHP showed promising results against HCT 116, as discussed earlier 19 . The present study investigates the induction of p53-independent apoptosis by DIHP using the HCT 116 cell line, with its efficacy assessed in both HCT 116 xenograft and AOM/DSS induced CAC mouse model, along with acute toxicity and PK studies. Our findings revealed that DIHP significantly induces p53-independent apoptosis in HCT 116 cells and effectively suppresses tumor growth in both in vivo models, without causing any mortality. These findings highlight the potential of DIHP as a promising therapeutic agent against CRC. 2. MATERIAL AND METHODS 2.1. Chemicals, antibodies, and reagents The chemicals including trypsin, RPMI 1640 medium (cat. no. 31800022; Gibco™), Fetal bovine serum (FBS) (cat. no. A5256701; Gibco™), penicillin-streptomycin (cat. no. 15140122: Gibco™), phosphate-buffered saline (PBS) (cat. no. 70011044; Gibco™) and Dimethyl sulfoxide (DMSO) (cat. no. D4540; Sigma Aldrich) were used for media preparation and cell culture. 5 fluorouracil (5-FU) (cat. no. F6627; Sigma Aldrich) was used as a standard control. Cytotoxicity was determined by using SRB dye (cat. no. A14769; Thermo Fisher Scientific), Trichloroacetic acid (TCA) (cat. no. 90544; Sisco Research Laboratories Pvt. Ltd.), and Glacial acetic acid (cat. no. 85801; Sisco Research Laboratories Pvt. Ltd.). Mitochondrial membrane potential and nuclear morphology were assessed by using rhodamine 123 (Rh123) (cat. no. R302; Thermo Fisher Scientific) and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (cat. no. D9542; Sigma Aldrich) respectively. For western blotting, the chemicals used were acrylamide (cat. no. 164855000; Thermo Fisher Scientific), N, N- methylene bisacrylamide (cat.no. 164791000; Thermo Fisher Scientific), Ammonium persulfate (cat. no. 327081000; Thermo Fisher Scientific), bromophenol blue (cat. no. A18469.09; Thermo Fisher Scientific), Coomassie Brilliant Blue (cat. no. 20278; Thermo Fisher Scientific), Triton X100 (cat. no. A16046.AE; Thermo Fisher Scientific), Trizma base (cat. no. T1503; Sigma Aldrich), EDTA (cat. no. 15575020; Thermo Fisher Scientific), sodium bicarbonate (cat. no. 0219549701; MP biomedical), β mercaptoethanol (cat. no. M6250; Sigma Aldrich), RIPA buffer (cat. no. 89901; Thermo Fisher Scientific), (N,N,N',N'-Tetramethylethylenediamine) TEMED (cat. no. 17919; Thermo Fisher Scientific) HEPES (cat. no. 15630056; Thermo Fisher Scientific), phenyl methyl sulfonyl fluoride (PMSF) (cat. no. P7626; Sigma Aldrich), protease inhibitor cocktail (cat. no. 78429; Thermo Fisher Scientific), methanol (cat. no. RANKM0276; Rankem), glycerol (cat. no. 49767; Sigma Aldrich), and glycine (cat. no. H5073; Promega) the chemiluminescent horseradish peroxidase (HRP) substrate (cat. no. 516531; Merck Millipore), and bovine serum albumin (BSA) (cat. no. B14; Thermo Fisher Scientific). Annexin V- FITC kit (cat. no. V13242; Thermo Fisher Scientific), and propidium iodide (PI) (cat. no. P1304MP; Thermo Fisher Scientific) were used in cell cycle analysis. Antibodies used were cleaved caspases-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), Caspase-9 (Cell Signalling Technology Cat# 9508, RRID: AB_2068620) and Caspase-8 (Cell Signaling Technology Cat# 9496, RRID: AB_561381), TRAIL (TNF-related apoptosis-inducing ligand) (Cell Signaling Technology Cat# 3219, RRID: AB_2205818), B-cell lymphoma-extra-large (Bcl-xL) (Cell Signaling Technology Cat# 2764, RRID: AB_2228008), Bcl-2-associated X protein (Bax) (Cell Signaling Technology Cat# 5023, RRID: AB_10557411), β-actin (Cell Signaling Technology Cat# 4967, RRID: AB_330288), poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology Cat# 9532, RRID: AB_659884), cytochrome c (Cell Signaling Technology Cat# 11940, RRID: AB_2637071), phosphatidylinositol 3-kinase (PI3K) (Cell Signaling Technology Cat# 4249, RRID: AB_2165248), phosphorylated AKT (p-AKT) (Cell Signaling Technology Cat# 9271, RRID: AB_329825), p-p53(Cell Signaling Technology Cat# 9284, RRID: AB_331464), Cyclin dependent kinase 1 (CDK1) (Cell Signaling Technology Cat# 9112, RRID: AB_2074654), Cyclin B1 (Cell Signaling Technology Cat# 12231, RRID: AB_2783553), BH3-interacting domain death agonist (BID) (Cell Signaling Technology Cat# 2002, RRID: AB_10692485), Signal Transducer and Activator of Transcription 3 (STAT3) (Cell Signaling Technology Cat# 4904, RRID: AB_331269), protein ladder (cat. no. 26619; Thermo Fisher Scientific), Tween-80 (cat. no. 822187; Merck Millipore), PEG-400 (cat. no. 8170035000; merck Millipore) and DMSO (cat. no. 1029525011; Merck Millipore) were used for the preparation of drug formulation. Enzyme-linked immune sorbent assay (ELISA) kit (Invitrogen) was used to estimate the levels of different pro-inflammatory cytokines. All other reagents/solvents were of analytical grade or above. 2.2. Cell culture and optimal growth conditions HCT 116 was obtained from the National Cancer for Cell Science Pune (NCCS). The literature is followed in maintaining and propagating the cell lines serially 20 . The human cancer cell lines were grown using cell culture flasks at 37°C, 5% CO 2 , and 95% relative humidity (RH), complete growth medium RPMI-1640 with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, and 100 units/ml penicillin. Bone-marrow-derived macrophage cells (BMDMC) were isolated from wild-type mice by flushing femurs and tibias with RPMI supplemented with antibiotics, 10% FBS, and 30 ng/mL Macrophage Colony Stimulating Factor (M-CSF). Cells were cultured at 37°C with 5% CO 2 and passed through a 70 µm strainer. On day three, the medium was replaced with M-CSF free media, and cells were incubated until they reached confluence 21 . 2.3. SRB assay-based in vitro cell viability evaluation in human cancer cell lines The ideal number of cells in each well was seeded in 96-well flat-bottom plates (cat. no. 167008; Thermo Fisher Scientific) following earlier literature to perform the SRB assay 22 . A 96-well plate was seeded with a 1 x 10 4 HCT 116 cell density in 100 µL/well. The test chemicals were introduced into the cells at different concentrations (2.5, 5, 10, and 20 µM), and the plates were incubated for 48 hours. Subsequently, 100 µL/well of ice-cold TCA (10% w/v) was used to fix the cells for an hour at 4°C. After an hour, the plates were given three water rinses before being allowed to air dry. Next, 100 µL of 0.4% SRB dye was added, and the plates were incubated at room temperature. After that, plates were washed three times with 1% glacial acetic acid to remove the unbound SRB. After allowing the dye to dry at room temperature, 100 µL of 10 mM Tris buffer (pH 10.4) was added to each well to dissolve the bound dye. The protein-bound dye was dissolved after shaking the plates for five minutes. A Thermo Scientific microplate reader computed cell viability using an OD measurement at 540nm. The Selectivity Index (SI) was calculated as the ratio of the 50% inhibitory concentration (IC 50 ) of the compound in normal cells to its 50% inhibitory concentration (IC 50 ) in cancer cells 23 . A higher SI indicates greater selectivity of the compound towards cancer cells over normal cells. 2.4. Morphological studies using 4,6-diamidino-2-phenylindole (DAPI) staining Nuclear condensation and fragmentation were investigated using DAPI labelling in HCT 116 cells to indicate the induction of apoptosis. For 48 hours, the cells were treated with 2.5, 5, and 7.5µM of DIHP. 5-FU at a dose of 10 µM was administered to cells used as a positive control. Cells were incubated for 48 hours before being cleaned with PBS. Cells were preserved at 4°C using a 3:1 methanol/acetic acid fixing solution. Following a PBS wash, cells were stained for 30 minutes using a 10 µM working DAPI solution in the dark to determine the nuclear morphological changes. Nuclear morphological changes associated with apoptosis were observed under a fluorescent microscope after the cells had been treated for 30 minutes and then rinsed with PBS 24 . Morphological analysis of the genome for DIHP-treated groups, standard, and negative control was examined under a fluorescence microscope. 2.5. Assessment of mitochondrial membrane depolarization The loss of the mitochondrial membrane potential brought on by the cytoplasmic release of apoptogenic stimuli causes cell death. 80,000 cells were seeded in each well of a six-well plate, and the cells were incubated for 24 hours to allow for the development of their morphology. The plates were incubated for 48 hours after the cells were treated with DIHP at 2.5, 5, and 7.5 µM doses and 5-FU as a standard at 10 µM concentration. After two PBS washes, each well was added with 1 mL of 200 nM Rhodamine-123 dye, and the plate was incubated for 15 minutes to allow the dye to be absorbed by the cells. Following a 15-minute duration, the wells underwent three PBS washes, 1 mL of PBS was added to each of the six wells, and fluorescence microscopy images were captured 25 . 2.6. Apoptosis and necrosis assessment by annexin V-fluorescein isothiocyanate (FITC)/PI dual staining The Annexin V FITC analysis was performed using a flow cytometer to investigate the induction of apoptosis by DIHP. HCT 116 cells were seeded in six-well plates at a density of 1.5 x 10 5 cells per well. DIHP was administered at a concentration of 2.5, 5, and 7.5 µM. 5-FU was used as a standard at a 10 µM concentration. After incubating for 48 hours, the cells were collected, labelled with Annexin V (a phosphatidylserine-binding protein) combined with FITC dye, and analyzed using a BD FACS system. 2.7. Flow cytometric assessment of cell cycle phases Cell cycle assay was performed using flow cytometry using propidium iodide stain. The HCT 116 cells were seeded in six-well plates at a 1.5 x 10 5 cells/mL/well density for 24 hours. Following this, the plate was incubated for 48 hours and treated with varying doses (2.5, 5, and 7.5 µM) of DIHP and 5-FU at 10 µM as a standard. Cells were trypsinized after 48 hours and washed with PBS. The cells were preserved in 70% ethanol for 24 hours. After 90 minutes at 37°C and digestion with RNase (0.1 mg/mL), cells were stained with PI (50 µg/mL) 26 . A flow cytometer was used to examine the cells. To ascertain the ultimate result of DNA dispersion in various cell cycle phases, BD FACS technology was used 27 . The data were analyzed using FlowJo software, version 10.8.1 (BD Biosciences). 2.8. Western blot analysis of DIHP effect in vitro DIHP treatment was given to HCT 116 cells for 48 hours at various doses (2.5, 5, and 7.5 µmol/L). Following a PBS wash and centrifugation, a RIPA buffer solution with a protease inhibitor cocktail was added to the cells for 45 minutes. Cell lysates were centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatants were transferred to fresh tubes for quantification, followed by the addition of Laemmli buffer 28 . The protein samples were loaded and separated using polyacrylamide electrophoresis at 70 V until the separation was complete, and then transferred to a PVDF membrane for two hours at 100 V. Using 5% Bovine serum albumin (BSA) for one hour inhibited the non-specific binding of antibodies in the PVDF membrane. After overnight incubation at 4°C with the primary antibody, the membrane was incubated for an additional two hours with the secondary antibody conjugated with HRP. TBST was used three times for five minutes to wash the protein blots. The ECL plus chemiluminescent (HRP) substrate was used to locate the protein on the PVDF membrane using a chemidoc imaging system (Syngene, model G: BOX, XT-4), and band intensities were quantified by Image J software. 2.9. Acute toxicity study An acute study was conducted using male and female Swiss Albino mice (IAEC approval no. 327/82/2/2023) following OECD guidelines 29 . The initial dose of the DIHP molecule was set at 50 mg/kg, which was administered orally, and no deaths were recorded following this administration. Following the guidelines, the dose was subsequently increased to 300 mg/kg, with again no mortality observed. The dosage was then raised to 2000 mg/kg, and no fatalities were noted at this dose. Additionally, the effect of DIHP on serum biochemical parameters like liver function test, metabolic function test, and kidney function test was studied in both male and female Swiss Albino mice. 2.10. Pharmacokinetic study The PK study was conducted on healthy female BALB/c mice (25 to 30 g) with (IAEC approval no. 339/83/8/2023), which were housed in an individually ventilated caging system under controlled conditions (12-h light/dark cycle, 25 ± 2°C temperature, 50 ± 20% humidity) and maintained on a standard pellet diet with free access to water. The mice were randomly divided into ten groups, each containing five animals 30 , with the first five groups receiving DIHP at a dose of 20 mg/kg and the remaining five groups receiving 40 mg/kg of DIHP, both administered orally through oral gavage. Before dosing, the animals were fasted overnight with water provided ad libitum. The dose formulation consisted of 5% DMSO, 15% PEG 400, 10% Tween-80, and 70% water (v/v), administered at a volume of 10 mL/kg. Blood samples were collected at predetermined time intervals (0, 0.25, 0.5, 1, 1.5, 3, 6, 10, and 24 h) from the retro-orbital plexus into microcentrifuge tubes containing 5% (w/v) disodium EDTA. Plasma was obtained by centrifugation at 14000 rpm for 10 min and stored at -80°C until analysis. For LC-MS/MS quantitation, plasma samples were treated with acetonitrile containing diazepam (100 ng/mL) as an internal standard, vortexed, and centrifuged 31 . The resulting supernatant was analyzed to quantify DIHP using a matrix-match calibration curve. The key PK parameters, including maximum plasma concentration (Cmax), time to reach maximum plasma concentration (Tmax), elimination half-life (T1/2), area under the curve for plasma concentration from zero to the last measurable plasma sample time (AUC0-t), area under the curve for plasma concentration from zero to time infinity (AUC0-∞), volume of distribution (Vd) and clearance (Cl), were then calculated using non-compartmental analysis with PK Solutions software (Summit Research Services, USA). 2.11. In vivo study for xenograft and AOM/DSS induced CAC models The in vivo experiment initially was conducted in xenograft model development in athymic nude mice (NU/J Foxn1nu https://www.jax.org/strain/002019 ) using HCT 116 cells. The experiment was conducted with the approval of the Institutional Animal Ethics Committee (IAEC) of CSIR-IIIM (IAEC Approval No: 403/85/8/2024 dt.: 29.07.2024) and performed adhering to the guidelines of CCSEA (Committee for Control and Supervision of Experiments on Animals, https://ccsea.gov.in ). A total of 24 athymic nude mice aged 6–8 weeks with a body weight of 25–27 grams were used for the experiment. Further, an AOM/DSS-induced CAC model was developed using Male BALB/c mice (eight to ten weeks old) with IAEC approval no. 329/82/2/2023 following the guidelines mentioned above. All animals were housed in IVC’s system under controlled environmental conditions (Temperature: 22 ± 2℃, Relative Humidity: 50–60%, and light cycle: 12h light/dark), provided with ad libitum feed (Gamm Irradiated) and drinking water (Autoclaved RO water). 2.12. Tumor inoculation The HCT-116 cells were grown in RPMI media with 10% FBS (Fetal Bovine Serum), trypsinized, and resuspended in PBS. The cell suspension (5 × 106 cells/100 µL) and Matrigel at a 1:1 ratio was injected subcutaneously into the right flank region of each mouse. The tumor volume was calculated based on the formula TV (mm3) = (L x W2)/2, where L is the length (largest reading) and W is the Width (shortest reading). Once the tumor volume reached 120–150 mm³, the mice were randomized to four groups with six animals per group (n = 6). 2.13. In vivo experimentation for HCT 116 xenograft study The experimental groups are control (vehicle), 5-FU (22 mg/kg b.w.), DIHP (20 mg/kg b.w.), and DIHP (40 mg/kg b.w.), respectively. DIHP was prepared using 5% DMSO, 15% PEG 400, 10% Tween-80, and distilled water. The standard drug 5-FU was administered intraperitoneally twice a week, and DIHP was given orally on alternate days for 21 days of the experimental period. The tumors were measured twice a week using a digital vernier caliper. Simultaneously, the body weight was recorded once a week and on the last day of the experiment. Tumor volume ≥ 2000 m3, weight loss > 15%, Tumor ulceration with infection, and functional abnormality due to tumor (no movement, feed, or water intake) were considered humane endpoints of this study. At the end of the experiment, animals were sacrificed by CO2 asphyxiation with the displacement percentage of 40%, and confirmed by respiratory cessation and corneal opacification. Tumors were removed and weighed using a calibrated analytical balance. 2.14. AOM/DSS-induced CAC mouse model A model of colorectal tumor associated with colitis in mice was developed as previously mentioned 32 . Five groups of BALBc mice (n = 6) with (IAEC approval no. 329/82/2/2023), consisting of AOM/DSS, AOM/DSS with 5-ASA, AOM/DSS with DIHP 20 mg/kg and 40 mg/kg, and control groups, were randomly grouped. Briefly, the animals were maintained on normal water and feed for one week after receiving an intraperitoneal injection of AOM (10 mg/kg body weight; Sigma-Aldrich, USA) on the first day. The animals were subsequently given 2.5% DSS (36–50 kDa; MP Biomedicals) in their drinking water for a week, followed by two weeks of regular water. There were three iterations of this cycle. During the induction of the CAC model, the animals of two groups were given the doses of 20 mg/kg and 40 mg/kg body weight of DIHP orally for two weeks. 5-ASA was administered orally (75 mg/kg) as a standard. The body weight of animals was measured weekly. At 12 weeks, the mice were euthanized, and the colons were isolated for additional examination. Every colon was longitudinally dissected to check for the development of a tumor. Colon from each animal of all the groups was investigated to determine the variations in colon length. A portion of the colonic tissues was processed, embedded in paraffin, and fixed in 10% neutral buffered formalin. Using standard techniques, the sections (5 µM thick) were produced and stained with eosin and haematoxylin. Additionally, fresh colonic tissue lysates were obtained for further examination, such as western blot. Every animal in every group had their blood extracted, and serum was collected to estimate the levels of cytokines using ELISA (Invitrogen, USA). 2.15. Western blotting analysis of colon tissue The colonic tissue was collected, and RIPA lysis buffer was used to lyse the mouse colonic tissues. The supernatants were collected and protein concentrations were determined using the Bradford assay with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, USA) following centrifugation at 14,000 g for 25 minutes at 4°C. Samples that had been electrophoresed were separated using SDS-PAGE, then transferred to PVDF membranes (Millipore) and blocked for one hour at room temperature using 5% Bovine Serum Albumin (BSA) (HIMEDIA). The membranes were first cleaned in TBST and then incubated with primary antibodies for an entire night at 4°C. The membranes were then washed with TBST three times before being incubated with secondary antibodies labelled with Horse Reddish Peroxidase (HPR). The ECL plus chemiluminescent (HRP) substrate was used to visualize the protein bands on the PVDF membrane using a chemidoc imaging system (Syngene, model G: BOX, XT-4), and band intensities were quantified by Image J software. 2.16. Measurement of cytokine levels The effect of DIHP on different pro-inflammatory cytokine level was assessed in AOM/DSS-induced CAC mouse model using ELISA. On the day of termination, serum was taken for additional analysis from the blood of all five animal groups following centrifugation at 850 × g for 20 minutes at 4°C. Following the manufacturer’s instructions, the concentration of pro-inflammatory cytokines (TNF- α, IL-6 and IL1- β) in the blood serum was measured using an ELISA kit (Invitrogen, USA). 2.17. Haematoxylin-eosin staining (HE) and histological analysis Colon tissues that have been preserved in 10% formaldehyde solution for more than 24 hours were placed in ethanol for gradient dehydration for an hour at a time after being cleaned three times with PBS. The colon tissue was then removed and left to stand in xylene for an hour in xylene I and II solutions for transparent treatment. Thereafter, paraffin embedding was done at a temperature of 56–58 ºC. After allowing the samples to cool to ambient temperature, they were cut into sections, put in warm water to unfold, mounted on glass slides, and dried. The sections were resoaked in xylene I and II solutions for the dewaxing process, and then submerged for one minute in 100%, 95%. 85%, and 75% ethanol to rehydrate them. After staining the portions for five minutes with haematoxylin, they were separated for 10–15 seconds with a solution of one percent HCL, twice washed with water, and then stained for two minutes with eosin. Subsequently, a second round of ethanol dehydration treatment, xylene I and II were soaked for three and five minutes, respectively. Following that, the film was examined under a microscope and sealed with neutral gum. For histological examination, HE staining was done. The colon tissues underwent paraffin embedding, dewaxed, rehydrated, and HE staining. The procedure for staining was previously detailed 32 . 2.18. Statistical analysis In vitro results are presented as mean ± SD for three independent experiments, whereas in vivo data were shown as ± S.E.M., n = 6. Dunnett's test and one or two-way ANOVA were used for statistical analysis using GraphPad Prism 8 software. Statistical significance was expressed as ns p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001. 3. RESULTS 3.1. Screening of various semi-synthetic derivatives of parthenin for their in vitro cytotoxicity Our published article has previously reported the synthesis of semi-synthetic derivatives of parthenin and their screening against various cancer cell lines. Among the tested cancer cell lines, the parent molecule (parthenin) exhibited an IC 50 of 6 ± 0.07µM, while the semi-synthetic derivative coded as 6, which is DIHP, demonstrated a lower IC 50 of 5 ± 0.08µM against the HCT 116 cell line, indicating enhanced anti-cancer activity compared to the parent compound 19 . We subsequently evaluated both the parent and its derivative for toxicity against normal primary bone marrow-derived mononuclear cells (BMDMC). We found that the selectivity index for parthenin was 3.9 ± 0.05, which increased to 9.5 ± 0.08 for DIHP. The replacement of the Michael acceptor in parthenin with dispiro-indanedione enhances efficacy and significantly reduces toxicity. 3.2. Morphological studies using 4,6-diamidino-2-phenylindole (DAPI) staining: DIHP induced nuclear morphological changes in HCT 116 cells through its cytotoxic effect. We observed various morphological changes, including membrane blebbing and irregular patterns with some projections in the cell shape, in DIHP-treated cells compared to the control cells. Cells in the untreated group exhibited intact nuclei with normal morphology, whereas those treated with DIHP showed progressive changes in nuclear morphology indicative of apoptosis. We observed mild chromatin condensation and fragmentation at the lower concentration of 2.5 µM. These apoptotic features became more pronounced at the IC 50 concentration (5 µM) and further amplified at 7.5 µM, indicating a dose-dependent induction of apoptosis by DIHP. Similar nuclear alterations were also evident in the standard treatment group (5-FU) (Fig. 1 A). These changes in nuclear structure provide strong evidence that DIHP could trigger apoptosis. 3.3 Effect of DIHP on mitochondrial membrane potential (MMP) and release of cytochrome c in HCT 116 cells The disruption of MMP is an indicator of early apoptotic events. As shown in (Fig. 1 B & C), the control group exhibited the highest fluorescence intensity of 100%, showing intact integrity of the mitochondrial membrane. Treatment with DIHP resulted in a significant, dose-dependent reduction in fluorescence intensity to 50% ±1.22, 39% ±1.76, and 9% ±1.42 at concentrations of 2.5, 5, and 7.5 µM, respectively (P < 0.05), indicating progressive mitochondrial depolarization and loss of MMP (ΔΨm). Similarly, cells treated with the standard compound 5-FU showed a significant decrease in MMP, with fluorescence reduced to 11% ±1.49 relative to the control. We further evaluated the effect of DIHP on mitochondrial integrity by performing western blotting to assess cytosolic cytochrome c, a hallmark of mitochondrial membrane disintegration and initiation of the intrinsic apoptotic pathway. The results (Fig. 1 D & E) showed that DIHP treatment significantly increases the level of cytosolic cytochrome c, confirming mitochondrial outer membrane permeabilization. These findings prove that DIHP induces apoptosis via mitochondrial dysfunction and activation of the intrinsic apoptotic signalling cascade. 3.4. DIHP induces apoptosis via a p53 independent pathway Western blot analysis revealed that DIHP treatment significantly upregulates pro-apoptotic proteins such as Bax, caspase-9, caspase-3, and PARP, and a significant reduction in the anti-apoptotic protein Bcl-xL, confirming the induction of apoptosis (Fig. 2 A & B). Notably, there was no effect of DIHP on the expression of P-p53 relative to control, whereas the standard drug 5-FU induced phosphorylation of p53 at serine 46 . This clearly indicates the induction of apoptosis predominantly through the p53-independent pathway by DIHP. Further western blot analysis revealed a significant increase of TRAIL protein expression after the treatment with DIHP in HCT 116 cells compared to the control. We observed upregulation of downstream apoptotic proteins in the TRAIL pathway, including cleaved caspase-8 p41/p43 and the truncated form of BID (t-BID) (Fig. 2 C & D), confirming the induction of apoptosis via TRAIL-mediated pathway. Further evaluation of DIHP on the survival signalling pathways revealed the suppression of phosphorylation of the NF-κB p65 subunit and P-STAT3, critical transcription factors within the NF-κB and JAK/STAT pathways, respectively. Moreover, the treatment with DIHP led to a significant reduction in the protein expression of key components of the PI3K/AKT pathway, specifically PI3K p110α and phosphorylated AKT at serine 473 (Fig. 3 A & 3 B). These results revealed the effect of DIHP on apoptotic and cell survival pathways in HCT 116 cells, demonstrating that it modulates key pro-apoptotic proteins and inhibits major survival pathways, including PI3K/AKT, NF-κB, and JAK/STAT, thereby inducing apoptosis and suppressing tumor cell survival pathways, showing its potent anticancer potential. 3.5. Estimation of DIHP-induced cellular apoptosis annexin V-FITC/PI: Treatment of HCT 116 cells with different concentrations of DIHP increases the apoptotic cell populations in a dose-dependent manner, as evidenced by flow cytometric analysis using Annexin V-FITC/PI staining. Representative dot plots and quantification (Fig. 3 D) revealed a significant increase in both early and late apoptotic cells, validating the induction of apoptosis by DIHP at all the given concentrations. DIHP caused a dose-dependent and statistically significant increase in the percentage of apoptotic cells, particularly in the late apoptotic phase, compared to the control. Specifically, the proportion of late apoptotic cells increased from 0.41% ±0.42 in controls to 1.44% ±0.41, 7.46% ±0.37, and 21.7% ±0.35 at 2.5, 5, and 7.5 µM of DIHP, respectively. This indicates the induction of apoptosis by DIHP at all the doses. The standard drug, 5-FU at 10 µM, induced a late apoptosis rate of 12.51% ±0.48. These results indicate that DIHP promotes apoptotic cell death in a dose-dependent manner in HCT 116 cells. 3.6. Effects of DIHP on the cell cycle The exposure of different concentrations of DIHP to HCT 116 cells led to a dose-dependent arrest of cells in the G2/M phase, increasing from 29.51% ± 0.99 at 2.5 µM to 38.07% ±1.09 and 50.85% ±0.99 at 5 and 7.5 µM, respectively (Fig. 3 E). These findings indicate that DIHP significantly induces cell cycle arrest at the G2/M phase. As a positive control, cells treated with 10 µM of 5-FU exhibited characteristic cell cycle distributions of 65.6% ±1.05, 10.26% ±1.01, and 22.10% ±0.99 in the G1, S, and G2/M phases, respectively, consistent with its well-established effects on cell cycle regulation. Additionally, western blot analysis further validated the G2/M arrest, showing a dose-dependent downregulation of G2/M regulatory proteins, cyclin B, and CDK1 (Fig. 3 A & C). The reduced expression of these proteins supports the arrest of cell cycle progression by DIHP in HCT 116 cells. These data demonstrate that DIHP induces G2/M phase cell cycle arrest in this cell line, disrupting cellular proliferation and contributing to its antitumor activity. 3.7. Effect of DIHP on different parameters in acute toxicity Administration of increasing oral doses of DIHP up to 2000 mg/kg did not result in any recorded mortality in either sex, indicating that the median lethal dose (LD 50 ) of DIHP is greater than 2000 mg/kg. Throughout the study period, there were no observable changes in parameters such as feed and water intake, as well as body weight, which remained consistent across all groups, indicating the least acute toxicity of DIHP (Fig. S1 & S2). In addition, comprehensive biochemical analyses revealed that DIHP did not affect serum parameters indicative of liver function, metabolic activity, or kidney function in both male and female mice (Table. S1 a & b). All measured values remained within the normal physiological range, suggesting the compound does not induce acute hepatic, renal, or metabolic disturbances. Importantly, these findings highlight the improved safety profile of DIHP compared to its parent molecule, Parthenin, as no adverse effects were observed up to 2000mg/kg. The absence of toxicity at all the given doses highlights the potential of DIHP as a safe candidate for further investigation in colorectal cancer therapy. 3.8. Pharmacokinetic profile of the DIHP The administration of DIHP at 20 mg/kg and 40 mg/kg doses achieved peak plasma concentrations (Cmax) of approximately 124 ± 32.03 ng/ml and 237 ± 22.78 ng/ml, respectively, within 15 minutes, indicating rapid systemic absorption. Both Cmax and area under the concentration-time curve (AUC) values increased proportionally with the administered dose, demonstrating dose-dependent pharmacokinetics. Despite this, DIHP exhibited a short elimination half-life, characterized by rapid clearance from the systemic circulation, coupled with extensive tissue distribution. (Fig. S3) illustrates the plasma concentration-time profile of DIHP, with key pharmacokinetic parameters detailed in (Table 1 ). These pharmacokinetic characteristics of DIHP, marked by rapid absorption, dose-proportional plasma exposure, and rapid elimination, underscore its suitability for therapeutic applications requiring quick systemic availability and targeted efficacy. Table 1 Main pharmacokinetic parameters of DIHP after oral administration in BALB/c mice. Cmax: maximum plasma concentration; Tmax: time to reach maximum plasma concentration; T 1/2 : elimination half-life; AUC 0 − t : area under the curve for plasma concentration from zero to the last measurable plasma sample time; AUC 0− ∞ : area under the curve for plasma concentration from zero to time infinity; Vd: volume of distribution; Cl: clearance. Data are presented as mean ± standard error of the mean (SEM, n = 5) PK parameters DIHP PO (20 mg/kg) PO (40 mg/kg) C max (ng/mL) 124 ± 32.03 237 ± 22.78 T max (h) 0.25 ± 0.05 0.25 ± 0.06 T ½ (h) 2.3 ± 0.42 1.6 ± 0.33 AUC 0 − t (ng.h/mL) 129 ± 16.67 315 ± 17.92 AUC 0−∞ (ng.h/mL) 141 ± 19.71 331 ± 20.67 V d (L/Kg) 477 ± 27.58 272 ± 48.61 Cl (L/h/Kg) 142 ± 15.76 121 ± 8.32 3.9. Effect of DIHP on xenograft mouse model We evaluated the in vivo efficacy of DIHP in HCT-116 xenografts in athymic nude mice. Compared to the control group, a statistically significant difference in tumor volume was observed from day 8 onwards in the DIHP and 5-FU treated groups. However, there was no significant difference between DIHP and 5-FU groups (Fig. 4 A). The DIHP and 5-FU treated groups showed a significant reduction in tumor mass compared to control animals, demonstrating a similar finding in tumor weight (Fig. 5 B). In addition, DIHP at 40 mg/kg body weight showed a better tumor inhibition percentage than the standard drug 5-FU at 20 mg/kg b.w. (Fig. 4 C). Furthermore, there were no significant changes in body weight relative to different treatment groups, showing that DIHP is safe and does not produce any signs of toxicity. Tumors in both standard and DIHP-treated groups had fewer tumor burdens compared to the control, which had large tumors. Upon removal, tumors from each group displayed reduced size in both standard and DIHP-treated groups, indicating the role of DIHP in tumour regression (Fig. 4 D-F). These results emphasize a significant reduction of tumor growth and tumor mass in the HCT 116 xenograft mice treated with DIHP at 20 mg/kg and 40 mg/kg doses, respectively, indicating its potential as a therapeutic agent for managing colorectal cancer. 3.10. DIHP alleviated AOM/DSS-induced colorectal tumorigenesis in mice To investigate whether DIHP could mitigate AOM/DSS induced CAC model, mice treated with AOM were administered 3 cycles of 2.5% DSS for seven days followed by two weeks of normal water, with DIHP given by oral gavage (Fig. 5 A) Body weight loss and Disease activity index (DAI) score, encompassing loss of body weight, stool blood and stool consistency were observed during the experiment. Treatment with DIHP improved loss of body weight and DAI score (P < 0.05). Further, the reduced colon length observed in the CAC group was restored by DIHP administration (P < 0.05). We also observed that the colon and rectum of mice receiving AOM/DSS developed numerous tumors that were significantly reduced in 5-ASA and DIHP-treated groups (Fig. 5 B & G). DIHP treatment markedly reduced pathological alterations in intestinal tissues, as demonstrated by decreased immune cell infiltration and fewer dysplastic cells. (Fig. 6 A). 3.11. DIHP attenuated intestinal inflammation in AOM/DSS treated mice Pro-inflammatory cytokines, viz. TNF-α, IL-6, and IL-1β play a vital role in the progression of CAC by activating STAT3 and NF-κB pathways involved in cell survival and proliferation. Suppressing these cytokines can improve disease outcome by reducing chronic inflammation and tumor development 33 . In order to evaluate the effect of DIHP on intestinal inflammation in the AOM/DSS-induced CAC mouse model, we assessed different pro-inflammatory cytokines in their blood serum. Our findings demonstrate that there was a significant reduction of pro-inflammatory cytokines in 5-ASA and DIHP treated groups compared to the diseased group (Fig. 6 B-D). 3.12. Effect of DIHP on the expression of PI3K/AKT, NF-κB, STAT3, and ERK The effect of DIHP on different cell survival and oncogenic pathways was evaluated. The NF-κB signalling pathway is reported to be essential for the development of tumors and the diseases associated with inflammation 34 . The level of NF-κB signalling components (phosphorylated p65-NF-κB) was found to be increased in AOM/DSS-treated mice when compared to the control group, suggesting that AOM/DSS encouraged the activation of NF-κB signalling. Remarkably, DIHP significantly reduced NF-κB signalling pathway activation, as shown by the decreased expression levels of phosphorylated p65-NF-κB. DIHP also significantly reduces the expression level of its upstream signalling pathway i.e., PI3K/AKT, which is known to play a crucial role in the activation of cell survival pathways. This downregulation of PI3K and AKT suggests that DIHP effectively disrupts the signalling cascades that promote cell survival and proliferation in HCT 116 cell. Furthermore, STAT3 and ERK signalling pathways, which are important for cell survival and proliferation, are also downregulated by DIHP (Fig. 7 A & B). These findings indicate that targeting these pathways may be a vital mechanism through which DIHP exerts its therapeutic effects in CAC. 4. DISCUSSION CRC is a significant health concern, representing approximately 10% of all cancer cases and being the world’s second leading cause of cancer-related fatalities 35 . The median overall survival of individuals with metastatic colorectal cancer remains a critical concern, even though there have been advancements in treatment strategies that emphasize the need to improve treatment efficacy 36 . In our study, we employed the HCT 116 cell line to investigate the effect of DIHP on the induction of the p53-independent apoptotic pathway. Most of patients with CRC limit the efficacy of chemotherapeutic regimens due to mutation of the p53 gene, which attenuates the induction of apoptosis in response to irinotecan and 5-FU 37 . The design of successful treatment approaches against CRC resistant to chemotherapy may be improved by elucidating pathways to unleash the death program in tumor cells. Medicinal plants have gained significant attention in cancer treatment due to their rich array of bioactive compounds that exhibit anticancer properties. Parthenin, a sesquiterpene lactone isolated from Parthenium hysterophorus , has shown potential anticancer activity 18 , 38 . However, its clinical application is hampered by its toxicity, including chromosomal aberration and other adverse reactions 18 . To overcome these issues, we have synthesized new derivatives of parthenin aimed at improving its therapeutic efficacy while reducing its toxicity profile 19 . The present exploration of these derivatives highlights the importance of medicinal plants in providing innovative solutions for treating CRC. We initially screened various derivatives of Parthenin against HCT 116 cell line. Among these derivatives, DIHP, designated as “6” in our previous study, exhibits enhanced anticancer activity, with an IC 50 of 5 ± 0.08µM, lower than the IC 50 of the parent compound 19 . We screened both parthenin and DIHP against the primary BMDMC to assess their toxicity. Notably, the selectivity index for DIHP was 9.5 ± 0.08, significantly higher than that of the parent molecule, which had a selectivity index of only 3.9 ± 0.05. In the DAPI assay, we directly observed the apoptotic effect of DIHP 39 . DAPI staining revealed various abnormalities and morphological alterations in DIHP-treated cells, including membrane blebbing, enlarged and flattened cells, and irregular shapes, in contrast to untreated cells (Fig. 1 A). The loss of mitochondrial membrane potential (MMP) and cytochrome c release are the key events for initiating apoptosis 40 . Our results showed the effect of DIHP in inducing mitochondrial membrane potential loss and confirming the release of cytochrome c, indicating the initiation of apoptosis (Fig. 1 B-E). The induction of apoptosis by differentiating apoptotic and necrotic cell populations using Annexin V-FITC/PI assay allows the assessment of apoptosis 41 . Our findings indicated a dose-dependent increase in the extent of apoptosis, confirming the effectiveness of DIHP in triggering apoptotic pathways in HCT 116 cells (Fig. 3 D). Flow cytometry was used to analyse the DNA content 42 and determine the distribution of HCT 116 cells across different phases of the cell cycle. It was observed that DIHP induced cell cycle arrest at the G2/M phase (Fig. 3 E). This was further validated by western blot analysis, which showed a significant reduction in the expression of key G2/M regulators, CDK1 and cyclin B, confirming the effect of DIHP on arresting cell cycle progression at G2/M phase (Fig. 3 A & C). A key implication of our findings is that DIHP effectively induces apoptosis by circumventing the p53-dependent pathway, offering a potential therapeutic strategy to target cancer cells with a mutated p53 gene. Our result showed the induction of TRAIL-mediated p53-independent apoptosis by treatment with DIHP. The p53 gene, which is involved in regulating apoptosis, is mutated in the vast majority of colorectal cancers 43 , 44 . Mutation in the p53 gene, a critical tumor suppressor, often renders conventional chemotherapy and targeted therapies ineffective 45 . The BH-3 domain-only protein BID is activated by TRAIL through caspase–8. BID-mediated mitochondrial activation of caspases (9, 7, and 3), is triggered by TRAIL; however, Bcl-xL expression inhibits this death signalling pathway 46 , 47 . Our data indicate that DIHP induces TRAIL-mediated apoptosis of HCT 116 cells independently of p53 by inhibiting STAT3 signalling. The JAK/STAT pathway is regulated by a balance between activating protein tyrosine kinases and suppressive protein tyrosine phosphatases. In colorectal cancer, mutations in tyrosine phosphatases or activation of receptor tyrosine kinases lead to constitutive STAT3 activation, promoting tumor progression 47 – 49 . Our data demonstrate that DIHP suppresses STAT3, downregulating survival proteins like Bcl-xL, and cooperates with TRAIL to facilitate p53-independent apoptosis in HCT 116 cells (Fig. 2 A-D). This highlights the role of DIHP in upregulating TRAIL-mediated apoptosis via STAT3 suppression through a distinct p53-independent mechanism. The PK profile reveals absorption and a plasma concentration of drugs, emphasizing its potential for targeted applications necessitating quick systemic availability 50 . Moreover, DIHP has shown a favourable PK profile supporting its therapeutic application. Furthermore, the acute toxicity of the DIHP molecule indicates an LD 50 greater than 2000 mg/kg, significantly higher than that of its parent compound, parthenin, which has an LD 50 of only 42 mg/kg 51 . This stark contrast in toxicity levels demonstrates that DIHP is safer than parthenin, suggesting its potential for safe use. These findings support the clinical utility of DIHP as an alternate option for cancer therapy, enabling higher dosing regimens without the severe toxicity associated with parthenin. Using xenograft mouse models in colon cancer research offers significant advantages, including the ability to mimic human tumor behaviour and response to therapies closely 52 . This model allows for a more accurate assessment of the efficacy and safety of potential therapeutic agents like DIHP. In our study, DIHP significantly reduced the tumor growth in the xenograft mouse model in addition to its anti-apoptotic effects. The combination of effective tumor reduction and low toxicity underscores the therapeutic potential of the DIHP molecule in treating CRC. The progression of inflammation-dysplasia-carcinoma is a common pathway in CAC 53 . This sequence of inflammation to carcinoma progression can be developed in mice using AOM/DSS, which mimics human CAC 54 . Emerging research indicates that controlling AOM/DSS-induced CAC is achieved through reducing inflammation 55 . The current study demonstrated that animals in DIHP and 5-ASA treated groups experienced less body weight loss compared to the diseased group, and the reduction of DAI score was improved by DIHP in a dose-dependent way. Chronic inflammation causes anatomic abnormalities resulting in the shortening of the colon 56 . This effect was consistent with the colon length of the disease group. However, treatment with 5-ASA and DIHP prevented the shortening of colon length. The administration of DIHP resulted in a significant decrease in tumor burden and resolved colon inflammation. When compared to disease control, the number of tumors decreased in the DIHP-treated mice, indicating its potential in preventing tumor progression. Histopathological analysis demonstrated that DIHP treatment significantly alleviated intestinal tissue damage. The pronounced immune cell infiltration and presence of dysplastic cells observed in the CAC group were notably reduced in both the 5-ASA and DIHP-treated groups, indicating the protective effect of DIHP on AOM/DSS-induced colon injury (Fig. 7 A). DSS, a heparin-like polysaccharide used in AOM/DSS-induced CAC model, can compromise intestinal permeability by weakening epithelial integrity. This triggers immune cell infiltration and intestinal inflammation 57 . The colonic tissues of mice treated with AOM/DSS exhibited inflammation, as indicated by impaired intestinal permeability and elevated inflammatory cytokine levels 58 . Our results showed that DIHP-treated groups significantly reduced the level of pro-inflammatory cytokines (Fig. 7 B-D) to resolve the intestinal inflammation. The development of colorectal tumors associated with colitis is significantly influenced by the NF-κB signalling pathway. It is previously reported that the NF-κB signalling pathway activation is responsible for colitis to develop cancer 59 . According to our findings, mice treated with AOM/DSS had higher than normal levels of NF-κB, which is significantly reduced by the treatment of DIHP, indicating its anti-inflammatory properties by blocking the NF-κB signalling pathway. The upstream pathway PI3K/AKT regulates NF-κB and promotes tumor progression and metastasis 60 . STAT3 and ERK are also involved in the pathogenesis of CAC by promoting chemoresistance and survival signals 61 , 62 . Our results demonstrated that DIHP significantly reduced the expression of key proteins involved in inflammation and supresses the pro-inflammatory cytokines. Overall, this study demonstrates that DIHP induces p53 independent apoptosis in HCT 116 cells and suppresses tumor progression in HCT 116 xenograft and AOM/DSS-induced CAC mouse models with minimal toxicity and favourable PK profile emphasizing the potential of DIHP in the treatment of CRC. 5. CONCLUSION This study explores the anti-cancer potential of DIHP in CRC by inducing p53-independent apoptosis through inhibition of STAT3 and activation of the TRAIL pathway in the HCT 116 cell line. DIHP significantly reduces tumor growth in HCT 116 xenograft models and protects against colon injury and tumorigenesis in AOM/DSS-induced CAC by suppressing inflammation and different survival pathways, including PI3K/AKT, NF-κB, STAT3, and ERK. These findings highlight the multifaceted therapeutic potential of DIHP against CRC. Declarations Author statements The authors declare that they have no conflicts of interest related to this manuscript. Acknowledgements M.A and D.K, acknowledge the University Grant Commission for providing fellowship. This research was funded by the Ramanujan fellowship grant of Dr. Sanket Shukla (SB/S2/RJN-078/2019). This manuscript has been given the institutional publication number CSIR-IIIM/IPR/00866, Dated 29/01/2025. 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Nat Cell Biol. 2021;23(4):377–90. Additional Declarations No competing interests reported. Supplementary Files Supplementrydata.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8268375","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":578778557,"identity":"d863053f-c91a-4f5b-b8c9-563c2ffa9f87","order_by":0,"name":"Manzoor Ahmed","email":"","orcid":"","institution":"Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Manzoor","middleName":"","lastName":"Ahmed","suffix":""},{"id":578778558,"identity":"c5a48503-03e8-4b21-8a92-154059de5113","order_by":1,"name":"Diljeet Kumar","email":"","orcid":"","institution":"Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Diljeet","middleName":"","lastName":"Kumar","suffix":""},{"id":578778559,"identity":"e213814c-5995-4cf8-8000-576ce2b2b22e","order_by":2,"name":"Chetan Paul Singh","email":"","orcid":"","institution":"Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chetan","middleName":"Paul","lastName":"Singh","suffix":""},{"id":578778560,"identity":"9b824c72-690e-4268-a7f5-c1e395ade4dc","order_by":3,"name":"Samriti Dogra","email":"","orcid":"","institution":"Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Samriti","middleName":"","lastName":"Dogra","suffix":""},{"id":578778561,"identity":"18d4360d-b818-41f0-9086-db177ab9dc9f","order_by":4,"name":"Diksha Manhas","email":"","orcid":"","institution":"Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Diksha","middleName":"","lastName":"Manhas","suffix":""},{"id":578778562,"identity":"bd8c68cf-7870-4d96-bc86-a3aff79f2b53","order_by":5,"name":"Yogesh P. 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10:02:29","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":171105,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/b2d77e30429891bc9a8b8a0d.html"},{"id":101171835,"identity":"91767d82-0e5a-466b-b36b-186af2b63de9","added_by":"auto","created_at":"2026-01-27 00:11:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":333005,"visible":true,"origin":"","legend":"\u003cp\u003eApoptotic changes induced by DIHP in HCT 116 cells. \u003cstrong\u003eA.\u003c/strong\u003e images of HCT 116 cells stained with DAPI dye and observed under a fluorescence microscope (20 X magnification) after 48 hours of treatment with DIHP. \u003cstrong\u003eB.\u003c/strong\u003e Loss of mitochondrial membrane potential in response to DIHP. HCT 116 cells were treated with DIHP at various concentration for 48 hours. Later the cells were stained with rhodamine-123 for 20 minutes and analysed under a fluorescence microscope. Scale bar: 50 μm \u003cstrong\u003eC.\u003c/strong\u003e Bar graph of fluorescence intensity \u003cstrong\u003eD.\u003c/strong\u003eCytochrome c expression was analysed by western blot. Β-actin was used as a loading control. E Graph showing relative protein expression. Data were expressed as mean ± SD, **P ≤ 0.01, ***P ≤ 0.001 represent significance difference compared to the control.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/de243f3171a8df468d6fb7e1.png"},{"id":101206795,"identity":"d1e94e64-a1d0-4733-85fa-6b4fedb27045","added_by":"auto","created_at":"2026-01-27 09:56:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":405104,"visible":true,"origin":"","legend":"\u003cp\u003eDIHP induce apoptosis of HCT 116 cells via Apo2L/TRAIL pathway. \u003cstrong\u003eA\u003c/strong\u003e. Western blot analyses of Bax, Bcl-xL, Caspase 9 in whole-cell lysates of HCT 116 cells after treatment with DIHP at a concentration of 2.5 \u003cem\u003eμ\u003c/em\u003eM, 5 \u003cem\u003eμ\u003c/em\u003eM and 7.5 \u003cem\u003eμ\u003c/em\u003eM. \u003cstrong\u003eB. \u003c/strong\u003eThe bar graph illustrates the levels of protein expression determined by image J analysis. \u003cstrong\u003eC.\u003c/strong\u003e Western blot analysis of TRAIL, caspase-8, STAT3 and tBID, and β- actin 5-FU was taken as a standard. \u003cstrong\u003eD\u003c/strong\u003e. The bar graph illustrates the levels of protein expression determined by image J analysis. Data are presented as mean ± SD. The significance level is indicated as follows: ns P \u0026gt; 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 representing significant differences compared to the control.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/1cf9353f68205f35303d26a0.png"},{"id":101206555,"identity":"112e1bb5-d1a6-4d4b-ab0d-a45b421c403d","added_by":"auto","created_at":"2026-01-27 09:56:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":468412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eWestern blot analyses of PI3K, AKT and NF-ƙB\u003cstrong\u003e B. \u003c/strong\u003eQuantification of protein expression via densitometry analysis. 5fu at 10 \u003cem\u003eμ\u003c/em\u003eM concentration was used as a standard. \u003cstrong\u003eA, C\u003c/strong\u003e. Western blot of specific cyclin (Cyclin B) and CKD (CDK1) of G2/M phase. The graph demonstrating the expression levels of CDK1 and cyclin B. \u003cstrong\u003eD. \u003c/strong\u003eApoptosis induction in HCT-116 cells as shown by flow cytometric analysis after treatment with DIHP at 2.5 \u003cem\u003eμ\u003c/em\u003eM, 5 \u003cem\u003eμ\u003c/em\u003eM and 7.5\u003cem\u003e μ\u003c/em\u003eM concentrations using annexin V-FITC. The apoptotic index of cells treated with DIHP increased in a concentration-dependent manner. \u003cstrong\u003eE\u003c/strong\u003e. Cell cycle arrest in DIHP treated HCT-116 cells. Following treatment with DIHP for 48 h, the cell population distribution in the cell cycle was analyzed by flow cytometry after staining with the PI dye. Control, 5fu (3 \u003cem\u003eμ\u003c/em\u003eM), DIHP 2.5 \u003cem\u003eμ\u003c/em\u003eM, DIHP 5 \u003cem\u003eμ\u003c/em\u003eM, AND DIHP 7.5 \u003cem\u003eμ\u003c/em\u003eM are shown in the figure. Histogram represents the quantification of cell cycle phase data. Data were expressed as mean ± SD. Statistical significance was demonstrated at *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 compared with untreated control Cell cycle arrest in DIHP treated HCT 116 cells.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/388b4b716ed399a302ecb66a.png"},{"id":101171859,"identity":"73f8409c-4abb-4560-9611-64b0b6c01fd7","added_by":"auto","created_at":"2026-01-27 00:11:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":282576,"visible":true,"origin":"","legend":"\u003cp\u003eDIHP shows good tolerability and positive impacts on different parameters in vivo, as evidenced by the following observations. \u003cstrong\u003eA.\u003c/strong\u003e Treatment with DIHP resulted in a significant decrease in tumor volume in xenograft mouse models.\u003cstrong\u003e B.\u003c/strong\u003eThe tumor weight in DIHP-treated groups was significantly lower compared to the control group.\u003cstrong\u003e C. \u003c/strong\u003eA higher percentage of tumor inhibition was observed in both DIHP-treated and standard treatment groups compared to controls. \u003cstrong\u003eD. \u003c/strong\u003eThere were no significant changes in body weight among the different treatment groups, indicating the safety of DIHP.\u003cstrong\u003e E. \u003c/strong\u003eImages illustrate reduced tumor growth in DIHP-treated and standard groups compared to the control group, which exhibited large tumor growth \u003cstrong\u003eF. \u003c/strong\u003eTumors were excised from each group to assess size changes, showing reduced tumor sizes in both standard and DIHP treated groups. Statistically significant difference between treatment groups and saline control was found based on ANOVA analysis; ns P \u0026gt; 0.05, *P ≤ 0.05, **P ≤ 0.01. All error bars represent SEM.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/cc37207eab06be8df9a27258.png"},{"id":101397696,"identity":"4b2554ce-1a49-42a4-a819-e8b112aee50d","added_by":"auto","created_at":"2026-01-29 09:35:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":276504,"visible":true,"origin":"","legend":"\u003cp\u003ePharmacodynamic validation of DIHP in AOM/DSS induced animal model: \u003cstrong\u003eA. \u003c/strong\u003eSchematic timeline for AOM/DSS induced CAC. \u003cstrong\u003eB.\u003c/strong\u003e Various mouse group with variations in their body weight. \u003cstrong\u003eC.\u003c/strong\u003e Colon lengths of mice in each group at 12 weeks. \u003cstrong\u003eD.\u003c/strong\u003e Bar graph depicting the length of colon among different groups. \u003cstrong\u003eE.\u003c/strong\u003e The number of tumors in CAC and DIHP treated groups. \u003cstrong\u003eF.\u003c/strong\u003eBar graph showing the number of tumors. \u003cstrong\u003eG.\u003c/strong\u003e Variation in the mouse disease activity index (DAI) among different groups. Data were expressed as mean ± SD. Statistical significance was demonstrated at ns P \u0026gt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 compared with CAC group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/e71476c198c127f87825543f.png"},{"id":101171839,"identity":"bad74433-ddd1-456a-8f41-87f2252bead4","added_by":"auto","created_at":"2026-01-27 00:11:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":475592,"visible":true,"origin":"","legend":"\u003cp\u003eDIHP alleviates pathological changes and suppresses inflammation in CAC model.\u003cstrong\u003e A. \u003c/strong\u003eRepresentative histopathological images of colon tissues stained with H\u0026amp;E. Immune cell infiltration is indicated by yellow arrows, and dysplastic cells are marked with black arrows in each experimental group. \u003cstrong\u003eB-D. \u003c/strong\u003eQuantification of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in blood serum, measured by ELISA. Data are expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test. ****P \u0026lt; 0.001 versus CAC group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/0b410ac8ad356bf4038d1567.png"},{"id":101207035,"identity":"d6cc4766-febb-4a45-8b8b-a74d8c5d14ca","added_by":"auto","created_at":"2026-01-27 09:57:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":262144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eRepresentative Western blots showing the protein expression levels of PI3K, AKT, NF-κB, ERK, and STAT3 in control, CAC, 5-ASA, and DIHP groups. β-actin was used as loading control. \u003cstrong\u003eB.\u003c/strong\u003eQuantification of protein expression via densitometry analysis. 5-ASA at 75 mg/kg was used as a standard. Data were expressed as mean ± SD. Statistical significance was demonstrated at; ns P \u0026gt; 0.05, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 compared to the diseased control.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/eeee67c644fd00b627412ff2.png"},{"id":106227052,"identity":"7f7102f8-5fb6-4c20-ad77-82cab6f014ac","added_by":"auto","created_at":"2026-04-06 11:27:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3875402,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/6d1a57dd-1690-4df0-9ae5-b8281c9f9065.pdf"},{"id":101206765,"identity":"4fbe8153-37e1-4bdd-a6d9-aace13ea2d20","added_by":"auto","created_at":"2026-01-27 09:56:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":123486,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementrydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8268375/v1/4bae7dc35dfc1a1f287f9332.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dispiro-indanedione hybrid of parthenin induces p53-independent apoptosis and suppresses tumor progression in colorectal cancer models","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eCancer greatly impacts the life expectancy of every country, which is one of the significant causes of mortality. In 2024, colorectal cancer (CRC) ranked as the second leading cause of cancer-related deaths, with an estimated 903,859 deaths, and the third most commonly diagnosed cancer worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. While advances in therapy have improved the median overall survival for patients with metastatic colorectal cancer, current research is focused on more effective strategies that overcome drug resistance and reduce adverse side effects\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDuring the development of CRC, many genetic defects occur that confer treatment resistance and reduce cellular vulnerability to apoptosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The p53 tumor suppressor gene, necessary for inducing apoptosis in response to chemotherapy, is either absent or inactive in most of the colorectal malignancies,\u003csup\u003e4,5\u003c/sup\u003e limiting the efficacy of both targeted and conventional treatment. Consequently, as alternative treatment approaches, p53-independent apoptotic mechanisms such as caspase activation and mitochondrial outer membrane permeabilization (MOMP) attract more attention\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Researchers have also made efforts with other death receptors, such as Fas and TRAIL, which have shown good activity in preclinical studies\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Notably, TRAIL induces apoptosis through DR4/DR5 receptors, leading to caspase-8 activation, which further cleaves BID, a pro-apoptotic BH3-only Bcl-2 family member into, its truncated form tBID. This induces permeability of the mitochondrial membrane by activating BAX\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, through which cytochrome c is released into the cytosol for the induction of apoptosis. However, clinical translation faces challenges, including chemoresistance and side effects. Proteins like Bcl-xL and upregulated STAT3 can inhibit TRAIL-mediated, p53-independent apoptosis, emphasizing the need for new drugs that activate alternative apoptotic pathways in CRC with minimal toxicity.\u003c/p\u003e \u003cp\u003eCancer research uses animal models to assess pharmacological treatments, especially human tumor xenografts using HCT 116 cells in nude mice. Before proceeding on to human clinical trials, these models provide an accurate preclinical evaluation by considering the tumor microenvironment and non-neoplastic cells, which gives insight into drug efficacy, toxicity, and safety\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCAC is a form of CRC, initiated by chronic inflammation, which ultimately progresses to carcinoma. This chronic inflammation leads to the activation of PI3K/AKT, NF-κB, STAT3, and ERK pathways, which are responsible for cellular proliferation and apoptotic halt\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. It also induces the release of pro-inflammatory cytokines like IL-1, IL-6, and TNF-α, providing a favourable tumor microenvironment that supports the progression and development of CRC\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Currently, the drugs such as 5-aminosalicyclic acid (5-ASA), immunosuppressant etc, used for the treatment, increases the susceptibility of the patients to infection and cause adverse side effects\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Total colectomy is recommended after the detection of a malignant tumor, but this does not always eliminate the risk of developing cancer\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Due to the limitations of current therapies, alternative drugs with the potential to prevent tumor progression and minimal side effects should be developed to inhibit the development of CAC.\u003c/p\u003e \u003cp\u003eParthenin, a sesquiterpene lactone, has been recognized for its anti-inflammatory and anti-cancer activity. However, its toxicity remains a significant concern. Parthenin is a potent allergen causing contact dermatitis, allergic reactions, and skin inflammation. It induces oxidative stress, inflammatory responses, chromosomal aberrations, and nuclear alterations, indicating genotoxic potential. These combined effects highlight its dermatological and systemic toxicity\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To address this issue, we have synthesized a series of novel derivatives of parthenin to reduce toxicity while enhancing therapeutic benefits. Among the series of derivatives, DIHP showed promising results against HCT 116, as discussed earlier\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present study investigates the induction of p53-independent apoptosis by DIHP using the HCT 116 cell line, with its efficacy assessed in both HCT 116 xenograft and AOM/DSS induced CAC mouse model, along with acute toxicity and PK studies. Our findings revealed that DIHP significantly induces p53-independent apoptosis in HCT 116 cells and effectively suppresses tumor growth in both \u003cem\u003ein vivo\u003c/em\u003e models, without causing any mortality. These findings highlight the potential of DIHP as a promising therapeutic agent against CRC.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals, antibodies, and reagents\u003c/h2\u003e \u003cp\u003eThe chemicals including trypsin, RPMI 1640 medium (cat. no. 31800022; Gibco\u0026trade;), Fetal bovine serum (FBS) (cat. no. A5256701; Gibco\u0026trade;), penicillin-streptomycin (cat. no. 15140122: Gibco\u0026trade;), phosphate-buffered saline (PBS) (cat. no. 70011044; Gibco\u0026trade;) and Dimethyl sulfoxide (DMSO) (cat. no. D4540; Sigma Aldrich) were used for media preparation and cell culture. 5 fluorouracil (5-FU) (cat. no. F6627; Sigma Aldrich) was used as a standard control. Cytotoxicity was determined by using SRB dye (cat. no. A14769; Thermo Fisher Scientific), Trichloroacetic acid (TCA) (cat. no. 90544; Sisco Research Laboratories Pvt. Ltd.), and Glacial acetic acid (cat. no. 85801; Sisco Research Laboratories Pvt. Ltd.). Mitochondrial membrane potential and nuclear morphology were assessed by using rhodamine 123 (Rh123) (cat. no. R302; Thermo Fisher Scientific) and 4\u0026prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (cat. no. D9542; Sigma Aldrich) respectively. For western blotting, the chemicals used were acrylamide (cat. no. 164855000; Thermo Fisher Scientific), N, N- methylene bisacrylamide (cat.no. 164791000; Thermo Fisher Scientific), Ammonium persulfate (cat. no. 327081000; Thermo Fisher Scientific), bromophenol blue (cat. no. A18469.09; Thermo Fisher Scientific), Coomassie Brilliant Blue (cat. no. 20278; Thermo Fisher Scientific), Triton X100 (cat. no. A16046.AE; Thermo Fisher Scientific), Trizma base (cat. no. T1503; Sigma Aldrich), EDTA (cat. no. 15575020; Thermo Fisher Scientific), sodium bicarbonate (cat. no. 0219549701; MP biomedical), β mercaptoethanol (cat. no. M6250; Sigma Aldrich), RIPA buffer (cat. no. 89901; Thermo Fisher Scientific), (N,N,N',N'-Tetramethylethylenediamine) TEMED (cat. no. 17919; Thermo Fisher Scientific) HEPES (cat. no. 15630056; Thermo Fisher Scientific), phenyl methyl sulfonyl fluoride (PMSF) (cat. no. P7626; Sigma Aldrich), protease inhibitor cocktail (cat. no. 78429; Thermo Fisher Scientific), methanol (cat. no. RANKM0276; Rankem), glycerol (cat. no. 49767; Sigma Aldrich), and glycine (cat. no. H5073; Promega) the chemiluminescent horseradish peroxidase (HRP) substrate (cat. no. 516531; Merck Millipore), and bovine serum albumin (BSA) (cat. no. B14; Thermo Fisher Scientific). Annexin V- FITC kit (cat. no. V13242; Thermo Fisher Scientific), and propidium iodide (PI) (cat. no. P1304MP; Thermo Fisher Scientific) were used in cell cycle analysis. Antibodies used were cleaved caspases-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), Caspase-9 (Cell Signalling Technology Cat# 9508, RRID: AB_2068620) and Caspase-8 (Cell Signaling Technology Cat# 9496, RRID: AB_561381), TRAIL (TNF-related apoptosis-inducing ligand) (Cell Signaling Technology Cat# 3219, RRID: AB_2205818), B-cell lymphoma-extra-large (Bcl-xL) (Cell Signaling Technology Cat# 2764, RRID: AB_2228008), Bcl-2-associated X protein (Bax) (Cell Signaling Technology Cat# 5023, RRID: AB_10557411), β-actin (Cell Signaling Technology Cat# 4967, RRID: AB_330288), poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology Cat# 9532, RRID: AB_659884), cytochrome c (Cell Signaling Technology Cat# 11940, RRID: AB_2637071), phosphatidylinositol 3-kinase (PI3K) (Cell Signaling Technology Cat# 4249, RRID: AB_2165248), phosphorylated AKT (p-AKT) (Cell Signaling Technology Cat# 9271, RRID: AB_329825), p-p53(Cell Signaling Technology Cat# 9284, RRID: AB_331464), Cyclin dependent kinase 1 (CDK1) (Cell Signaling Technology Cat# 9112, RRID: AB_2074654), Cyclin B1 (Cell Signaling Technology Cat# 12231, RRID: AB_2783553), BH3-interacting domain death agonist (BID) (Cell Signaling Technology Cat# 2002, RRID: AB_10692485), Signal Transducer and Activator of Transcription 3 (STAT3) (Cell Signaling Technology Cat# 4904, RRID: AB_331269), protein ladder (cat. no. 26619; Thermo Fisher Scientific), Tween-80 (cat. no. 822187; Merck Millipore), PEG-400 (cat. no. 8170035000; merck Millipore) and DMSO (cat. no. 1029525011; Merck Millipore) were used for the preparation of drug formulation. Enzyme-linked immune sorbent assay (ELISA) kit (Invitrogen) was used to estimate the levels of different pro-inflammatory cytokines. All other reagents/solvents were of analytical grade or above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell culture and optimal growth conditions\u003c/h2\u003e \u003cp\u003eHCT 116 was obtained from the National Cancer for Cell Science Pune (NCCS). The literature is followed in maintaining and propagating the cell lines serially\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The human cancer cell lines were grown using cell culture flasks at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 95% relative humidity (RH), complete growth medium RPMI-1640 with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, and 100 units/ml penicillin. Bone-marrow-derived macrophage cells (BMDMC) were isolated from wild-type mice by flushing femurs and tibias with RPMI supplemented with antibiotics, 10% FBS, and 30 ng/mL Macrophage Colony Stimulating Factor (M-CSF). Cells were cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e and passed through a 70 \u0026micro;m strainer. On day three, the medium was replaced with M-CSF free media, and cells were incubated until they reached confluence\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. SRB assay-based \u003cem\u003ein vitro\u003c/em\u003e cell viability evaluation in human cancer cell lines\u003c/h2\u003e \u003cp\u003eThe ideal number of cells in each well was seeded in 96-well flat-bottom plates (cat. no. 167008; Thermo Fisher Scientific) following earlier literature to perform the SRB assay\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. A 96-well plate was seeded with a 1 x 10\u003csup\u003e4\u003c/sup\u003e HCT 116 cell density in 100 \u0026micro;L/well. The test chemicals were introduced into the cells at different concentrations (2.5, 5, 10, and 20 \u0026micro;M), and the plates were incubated for 48 hours. Subsequently, 100 \u0026micro;L/well of ice-cold TCA (10% w/v) was used to fix the cells for an hour at 4\u0026deg;C. After an hour, the plates were given three water rinses before being allowed to air dry. Next, 100 \u0026micro;L of 0.4% SRB dye was added, and the plates were incubated at room temperature. After that, plates were washed three times with 1% glacial acetic acid to remove the unbound SRB. After allowing the dye to dry at room temperature, 100 \u0026micro;L of 10 mM Tris buffer (pH 10.4) was added to each well to dissolve the bound dye. The protein-bound dye was dissolved after shaking the plates for five minutes. A Thermo Scientific microplate reader computed cell viability using an OD measurement at 540nm. The Selectivity Index (SI) was calculated as the ratio of the 50% inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of the compound in normal cells to its 50% inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) in cancer cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. A higher SI indicates greater selectivity of the compound towards cancer cells over normal cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Morphological studies using 4,6-diamidino-2-phenylindole (DAPI) staining\u003c/h2\u003e \u003cp\u003eNuclear condensation and fragmentation were investigated using DAPI labelling in HCT 116 cells to indicate the induction of apoptosis. For 48 hours, the cells were treated with 2.5, 5, and 7.5\u0026micro;M of DIHP. 5-FU at a dose of 10 \u0026micro;M was administered to cells used as a positive control. Cells were incubated for 48 hours before being cleaned with PBS. Cells were preserved at 4\u0026deg;C using a 3:1 methanol/acetic acid fixing solution. Following a PBS wash, cells were stained for 30 minutes using a 10 \u0026micro;M working DAPI solution in the dark to determine the nuclear morphological changes. Nuclear morphological changes associated with apoptosis were observed under a fluorescent microscope after the cells had been treated for 30 minutes and then rinsed with PBS\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Morphological analysis of the genome for DIHP-treated groups, standard, and negative control was examined under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Assessment of mitochondrial membrane depolarization\u003c/h2\u003e \u003cp\u003eThe loss of the mitochondrial membrane potential brought on by the cytoplasmic release of apoptogenic stimuli causes cell death. 80,000 cells were seeded in each well of a six-well plate, and the cells were incubated for 24 hours to allow for the development of their morphology. The plates were incubated for 48 hours after the cells were treated with DIHP at 2.5, 5, and 7.5 \u0026micro;M doses and 5-FU as a standard at 10 \u0026micro;M concentration. After two PBS washes, each well was added with 1 mL of 200 nM Rhodamine-123 dye, and the plate was incubated for 15 minutes to allow the dye to be absorbed by the cells. Following a 15-minute duration, the wells underwent three PBS washes, 1 mL of PBS was added to each of the six wells, and fluorescence microscopy images were captured\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Apoptosis and necrosis assessment by annexin V-fluorescein isothiocyanate (FITC)/PI dual staining\u003c/h2\u003e \u003cp\u003eThe Annexin V FITC analysis was performed using a flow cytometer to investigate the induction of apoptosis by DIHP. HCT 116 cells were seeded in six-well plates at a density of 1.5 x 10\u003csup\u003e5\u003c/sup\u003e cells per well. DIHP was administered at a concentration of 2.5, 5, and 7.5 \u0026micro;M. 5-FU was used as a standard at a 10 \u0026micro;M concentration. After incubating for 48 hours, the cells were collected, labelled with Annexin V (a phosphatidylserine-binding protein) combined with FITC dye, and analyzed using a BD FACS system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Flow cytometric assessment of cell cycle phases\u003c/h2\u003e \u003cp\u003eCell cycle assay was performed using flow cytometry using propidium iodide stain. The HCT 116 cells were seeded in six-well plates at a 1.5 x 10\u003csup\u003e5\u003c/sup\u003e cells/mL/well density for 24 hours. Following this, the plate was incubated for 48 hours and treated with varying doses (2.5, 5, and 7.5 \u0026micro;M) of DIHP and 5-FU at 10 \u0026micro;M as a standard. Cells were trypsinized after 48 hours and washed with PBS. The cells were preserved in 70% ethanol for 24 hours. After 90 minutes at 37\u0026deg;C and digestion with RNase (0.1 mg/mL), cells were stained with PI (50 \u0026micro;g/mL)\u003csup\u003e26\u003c/sup\u003e. A flow cytometer was used to examine the cells. To ascertain the ultimate result of DNA dispersion in various cell cycle phases, BD FACS technology was used\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The data were analyzed using FlowJo software, version 10.8.1 (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Western blot analysis of DIHP effect \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eDIHP treatment was given to HCT 116 cells for 48 hours at various doses (2.5, 5, and 7.5 \u0026micro;mol/L). Following a PBS wash and centrifugation, a RIPA buffer solution with a protease inhibitor cocktail was added to the cells for 45 minutes. Cell lysates were centrifuged at 12,000 rpm for 20 minutes at 4\u0026deg;C. The supernatants were transferred to fresh tubes for quantification, followed by the addition of Laemmli buffer\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The protein samples were loaded and separated using polyacrylamide electrophoresis at 70 V until the separation was complete, and then transferred to a PVDF membrane for two hours at 100 V. Using 5% Bovine serum albumin (BSA) for one hour inhibited the non-specific binding of antibodies in the PVDF membrane. After overnight incubation at 4\u0026deg;C with the primary antibody, the membrane was incubated for an additional two hours with the secondary antibody conjugated with HRP. TBST was used three times for five minutes to wash the protein blots. The ECL plus chemiluminescent (HRP) substrate was used to locate the protein on the PVDF membrane using a chemidoc imaging system (Syngene, model G: BOX, XT-4), and band intensities were quantified by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Acute toxicity study\u003c/h2\u003e \u003cp\u003eAn acute study was conducted using male and female Swiss Albino mice (IAEC approval no. 327/82/2/2023) following OECD guidelines\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The initial dose of the DIHP molecule was set at 50 mg/kg, which was administered orally, and no deaths were recorded following this administration. Following the guidelines, the dose was subsequently increased to 300 mg/kg, with again no mortality observed. The dosage was then raised to 2000 mg/kg, and no fatalities were noted at this dose. Additionally, the effect of DIHP on serum biochemical parameters like liver function test, metabolic function test, and kidney function test was studied in both male and female Swiss Albino mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Pharmacokinetic study\u003c/h2\u003e \u003cp\u003eThe PK study was conducted on healthy female BALB/c mice (25 to 30 g) with (IAEC approval no. 339/83/8/2023), which were housed in an individually ventilated caging system under controlled conditions (12-h light/dark cycle, 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C temperature, 50\u0026thinsp;\u0026plusmn;\u0026thinsp;20% humidity) and maintained on a standard pellet diet with free access to water. The mice were randomly divided into ten groups, each containing five animals\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, with the first five groups receiving DIHP at a dose of 20 mg/kg and the remaining five groups receiving 40 mg/kg of DIHP, both administered orally through oral gavage. Before dosing, the animals were fasted overnight with water provided ad libitum. The dose formulation consisted of 5% DMSO, 15% PEG 400, 10% Tween-80, and 70% water (v/v), administered at a volume of 10 mL/kg. Blood samples were collected at predetermined time intervals (0, 0.25, 0.5, 1, 1.5, 3, 6, 10, and 24 h) from the retro-orbital plexus into microcentrifuge tubes containing 5% (w/v) disodium EDTA. Plasma was obtained by centrifugation at 14000 rpm for 10 min and stored at -80\u0026deg;C until analysis. For LC-MS/MS quantitation, plasma samples were treated with acetonitrile containing diazepam (100 ng/mL) as an internal standard, vortexed, and centrifuged\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The resulting supernatant was analyzed to quantify DIHP using a matrix-match calibration curve. The key PK parameters, including maximum plasma concentration (Cmax), time to reach maximum plasma concentration (Tmax), elimination half-life (T1/2), area under the curve for plasma concentration from zero to the last measurable plasma sample time (AUC0-t), area under the curve for plasma concentration from zero to time infinity (AUC0-\u0026infin;), volume of distribution (Vd) and clearance (Cl), were then calculated using non-compartmental analysis with PK Solutions software (Summit Research Services, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. \u003cem\u003eIn vivo\u003c/em\u003e study for xenograft and AOM/DSS induced CAC models\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e experiment initially was conducted in xenograft model development in athymic nude mice (NU/J Foxn1nu \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.jax.org/strain/002019\u003c/span\u003e\u003cspan address=\"https://www.jax.org/strain/002019\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using HCT 116 cells. The experiment was conducted with the approval of the Institutional Animal Ethics Committee (IAEC) of CSIR-IIIM (IAEC Approval No: 403/85/8/2024 dt.: 29.07.2024) and performed adhering to the guidelines of CCSEA (Committee for Control and Supervision of Experiments on Animals, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccsea.gov.in\u003c/span\u003e\u003cspan address=\"https://ccsea.gov.in\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A total of 24 athymic nude mice aged 6\u0026ndash;8 weeks with a body weight of 25\u0026ndash;27 grams were used for the experiment.\u003c/p\u003e \u003cp\u003eFurther, an AOM/DSS-induced CAC model was developed using Male BALB/c mice (eight to ten weeks old) with IAEC approval no. 329/82/2/2023 following the guidelines mentioned above. All animals were housed in IVC\u0026rsquo;s system under controlled environmental conditions (Temperature: 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃, Relative Humidity: 50\u0026ndash;60%, and light cycle: 12h light/dark), provided with ad libitum feed (Gamm Irradiated) and drinking water (Autoclaved RO water).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Tumor inoculation\u003c/h2\u003e \u003cp\u003eThe HCT-116 cells were grown in RPMI media with 10% FBS (Fetal Bovine Serum), trypsinized, and resuspended in PBS. The cell suspension (5 \u0026times; 106 cells/100 \u0026micro;L) and Matrigel at a 1:1 ratio was injected subcutaneously into the right flank region of each mouse. The tumor volume was calculated based on the formula TV (mm3) = (L x W2)/2, where L is the length (largest reading) and W is the Width (shortest reading). Once the tumor volume reached 120\u0026ndash;150 mm\u0026sup3;, the mice were randomized to four groups with six animals per group (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. \u003cem\u003eIn vivo\u003c/em\u003e experimentation for HCT 116 xenograft study\u003c/h2\u003e \u003cp\u003eThe experimental groups are control (vehicle), 5-FU (22 mg/kg b.w.), DIHP (20 mg/kg b.w.), and DIHP (40 mg/kg b.w.), respectively. DIHP was prepared using 5% DMSO, 15% PEG 400, 10% Tween-80, and distilled water. The standard drug 5-FU was administered intraperitoneally twice a week, and DIHP was given orally on alternate days for 21 days of the experimental period. The tumors were measured twice a week using a digital vernier caliper. Simultaneously, the body weight was recorded once a week and on the last day of the experiment. Tumor volume\u0026thinsp;\u0026ge;\u0026thinsp;2000 m3, weight loss\u0026thinsp;\u0026gt;\u0026thinsp;15%, Tumor ulceration with infection, and functional abnormality due to tumor (no movement, feed, or water intake) were considered humane endpoints of this study. At the end of the experiment, animals were sacrificed by CO2 asphyxiation with the displacement percentage of 40%, and confirmed by respiratory cessation and corneal opacification. Tumors were removed and weighed using a calibrated analytical balance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. AOM/DSS-induced CAC mouse model\u003c/h2\u003e \u003cp\u003eA model of colorectal tumor associated with colitis in mice was developed as previously mentioned\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Five groups of BALBc mice (n\u0026thinsp;=\u0026thinsp;6) with (IAEC approval no. 329/82/2/2023), consisting of AOM/DSS, AOM/DSS with 5-ASA, AOM/DSS with DIHP 20 mg/kg and 40 mg/kg, and control groups, were randomly grouped. Briefly, the animals were maintained on normal water and feed for one week after receiving an intraperitoneal injection of AOM (10 mg/kg body weight; Sigma-Aldrich, USA) on the first day. The animals were subsequently given 2.5% DSS (36\u0026ndash;50 kDa; MP Biomedicals) in their drinking water for a week, followed by two weeks of regular water. There were three iterations of this cycle. During the induction of the CAC model, the animals of two groups were given the doses of 20 mg/kg and 40 mg/kg body weight of DIHP orally for two weeks. 5-ASA was administered orally (75 mg/kg) as a standard. The body weight of animals was measured weekly. At 12 weeks, the mice were euthanized, and the colons were isolated for additional examination. Every colon was longitudinally dissected to check for the development of a tumor. Colon from each animal of all the groups was investigated to determine the variations in colon length. A portion of the colonic tissues was processed, embedded in paraffin, and fixed in 10% neutral buffered formalin. Using standard techniques, the sections (5 \u0026micro;M thick) were produced and stained with eosin and haematoxylin. Additionally, fresh colonic tissue lysates were obtained for further examination, such as western blot. Every animal in every group had their blood extracted, and serum was collected to estimate the levels of cytokines using ELISA (Invitrogen, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Western blotting analysis of colon tissue\u003c/h2\u003e \u003cp\u003eThe colonic tissue was collected, and RIPA lysis buffer was used to lyse the mouse colonic tissues. The supernatants were collected and protein concentrations were determined using the Bradford assay with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, USA) following centrifugation at 14,000 g for 25 minutes at 4\u0026deg;C. Samples that had been electrophoresed were separated using SDS-PAGE, then transferred to PVDF membranes (Millipore) and blocked for one hour at room temperature using 5% Bovine Serum Albumin (BSA) (HIMEDIA). The membranes were first cleaned in TBST and then incubated with primary antibodies for an entire night at 4\u0026deg;C. The membranes were then washed with TBST three times before being incubated with secondary antibodies labelled with Horse Reddish Peroxidase (HPR). The ECL plus chemiluminescent (HRP) substrate was used to visualize the protein bands on the PVDF membrane using a chemidoc imaging system (Syngene, model G: BOX, XT-4), and band intensities were quantified by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. Measurement of cytokine levels\u003c/h2\u003e \u003cp\u003eThe effect of DIHP on different pro-inflammatory cytokine level was assessed in AOM/DSS-induced CAC mouse model using ELISA. On the day of termination, serum was taken for additional analysis from the blood of all five animal groups following centrifugation at 850 \u0026times; g for 20 minutes at 4\u0026deg;C. Following the manufacturer\u0026rsquo;s instructions, the concentration of pro-inflammatory cytokines (TNF- α, IL-6 and IL1- β) in the blood serum was measured using an ELISA kit (Invitrogen, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17. Haematoxylin-eosin staining (HE) and histological analysis\u003c/h2\u003e \u003cp\u003eColon tissues that have been preserved in 10% formaldehyde solution for more than 24 hours were placed in ethanol for gradient dehydration for an hour at a time after being cleaned three times with PBS. The colon tissue was then removed and left to stand in xylene for an hour in xylene I and II solutions for transparent treatment. Thereafter, paraffin embedding was done at a temperature of 56\u0026ndash;58 \u0026ordm;C. After allowing the samples to cool to ambient temperature, they were cut into sections, put in warm water to unfold, mounted on glass slides, and dried. The sections were resoaked in xylene I and II solutions for the dewaxing process, and then submerged for one minute in 100%, 95%. 85%, and 75% ethanol to rehydrate them. After staining the portions for five minutes with haematoxylin, they were separated for 10\u0026ndash;15 seconds with a solution of one percent HCL, twice washed with water, and then stained for two minutes with eosin. Subsequently, a second round of ethanol dehydration treatment, xylene I and II were soaked for three and five minutes, respectively. Following that, the film was examined under a microscope and sealed with neutral gum. For histological examination, HE staining was done. The colon tissues underwent paraffin embedding, dewaxed, rehydrated, and HE staining. The procedure for staining was previously detailed\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18. Statistical analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for three independent experiments, whereas \u003cem\u003ein vivo\u003c/em\u003e data were shown as \u0026plusmn;\u0026thinsp;S.E.M., n\u0026thinsp;=\u0026thinsp;6. Dunnett's test and one or two-way ANOVA were used for statistical analysis using GraphPad Prism 8 software. Statistical significance was expressed as ns p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Screening of various semi-synthetic derivatives of parthenin for their \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity\u003c/h2\u003e \u003cp\u003eOur published article has previously reported the synthesis of semi-synthetic derivatives of parthenin and their screening against various cancer cell lines. Among the tested cancer cell lines, the parent molecule (parthenin) exhibited an IC\u003csub\u003e50\u003c/sub\u003e of 6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u0026micro;M, while the semi-synthetic derivative coded as 6, which is DIHP, demonstrated a lower IC\u003csub\u003e50\u003c/sub\u003e of 5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u0026micro;M against the HCT 116 cell line, indicating enhanced anti-cancer activity compared to the parent compound\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We subsequently evaluated both the parent and its derivative for toxicity against normal primary bone marrow-derived mononuclear cells (BMDMC). We found that the selectivity index for parthenin was 3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, which increased to 9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 for DIHP. The replacement of the Michael acceptor in parthenin with dispiro-indanedione enhances efficacy and significantly reduces toxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Morphological studies using 4,6-diamidino-2-phenylindole (DAPI) staining:\u003c/h2\u003e \u003cp\u003eDIHP induced nuclear morphological changes in HCT 116 cells through its cytotoxic effect. We observed various morphological changes, including membrane blebbing and irregular patterns with some projections in the cell shape, in DIHP-treated cells compared to the control cells. Cells in the untreated group exhibited intact nuclei with normal morphology, whereas those treated with DIHP showed progressive changes in nuclear morphology indicative of apoptosis. We observed mild chromatin condensation and fragmentation at the lower concentration of 2.5 \u0026micro;M. These apoptotic features became more pronounced at the IC\u003csub\u003e50\u003c/sub\u003e concentration (5 \u0026micro;M) and further amplified at 7.5 \u0026micro;M, indicating a dose-dependent induction of apoptosis by DIHP. Similar nuclear alterations were also evident in the standard treatment group (5-FU) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These changes in nuclear structure provide strong evidence that DIHP could trigger apoptosis.\u003c/p\u003e \u003cp\u003e3.3 Effect of DIHP on mitochondrial membrane potential (MMP) and release of cytochrome c in HCT 116 cells\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe disruption of MMP is an indicator of early apoptotic events. As shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u0026amp; C), the control group exhibited the highest fluorescence intensity of 100%, showing intact integrity of the mitochondrial membrane. Treatment with DIHP resulted in a significant, dose-dependent reduction in fluorescence intensity to 50% \u0026plusmn;1.22, 39% \u0026plusmn;1.76, and 9% \u0026plusmn;1.42 at concentrations of 2.5, 5, and 7.5 \u0026micro;M, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating progressive mitochondrial depolarization and loss of MMP (ΔΨm). Similarly, cells treated with the standard compound 5-FU showed a significant decrease in MMP, with fluorescence reduced to 11% \u0026plusmn;1.49 relative to the control.\u003c/p\u003e \u003cp\u003eWe further evaluated the effect of DIHP on mitochondrial integrity by performing western blotting to assess cytosolic cytochrome c, a hallmark of mitochondrial membrane disintegration and initiation of the intrinsic apoptotic pathway. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD \u0026amp; E) showed that DIHP treatment significantly increases the level of cytosolic cytochrome c, confirming mitochondrial outer membrane permeabilization. These findings prove that DIHP induces apoptosis via mitochondrial dysfunction and activation of the intrinsic apoptotic signalling cascade.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4. DIHP induces apoptosis via a p53 independent pathway\u003c/h2\u003e \u003cp\u003eWestern blot analysis revealed that DIHP treatment significantly upregulates pro-apoptotic proteins such as Bax, caspase-9, caspase-3, and PARP, and a significant reduction in the anti-apoptotic protein Bcl-xL, confirming the induction of apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp; B). Notably, there was no effect of DIHP on the expression of P-p53 relative to control, whereas the standard drug 5-FU induced phosphorylation of p53 at serine\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This clearly indicates the induction of apoptosis predominantly through the p53-independent pathway by DIHP.\u003c/p\u003e \u003cp\u003eFurther western blot analysis revealed a significant increase of TRAIL protein expression after the treatment with DIHP in HCT 116 cells compared to the control. We observed upregulation of downstream apoptotic proteins in the TRAIL pathway, including cleaved caspase-8 p41/p43 and the truncated form of BID (t-BID) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC \u0026amp; D), confirming the induction of apoptosis via TRAIL-mediated pathway.\u003c/p\u003e \u003cp\u003eFurther evaluation of DIHP on the survival signalling pathways revealed the suppression of phosphorylation of the NF-κB p65 subunit and P-STAT3, critical transcription factors within the NF-κB and JAK/STAT pathways, respectively. Moreover, the treatment with DIHP led to a significant reduction in the protein expression of key components of the PI3K/AKT pathway, specifically PI3K p110α and phosphorylated AKT at serine\u003csup\u003e473\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results revealed the effect of DIHP on apoptotic and cell survival pathways in HCT 116 cells, demonstrating that it modulates key pro-apoptotic proteins and inhibits major survival pathways, including PI3K/AKT, NF-κB, and JAK/STAT, thereby inducing apoptosis and suppressing tumor cell survival pathways, showing its potent anticancer potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Estimation of DIHP-induced cellular apoptosis annexin V-FITC/PI:\u003c/h2\u003e \u003cp\u003eTreatment of HCT 116 cells with different concentrations of DIHP increases the apoptotic cell populations in a dose-dependent manner, as evidenced by flow cytometric analysis using Annexin V-FITC/PI staining. Representative dot plots and quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) revealed a significant increase in both early and late apoptotic cells, validating the induction of apoptosis by DIHP at all the given concentrations.\u003c/p\u003e \u003cp\u003eDIHP caused a dose-dependent and statistically significant increase in the percentage of apoptotic cells, particularly in the late apoptotic phase, compared to the control. Specifically, the proportion of late apoptotic cells increased from 0.41% \u0026plusmn;0.42 in controls to 1.44% \u0026plusmn;0.41, 7.46% \u0026plusmn;0.37, and 21.7% \u0026plusmn;0.35 at 2.5, 5, and 7.5 \u0026micro;M of DIHP, respectively. This indicates the induction of apoptosis by DIHP at all the doses. The standard drug, 5-FU at 10 \u0026micro;M, induced a late apoptosis rate of 12.51% \u0026plusmn;0.48. These results indicate that DIHP promotes apoptotic cell death in a dose-dependent manner in HCT 116 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Effects of DIHP on the cell cycle\u003c/h2\u003e \u003cp\u003eThe exposure of different concentrations of DIHP to HCT 116 cells led to a dose-dependent arrest of cells in the G2/M phase, increasing from 29.51% \u0026plusmn; 0.99 at 2.5 \u0026micro;M to 38.07% \u0026plusmn;1.09 and 50.85% \u0026plusmn;0.99 at 5 and 7.5 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These findings indicate that DIHP significantly induces cell cycle arrest at the G2/M phase.\u003c/p\u003e \u003cp\u003eAs a positive control, cells treated with 10 \u0026micro;M of 5-FU exhibited characteristic cell cycle distributions of 65.6% \u0026plusmn;1.05, 10.26% \u0026plusmn;1.01, and 22.10% \u0026plusmn;0.99 in the G1, S, and G2/M phases, respectively, consistent with its well-established effects on cell cycle regulation.\u003c/p\u003e \u003cp\u003eAdditionally, western blot analysis further validated the G2/M arrest, showing a dose-dependent downregulation of G2/M regulatory proteins, cyclin B, and CDK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; C). The reduced expression of these proteins supports the arrest of cell cycle progression by DIHP in HCT 116 cells. These data demonstrate that DIHP induces G2/M phase cell cycle arrest in this cell line, disrupting cellular proliferation and contributing to its antitumor activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Effect of DIHP on different parameters in acute toxicity\u003c/h2\u003e \u003cp\u003eAdministration of increasing oral doses of DIHP up to 2000 mg/kg did not result in any recorded mortality in either sex, indicating that the median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) of DIHP is greater than 2000 mg/kg. Throughout the study period, there were no observable changes in parameters such as feed and water intake, as well as body weight, which remained consistent across all groups, indicating the least acute toxicity of DIHP (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e \u0026amp; S2).\u003c/p\u003e \u003cp\u003eIn addition, comprehensive biochemical analyses revealed that DIHP did not affect serum parameters indicative of liver function, metabolic activity, or kidney function in both male and female mice (Table. S1 a \u0026amp; b). All measured values remained within the normal physiological range, suggesting the compound does not induce acute hepatic, renal, or metabolic disturbances.\u003c/p\u003e \u003cp\u003eImportantly, these findings highlight the improved safety profile of DIHP compared to its parent molecule, Parthenin, as no adverse effects were observed up to 2000mg/kg. The absence of toxicity at all the given doses highlights the potential of DIHP as a safe candidate for further investigation in colorectal cancer therapy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Pharmacokinetic profile of the DIHP\u003c/h2\u003e \u003cp\u003eThe administration of DIHP at 20 mg/kg and 40 mg/kg doses achieved peak plasma concentrations (Cmax) of approximately 124\u0026thinsp;\u0026plusmn;\u0026thinsp;32.03 ng/ml and 237\u0026thinsp;\u0026plusmn;\u0026thinsp;22.78 ng/ml, respectively, within 15 minutes, indicating rapid systemic absorption. Both Cmax and area under the concentration-time curve (AUC) values increased proportionally with the administered dose, demonstrating dose-dependent pharmacokinetics. Despite this, DIHP exhibited a short elimination half-life, characterized by rapid clearance from the systemic circulation, coupled with extensive tissue distribution. (Fig. S3) illustrates the plasma concentration-time profile of DIHP, with key pharmacokinetic parameters detailed in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These pharmacokinetic characteristics of DIHP, marked by rapid absorption, dose-proportional plasma exposure, and rapid elimination, underscore its suitability for therapeutic applications requiring quick systemic availability and targeted efficacy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMain pharmacokinetic parameters of DIHP after oral administration in BALB/c mice. Cmax: maximum plasma concentration; Tmax: time to reach maximum plasma concentration; T\u003csub\u003e1/2\u003c/sub\u003e: elimination half-life; AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/sub\u003e: area under the curve for plasma concentration from zero to the last measurable plasma sample time; AUC\u003csub\u003e0\u0026minus; \u0026infin;\u003c/sub\u003e: area under the curve for plasma concentration from zero to time infinity; Vd: volume of distribution; Cl: clearance. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM, n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePK parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eDIHP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePO (20 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePO (40 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003emax\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(ng/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e124\u0026thinsp;\u0026plusmn;\u0026thinsp;32.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e237\u0026thinsp;\u0026plusmn;\u0026thinsp;22.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003emax\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(h)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003e\u0026frac12;\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(h)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAUC\u003c/b\u003e\u003csub\u003e\u003cb\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(ng.h/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e129\u0026thinsp;\u0026plusmn;\u0026thinsp;16.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e315\u0026thinsp;\u0026plusmn;\u0026thinsp;17.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAUC\u003c/b\u003e\u003csub\u003e\u003cb\u003e0\u0026minus;\u0026infin;\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(ng.h/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e141\u0026thinsp;\u0026plusmn;\u0026thinsp;19.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e331\u0026thinsp;\u0026plusmn;\u0026thinsp;20.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eV\u003c/b\u003e\u003csub\u003e\u003cb\u003ed\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(L/Kg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e477\u0026thinsp;\u0026plusmn;\u0026thinsp;27.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e272\u0026thinsp;\u0026plusmn;\u0026thinsp;48.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCl\u003c/b\u003e \u003cb\u003e(L/h/Kg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e142\u0026thinsp;\u0026plusmn;\u0026thinsp;15.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e121\u0026thinsp;\u0026plusmn;\u0026thinsp;8.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Effect of DIHP on xenograft mouse model\u003c/h2\u003e \u003cp\u003eWe evaluated the in vivo efficacy of DIHP in HCT-116 xenografts in athymic nude mice. Compared to the control group, a statistically significant difference in tumor volume was observed from day 8 onwards in the DIHP and 5-FU treated groups. However, there was no significant difference between DIHP and 5-FU groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The DIHP and 5-FU treated groups showed a significant reduction in tumor mass compared to control animals, demonstrating a similar finding in tumor weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, DIHP at 40 mg/kg body weight showed a better tumor inhibition percentage than the standard drug 5-FU at 20 mg/kg b.w. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFurthermore, there were no significant changes in body weight relative to different treatment groups, showing that DIHP is safe and does not produce any signs of toxicity. Tumors in both standard and DIHP-treated groups had fewer tumor burdens compared to the control, which had large tumors. Upon removal, tumors from each group displayed reduced size in both standard and DIHP-treated groups, indicating the role of DIHP in tumour regression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). These results emphasize a significant reduction of tumor growth and tumor mass in the HCT 116 xenograft mice treated with DIHP at 20 mg/kg and 40 mg/kg doses, respectively, indicating its potential as a therapeutic agent for managing colorectal cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.10. DIHP alleviated AOM/DSS-induced colorectal tumorigenesis in mice\u003c/h2\u003e \u003cp\u003eTo investigate whether DIHP could mitigate AOM/DSS induced CAC model, mice treated with AOM were administered 3 cycles of 2.5% DSS for seven days followed by two weeks of normal water, with DIHP given by oral gavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) Body weight loss and Disease activity index (DAI) score, encompassing loss of body weight, stool blood and stool consistency were observed during the experiment. Treatment with DIHP improved loss of body weight and DAI score (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Further, the reduced colon length observed in the CAC group was restored by DIHP administration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). We also observed that the colon and rectum of mice receiving AOM/DSS developed numerous tumors that were significantly reduced in 5-ASA and DIHP-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB \u0026amp; G). DIHP treatment markedly reduced pathological alterations in intestinal tissues, as demonstrated by decreased immune cell infiltration and fewer dysplastic cells. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.11. DIHP attenuated intestinal inflammation in AOM/DSS treated mice\u003c/h2\u003e \u003cp\u003ePro-inflammatory cytokines, viz. TNF-α, IL-6, and IL-1β play a vital role in the progression of CAC by activating STAT3 and NF-κB pathways involved in cell survival and proliferation. Suppressing these cytokines can improve disease outcome by reducing chronic inflammation and tumor development\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In order to evaluate the effect of DIHP on intestinal inflammation in the AOM/DSS-induced CAC mouse model, we assessed different pro-inflammatory cytokines in their blood serum. Our findings demonstrate that there was a significant reduction of pro-inflammatory cytokines in 5-ASA and DIHP treated groups compared to the diseased group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.12. Effect of DIHP on the expression of PI3K/AKT, NF-κB, STAT3, and ERK\u003c/h2\u003e \u003cp\u003eThe effect of DIHP on different cell survival and oncogenic pathways was evaluated. The NF-κB signalling pathway is reported to be essential for the development of tumors and the diseases associated with inflammation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The level of NF-κB signalling components (phosphorylated p65-NF-κB) was found to be increased in AOM/DSS-treated mice when compared to the control group, suggesting that AOM/DSS encouraged the activation of NF-κB signalling. Remarkably, DIHP significantly reduced NF-κB signalling pathway activation, as shown by the decreased expression levels of phosphorylated p65-NF-κB.\u003c/p\u003e \u003cp\u003eDIHP also significantly reduces the expression level of its upstream signalling pathway i.e., PI3K/AKT, which is known to play a crucial role in the activation of cell survival pathways. This downregulation of PI3K and AKT suggests that DIHP effectively disrupts the signalling cascades that promote cell survival and proliferation in HCT 116 cell. Furthermore, STAT3 and ERK signalling pathways, which are important for cell survival and proliferation, are also downregulated by DIHP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA \u0026amp; B). These findings indicate that targeting these pathways may be a vital mechanism through which DIHP exerts its therapeutic effects in CAC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eCRC is a significant health concern, representing approximately 10% of all cancer cases and being the world\u0026rsquo;s second leading cause of cancer-related fatalities\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The median overall survival of individuals with metastatic colorectal cancer remains a critical concern, even though there have been advancements in treatment strategies that emphasize the need to improve treatment efficacy\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In our study, we employed the HCT 116 cell line to investigate the effect of DIHP on the induction of the p53-independent apoptotic pathway. Most of patients with CRC limit the efficacy of chemotherapeutic regimens due to mutation of the p53 gene, which attenuates the induction of apoptosis in response to irinotecan and 5-FU\u003csup\u003e37\u003c/sup\u003e. The design of successful treatment approaches against CRC resistant to chemotherapy may be improved by elucidating pathways to unleash the death program in tumor cells.\u003c/p\u003e \u003cp\u003eMedicinal plants have gained significant attention in cancer treatment due to their rich array of bioactive compounds that exhibit anticancer properties. Parthenin, a sesquiterpene lactone isolated from \u003cem\u003eParthenium hysterophorus\u003c/em\u003e, has shown potential anticancer activity\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, its clinical application is hampered by its toxicity, including chromosomal aberration and other adverse reactions\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To overcome these issues, we have synthesized new derivatives of parthenin aimed at improving its therapeutic efficacy while reducing its toxicity profile\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The present exploration of these derivatives highlights the importance of medicinal plants in providing innovative solutions for treating CRC.\u003c/p\u003e \u003cp\u003eWe initially screened various derivatives of Parthenin against HCT 116 cell line. Among these derivatives, DIHP, designated as \u0026ldquo;6\u0026rdquo; in our previous study, exhibits enhanced anticancer activity, with an IC\u003csub\u003e50\u003c/sub\u003e of 5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u0026micro;M, lower than the IC\u003csub\u003e50\u003c/sub\u003e of the parent compound\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We screened both parthenin and DIHP against the primary BMDMC to assess their toxicity. Notably, the selectivity index for DIHP was 9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, significantly higher than that of the parent molecule, which had a selectivity index of only 3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05. In the DAPI assay, we directly observed the apoptotic effect of DIHP\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. DAPI staining revealed various abnormalities and morphological alterations in DIHP-treated cells, including membrane blebbing, enlarged and flattened cells, and irregular shapes, in contrast to untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The loss of mitochondrial membrane potential (MMP) and cytochrome c release are the key events for initiating apoptosis\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Our results showed the effect of DIHP in inducing mitochondrial membrane potential loss and confirming the release of cytochrome c, indicating the initiation of apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E). The induction of apoptosis by differentiating apoptotic and necrotic cell populations using Annexin V-FITC/PI assay allows the assessment of apoptosis\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Our findings indicated a dose-dependent increase in the extent of apoptosis, confirming the effectiveness of DIHP in triggering apoptotic pathways in HCT 116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Flow cytometry was used to analyse the DNA content\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and determine the distribution of HCT 116 cells across different phases of the cell cycle. It was observed that DIHP induced cell cycle arrest at the G2/M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This was further validated by western blot analysis, which showed a significant reduction in the expression of key G2/M regulators, CDK1 and cyclin B, confirming the effect of DIHP on arresting cell cycle progression at G2/M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; C).\u003c/p\u003e \u003cp\u003eA key implication of our findings is that DIHP effectively induces apoptosis by circumventing the p53-dependent pathway, offering a potential therapeutic strategy to target cancer cells with a mutated p53 gene. Our result showed the induction of TRAIL-mediated p53-independent apoptosis by treatment with DIHP. The p53 gene, which is involved in regulating apoptosis, is mutated in the vast majority of colorectal cancers\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Mutation in the p53 gene, a critical tumor suppressor, often renders conventional chemotherapy and targeted therapies ineffective\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The BH-3 domain-only protein BID is activated by TRAIL through caspase\u0026ndash;8. BID-mediated mitochondrial activation of caspases (9, 7, and 3), is triggered by TRAIL; however, Bcl-xL expression inhibits this death signalling pathway\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Our data indicate that DIHP induces TRAIL-mediated apoptosis of HCT 116 cells independently of p53 by inhibiting STAT3 signalling.\u003c/p\u003e \u003cp\u003eThe JAK/STAT pathway is regulated by a balance between activating protein tyrosine kinases and suppressive protein tyrosine phosphatases. In colorectal cancer, mutations in tyrosine phosphatases or activation of receptor tyrosine kinases lead to constitutive STAT3 activation, promoting tumor progression\u003csup\u003e\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Our data demonstrate that DIHP suppresses STAT3, downregulating survival proteins like Bcl-xL, and cooperates with TRAIL to facilitate p53-independent apoptosis in HCT 116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). This highlights the role of DIHP in upregulating TRAIL-mediated apoptosis via STAT3 suppression through a distinct p53-independent mechanism.\u003c/p\u003e \u003cp\u003eThe PK profile reveals absorption and a plasma concentration of drugs, emphasizing its potential for targeted applications necessitating quick systemic availability\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Moreover, DIHP has shown a favourable PK profile supporting its therapeutic application. Furthermore, the acute toxicity of the DIHP molecule indicates an LD\u003csub\u003e50\u003c/sub\u003e greater than 2000 mg/kg, significantly higher than that of its parent compound, parthenin, which has an LD\u003csub\u003e50\u003c/sub\u003e of only 42 mg/kg\u003csup\u003e51\u003c/sup\u003e. This stark contrast in toxicity levels demonstrates that DIHP is safer than parthenin, suggesting its potential for safe use. These findings support the clinical utility of DIHP as an alternate option for cancer therapy, enabling higher dosing regimens without the severe toxicity associated with parthenin.\u003c/p\u003e \u003cp\u003eUsing xenograft mouse models in colon cancer research offers significant advantages, including the ability to mimic human tumor behaviour and response to therapies closely\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. This model allows for a more accurate assessment of the efficacy and safety of potential therapeutic agents like DIHP. In our study, DIHP significantly reduced the tumor growth in the xenograft mouse model in addition to its anti-apoptotic effects. The combination of effective tumor reduction and low toxicity underscores the therapeutic potential of the DIHP molecule in treating CRC.\u003c/p\u003e \u003cp\u003eThe progression of inflammation-dysplasia-carcinoma is a common pathway in CAC\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. This sequence of inflammation to carcinoma progression can be developed in mice using AOM/DSS, which mimics human CAC\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Emerging research indicates that controlling AOM/DSS-induced CAC is achieved through reducing inflammation\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The current study demonstrated that animals in DIHP and 5-ASA treated groups experienced less body weight loss compared to the diseased group, and the reduction of DAI score was improved by DIHP in a dose-dependent way. Chronic inflammation causes anatomic abnormalities resulting in the shortening of the colon\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. This effect was consistent with the colon length of the disease group. However, treatment with 5-ASA and DIHP prevented the shortening of colon length. The administration of DIHP resulted in a significant decrease in tumor burden and resolved colon inflammation. When compared to disease control, the number of tumors decreased in the DIHP-treated mice, indicating its potential in preventing tumor progression. Histopathological analysis demonstrated that DIHP treatment significantly alleviated intestinal tissue damage. The pronounced immune cell infiltration and presence of dysplastic cells observed in the CAC group were notably reduced in both the 5-ASA and DIHP-treated groups, indicating the protective effect of DIHP on AOM/DSS-induced colon injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eDSS, a heparin-like polysaccharide used in AOM/DSS-induced CAC model, can compromise intestinal permeability by weakening epithelial integrity. This triggers immune cell infiltration and intestinal inflammation\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The colonic tissues of mice treated with AOM/DSS exhibited inflammation, as indicated by impaired intestinal permeability and elevated inflammatory cytokine levels\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Our results showed that DIHP-treated groups significantly reduced the level of pro-inflammatory cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-D) to resolve the intestinal inflammation.\u003c/p\u003e \u003cp\u003eThe development of colorectal tumors associated with colitis is significantly influenced by the NF-κB signalling pathway. It is previously reported that the NF-κB signalling pathway activation is responsible for colitis to develop cancer\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. According to our findings, mice treated with AOM/DSS had higher than normal levels of NF-κB, which is significantly reduced by the treatment of DIHP, indicating its anti-inflammatory properties by blocking the NF-κB signalling pathway. The upstream pathway PI3K/AKT regulates NF-κB and promotes tumor progression and metastasis\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. STAT3 and ERK are also involved in the pathogenesis of CAC by promoting chemoresistance and survival signals\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Our results demonstrated that DIHP significantly reduced the expression of key proteins involved in inflammation and supresses the pro-inflammatory cytokines.\u003c/p\u003e \u003cp\u003eOverall, this study demonstrates that DIHP induces p53 independent apoptosis in HCT 116 cells and suppresses tumor progression in HCT 116 xenograft and AOM/DSS-induced CAC mouse models with minimal toxicity and favourable PK profile emphasizing the potential of DIHP in the treatment of CRC.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThis study explores the anti-cancer potential of DIHP in CRC by inducing p53-independent apoptosis through inhibition of STAT3 and activation of the TRAIL pathway in the HCT 116 cell line. DIHP significantly reduces tumor growth in HCT 116 xenograft models and protects against colon injury and tumorigenesis in AOM/DSS-induced CAC by suppressing inflammation and different survival pathways, including PI3K/AKT, NF-κB, STAT3, and ERK. These findings highlight the multifaceted therapeutic potential of DIHP against CRC.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest related to this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.A and D.K, acknowledge the University Grant Commission for providing fellowship. This research was funded by the Ramanujan fellowship grant of Dr. Sanket Shukla (SB/S2/RJN-078/2019). This manuscript has been given the institutional publication number CSIR-IIIM/IPR/00866, Dated 29/01/2025.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.A, S.K.S and Z.A conceptualised the manuscript; all authors provided significant inputs. M.A and D.K performed \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. HCT 116 xenograft study conducted by S.D and R.P. D.M performed the PK study. DIHP synthesized by C.P.S and Y.P.B. All authors wrote, reviewed, edited and approved this final manuscript.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFadlallah H, El Masri J, Fakhereddine H, et al. Colorectal cancer: Recent advances in management and treatment. World J Clin Oncol. 2024;15(9):1136.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Shen X, Chen G, Du J. Drug resistance in colorectal cancer: from mechanism to clinic. 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Nat Cell Biol. 2021;23(4):377\u0026ndash;90.\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":"HCT 116, DIHP, Apoptosis, p53, Colorectal cancer","lastPublishedDoi":"10.21203/rs.3.rs-8268375/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8268375/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) is the second leading cause of cancer-related death worldwide, with mutation of the p53 gene found in up to 80% of advanced CRC cases, resulting in apoptotic halt and significant drug resistance. The development of new drugs targeting the mutated p53 gene in CRC patients is limited. This study explored the anticancer potential of a dispiro-indanedione hybrid of parthenin (DIHP) in a p53-independent manner using HCT 116 (human colorectal carcinoma cell line). DIHP induced apoptosis by blocking JAK2-STAT3-dependent survival protein like Bcl-xL, in cooperation with TRAIL (Apo2L), leading to dose-dependent cell death and cell cycle arrest. Acute toxicity and pharmacokinetic (PK) studies revealed DIHP is significantly less toxic than its parent parthenin and exhibits a favorable PK profile respectively. Efficacy assessments in both HCT 116 xenograft and AOM (Azoxymethane)/DSS (Dextran Sulfate Sodium) induced colitis-associated colorectal cancer (CAC) mouse models demonstrated significant tumor growth suppression and improved survival. Mechanistically, DIHP downregulated key oncogenic survival pathways including PI3K/AKT, NF-κB, STAT3, and ERK. Additionally, DIHP attenuated pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in the CAC model. These findings highlight the potential of DIHP in combating CRC, providing a new opportunity for the development of anticancer treatment with improved efficacy and possible therapeutic benefits.\u003c/p\u003e","manuscriptTitle":"Dispiro-indanedione hybrid of parthenin induces p53-independent apoptosis and suppresses tumor progression in colorectal cancer models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 00:11:15","doi":"10.21203/rs.3.rs-8268375/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":"bc4a92dc-45bd-436a-aa35-5b34ef6499e4","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-06T11:23:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-27 00:11:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8268375","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8268375","identity":"rs-8268375","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}