Influence of cobalt (III) Schiff base complexes on in-vitro anti-proliferative, oxidative stress and gene expression analysis in HeLa and HepG2 cell lines

Influence of cobalt (III) Schiff base complexes on in-vitro anti-proliferative, oxidative stress and gene expression analysis in HeLa and HepG2 cell lines | 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 Influence of cobalt (III) Schiff base complexes on in-vitro anti-proliferative, oxidative stress and gene expression analysis in HeLa and HepG 2 cell lines Gowdhami Balakrishnan, Vimala R.T.V, Mohamed Asik Rajmohamed, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1568074/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 The development of new medicines for the successful treatment of cervical and liver malignancies is critical in order to address the disadvantages of current chemotherapeutics, such as increased resistance. Metal ion-based chemical complexes have recently emerged as a prominent method for cancer therapy. As a result, the study aims to create novel anticancer medicines based on leads acquired through combinatorial chemistry of metal complexes. Cobalt (III) Schiff base trans- [Co(salen)(DA)2](ClO4) (complex 1) and trans-[Co (salophen)(DA)2](ClO4) (complex 2)] where, the salen and salopen were N, N′-bis(salicylidene) ethylenediamine, and N, N′-bis(salicylidene)-1,2- phenylenediamine, DA: dodecylamine) were synthesized as an alternative to the existing drugs, and their cytotoxic effect were evaluated against human cervical (HeLa) and liver cancer cell lines (HepG2) using MTT viability assay, and apoptotic morphological staining which including Acridine Orange/Ethidium Bromide (AO/EB), Hoechst 33528, Annexin V-Cy3 assay, JC-1 staining Comet assay, and reactive oxygen species (ROS) assay. Further the apoptosis was confirmed with immunocytochemistry and real-time reverse transcription-polymerase chain reaction ( RT-PCR). The results indicated that cobalt (III) complexes reduced the viability of the HeLa and HepG2 cell lines at their half minimum lethal doses, with cell death induced mainly via apoptotic pathway. Hence, the cobalt complexes 1 and 2 could be act as an effective drug for cervical and liver cancers. Anti-cervial and liver cancer drug cobalt (III) Schiff base complexes apoptosis DNA damage reactive oxygen species immune-fluorescence RT-PCR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Cervical and liver cancers are the most frequent global tumors that cause mortality. According to the Globocan report 2020, globally 19.2 million new cancer cases and 9.9 million deaths were reported, with liver cancer accounting for 0.9 million (4.7%) cases and 0.83 million deaths (8.3%), and cervical cancer accounting for 0.6 million (3.1%) new cases and 0.34 million deaths (3.4%), where lung cancer is the fifth most common cancer in males and cervical cancer is the fourth most common cancer in females (Sung et al., 2021). Cervical cancer is mostly caused by human papillomavirus (HPV) infection in females, therefore early diagnosis of HPV and HPV vaccination might assist to reduce patient death (Bogani et al., 2018). Currently, treatment methods such as chemo, hormone and immunotherapies in combination with surgery is widely used by physicians against the cervical and liver cancer which may help only before it has spread to other organs (Saini et al.,2020; Sudhakar, 2009 ,). In addition, commonly prescribing anticancer pills such as paclitaxel, etoposide, and cisplatin are inducing severe side effects and create drug resistant to the body (Longley and Johnston, 2005 ). Despite the fact that there are several strategies available for developing potential anticancer drugs, metal complexes have received a lot of attention in recent decades due to their potential properties such as redox activity, reactivity towards the organic substrate, hydrophilicity, solubility, bioavailability, and reduced in vivo toxicity (Nikolic et al., 2016 ; Frezza et al., 2010 ). Aside from that, when Schiff bases bind to metal complexes, they exert a potent chemotherapeutic effect [Kamal et al, 2017 ]. Furthermore, the ligand-mixed cobalt complexes can act as DNA cleavers, free radicals scavengers by activating antioxidant enzymes (e.g., SOD, CAT, GPx1), apoptotic enzymes, and mitochondrial redox regulation (Bagrezaei et al., 2018 ; Sheikhrezaei et al., 2018). Several studies show that coordinated cobalt (III) complexes are very cytotoxic and have anticancer properties (Wang et al., 2017 ). Cobalt (III) complexes induce DNA cleavage via the singlet oxygen cleavage pathway, as well as cytotoxicity against anti-breast cancer cells (Thamilarasan et al, 2016 ). By infiltrating cancer cells and binding to DNA, it enhances the chemotoxic impact of the complexes, causing cell death. As a result, cell metabolism is inhibited which activates caspases and causes apoptosis (Bauer et al., 2002 , Ahamed et al., 2016 ). As a result, cobalt (III) complexes 1 and 2 were synthesized and reported before in order to confirm their anticancer properties. The molecular processes behind the anticancer capability of cobalt (III) complexes against HeLa and HepG2 cell lines were investigated, and the results proved that the compound had the DNA intercalating and groove binding characteristics (Ambika et al., 2019 ). Our recent findings demonstrated the antiproliferative efficiency of the cobalt (III) Schiff base complexes 1 and 2 against human lung cancer cell lines (A549) and breast cancer cells (MCF-7) (Gowdhami et al., 2021). The current study has now been expanded to include cytotoxic effects on cervical cancer cell lines (HeLa) and liver cancer cell lines (HepG2). Furthermore, by comparing HeLa and HepG2 cell lines to normal mouse fibroblast (L929) cells, we verified the cytotoxicity and apoptosis up on evaluating several parameters such as DNA damage, chromosomal aberrations, cellular ROS production, micronuclei formation, and proapoptotic and antiapoptotic gene expression. Materials And Methods Two cobalt (III) Schiff base complexes, trans-[Co(salen)(DA)2] (ClO4) (1) and trans- [Co(salophen)(DA)2] (ClO4) (2) (where salen: N,N′ -bis(salicylidene)ethylenediamine, salophen: N,N′ -bis(salicylidene)- 1,2 phenylenediamine, DA: dodecylamine) were synthesized according to the methodology mentioned by Ambika et al., 2019 (Figure 1). Cell culture Human cervical cancer cells (HeLa), human liver cancer cells (HepG2), and normal mouse fibroblast cells (L929) were supplied by the National Centre for Cell Science in Pune, India, for this work. The cells were grown in DMEM medium supplemented with 10% FBS and 100 U/mL, penicillin and streptomycin (Antibiotics-Sigma, St. Louis, USA). The culture was maintained in a humidified atmosphere. MTT assay The MTT [3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide] test was mostly utilized to estimate the IC50 concentration of complexes (1 and 2). Cobalt complexes were dissolved in a buffer containing 100% DMSO (Sigma, St. Louis, USA). Then, the complexes were treated with cultured HeLa, HepG2, and L929 cells (96 well plates in each well 5000 cells)) separately for 24, 48, and 72 h. The positive control Cisplatin was dissolved in 0.02% DMSO. Then, 20 µL of MTT (5 mg/mL in PBS) solution was added to each well and the microtitre plates were kept for incubation for 4 h at 37 ºC by covering it with aluminium foil. The formazan product appeared in purple colour was dissolved in 100 µL DMSO and the solution absorbance at the wavelength of 570 nm (sample) and 630 nm (reference) was taken (Bio-Rad, California, USA). Finally, means and the standard deviations for three replicates were calculated to analyze the inhibition in terms of percentage using the formulae, Assays using Acridine Orange (AO) and Ethidium Bromide (EB) The experiment was conducted according to the Spector et al. method (1998). HeLa and HepG2 cells grown in a 6-well plate (Tarson, India) were incubated with the complexes (IC50 doses) for 24 hours. AO/EB staining was performed in both control and treated cells (5000 cells in a µL suspension) and the morphological changes were examined with the fluorescent microscope (Carl Zeiss, Jena, Germany). Based on the cytoplasm and nucleus appearance, the cells were categorized as alive or dead, and if dead, whether they died of apoptosis or necrosis, based on cytoplasm and nuclear morphology (Kumar et al., 2008). The data was obtained from the test that was performed in triplicate. Hoechst 33528 staining For 24 hours, HeLa and HepG2 cells were treated with the complexes (IC50 concentrations) and the cells were then stained with Hoechst 33528 for microscopic examination using fluorescent microscope where 100 cells randomly examined for the normal and abnormal nuclei of control vs treated cells (Rajendiran et al., 2007). The experiments were carried out in triplicate for the statistical analysis. Annexin V-Cy3 and 6-CFDA staining One of the first apoptotic characteristics is phosphoidylserine relocation from the internal to exterior surface of the cell membrane. The presence of phosphatidyl serine at the cell surface may be determined by AnnexinV-Cy3 staining (Spector et al., 1998). 6-CFDA is utilized to distinguish between apoptotic and live cells. The complexes' (IC50 doses) were administered to HeLa and HepG2 cells for 24 hours. The cells were harvested and washed with PBS binding buffer (0.1 M HEPES/NaOH, 25 mMCaCl, 1.4 M NaCl, pH 7.4) and suspended in 50 µL of Ann V-Cy3 and 6- CFDA. The plates were then incubated in the dark for 10 minutes . The cells were washed with annexin binding buffer in order to remove the excess cell staining and background fluorescents, and the Ann V-Cy3 (red) and 6-CFDA (green) labeled cells were observed under the fluorescent microscope. The results grouped upon counting the living (express only green) and early apoptotic (express both red and green) cells and the percentage of cells in live and early apoptotic cells was statistically analyzed. Mitochondrial membrane potential assessment (JC1 staining) Membrane potential (ΔΨ) was evaluated using the fluorescent probe JC-1; in healthy mitochondria, JC-1 monomers concentrate and release green fluorescence; in unhealthy mitochondria, the dye forms J-aggregates and emits orange fluorescent into the cytosol owing to loss of ΔΨ (Reers et al., 1991). HeLa and HepG2 cells were cultured in cover slips and the complexes were treated with the IC50 concentrations and incubated for 12 and 24 hours. The morphological changes were observed under a fluorescent microscope. Comet assay The IC50 doses of the complexes were administered to HeLa and HepG2 cells for 24 hours. (Singh et al. 1988). Clean glass slides were pre-coated with a typical 1% melting point agarose layer and the individually separated cells in 1% low melting agarose was coated over the solidified layer, and again the 1% normal melting agarose coated on the solidified layers and allowed to cool down. Later, the slides were immersed in a lysis solution (2.5 M NaCl, 100 mM Na2EDTA, Triton X-100, 0.2 mMNaOH [pH 10], and 10 mMTris) and incubated overnight to lyse the cell and enable DNA unfolding. The slides were then incubated for 20 mins with alkaline electrophoresis buffer (300 mM NaOH and 1 mM Na2-EDTA [pH = 13]) to unwind the DNA followed by 15 mins of electrophoresis at 0.8v/cm. The slides were observed under the fluorescence microscope after being stained with ethidium bromide (EtBr), and the percentage of DNA damage was calculated using CASP software. Measurement of ROS by DCFH-DA staining The oxidative stress agents produce reactive oxygen species (ROS) which can be measured with DCFH-DA staining where DCFH-DA is oxidized by ROS and generate to 2′, 7′- dichlorofluorescin diacetate which produce high green fluorescent (Wang and Joseph, 1999). HeLa and HepG2 cells were treated for 12 hours with the complexes 24 hours IC50 concentrations. After incubation, the DCFH-DA (5 µM) was added and incubated for 30 minutes at 37 ºC. The fluorescence intensity was recorded using a spectrofluorometer (Fluoroskan ascent, thermo scientific, USA), and the ROS production was observed using a fluorescent microscope. Immunofluorescence assay for Bcl2 and Bax proteins HeLa and HepG2 cells in cover slips were treated with the complexes (at IC50 concentrations) and incubated for 24 hours. The cells were fixed with 4% paraformaldehyde (prepared in PBS buffer) for 15 minutes at room temperature, washed twice with PBS ,and then permeabilized with 0.1 % Triton X-100. After the Triton X-100 was removed, the antigens were blocked for 1 hour using the blocking buffer (0.1% Triton X-100 + 5% BSA in PBS) . After washing, the cells were incubated overnight at 4 ºC with the primary antibodies (Bcl-2 and Bax). Later the cells were washed and incubated in dark for 30 min with the secondary antibody (Rabbit anti-Mouse IgM 2º Ab conjugated with FITC- Sigma, USA). Finally, the nucleus was stained with 4, 6-diamidino-2- phenylindole (DAPI; Sigma, USA) and the expression of BAX and Bcl2 was seen under a fluorescent microscope. Expression analysis of apoptosis-related genes by RT-PCR Thr RNA was isolated using Trizol method from control and treated HeLa and HepG2 cells, and it was quantified using ΔΔCt method. RNase-free DNase I (2 units) was added to the extracted RNA for 30 min at 37 °C to remove genomic DNAs. Multiscribe reverse transcriptase was used to create cDNA in the presence of dNTP, random primers, and 10x RT buffer at 25 °C for 10 minutes, followed by 120 minutes at 37 °C. A SYBR Green Master Mix (iScriptcDNA Kit) and a 7900 RT- PCR detection kit were used for RT-PCR (Roche, Light cycler 96, Germany). The RT-PCR was performed under the following conditions: First, 95 °C for 15 min; then, 40 cycles of 94 °C for 0.5-1 min, 50-58 °C for 0.5-1 min, and 72 °C for 1 min. GAPDH was used as a control (Murugadas et al., 2016; Gayathri et al., 2020). The experiment was triplicated for the statistical analysis. Statistics Data are calculated as mean ± standard deviation. Graph Pad Prism-6.0 was used to perform statistical analysis. Results Anti-proliferative activity of cobalt (III) complexes assessed by MTT assay The cobalt complexes 1 and 2 were synthesized according to the method described by us earlier (Ambika et al. 2019). Fig.1 shows the proposed structure of the complexes 1 and 2. MTT tests were performed on HeLa, HepG2, and L929 cells at various doses at different time intervals (24, 48, and 72 hours) to determine the percentage inhibition of cobalt (III) complexes 1 and 2. Furthermore, the cytotoxic evaluations of the complexes were assessed based on the concentration of exposure to reduce the cell survival rate at 50% (IC50 Complexes 1 and 2 were shown to be more effective against Hela and HepG2 cells than conventional Cisplatin based on the obtained IC50 values at 24, 48, and 72 hours). [HeLa: Complex 1 : IC50 values respectively in 22.15±0.6 mM (24 h); 18.51±0.5 mM (48 h); 12.53±0.1 mM (72 h); Complex 2 : 10.25±0.3 mM (24 h): 7.4±0.2 mM (48 h) 5.2±0.2 mM (72 h); HepG2: Complex 1 : 56.5±0.6 mM (24 h); 49.41±0.5 mM (24 h); 38.53±0.1 mM (24 h); Complex 2 : 40.25±0.3 mM (24 h); 34.2±0.2 mM (24 h); 28.28±0.2 mM (24 h)]. However, normal L929 cells causes less cytotoxicity for both cobalt (III) complexes 1 and 2 only at very high concentrations. The IC50 of cisplatin was, was 24 h, 142.2±0.5; 48 h, 124.6±0.6; 72 h, 102.8±0.6mM [Table 1; Fig. 2(A) and 2(B)]. Apoptosis-associated changes as revealed in AO/EtBr and Hoechst 33528 staining AO/EtBr staining was used to evaluate the cellular viability and apoptosis-associated changes in the cell membrane. Highly structured green fluorescing nuclei indicate viable and healthy cells (Fig 3). The affected cells manifested different grades of changes such as chromatin condensation in the nuclei: apoptosis at early stage-green fluorescing nuclei with perinuclear chromatin condensation; apoptosis at late stage - orange to red fluorescing nuclei with highly condensed chromatin; necrosis- swollen cells with fluorescing nuclei (red). Both qualitative and quantitative analysis indicated that cobalt (III) complexes at 24 hours of IC50 treatment, induced cell death to a large extent and necrosis to a marginal extent (Fig 4). Nuclear staining of cells treated with complexes with IC50 concentrations at 24 hours using Hoechst 33258 revealed normal nuclei in control cells, but the abnormal nuclei in treated cells about half of the papulation, including chromatin condensation/shrinkage, nuclear fragmentation into dot- like bodies, apoptotic body formation, etc. (Fig. 5). Quantitative analysis showed that normal nuclei were considerably reduced in treated HeLa and HepG2 cells on comparing with control as follows: complex 1= 52.0 ± 0.81 %; complex 2 = 48.3 ± 0.47 % in HeLa cells and complex 1= 51.33 ± 0.47 % and complex 2 = 53.33 ± 0.47 % in HepG2 cells. Control was showed 93 ± 0.81% normal nuclei in HeLa and 92.66±1.24 % in HepG2 cells (Fig. 6) . Change in mitochondrial transmembrane potential (MMP) MMP (ΔΨm) changes resulted in an electrochemical gradient which was an early stage of apoptosis and was detected by JC-1 dye. In our study, orange fluorescing cells were abundant in control cells indicating that there is no alteration in MMP. Treatment showed more than fifty percentage of the cells exhibit green fluorescent representing change in MMP, indicating an early step in apoptosis. Complexes 1 and 2 had significant influence in both HeLa and HepG2 cell lines (Fig. 7). DNA damage induced by the treatment, as revealed in Comet assay Cobalt (III) complexes mediated DNA damage in HeLa and HepG2 cells was demonstrated with comet assay. There were very few comets in the control cells. The frequency of comets and the comet tail length increased dramatically in the cells treated with cobalt (III) complexes (Fig. 8). The geometric and densitometric characteristics were analysed with CASP software to determine the head DNA % (intact DNA) and tail DNA % (strand breaks) areas. Furthermore, treated HeLa and HepG2 cells exhibit longer tail lengths, indicating that the compound promotes apoptosis. In HeLa and HepG2 have 98 % head DNA and 2% tail DNA, while control cells have 95% head DNA and 5% tail DNA (Fig. 9). Apoptosis vs necrosis, as revealed in AnnexinV-Cy3 – 6-CFDA staining As a sign of early apoptosis, the phosphatidyl serine shift to outer plasma membrane was examined with the control and treated cobalt (III) complexes on HeLa and HepG2 cells using annexin V-cy3 assay where the Annexin V positive (red fluorescence) and 6-CFDA positive (green fluorescence) cells was used to differentiate the viable, early apoptosis and necrosis cells . The result suggested that the complexes might preponderantly activate apoptosis rather than the necrosis (Fig. 10). The pictures reveal a large number of early apoptotic cells (Annexin V-Cy3 positive, 6-CFDA positive), a few necrotic cells (Annexin V-Cy3 positive, 6-CFDA negative), and a substantial proportion of viable cells (Annexin V-Cy3 positive, 6-CFDA negative) (Annexin V-Cy3 negative, 6- CFDA positive) (Fig 11). The complexes 1 and 2 were found to be a potential agents inducing apoptosis against HeLa and HepG2 cells. Generation of ROS on treatment with cobalt (III) complexes Mitochondria are important sites for energy metabolism where there is a strong association between oxidative stress and mitochondrial activity. As a result, the ROS productions induced by the cobalt (III) complexes (IC50 treatment for 12 hours) were measured in HeLa and HepG2 cells. The elevated ROS generation was observed using both spectroscopic measurement (reported as fold change) and fluorescent microscopy analysis (high contrast green-fluorescent images) (Fig 12 A, B). Effect of the complexes on pro-apoptotic and anti-apoptotic protein expression The effect of cobalt (III) complexes on the expression levels of Bcl-2 (pro-apoptotic protein) and Bax (anti-apoptotic protein) in HeLa and HepG2 cells was studied using immunofluorescent protocols. The fluorescence intensity was enhanced in Bax but decreased in Bcl-2 .The result revealed that the expression of Bax was upregulated and that of BCl2 was downregulated in the complexes, 1 and 2 (Fig. 13A, B). Effect of the complexes on the expression of apoptosis-associated genes For the development of chemotherapeutic drugs, sufficient knowledge of the cell signaling apoptotic pathway is necessary. Furthermore, chemotherapeutic drugs are directly associated to proapoptotic and antiapoptotic protein ratio which aids in the induction of the cancer cell death. As a result, we used RT-PCR to evaluate the influence of cobalt (III) complexes on the expression of pro- apoptotic (Cas-3, BAX, BID, and Cas-8), anti-apoptotic (Bcl-2 and GRP-78), and antioxidant gene (CAT, GPX1, and SOD) expressions. The expression of pro-apoptotic genes were upregulated (fold change for HeLa, Complex 1: BID (1.24), Bax (1.2), caspase-3 (1.5), and caspase- 8 (1.12) and complex 2: BID (1.05), Bax (1.12), caspase-3 (1.1), and caspase-8 (1.16) and HepG2, Complex: 1 BID (1.05), Bax (1.115), caspase-3 (1.11), and caspase- 8 (1.16) and complex 2: BID (1.45), Bax (1.24), caspase-3 (1.42), and caspase–8 (1.12)) compared to the control (normalized as 1 for all groups) in treated HeLa and HepG2 cells. Compared to control, anti-apoptotic genes (fold change for HeLa, Complex 1: Bcl-2 (0.84) and GRP- 78 (0.73), complex 2: Bcl-2 (0.64) and GRP-78 (0.69) and for HepG2, Complex 1: Bcl-2 (0.95) and GRP-78 (0.92), complex 2: Bcl-2 (0.84) and GRP-78 (0.91)) (Fig.14) and antioxidant genes (fold change for HeLa, Complex 1: SOD (0.64), CAT (0.84), and GPX1 (0.91) and complex 2: SOD (0.44), CAT (0.64), and GPX1 (0.45), and HepG2 Complex 1: SOD (0.84), CAT (0.81), and GPX1 (0.81) and complex 2: SOD (0.45), CAT (0.80), and GPX1 (0.54)) were down-regulated (Fig. 15). The antioxidant enzymes which play an important role in the defense mechanism against oxidative stress. In contrast, both cell lines have shown efficient gene expression succumbed to cell death. Discussion The structural and functional alteration of an existing chemical may allow for the discovery of a viable lead therapeutic molecule. The structure of the molecule may be altered by combining the ligands with the metal complexes in order to cause apoptotic cell death while also refining its properties such as enhancing solubility, improving absorbability, reducing toxicity, and providing other advantages (Jaividhya et al., 2012). Furthermore, current research has highlighted cobalt complexes as cytotoxic agents since they do not cause adverse effects (Riyasdeen.,et al., 2012). Further, our previous research shown the synthesis of novel cobalt complexes and their characterization using various techniques such as infra-red (IR) spectroscopy, NMR spectroscopy, ESI-MS, and UV-Visible spectroscopy (Ambika et al., 2019). In the absorption spectra, our study has observed the binding ability of the complexes to DNA through intercalating agent which will give the result of hypochromism. It is suggested the intercalation is mostly caused by aromatic functional group of salicylaldehyde. These unique features are believed to be significant in inducing the apoptotic cell death. As a result, the cytotoxic impact on cancer cell lines must be assessed. Further, complex 2 was found to have higher Kb values than complex 1 ( ( 1.31 × 10 4 and 3.15 × 10 4 M −Mn for complex 1 and complex 2). In this study, we explored the anticancer potential of cobalt (III) Schiff base complexes against human cervical and liver cancer cell lines, as well as validating our previous findings with A549 and MCF7 cells (Gowdhami et al., 2021). In order to confirm the cell cytotoxicity, various experiments on HeLa and HepG2 cells were conducted with the cobalt (III) Schiff base complexes. Initially, MTT assay was used to assess the cell viability and growth, and it was revealed that the complexes reduce the survival percentage of HeLa and HepG2 cells by stimulating the cell death at different time intervals at the IC50 concentration. It is hypothesized that changing the surface property of cobalt complexes may interact with DNA via modifying the hydrophobic forces (Alhadlaq et al., 2015; Mahmoudi et al., 2009). Further, AO/EB and Hoechst 33258 staining were performed to confirm the cell death. The morphological changes reported including chromatin fragmentation, vacuolation, cytoplasmic blebbing, multi-nucleation, and apoptotic body formation. In addition, cobalt (III) complexes showed better antiproliferative activity against HeLa and HepG2 cells than previously reported copper complex (Priyanga et al., 2019). In contrast, cobalt (III) complexes showed remarkable effect in their cytotoxic ability due to the DNA binding interactions and antiproliferative activity. Moreover, comet assay was performed as a one of the major techniques for measuring the DNA damage (Riyasdeen et al., 2012). When the HeLa and HepG2 cells were treated with cobalt (III) complexes, the tail DNA percentage was increased, resulting in apoptosis. It is proposed that the complexes produce ROS and chelate the DNA by different factors such as vanderwaal interactions, hydrophobic effects and so on, indicating the cells died of apoptosis (kumar and Arunachalam, 2008). Further, cellular stresses create aberrant circumstances that activate tumor suppressor protein p53. The activation of p53 (apoptotic protein) significantly increases p21 expression, which leads to release of apoptogenic factors, resulting in apoptosis (Cory and Adams 2002). As a consequence, our data show that cobalt (III) complexes treatment causes DNA damage in HeLa and HepG2 cells, resulting in cell death. AnnexinV-Cy3 staining was used to confirm the early apoptosis. According to the Mario and Kroemer (2013), one of the earliest processes in apoptosis is the translocation of phosphatidyl serine from the inner to outer leaflet of the plasma membrane. Cobalt (III) complexes have been shown to trigger apoptotic cell death, and mitochondrial silencing has also been connected to apoptosis. Mitochondria are maintaining the redox balance by regulating cell viability and generating ROS (Teodoro et al., 2011). Also, MMP alterations promote the mitochondrial swelling and readily permeabilize the outer membrane easily (Youle&Strasser, 2008; Kitsis & Molkentin, 2010). ROS is strongly associated with the MMP loss, indicating cell death where increasing ROS level result in cell cycle disruption, and cell signaling pathway. In addition, ROS triggers apoptosis in endoplasmic reticulum by stimulating various processes like hypoxia (Min et al., 2014). To validate this, we used DCFH-DA where the cell treated with cobalt (III) complexes and measure ROS level in terms of fluorescence. Under normal physiological conditions, intracellular ROS production is to be maintained to hinder the cell damage through neutralizing the antioxidants which specifically scavenge different kind of enzymes (Murugadas et al., 2016). ROS are highly reactive in general due to an unpaired electron in their outer cell. Increased level of reactive oxygen species will cause mitochondrial damage, increased cellular receptor signaling, cyclooxygenases, peroxisome activity, increased activity of oxidases, oncogene activity with invading immune cell (Liou and Storz, 2010). In General, the antioxidant activity of CAT, GPX1, and SOD are involved in the reduction of ROS level, with the different SOD enzymes being very specialized in regulating biological processes (Copin et al., 2000). Catalase also inhibits hepatic metastasis and decreases matrix metalloproteinase activity. Catalase also attenuates MMP expression at both the baseline and MnSOD-dependent levels. SOD cleaves the superoxide anion into oxygen and hydrogen peroxide in the presence of metal ions as cofactors and exists in eukaryotes and prokaryotes (Nishikawa et al., 2002). In the presence of several secondary enzymes and cofactors, GPX1, a seleno protein participates in antioxidant process with high efficiency. Over expression of this enzyme defend cells against oxidative damage and repress apoptosis caused by H2O2 (Lewis et al., 2006). The results of our study showed that CAT, GPX1, and SOD expression were down regulated at cobalt (III) complexes treated with IC50 for 24 hours, mean time the apoptotic genes were upregulated. It confirmed the neutralizing effect of the living system stimulated by the complexes. According to the findings, the complexes induce the apoptosis by increasing ROS production and reducing antioxidant gene expression (Gentile et al., 2017). Similarly, homeostatic conditions have to be sustained by equally balancing level of antiapoptotic and proapoptotic genes. Hence, our study tested Bcl-2, GRP78 (antiapoptotic gene), Cas-3, Bax, Cas-8, and BID gene expression levels (proapoptotic gene). The result showed that Bax, BID, Cas-3, and Cas- 8 gene expressions were found to be up regulated and Bcl-2 gene and GRP78 expression were found to be down regulated. It is suggested that Bcl-2 family proteins hinders the membrane permeability and prevent the interaction of other proteins. According to the results of the immunofluorescence test, there was a decrease in BCl2 and an increase in Bax fluorescence in the cobalt (III) complexes-treated cells. Caspase-8 can directly activate downstream caspases in the death receptor pathway, where mitochondrial damage is not needed in certain cells (type I cells). In other circumstances, however, caspase-8 cleaves and activates Bid to its truncated form (tBid), which recruits the mitochondrial pathway, which is a key step for cell death (Jost et al., 2009). For example, the BH3 proteins like Bid, conjugate with all prosurvival proteins where the prosurvival proteins bind and activate Bax and Bak. It involves in BH3: groove interaction and activate apoptosis (Lindsay et al., 2010). Further, the intrinsic pathway is mainly involved in mitochondrial cytochrome-c activation which results apoptosome construction consists of procaspase-9, cytochrome-c and Apaf-1 that activate caspase-9 which subsequently triggers caspase-3. As a result, it leads DNA fragmentation finally (Ergun et al., 2014). GRP78 triggers pro-survival pathways and transmit signals by inducing tumor proliferation, survival, anti-apoptosis (Casas., 2017; roller et al., 2013). Henceforth, over all the result is concluded that the role of apoptotic and antiapoptotic proteins has important potential for cancer therapy. Conclusion In conclusion, the cobalt complexes repressed the growth of cervical and liver cancer cells compared to normal cells. According to cytological labelling, cobalt (III) complexes promote apoptosis in the both the cancer cell lines by destroying DNA, altering mitochondrial membrane potential and causing oxidative stress. According to the molecular level analysis, it has been found that increased pro-apoptotic proteins while decreasing anti-apoptotic proteins expression, where cobalt complexes 1 and 2 are more effective anti-cancer chemotherapeutic drugs than cisplatin which might be investigated further as an anti-cancer chemotherapeutic treatment Declarations Acknowledgments We express our sincere gratitude to the Dr. R. Thirumurugan, Co-ordinator of National Centre for Alternatives to Animal Experiments (NCAAE), for his continuous support, encouragement, and guidance during the course of this research. This work was financially supported by National Centre for Alternatives to Animal Experiments (NCAAE) under UGC-CPEPA scheme, Government of India (F.No.2-1/2013 (NS/PE)). The research facility provided by Mahatma Gandhi-Doerenkamp Centre, established by Doerenkamp-Zbinden Foundation, and the Department of Science and Technology, Government of India, under DST-Promotion of University Research and Scientific Excellence (PURSE) scheme-Phase II, RashtriyaUchchatar Shiksha Abhiyan (RUSA)-2.O by the Department of Animal Science is heartily acknowledged. The authors thank the Management of Bishop Heber College (Autonomous), Tiruchirappalli-620 017, Tamil Nadu, India, for the support (F.No: MRP/1014/2020 dated : 23.12.2020) and facilities provided through Material Chemistry Lab, PG and Research Department of Chemistry and DST-FIST Instrumentation Centre (HAIF) at Bishop Heber College. Conflicts of interest The authors declare no conflicts of interest. Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors. References Ahamed, M., Akhtar M.J., Alhadlaq, H.A., Alshamsan, A., 2016. 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Experimental Cell Research. 175: 184–191. Spector, D.L., Goldman, R.D., Leinwand. 1998. Cells: a laboratory manual, Cold Spring Harbor Laboratory Sudhakar A., 2009. History of Cancer, Ancient and Modern Treatment Methods. Journal of Cancer Science and Therapy. 1(2): 1–4. Teodoro, J.S, Simões, A.M., Duarte, F.V., Rolo, A.P, Murdoch, R.C., Hussain, S.M., Palmeira, C.M., 2011. Assessment of the toxicity of silver nanoparticles in vitro: a mitochondrial perspective. Toxicology In vitro.25 (3):664-70. Thamilarasan, V., Sengottuvelan, N., Sudha, A., Srinivasan, P., Chakkaravarthi, G., 2016. Cobalt (III) complexes as potential anticancer agents: Physicochemical, structural, cytotoxic activity and DNA/protein interactions. The Journal of Photochemistry and Photobiology B. 162:558-569. Wang, H., Joseph, J.A., 1999.Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Biology and Medicine.27 (5-6):612-616. Wang, T., Fang, Y., Zhao, M., Li, M.X., Ji, Y.M., Han, Q.X., 2017. Cu (II), Ga (III) and In (III) complexes of 2-acetylpyridine N (4)-phenylthiosemicarbazone: synthesis, spectral characterization and biological activities. Medicinal Chemistry Communications.8 (11):2125- 2132. Youle, R.J, Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology. 9:47-59. Tables Table 1. MTT assay showing the effect of cobalt complexes (1-2) and cisplatin on HeLa, HepG2, and L929 cells. The mean and standard deviation of three triplicates are represented by each data point. Cell line Compound 24 h 48 h 72 h HeLa(µM) Complex1 22.15±0.6 18.51±0.5 12.53±0.1 Complex2 10.25±0.3 7.4±0.2 5.2±0.2 Cisplatin 26.15±0.4 21.52±0.1 18.43±0.1 HepG2(µM) Complex1 56.5±0.6 49.41±0.5 38.53±0.1 Complex2 40.25±0.3 34.2±0.2 28.28±0.2 Cisplatin 31.15±0.4 26.52±0.1 18.43±0.1 L929(µM) Complex 1 124.4±0.5 104.6±0.9 82.9±0.3 Complex 2 116±0.5 95.8±0.6 78.58±0.4 Cisplatin 142.2±0.5 124.6±0.6 102.8±0.6 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1568074","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":100669832,"identity":"8602ccd8-de91-4f0a-b48b-d1787858bf14","order_by":0,"name":"Gowdhami Balakrishnan","email":"","orcid":"","institution":"Bharathidasan University","correspondingAuthor":false,"prefix":"","firstName":"Gowdhami","middleName":"","lastName":"Balakrishnan","suffix":""},{"id":100669833,"identity":"6efb45ea-6c9b-4ec3-a67b-594f0e22863e","order_by":1,"name":"Vimala R.T.V","email":"","orcid":"","institution":"Bharathidasan 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2","display":"","copyAsset":false,"role":"figure","size":247078,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Antiproliferative effect of cobalt complexes with HeLa, HepG2, and L929 cells for 24, 48, and 72 hours (The mean and standard deviation of three triplicates are represented by each data point).\u003c/p\u003e\u003cp\u003e(B) Bar diagram showing the treatment effect of cobalt complexes and cisplatin with HeLa, HepG2, and L929 cells (The mean and standard deviation of three triplicates is represented by each data point).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig02.png","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/cf085d45119172baab2e4d76.png"},{"id":20785138,"identity":"4cd8a309-89b2-4705-9999-77af7e394549","added_by":"auto","created_at":"2022-04-26 15:10:41","extension":"jpeg","order_by":3,"title":"Figure 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6","display":"","copyAsset":false,"role":"figure","size":20448,"visible":true,"origin":"","legend":"\u003cp\u003e\tTreatment percentage of normal and abnormal nuclei at 24 h (The mean and standard deviation of three triplicates is represented by each data point).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/15f386a10946e12701383e26.png"},{"id":20783304,"identity":"6e2cbd6a-9cbe-4088-86c1-71ff75646a62","added_by":"auto","created_at":"2022-04-26 14:55:42","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":178767,"visible":true,"origin":"","legend":"\u003cp\u003eJC-1 staining of control and treated HeLa and HepG2 cells for mitochondrial membrane 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9","display":"","copyAsset":false,"role":"figure","size":10921,"visible":true,"origin":"","legend":"\u003cp\u003eDNA damage induction in HeLa and HepG2 cells by cobalt (III) complexes. Intact (0-20%), somewhat injured (20-40%), damaged (40-60%), severely damaged (60-80%), and dead (80-100%) (The mean and standard deviation of three triplicates is represented by each data point).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/3f2c56c5916f8d6f34c2e231.png"},{"id":20783303,"identity":"41101d1c-69ae-4d22-b135-8ab7b161fe28","added_by":"auto","created_at":"2022-04-26 14:55:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":56910,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of cobalt (III) complexes on HeLa and HepG2 cells using Annexin V-Cy3 - 6- CFDA staining.\u003c/p\u003e","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/f4d55f98e0a542f9074a36b6.png"},{"id":20784097,"identity":"09a66a27-f104-4646-80f9-e05cca399f81","added_by":"auto","created_at":"2022-04-26 15:00:41","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":11239,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of cell death induced by Cobalt (III) complexes against HeLa and HepG2 cells (The mean and standard deviation of three replicates is shown by each data point).\u003c/p\u003e","description":"","filename":"Onlinefloatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/ded04469e35cbe5a1e6a5452.png"},{"id":20783301,"identity":"3d7c8d24-0e93-440b-9d20-2eb7b749c850","added_by":"auto","created_at":"2022-04-26 14:55:41","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":234952,"visible":true,"origin":"","legend":"\u003cp\u003eA. Influence of cobalt (III) complexes on HeLa and HepG2 cells using DCFH-DA fluorescence staining.\u003c/p\u003e\u003cp\u003eB. Influence of cobalt (III) complexes on HeLa and HepG2 cells as revealed by ROS assay (The mean and standard deviation of three replicates is shown by each data point).\u003c/p\u003e","description":"","filename":"Fig12.png","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/6909faf5059a9b2fcfaaaa57.png"},{"id":20782063,"identity":"fca61504-455a-4152-b27f-b69f5b264d06","added_by":"auto","created_at":"2022-04-26 14:50:41","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":1097211,"visible":true,"origin":"","legend":"\u003cp\u003eA. Influence of Bax gene expression against HeLa and HepG2cells treated with cobalt (III) complexes.\u003c/p\u003e\u003cp\u003eB. Influence of BCl2 gene expression against HeLa and HepG2 cells treated with cobalt (III) complexes.\u003c/p\u003e","description":"","filename":"Fig13.png","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/7c53485524d2a194d045291e.png"},{"id":20784094,"identity":"a7b4caa2-e763-4baf-beea-fad496cb7f4e","added_by":"auto","created_at":"2022-04-26 15:00:41","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":102330,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of pro- and anti-apoptotic genes in HeLa and HepG2 cells exposed to cobalt (III) complexes. G) Superoxide dismutase; H) Catalase; I) GPX1. As a control, GAPDH was utilized. The mean and standard deviation of three replicates is shown by each data point.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig14.png","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/e89e0090b374b5223f1b56c3.png"},{"id":20782069,"identity":"1f9f8330-e949-40ae-ba42-11d08d70e163","added_by":"auto","created_at":"2022-04-26 14:50:41","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":60177,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of antioxidant genes in HeLa and HepG2 cells exposed to cobalt (III) complexes. G) Superoxide dismutase; H) Catalase; I) GPX1. As a control, GAPDH was utilized. The mean and standard deviation of three replicates is shown by each data point.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig15.png","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/bac6e1bee2e110444c608d5a.png"},{"id":22083234,"identity":"b8fd199b-000d-4ca9-8e16-ecbdccbd2c83","added_by":"auto","created_at":"2022-05-31 16:11:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3043417,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1568074/v1/7027fe0d-70f9-4192-8d8c-7b093c666e89.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eInfluence of cobalt (III) Schiff base complexes on\u003cem\u003e in-vitro\u003c/em\u003e anti-proliferative, oxidative stress and gene expression analysis in HeLa and HepG\u003csub\u003e2\u003c/sub\u003e cell lines\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCervical and liver cancers are the most frequent global tumors that cause mortality. According to the Globocan report 2020, globally 19.2\u0026nbsp;million new cancer cases and 9.9\u0026nbsp;million deaths were reported, with liver cancer accounting for 0.9\u0026nbsp;million (4.7%) cases and 0.83\u0026nbsp;million deaths (8.3%), and cervical cancer accounting for 0.6\u0026nbsp;million (3.1%) new cases and 0.34\u0026nbsp;million deaths (3.4%), where lung cancer is the fifth most common cancer in males and cervical cancer is the fourth most common cancer in females (Sung et al., 2021). Cervical cancer is mostly caused by human papillomavirus (HPV) infection in females, therefore early diagnosis of HPV and HPV vaccination might assist to reduce patient death (Bogani et al., 2018). Currently, treatment methods such as chemo, hormone and immunotherapies in combination with surgery is widely used by physicians against the cervical and liver cancer which may help only before it has spread to other organs (Saini et al.,2020; Sudhakar, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e,). In addition, commonly prescribing anticancer pills such as paclitaxel, etoposide, and cisplatin are inducing severe side effects and create drug resistant to the body (Longley and Johnston, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the fact that there are several strategies available for developing potential anticancer drugs, metal complexes have received a lot of attention in recent decades due to their potential properties such as redox activity, reactivity towards the organic substrate, hydrophilicity, solubility, bioavailability, and reduced in vivo toxicity (Nikolic et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Frezza et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Aside from that, when Schiff bases bind to metal complexes, they exert a potent chemotherapeutic effect [Kamal et al, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e]. Furthermore, the ligand-mixed cobalt complexes can act as DNA cleavers, free radicals scavengers by activating antioxidant enzymes (e.g., SOD, CAT, GPx1), apoptotic enzymes, and mitochondrial redox regulation (Bagrezaei et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sheikhrezaei et al., 2018).\u003c/p\u003e\u003cp\u003eSeveral studies show that coordinated cobalt (III) complexes are very cytotoxic and have anticancer properties (Wang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cobalt (III) complexes induce DNA cleavage via the singlet oxygen cleavage pathway, as well as cytotoxicity against anti-breast cancer cells (Thamilarasan et al, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). By infiltrating cancer cells and binding to DNA, it enhances the chemotoxic impact of the complexes, causing cell death. As a result, cell metabolism is inhibited which activates caspases and causes apoptosis (Bauer et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Ahamed et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAs a result, cobalt (III) complexes 1 and 2 were synthesized and reported before in order to confirm their anticancer properties. The molecular processes behind the anticancer capability of\u003c/p\u003e\u003cp\u003ecobalt (III) complexes against HeLa and HepG2 cell lines were investigated, and the results proved that the compound had the DNA intercalating and groove binding characteristics (Ambika et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our recent findings demonstrated the antiproliferative efficiency of the cobalt (III) Schiff base complexes 1 and 2 against human lung cancer cell lines (A549) and breast cancer cells (MCF-7) (Gowdhami et al., 2021).\u003c/p\u003e\u003cp\u003eThe current study has now been expanded to include cytotoxic effects on cervical cancer cell lines (HeLa) and liver cancer cell lines (HepG2). Furthermore, by comparing HeLa and HepG2 cell lines to normal mouse fibroblast (L929) cells, we verified the cytotoxicity and apoptosis up on evaluating several parameters such as DNA damage, chromosomal aberrations, cellular ROS production, micronuclei formation, and proapoptotic and antiapoptotic gene expression.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eTwo cobalt (III) Schiff base complexes, trans-[Co(salen)(DA)2] (ClO4) (1) and trans- [Co(salophen)(DA)2] (ClO4) (2) (where salen: N,N\u0026prime; -bis(salicylidene)ethylenediamine, salophen:\u0026nbsp;N,N\u0026prime; -bis(salicylidene)- 1,2 phenylenediamine, DA: dodecylamine) were synthesized according to the methodology mentioned by Ambika et al., 2019 (Figure\u0026nbsp;1).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHuman cervical cancer cells (HeLa), human liver cancer cells (HepG2), and normal mouse\u0026nbsp;fibroblast cells (L929) were supplied by the National Centre for Cell Science in Pune, India, for this work. The cells were grown in DMEM medium supplemented with 10% FBS and 100 U/mL, penicillin and streptomycin (Antibiotics-Sigma, St. Louis, USA). The culture was maintained in a humidified atmosphere.\u003c/p\u003e\n\u003ch3\u003eMTT assay\u003c/h3\u003e\n\u003cp\u003eThe MTT [3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide] test was mostly utilized to estimate the IC50\u0026nbsp;concentration of complexes (1 and 2). Cobalt complexes were dissolved\u0026nbsp;in a buffer containing 100% DMSO (Sigma, St. Louis, USA). Then, the complexes were treated with cultured HeLa, HepG2, and L929 cells (96 well plates in each well 5000 cells)) separately for 24, 48,\u0026nbsp;and 72 h. The positive control Cisplatin was dissolved in 0.02% DMSO. Then, 20 \u0026micro;L of MTT (5 mg/mL in PBS) solution was added to each well and the microtitre plates were kept for incubation for 4\u0026nbsp;h\u0026nbsp;at\u0026nbsp;37\u0026nbsp;\u0026ordm;C\u0026nbsp;by\u0026nbsp;covering\u0026nbsp;it\u0026nbsp;with\u0026nbsp;aluminium\u0026nbsp;foil.\u0026nbsp;The\u0026nbsp;formazan\u0026nbsp;product\u0026nbsp;appeared\u0026nbsp;in\u0026nbsp;purple\u0026nbsp;colour\u0026nbsp;was\u003c/p\u003e\n\u003cp\u003edissolved in 100 \u0026micro;L DMSO and the solution absorbance at the wavelength of 570 nm (sample) and 630 nm (reference) was taken (Bio-Rad, California, USA). Finally, means and the standard deviations for three replicates were calculated to analyze the inhibition in terms of percentage using the formulae,\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"539\" 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alt=\"image\"\u003e\u003c/p\u003e\n\u003ch3\u003eAssays using Acridine Orange (AO) and Ethidium Bromide (EB)\u003c/h3\u003e\n\u003cp\u003eThe experiment was conducted according to the Spector et al. method (1998). HeLa and HepG2\u0026nbsp;cells grown in a 6-well plate (Tarson, India) were incubated with the complexes (IC50 doses)\u0026nbsp;for 24 hours. AO/EB staining was performed in both control and treated cells (5000 cells in a \u0026micro;L suspension) and the morphological changes were examined with the fluorescent microscope (Carl Zeiss, Jena, Germany). Based on the cytoplasm and nucleus appearance, the\u0026nbsp;cells were categorized as alive or dead, and if dead, whether they died of apoptosis or necrosis, based on cytoplasm and nuclear morphology (Kumar et al., 2008). The data was obtained from the test that was performed in triplicate.\u003c/p\u003e\n\u003ch3\u003eHoechst 33528 staining\u003c/h3\u003e\n\u003cp\u003eFor 24 hours, HeLa and HepG2\u0026nbsp;cells were treated with the complexes (IC50 concentrations)\u0026nbsp;and the cells were then stained with Hoechst 33528 for microscopic examination using fluorescent microscope where 100 cells randomly examined for the normal and abnormal nuclei of control vs treated cells (Rajendiran et al., 2007). The experiments were carried out in triplicate for the statistical analysis.\u003c/p\u003e\n\u003ch3\u003eAnnexin V-Cy3 and 6-CFDA staining\u003c/h3\u003e\n\u003cp\u003eOne of the first apoptotic characteristics is phosphoidylserine relocation from the internal to exterior surface of the cell membrane. The presence of phosphatidyl serine at the cell surface may be determined by AnnexinV-Cy3 staining (Spector et al., 1998). 6-CFDA is utilized to distinguish between apoptotic and live cells. The complexes\u0026apos; (IC50 doses) were administered to HeLa and HepG2\u0026nbsp;cells for 24 hours. The cells were harvested and washed with PBS binding buffer (0.1 M HEPES/NaOH, 25 mMCaCl, 1.4 M NaCl, pH 7.4) and suspended in 50 \u0026micro;L of Ann V-Cy3 and 6- CFDA. The plates were then incubated in the dark for 10 minutes\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThe cells were washed with annexin binding buffer in order to remove the excess cell staining and background fluorescents, and\u003c/p\u003e\n\u003cp\u003ethe Ann V-Cy3 (red) and 6-CFDA (green) labeled cells were observed under the fluorescent microscope. The results grouped upon counting the living (express only green) and early apoptotic (express both red and green) cells and the percentage of cells in live and early apoptotic cells was statistically analyzed.\u003c/p\u003e\n\u003ch3\u003eMitochondrial membrane potential assessment (JC1 staining)\u003c/h3\u003e\n\u003cp\u003eMembrane potential (\u0026Delta;\u0026Psi;) was evaluated using the fluorescent probe JC-1; in healthy mitochondria, JC-1 monomers concentrate and release green fluorescence; in unhealthy mitochondria, the dye forms J-aggregates and emits orange fluorescent into the cytosol owing to loss of \u0026Delta;\u0026Psi; (Reers et al., 1991). HeLa and HepG2\u0026nbsp;cells were cultured in cover slips and the complexes were treated with the IC50\u0026nbsp;concentrations and incubated for 12 and 24 hours. The morphological changes were observed\u0026nbsp;under a fluorescent\u0026nbsp;microscope.\u003c/p\u003e\n\u003ch3\u003eComet assay\u003c/h3\u003e\n\u003cp\u003eThe IC50 doses of the complexes were administered to HeLa and HepG2\u0026nbsp;cells for 24 hours.\u0026nbsp;(Singh et al. 1988). Clean glass slides were pre-coated with a typical 1% melting point agarose layer and the individually separated cells in 1% low melting agarose was coated over the solidified layer, and again the 1% normal melting agarose coated on the solidified layers and allowed to cool down. Later, the slides were immersed in a lysis solution (2.5 M NaCl, 100 mM Na2EDTA, Triton X-100,\u003c/p\u003e\n\u003cp\u003e0.2 mMNaOH [pH 10], and 10 mMTris) and incubated overnight to lyse the cell and enable DNA unfolding. The slides were then incubated for 20 mins with alkaline electrophoresis buffer (300 mM NaOH and 1 mM Na2-EDTA [pH = 13]) to unwind the DNA followed by 15 mins of electrophoresis at 0.8v/cm. The slides were observed under the fluorescence microscope after being stained with ethidium bromide (EtBr), and the percentage of DNA damage was calculated using CASP software.\u003c/p\u003e\n\u003ch3\u003eMeasurement of ROS by DCFH-DA staining\u003c/h3\u003e\n\u003cp\u003eThe oxidative stress agents produce reactive oxygen species (ROS) which can be measured with DCFH-DA staining where DCFH-DA is oxidized by ROS and generate to 2\u0026prime;, 7\u0026prime;- dichlorofluorescin diacetate which produce high green fluorescent (Wang and Joseph, 1999). HeLa and HepG2\u0026nbsp;cells were treated for 12 hours with the complexes 24 hours IC50\u0026nbsp;concentrations. After\u0026nbsp;incubation, the DCFH-DA (5 \u0026micro;M) was added and incubated for 30 minutes at 37 \u0026ordm;C. The fluorescence intensity was recorded using a spectrofluorometer (Fluoroskan ascent, thermo scientific, USA), and the ROS production was observed using a fluorescent\u0026nbsp;microscope.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence assay for Bcl2\u0026nbsp;and Bax proteins\u003c/h3\u003e\n\u003cp\u003eHeLa and HepG2 cells in cover slips were treated with the complexes (at IC50 \u0026nbsp;concentrations) and incubated for 24 hours. The cells were fixed with 4% paraformaldehyde (prepared in PBS buffer) for 15 minutes at room temperature, washed twice with PBS ,and then permeabilized with 0.1 % Triton X-100. After the Triton X-100 was removed, the antigens were blocked for 1 hour using the blocking buffer (0.1% Triton X-100 + 5% BSA in PBS) . After washing, the cells were incubated overnight at 4 \u0026ordm;C with the primary antibodies (Bcl-2 and Bax). Later the cells were washed and incubated in dark for 30 min with the secondary antibody (Rabbit anti-Mouse IgM 2\u0026ordm; Ab conjugated with FITC- Sigma, USA). Finally, the nucleus was stained with 4, 6-diamidino-2- phenylindole (DAPI; Sigma, USA) and the expression of BAX and Bcl2 was seen under a fluorescent microscope.\u003c/p\u003e\n\u003ch3\u003eExpression analysis of apoptosis-related genes by RT-PCR\u003c/h3\u003e\n\u003cp\u003eThr RNA was isolated using\u0026nbsp;\u003ca href=\"http://scholar.google.co.in/scholar?q=trizol%2BRNA%2BISOLATION%2BMETHODS\u0026hl=en\u0026as_sdt=0\u0026as_vis=1\u0026oi=scholart\"\u003eTrizol\u0026nbsp;\u003c/a\u003emethod from control and treated HeLa and HepG2\u0026nbsp;cells,\u0026nbsp;and it was quantified using \u0026Delta;\u0026Delta;Ct method. RNase-free DNase I (2 units) was added to the extracted RNA for 30 min at 37 \u0026deg;C to remove genomic DNAs. Multiscribe reverse transcriptase was used to create cDNA in the presence of dNTP, random primers, and 10x RT buffer at 25 \u0026deg;C for 10 minutes, followed by 120 minutes at 37 \u0026deg;C. A SYBR Green Master Mix (iScriptcDNA Kit) and a 7900 RT- PCR detection kit were used for RT-PCR (Roche, Light cycler 96, Germany). The RT-PCR was performed under the following conditions: First, 95 \u0026deg;C for 15 min; then, 40 cycles of 94 \u0026deg;C for 0.5-1 min, 50-58 \u0026deg;C for 0.5-1 min, and 72 \u0026deg;C for 1 min. GAPDH was used as a control (Murugadas et al., 2016; Gayathri et al., 2020). The experiment was triplicated for the statistical analysis.\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eData are calculated as mean \u0026plusmn; standard deviation. Graph Pad Prism-6.0 was used to perform statistical analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAnti-proliferative activity of cobalt (III) complexes assessed by MTT assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cobalt complexes 1 and 2 were synthesized according to the method described by us earlier (Ambika et al. 2019). Fig.1 shows the proposed structure of the complexes 1 and 2.\u003c/p\u003e\n\u003cp\u003eMTT tests were performed on HeLa, HepG2, and L929 cells at various doses at different time\u0026nbsp;intervals (24, 48, and 72 hours) to determine the percentage inhibition of cobalt (III) complexes 1 and\u003c/p\u003e\n\u003cp\u003e2. Furthermore, the cytotoxic evaluations of the complexes were assessed based on the concentration of exposure to reduce the cell survival rate at 50% (IC50 Complexes 1 and 2 were shown to be more effective against Hela and HepG2 cells than conventional Cisplatin based on the obtained \u0026nbsp;IC50 values at 24, 48, and 72 hours). [HeLa: Complex \u003cstrong\u003e1\u003c/strong\u003e: IC50 values respectively in 22.15\u0026plusmn;0.6 mM (24 h); 18.51\u0026plusmn;0.5 mM (48 h); 12.53\u0026plusmn;0.1 mM (72 h); Complex \u003cstrong\u003e2\u003c/strong\u003e: 10.25\u0026plusmn;0.3 mM (24 h): 7.4\u0026plusmn;0.2 mM (48 h) 5.2\u0026plusmn;0.2 mM (72 h); HepG2: Complex \u003cstrong\u003e1\u003c/strong\u003e: 56.5\u0026plusmn;0.6 mM (24 h); 49.41\u0026plusmn;0.5 mM (24 h); 38.53\u0026plusmn;0.1 mM (24 h); Complex \u003cstrong\u003e2\u003c/strong\u003e: 40.25\u0026plusmn;0.3\u0026nbsp;mM (24 h); 34.2\u0026plusmn;0.2\u0026nbsp;mM (24 h); 28.28\u0026plusmn;0.2\u0026nbsp;mM (24 h)]. However, normal L929 cells causes less cytotoxicity for both cobalt (III) complexes 1 and 2 only at very high\u0026nbsp;concentrations. The IC50\u0026nbsp;of cisplatin was, was 24 h, 142.2\u0026plusmn;0.5; 48 h, 124.6\u0026plusmn;0.6; 72 h, 102.8\u0026plusmn;0.6mM\u0026nbsp;[Table 1; Fig. 2(A) and\u0026nbsp;2(B)].\u003c/p\u003e\n\u003ch3\u003eApoptosis-associated changes as revealed in AO/EtBr and Hoechst 33528 staining\u003c/h3\u003e\n\u003cp\u003eAO/EtBr staining was used to evaluate the cellular viability and apoptosis-associated changes in the cell membrane. Highly structured green fluorescing nuclei indicate viable and healthy cells (Fig 3). The affected cells manifested different grades of changes such as chromatin condensation in the nuclei: apoptosis at early stage-green fluorescing nuclei with perinuclear chromatin condensation; apoptosis at late stage - orange to red fluorescing nuclei with highly condensed chromatin; necrosis- swollen cells with fluorescing nuclei (red). Both qualitative and quantitative analysis indicated that cobalt (III) complexes at 24 hours of IC50\u0026nbsp;treatment, induced cell death to a large extent and necrosis\u0026nbsp;to a marginal extent (Fig 4).\u003c/p\u003e\n\u003cp\u003eNuclear staining of cells treated with complexes with IC50\u0026nbsp;concentrations at 24 hours using\u0026nbsp;Hoechst 33258 revealed normal nuclei in control cells, but the abnormal nuclei in treated cells about half of the papulation, including chromatin condensation/shrinkage, nuclear fragmentation into dot- like bodies, apoptotic body formation, etc. (Fig. 5).\u003c/p\u003e\n\u003cp\u003eQuantitative analysis showed that normal nuclei were considerably reduced in treated HeLa and HepG2\u0026nbsp;cells on comparing with control as follows: complex 1= 52.0 \u0026plusmn; 0.81 %; complex 2 = 48.3\u003c/p\u003e\n\u003cp\u003e\u0026plusmn; 0.47 % in HeLa cells and complex 1= 51.33 \u0026plusmn; 0.47 % and complex 2 = 53.33 \u0026plusmn; 0.47 % in HepG2\u0026nbsp;cells. Control was showed 93 \u0026plusmn; 0.81% normal nuclei in HeLa and 92.66\u0026plusmn;1.24 % in HepG2\u0026nbsp;cells (Fig.\u0026nbsp;6)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003ch3\u003eChange in mitochondrial transmembrane potential (MMP)\u003c/h3\u003e\n\u003cp\u003eMMP (\u0026Delta;\u0026Psi;m) changes resulted in an electrochemical gradient which was an early stage of apoptosis and was detected by JC-1 dye. In our study, orange fluorescing cells were abundant in control cells indicating that there is no alteration in MMP. Treatment showed more than fifty percentage of the cells exhibit green fluorescent representing change in MMP, indicating an early step in apoptosis. Complexes 1 and 2 had significant influence in both HeLa and HepG2\u0026nbsp;cell lines (Fig. 7).\u003c/p\u003e\n\u003ch3\u003eDNA damage induced by the treatment, as revealed in Comet assay\u003c/h3\u003e\n\u003cp\u003eCobalt (III) complexes mediated DNA damage in HeLa and HepG2\u0026nbsp;cells was demonstrated\u0026nbsp;with comet assay. There were very few comets in the control cells. The frequency of comets and the comet tail length increased dramatically in the cells treated with cobalt (III) complexes (Fig. 8). The geometric and densitometric characteristics were analysed with CASP software to determine the head DNA % (intact DNA) and tail DNA % (strand breaks) areas. Furthermore, treated HeLa and HepG2 cells exhibit longer tail lengths, indicating that the compound promotes apoptosis. In\u0026nbsp;HeLa and HepG2\u0026nbsp;have 98 % head DNA and 2% tail DNA, while control cells have 95% head DNA and 5%\u0026nbsp;tail DNA (Fig.\u0026nbsp;9).\u003c/p\u003e\n\u003ch3\u003eApoptosis vs necrosis, as revealed in AnnexinV-Cy3 \u0026ndash; 6-CFDA staining\u003c/h3\u003e\n\u003cp\u003eAs a sign of early apoptosis, the phosphatidyl serine shift to outer plasma membrane was examined with the control and treated cobalt (III) complexes on HeLa and HepG2 cells using annexin V-cy3 assay where the Annexin V positive (red fluorescence) and 6-CFDA positive (green fluorescence) cells was used to differentiate the viable, early apoptosis and necrosis cells . The result suggested that the complexes might preponderantly activate apoptosis rather than the necrosis (Fig. 10). The pictures reveal a large number of early apoptotic cells (Annexin V-Cy3 positive, 6-CFDA positive), a few necrotic cells (Annexin V-Cy3 positive, 6-CFDA negative), and a substantial proportion of viable cells (Annexin V-Cy3 positive, 6-CFDA negative) (Annexin V-Cy3 negative, 6- CFDA positive) (Fig 11). The complexes 1 and 2 were found to be a potential agents inducing apoptosis against HeLa and HepG2\u0026nbsp;cells.\u003c/p\u003e\n\u003ch3\u003eGeneration of ROS on treatment with cobalt (III) complexes\u003c/h3\u003e\n\u003cp\u003eMitochondria are important sites for energy metabolism where there is a strong association between oxidative stress and mitochondrial activity. As a result, the ROS productions induced by the cobalt (III) complexes (IC50 treatment for 12 hours) were measured in HeLa and HepG2\u0026nbsp;cells. The\u0026nbsp;elevated ROS generation was observed using both spectroscopic measurement (reported as fold change) and fluorescent microscopy analysis (high contrast green-fluorescent images) (Fig 12 A, B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of the complexes on pro-apoptotic and anti-apoptotic protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of cobalt (III) complexes on the expression levels of Bcl-2 (pro-apoptotic protein) and Bax (anti-apoptotic protein) in HeLa and HepG2\u0026nbsp;cells was studied using immunofluorescent\u0026nbsp;protocols. The fluorescence intensity was enhanced in Bax but decreased in Bcl-2 .The result revealed that the expression of Bax was upregulated and that of BCl2\u0026nbsp;was downregulated in the complexes, 1\u0026nbsp;and 2 (Fig. 13A, B).\u003c/p\u003e\n\u003ch3\u003eEffect of the complexes on the expression of apoptosis-associated genes\u003c/h3\u003e\n\u003cp\u003eFor the development of chemotherapeutic drugs, sufficient knowledge of the cell signaling apoptotic pathway is necessary. Furthermore, chemotherapeutic drugs are directly associated to proapoptotic and antiapoptotic protein ratio which aids in the induction of the cancer cell death. As a result, we used RT-PCR to evaluate the influence of cobalt (III) complexes on the expression of pro- apoptotic (Cas-3, BAX, BID, and Cas-8), anti-apoptotic (Bcl-2 and GRP-78), and antioxidant gene (CAT, GPX1, and SOD) expressions.\u003c/p\u003e\n\u003cp\u003eThe expression of pro-apoptotic genes were upregulated (fold change for HeLa, Complex 1: BID\u0026nbsp;(1.24),\u0026nbsp;Bax\u0026nbsp;(1.2),\u0026nbsp;caspase-3\u0026nbsp;(1.5),\u0026nbsp;and\u0026nbsp;caspase-\u0026nbsp;8\u0026nbsp;(1.12)\u0026nbsp;and\u0026nbsp;complex\u0026nbsp;2:\u0026nbsp;BID\u0026nbsp;(1.05),\u0026nbsp;Bax\u0026nbsp;(1.12),\u003c/p\u003e\n\u003cp\u003ecaspase-3\u0026nbsp;(1.1),\u0026nbsp;and\u0026nbsp;caspase-8\u0026nbsp;(1.16)\u0026nbsp;and\u0026nbsp;HepG2,\u0026nbsp;Complex:\u0026nbsp;1 \u0026nbsp;BID\u0026nbsp;(1.05),\u0026nbsp;Bax\u0026nbsp;(1.115),\u0026nbsp;caspase-3\u003c/p\u003e\n\u003cp\u003e(1.11), and caspase- 8 (1.16) and complex 2: BID (1.45), Bax (1.24), caspase-3 (1.42), and caspase\u0026ndash;8 (1.12)) compared to the control (normalized as 1 for all groups) in treated HeLa and HepG2\u0026nbsp;cells.\u0026nbsp;Compared to control, anti-apoptotic genes (fold change for HeLa, Complex 1: Bcl-2 (0.84) and GRP- 78\u0026nbsp;(0.73),\u0026nbsp;complex\u0026nbsp;2:\u0026nbsp;Bcl-2\u0026nbsp;(0.64)\u0026nbsp;and\u0026nbsp;GRP-78\u0026nbsp;(0.69)\u0026nbsp;and\u0026nbsp;for\u0026nbsp;HepG2,\u0026nbsp;Complex\u0026nbsp;1:\u0026nbsp;Bcl-2\u0026nbsp;(0.95)\u0026nbsp;and\u003c/p\u003e\n\u003cp\u003eGRP-78 (0.92), complex 2: Bcl-2 (0.84) and GRP-78 (0.91)) (Fig.14) and antioxidant genes (fold change\u0026nbsp;for\u0026nbsp;HeLa,\u0026nbsp;Complex\u0026nbsp;1:\u0026nbsp;SOD\u0026nbsp;(0.64),\u0026nbsp;CAT\u0026nbsp;(0.84),\u0026nbsp;and\u0026nbsp;GPX1\u0026nbsp;(0.91)\u0026nbsp;and\u0026nbsp;complex\u0026nbsp;2:\u0026nbsp;SOD\u003c/p\u003e\n\u003cp\u003e(0.44),\u0026nbsp;CAT\u0026nbsp;(0.64),\u0026nbsp;and\u0026nbsp;GPX1\u0026nbsp;(0.45),\u0026nbsp;and\u0026nbsp;HepG2 \u0026nbsp;Complex\u0026nbsp;1:\u0026nbsp;SOD\u0026nbsp;(0.84),\u0026nbsp;CAT\u0026nbsp;(0.81),\u0026nbsp;and\u0026nbsp;GPX1\u003c/p\u003e\n\u003cp\u003e(0.81) and complex 2: SOD (0.45), CAT (0.80), and GPX1 (0.54)) were down-regulated (Fig. 15). The antioxidant enzymes which play an important role in the defense mechanism against oxidative stress. In contrast, both cell lines have shown efficient gene expression succumbed to cell\u0026nbsp;death.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe structural and functional alteration of an existing chemical may allow for the discovery of a viable lead therapeutic molecule. The structure of the molecule may be altered by combining the ligands with the metal complexes in order to cause apoptotic cell death while also refining its properties such as enhancing solubility, improving absorbability, reducing toxicity, and providing other advantages (Jaividhya et al., 2012). Furthermore, current research has highlighted cobalt complexes as cytotoxic agents since they do not cause adverse effects (Riyasdeen.,et al., 2012). Further, our previous research shown the synthesis of novel cobalt complexes and their characterization using various techniques such as infra-red (IR) spectroscopy, NMR spectroscopy, ESI-MS, and UV-Visible spectroscopy (Ambika et al., 2019). In the absorption spectra, our study has observed the binding ability of the complexes to DNA through intercalating agent which will give the result of hypochromism. It is suggested the intercalation is mostly caused by aromatic functional group of salicylaldehyde. These unique features are believed to be significant in inducing the apoptotic cell death. As a result, the cytotoxic impact on cancer cell lines must be assessed. Further, complex 2 was found to have higher Kb values than complex 1 (\u003cstrong\u003e(\u003c/strong\u003e1.31 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e and 3.15 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;Mn\u003c/sup\u003e for complex 1 and complex 2).\u003c/p\u003e\n\u003cp\u003eIn this study, we explored the anticancer potential of cobalt (III) Schiff base complexes against human cervical and liver cancer cell lines, as well as validating our previous findings with A549 and MCF7 cells (Gowdhami et al., 2021). In\u0026nbsp;order to confirm the cell cytotoxicity, various experiments on HeLa and HepG2 cells were conducted with the cobalt (III) Schiff base complexes. Initially, MTT assay was used to assess the cell viability and growth, and it was revealed that the complexes reduce the survival percentage of HeLa and HepG2 cells by stimulating the cell death at different time intervals at the IC50 concentration. It\u0026nbsp;is hypothesized that changing the surface property of cobalt complexes may interact with DNA via modifying the hydrophobic forces (Alhadlaq et al., 2015; Mahmoudi et al., 2009). Further, AO/EB and Hoechst 33258 staining were performed to confirm the cell death. The morphological changes reported including chromatin fragmentation, vacuolation, cytoplasmic blebbing, multi-nucleation, and apoptotic body formation. In addition, cobalt (III) complexes showed better antiproliferative activity against HeLa and HepG2 \u0026nbsp;cells than previously reported copper complex (Priyanga et al., 2019). In\u0026nbsp;contrast, cobalt (III) complexes showed remarkable effect in their cytotoxic ability due to the DNA binding interactions and antiproliferative\u0026nbsp;activity.\u003c/p\u003e\n\u003cp\u003eMoreover, comet assay was performed as a one of the major techniques for measuring the DNA damage (Riyasdeen et al., 2012). When the HeLa and HepG2 cells were treated with cobalt (III) complexes, the tail DNA percentage was increased, resulting in apoptosis. It\u0026nbsp;is proposed that the complexes produce ROS and chelate the DNA by different factors such as vanderwaal interactions, hydrophobic effects and so on, indicating the cells died of apoptosis (kumar and Arunachalam, 2008). Further, cellular stresses create aberrant circumstances that activate tumor suppressor protein p53. The activation of p53 (apoptotic protein) significantly increases p21 expression, which leads to release of apoptogenic factors, resulting in apoptosis (Cory and Adams 2002). As a consequence, our data show that cobalt (III) complexes treatment causes DNA damage in HeLa and HepG2 cells, resulting in cell death. AnnexinV-Cy3 staining was used to confirm the early apoptosis. According to the Mario and Kroemer (2013), one of the earliest processes in apoptosis is the translocation of phosphatidyl serine from the inner to outer leaflet of the plasma membrane. Cobalt (III) complexes have been shown to trigger apoptotic cell death, and mitochondrial silencing has also been connected to apoptosis.\u003c/p\u003e\n\u003cp\u003eMitochondria are maintaining the redox balance by regulating cell viability and generating ROS (Teodoro et al., 2011). Also, MMP alterations promote the mitochondrial swelling and readily permeabilize the outer membrane easily (Youle\u0026amp;Strasser, 2008; Kitsis \u0026amp; Molkentin, 2010). ROS is strongly associated with the MMP loss, indicating cell death where increasing ROS level result in cell cycle disruption, and cell signaling pathway. In addition, ROS triggers apoptosis in endoplasmic reticulum by stimulating various processes like hypoxia (Min et al., 2014). To validate this, we used DCFH-DA where the cell treated with cobalt (III) complexes and measure ROS level in terms of fluorescence.\u003c/p\u003e\n\u003cp\u003eUnder normal physiological conditions, intracellular ROS production is to be maintained to hinder the cell damage through neutralizing the antioxidants which specifically scavenge different kind of enzymes (Murugadas et al., 2016). ROS are highly reactive in general due to an unpaired electron in their outer cell. Increased level of reactive oxygen species will cause mitochondrial damage, increased cellular receptor signaling, cyclooxygenases, peroxisome activity, increased activity of oxidases, oncogene activity with invading immune cell (Liou and Storz, 2010). In\u0026nbsp;General, the antioxidant activity of CAT, GPX1, and SOD are involved in the reduction of ROS level, with the different SOD enzymes being very specialized in regulating biological processes (Copin et al., 2000). Catalase also inhibits hepatic metastasis and decreases matrix metalloproteinase activity. Catalase also attenuates MMP expression at both the baseline and MnSOD-dependent levels. SOD cleaves the superoxide anion into oxygen and hydrogen peroxide in the presence of metal ions as cofactors and exists in eukaryotes and prokaryotes (Nishikawa et al., 2002). In the presence of several secondary enzymes and cofactors, GPX1, a seleno protein participates in antioxidant process with high efficiency. Over expression of this enzyme defend cells against oxidative damage and repress apoptosis caused by H2O2\u0026nbsp;(Lewis et al., 2006). The results of our study showed that CAT, GPX1, and\u0026nbsp;SOD expression were down regulated at cobalt (III) complexes treated with IC50 for 24 hours, mean time the apoptotic genes were upregulated. It\u0026nbsp;confirmed the neutralizing effect of the living system stimulated by the complexes. According to the findings, the complexes induce the apoptosis by increasing ROS production and reducing antioxidant gene expression (Gentile et al.,\u0026nbsp;2017).\u003c/p\u003e\n\u003cp\u003eSimilarly, homeostatic conditions have to be sustained by equally balancing level of antiapoptotic and proapoptotic genes. Hence, our study tested Bcl-2, GRP78 (antiapoptotic gene), Cas-3, Bax, Cas-8, and BID gene expression levels (proapoptotic gene). The result showed that Bax, BID, Cas-3, and Cas- 8 gene expressions were found to be up regulated and Bcl-2 gene and GRP78 expression were found to be down regulated. It is suggested that Bcl-2 family proteins hinders the\u003c/p\u003e\n\u003cp\u003emembrane permeability and prevent the interaction of other proteins. According to the results of the immunofluorescence test, there was a decrease in BCl2 and an increase in Bax fluorescence in the cobalt (III) complexes-treated cells.\u003c/p\u003e\n\u003cp\u003eCaspase-8 can directly activate downstream caspases in the death receptor pathway, where mitochondrial damage is not needed in certain cells (type I cells). In other circumstances, however, caspase-8 cleaves and activates Bid to its truncated form (tBid), which recruits the mitochondrial pathway, which is a key step for cell death (Jost et al., 2009). For example, the BH3 proteins like Bid, conjugate with all prosurvival proteins where the prosurvival proteins bind and activate Bax and Bak. It involves in BH3: groove interaction and activate apoptosis (Lindsay et al., 2010). Further, the intrinsic pathway is mainly involved in mitochondrial cytochrome-c activation which results apoptosome construction consists of procaspase-9, cytochrome-c and Apaf-1 that activate caspase-9 which subsequently triggers caspase-3. As a result, it leads DNA fragmentation finally (Ergun et al., 2014). GRP78 triggers pro-survival pathways and transmit signals by inducing tumor proliferation, survival, anti-apoptosis (Casas., 2017; roller et al., 2013). Henceforth, over all the result is concluded that the role of apoptotic and antiapoptotic proteins has important potential for cancer therapy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn conclusion, the cobalt complexes repressed the growth of cervical and liver cancer cells compared to normal cells. According to cytological labelling, cobalt (III) complexes promote apoptosis in the both the cancer cell lines by destroying DNA, altering mitochondrial membrane potential and causing oxidative stress. According to the molecular level analysis, it has been found that increased pro-apoptotic proteins while decreasing anti-apoptotic proteins expression, where cobalt complexes 1 and 2 are more effective anti-cancer chemotherapeutic drugs than cisplatin which might be investigated further as an anti-cancer chemotherapeutic treatment\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eAcknowledgments\u003c/h3\u003e\n\u003cp\u003eWe express our sincere gratitude to the Dr. R. Thirumurugan, Co-ordinator of National Centre for Alternatives to Animal Experiments (NCAAE), for his continuous support, encouragement, and guidance during the course of this research.\u0026nbsp;This work was financially supported by National Centre for Alternatives to Animal Experiments (NCAAE) under UGC-CPEPA scheme, Government of India (F.No.2-1/2013 (NS/PE)). The research facility provided by Mahatma Gandhi-Doerenkamp Centre, established by Doerenkamp-Zbinden Foundation, and the Department of Science and Technology, Government of India, under DST-Promotion of University Research and Scientific Excellence (PURSE) scheme-Phase II, RashtriyaUchchatar Shiksha Abhiyan (RUSA)-2.O\u0026nbsp;by the Department of Animal Science is heartily acknowledged. The authors thank the Management of Bishop Heber College (Autonomous), Tiruchirappalli-620 017, Tamil Nadu, India, for the support (F.No: MRP/1014/2020 dated : 23.12.2020) and facilities provided through Material Chemistry Lab, PG and Research Department of Chemistry and DST-FIST Instrumentation Centre (HAIF) at Bishop Heber College.\u003c/p\u003e\n\u003ch3\u003eConflicts of interest\u003c/h3\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhamed, M., Akhtar M.J., Alhadlaq, H.A., Alshamsan, A., 2016. Copper ferrite nanoparticle- induced cytotoxicity and oxidative stress in human breast cancer MCF- 7 cells. Colloids and Surfaces B\u003cem\u003e: \u003c/em\u003eBiointerfaces\u003cem\u003e.\u003c/em\u003e142:46\u0026ndash;54.\u003c/li\u003e\n\u003cli\u003eAlhadlaq, H.A, Akhtar, M.J, Ahamed, M., 2015. Zinc ferrite nanoparticle-induced cytotoxicity and oxidative stress in different human cells. Cell \u003cem\u003e\u0026amp; \u003c/em\u003eBioscience\u003cem\u003e. \u003c/em\u003e5,\u003c/li\u003e\n\u003cli\u003eAmbika, S., Manojkumar, Y., Arunachalam, S., Gowdhami, B., MeenakshiSundaram, K. K., Solomon, R.V., Venuvanalingam, P., Akbarsha, M.A., Sundararaman, M., 2019. Biomolecular interaction, anti-Cancer and anti-Angiogenic properties of cobalt (III) Schiff Base complexes. Scientific. Reports. 9:2721.\u003c/li\u003e\n\u003cli\u003eBagrezaei, F., Hassanshahi, G., Mahmoodi, M., Khanamani Falahati-Pour,S., Mirzaei, M.R., 2018.Expression of Inhibitor of Apoptosis Gene Family Members in Bladder Cancer Tissues and the 5637 Tumor Cell Line. 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The Bcl2 family: regulators of the cellular life-or-death switch. Nature Reviews Cancer. 2: 647\u0026ndash;656.\u003c/li\u003e\n\u003cli\u003eErgun, S., Arman, K., Temiz, E., Bozgeyik, I., Yumrutaş, \u0026Ouml;., Safdar, M., Dağlı, H., Arslan A., Oztuzcu, S., 2014. Expression patterns of miR-221 and its target Caspase-3 in different cancer cell lines. Molecular Biology Reports. 41(9):5877-81.\u003c/li\u003e\n\u003cli\u003eFrezza, M., Hindo, S., Chen, D., Davenport, A., Schmitt, S., Tomco, D., Dou, Q.P., 2010. Novel metals and metal complexes as platforms for cancer therapy. Current Pharmaceutical Design.16 (16):1813-25.\u003c/li\u003e\n\u003cli\u003eGayathri, L., Akbarsha, M.A, Ruckmani, K., 2020. \u003cem\u003eIn vitro \u003c/em\u003estudy on aspects of molecular mechanisms underlying invasive \u003cem\u003easpergillosis \u003c/em\u003ecaused by gliotoxin and fumagillin, alone and in combination. 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Bcl-2 proteins and mitochondria--specificity in membrane targeting for death.\u003cem\u003eBiochimicaEtBiophysicaActa\u003c/em\u003e. 1813(4):532-9.\u003c/li\u003e\n\u003cli\u003eLiou, G.Y., Storz, P., 2010. Reactive oxygen species in cancer. Free Radical Research. 44(5).\u003c/li\u003e\n\u003cli\u003eLongley, D.B, Johnston, P.G., 2005.Molecular mechanisms of drug resistance. Journal of Pathology. 205: 275\u0026ndash;29\u003c/li\u003e\n\u003cli\u003eMahmoudi, M., Simchi, A., Milani, A., Stroeve, P., 2009. Cell toxicity of super paramagnetic iron oxide nanoparticles. Journal of Colloid and Interface 336:510-518.\u003c/li\u003e\n\u003cli\u003eMari\u0026ntilde;o., G., Kroemer, G., 2013. Mechanisms of apoptotic phosphatidylserine exposure. Cell Research.23 (11):\u003c/li\u003e\n\u003cli\u003eMin, M., Mevissen, T.E., De Luca, M., Komander, D., Lindon, C.,2015 .Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages. 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The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology. 9:47-59.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eMTT assay showing the effect of cobalt complexes (1-2) and cisplatin on HeLa, HepG2, and L929 cells. The mean and standard deviation of three triplicates are represented by each data point.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.699386503067483%\"\u003e\n \u003cp\u003eCell line\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.609406952965234%\"\u003e\n \u003cp\u003eCompound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.427402862985684%\"\u003e\n \u003cp\u003e24 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.83640081799591%\"\u003e\n \u003cp\u003e48 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.427402862985684%\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"22.699386503067483%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eHeLa(\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.609406952965234%\"\u003e\n \u003cp\u003eComplex1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.427402862985684%\"\u003e\n \u003cp\u003e22.15\u0026plusmn;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.83640081799591%\"\u003e\n \u003cp\u003e18.51\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.427402862985684%\"\u003e\n \u003cp\u003e12.53\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"24.074074074074073%\"\u003e\n \u003cp\u003eComplex2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e10.25\u0026plusmn;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.66137566137566%\"\u003e\n \u003cp\u003e7.4\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e5.2\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"24.074074074074073%\"\u003e\n \u003cp\u003eCisplatin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e26.15\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.66137566137566%\"\u003e\n \u003cp\u003e21.52\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e18.43\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"22.699386503067483%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eHepG2(\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.609406952965234%\"\u003e\n \u003cp\u003eComplex1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.427402862985684%\"\u003e\n \u003cp\u003e56.5\u0026plusmn;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.83640081799591%\"\u003e\n \u003cp\u003e49.41\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.427402862985684%\"\u003e\n \u003cp\u003e38.53\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"24.074074074074073%\"\u003e\n \u003cp\u003eComplex2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e40.25\u0026plusmn;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.66137566137566%\"\u003e\n \u003cp\u003e34.2\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e28.28\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"24.074074074074073%\"\u003e\n \u003cp\u003eCisplatin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e31.15\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.66137566137566%\"\u003e\n \u003cp\u003e26.52\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e18.43\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"22.699386503067483%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eL929(\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.609406952965234%\"\u003e\n \u003cp\u003eComplex 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.427402862985684%\"\u003e\n \u003cp\u003e124.4\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.83640081799591%\"\u003e\n \u003cp\u003e104.6\u0026plusmn;0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.427402862985684%\"\u003e\n \u003cp\u003e82.9\u0026plusmn;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"24.074074074074073%\"\u003e\n \u003cp\u003eComplex 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e116\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.66137566137566%\"\u003e\n \u003cp\u003e95.8\u0026plusmn;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e78.58\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"24.074074074074073%\"\u003e\n \u003cp\u003eCisplatin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e142.2\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.66137566137566%\"\u003e\n \u003cp\u003e124.6\u0026plusmn;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.132275132275133%\"\u003e\n \u003cp\u003e102.8\u0026plusmn;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\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":"Anti-cervial and liver cancer drug, cobalt (III) Schiff base complexes, apoptosis, DNA damage, reactive oxygen species, immune-fluorescence, RT-PCR","lastPublishedDoi":"10.21203/rs.3.rs-1568074/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1568074/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of new medicines for the successful treatment of cervical and liver malignancies is critical in order to address the disadvantages of current chemotherapeutics, such as increased resistance. Metal ion-based chemical complexes have recently emerged as a prominent method for cancer therapy. As a result, the study aims to create novel anticancer medicines based on leads acquired through combinatorial chemistry of metal complexes. Cobalt (III) Schiff base trans- [Co(salen)(DA)2](ClO4) (complex 1) and trans-[Co (salophen)(DA)2](ClO4) (complex 2)] where, the salen and salopen were N, N′-bis(salicylidene) ethylenediamine, and N, N′-bis(salicylidene)-1,2- phenylenediamine, DA: dodecylamine) were synthesized as an alternative to the existing drugs, and their cytotoxic effect were evaluated against human cervical (HeLa) and liver cancer cell lines (HepG2) using MTT viability assay, and apoptotic morphological staining which including Acridine Orange/Ethidium Bromide (AO/EB), Hoechst 33528, Annexin V-Cy3 assay, JC-1 staining Comet assay, and reactive oxygen species (ROS) assay. Further the apoptosis was confirmed with immunocytochemistry and real-time reverse transcription-polymerase chain reaction \u003cstrong\u003e(\u003c/strong\u003eRT-PCR). The results indicated that cobalt (III) complexes reduced the viability of the HeLa and HepG2 cell lines at their half minimum lethal doses, with cell death induced mainly via apoptotic pathway. Hence, the cobalt complexes 1 and 2 could be act as an effective drug for cervical and liver cancers.\u003c/p\u003e","manuscriptTitle":"Influence of cobalt (III) Schiff base complexes on in-vitro anti-proliferative, oxidative stress and gene expression analysis in HeLa and HepG2 cell lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-26 14:50:39","doi":"10.21203/rs.3.rs-1568074/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":"cf7af128-3507-4636-80a0-900e1895f7db","owner":[],"postedDate":"April 26th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-05-31T16:11:32+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-26 14:50:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1568074","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1568074","identity":"rs-1568074","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}