Synergistic Apoptotic Effects of Metformin and Atorvastatin Through Bax/Bcl-2 and AMPK/ERK Modulation in OVCAR3 Cells

Synergistic Apoptotic Effects of Metformin and Atorvastatin Through Bax/Bcl-2 and AMPK/ERK Modulation in OVCAR3 Cells | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 12 April 2025 V1 Latest version Share on Synergistic Apoptotic Effects of Metformin and Atorvastatin Through Bax/Bcl-2 and AMPK/ERK Modulation in OVCAR3 Cells Authors : Zunaira Zahid , Kazim Sahin , Muhammed Tokmak , Fusun Erten , Besir Er , and Hasan Gencoglu 0000-0002-7716-552X [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174443767.73020504/v1 292 views 145 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Ovarian cancer remains one of the deadliest gynecological malignancies due to late-stage diagnosis and inherent resistance to conventional therapies, highlighting a critical need for novel treatment strategies. This study aimed to elucidate the apoptotic and signaling mechanisms underlying the individual and combined anticancer effects of MET and ATOR on ovarian cancer cells. The OVCAR-3 ovarian cancer cell line was exposed to varying concentrations of MET (2–256 mM) and ATOR (0.78–50 mM), alone or in combination, for 24, 48, and 72 hours. Cell viability was assessed via the MTS assay, while expression of apoptotic and signaling proteins (Bax, Bcl-2, Caspase 3, AMPK, ERK) was analyzed using Western blotting. Both MET and ATOR significantly reduced OVCAR-3 cell viability in a dose- and time-dependent manner, with calculated IC50 values of 12.77 mM (MET, p<0.0001) and 1.51 mM (ATOR, p<0.0001) at 72 hours. Combination treatment exhibited a pronounced synergistic effect, markedly enhancing apoptosis by increasing pro-apoptotic Bax (p<0.0001) and Caspase 3 (p<0.0001) and reducing anti-apoptotic Bcl-2 expression (p<0.0001). Furthermore, MET and ATOR synergistically modulated critical signaling pathways by significantly activating AMPK (p<0.0001) and suppressing ERK (p<0.0001). MET and ATOR combination demonstrated substantial synergistic anticancer efficacy in ovarian cancer cells, mediated through enhanced apoptotic signaling and modulation of AMPK/ERK pathways. These findings emphasize justification for further preclinical and clinical exploration of MET and ATOR as viable, cost-effective treatment options to improve therapeutic outcomes in ovarian cancer studies. Synergistic Apoptotic Effects of Metformin and Atorvastatin Through Bax/Bcl-2 and AMPK/ERK Modulation in OVCAR3 Cells Zunaira Zahid 1 , Kazim Sahin 2 , Muhammed Tokmak 2 , Fusun Erten 3 , Besir Er 1 , Hasan Gencoglu 1* 1 Department of Biology, Faculty of Science, Firat University, Elazig, 23119, Turkiye 2 Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Firat University, Elazig, 23119, Turkiye 3 Department of Veterinary Science, Pertek Sakine Genc Vocational School, Munzur University, Tunceli, 62000, Turkiye *Corresponding author: Hasan Gencoglu, Ph.D. Assoc. Prof. of Molecular Biology and Genetics, Faculty of Science, Firat University, 23119, Elazig, Turkey Phone: +904242370000/3774; Fax: +90424 2388173 E-mail: [email protected] Abstract Ovarian cancer remains one of the deadliest gynecological malignancies due to late-stage diagnosis and inherent resistance to conventional therapies, highlighting a critical need for novel treatment strategies. This study aimed to elucidate the apoptotic and signaling mechanisms underlying the individual and combined anticancer effects of MET and ATOR on ovarian cancer cells. The OVCAR-3 ovarian cancer cell line was exposed to varying concentrations of MET (2–256 mM) and ATOR (0.78–50 mM), alone or in combination, for 24, 48, and 72 hours. Cell viability was assessed via the MTS assay, while expression of apoptotic and signaling proteins (Bax, Bcl-2, Caspase 3, AMPK, ERK) was analyzed using Western blotting. Both MET and ATOR significantly reduced OVCAR-3 cell viability in a dose- and time-dependent manner, with calculated IC50 values of 12.77 mM (MET, p<0.0001) and 1.51 mM (ATOR, p<0.0001) at 72 hours. Combination treatment exhibited a pronounced synergistic effect, markedly enhancing apoptosis by increasing pro-apoptotic Bax (p<0.0001) and Caspase 3 (p<0.0001) and reducing anti-apoptotic Bcl-2 expression (p<0.0001). Furthermore, MET and ATOR synergistically modulated critical signaling pathways by significantly activating AMPK (p<0.0001) and suppressing ERK (p<0.0001). MET and ATOR combination demonstrated substantial synergistic anticancer efficacy in ovarian cancer cells, mediated through enhanced apoptotic signaling and modulation of AMPK/ERK pathways. These findings emphasize justification for further preclinical and clinical exploration of MET and ATOR as viable, cost-effective treatment options to improve therapeutic outcomes in ovarian cancer studies. Keywords: Metformin, Atorvastatin, Apoptosis, AMPK, ERK, OVCAR-3 Significance Ovarian cancer is among the most lethal gynecological cancers, often diagnosed late and associated with resistance to standard treatments, emphasizing the urgent need for alternative therapeutic strategies. Drug redesign utilising established drugs such as Metformin (MET) and atorvastatin (ATOR), particularly in diabetic and overweight cancer patients, has emerged as a promising therapeutic approach. This study identifies a synergistic apoptotic mechanism exerted by MET and ATOR in ovarian cancer cells (OVCAR-3), highlighting their potential to enhance apoptosis via key apoptotic (Bax, Bcl-2, Caspase-3) and metabolic signaling pathways (AMPK/ERK). These findings underscore the therapeutic promise of drug repurposing, particularly combination treatments that target multiple pathways simultaneously, potentially overcoming chemoresistance and improving patient outcomes. This research establishes a foundation for further preclinical and clinical investigations into metformin and atorvastatin as effective, affordable adjuncts to current ovarian cancer therapies. Introduction Ovarian cancer exhibits the peak fatality rate among all the cancers of gynecology, with a 5-year overall only 46% rate of survival. Consequently, the cancer of ovaries ranks as the fifth leading and the main cause of deaths of women suffering from Cancer (Siegel et al. 2016). The highest rates of mortality among all female reproductive tract cancers are associated with ovarian cancer, which has been shown to have a poor prognosis due to advanced stages at diagnosis. This is because metastatic behavior is unusual for the typical characteristics of this type of cancer (Momenimovahed et al. 2019). Ovarian cancer includes several subtypes; however, they are treated as a single disease entity. Similar to other forms of cancer, there exists considerable diversity within and across subtypes of tumours, leading to therapeutic inefficacy (Kossaï et al. 2018). Galega officinalis , also referred to as goat rue or French lilac, is utilized to alleviate polyuria and other symptoms associated with diabetes. Galega officinalis is the origin of metformin (1,1-dimethylbiguanide hydrochloride), a substance that was discovered over a hundred years ago and has been proven to have powerful benefits in reducing glucose levels. Metformin was initially prescribed in 1957 for type 2 diabetes (T2D) treatment. Due to its compelling clinical evidence, few contraindications and typically well-tolerated side effects, it remains the preferred medicine for patients suffering from type 2 diabetes (Urpilainen et al. 2020). Metformin is a first-line antidiabetic drug that reduces levels of insulin. It has anticancer properties because insulin has mitogenic and pro-survival actions, and tumor cells usually express large amounts of the insulin receptor (Podhorecka et al. 2017). Metformin may inhibit the development, survival, and spread of several tumor cell types, including those from liver, lung, endometrial, kidney, and colorectal malignancies (Podhorecka et al. 2017). Previous data demonstrated that metformin triggers both apoptosis and necrosis in the SKOV-3. Furthermore, besides its affective usage on women’s polycystic ovary syndrome (PCOS) disease, it has been verified that apoptosis in SKOV-3 ovarian cancer cells by the treatment of metformin is linked to changes in the activation of BIRC5 mRNA (Rogalska et al. 2014). Metformin was first recommended for diabetes mellitus treatment and is widely used due to its low contraindications, and tolerable side effects. Metformin has anticancer effects in addition to its antidiabetic ones, it lowers insulin levels, which are frequently raised in tumor cells that have high insulin receptor expression. Research has revealed that the proliferation, persistence, and spread of ovarian cancer and a variety of other tumor types can be inhibited by metformin. It specifically causes necrosis and apoptosis in cancer cell lines and exhibits anti-proliferative properties in ovarian cancer. According to these results, metformin shows potential to treat ovarian cancer and possibly other cancers. Many studies have shown that statins and MET may improve the efficacy of traditional chemotherapy, even with the lack of strong clinical evidence to support their use as monotherapy for cancer treatment. Research has shown that the combined action of ATOR and MET dramatically inhibits proliferation and induces apoptosis in human prostate cancer cells (Wang et al. 2017). Furthermore, in non-small cell lung cancer, ATOR and MET have both separate and combined antiproliferative effects (Salim et al. 2024). The available evidence is insufficient to confirm that metformin has positive effects and can be a standalone preventative medication for ovarian cancer (Kossaï et al. 2018). Moreover, the relationship between metformin and longevity following ovarian cancer is intricate. Furthermore, statins such as ATOR can enhance anti-tumor activity when they synergistically interact with other medicines. The occurrence of medication combinations is uncommon (Xia et al. 2023). In addition, a study has revealed the anti-proliferation effect on the OVCAR 3 cell line, and it was observed that when metformin is administered, AMPK activity increases in a time and dose-dependent manner, suggesting the stimulation of AMPK in peripheral tissues. Reduced phosphorylation of p70S6K and S6 kinase, which is known to lower mRNA translation and protein formation, is connected to this increase in P-AMPK. These actions might be part of the reason that metformin has been shown to have antineoplastic properties (Gotlieb et al. 2008). However, the proof of metformin’s effects on ovarian cancer remains limited and ambiguous. Metformin was first used in clinical practice in the 1950s to treat type II diabetes mellitus, but it is now widely utilized to treat other insulin-resistant diseases, particularly polycystic ovarian syndrome (Shafiee et al. 2014). It was discovered that nearly half of diabetic individuals who received metformin lived for five years longer than nondiabetic patients who did not receive metformin (Zhang and Li 2014). Current research suggests that ovarian, endometrial, and cervical cancer can be treated successfully (Milewicz et al. 2013). Preclinical research on ovarian cancer cell lines has revealed that metformin can limit their proliferation (Dilokthornsakul et al. 2013). Therefore, the role of metformin in other types of cancer may increase the prospects for new therapies in the fight against ovarian cancer. Statins, which are established lipid-lowering agents, have demonstrated significant efficacy in the treatment of cancer. As a reductase (HMG-CoA) inhibitor, atorvastatin may affect survival, migration, and proliferation of cancer cells (Shaghaghi et al. 2022). Atorvastatin induced G1 arrest and apoptosis in the ovarian cancer cell lines SKOV-3 and Hey. The impact of ATOR on different proteins involved in apoptosis, like MCL-1, BCL-2, and PARP, has been reported in all cell lines of cancer. ATOR reduced the level of MCL-1 in both cell lines, but it enhanced the level of BCL-2 in Hey cells and decreased it in SKOV3 cells. In addition, the administration of ATOR resulted in an upregulation of cleaved PARP protein expression, suggesting that ATOR stops the growth and spread of tumors by inducing mitochondrial apoptosis. Furthermore, the antiproliferative effects of atorvastatin were also detected. These findings indicate that ATOR inhibits the mTOR pathway. However, depending on the specific type of ovarian cancer cell, ATOR may focus on a distinct pathway to decrease cell growth (Jones et al. 2017). A recent study conducted in 2024 demonstrated that repurposing ATOR and MET has an antiproliferative effect on non-small cell lung cancer, both alone and in combination, and that they may be used via a similar mechanism (Salim et al. 2024). By activating caspases 3/7, decreasing Bcl-2 and Bcl-xL expression, and upregulating Bax and Bad expression, MET has been shown to cause cell death in OVCAR-3 and OVCAR-4 cells (Yasmeen et al. 2011). ATOR has been proven to have apoptotic effects on cell lines of ovarian cancer such as A2780 and IGROV-1, which means that it is highly effective at slowing the proliferation and multiplication of ovarian cancer cells while also initiate the programmed cell death (Göbel et al. 2020). Medications such as statins that block the HMG-CoA reductase enzyme show potential anticancer effects because they affect vital signaling proteins (Otahal et al. 2020). When paired with conventional medications, they improve the prognosis of cancer patients. Research has suggested that statin therapy can slow the growth of tumors and colony formation in patients with breast cancer as well as the spread of invasive breast cancer (Marti et al. 2021). Moreover, statins cause apoptosis and halt the proliferation of cells in colon cancer (Cai and Gao 2021). The results also demonstrated antitumor activity in lung carcinoma and increased the sensitivity of drug-resistant neoplastic cells to chemotherapeutic agents. Based on these findings, statins may be employed as adjuvant therapies to improve the outcome of cancer therapies. This study aimed at examining the efficacy in more detail with respect to the synergistic effects of metformin and atorvastatin on OVCAR-3 cells upon cell survival, cell death, and the levels of apoptotic markers in the ovarian malignant cell line OVCAR-3. Ovarian cancer cells were used in our study because this kind of cancer is one of the most challenging issues in gynecological cancer treatment (Denel and Marczak 2013), and finding new medications that effectively treat ovarian cancer, especially in the patients who that are resistant to existing forms of therapy, is still a difficult priority. Materials and Methods In this study, the OVCAR-3 ovarian cancer cell line was used. OVCAR-3 was originally isolated in 1982 from the malignant ascites of a patient with progressive adenocarcinoma of the ovary. The cells were originally purchased from American Type Culture Collection, Manassas, VA, USA. DMEM/F12 (Sigma-Aldrich, Munich, Germany), with the addition of 20% fetal bovine serum and 1% penicillin-streptomycin, was utilized in the culturing of the cells; and incubation under standard conditions included incubation at 37°C in an atmosphere with humidified air containing 5% CO₂.Metformin: 1,1-Dimethylbiguanide hydrochloride was provided by Sigma-Aldrich in white solid form, with a molecular weight of 165.62 g/mol. In the study, powdered metformin was dissolved in DMEM-F12 to form a main stock at a dose of 300 mM.Atorvastatin: It was provided by Sigma-Aldrich in white powder, its molecular weight of 558.64 g/mol. During the experiment, atorvastatin powder was dissolved in DMEM-F12 to prepare the main stock solution at the concentration of 200 mM. The main stock was made on the same day that culture medium will dilute it. The OVCAR3 cell line that was used in the study was provided as an early passage (pn: 12-16). OVCAR3 cells were cultured in T25 or T75 flasks in a medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in DMEM-F12 medium (Serox, Manheim, Germany). Both media were fortified with 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) against possible pathogens. The cells were kept in an incubator with 5% CO2, and 95% humidity at 37°C. DMEM-F12 contains phenol red, which acts as a pH indicator and provides a quick way to ensure that cells grow in the appropriate environment; If the density of the cells is too high or the medium is not regularly modified, acidic metabolites would have formed and the medium would have changed from orange/red to yellow. The culture medium sub-cultured and changed every 3–4 days. Passage of Cells The cells were subcultured once they reached 60–70% confluence to encourage additional proliferation or for use in research. Trypsination was used to carry out this procedure. Using a 0.1%/0.02% trypsin/EDTA solution, the cells were extracted from the tissue culture plastic. The adhesion sites between the cells and the developing surface were broken down by trypsin, and the Ca2+ chelation required for Ca2+-dependent adhesion was achieved by EDTA.Aspiration was used to remove the growth medium, and as serum inhibited trypsin, after that the cells were then washed with sterile PBS (phosphate-buffered saline) to remove any remaining serum in the vial. After that, trypsin was added to the cells, to ensure that the entire vial was covered, and the cells were incubated for three minutes at 37°C. Trypsin was introduced to the cells and inactivated by gently tapping the vial’s sides with the palm of the hand after it had been incubated. The cells were then placed in a serum-containing solution.After being collected, the cells were centrifuged for five minutes after being added to a sterile 15 ml Falcon tube. After aspirating the supernatant, the pellet that formed was resuspended in a culture medium. The cells were employed in experiments or replanted in fresh flasks. The Zeiss Axiovert A1 microscope (Oberkochen, Germany) was utilized for trypsin-induced cell passage and imaging. Determination of the Number of Cells The number of cells were counted by using an Improved Neubauer Hemocytometer. The hemocytometer was covered with coverslip, and its fixed depth was 0.1 mm. Trypsin treatment was used for cell extraction from the flask and re-suspended in 1ml growth media. A 10 μl aliquot was extracted from the cell suspension and further diluted to a dilution rate of 1:15 by mixing it with the growth media. Then, 10 μL was added to the hemocytometer chamber, and using a light microscope with an x100 magnification, the cells were counted in four distinct squares with a side length of 1 mm. After calculating the average number of cells, the following formula was used to get the number of cells per milliliter of the suspension: Number of cells per ml of cell suspension = average number of cells per 0.1mm3 x dilution factor x 104 Determination of viability and analysis of the IC50 The MTS test was used in this investigation to measure the dose-dependent inhibitory action of MET and ATOR on an equivalent number of OVCAR3 cells at 24, 48, and 72 hours. In the present experiment, proliferating cells transformed MTS, a tetrazolium salt, into dark pink/red formazan crystals with elevated activity of the mitochondrial dehydrogenase enzyme. Using an ELISA reader, at 492 nm the formazan’s absorbance was measured. Accepting viability of cells that are untreated as 100% and calculating the viability of treated cells as a percentage (%) with respect to these cells, which allowed for the presentation of the numerical data. In light of this, the amount of MET and ATOR medication needed to block the regular proliferation process was quantitatively demonstrated. Western Blot Analysis In OVCAR-3 cells, the protein expression of Bax, Bcl-2, Caspase 3, AMPK, and ERK was measured by Western blot analysis. In brief, OVCAR-3 cells from each experimental group (negative control, metformin-treated, atorvastatin-treated, and combination-treated) were lysed in lysis medium containing enzyme inhibitors to extract total protein. The protein concentration was measured with a Qubit 4.0 fluorometer (Invitrogen, CA, USA). 20 micrograms of protein from each sample were separated by SDS page, which was then transferred to a nitrocellulose membrane using the Trans-Blot Turbo Transfer System. Primary antibodies (Abcam, Cambridge, UK) were used, that were specific to bind to Bax, Bcl-2, Caspase 3, AMPK, ERK, and β-actin was used as a loading control. The membranes were blocked for 1 hour at room temperature before being incubated overnight at 4°C. After being washed with TBS-T, the membranes were treated with a secondary rabbit antibody at room temperature for 1 hour. To ensure reproducibility, the experiments were performed in triplicate. Protein bands were detected using enhanced chemiluminescence (ECL) and evaluated densitometrically with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Protein levels were adjusted for β-actin and expressed as values relative values against the negative control group. Statistical Analyses Each trial will be performed independently and repeated 3 times. For the statistical analysis the GraphPad Prism 8.0 software package (GraphPad Software, Inc., La Jolla, CA, USA) was used. The “t” test was utilized for comparisons between two groups and ANOVA analysis was used for comparisons between more than one group. These data are expressed as the mean ± standard deviation (SD) and p < .05 was considered statistically significant. Results The IC50 concentration was determined by using the MTS test. Metformin as a monotherapy in OVCAR-3 showed substantial impacts at concentrations of 8 mM or greater compared with those in negative control samples. Atorvastatin administration substantially reduced the relative viability of the cells beginning at 0.78 mM and greater when compared to negative control samples. Effects of Metformin on OVCAR-3 Cell Survival Metformin was administered to OVCAR-3 cells at concentrations ranging from 2 mM to 256 mM for 24, 48, and 72 hours (Fig. 1A). Metformin displayed a dose-dependent reduction in cell viability. At 24 hours, the cell viability remained above 80% at concentrations between 2 mM and 16 mM. A significant decline was observed at 32 mM and above, with viability decreasing to 60% at 64 mM and below 40% at 128 mM. \RL At 48 hours, metformin’s inhibitory effects became more evident. The viability decreased to 70% at 16 mM and to 50% at 64 mM. At the highest concentration tested (256 mM), cell viability dropped to approximately 20%. After 72 hours, metformin’s cytotoxicity was the most pronounced. The viability fell to 60% at 32 mM, below 30% at 128 mM, and to approximately 10% at 256 mM. These results highlight metformin’s time- and dose-dependent effects on OVCAR-3 cell survival. The IC50 value for the metformin group was 12,77 mM at the end of 72 hours. Effect of Atorvastatin on OVCAR-3 Cell Survival OVCAR-3 cells were treated with atorvastatin at concentrations ranging from 0.78 mM to 50 mM for 24, 48, and 72 hours (Fig. 1B). Cell viability measurements revealed a dose-dependent reduction in viability across all time points. At 24 hours, lower concentrations of atorvastatin (0.78 mM to 6.25 mM) had minimal impact on cell viability, maintaining levels above 80%. Significant reductions were observed at 12.5 mM and higher concentrations, where the cell viability dropped to around 50% at 25 mM and 30% at 50 mM. \RL At 48 hours, the cytotoxic effects became more pronounced. Viability began to decline at 6.25 mM, with a significant decrease observed at 12.5 mM (approximately 60% viability). At the highest concentration (50 mM), the cell viability was reduced to nearly 20%. After 72 hours, atorvastatin had a significant inhibitory effect. The viability remained above 90% at concentrations below 3.125 mM but dropped to 70% at 6.25 mM. Higher concentrations (12.5 mM to 50 mM) caused a steep decline, reducing viability to 40% and 10%, respectively. These results suggest that atorvastatin’s cytotoxicity increases with both dose and exposure time. The IC50 value for the atorvastatin group was 1.510 mM at the end of 72 hours. Effect of Atorvastatin and Metformin on the Expression of Apoptotic factors Effect of Metformin and Atorvastatin on Bax To examine the effects of metformin, atorvastatin, and their combination on apoptosis, western blotting was performed to examine the expression of the pro-apoptotic factor Bax in control and treated cells (Fig. 2). The results demonstrated that the IC50 value of atorvastatin (1.510 mM), the IC50 value of metformin (12.77 mM) and the combination (Met + Ator) significantly increased Bax expression in the cells. Metformin treatment enhanced the Bax level in dose dependent manner, atorvastatin treatment resulted in a similar upregulation and the combination treatment (Met + Ator) resulted in the greatest increase compared with the Negative Control (NC) group. These findings showed that metformin and atorvastatin, as well as their combination, activate apoptotic pathways via Bax overexpression. The maximum upregulation of Bax was recorded in the combination treatment (Met + Ator) group (p<0.0001), followed by the metformin (p<0.0001) and atorvastatin (p<0.0001) treated groups. The lowest expression was observed in the negative control (NC) group . Compared to control group, there were statistical differences among the metformin (p<0.0001); atorvastatin (p<0.0001); Met+Ator (p<0.0001) groups. Effect of Metformin and Atorvastatin on Bcl-2 The activity of Bcl-2 was considerably reduced when the cells were treated with metformin at the tested IC50 (12.77 mM), atorvastatin at the tested IC50 (1.510 mM), and a combination of the metformin and atorvastatin. Metformin and atorvastatin treatments significantly reduced the Bcl-2 protein level, and the combination treatment compared to the NC group (Fig. 3). The maximum Bcl-2 expression was recorded in the negative control (NC) group, while the negligible expression was detected in the combination treatment (Met + Ator) group (p<0.0001), followed by the metformin (p<0.0001) and atorvastatin (p<0.0001) treated groups. Effect of Metformin and Atorvastatin on Caspase 3 The enhanced activity of Caspase 3 in the control and treated cells was documented in the concentration- dependent manner. The tested IC50 value of atorvastatin (1.510 mM) and metformin (12,77 mM) and the combination (Met + Ator) considerably increased the expression of Caspase 3. Treatment with metformin resulted in a significant increase in Caspase 3 protein levels, atorvastatin treatment resulted in similar upregulation and the combination treatment (Met + Ator) resulted in the highest increase compared to the negative control (NC) group. These results suggest that metformin and atorvastatin, as well as their combination, activate apoptotic pathways via Caspase 3 overexpression (Fig. 4). The highest level of caspase 3 expression was observed in the combination treatment (Met+Ator) group (p<0.0001), followed by the atorvastatin (p<0.0001) and metformin (p<0.0001) treated groups. The n egative control (NC) group presented the weakest expression of Caspase 3. Effect of Metformin and Atorvastatin on ERK To assess the level of ERK in control and treated cells, Western blotting was performed. ERK expression was considerably reduced when the cells were treated with metformin at the tested IC50 (12,77 mM), atorvastatin at the tested IC50 (1.510 mM), and a combination of the metformin and atorvastatin. Metformin and Atorvastatin treatments significantly reduced ERK protein levels and the combination treatment compared to the NC group (Fig. 5). The negative control (NC) group presented the maximum expression of ERK, while ERK was least abundant in the combination treatment (Met + Ator) group (p<0.0001), followed by the metformin (p<0.0001) and atorvastatin (p<0.0001) treated groups. Effect of Metformin and Atorvastatin on AMPK The efficacy of metformin, atorvastatin, and their combination on apoptosis was determined. The IC50 value of atorvastatin (1.510 mM), the IC50 value of metformin (12,77 mM) and the combination (Met + Ator) significantly increased AMPK expression in the treated cells in a dose-dependent manner. AMPK activity was elevated in the cells treated with metformin and atorvastatin treatment presented the same results and the combination treatment (Met + Ator) resulted in the highest increase compared to the negative control (NC) group. These findings showed that metformin and atorvastatin as monotherapy, as well as their combination, activate apoptotic pathways via AMPK overexpression (Fig. 6). The highest level of AMPK expression was observed in the combination treatment (Met + Ator) (p<0.0001) group, followed by the metformin (p<0.0001) and atorvastatin (p<0.0001) treated groups. The lowest expression was observed in the negative control (NC) group. Discussion The present study evaluated the effectiveness of metformin (Met) and atorvastatin (Ator) on the cell viability and the expression of apoptotic markers in human ovarian cancer cells (OVCAR-3). The findings showed that both drugs, administered individually and in combination, significantly decreased cell viability and altered key apoptotic markers, including Bax, Bcl-2, Caspase-3, AMPK, and ERK. The results show that Met and Ator, particularly when combined, have strong anticancer effects on ovarian cancer cells through the induction of apoptosis and disruption of metabolic pathways, which are critical for tumor survival. In our study, an MTS assay was performed that revealed that both metformin and atorvastatin reduced the viability of OVCAR-3 cells in a dose and time-dependent manner. Atorvastatin produced a more pronounced cytotoxic effect with an IC50 value of 1.510 mM at 72 hours, than metformin with an IC50 value of 12.77 mM for metformin. This agrees with previous studies showing that statins, including atorvastatin, induce cytotoxicity in ovarian cancer cells through the inhibition of the mevalonate pathway, which is important for the proliferation and survival of cancer cells. For instance, Göbel et al. (2020) demonstrated that atorvastatin inhibits the growth of ovarian cancer cells by disrupting the mevalonate pathway, thus decreasing cell viability and increasing apoptosis (Göbel et al. 2020) . Similarly, the cytotoxic effects of metformin are consistent with its established role in activating AMPK, leading to the inhibition of mTOR signaling and subsequent suppression of cancer cell proliferation. Gotlieb et al. (2008) reported that metformin reduces ovarian cancer cell viability through the activation of AMPK and the inhibition of mTOR, which is consistent with our results (Gotlieb et al. 2008) . Additionally, Kim et al. (2019) demonstrated that the combined treatment of metformin and simvastatin leads to reduced cell proliferation by inducing apoptosis in endometrial cancer cells via the inhibition of the mTOR signaling pathway (Kim et al. 2019). A study revealed that this combination has synergistic benefits in suppressing tumor cell growth, alleviating hypoxia, decreasing angiogenesis, enhancing vascular normalization and boosting apoptosis in cancer cells (Liu et al. 2022) , thus providing a rationale for the effects observed in our study with atorvastatin and metformin. Additionally, it has been reported that simvastatin induces apoptosis in breast cancer cells through inhibition of the mevalonate pathway and the downregulation of Bcl-2 expression, which agrees with our results using atorvastatin in ovarian cancer cells (Spampanato et al. 2012) . Western blot analysis revealed that Met and Ator significantly increased the expression of Bax, a proapoptotic protein, and decreased Bcl-2, an antiapoptotic protein. These modifications suggest a change in pro-apoptotic signaling via the intrinsic mitochondrial pathway. These result support previous studies in which metformin was shown to induce apoptosis in ovarian cancer cells via modulation of the Bax/Bcl-2 expression ratio (Rogalska et al. 2014). A previous study by Yasmeen et al. (2011) reported that metformin can induce the apoptosis of OVCAR-3, and OVCAR4 cells by significantly increasing Bax and decreasing bcl-2 concentrations (Yasmeen et al. 2011) . Similarly, atorvastatin has been reported to induce apoptosis and reduce the expression of the Bcl-2 family of proteins in breast cancer cells (Wood et al. 2013) . Moreover, Jones et al. (2007) found that atorvastatin decreases Bcl-2 expression and increases Bax levels in models of ovarian cancer cells, which supports our findings (Jones et al. 2017) . The significant increase in the expression of Caspase-3 underscores the initiation of apoptotic mechanisms. Caspase-3 is a primary executioner that plays a critical role during the terminal phases of apoptosis. Compared to monotherapy, combination therapy yielded the highest levels of Caspase-3, suggesting an enhanced apoptotic response. These results are consistent with previous studies showing that metformin and atorvastatin induce the activation of Caspase-3 in ovarian cancer cells. For instance, Rogalska et al. (2014) demonstrated that metformin induces apoptosis in ovarian cancer cells through the activation of Caspase-3/7 (Rogalska et al. 2014) . Similarly, Jones et al. (2017) reported that atorvastatin enhances caspase-3 activity in ovarian cancer cells, thus confirming our results (Jones et al. 2017) . The activation of AMPK and the downregulation of ERK in our study suggest that both drugs inhibit metabolic and proliferative signaling pathways. Metformin activates AMPK, which in turn inhibits mTOR, an essential regulator of cell growth and survival. Metformin activates AMPK in OVCAR-3 cells, as shown by Gotlieb et al. (2008) which leads to reduced cell proliferation (Gotlieb et al. 2008). Similarly, the inhibition of ERK, a component of the MAPK signaling pathway, is consistent with studies showing that atorvastatin blocks ERK signaling, thus reducing the proliferation and survival of cancer cells. Jones et al. (2017) reported that atorvastatin inhibits ERK phosphorylation in ovarian cancer cells, which aligns with our findings (Jones et al. 2017). In our study the combination of metformin and ator had a greater impact on apoptotic markers and cell survival than either drug alone, likely due to the synergistic effect of these two drugs acting through complementary mechanisms: metformin mainly affects the AMPK/mTOR signaling pathway and triggers metabolic stress, whereas atorvastatin is an inhibitor of the mevalonate pathway, which hinders cholesterol synthesis and prenylation of proteins that are pivotal for cancer cells. Concurrent targeting of metabolic and proliferative pathways represents a powerful approach toward overcoming the resistance mechanisms commonly encountered in ovarian cancer cells. Similarly, the synergistic effect of metformin and statin i.e. simvastatin has been observed in other malignancies. Kim et al. (2019) revealed that the combination of metformin and simvastatin resulted in the inhibition of endometrial cancer cells by inducing apoptosis and through mTOR pathway inhibition (Kim et al. 2019) . Another study conduct ed which indicated that the combination of metformin with atorvastatin considerably inhibits the growth of prostate cancer cells as well as in vivo tumor progression (Wang et al. 2017) . A recent study conducted in 2024 gives credence to such observations by showing that the combination of metformin and simvastatin enhances apoptosis and suppresses tumor growth in ovarian cancer through the targeting of the AMPK/mTOR and PI3K/AKT pathways (Mikhael et al. 2024) . Our study results are in accordance with those of previous reports confirming the anticancer effects of metformin and atorvastatin in ovarian and other malignancies. For instance, metformin has been shown to induce the activation of Caspase-3/7 and modulate Bcl-2 family proteins to cause apoptosis in ovarian cancer cells (Rogalska et al. 2014) . Moreover, Another clinical data substantiates the anti-cancer properties of metformin in patients with breast cancer, suggesting its potential utility as a dual-function treatment for both cancer and metabolic conditions, which is particularly significant for ovarian cancer patients (Chae et al. 2016) . Similarly, atorvastatin has been shown to inhibit ovarian cancer cell growth and induce apoptosis through the mTOR and MAPK signaling pathways (Jones et al. 2017). The synergistic effects observed in this study agree with evidence showing that co-administration of metformin with statins results in improved anticancer efficacy in many cancer models (Kim et al. 2019; Salim et al. 2024). The present findings thus indicate a great potential for repurposing drugs, as metformin and atorvastatin, for the treatment of cancer, particularly ovarian cancer, where resistance to conventional therapeutics is a major challenge. Although the findings are promising, there are several limitations to this study. The experiments were performed in vitro; thus, the results may not reflect the complexity of the tumor microenvironment in vivo. Additionally, because the study was conducted with a single ovarian cancer cell line, OVCAR-3, further studies are needed to establish these findings in other models of ovarian cancer and in vivo. Subsequent investigations should be carried out to evaluate the effects of metformin and atorvastatin in additional ovarian cancer cell lines and animal models. Detailed molecular mechanisms, such as metabolic reprogramming and epigenetic alterations, leading to synergistic effects should be defined; in addition, the potential clinical applications of this combination therapy in patients with diabetes, hyperlipidemia, or obesity-related ovarian cancer should be explored. In conclusion, the approach of combining MET and ATOR to target apoptotic markers in the OVCAR-3 cell line, including Bax, Bcl-2, Caspase 3, AMPK, and ERK, is novel. Our study revealed that metformin and atorvastatin, alone and in combination, have potent anticancer effects on OVCAR-3 cells by inducing apoptosis and modulating major metabolic and proliferative pathways. The combination therapy resulted in better efficacy and is potentially a good strategy for the treatment of ovarian cancer. These data provide a solid basis for future preclinical and clinical studies to test the therapeutic potential of this two-drug approach. Author Contributions HG conceived and designed the research. Material preparation, data collection and analysis were performed HG, MT, and ZZ. The first draft of the manuscript was written by ZZ. MT and ZZ conducted experiments. FE and BE assisted in the laboratory experiments. HG and MT analyzed the data. ZZ wrote the manuscript. KS edited the manuscript. All authors read and approved the manuscript. Funding This work was supported by project number FF.24.32 by Firat University Scientific Research Projects Coordination Unit (FÜBAP). Data Availability The datasets created and/or examined in this study can be obtained from the corresponding author upon reasonable request. Competing Interests The authors declare no competing interests References Cai S, Gao Z (2021) Atorvastatin inhibits proliferation and promotes apoptosis of colon cancer cells via COX-2/PGE2/β-Catenin Pathway. J BUON 26:1219-1225. Chae YK, Arya A, Malecek MK, Shin DS, Carneiro B, Chandra S, Kaplan J, Kalyan A, Altman JK, Platanias L, Giles F (2016) Repurposing metformin for cancer treatment: Current clinical studies. Oncotarget 7:40767-40780. https://doi.org/10.18632/oncotarget.8194 Denel M, Marczak A (2013) Panels of protein biomarkers and non-protein markers in the diagnosis of the ovarian cancer. Przegląd Menopauzalny 12:404 408. https://doi.org/10.5114/pm.2013.38595 Dilokthornsakul P, Chaiyakunapruk N, Termrungruanglert W, Pratoomsoot C, Saokaew S, Sruamsiri R (2013) The effects of metformin on ovarian cancer: A systematic review. Int J Gynecol Cancer 23:1544-1551. https://doi.org/10.1097/IGC.0b013e3182a80a21 Göbel A, Zinna VM, Dell’Endice S, Jaschke N, Kuhlmann JD, Wimberger P, Rachner TD (2020) Anti-tumor effects of mevalonate pathway inhibition in ovarian cancer. BMC Cancer 20:703. https://doi.org/10.1186/s12885-020-07164-x Gotlieb WH, Saumet J, Beauchamp MC, Gu J, Lau S, Pollak MN, Bruchim I (2008) In vitro metformin anti-neoplastic activity in epithelial ovarian cancer. Gynecol Oncol 110:246-250. https://doi.org/10.1016/j.ygyno.2008.04.008 Jones HM, Fang Z, Sun W, Clark LH, Stine JE, Tran AQ, Sullivan SA, Gilliam TP, Zhou C, Bae-Jump VL (2017) Atorvastatin exhibits anti-tumorigenic and anti-metastatic effects in ovarian cancer in vitro. Am J Cancer Res 7:2478-2490. Kim JS, Turbov J, Rosales R, Thaete LG, Rodriguez GC (2019) Combination simvastatin and metformin synergistically inhibits endometrial cancer cell growth. Gynecol Oncol 154:432-440. https://doi.org/10.1016/j.ygyno.2019.05.022 Kossaï M, Leary A, Scoazec JY, Genestie C (2018) Ovarian Cancer: A Heterogeneous Disease. Pathobiology 85:41-49. https://doi.org/10.1159/000479006 Liu J, Wang H, Zhang M, Li Y, Wang R, Chen H, Wang B, Gao X, Song S, Wang Y, Ren Y, Li J, Liu P (2022) Metformin and simvastatin synergistically suppress endothelin 1‐induced hypoxia and angiogenesis in multiple cancer types. Cancer Sci 114:640-653. https://doi.org/10.1111/cas.15602 Marti JLG, Beckwitt CH, Clark AM, Wells A (2021) Atorvastatin facilitates chemotherapy effects in metastatic triple-negative breast cancer. Br J Cancer 125:1285-1298. https://doi.org/10.1038/s41416-021-01529-0 Mikhael S, Kurdi A, Khoueiry-Zgheib N, Tahtouh R, Nasr R, Hilal G (2024) Evaluating synergistic effects of metformin and simvastatin on ovarian cancer cells. PLOS ONE 19:e0298127 . https://doi.org/10.1371/journal.pone.0298127 Milewicz T, Kiałka M, Mrozińska S, Ociepka A, Krzysiek J (2013) [Metformin—New treatment strategies for gynecologic neoplasms]. Przegl Lek 70:81-84. Momenimovahed Z, Tiznobaik A, Taheri S, Salehiniya H (2019) Ovarian cancer in the world: Epidemiology and risk factors. Int J Womens Health 11:287-299. https://doi.org/10.2147/IJWH.S197604 Otahal A, Aydemir D, Tomasich E, Minichsdorfer C (2020) Delineation of cell death mechanisms induced by synergistic effects of statins and erlotinib in non-small cell lung cancer cell (NSCLC) lines. Sci Rep 10:959. https://doi.org/10.1038/s41598-020-57707-2 Podhorecka M, Ibanez B, Dmoszyńska A (2017) Metformin—Its potential anti-cancer and anti-aging effects. Postepy Hig Med Dosw 71:170-175. https://doi.org/10.5604/01.3001.0010.3801 Rogalska A, Forma E, Ciesielski P, Bryś M, Krześlak A, Marczak A (2014) Effect of metformin on apoptosis induction in ovarian cancer cells. Przegląd Menopauzalny 13:155-161. https://doi.org/10.5114/pm.2014.43817 Salim EI, Elsebakhy S, Hessien M (2024) Repurposing of atorvastatin and metformin denotes their individual and combined antiproliferative effects in non-small cell lung cancer. Fundam Clin Pharmacol. https://doi.org/10.1111/fcp.12981 Shafiee MN, Khan G, Ariffin R, Abu J, Chapman C, Deen S, Nunns D, Barrett DA, Seedhouse C, Atiomo W (2014) Preventing endometrial cancer risk in polycystic ovarian syndrome (PCOS) women: Could metformin help? Gynecol Oncol 132:248-253. https://doi.org/10.1016/j.ygyno.2013.10.028 Shaghaghi Z, Alvandi M, Farzipour S, Dehbanpour MR, Nosrati S (2022) A review of effects of atorvastatin in cancer therapy. Med Oncol 40:27. https://doi.org/10.1007/s12032-022-01892-9 Siegel RL, Miller KD, Jemal A (2016) Cancer statistics, 2016. CA Cancer J Clin 66:7-30. https://doi.org/10.3322/caac.21332 Spampanato C, De Maria S, Sarnataro M, Giordano E, Zanfardino M, Baiano S, Cartenì M, Morelli F (2012) Simvastatin inhibits cancer cell growth by inducing apoptosis correlated to activation of Bax and down-regulation of BCL-2 gene expression. Int J Oncol 40:935-941. https://doi.org/10.3892/ijo.2011.1273 Urpilainen E, Puistola U, Boussios S, Karihtala P (2020) Metformin and ovarian cancer: The evidence. Ann Transl Med 8:24. https://doi.org/10.21037/atm-20-1060 Wang ZS, Huang HR, Zhang LY, Kim S, He Y, Li DL, Farischon C, Zhang K, Zheng X, Du ZY, Goodin S (2017) Mechanistic Study of Inhibitory Effects of Metformin and Atorvastatin in Combination on Prostate Cancer Cells in Vitro and in Vivo. Biol Pharm Bull 40:1247-1254. https://doi.org/10.1248/bpb.b17-00077 Wood WG, Igbavboa U, Muller WE, Eckert GP (2013) Statins, Bcl-2 and Apoptosis: Cell Death or Cell Protection? Mol Neurobiol 48:308-314. https://doi.org/10.1007/s12035-013-8496-5 Xia L, Ding S, Wang X, Zhang X, Zhu L, Zhang H, Li H (2023) Advances in ovarian cancer treatment using a combination of statins with other drugs. Front Pharmacol 13:1048484. https://doi.org/10.3389/fphar.2022.1048484 Yasmeen A, Beauchamp MC, Piura E, Segal E, Pollak M, Gotlieb WH (2011) Induction of apoptosis by metformin in epithelial ovarian cancer: Involvement of the Bcl-2 family proteins. Gynecol Oncol 121:492-498. https://doi.org/10.1016/j.ygyno.2011.02.021 Zhang ZJ, Li S (2014) The prognostic value of metformin for cancer patients with concurrent diabetes: A systematic review and meta-analysis. Diabetes Obes Metab 16:707-710. https://doi.org/10.1111/dom.12267 FIGURE LEGENDS Fig. 1. The cell survival rates (percent) of OVCAR3 cancer line 24, 48 and 72 h after treatment with (A) Metformin and (B) Atorvastatin. All groups were compared to their corresponding negative control. Fig. 2. Bax Expression in Met and Ator treated OVCAR 3 cell groups. The difference between groups is indicated by different symbols (*,#,&). Statistical significance between the groups is as follows: (****p<0.0001) for the NC group; (****p<0.0001) for Metformin-treated cells (Met); (****p<0.0001) for Atorvastatin-treated cells (Ator); (****p<0.0001) for the Combination treatment (Met + Ator), compared to the NC group. (###p<0.0001) for Atorvastatin-treated cells (Ator); (####p<0.0001) for the Combination treatment (Met + Ator), compared to the Metformin-treated cells (Met) group. (&&&&p<0.0001) for the Combination treatment (Met + Ator), compared to the Atorvastatin-treated cells (Ator) group. Blotting was performed at least 3 times in duplicate. Western blot analysis was performed with the included Beta actin to ensure equal protein loading. All data expressed as mean ± standard error of the mean was analyzed by one-way ANOVA with Tukey post hoc analysis for multiple comparisons. Fig. 3. Bcl-2 Expression in Met and Ator treated OVCAR 3 cell groups. The difference between groups is indicated by different symbols (*,#,&). Statistical significance between the groups is as follows: (****p<0.0001) for the NC group; (****p<0.0001) for Metformin-treated cells (Met); (****p<0.0001) for Atorvastatin-treated cells (Ator); (****p<0.0001) for the Combination treatment (Met + Ator), compared to the NC group. (####p<0.0001) for Atorvastatin-treated cells (Ator); (####p<0.0001) for the Combination treatment (Met + Ator), compared to the Metformin-treated cells (Met) group. (&&p<0.0001) for the Combination treatment (Met + Ator), compared to the Atorvastatin-treated cells (Ator) group. Blotting was performed at least 3 times in duplicate Western blot analysis was performed with the included Beta-actin to ensure equal protein loading. All data expressed as mean ± standard error of the mean was analyzed by one-way ANOVA with Tukey post hoc analysis for multiple comparisons. Fig. 4. Caspase 3 Expression in Met and Ator treated OVCAR 3 cell groups. Blotting was performed at least 3 times in duplicate Western blot analysis was performed with the included Beta-actin to ensure equal protein loading. All data expressed as mean ± standard error of the mean was analyzed by one-way ANOVA with Tukey post hoc analysis for multiple comparisons. The difference between groups is indicated by different symbols (*,#,&). Statistical significance between the groups is as follows: (****p<0.0001) for the NC group; (****p<0.0001) for Metformin-treated cells (Met); (****p<0.0001) for Atorvastatin-treated cells (Ator); (****p<0.0001) for the Combination treatment (Met + Ator), compared to the NC group. (####p<0.0001) for the Combination treatment (Met + Ator), compared to the Metformin-treated cells (Met) group. (&&p<0.0001) for the Combination treatment (Met + Ator), compared to the Atorvastatin-treated cells (Ator) group. Fig. 5. ERK Expression in Met and Ator treated OVCAR 3 cell groups. Blotting was performed at least 3 times in duplicate Western blot analysis was performed with the included Beta-actin to ensure equal protein loading. All data expressed as mean ± standard error of the mean was analyzed by one-way ANOVA with Tukey post hoc analysis for multiple comparisons. The difference between groups is indicated by different symbols (*,#,&). Statistical significance between the groups is as follows: (****p<0.0001) for the NC group; (****p<0.0001) for Metformin-treated cells (Met); (****p<0.0001) for Atorvastatin-treated cells (Ator); (****p<0.0001) for the Combination treatment (Met + Ator), compared to the NC group. (##p<0.0001) for Atorvastatin-treated cells (Ator); (####p<0.0001) for the Combination treatment (Met + Ator), compared to the Metformin-treated cells (Met) group. (&&p<0.0001) for the Combination treatment (Met + Ator), compared to the Atorvastatin-treated cells (Ator) group. Fig. 6. AMPK Expression in Met and Ator treated OVCAR 3 cell groups. Blotting was performed at least 3 times in duplicate Western blot analysis was performed with the included Beta-actin to ensure equal protein loading. All data expressed as mean ± standard error of the mean was analyzed by one-way ANOVA with Tukey post hoc analysis for multiple comparisons. The difference between groups is indicated by different symbols (*,#,&). Statistical significance between the groups is as follows: (****p<0.0001) for the NC group; (****p<0.0001) for Metformin-treated cells (Met); (****p<0.0001) for Atorvastatin-treated cells (Ator); (****p<0.0001) for the Combination treatment (Met + Ator), compared to the NC group. (##p<0.0001) for Atorvastatin-treated cells (Ator); (####p<0.0001) for the Combination treatment (Met + Ator), compared to the Metformin-treated cells (Met) group. (&&&&p<0.0001) for the Combination treatment (Met + Ator), compared to the Atorvastatin-treated cells (Ator) group FIGURES Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Information & Authors Information Version history V1 Version 1 12 April 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords ampk apoptosis atorvastatin erk metformin ovcar-3 Authors Affiliations Zunaira Zahid Firat Universitesi Fen Fakultesi View all articles by this author Kazim Sahin Firat Universitesi Veteriner Fakultesi View all articles by this author Muhammed Tokmak Firat Universitesi Veteriner Fakultesi View all articles by this author Fusun Erten Munzur Universitesi View all articles by this author Besir Er Firat Universitesi Fen Fakultesi View all articles by this author Hasan Gencoglu 0000-0002-7716-552X [email protected] Firat Universitesi Fen Fakultesi View all articles by this author Metrics & Citations Metrics Article Usage 292 views 145 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Zunaira Zahid, Kazim Sahin, Muhammed Tokmak, et al. Synergistic Apoptotic Effects of Metformin and Atorvastatin Through Bax/Bcl-2 and AMPK/ERK Modulation in OVCAR3 Cells. Authorea . 12 April 2025. DOI: https://doi.org/10.22541/au.174443767.73020504/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.174443767.73020504/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a0076c44ad6bc13d',t:'MTc3OTU3NDU2NA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();