Synergistic Anticancer Effects of Metformin and Doxorubicin in Ovarian Cancer Cells Through Dual Apoptotic Pathway Activation and Oxidative Stress Enhancement

This study aimed to evaluate the antiproliferative, apoptotic, and oxidative stress-inducing effects of the combination of metformin and doxorubicin (adriamycin) in OVCAR3 and SKOV3 ovarian cancer cell lines and to investigate the potential synergistic interactions between the two agents. Cell viability was assessed using the MTT assay. Apoptosis was quantified via Annexin V/PI staining followed by flow cytometry. Caspase-8 and caspase-9 activities were measured using colorimetric assays. Oxidative stress parameters, including reactive oxygen species (ROS) and nitric oxide (NO), were determined using DCFH-DA fluorescence and the Griess assay, respectively. The mRNA expression levels of apoptosis-related genes (Bcl-2, Survivin, Bax, and Caspase-3) were analyzed by qRT-PCR. Drug interaction and synergy were evaluated using the Chou-Talalay combination index (CI) model and the highest single agent (HSA) model. Prognostic relevance of target genes and protein interaction networks was examined through TCGA and STRING databases. The metformin-doxorubicin combination demonstrated strong synergistic antiproliferative effects in both cell lines (CI < 0.7 in OVCAR3). The combination significantly increased apoptosis compared with single-agent treatments, yielding a total apoptotic rate of 62.5 ± 4.2% in OVCAR3. Caspase-8 and caspase-9 activities were elevated by 5.6 ± 0.7-fold and 7.3 ± 0.8-fold, respectively. Combination treatment also induced marked oxidative stress, increasing NO levels to 12.4 ± 1.1 µM and ROS levels to 412 ± 25% in OVCAR3 cells. qRT-PCR analyses revealed downregulation of anti-apoptotic Bcl-2 (0.28 ± 0.04-fold) and Survivin (0.25 ± 0.03-fold), along with upregulation of pro-apoptotic Bax (5.8 ± 0.6-fold) and Caspase-3 (6.5 ± 0.7-fold). Bioinformatic analyses indicated that high Bcl-2 and Survivin expression correlated with poorer overall survival in ovarian cancer patients. Metformin enhances the anticancer efficacy of doxorubicin through synergistic activation of intrinsic and extrinsic apoptotic pathways, induction of oxidative and nitrosative stress, and transcriptional regulation of key apoptotic markers. These findings support the potential use of metformin as an adjuvant agent to strengthen doxorubicin-based chemotherapy in ovarian cancer.