Induction of apoptosis by metformin in epithelial ovarian cancer: Involvement of the Bcl-2 family proteins
Research Highlights
► Metformin induces apoptosis in ovarian cancer cells in an AMPK-independent manner, an effect enhanced by cisplatin. ► Metformin blocks cell cycle progression in the S and G2/M phase. ► Metformin modulates protein expression from the Bcl-2 family.
Introduction
Ovarian cancer is the leading cause of death among all gynecological cancers and the fifth most common cause of cancer-related death in western countries [1]. The lack of symptoms of this disease in its early stages makes early diagnosis extremely difficult. Patients with advanced ovarian cancer are initially treated by a combination of debulking surgery and standard chemotherapy [2]. Despite an initial 70–80% response rate, most patients will relapse within 1–2 years and develop resistance to chemotherapy. In fact, the overall 5-year survival rate is less than 30% [3]. The identification of new drugs or novel therapeutic strategies with the ability to re-sensitize ovarian carcinoma cells to existing chemotherapy has become a major challenge.
Metformin is an oral biguanide which lowers circulating levels of glucose and insulin and is commonly used for the treatment of type II diabetes. Two population studies provided preliminary evidence that metformin may reduce cancer risk and improve prognosis in patients with type II diabetes [4], [5]. This protective effect of metformin on cancer risk was recently confirmed in a cohort study comprising 4000 patients with type II diabetes [6]. Moreover, it was recently reported that diabetic patients with breast cancer treated with metformin along with neoadjuvant chemotherapy have a better pathologic complete response (pCR) rate in comparison to patients not receiving metformin [7]. Recent data further demonstrated that the key mechanism of action of metformin is by activating the AMPK–LKB1 pathway [8], [9]. Other AMPK activators have displayed growth inhibitory effects in various cancer cell types [10], [11], [12]. Therefore, metformin might exhibit two potential anti-neoplastic effects: reducing circulating insulin levels and directly inhibiting growth through the AMPK–LKB1 pathway.
We have previously demonstrated that metformin decreases ovarian cancer cell survival in a dose- and time-dependent manner, partly through AMPK activation [13]. Moreover, we observed that the effect of metformin is potentiated by the addition of cisplatin. We therefore decided to evaluate whether, in addition to its antiproliferative effect, metformin could stimulate apoptosis in human ovarian cancer cells. We also sought out to identify the pathways involved in this effect.
Bcl-2 family proteins are crucial for apoptosis commitment, mainly via the control of the mitochondrial pathway which is frequently triggered in response to chemotherapeutic agents. Elevated levels of Bcl-2 in tumor cells may contribute to chemoresistance by stabilizing the mitochondrial membrane against apoptotic insult. Thus, Bcl-2 or Bcl-xl may be good therapeutic targets [14], [15]. Although controversial, the majority of studies exhibit evidence of an increased expression of Bcl-2 and Bcl-xL in ovarian cancer [16], [17], [18]. Moreover, Anderson et al. recently demonstrated that Bcl-2 levels are elevated in the urine of patients with epithelial ovarian cancer in two different cohorts [19]. We next examined whether metformin, alone or in combination with cisplatin, modulates the pro- and anti-apoptotic protein members of the Bcl-2 family.
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Cells lines and treatment
The ovarian cancer cell lines OVCAR-3, (American Tissue Culture Collection, Manassas, VA) and OVCAR-4 were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 10 μg/ml gentamicin. The cells were routinely passaged every 5–7 days. All cells were maintained at 37 °C in a 5% CO2, 95% air atmosphere incubator. Assays were performed in medium containing 1% FBS. Metformin was obtained from Sigma-Aldrich (cat#D150959) and kept as a stock solution of 1 M in RPMI without
Metformin induces apoptosis of epithelial ovarian cancer cells in an AMPK-independent manner
Flow cytometry analysis using annexin V labeling was carried out to measure apoptosis in our cell lines in the presence of metformin. As shown in Fig. 1A, metformin induces apoptosis dose-dependently in both cell lines with a more pronounced effect observed in OVCAR-3 cells. As an additional indication of apoptosis occurring in those cells, caspases-3/7 activity, which play key effector roles in apoptosis, were measured. As shown in Fig. 1B, caspases-3/7 activity was also increased in a
Discussion
Epithelial ovarian cancer is the leading cause of death among gynecological cancers and close to 70% of patients with advanced-stage disease will experience recurrence [22], [23]. This is caused by the development of resistance to current therapies, implying the need to develop novel therapeutic modalities with innovative mechanisms of action.
Metformin has been used for several decades for the treatment of type 2 diabetes and has a proven track record of being highly effective with minimal
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was made possible in part by grants from the Montreal-Israel Cancer Research Foundation, the Gloria Shapiro fund, the Turqwise fund for ovarian cancer, the Greenberg Foundation, and the Friends for Life charity.
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These authors contributed equally to this work.