Metformin targets ovarian cancer stem cells in vitro and in vivo
Highlights
► Metformin has anti-ovarian cancer activity in vitro and in vivo. ► Metformin restricts the growth of primary human ovarian cancer stem cells. ► Clinical trials to assess the impact of metformin on ovarian cancer outcomes are indicated.
Introduction
Ovarian cancer is the deadliest gynecologic malignancy and the fifth most deadly malignancy of women in the United States [1]. Although multi-modality treatment with cytoreductive surgery and platinum-taxane based chemotherapy have prolonged survival, the overall cure rate of the disease has not changed dramatically. There have been few new developments in the treatment of ovarian cancer patients since the standardization of platinum and paclitaxel, which has been the mainstay of ovarian cancer adjuvant therapy for the last 20 years. Novel therapeutics are needed. One way forward is suggested by recent work implicating cancer stem cells (CSC) as the source of therapeutic resistance and disease recurrence.
The cancer stem cell hypothesis suggests that cancer stem cells are a rare population of inherently chemo-resistant cancer cells able to regenerate the various cell types within a tumor, thereby causing a disease relapse [2], [3]. We have recently demonstrated that a population of ovarian CSC can be isolated based on aldehyde dehydrogenase (ALDH) activity [4]. ALDH(+) cells are inherently resistant to chemotherapy. Small numbers of ALDH(+) cells can initiate tumors in mice, while a 10–50 fold excess of ALDH(−) cells cannot. Interestingly, cells which express both ALDH and CD133 possess greater tumor initiation capacity [4].
Drugs that target cancer stem cells may offer a great promise. Metformin, a traditional type 2 diabetes medication, has also been associated with transcriptional repression of the epithelial–mesenchymal transition, a cellular phenotype associated with CSC [5]. Metformin has recently been shown to target CSC in breast cancer. Metformin inhibited cellular transformation and selectively killed breast cancer stem cells in vitro and in vivo [6]. Metformin selectively kills chemotherapy-resistant CSC in breast cancer cell lines [7]. Another study in breast cancer demonstrated that metformin synergistically interacts with trastuzumab, the anti-HER2 monoclonal antibody, to suppress the self-renewal and proliferation of CSC in HER-2 positive carcinomas [8]. Finally, the combination metformin and standard chemotherapy reduced tumor mass and prevented relapse in cell line xenograft mouse models of prostate and lung cancer [7]. All these data strongly implicate metformin as a CSC targeting agent.
Metformin may have similar activity in ovarian cancer. In vitro studies have demonstrated anti-proliferative and pro-apoptotic effects of metformin in ovarian cancer [9]. Metformin therapy has been associated with anti-proliferative effects through both AMPK-dependent and AMPK-independent pathways and increased tumor cell apoptosis and decreased metastasis [10], [11]. In our study, we provide evidence that a major mechanism for metformin's ability to inhibit the growth of ovarian cancer lies in its effect on ovarian cancer stem cells.
Section snippets
Cell lines and cytotoxic assays
A2780 cells were obtained from Dr. Susan Murphy (Duke University, Durham, NC). SKOV3 cells were obtained from Dr. Rebecca Liu (University of Michigan, Ann Arbor, MI). In order to achieve ~ 60% confluency on the culture plate, 1.8 × 105 SKOV3 cells and 2.5 × 105 A2780 cells were plated in replicate in RPMI-10 (10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen)) and rested for 24 h. Cells were then treated with cisplatin (APP Pharmaceuticals) 1.5 μg/mL for SKOV3 cells or 0.5 μg/mL for
Metformin as an anti-neoplastic in ovarian cancer
Some in vitro studies of metformin's impact on ovarian cancer cell lines used supra-physiologic doses (50 mM) of metformin [9]. We performed dose-titration studies examining the effects of physiologic doses of metformin on ovarian cancer cell lines. Metformin therapy inhibited the proliferation of SKOV3 cells at doses of 1 mM and higher (Fig. 1A and data not shown). Metformin inhibited the growth of A2780 cells in doses of 300 μM and higher. We estimated an IC50 of 1–3 mM in two cell lines; a dose
Discussion
Metformin has been shown to be active against ovarian cancer cells in vitro and in vivo. We demonstrate here that metformin acts on ovarian cancer stem cells, reducing the percentage of ALDH(+) CSC in vitro and in vivo, and inhibiting the growth of ovarian tumor spheres. Metformin was active against primary human CSC in vitro and, metformin therapy alone slows the growth of ovarian CSC in vivo.
Our results are most consistent with metformin having anti-proliferative rather than cytotoxic
Conflict of interest statement
None of the authors have a conflict of interest.
Acknowledgments
This work was supported by a grant from the Michigan Institute for Clinical Health Research and the National Institutes of Health New Innovator Directors Award grant #00440377.
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