Elsevier

Gynecologic Oncology

Volume 127, Issue 2, November 2012, Pages 390-397
Gynecologic Oncology

Metformin targets ovarian cancer stem cells in vitro and in vivo

https://doi.org/10.1016/j.ygyno.2012.07.115Get rights and content

Abstract

Purpose

Studies in non-gynecologic tumors indicate that metformin inhibits growth of cancer stem cells (CSC). Diabetic patients with ovarian cancer who are taking metformin have better outcomes than those not taking metformin. The purpose of this study was to directly address the impact of metformin on ovarian CSC.

Methods

The impact of metformin on ovarian cancer cell line growth and viability was assessed with trypan blue staining. Aldehyde dehydrogenase (ALDH) expressing CSC were quantified using FACS®. Tumor sphere assays were performed to determine the impact of metformin on cell line and primary human ovarian tumor CSC growth in vitro. In vivo therapeutic efficacy and the anti-CSC effects of metformin were confirmed using both tumor cell lines and ALDH(+) CSC tumor xenografts.

Results

Metformin significantly restricted the growth of ovarian cancer cell lines in vitro. This effect was additive with cisplatin. FACS analysis confirmed that metformin reduced ALDH(+) ovarian CSC. Consistent with this, metformin also inhibited the formation of CSC tumor spheres from both cell lines and patient tumors. In vivo, metformin significantly increased the ability of cisplatin to restrict whole tumor cell line xenografts. In addition, metformin significantly restricted the growth of ALDH(+) CSC xenografts. This was associated with a decrease in ALDH(+) CSC, cellular proliferation, and angiogenesis.

Conclusions

Metformin can restrict the growth and proliferation of ovarian cancer stem cells in vitro and in vivo. This was true in cell lines and in primary human CSC isolates. These results provide a rationale for using metformin to treat ovarian cancer patients.

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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