The anti-proliferative effect of metformin in triple-negative MDA-MB-231 breast cancer cells is highly dependent on glucose concentration: Implications for cancer therapy and prevention

https://doi.org/10.1016/j.bbagen.2014.01.023Get rights and content

Highlights

  • Metformin inhibited the growth of MDA-MB-231 cells in normoglycemic conditions.

  • Metformin caused a striking activation of AMPK in normoglycemic conditions.

  • Metformin inhibited mTOR, ERK, and p70S6K signaling in normoglycemic conditions.

  • These metformin effects are AMPK-dependent.

  • Metformin had no effect on MDA-MB-231 cells in hyperglycemic conditions.

Abstract

Background

Metformin has been shown to have a strong anti-proliferative effect in many breast cancer cell lines, mainly due to the activation of the energy sensing kinase, AMP-activated protein kinase (AMPK). MDA-MB-231 cells are aggressive and invasive breast cancer cells that are known to be resistant to several anti-cancer agents as well as to the anti-proliferative effect of metformin. As metformin is a glucose lowering drug, we hypothesized that normoglycemia will sensitize MDA-MB-231 cells to the anti-proliferative effect of metformin.

Methods

MDA-MB-231 cells were treated with increasing metformin concentrations in hyperglycemic or normoglycemic conditions. The growth inhibitory effect of metformin was assessed by MTT assay. The expression of several proteins involved in cell proliferation was measured by Western blotting.

Results

In agreement with previous studies, treatment with metformin did not inhibit the growth of MDA-MB-231 cells cultured in hyperglycemic conditions. However, metformin significantly inhibited MDA-MB-231 growth when the cells were cultured in normoglycemic conditions. In addition, we show that metformin-treatment of MDA-MB-231 cells cultured in normoglycemic conditions and not in hyperglycemic conditions caused a striking activation of AMPK, and an AMPK-dependent inhibition of multiple molecular signaling pathways known to control protein synthesis and cell proliferation.

Conclusion

Our data show that normoglycemia sensitizes the triple negative MDA-MB-231 breast cancer cells to the anti-proliferative effect of metformin through an AMPK-dependent mechanism.

General significance

These findings suggest that tight normoglycemic control may enhance the anti-proliferative effect of metformin in diabetic cancer patients.

Introduction

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths among females worldwide [1]. In the United States, over 200,000 new breast cancer cases are diagnosed every year and approximately 40,000 of these diagnosed patients will die from the disease [1]. Breast cancers are usually classified according to their expression of estrogen receptors (ER), progesterone receptors (PR), or human epidermal growth factor receptor (HER2) [2]. Most of the current successful therapies for breast cancer, which include anti-estrogen therapies, aromatase inhibitors, or Herceptin, target these receptors [3]. Triple-negative breast cancers (TNBCs) which represent about 15% of breast cancer cases [4] do not express any of these receptors and are thus more difficult to treat with existing therapies. Moreover, TNBCs are more likely to metastasize thus resulting in poorer prognosis [5]. MDA-MB-231 cells are aggressive and invasive TNBC cells that are known to be resistant to several anti-cancer agents [6]. In addition to being triple-negative, MDA-MB-231 cells express a mutant p53 and lack the tumor-suppressor kinase LKB1, which make them even more resistant to treatment [7].

Several epidemiological studies have suggested that diabetes is associated with an increased risk of breast cancer [8], [9]. Indeed, hyperglycemia and hyperinsulinemia are thought to promote the growth of cancer cells in diabetic patients [10]. Interestingly, metformin, the most commonly prescribed oral anti-diabetic medication, has been shown to have strong anti-proliferative and/or pro-apoptotic properties in several breast cancer cell lines. Indeed, metformin has been shown to inhibit the proliferation of BT474, BT20, MDA-MB-453, T47D, and MCF7 breast cancer cells independent of estrogen receptor, HER2, or p53 status [11]. In addition, metformin has been shown to induce caspase-dependent cell death in several breast cancer cell lines including MCF7, T47D, MDA-MB-453, and BT474 [12]. While the importance of a commonly prescribed and well-tolerated drug that may also function as a cancer therapy and/or in cancer prevention is of extreme clinical significance [13], several investigators have shown that metformin does not inhibit the proliferation or induce cell apoptosis of the aggressive MDA-MB-231 cancer cells [11], [12]. Although definitive evidence is not available to explain why the LKB1-deficient MDA-MB-231 cancer cells are resistant to metformin, it has been proposed that metformin signals through LKB1 to exert both its anti-diabetic [14] and anti-proliferative [15] effects.

LKB1 is an important upstream kinase of the energy sensing enzyme AMP-activated protein kinase (AMPK) [16] and the activation of AMPK is the most widely accepted mechanism to explain the anti-cancer effects of metformin [17]. While likely not completely delineated, it is thought that metformin activates AMPK via an LKB1-dependent mechanism, which inhibits the mammalian target of rapamycin (mTOR) resulting in a strong inhibition of cell proliferation in several cancer cell lines [18], [19], [20]. Consistent with this, it has been suggested that metformin is not able to inhibit the proliferation of MDA-MB-231 cells because these cells do not express LKB1 and thus metformin fails to activate AMPK [18]. However, most of the previous studies have tested the effectiveness of metformin in inhibiting proliferation of cancer cells using culture conditions that contain high concentrations of glucose [21]. Importantly, given the fact that metformin-treated breast cancer diabetic patients have better clinical outcomes than non-metformin-treated patients [22], it is possible that normal serum concentrations of glucose may be involved in the degree of effectiveness of metformin treatment of cancer patients, potentially even in LKB1-deficient cells.

Of particular importance to understanding the role of metformin and serum concentrations of glucose and insulin in cancer patients, high concentrations of glucose and insulin have been shown to enhance the proliferation of MDA-MB-231 cells [23]. On the other hand, the anti-proliferative effect of metformin was dramatically enhanced when pancreatic cancer cells were cultured in media with physiological concentrations of glucose [21]. Based on these previous observations, we hypothesized that normal physiological concentrations of glucose sensitize MDA-MB-231 cells to the anti-proliferative effect of metformin. In the current work, we demonstrate for the first time that metformin significantly inhibits the proliferation of MDA-MB-231 cells when they are cultured in normoglycemic conditions. In addition, we show that the concentrations of metformin that are needed to prevent proliferation of MDA-MB-231 cells are much lower than those used in previous studies using other cell types. Together, our data show that LKB1-deficient MDA-MB-231 cells are not metformin-resistant in normoglycemic conditions and not only highlight the potential importance of good glycemic control as a requirement for the anti-proliferative action of metformin, but also question the involvement of LKB1 in these processes.

Section snippets

Materials

Dulbecco's modified Eagle's medium (DMEM) base, metformin, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). OCT-1 primary antibody was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). BrdU cell proliferation assay kit and other primary and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Chemiluminescence Western blotting detection reagents were purchased from

Metformin inhibits MDA-MB-231 cell growth in normoglycemic but not in hyperglycemic conditions

To assess the effect of metformin on the growth of the triple-negative MDA-MB-231 breast cancer cells, the cells were cultured in 96-well plates in conventional high glucose medium containing 10% fetal bovine serum. After 24 h, the medium was changed to serum free medium supplemented with 25 mM glucose (hyperglycemic conditions) or 5 mM glucose (normoglycemic conditions). Thereafter, the cells were treated with increasing concentrations of metformin (from 0.1 mM to 5 mM) for 48 h or 96 h. In

Discussion

Several lines of evidence drawn from in vitro, in vivo, and epidemiological studies have suggested that metformin may be of benefit to diabetic cancer patients [33], [34]. Of interest, breast cancer risk has been shown to be lower in diabetic patients treated with metformin than in those treated with other anti-diabetic medications [35]. In addition, metformin-treated diabetic breast cancer patients have better clinical outcomes than non metformin-treated patients [22]. On the other hand, some

Acknowledgements

This study was supported by the Canadian Institutes of Health Research (CIHR) (J. R. B. Dyck). BNMZ is the recipient of a CIHR post-doctoral fellowship and the Alberta Innovates — Health Solutions (AIHS) clinician fellowship. DB is the recipient of AIHS summer studentship.

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