Metformin Enhances the Antiproliferative and Apoptotic Effect of Bicalutamide in Prostate Cancer

AJ Colquhoun; NA Venier; AD Vandersluis; R Besla; LM Sugar; A Kiss; NE Fleshner; M Pollak; LH Klotz; V Venkateswaran

Disclosures

Prostate Cancer Prostatic Dis. 2012;15(4):346-352. 

In This Article

Abstract and Introduction

Abstract

Background: Prostate cancer incidence and mortality vary dramatically by geographical location. Both are higher in developed countries. Some attribute this to westernized lifestyles of high-energy diets and limited physical activity with consequent obesity. Obesity and obesity-related diseases like diabetes cause hyperinsulinaemia, which upregulates pro-survival cell signalling. Previous work revealed diet-induced hyperinsulinaemia enhances prostate cancer xenograft growth in vivo. Metformin, an antidiabetic medication, reduces hyperinsulinaemia and also exhibits antineoplastic properties. Herein, we assess the potential additive benefit of combining bicalutamide antiandrogen therapy with metformin, in vitro and in vivo.

Methods: Using clonogenic assays, we assessed the effect of bicalutamide and/or metformin on clonogenicity in prostate cancer cell lines. Western blot and cell cycle analyses were used to elucidate mechanisms of interaction between the drugs in androgen receptor (AR)-positive (LNCaP) and AR-negative (PC3) cell lines. The combination treatment regimen was assessed in vivo using an LNCaP murine xenograft model.

Results: Micromolar bicalutamide or millimolar metformin caused a significant dose-dependent reduction in clonogenicity (P<0.001). Combination treatment further significantly reduced clonogenicity (P<0.005) with greater effects in AR-positive cells. Western blot and cell cycle analyses suggested differing mechanisms of interaction in AR-positive and -negative cell lines. Following combination treatment, LNCaP cells exhibited an altered cell proliferation (decreased phospho mammalian target of rapamycin expression) and perturbed cell cycle kinetics (G1/S cell cycle arrest). PC3 cells showed evidence of enhanced apoptosis (increased Bcl-2-associated X protein and decreased total caspase 3 expression). Markedly diminished tumour growth occurred following combination treatment in vivo (P<0.001).

Conclusions: Combining bicalutamide and metformin significantly reduces prostate cancer cell growth further than either monotherapy. In AR-positive cells, this effect appeared to be mediated by reducing proliferation rates, whereas in AR-negative cells the combination treatment appeared to promote apoptosis. This combination drug regimen may improve prostate-cancer-specific survival by the direct antineoplastic properties outlined.

Introduction

The incidence of prostate cancer has risen dramatically over the past three decades, with over 215 000 new diagnoses and 32 000 deaths now occurring per annum in the United States of America.[1] Simultaneously, the incidence of obesity has risen dramatically, particularly in so called developed countries.[2] It is estimated that by 2020, 40% of the American population will be classified as obese (body mass index >30 kg m−2). A strong correlation has been described between obesity and colorectal and endometrial cancer.[3] However, evidence of a direct link between prostate cancer diagnosis and obesity is less conclusive.[4] This may be explained, in part, by the differential effect of obesity on the development of high- and low-grade prostatic tumours. Several authors report obesity to be a risk factor for high-grade disease, whereas excess weight appears protective against low-grade tumours.[5–8] Given that obese individuals are known to exhibit hyperinsulinaemia, one plausible mechanism by which obesity may influence the development and/or progression of prostate cancer relates to the proproliferative effects of insulin and the related insulin-like growth factors (IGF). Pre-clinical studies from our own laboratory showed mice bearing prostate cancer tumour xenografts fed on a high-carbohydrate high-fat, designed to induce hyperinsulinaemia, exhibited a significantly greater tumour growth than mice fed on an isocaloric low-carbohydrate high-fat diet.[9]

A rapid escalation in both obesity and diabetes diagnoses has occurred over the last 30 years.[10,11] The incidence of non-insulin-dependent diabetes has particularly increased.[12] Metformin, an oral biguanide, has been used for several decades to treat non-insulin-dependent diabetes. Although classically described as an insulin-sensitizing agent, there is no clear consensus regarding its modes of action. In the liver, metformin reduces serum glucose levels by inhibiting glycogenolysis and gluconeogenesis.[13,14] Its activity in peripheral tissues is less clearly defined. Inhibition of hepatic glycogenolysis and gluconeogenesis is mediated by the activation of adenosine monophosphate kinase (AMPK). AMPK is a highly conserved protein kinase, which functions to maintain both whole-body and cellular energy homeostasis.[15] The net effect of metformin use is to lower serum glucose levels and consequently inhibit the development of chronic hyperinsulinaemia.

As well as exhibiting antidiabetic activity, metformin has recently been shown to exert antineoplastic effects in a number of tumour systems, including the prostate, ovarian, breast, colorectal and endometrial carcinoma models.[15–22] Potential mechanisms of action for these antineoplastic effects include direct antiproliferative activity mediated by metformin-induced activation of AMPK, with a subsequent downstream inhibition of the mammalian target of rapamycin (mTOR)/S6 kinase pathway, or direct inhibition of insulin/IGF-mediated cellular proliferation.

Given the accumulating data regarding the antineoplastic capacity of metformin in pre-clinical prostate cancer models, we set out to assess the use of metformin as an adjunct to bicalutamide, an established hormonal therapy used in the treatment of prostate cancer. Bicalutamide is a non-steroidal antiandrogen, which functions by blocking the androgen receptor (AR). It exerts its effect predominantly through induction of a G1/S phase arrest of the cell cycle.[23,24] We hypothesized that combining metformin with bicalutamide may have an additive antiproliferative effect by each drug's differing mechanisms of action. Thus, we sought to determine whether combining metformin and bicalutamide would inhibit prostate cancer cell growth using in vitro and in vivo prostate cancer models. We also proposed to assess whether any potential beneficial effect was AR-dependent by utilizing prostate cancer cell lines of differing AR expression.

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