Metformin decreases growth of pancreatic cancer cells by decreasing reactive oxygen species: Role of NOX4

https://doi.org/10.1016/j.bbrc.2015.07.118Get rights and content

Highlights

  • We studied the role of metformin in reducing ROS and pancreatic cell growth.

  • Metformin-induced decrease in cell survival is associated with reduced ROS.

  • Metformin increased expression of MnSOD, Catalase, and decreased NOX2 and NOX4.

  • Overexpression of NOX4 partially abrogated the anti-survival effects of metformin.

  • Metformin inhibits cell survival by reducing ROS, in part through reduced NOX4.

Abstract

Background

Retrospective epidemiologic studies show that metformin reduced the incidence of pancreatic cancer in diabetic patients. One potential mechanism may be by altering reactive oxygen species (ROS) and apoptosis. In this in vitro study, we explore the role of ROS and apoptosis in mediating the growth-inhibitory effects of metformin in pancreatic cancer cells.

Methods

We cultured MIA PaCa and Panc1 pancreatic cancer cells in medium containing physiological concentrations of glucose (5 mM) or supra-physiological concentrations of glucose and then treated cells with metformin. Cell viability, ROS production, apoptosis, and protein levels of manganese superoxide dismutase (MnSOD), NADPH oxidase (NOX) 2, and NOX4 were measured.

Results

Metformin decreased viability of MIA PaCa and Panc1 cells under physiological glucose conditions in comparison to untreated cells; metformin did not have any effect on human pancreatic normal epithelial (HPNE) cells. The decrease in cell survival was associated with decreased intracellular ROS, increased protein levels of MnSOD, and decreased levels of NOX2 and NOX4 proteins in MIA PaCa and Panc1 cells but not HPNE cells. Transfecting MIA PaCa and Panc1 cells with pcDNA3NOX4 protected against the anti-survival effects of metformin.

Conclusion

Our findings suggest that metformin decreases cell survival by reducing ROS production, in part through down regulation of NOX4 protein expression.

Introduction

There is growing evidence that metabolic disorders such as increased body mass weight, obesity, and type 2 diabetes are risk factors for pancreatic cancer [1], [2], [3], [4], [5], [6]. The prevalence of people both overweight and obese has increased over the past several decades with a third of the adult population now obese [7], [8]. In a wide range of species, calorie restriction (CR), resulting in lower body weight, is well-recognized for extending life span and reducing incidence of many chronic diseases including several types of cancers [2], [3], [4], [5], [6], [7], [8]. Our recent study demonstrated that CR delayed the progression of early PanIn lesions to pancreatic tumors in the KrasG12D; Pdx-1Cre mouse model of pancreatic cancer [9].

The benefits of CR may also be attained by CR mimetics such as metformin. Metformin, a member of the biguanide family, is one potential compound that mimics many of the cell survival and energy metabolism pathways altered by CR. Metformin has several advantages for use as a chemopreventative agent; it is the most widely used drug for treatment of type-2 diabetes and has been used for decades without negative side effects [10]. There is a growing knowledge base that suggests that anti-diabetic drugs such as metformin may protect against cancer. Retrospective and epidemiological studies have reported that diabetic patients receiving metformin had a lower incidence of pancreatic cancer [11], [12], [13] and better survival [12]. The mechanisms for the antitumor effects of metformin are not well understood. Metformin may act through systemic effects by improving insulin resistance and/or cell-direct effects. At the cellular level, metformin inhibits mitochrondrial respiratory chain complex I resulting in reduced ATP levels, which stimulate an increase in the activation of AMPK and decrease mTOR signaling [14].

The inhibition of complex 1 raises the possibility that metformin may inhibit growth of cancer cells through decreased ATP and ROS resulting in mitochondrial damage. Since ROS is necessary for survival and growth KrasG12D-driven pancreatic tumorigenesis [15], [16], reducing ROS levels below survival requirements may decrease tumor cell proliferation and induce apoptosis.

Increased production of the intracellular level of ROS in pancreatic cancer results from the imbalance of the antioxidant scavenger enzymes that remove ROS and the NOX system that produce ROS. The antioxidant enzymes include manganese superoxide dismutase (MnSOD), copper and zinc (Cu/Zn)SOD, catalase, and glutathione peroxidase. Protein expression of NOX2 and NOX4 are increased in pancreatic cancer compared to normal tissue [17]. There is evidence that metformin reduces intracellular ROS in endothelial and smooth muscle cells [18], [19], [20]. However, the potential role of metformin in reducing oxidative stress and cell growth in cancer is not well known.

In this study, we demonstrate the growth-inhibitory and proapoptotic effects of metformin in human pancreatic cancer cell lines and that these effects are associated with increased protein expression of MnSOD and decreased NOX2 and NOX4. Moreover, we provide evidence that metformin reduces oxidative stress by down regulating NOX4.

Section snippets

Cell lines, reagents, and antibodies

Pancreatic cancer cell lines, MIA PaCa and Panc1 cells were grown in DMEM and 5% dialyzed FBS. hTERT-HPNE cells were grown in medium 75% DMEM, 25% Medium M3 Base, 5% dialyzed FBS. Recent studies have reported that the pancreatic cancer cells cultured in physiological concentrations of glucose have increased sensitivity to metformin [20], [21], [22]. Pancreatic cancer cell lines were incubated with 5 mM glucose (physiological concentration) or 22 mM glucose (supra-physiological concentration)

Metformin produces a concentration- and time-dependent decrease in cell survival when incubated in physiological concentrations of glucose

Pancreatic cancer cells cultured in a physiological concentration of glucose (5 mM) were more sensitive to the growth inhibitory effects of metformin than those cultured in diabetic-like conditions with supra-physiological concentrations (22 mM) [21], [22]. Survival of MIA PaCa and Panc1 cells decreased at all concentrations of metformin after 48 h of incubation (Fig. 1A and B). A concentration of 1 mM of metformin significantly killed 27% Panc1 cells after 48 h and 35% of the MIA PaCa cells

Discussion

Population studies have found that metformin lowers risk of pancreatic cancer in type 2 diabetic patients [11], [12], [13]. Preclinical studies show that metformin inhibited growth of pancreatic cancer cells in vitro [22], [24], [31], [34] and in vivo grown as xenograft [31], [32], or orthotopic tumors in mice [33]. Similar to previous studies [23], [24], our data also show that metformin inhibits survival of pancreatic cells and that cells cultured in a physiological concentration of glucose

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