(M1530-05-30) Metformin Induced-Oxidative Stress Modulates LNCaP Cellular Redox Status and Suppressed Cell Growth

Purpose: To evaluate the ability of metformin to regulate cellular redox homeostasis and suppress LNCaP prostate cancer cell growth. Prostate cancer is the second leading cause of cancer death in American men. Despite multiple treatment options, there are several challenges that affect treatment outcome; these include patient age, cancer resistance and treatment-related side effects. Although, treatment options focus on cellular targets such as androgen synthesis, cell cycle phases, and immune cells. These cellular targets bypass the underlying supportive metabolic networks that contribute to a cancer cell's ability to evade apoptosis, proliferate, and develop chemoresistance. Notably, several preclinical and clinical studies have indicated the potential of metformin in prostate cancer therapy. These studies demonstrated metformin's pleiotropic effects on several molecular and metabolic pathways such as androgen signaling, cell cycle, and cellular bioenergetics, including its unique ability to activate the energy sensor 5' -AMP-activated protein kinase (AMPK). We previously demonstrated that metformin alters mitochondrial function in LNCaP prostate cancer cells. In light of this, we hypothesized that metformin might modulate cell growth and survival by regulating redox homeostasis in LNCaP prostate cancer cells.

Methods: Using Seahorse XP extracellular analysis, we measured LNCaP prostate cancer cell bioenergetics (oxygen consumption rate -OCR) in the presence of metformin. Liquid Chromatography/Mass Spectrophotometry (LC/MS), 2,7- dichlorofluoresin diacetate (DCFDA) assay, and MTT assay (doxorubicin was added as a positive control) were used to assess the effects of metformin on cellular redox status, redox-related metabolites, and prostate cancer cell survival. Survival gene-related mRNA expression and transsulfuraion-related enzymes mRNA expressions were measured using RT-qPCR.

Results: Metformin altered LNCaP prostate cancer bioenergetics by suppressing oxidative phosphorylation as denoted by lowering OCR levels. LC/MS analysis of metformin-treated cells showed a significant increase in homocysteine level (HCY) whereas glutathione (GSH) and cystathionine (CYS) levels were reduced, and this was supported by the reduction in GSH/GSSG ratio. These lower levels of GSH and CYS were consistent with a compensatory increase in the gene expression of transsulfuration-related enzymes, cystathionine beta synthase (CBS) and cystathionine gamma-lyase (CSE). Additionally, metformin increased the gene expressions of the glutathione-related enzymes, γ-glutamyl cysteine ligase (GCL) and glutathione synthetase (GSS). Overall, these findings were supported by the DCFDA assay results which showed that metformin significantly increased reactive oxygen species (ROS) production. Notably, metformin significantly downregulated survivin mRNA expression, which was denoted by the reduced proliferative effects observed with MTT assays. Moreover, our findings were similiar to doxorubicin effects (an ROS inducer), which suggested that metformin-induced ROS may contribute to its antiproliferative effects in LNCaP prostate cancer cells.

Conclusion: Metformin modulates LNCaP prostate cancer redox homeostasis by inducing oxidative stress via ROS production and deceased cell viability.

Acknowledgements: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profits sectors. We thank Ms. Natasha Rose for technical assistance and training

Metformin downregulates oxidative phosphorylation

Metformin modulates redox-related metabolites and mRNA expression

Metformin suppressed cell viability and increase ROS production