(M1030-03-15) Metformin Induced-Oxidative Stress Modulates Cellular Redox Homeostasis and Suppresses Cell Growth in Early and Late-Stage Prostate Cancer Cells

Purpose: To evaluate the ability of metformin to modulate cellular redox status and suppress cell growth among early and late-stage prostate cancer cells Prostate cancer is the second leading cause of cancer death in American men. Despite several treatment options, there are challenges that affect treatment outcome such as treatment-related side effects and drug resistance. Although, these current treatment options focus on cellular targets such as androgen synthesis and cell cycle phases. Cancer cells use underlying supportive metabolic networks to bypass cellular targets to promote survival, evade apoptosis, and develop immunity to treatment. Several preclinical and clinical studies have indicated the potential for metformin in prostate cancer therapy. These studies demonstrate metformin’s pleiotropic effects on several molecular and metabolic pathways such as androgen signaling cell cycle and immune cells. In previous research we demonstrated that metformin alters mitochondrial function and suppresses cell survival in early-stage prostate cancer cells. Metformin’s effects on redox status of late-stage prostate cancer remain largely unexplored. We hypothesized that early-stage prostate cancer will exhibit a higher sensitivity to metformin than late-stage prostate cancer survival by altering redox homeostasis and suppressing cell survival. In this study we utilized LNCaP (androgen sensitive) and PC3 (androgen insensitive) cells as early and late-stage prostate cancer cells, respectively.

Methods: Using Liquid Chromatography/ Mass Spectrophotometry (LC/MS), 2,7- Dichlorofluoresin diacetate (DCFDA) assay, and MTT assay (1µM 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. Glutathione and transsulfuration-related enzymes mRNA expressions were measured using RT-qPCR.

Results: LC/MS analysis of metformin-treated cells showed that glutathione (GSH) levels decreased in both LNCaP and PC3 cells. In contrast, LNCaP cells showed reduced levels of cystathionine and increased homocysteine, while PC3 cells exhibited a slight decrease in cystathionine with no change in homocysteine. Although these metabolites were differentially altered in both cell types, reduced GSH levels were further corroborated by the reduction in GSH/GSSG ratio. Notably, lower levels of GSH and CYS were consistent with a compensatory increase in transsulfuration-related enzymes, cystathionine beta synthase (CBS), and cystathionine gamma-lyase (CSE) were observed in LNCaP cells. These findings were consistent in metformin-treated PC3 cells with the exception of CBS expression. Metformin increased gene expression of the glutathione-related enzymes, γ-glutamyl cysteine ligase (GCL) and glutathione synthetase (GSS) in both LNCaP and PC3 cells. DCFDA assay showed a dose differential effect of metformin on reactive oxygen species (ROS) production, where ROS levels increased in LNCaP but decreased in PC3 cells. Metformin also significantly downregulated survivin mRNA expression which was denoted by the reduced proliferative effects observed with MTT assay in LNCaP cells. Such associations were not observed in PC3 cells although metformin exerted antiproliferative action in PC3 cells.  Moreover, these findings were supported by doxorubicin’s effects (a ROS inducer) which indicated that metformin-induced ROS may contribute to its anti-proliferative effects by reducing the cells’ GSH levels.

Conclusion: This study demonstrates that metformin modulates redox homeostasis by inducing oxidative stress by lowering cellular antioxidant capacity and decreasing cell viability. Such redox changes were uniquely distinct among cell types where metformin increased a higher OS in LNCaP cells than PC3 cells which also suggests metformin might exert a greater anticancer effect in early-stage prostate cancer cells than late-stage prostate cancer cells.

References: Dixon S, Tran A, Schrier MS, Dong J, Deth RC, Castejon A, et al. Metformin-induced oxidative stress inhibits LNCaP prostate cancer cell survival. Mol Biol Rep. 2024;51:729

Acknowledgements: We acknowledge Nova Southeastern University for the implementation of the research and Ms. Sarai Harris for her assistance with synthesizing and reviewing this abstract.

Metformin decrease cellular antioxidant capacity

Metformin upregulates mRNA expression of redox-related enzymes

Metformin regulate ROS production and suppressed cell growth