Molecular mechanisms of metformin in prostate cancer - Mitochondrial toxicity and apoptosis

Objectives: Metformin is a medication that may be used to counter adverse effects of ADT such as hyperinsulinaemia. Metformin has also been shown to be a potential therapeutic agent for prostate cancer. In vitro, metformin inhibits mitochondria in prostate cancer cell lines leading to apoptosis. We explored the effects of metformin in cells treated under conditions that mimic ADT such as hyperinsulinaemia. Methods: LNCaP, DuCaP and MSK3 (CRPC model) cell lines were treated with insulin (10 nM) dihydrotestosterone (DHT) (10 nM), or enzalutamide (10 μM) with and without metformin (5 mM). Microarray was performed on a custom 180k Agilent oligo microarray. Growth was assessed using real-time cell microscopy and DNA quantitation, gene expression by QRT-PCR, apoptosis using FACS of Annexin V/PI and Autophagy using CYTO-ID (Enzo). Results: Microarray of LNCaP cells ±10 nM insulin, ±10 nM DHT, ±5 mM metformin followed by Ingenuity pathway analysis identified apoptosis and autophagy pathways modulated by metformin. QRT-PCR revealed Bcl2, Bcl-XL and ATG12, upregulated by androgens, were dose-dependently downregulated by metformin in all cell lines. Increased rates of apoptosis with metformin treatment, measured by FACS accompanied decreases in autophagy, using CYTO-ID stain. Androgen deprivation was associated with a 15% reduction in maximal mitochondrial respiration, which was restored with 10 nM DHT. Metformin dose-dependently reduced mitochondrial activity, which was accompanied by an overall decreased glycolytic capacity. Conclusion: Our results suggest metformin counteracts survival pathways in androgen-deprived PCa cells even in the presence of insulin. These results suggest a susceptibility of PCa cells to metformin, irrespective of androgen dependence.