Evaluation of mitochondrial bioenergetics and oxidative stress in human prostate cells treated with docosahexaenoic acid and melatonin

Abstract

Prostate is regulated by sexual steroids and influence of exogenous factors, such as diet, remain unknown. Several studies show that not only quantity, but lipid quality can influence intracellular homeostasis leading to carcinogenesis. Therefore, is known that prostate tumor cells change energetic metabolism from glycolysis to fatty acid oxidation. Docosahexaenoic acid (DHA) is an essential É-3 polyunsaturated fatty acid (PUFA) and its connection with prostate cancer is still unclear. Some authors have described a high DHA concentration inside prostate tumors, while others have investigated its therapeutic potential due to oxidative stress increase in mitochondria leading to cell death. However, DHA intake also occurs at normal conditions throughout diet e its effects on normal prostate cells can involve mechanisms that affect gland biology. Melatonin is a pineal hormone that have antimitogenic and antioxidant properties and apparently has an effect on cell fatty acid input. This indole can cross plasmatic membrane, directly counteract oxygen reactive species and positively influence mitochondrial physiology as well. The aim is to determine if and how DHA affects proliferation and survivor of normal prostate cells, as well as mitochondrial homeostasis, and role of melatonin. PTNA1 cells will be tested at normal condition (C), with melatonin (M), with DHA (DHA) and both (DHA+M). First, DHA concentration which affects intracellular redox state will be determined. Following this step physiological, supraphysiological and pharmacological melatonin concentrations will be tested for antioxidant property evaluation under oxidant condition provoked by DHA. Luzindole will be applied as antagonist for MT1 and MT2 membrane receptors. These experiments will be monitored by exclusion test with Trypan Blue, cell proliferation assay (MTT) and GSH/GSSG rate. Melatonin intracellular levels will be assessed by HPLC-UV. Mitochondrial bioenergetics will be evaluated according to oxygen consumption as well as respiratory complex activities and mitochondrial potential membrane after mitochondria isolation. Hydrogen peroxide levels will be determined with Amplex Red method and superoxide with MitoSOX assay, both performed with fluorescent spectrophotometer. Antioxidant profile will be assessed with biochemistry specific assays for Catalase, Glutathione peroxidase, Superoxide dismutase and Glutathione-S-transferase activities. Lipoperoxidation will be determined according to MDA levels. AKT, pAKT, mTOR and pmTOR will be assessed with Western Blotting for cell survivor pathways analysis. Western Blotting also will be applied for MT1 receptor at mitochondrial fraction. Ultrastructure will be performed in ultrathin sections after historresin inclusion for transmition electronic microscopy. This study will provide new insights about mechanisms linked to prostate cells survivor submitted to PUFAs and melatonin treatment. (AU)