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Resveratrol Attenuates Doxorubicin Cytotoxicity and Mitochondrial Dysfunction and Human Proximal Tubular Epithelial Cells

Doxorubicin (DOX, Adriamycin), is a cancer chemotherapy employed in the treatment regimen for breast, Hodgkin and Non‐Hodgkin lymphomas, small cell lung cancer and acute lymphoid leukemia. The U.S. FDA issued a Black Box warning for Cardiomyopathy in patients treated with doxorubicin. Other adverse effects include renal impairment. The cellular mechanisms for doxorubicin mediated renal cytotoxicity are not known. Resveratrol (RES) is a phytoalexin found in many fruits including grapes, blueberries, cranberries, dark chocolate and nuts. Antioxidant and anticancer properties have been associated with RES in cell culture and in human clinical studies in cancer patients and in vivo rodent models. Our studies tested the hypothesis that RES protection for DOX renal cytotoxicity, was mediated by maintaining mitochondrial function. All studies were conducted in human noncancerous renal proximal tubular epithelial cells (HK‐2). HK‐2 cells were plated and equilibrated for 48 h. Cells were next pre‐incubated for 1 h with 0 (DMSO) or 5 or 7.5 uM RES followed by a 24 h co‐incubation with 0–5 uM DOX. All results were obtained from 3 independent experiments. Western analysis for protein carbonylation and 4‐hydroxynooneal (4‐HNE) were used as an indicator of oxidative stress. Mitochondrial function was assessed using a Seahorse analyzer system. Prior to examining mitochondrial function, cell number and substrate concentrations were optimized based on the manufacturer’s recommendations. Mitochondrial Stress Test was conducted following the treatments described above and each well was normalized by protein. Viability was assessed using MTT leakage and tyrpan blue exclusion cell counts. RES did not alter cell viability as indicated by comparable MTT values between DMSO and RES groups (p>0.05). DOX was cytotoxic to HK‐2 cells within 24 h. Pretreatment with RES provided protection from DOX to HK‐2 cells. Basal mitochondrial respiration assessed as oxygen consumption rate (OCR) was diminished by 4 uM DOX. Mitophagy mediated by DOX and the effect of RES compared protein expression of LC3BI, LC3BII and the ratio of LC3BII/I. In summary, RES attenuated DOX renal cytotoxicity by maintaining mitochondrial function. Further studies are needed to probe changes in specific mitochondrial proteins mediated by DOX.