Doxorubicin Cytotoxicity and Mitochondrial Changes in Human Proximal Tubular Epithelial Cells (HK‐2) is Partially Prevented by Resveratrol

Abstract

The cancer chemotherapy agent Doxorubicin (DOX, Adriamycin), is FDA approved for use in the treatment regimen for acute lymphoid leukemia, breast, Hodgkin and Non-Hodgkin lymphomas, small cell lung cancer and advanced non-small cell lung cancer. Serious adverse effects include cardiomyopathy, congestive heart failure and renal dysfunction. The cellular mechanisms for doxorubicin mediated renal cytotoxicity have not been entirely identified. The trans-stilbene resveratrol (RES) is a phytoalexin found in Japanese knotweed, grape skins, blueberries, cranberries, dark chocolate and nuts. Antioxidant and anticancer properties have been associated with RES in cell culture and in human clinical studies. Our studies examined RES protection for DOX renal cytotoxicity 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 up to 10 uM RES followed by a 24 h co-incubation with 0-5 uM DOX. All results were obtained from 3 independent experiments. Western analysis probed for protein carbonylation was 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. Mitochondrial Stress Test was conducted following the treatments described above and each well was normalized by protein. Viability was assessed using MTT leakage and trypan 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 was probed using protein expression of LC3BI, LC3BII and the ratio of LC3BII/I following DOX exposure. In summary, RES attenuated DOX renal cytotoxicity by maintaining mitochondrial OCR near normal.