EPIGENETIC MECHANISM FOR OXIDATIVE STRESS-INDUCED DOXORUBICIN RESISTANCE IN KIDNEY CANCER CELLS
Logeswari Ponnusamy, Prathap Kumar S Mahalingaiah, Kamaleshwar Singh.
Texas Tech University, Lubbock, TX.
Human renal cell carcinoma (RCC) is the common form of kidney cancer. Regardless of therapeutic advancement, treatment to some patients fail due to rapidly developing resistance. While reports suggest the role of reactive oxygen species (ROS) in chemoresistance, response of cancer cells with chronic exposure to oxidative-stress to chemotherapeutics is unknown. Hence, the objective of this study was to evaluate the impact of chronic exposure to elevated levels of oxidative stress in human kidney cancer cells. Caki-1 renal carcinoma cells were chronically exposed to 25 µM and 250 µM direct-ROS source hydrogen peroxide (H2O2). The effects of ROS on sensitivity to chemotherapeutic doxorubicin was measured by cytotoxicity assay, cell cycle-analysis, and anchorage-independent growth. Expression of genes/proteins involved in drug transport, survival, and DNA repair were measured by qRT-PCR and western blot. Results showed that doxorubicin cytotoxicity was significantly less in Caki-1 cells chronically exposed to H2O2 than control cells. Changes in gene/protein expression further confirmed increased resistance to doxorubicin. Down regulation of the MSH2 gene suggests that loss of DNA repair-dependent apoptosis could be a potential mechanism for increased resistance observed. To further evaluate the role of the epigenetic process of DNA-methylation in ROS-induced resistance, cells were pretreated with demethylating agent 5-aza-2’-deoxycytidine and then treated with doxorubicin. Pretreatment with 5-aza-2’-deoxycytidine significantly restored MSH2 gene expression and resensitized resistant Caki-1 cells to doxorubicin. This suggests that epigenetic silencing of the MSH2 gene and therefore DNA repair-dependent apoptosis could be a potential mechanism for ROS-induced resistance. This study provides, for the first time, direct-evidence for the role of oxidative-stress in chemoresistance in renal carcinoma cells