Abstract 4571: Chronic oxidative stress increases resistance in renal carcinoma cells to doxorubicin-induced cytotoxicity potentially through epigenetic mechanism

Abstract

Abstract Human renal cell carcinoma (RCC) is the most common form of kidney cancer. Regardless of therapeutic advancement, patients with metastatic RCC are failed to clinical treatment due to rapidly developing resistance. Resistance to chemotherapy can be of innate or acquired overtime following exposure to drug and can arise through a number of different intrinsic and extrinsic factors and their interactions. While some reports suggest role of ROS in developing drug resistance, response of cancer cells with chronic exposure to oxidative stress to chemotherapeutic drugs is not known. 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 human renal carcinoma cells were given chronic (6 months) exposure to direct ROS source hydrogen peroxide (25 μM and 200 μM). Using these cells the effect of oxidative stress on sensitivity to doxorubicin-induced cytotoxicity was measured by MTT assay. The result was further confirmed by anchorage independent growth on soft agar and cell cycle analysis using flow cytometer. To understand the molecular mechanism, the expression of genes involved in drug resistance, cell survival, and DNA repair dependent apoptosis were measured by qRT- PCR. Results of MTT, soft agar assay, and cell cycle analysis showed that doxorubicin-induced cytotoxicity was significantly less in Caki-1 cells chronically exposed to H2O2 when compare to H2O2 unexposed control cells. The alterations in the expression of genes involved in drug transport further confirmed the increased resistance to doxorubicin-induced cytotoxicity in cells exposed to oxidative stress. Decreased expression of MSH2 gene in cells exposed to H2O2-induced oxidative stress suggests that loss of DNA repair-dependent apoptosis could be a potential mechanism for increased resistance to doxorubicin-induced cytotoxicity. To further evaluate the role of epigenetic process of DNA methylation in oxidative stress-induced drug resistance, cells were pretreated with demethylating agent 5-aza-2′ deoxycytidine (5-aza 2dC), and then treated with doxorubicin. The pretreatment with 5-aza 2dC significantly resensitized the chronic ROS adapted Caki-1 cells to doxorubicin-induced cytotoxicity and restored the expression of MSH2 gene in these cells. This suggests that epigenetic silencing of MSH2 and therefore DNA repair-dependent apoptosis could be a potential mechanism for oxidative stress-induced resistance to chemotherapeutic drugs in renal cancer cells. In summary, this study for the first time provides direct evidence for the role of oxidative stress in chemoresistance in renal carcinoma cells. Additionally, the findings of this study also suggest that epigenetic therapy may be a useful strategy in re-sensitizing the chemotherapeutic resistant RCC cells. Citation Format: Logeswari Ponnusamy, PrathapKumar S. Mahalingaiah, Kamaleshwar P. Singh. Chronic oxidative stress increases resistance in renal carcinoma cells to doxorubicin-induced cytotoxicity potentially through epigenetic mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4571. doi:10.1158/1538-7445.AM2015-4571