Epigenetic regulation of kidney cancer drug sensitivity, resistance, and fibrosis



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This dissertation consists of studies on epigenetic regulation of kidney cancer drug sensitivity, resistance, and fibrosis. Epigenetics alterations are predominant causes of kidney cancer development but their roles in drug sensitivity, acquisition of drug resistance, and a pathological process i.e. fibrogenesis are not fully uncovered. The first chapter of this dissertation has been published in the molecular and cellular biochemistry journal. Some patients respond better than others to the same chemotherapeutic drug. This variation in response to chemotherapy is called differential sensitivity. Two kidney cancer cell lines having wild-type tumor suppressors (p53 and VHL in Caki-1) and mutant-type tumor suppressors (p53 and VHL in 786-0) were exposed to doxorubicin and their differential sensitivity was determined. Caki-1 cells were less sensitive to doxorubicin than 786-0 cells. We found increased expression of drug transporters and DNA repair genes in Caki-1 but not in 786-0 cells forming the molecular basis of differential sensitivity to doxorubicin. Additionally, we used epigenetic drugs 5-aza-2-dc & TSA as well and observed cytotoxicity of epigenetic therapeutics individually or in combination with doxorubicin against renal cell carcinoma with wild-type tumor suppressor genes. This finding has clinical significance as doxorubicin combined with epigenetic drugs seems beneficial to kidney cancer patients. The second chapter is about acquisition of doxorubicin resistance in kidney cancer and its prevention by co-treatment of epigenetic therapeutics. Kidney cancer is one of the most drug-resistant cancers and the mechanism of acquisition of drug resistance is not fully known. As we found two kidney cancer cells of different genetic and epigenetic backgrounds differentially sensitive to doxorubicin, we were interested to see if differential sensitivity has any influence on the acquisition of doxorubicin resistance and the role of epigenetics. Doxorubicin-resistant cells were established by exposing them to a clinically relevant dose of doxorubicin for more than 3 months. Co-treatment with epigenetic therapeutics was done to see if doxorubicin exposure-associated epigenetic changes could be prevented by retaining drug sensitivity unchanged. We found 786-0 cells acquiring a higher level of doxorubicin resistance with more apparent changes in morphology compared to Caki-1 cells. Additionally, cancer cells with different tumor suppressor genes status acquiring a different level of resistance to doxorubicin and abrogation of epigenetic modifications by epigenetic therapeutics are helping to prevent the acquisition of resistance in both kidney cancer cell lines. The finding co-treatment with epigenetic therapeutics helping to prevent the acquisition of doxorubicin resistance has a potential clinical significance. The third chapter is about understanding epigenetic mechanisms linked to expression of key genes of renal fibrosis and possible application of epigenetic therapeutics against renal fibrosis. Normal human renal proximal tubular epithelial cells (HK2) in-vitro and folic acid model in-vivo were used to study these processes. HK2 cells constantly exposed to a very high dose of folic acid for more than a month were found transformed to myofibroblast-like phenotype and upon exposure to epigenetic therapeutic (5-aza-2-dc) tend to go back to normal cell phenotypes. Representative gene expression associated with fibrosis shows increased expression of mesenchymal marker genes in long-term folic acid-treated HK2 cells which upon exposure to 5-aza-2-dc reduced the level of mesenchymal marker genes and enhanced the level of epithelial marker genes. It indicates 5-aza-2-dc abrogates folic induced fibrogenic changes in HK2 cells. In the animal model, serum creatinine assay showed a significant increase in creatinine in folic acid only injected group while in folic acid followed by 5-aza-2-dc injected group it was equivalent to the level of the control group. Also, histopathological slides showed patchy interstitial fibrosis in folic acid only injected group of animals while in folic acid followed by 5-aza-2-dc injected group interstitial fibrosis was absent with presence of regenerating tissue. The finding of this study indicates potential involvement of epigenetic modifications in renal fibrosis and the use of 5-aza-2-dc could restore the renal function attenuating fibrosis.

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Epigenetics, Kidney Cancer