Browsing by Subject "Renal cell carcinoma"
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Item Induction of apoptosis and cell cycle arrest in renal carcinoma cells by phenethyl isothiocyanate and the mechanisms involved(2011-05) Khan, Maruf; DeGraffenried, Linda; Ciolino, Henry P.; Sanders, Bob G.; Nunez, Nomeli P.; Fischer, Susan M.Renal Cell Carcinoma (RCC) has low 5 year survival rate and is resistant to radiation and chemotherapy. Phenethyl Isothiocyanate (PEITC) is a naturally occurring phytochemical that has a variety of anti-cancer properties. Here we explore two anti-cancer properties of PEITC: induction of apoptosis and induction of cell cycle arrest in RCC cells and the underlying mechanisms. We used two human RCC cell lines Caki-1 and Caki-2. Survival and cell proliferation was assayed using Calcein AM. Annexin V staining was used to measure apoptosis. Caspase-3/7 induction was measured using a fluorescent substrate. Cell cycle was studied using Propidium Iodide staining. DNA damage was determined using phospho [gamma]-H2AX antibody. Protein expression and phosphorylation was determined using immunoblotting. PEITC significantly reduced survival of Caki-1 and Caki-2 cells and inhibited their proliferation as determined by Calcein AM. 15 and 20 [mu]M PEITC induced apoptosis in both cell lines and induced caspase-3/7 activity. Western blot analysis revealed caspase-8, caspase-9 and Bid cleavage as well as upregulation of the death receptors Fas and DR5. Lower doses (up to 10 [mu]M) arrested Caki-1 cells in G2/M phase, and this was associated with increased p38 and MK2 (Thr334) phosphorylation. The p38 inhibitor SB203850 inhibited this G2 arrest induced by PEITC. 15 and 20 [mu]M PEITC treatment resulted in increased [gamma]-H2AX phosphorylation suggesting DNA damage, but this was completely blocked by caspase inhibitor. In summary, our study shows that PEITC induces apoptosis in Caki-1 and Caki-2 cells by upregulating Fas and DR5 and activating the downstream apoptosis cascade. PEITC does not cause direct DNA damage to the cells; the observed DNA damage is a result of the apoptotic process and is blocked by caspase inhibitor. PEITC induces G2/M arrest in Caki-1 cells and the mechanism involves p38 phosphorylation which activates MK2. Inducing cell cycle arrest and apoptosis may play an important role in the anti-cancer properties of PEITC. Fully understanding the mechanism by which PEITC induces apoptosis and cell cycle arrest in RCC cells may lead to development of novel chemotherapeutic drugs against RCC.Item Regulation and function of tuberous sclerosis complex-2 tumor suppressor in renal cell carcinoma(2004-05) Liu, Yu, 1975-; Walker, Cheryl, 1955-; Richburg, John H.Tuberous sclerosis complex - 2 (TSC2) is an important tumor suppressor gene for multiple tumors associated with human TSC disease and rodent renal cell carcinoma (RCC) development. Germline mutation of Tsc2 in the Eker rats predisposes to spontaneous RCC, while von Hippel-Lindau (VHL) tumor suppressor gene is the major target for human RCC development. Using Eker rat animal model, I investigated the etiology of RCC development and the mechanism of TSC2 regulation by Akt phophorylation. In the first study, I characterized that VHL gene product pVhl has a unique distribution pattern in the rat nephron compared with human and mouse nephrons. I found that rat pVhl has two isoforms arising from different translation start sites. In contrast to human pVHL nuclear/cytoplasmic subcellular localization, both rat pVhl isoforms were expressed mainly in the membrane and cytosolic fraction. The difference of pVhl distribution/localization between human and rat may explain the species-specific function of Vhl in RCC development. In the second study, I provide evidence suggesting that human and rat RCC converge at the regulation of Hypoxiainducible Factor α (HIFα). Eker rat RCC-derived cells exhibited high basal levels of HIF activity, similar to human RCC cell lines with VHL alterations. HIF2α was stabilized in Tsc2 null rat cells as well as in Eker rat RCC. High activity of HIF2α resulted in HIFα mediated upregulation of Vascular Endothelial Growth Factor (VEGF) expression and further angiogenic phenotype of rat RCC. These data identified dysregulation of HIF2α as a convergent pathway for both human and rat RCC development. Finally, I present data demonstrating that TSC2 gene product tuberin is a phosphorylated protein which contains multiple Akt phosphorylation sites overlapping with 14-3-3 binding sites. Phosphorylation of tuberin correlated with Akt phosphorylation. In addition, tuberin associated with 14-3-3 via Akt mediated phosphorylation. Interaction between tuberin and 14-3-3 could be abrogated by a tuberin polypeptide with the Akt site phosphorylated. These data suggest that Akt phosphorylation mediates tuberin/14-3-3 association and inhibits tuberin function. Taken together, these studies place TSC2/tuberin in the Akt mediated signaling pathway and elucidate the tumorigenesis mechanism for RCC development associated with loss of TSC2 function.