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. / text