Abstract 4222: Induction of apoptosis and cell cycle arrest in renal cancer cells by phenethyl isothiocyanate (PEITC) and the mechanisms involved

Abstract

Abstract Background: 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 cell lines and the underlying mechanisms. Methods: 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 H2AX-γ antibody. Protein expression and phosphorylation was determined using immunoblotting. Results: PEITC significantly reduced survival of Caki-1 and Caki-2 cells and inhibited their proliferation as determined by Calcein AM. 15 and 20 μM PEITC induced apoptosis (revealed by Annexin V staining) in both cell lines and induced caspase-3/7 activity. Western blot analysis revealed caspase-8, caspase-9 and Bid cleavage suggesting that the apoptosis is mediated through the extrinsic pathway. Lower doses (up to 10 μM) arrested Caki-1 cells in G2/M phase, and this was associated with an increase p38 and MK2 (Thr334) phosphorylation (as revealed by Western Blot). The p38 inhibitor SB203850 inhibited this G2 arrest induced by PEITC. In order to determine if DNA damage was responsible for the apoptosis and G2 arrest, we used an antibody to H2AX-γ phosphorylation. 15 and 20 µM treatment resulted in increased H2AX-γ phosphorylation, but this was completely blocked by caspase-3 inhibitor. Conclusion: PEITC induces apoptosis in Caki-1 and Caki-2 cells by a mechanism that likely involves the extrinsic pathway. We will use immunoblotting and siRNA to determine which of the extrinsic receptors (DR4, DR5, TRAIL or FAS) is responsible for this apoptosis induction. 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-3 inhibitors. PEITC induces G2/M arrest in Caki-1 cells and the mechanism is dependent on 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. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4222. doi:10.1158/1538-7445.AM2011-4222