Isothiocyanates (ITCs) are phytochemicals highly expressed in cruciferous vegetables and these compounds are associated with the decreased incidence of cancers in populations consuming high levels of cruciferous vegetables. Several individual ITCs including phenethyl isothiocyanate (PEITC) inhibit tumor growth and angiogenesis and their anticancer activity has been linked to inhibition of cancer cell growth, survival and inflammation (NFB). It has also been demonstrated that PEITC induces reactive oxygen species (ROS) and that ROS is largely responsible for PEITC-induced cell death. To confirm PEITC-induced cancer cell death we have investigated the mechanism of action of PEITC in pancreatic cancer cell lines and PEITC induces ROS and inhibits growth and induces apoptosis (PARP cleavage). In addition, PEITC downregulates expression of several gene products including vascular endothelial growth factor (VEGF), cyclin D1 (CD1), Bcl2 and survivin and these have previously been reported in other studies. However, since these gene products are all regulated by specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, which are overexpressed in cancer cells and tumors, we investigated the effects of PEITC on Sp proteins and observed that PEITC decreased expression of Sp1, Sp3 and Sp4 in pancreatic cancer cells. These results demonstrate for the first time that an important underlying mechanism of action of ITCs likely involves targeting Sp transcription factors through an ROS-mediated mechanism and the pathways required for ITC-induced Sp downregulation were investigated and the results are presented in this paper.

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