This translational research project is based on the premise that isothiocyanates (ITCs; R-N=C=S) may be used to inhibit onset and/or progression of human prostate cancer, which is a serious public health concern especially for elderly men. Based on our preliminary data, we hypothesize that ITCs will be highly effective in suppressing growth of human prostate cancer cells due to their ability to (a) induce apoptosis involving activation of caspases and extraeellular signal-regulated kinases 1/2 (ERKI/2), and (b) arrest cells in G2/M phase by reducing the activity of the cyclin-dependent kinase1 (Cdk1)/cyclinB kinase complex. An interesting corollary to the above hypothesis is that the antiproliferative activity of the ITCs is significantly affected not only by chemical modification in their terminal R-group and alkyl chain length but also because of their reactivity with glutathione (GSH). We propose to test the above mentioned hypotheses through studies in following specific aims:
Specific Aim 1 : Determine the effects of the ITC analogues with varying terminal R-group and or alkyl chain length on proliferation, apoptosis induction and cell cycle distribution in PC-3 and LNCaP human prostate cancer cells, and a normal prostate epithelial cell line (PrEC). These studies will not only provide insights into the crucial structure-activity relationship but also identify the most effective natural ITC analogue for mechanistic and in vivo efficacy studies for specific aims 2-5. Among the ITC analogs screened to date in our preliminary studies, phenethy-ITC (PEITC) appears most promising due to its strong growth suppressive activity against cultured human prostate cancer cells. Studies for specific aims 2 through 5 will focus on PEITC if this agent is superior to other ITC analogues in terms of greater cytotoxicity to prostate cancer cells and lack of an effect on proliferation of normal prostate epithelial cell line, Specific Aim 2: Determine the impact of GSH conjugation on antiproliferative activity of ITCs using PC-3 and LNCaP cells. This objective will be achieved by (a) determining the effect of overexpression of Pi class GSH transferase (hGSTP1-1), through stable transfection in prostate cancer cells, on antiproliferative activity of the ITC, and (b) determining the effect of ethacrynic acid, an inhibitor of GST activity, on antiproliferative activity of the ITC against prostate cancer ceils.
Specific Aim 3 : Define the mechanism(s) of lTC-induced apoptosis using cultured PC-3 and LNCaP cells. In this specific aim, studies are designed to determine the role of caspases and ERKs and their associated signaling pathways in ITC-induced apoptosis.
Specific Aim 4 : Define the mechanism(s) of lTC-induced G2/M arrest using PC-3 and LNCaP. Specifically, studies are designed to test the hypothesis that G2/M arrest in ITC treated prostate cancer cells is attributable to reduced activity of the Cdk1/cyclinB kinase complex due to increased ubiquitin-proteasome mediated degradation of dual-specificity phosphatases Cdc25B and Cdc25C, and possibly Cdk1.
Specific Aim 5 : Determine the effect of oral administration of ITC on growth of PC-3 and LNCaP human prostate cancer xenografts in vivo in nude mice. The effect of the ITC administration will be assessed in terms of xenograft growth and tumor regression. Studies are also planned to determine whether ITC treatment leads to a sustained suppression of tumor growth even after cessation of its administration. Tumors from the vehicle treated control and ITC treated mice will be examined to determine the extent to which ITC-induced molecular changes observed in cells (aims 3 and 4) correlate with its effect in vivo. Studies proposed in this application may lead to identification of an ITC analogue that can be used to delay onset and/or progression of human prostate cancer.
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