Functional characterization of adaphostin mediated induction of cell death in human prostate cancer (PC-3) cell line Free

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Adaphostin (NSC 680410) is a new, novel member of the tryphostin family of protein kinase inhibitors. Both in vitro and in vivo several human tumor model systems displayed significant antiproliferative activity by adaphostin. It has recently entered phase-I clinical trials because of its better therapeutic index. However, the mechanisms by which adaphostin exercise this favorable effect is still unclear. Particularly, its efficacy to be used as an anticancer drug is very promising in bcr/abl-positive several leukemia cell lines. However, few adherent cell types have also shown considerable sensitivity to this compound. Little is known about the action of the molecule on adherent cell lines. To dissect out the mechanism of action, and to identify it’s molecular target, the present study was designed to look into its effect on several cell cycle and apoptosis related parameters in p53 deficient human prostate (PC-3) cancer cell line. Shortly after exposure of adaphostin in PC-3 cellular system clearly indicate that cells are undergoing death process. We asked here whether the decrease in cell viability is due to apoptosis. Our results indicate that IC₅₀ for adaphostin for the 48 hrs is 2-2.5 μM in this cell type. Here we are reporting that adaphostin -induced cell cycle arrest and apoptosis in a dose (1, 2, 4 μM for 24 h) and time (6, 12, 24, 36 48 h with 2.5 μM) dependent manner, which were associated with an increase in p21 ^waf1/cip1 and p27 ^kip1 levels. The cell cycle block was at G1-S checkpoint, as evidenced by flow cytometric observations and further with the alterations in the corresponding cyclins and cdks. Apoptosis was observed as early as 20 to 24 h at 2.5 μM on adaphostin treatment in PC-3 cells, as evidenced by Annexin V, propidium iodide staining with the activation of several caspases. We further investigated that upregulation of p38 MAP-Kinase might be the cause for the p21 ^waf1/cip1 and p27 ^kip1 upregulation as very low concentration of SB203580 completely reversed this phenomenon. To understand better we further developed adaphostin resistant PC-3 cell line, which was able to block this adaphostin, induced cell cycle arrest and apoptosis. Based on the adaphostin resistance cell line development, it seems to us signaling pathway could be understand better for the p21 ^waf1/cip1 and p27 ^kip1 upregulation process along with the pathways for the mechanism of its sensitivity.