The same panel of 5 metastatic human colon carcinoma cell lines were cultured in the presence of various doses of BV6 and measured for growth inhibition. Like LCL85, BV6 exhibited direct cytotoxicity in a dose dependent manner. Next, we used a sublethal dose of BV6 to determine whether BV6 sensitizes metastatic human technical support colon carcinoma cells to FasL induced apoptosis. Incu bation of tumor cells with BV6 and FasL revealed that BV6 significantly increases sensitivity of all 5 metastatic human colon carcinoma cells to FasL induced cell growth inhibition, and the growth inhibition pattern is strikingly similar to that induced by LCL85 and FasL, suggesting that LCL85 might sensitize meta static colon carcinoma cells to Fas mediated apoptosis by a mechanism similar to BV6.

BV6 targets IAP proteins to induce apoptosis We Inhibitors,Modulators,Libraries then analyzed the effects of LCL85 on IAP proteins in metastatic human colon carcinoma cells. SW620 cells were treated with LCL85 and analyzed for IAP protein levels at various time points. Among the 3 IAP proteins, Inhibitors,Modulators,Libraries xIAP protein levels dramatically decreased 12 h after LCL85 treatment. cIAP1 protein was also decreased, albeit at a smaller degree. cIAP2 protein level Inhibitors,Modulators,Libraries was not significantly changed by LCL85 treatment. To determine whether LCL85 also decreases xIAP protein levels in metastatic human breast cancer cells, MDA MB 231 cells were treated with LCL85, and ana lyzed for xIAP and cIAP protein levels. It is clear that LCL85 decreases xIAP and cIAP1 protein levels in a dose dependent manner. Next, SW620 cells were cultured in the presence of a sublethal dose of BV6 and FasL, and analyzed for apoptosis.

It is Inhibitors,Modulators,Libraries clear that BV6 dramatically increased SW620 cell sensitivity to FasL induced apoptosis. Our results thus revealed that LCL85 targets xIAP and cIAP1 to sensitize metastatic human colon carcinoma cells to Fas mediated apoptosis. RT PCR analysis indicated that LCL85 does not alter the mRNA levels of IAP proteins in human colon car cinoma cells. Proteasome inhibitor MG 132 blocked LCL85 induced xIAP degradation, whereas caspase inhibitor Z VAD did not block LCL85 induced xIAP degradation. Our data thus suggest that LCL85 mediates proteasome dependent degradation of xIAP protein. To determine the IAP protein levels in various human colon cancer cell lines, we analyzed xIAP and cIAP1 protein levels in 5 other human colon carcinoma cell lines.

Western Inhibitors,Modulators,Libraries blotting analysis indicated that xIAP and cIAP1 are expressed in all 5 cell lines at a level similar to that in LS411N and SW620. To validate the functions of xIAP and cIAP1 in Fas mediated 17-DMAG fda apoptosis in human colon carcinoma cells, SW620 cells were transfected with xIAP and cIAP1 specific siRNAs, respectively, and analyzed the tumor cell sensitivity to FasL induced apoptosis. Silencing xIAP or cIAP1 significantly increased the tumor cell to FasL induced apoptosis.