Next, SW620 cells were cultured in the presence of a sublethal dose of BV6 and selleck chem inhibitor FasL, and analyzed for apoptosis. It is clear that BV6 dramatically increased SW620 cell sensitivity to FasL induced apoptosis. Our results thus revealed that LCL85 targets xIAP Inhibitors,Modulators,Libraries 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 blotting analysis indicated that xIAP and Inhibitors,Modulators,Libraries 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 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. Our data thus suggest that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells, and ceramide Inhibitors,Modulators,Libraries sensitizes the tumor cell to Fas mediated apop tosis at least partially through inducing cIAP1 and xIAP degradation.

LCL85 also targets Bcl xL Ceramide has been shown to regulate Bcl x alternative splicing to decrease Bcl xL level, and to mediate Bak and Bax function in the intrinsic Inhibitors,Modulators,Libraries apoptosis pathway. In addition, Bcl 2 has been shown to activate Bak to induce C16 ceramide accumulation. We then analyzed these Bcl 2 family proteins. Western blot ting analysis revealed that only Bcl xL protein level is dramatically decreased by LCL85 in metastatic human colon cancer cells, and in the metastatic breast cancer cells, albeit to a less degree. Ceramide analog and Smac mimetic additively sensitize metastatic human colon carcinoma cells to apoptosis induction Our observations that LCL85 and BV6 both target IAP proteins suggest that they may act additively in sen sitization of tumor cell to apoptosis induction.

To test this hypothesis, SW620 and LS411N cells were treated with these Inhibitors,Modulators,Libraries two agents alone or in combination, and analyzed for the tumor cell sensitivity to FasL induced apoptosis. Although sublethal doses of LCL85 and BV6 are both effective in sensitization of tumor cells to FasL induced apoptosis, clearly, combined LCL85 and BV6 exhibited significantly greater selleck catalog effects than each agent alone on sensitization of these two tumor cells to FasL induced apoptosis.