Staining cells with Annexin V and PI revealed that LCL85 induces apoptosis of SW620 selleckchem Vandetanib and Inhibitors,Modulators,Libraries LS411N cells in a dose dependent manner. However, LCL85 alone at low doses only induced a small degree of apoptosis. In contrast, a sublethal dose of LCL85 dramatically increased SW620 and LS411N cell sensitivity to FasL induced apoptosis. To determine whether LCL85 sensitized apoptosis is tumor type dependent, we also tested the effects of LCL85 on metastatic human breast cancer cells. MDA MB 231 cells were treated with various doses of LCL85 in the absence or presence of FasL and analyzed for apoptosis. As in the human colon carcinoma cells, LCL85 induced MDA MB 231 apoptosis in a dose dependent manner, Inhibitors,Modulators,Libraries albeit at a low degree.

MDA MB 231 cells are resistant to FasL induced apoptosis, Inhibitors,Modulators,Libraries and LCL85 is effective in sensitizing MDA MB 231 cells to FasL induced apoptosis at a dose of 25 uM. These observa tions thus suggest that a sublethal dose of ceramide analog LCL85 is a potent apoptosis sensitizer. LCL85 increases cellular C16 ceramide level to sensitize colon carcinoma cells to apoptosis We next treated SW620 cells with a sublethal dose of LCL85 and measured the level of cellular ceramides and ceramide metabolites. Treatment of LCL85 increased C16 ceramide level in the tumor cells, suggesting that LCL85 might increase C16 ceramide level to sensitize human colon carcinoma cells to Fas mediated apoptosis. To test this hypothesis, SW620 cells were cultured in the presence of exogenous C16 ceramide and FasL.

Although exogenous C16 ceramide directly induced apoptosis in a dose dependent manner, albeit at a low level, exogenous C16 ceramide Inhibitors,Modulators,Libraries significantly increased SW620 cell sensi tivity to FasL induced apoptosis. There fore, LCL85 sensitizes human colon carcinoma cells to Fas mediated apoptosis at least partially through increa sing C16 ceramide level in the tumor cells. xIAP and cIAP1 are molecular targets of LCL85 We next sought to identify the targets of ceramide. To determine whether LCL85 alters Fas expression, we treated SW620 cells with LCL85 and analyzed cell surface Fas protein levels. Flow cytometry analysis indicated that LCL85 does not increase cell surface Fas protein level. As a positive Inhibitors,Modulators,Libraries control, Vorinostat significantly increased cell surface Fas protein level in SW620 cells. As a complimentary approach, SW620 cells were treated with C16 ceramide and analyzed for cell surface Fas expression level. C16 ceramide treatment did not alter cell surface Fas protein level. The above observations that LCL85 does not alter Fas level suggests that LCL85 may target mediators of the Fas mediated apoptosis signaling pathways. IAPs are po tent inhibitors of apoptosis, including Fas mediated they apop tosis.