Figure 7

Ceramide targets cIAP1 and xIAP to sensitize metastatic human colon and breast cancer cells to Fas-mediated apoptosis. A. Five metastatic human colon carcinoma cell lines were cultured in the absence or presence of BV6 (5 μM) and FasL at the indicated concentrations for 24 h. Cell growth rates were measured by MTT assays. *p < 0.05; **p < 0.01. B. SW620 cells were treated with LCL85 (5 μM) for approximately 24 h and analyzed for IAP protein levels at the indicated time points. C. MDA-MD-231 cells treated with LCL85 at the indicated doses for approximately 24 h and analyzed for xIAP and cIAP1 protein levels by Western blotting. D. SW620 cells were incubated with BV6 (5 μM) for 1 h and then cultured in the absence or presence of FasL overnight. Cells were stained with Annexin V and PI and analyzed by flow cytometry. The apoptotic cell death was indicated at the top right corner of each plot. Right panel: Quantification of apoptosis. % FasL-induced cell death is calculated by the formula: % Annexin V⁺ and PI⁺ cells in the presence of FasL - % Annexin V⁺ and PI⁺ cells in the absence of FasL. E. SW620 cells were treated with LCL85 (5 μM) and BV6 (5 μM), respectively, for the indicated time and analyzed for expression levels of cIAP1, cIAP2 and xIAP by RT-PCR. F. SW620 cells were pretreated with or without MG-132 (20 μM) or Z-VAD (25 μM) for 1 h followed by treatment with LCL85 for 12 h. Cells were lysed and analyzed for xIAP protein level by Western blotting.