Ceramide has also been shown to regulate apoptosis selleckbio through modulating key Inhibitors,Modulators,Libraries molecules of the Fas mediated apoptosis pathways. Inhibitors,Modulators,Libraries Elevation of acid ceramidase, the enzyme that converts ceramide to sphingosine and subsequently sphingosine 1 phosphate, has been frequently observed in apoptosis resistant cancer cells, including metastatic colon carcinoma cells. These observations thus suggest that targeting ceramide metabolism to increase ceramide accumulation might be an effective approach to overcome cancer cell resistance to Fas mediated apoptosis. In this study, we demonstrated that aromatic ceramide analog LCL85 ef fectively overcomes metastatic human colon and breast cancer cell resistance to Fas mediated apoptosis at least partially through inducing proteasomal degradation of cIAP1 and xIAP in vitro.
More significantly, we demon strated that LCL85 effectively suppresses colon and breast cancer metastasis in vivo. Our data determined that LCL85 is potentially an effective apoptosis sensitizer that warrants further development Inhibitors,Modulators,Libraries as an adjunct agent to increase the efficacy of FasL CTL based cancer immunotherapy. Methods Inhibitors,Modulators,Libraries Mice BALB c mice were obtained from National Cancer Institute. All studies are approved by the Georgia Regents University Institutional Animal Care and Use Committee. Cell lines All human cell lines established from primary and meta static colon and breast cancer tissues, and mouse breast cancer cell line 4 T1 were obtained from American Type Culture Collection. ATCC characterizes these cells by morphology, immunology, DNA fingerprint, and cyto genetics.
Murine Colon26 cells were kindly provided by Dr. William E. Carson, III. Reagents BV6 was kindly provided by Genentech. Ceramide analogs B13 and LCL85 were synthesized by Lipidomics Shared Resource at Medical University of South Carolina. FasL was provided by Drs. Steven Butcher and Lars Damstrup. C16 ceramide was obtained from Santa Cruz Biotech, and was dissolved in dodecane,ethanol as Inhibitors,Modulators,Libraries described. MG 132 and Z VAD FMK were obtained from Enzo Life Sciences. Western blotting analysis Western blotting analysis was performed as previously described. Anti cIAP1 was obtained from R D Sys tem. Anti Bax and cIAP2 antibodies were obtained from Santa Cruz Biotech. Anti Bak and xIAP antibodies were obtained from Cell Signaling Biotech, anti Bcl 2, and Bcl xL antibodies were obtained from BD Biosciences, and anti B actin was obtained from Sigma.
Cell viability assays Cell viability assay was carried out as previously described using the MTT cell proliferation assay kit. Apoptosis analysis Cells were treated ABT888 with BV6, LCL85, or C16 ceramide for 1 h, followed by incubation with FasL for approximately 24 h. Apoptosis analysis was as previously described. Briefly, cells were then collected and incubated with propidium iodide and Annexin V, and analyzed by flow cytometry.