Our data determined that LCL85 is potentially an effective apoptosis sensitizer http://www.selleckchem.com/products/AZD2281(Olaparib).html that warrants further development as an adjunct agent to increase the efficacy of FasL CTL based cancer immunotherapy. Methods 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 described. MG 132 and Z VAD FMK were obtained from Enzo Life Sciences. Western blotting analysis Western blotting Inhibitors,Modulators,Libraries 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, Inhibitors,Modulators,Libraries 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 with BV6, LCL85, or C16 ceramide for 1 h, followed by incubation with FasL for Inhibitors,Modulators,Libraries 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. The percentage of apoptosis was calculated by the formula % apoptosis % PI and AnnexinV double positive cells with FasL % PI and Annexin V double positive cells without FasL. Measurement of endogenous ceramide level Cellular levels of endogenous ceramides were measured by Lipidomics Shared Resource, MUSC, using high performance Inhibitors,Modulators,Libraries liquid chromatography mass spectrometry approach as previously described. Ceramide levels were normalized to the total cellular protein Inhibitors,Modulators,Libraries contents.
Cell surface protein analysis Tumor cells were stained with anti Fas, anti FasL, or anti CD8 mAbs. Isotype matched control IgG was used as a negative control. The stained cells were ana lyzed by flow cytometry. For FasL protein analysis, mouse lungs were digested in collagenase solution to make a single cell suspension. The cell suspension was selleck products stained with PE conjugated FasL or FITC conjugated CD8 mAb, or both mAbs and analyzed by flow cytometry.