Article Title: Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells
Figure Lengend Snippet: Lovastatin induced cell apoptosis, and sensitized cancer cells to TRAIL-induced apoptosis A. Representative cells to enable visualization and overlays of lipid rafts/cholesterol in non-malignant and cancer cell lines, using confocal microscopy. Non-malignant cells PZ-HPV-7 and MCF10A and prostate cancer cells C4-2, PC-3 and LNCaP were labeled with Alexa red Fluo555/565 – CTXB (lipid rafts, glycolipoprotein microdomains, GM) and green filipin (cholesterol, CH) and analyzed by confocal microscopy. B. The correlation of level of lipid rafts/cholesterol and the sensitivity of cells to lovastatin-induced apoptosis. Serum-starved cells were treated with lovastatin at 10 μM or DSMO control for 16 hours, with triplicate-wells for each cell line. Apoptotic cells were stained with Annexin V-FITC and PI, and detected by flow cytometry. Data were expressed as the ratio of lovastatin-treated cells to control. C. The effect of lovastatin on the level of cholesterol in the lipid rafts and apoptosis in prostate cancer cells. Serum-starved cells were treated with DSMO (control) or lovastatin at 10 μM for 16 hours, and then incubated with or without 500 μM cholesterol for 2 hours. Cells were stained with CTXB-Alexa 555/558 (GM) and filipin (CH), and monitored by confocal microscopy. The cells were processed for Annexin V-FITC and PI staining, and the percent apoptotic cells analyzed by flow cytometry. Each experiment was replicated 3 times. D. Prostate cancer cells are resistant to TRAIL-induced apoptosis. Prostate cancer cells LNCap, C4-2, CWR22rv, PC-3, DU145, non-malignant prostate epithelial cells PZ-HPV-7, keratinocytes, non small lung adenocarcinoma cells A549 and colon cancer Lovo cells were treated with TRAIL protein for 24 hours at a range of doses (n=5 for each cell line). Cell viability was measured by MTT assay at 72 hours after drug treatment. E. Lovastatin significantly enhanced TRAIL-induced apoptosis in prostate cancer cells, but not in normal cells. LNCaP, C4-2, PC-3, PZ-HPV-7 and keratinocytes were treated with lovastatin at 10 μM for 16 hours, before being treated with or without TRAIL(200 ng/mL) for 24 hours (n=4/group). The cells were processed for Annexin V-FITC and PI staining, and the percent apoptotic cells analyzed by flow cytometry.
Article Snippet: After rinsing in PBS, the cells were incubated with 1 ml of Filipin working solution (0.05 mg/ml in PBS, Sigma Aldrich, St. Louis, MO, USA) for 2 h at room temperature.
Techniques: Confocal Microscopy, Labeling, Staining, Flow Cytometry, Cytometry, Incubation, MTT Assay