| ForewordEmbelin is a novel cell permeable inhibitor of X-linked inhibitor of apoptosis (XIAP) which was discovered by screening a library of natural products derived from traditional Chinese medicine. They have multiple biological, pharmacological and medicinal properties including anti-inflammatory, antibacterial, antioxidant and antitumor.ObjectiveThe present study was undertaken to determine the molecular mechanisms by which embelin induces apoptosis in human leukemia cells and explore possible mechanisms.Methods1. Cell culture and group assignmentHuman leukemia cell line HL60 cells was grown and maintained in RPMI-1640 liqiud containing 10%fetal serum,100u/L penicillin and 100μg/ml streptomycin at an 37℃and 5%CO2 in a cell incubator.Cells were passaged every two or three days.Groups include embelin of 3,10,30,100 and 300μg/ml. Cell control and sovent control were designed.2.MTT assay to detect cell viabilityHL60 cells were seeded in 96 holes of the plate with 8 paralele holes for 1×105/ml. Dimethyl sulfoxide was added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution was be quantified by measuring at a certain wavelength (570nm) by a spectrophotometer.Cell proliferation rate%=(A of test group/A of control group)%,and corresponding cell proliferation inhibition rate%= [(A of control group-A of test group)/A of control group]%. 3. Annexin V-FITC and PI staining and FACS to detect cell apoptosis rateAnnexin V-FITC and PI were added to cells with final concentration of 50μg/ml and 100μg/ml respectively.Irritation wavelength of FACS is 488nm.In scatter plot, LL quadrant is normal cells (FITC-/PI-);RU quadrant is dead cells (FITC+/PI+); RL quadrant is apoptotic cells (FITC+/PI-)4.JC-1 staining and observation under microscopyCells were washed by PBS twice and was collected about 1×106 and 500μl JC-1 working fluid suppended cells.A drop of cell suspension was fixed in flims.Under microcopy,normal cells is yellow-greenish and apoptotic cells is greenish.5.Western Blot for analysis of XIAP,caspase-3 and caspase-9Samples were taken from cell culture after different treatment. Cells were broken down by cell lysis buffer and analyzed by Bradford protein analysis system. The proteins of the sample are separated using SDS-PAGE (SDS polyacrylamide gel electrophoresis) and transferred to a membrane overnight at 4℃. Blocking of non-specific binding was achieved by placing the membrane in a dilute solution of non-fat dry milk.XIAP (1:400),caspase-3 (1:500) and caspase-9 antibody (1: 500) were added and kept overnight at 4℃and then second antibody was added.Two hours later membrane was washed by phosphate buffer and developer was added before developed.6.Statistical analysisSPSS 15.0 software was used for analysis,F test was used for variations comparison and X2 test was used for ratio comparison.Results1. Embelin show inbibion on HL60 cells in a time-and dose-dependent manner and inhibition is obvious with concentration over 30μg/L (P<0.05). Embelin shows cytotoxity to HL60.2. After treatment of HL60 cells by embelin, specific apoptosis rate is (9.23±0.05)%,(25.86±0.30)%and (39.03±0.07)%respectively at 100μg/ml of embelin with 12-,24-and 48-hours'action correspondingly (P<0.05) 3. Wesren Blot shows embelin inhibits expression of XIAP.4. Wesren Blot shows embelin increses expression of caspase-3 and caspase-9.
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