Eeffect Of Betulinic Acid On Apoptosis Of Human Ovarian Cancer CAOV3 Cells

Objective:This article through studied the inhibit proliferation effect of betulinic acid[3β-Hydroxy-lup-20 (29)-en-28-oic acid] in ovarian cancer cellsand explain it’s mechanism of action, offerda theoretical foundation of the development and application ofclinicala nti-cancer drugs in future.Method:1.Through 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytet razoliumromide(MTT) experiment to detection the betulinic acid’s inhi biti-ve ability in CAOV3 cells of ovarian cancer,and measure the valu e of IC50,what is the half inhibitory concentration;2.Use fluorescent staining to observe The morphological character-istics of apoptotic cells;3. By immunofluorescence experiments to observe Smac protein in ovarian cancer cell plasma that has treated by betulinic acid; 4. Use western blot method to detection the fracture of PARP what is the specific substrate of Caspase-3;5. Analysis and test the activity of Caspase 3、Caspase-8、 Caspase-9 in vitro what is the key enzyme of apoptosis.Results:1.Betulinic acid has the effect of inhibit proliferation in CAOV3 cells of ovarian cancer, and it depends on the density, the inhibition concentration of value of IC50 is 27.7μg/ml;2. Observed the ovarian cancer cells CAOV3 that induced bybetulinic acid under the fluorescence microscope, we foundthe typical morphological characteristics of apoptosis, like the cell became round, cell membrane foamed,nuclei condensed and fragmented and so on.We addbetulinic acid in ovarian cancer cells with the concentrationin of 0、10、20、30μg/mL, afterobserved 24 hours, by DAPI fluorescent dye,when the drug concentration is 20μg/mL, we found the characteristic of apoptotic cell.3. By immunofluorescence experiments,we found when the ovarian cancer cells in betulinic acid with concentration of 10μg/mL, eight hours’s later, Smac protein that originally located in the mitochondria diffuseed to the the cytoplasm. The Smac protein is the keyprotein in cell apoptosis. When it released from mitochondria to the cytoplasm, it can react and incorporationwith IAPs. The lattercan no longer suppress the activity of caspase and restrain Caspase 3’s activity. It could play it’s originally effect of promoting apoptosis and cause the occurrence of apoptosis;4. Test with the western blot method, thebetulinic acid with the concentration of 0、10、20and30μg/mL respectively acted on ovariancancer cells CAOV3, PARP what is the specificity of substrate of Caspase 3 cracked more obvious withthe increased of theconcentration, and itis the most prominent when the concentration is 30μg/mL;5. By the Caspase activity detection method, we detected that whenovarian cancer cells affected by betulinic acid. Caspase-3 and Caspase-9 were activated, when the concentration is 10μg/ml, the activity of Caspase 9 and Caspase 3 were increased, anditdepended onthe concentration.Conclusion:1.Betulinic acidhas cell proliferatoninhibitionon to human ovarian cancer CAOV3 cells;2. It is through the pathway of induced thecell apoptosisof of Caspase3 and Caspase 9;...