Inhibiting Effect Of Artesunate On Ovarian Cancer Cell Growth And Impact On Activity Of Survivin And Caspase-3

ObjectiveArtemisinin is extracted from Artemisia annua in a class of sesquiterpene lactone compounds, we developed artemether, Artemisia ether, artesunate, dihydroartemisinin and other derivatives as a prototype. Artemisinin and its derivatives are a class of highly active anti-malarial drugs, primarily for the treatment of chloroquine-resistant falciparum malaria. In recent years, studies have found that artemisinin and its derivatives also have anti-tumor effect. Artesunate is an important derivative of artemisinin. Efferth found the anti-tumor effect of artesunate in 55 human tumor cell line from the National Cancer Institute, the most sensitive sort of cell were person leukemia and colon cancer cells, the moderately sensitive sort of cell were melanoma, breast cancer, ovarian cancerprostate cancer, central nervous system tumors and renal cancer, the worst sensitivity of sort was small cell lung cancer.In the present study, we use MTT assay, flow cytometry, transmission electron microscopy to observe the inhibiting effect of artesunate on ovarian cancer cell growth and the sensitizing effect of artesunate to cisplatin. Through the establishment of ovarian cancer xenografts in nude mice, we confirmed that the inhibiting effect of artesunate on ovarian cancer. We check the activity of Survivin and Caspase-3 in nude mice tumors by Western blot and discuss the mechanism of inhibiting effect of artesunate on ovarian cancer cell growth.Materials and Methods 1.Materaials(1) Drug preparationBefore use of artesunate with NaHCO3 solution then diluted with normal saline to the desired concentration,0-4℃refrigerator.(2) Cell culure.Human ovrian carcinoma cell line CAOV3 was grown as monolayer in DMEM medium with 10% fetal calf serum, 100U/ml penicillin,100μg/ml streptomycin at 37℃in a 5%CO2 atmosphere.(3) Mude miceBALB/C, nude mice,4-6-week-old, female, weight 18-22g, was purchased from the Experimental Animal Center Hayes Lake, raised in the Experimental Animal Center of China Medical University, SPF (specific pathogen free) grade laminar flow rack in the environment, through high pressure sterilization standards for animal feed and water free for consumption.2. Methods(1) Check the inhibiting effect of artesunate on human ovarian cancer cell growth and the synergistic action of artesunate with cisplatin by MTT assay;(2) Observe the morphology of artesunate on human ovarian cancer cell line CAOV3 by TEM (transmission electron microscope);(3) Check the cycle changes of human ovarian cancer cell line CAOV3 and apoptosis rate by flow cytometry;(4) Set up human ovarian cancer xenografts in nude mice to study the inhibiting effect of artesunate on ovarian cancer cell growth;(5) Check the activity of Survivin and Caspase-3 in nude mice tumors by Western blot;3.Statistics methodsExperimental data using SPSS 13.0 software, t-test and correlation analysis. P＜0.05 suggest significant difference.Results1. MTT experiments show that the inhibiting effect of artesunate on human ovarian cancer cell growth were in a dose-and time-dependent, with the increase in the concentration and time extension, inhibition rate increased gradually (P<0.01 or P<0.05). The inhibition rate of artesunate and cisplatin on ovarian cancer cell growth significantly increased (P<0.01);2. By TEM we can observe that membrane was integrate in normal ovarian cancer cell CAOV3, the nuclear membrane were more vague, the nuclear chromatin condensation within the different edge sets clear that the membrane has broken, showing the characteristic changes of apoptosis in the affected ovarian cancer cells by artesunate;3. Through flow cytometry, in the DNA histogram we can see that G1 hypodiploid cell populations appeared on the left of the peak-type that is apoptotic peak (sub G1 peak), and with the concentration of increase the apoptosis rate increased gradually, in the cell cycle(P＜0.01), G1 and S phase cells decreased, G2 and S phase cells increased significantly(P<0.01), showed that artesunate can inhibit ovarian cancer cell line CAOV3 cell proliferation, induction of apoptosis;4. Successful establishment of ovarian cancer xenografts in nude mice given three different doses of artesunate drug intervention can be drawn artesunate inhibit transplanted tumors, and with the increase in drug concentration inhibitory rate increased gradually, as compared with the control group was statistically significant (P <0.05 or P<0.01).5. Western blot assay in nude mice xenograft Survivin and Caspase-3 protein expression results showed that compared with the control group, the artesunate treatment group were significantly down-regulated expression of Survivin, and Caspase-3 expression increases.Conclusions 1. Artesunate can inhibit the growth of ovarian cancer cells;2.Artesunate has a synergistic effect with conventional chemotherapy cisplatin on inhibition of ovarian cancer cell growth;3.The inhibiting effect of cisplatin on ovarian cancer cell growth may be through inhibiting the expression of Survivin, activating Caspase-3 to promote apoptosis in ovarian cancer cell.