| Object: Endometrial carcinoma is about 7% of all female carcinoma and 20%-30% of female reproductive system tumor. Recently, incidence of endometrial carcinoma has stepped-up year by year in the world. More and more young women have been endometrial carcinoma patients. Surgical treatment of is the first choice for the endometrial carcinoma patients. Research verified that: for the cases of early recurrence, chemotherapy has preventive useness, and for late and recurrence cases, chemotherapy also has objective effective power. Chemotherapy resistance is the top issue for current tumor study. Etoposide(VP-16) association with cisplatin(CDDP) can decrease assistance and kill tumor cell. But the mechanism is not clear. In this study, we choose various concentration of VP-16 association with CDDP to interfere the growth of the ISHIKAWA cell in vitro. And compare the difference of cell growth and cell apoptosis after various medicine concentration treatment, for finding the chemotherapy resistance' mechanism deeply.Livin protein is a new member of IAPs (inhibitor of apoptosis proteins) protein family, and is a top apoptosis protein related to chemotherapy resistance. Livin can inhibit the activity of caspases. Livin produce marked effect in tumor cell' tolerance of medicine and inducing apoptosis. High expressed Livin can upgrade the tolerance of cancer cell facing chemotherapy medicine, therefore, decreasing the expression of Livin is a new anti-tumor road .In vivo and vitro studies certified, using smac antisense nuclotide, RNAi and so on ,varied techniques to degrade the expression of Livin can inhibit growth of tumor cell.Livin is high expressed in many malignant tumor tissue, playing comprehensive anti-apoptosis effect by complex signaling paths, and participating in many biological processes of tumor, for example, taking place, progression, relapse, medicine resisting, and so on. Over expression of Livin induces tolerance of tumor cell facing stimulate with promoting apoptosis effect. While medicine tolerance is our urgent problem we must explain as soon as possible.Caspase-3 is one of key executing enzyme in cell apoptosis. In executing phase of cell apoptosis , caspase-3 entirely or partly takes charge in hydrolyzing large number of bottom protein. Deversified apoptotic signal activate complex signal transmitting path, ultimately converging a common access, caspases protease cascade magnified reactivity. Activation of caspase-3 is conjunct path by which apoptosis finally processed. In this study, by western blotting, half-quantitative analysis the expression difference and correlativity of Livin and caspase-3 after different medicine treatment, probing into the mechanism of medicine resistance in angle of cell apoptosis.Methods:1 Cell culture ISHIKAWA cell line is bought from the Fourth hospital graduate school of He Bei Medicine University. ISHIKAWA cell frozen in liquid nitrogen were revived in routine method, and inoculated in RPMI-1640 blended .with 10% fetus cattle serum, cultured in culture bottle. The bottle was put in the incubator with the condition of 37℃,5%CO2. The growth of cells was observed every day. 70% of cells confluence were digested with EDTA .2 The choice of medicine concentration and experimental groupingThe PSC of VP-16 is 5μg/ml, of CDDP is 3μg/ml .Cells were divided into four groups referring to PSC of VP-16. (1)normal group .(2)VP-16 group: ISHIKAWA cells were exposed to VP-16 in 0.05μg/ml,0.5μg/ml,5μg/ml,50μg/ml,500μg/mlrespectively.(3)VP-16+CDDP group: ISHIKAWA cells were exposed to CDDP in PSC combined with VP-16 in 0.05μg/ml,0.5μg/ml,5μg/ml,50μg/ml,500μg/ml respectively. (4)CDDP group: ISHIKAWA cells were exposed to CDDP in PSC.3 MTT method was applied to detect the inhibitory effect on cell growth of each experiment group, calculating proliferous inhibitory ratio, estimating the combination effect by the formula of JINSHI.4 FCM was applied to detect the ISHIKAWA cells apoptosis ratio of each experimental group, to see the difference of cell growth among different medical treatment .5 Western blotting was used to determine the expression change of Livin protein and caspase-3 protein, probing into possible mechanism of chemotherapy resistant by knowing the change and correlativity of two intent protein.6 Statistical Methods: All datas are using computer statistical software SPSS(13.0)for statistical analysis. The experimental data with mean±standard deviation ( X±S ) . Among the groups using single-factor analysis of variance, Western Blotting data using quantity-one system, with a peak value of the area and optical density quantification. Criterion: p<0.05, to have statistical significance.Result:1 MTT result: There was a synergistic effect on ISHIKAWA cells when treated with CDDP in PSC combined with various concentrations of VP-16(0.05μg/ml,0.5μg/ml, 5μg/ml, 50μg/ml,500μg/ml). According to formula of JINSHI, Q data were 0.91(+),1.18(++),1.31(++),0.89(+),0.85(+) respectively. We can see that VP-16 in PSC combined with CDDP in PSC showed highest synergistic effect: Q=1.31(++).2 FCM result: Various concentration of VP-16 (0μg/ml ,0.5μg/ml,5μg/ml,50μg/ml ) incubated with ISHIKAWA cells for 24h, apoptosis rates detected by FCM were (9.6±3.1)%, (21.5±2.7 )%, (30.4±1.5)%, (43.4±1.8)%. Above corresponding concentrations of VP-16 combined with CDDP in PSC incubated with ISHIKAWA cells for 24h , apoptosis rates detected by FCM were(15.2±1.5)%, (28.7±2.8)%, (45.7±3.5)%, (47.2±2.1)%. From the result we can estimate that two drug(VP-16+ CDDP) combination groups have stronger power of killing cell than alone drug (VP-16) groups.There are statistically significant about apoptosis rates between the two drug (VP+ CDDP)combination groups and alone drug (VP-16) groups in low concentrations of VP-16(0.5μg/ml,5μg/ml) respectively, p<0.05. And there are not statistically significant in high concentrations of VP-16(50μg/ml), p>0.05.3 Western blotting results: Various concentration of VP-16 (0μg/ml,0.5μg/ml,5μg/ml,50μg/ml ) incubated with ISHIKAWA cells for 24h, Livin protein expressions were 0.18±0.01,0.14±0.01, 0.08±0.01, 0.05±0.01。Above corresponding concentrations of VP-16 combined with CDDP in PSC incubated with ISHIKAWA cells for 24h ,Livin protein expressions were 0.11±0. 01, 0.11±0. 01, 0.06±0.01, 0.04±0.01. From the result we can see Livin protein expressions of drug(VP-16+CDDP) combination groups were less than that alone drug (VP-16) groups. There are statistically significant between the two drug (VP-16+CDDP) combination groups and alone drug (VP-16) groups respectively. Various concentration of VP-16(0μg/ml ,0.5μg/ml,5μg/ml,50μg/ml ) incubated with ISHIKAWA cells for 24h, caspase-3 protein expressions were 0.69±0.08, 1.00±0.05, 1.15±0.04, 1.22±0.04), Above corresponding concentrations of VP-16 combined with CDDP in PSC incubated with ISHIKAWA cells for 24h ,caspase-3 protein expressions were 0.85±0.02, 1.05±0.04,1.18±0.03, 1.37±0.14. From the result we can see caspase-3 protein expressions of drug(VP-16+CDDP) combination groups were more than that alone drug (VP-16) groups. There are statistically significant between the two drug (VP-16+CDDP) combination groups and alone drug (VP-16) groups respectively.Conclusion:1 There was a synergistic effect on ISHIKAWA cells when treated with CDDP in PSC combined with various concentrations of VP-16. As the VP-16 concentration increased, the inhibitory rate of drugs on cells showing a rising trend along. And the group of VP-16 in PSC combined with CDDP in PSC appears the most obvious synergistic effect.2 With the VP-16 concentration increased, the cancer cell apoptosis rate has increased. two drug(VP-16+CDDP) combination groups have higher cell apoptosis rates than alone drug (VP-16) groups.There are statistically significant about apoptosis rates between the two drug (VP-16+CDDP)combination groups and alone drug (VP-16) groups in low concentrations of VP-16(0.5μg/ml,5μg/ml) respectively. And there are not statistically significant in high concentrations of VP-16(50μg/ml).3 Livin protein highly expressed in the blank control group. Livin protein expression reduced when ISHIKAWA cells treated with various concentration of VP-16, while expression reduced heavily when ISHIKAWA cells treated with various concentration of VP-16 and CDDP . There are statistically significant (p<0.05 )about the expression of Livin between the two drug (VP-16+CDDP)combination groups and alone drug (VP-16) groups respectively. caspase-3 protein expression with the Livin protein expression was a clear trend of negative correlation, single-drug experimental group compared with the combined drug difference between the experimental group was statistically significant respectively (p<0.05 ).
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