| Objective: Ovarian carcinoma is very late when it was been discovered because of lacking the effective diagnostic methods. The mortality rate of ovarian carcinoma is the first one in the malignant tumor of gynecology. At present, operation is the main method for the patients of ovarian carcinoma. But it is the very important way that we use multiple methods such as chemotherapy,radiotherapy...etc. Among these methods, many courses of chemotherapy is important .Especially it is significant for those patient of ovarian carcinoma in later period. It is easy for the cell of ovarian carcinoma to emerge the resistance during the courses of chemotherapy. At present almost doctors choose Cisplatin(CDDP) as the first choosing drug; meanwhile, they choose some other drugs as the compatibility. Among these compatibility drugs, Etoposide(VP-16) is often used。Trough a great deal of research,we have already had thorough understanding to TopoII. As the second choosing drug of ovary cancer chemotherapy, topoisomerase (topoII) inhibitor is often used in clinical.TopoII and TopoIII belong to Topoisomerase enzyme,both of them could adjust topological structure and promote DNA replication and transcribe. According to these research,we audaciously guess that the TopoIII inhibitor may get an application as a chemotherapy medicine at clinical. But the currently study of TopoIII is few, the development of TopoIII inhibitor also need more basal researches to provide theory.Glutathions S-transferase pi(GST-π)is a kind of multi-function medicine metabolism II mutually enzyme. GST-πnot only expresses in many kinds of tumor tissue , but also express too much in the diversiform medicine tumor cell . A great deal of research indicate that the GST-πhoists evidently in malignant tumor organization and relates nearly to medicine with tumor cell to cause the chemotherapy failure. Therefore, the examination of GST-πhas important clinical meaning to judging a sufferer whether he is drug-fast ,designing chemotherapy project to different medicine mechanism, consociation to use different chemotherapy medicine and raising a sufferer's existence rate.There has been research enunciated that the TopoIII has no expression in normal ovary cell, but expresses highly in the ovary tumor cell . CDDP could up-regulate the expression of topo II.To make clear the influence of the CDDP upon the TopoIIIa and GST-πexpression, in this study, western blot was used to observe the expression of TopoIIIa and GST-πafter different concentration drugs treatment.We can further understand CDDP function mechanism and provide theory basis to TopoIIIa inhibitor.Methods:1 Cell cultureHuman ovarian serous cystadenocarcinoma SKOV3 cells were freezed in our gynecology laboratory. SKOV3 cells were cultured in 1640 medium containing 10% fetal bovine serum,4 mmol/ml L-glutamine,1 mol/L HEPES,100U/ml Penicillin,100μg/ml Streptomycin , all of the cells were cultured at 37°C in a humidified chamber containing 5% CO2.2 Drug concentration and groupsAccording to the peak serum concentration(PSC), we divided these cells into six groups. We dealed with the first group cells by 1/5 PSC of CDDP(0.6μg/ml), the second group cells were dealed with by 2/5 PSC of CDDP(1.2μg/ml), the third were by one time PSC of. The other three groups cells were dealed with by VP-16 which in PSC combined with different concentration of CDDP(1/5PSC,2/5PSC,PSC)respectively.3 Nuclear extracts and Western Blot Analysis3.1 Preparation of Cellular ExtractsOvarian carcinoma cells were collected at 24h after different concentration of drugs treatment for target protein determinations, every groups were washed twice by PBS, nuclear extracts were produced at 4℃for 3 mins by lysing cells.The procedure of nuclear extracts refer to the description.The cellular debris was removed from the extract by centrifugation, and the supernatant was stored at–20℃for pre-emergency. The protein concentrations of nuclear extracts were determined using the methods of G-250.3.2 Western Blot AnalysisExtracted protein from every groups ovarian carcinoma cells combined with different concentrations drugs treatment were separated on 10%SDS-PAGE gels at 4℃. Proteins were transferred to a cellulose nitrate membrane using a semidry blotter in 100 mV current flow for 2-3 hours at 4℃. Western blot analysis was performed with an appropriate dilution of each antibody.First, confining liquid was used at 4℃for one hour; second, add to rabbit antihuman GST-π和TOPOIIIαpolyclone antibody respectively at 4℃overnight; third, add to goat antirabbit IgG antibody for 1 hours at 37℃.The reaction completed followed by DAB or ECL visualization using enhanced chemiluminescence. And the expression of transcription factor GST-π和TOPOIIIαwere assayed by Western blot on protein level, respectively.4 Data were analysed using the Satistical Package of the SAS V8.0. Statistical analysis was performed using univariate analyses, one-way ANOVA, experimental data use mean and standard deviation ( X±S). Western blot analysis use HPIAS-1000 by determing optical density and peak value areas of straps. A P value of less than 0.05 was considered as significant.Results: Western blot analysis showed that the expression of transcription factor GST-πand TOPOIIIαprotein. 1 Expression of GST-π: increased with concentration, It was found that the expression of GST-πin groups with different concentrations CDDP significantly increased compared with the control group (G3:2.17±0.05;G4:2.41±0.05;G5:2.78±0.04 vs control group: 1.76±0.03 respectively), there was a significant difference, P<0.01.And the expression of GST-πprotein have a orderly reinforcement to accompany with the concentration increasing of CDDP, P<0.01. However, the expressions of GST-πincreased by Western blot on protein level in VP-16 groups(2.31±0.03vs 1.76±0.03), P<0.01.GST-πprotein in CDDP and VP-16 groups compared with control group was a significant difference (G6:1.81±0.04;G7:1.98±0.04;G8:2.11±0.05 vs control group: 1.76±0.03 respectively), P<0.01.2 Expression of TOPOIIIαprotein:TOPOIIIαprotein increase after different concentration CDDP by western blot analysis compared with control group (G3:1.21±0.06;G4:1.41±0.06;G5:1.71±0.10 vs control group:0.66±0.02),P<0.01, but there were no significant difference in every groups with different concentration CDDP ,P>0.05. The expression of TOPOIIIαdecreased by Western blot on protein level in VP-16 groups(0.71±0.05 vs 0.91±0.07) ,P<0.01. TOPOIIIαprotein in CDDP and VP-16 groups compared with control group increased(G6:1.02±0.04;G7: 1.14±0.02;G8:1.27±0.04 vs control group:0.91±0.07),there was significant difference, P<0.01.Conclusion: The expression of TopoⅢand GST-πwere enhanced after cisplatin treatment in ovarian carcinoma cell SKOV3 in vitro. Etoposide can down-regulate the expression of TOPOIIIα, this has meaning of reference to the research of TopoIII inhibitor.The more GST-πexpress,the stronger the cell's resistance of drug is. The decrease of GST-π's expression show cisplatin and etoposide can decrease cell's resistance of drug . It is very significant for understanding CDDP function mechanism and decreasing the resistance of CDDP .raising cancer sufferer'existence rate and clinical doctors to find a most effective plan of chemotherapy.
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