| Objective: Ovarian cancer is one of the most common gynecologicmalignant tumors. The incidence rate in gynecological malignant tumor is inthe third place, but the mortality rate is in the first. As the highest death rate oftumor in gynecologic tumors, it is a serious threat to women’s health. Althoughthe operation and conventional chemotherapy, radiotherapy technology isimproved, the long-term survival rate of patients with advanced ovarian canceris still not exceeding30%. The carboplatin and paclitaxel chemotherapy afterthe cytoreductive surgery is the standard treatment. However, the tumor cellsproducing drug resistance to chemotherapy in patients with advanced ovariancancer is a major cause of treatment failure and death. Therefore, study on themechanism of drug resistance of ovarian cancer，looking for ways to reversingthe ovarian cancer is the key to improve the survival rate of ovarian cancerpatients. In recent years，many stadies make it clear that the following factorscan lead the ovarian cancer cells to become resistant to drugs including theenhencement of repairment capacity of DNA damage, decrease of the effectiveintracellular drug concentration,abnormality of drug target and abnormalitiesof intracellular signal transduction pathway.Among these the enhencement ofrepairment capacity of DNA damage and abnormalities of intracellular signaltransduction pathway has become hot spots by statistics.It was found that,Germany is antibiotic medicines of benzene AnSha, belongs to the Hsp90inhibitors. Our previous studies showed that,geldanamycin can be used to cutthe cisplatin resistance of resistant gene LRP and GST-PI in ovarian cancercell SKOV3/DDP and then improve ovarian cancer cells to cisplatinresistance.but the preclinical study found that,geldanamycin have higher livertoxicity and limits its clinical use. Foreign studies have shown that geldnamy- cin derivatives, amino-17-17-allyl to methoxyl, geldanamycin (17-AAG)can reverse many cancer drug resistance and blocking intracellular signaltransduction pathways in cancer drug resistant cells, and has lower livertoxicity.It used more in the research and application of breast cancer and lungcancer,but no studies in application of ovarian cancer.This study was toexplore that,17-AAG and cisplatin to human ovarian cancer cell SKOV3andof cisplatin resistant ovarian cancer cell SKOV3/DDP resistance effect and itsmechanism, and to probe into cause ovarian cancer chemotherapy drugresistance mechanism, provide theoretical basis for improving ovarian cancerchemotherapy drug resistance. This study was to explore the effect andmechanism of drug resistance of the joint application of the17-AAGand Cisplatin tohuman ovarian cancer cell SKOV3and cisplatin resistantovarian cancer cell SKOV3/DDP, to probe into the drug resistance mechanismof ovarian cancer chemotherapy, and to provide theoretical basis forimproving ovarian cancer chemotherapy drug resistance.Methods:1Detecting proliferation inhibition in different concentrations of cisplatin(2,4,8,16,32μg/ml), geldanamycin derivative17-AAG (1.2,2.4,4.8,9.6,19.2μg/ml) on human ovarian cancer SKOV3and SKOV3/DDP on cisplatin cell,17-AAG combined with cisplatin (1.2μg/ml17-AAG+2μg/ml cisplatin,2.4μg/ml17-AAG+2μg/ml cisplatin,4.8μg/ml17-AAG+2μg/ml cisplatin,9.6μg/ml17-AAG+2μg/ml cisplatin,19.2μg/ml17-AAG+2μg/ml cisplatin) onhuman ovarian cancer SKOV3cell,17-AAG combined with cisplatin (1.2μg/ml17-AAG+3μg/ml cisplatin,2.4μg/ml17-AAG+3μg/ml cisplatin,4.8μg/ml17-AAG+3μg/ml cisplatin,9.6μg/ml17-AAG+3μg/ml cisplatin,19.2μg/ml17-AAG+3μg/ml cisplatin) on SKOV3/DDP cell used by MTT.2Detecting change df cell cycle and apoptosis rate use by flow cytometryin SKOV3and SKOV3/DDP cells treated with17-AAG, cisplatin,17-AAGcombined with cisplatin.3Checking the repairment of DNA genes ERCC1, XRCC1and theexpression of the tumor suppressor gene BRCA1, BRCA2mRNA.Use by RT-PCR in SKOV3and SKOV3/DDP cells treated with17-AAG, cisplatin,17-AAG combined with cisplatin.4All the date statistical analysis using software SPSS13.0. Experimentalresults withx±s, mean differences between the two groups compared with ttest, multiple mean comparison using single factor analysis of variancebetween groups, P<0.05for the difference is significant of statistically.Results:1Geldanamycin derivative17-AAG (1.2,2.4,4.8,9.6,19.2μ g/ml) cansignificantly inhibit the proliferation of SKOV3/DDP cells, and also inhibitproliferation of SKOV3, but the proliferation of SKOV3/DDP is relativelyweak. With the increasing of17-AAG concentration, the effect of inhibition ofSKOV3and the proliferation of SKOV3/DDP gradually increase, in adose-dependent manner. Different concentrations of cisplatin (2,4,8,16,32g/mlSKOV3and SKOV3/DDP) treat cells, after48hours, inhibiting effect on twokinds of cells proliferation is significant, dose-dependent. Inhibition ofcisplatin on SKOV3cells with SKOV3/DDP is significant.17-AAG combinedwith cisplatin also significantly inhibit the proliferation of SKOV3andSKOV3/DDP cells. It has strong effects on SKOV3/DDP cells. Separatedapplication of inhibition is higher than that of17-AAG and cisplatin, and ithas the reversal of drug resistance to cisplatin.2Fluid cytology for detecting the applications of cisplatin,17-AAG, and17-AAG combined with cisplatin in SKOV3and SKOV3/DDP cells of cell cycle arrest and apoptosis rate.(1) In SKOV3cells，theproportion of cells which17-AAG,17-AAG combined with cisplatin was inearly DNA G0/G1phase were45.37%±6.24%，53.60%±9.77%，and48.50%±8.3%, in DNA G2/M phase,33.27%±3.83%，24.83%±2.72%，and23.69%±0.72%, in DNA’S phase,21.35%±0.17%，14.33%±0.42%，and23.03%±0.004%. Our study showed that the treatments ofcisplatin,17-AAG, and17-AAG combined with cisplatin to the distribution of cellcycle of SKOV3and SKOV3/DDP had no significant effect by statisticanalysis.(2) Apoptosis rate in SKOV3cells of all groups were10.54%±2.19%, 3.34%±0.90%and15.65%±0.96%. In SKOV3/DDP cells,7.61%±1.50%,20.38%±6.50%and29.22%±2.74%.Apoptosis rate of17-AAG combined withcisplatin was the highest both in SKOV3and SKOV3/DDP cells, it hadsignificant differences (P<0.05).This showed that cisplatin,17-AAG, and17-AAG combined with cisplatin had effect apoptosis of ovarian cancercells SKOV3and SKOV3/DDP. It had no significant differences to cell cycleand the inhibitory effect of17-AAG combined with cisplatin to SKOV3andSKOV3/DDP cells had significant differences (P<0.05).3On the level of the detection of gene RT-PCR, DNA repair genesERCC1, XRCC1mRNA, suppressor the change of tumor gene BRCA1,BRCA2mRNA in SKOV3and SKOV3/in DDP cells. Results showed thatafter48hour effect of17-AAG and17-AAG combined with cisplatin onSKOV3/DDP, DNA repairing gene ERCC1, XRCC1mRNA expression waslower than that of the control group and cisplatin group. It has no significantchange in comparison cisplatin group and the control group. However, thetumor suppressor gene wihch BRCA1, BRCA2expression increase.Compared with control group, it has significant difference (P<0.05).17-AAGcombined with cisplatin group down-regulation of ERCC1, XRCC1mRNAtwo DNA repaired gene, upregulation of tumor suppressor gene BRCA1andBRCA2mRNA,the role of two mRNA was significantly higher than that ofsingle drug17-AAG and cisplatin group. After48h treatment of the SKOV3cells, in cisplatin group, ERCC1, XRCC1, BRCA1, BRCA2mRNA increase inexpression in SKOV3cells. The expression of ERCC1, XRCC1mRNAdecrease and BRCA1, BRCA2mRNA increase in the17-AAG group and17-AAG combined with cisplatin group than cisplatin group.Conclusion:117-AAG combined with cisplatin had obvious role in inducing cellapoptosis both in SKOV3/DDP and SKOV3cells, could increase cellapoptosis rate significantly. The apoptosis inducing effect of two medicinecombined had in SKOV3/DDP and SKOV3cells was significantly strongerthan17-AAG and cisplatin alone, however, the apoptosis inducing effect of 17-AAG combined with cisplatin was weaker in SKOV3cells than in SKOV3/DDP cells.217-AAG combined with cisplatin have inhibit apoptosis effect onSKOV3/DDP cells. Apoptosis of the combination of the two drugs onSKOV3/DDP cells was significantly stronger than that of17-AAG andcisplatin used alone, and there is no without obvious inhibition of apoptosis inSKOV3. Visible17-AAG combined with cisplatin has inhibitory effect onapoptosis of drug-resistant ovarian carcinoma cell SKOV3/DDP, and it can beused as effective treatment of drug resistant ovarian cancer.Cisplatin,17-AAG,17-AAG combine with cisplatin have no affection in cellsof SKOV3ã€SKOV3/DDP.317-AAG enhanced sensitivity to cisplatin. Reversing the resistance ofovarian cancer.17-AAG improve cisplatin resistance that may be can cutthrough down-regulate DNA repaired gene ERCC1, XRCC1and up-regulatesuppressor gene BRCA1, BRCA2. |
