| Objective: Ovarian cancer is the most common malignant tumor fromgynecological cancers.It is the leading cause of death in gynecological cancers.75%pations are diagnosed at an advanced stage because of the occultation ofthe early symptoms. Tumor cytoreductive surgery after neoadjuvantchemotherapy is the main method of treatment of ovarian cancer, but theoverall actuarial5year survival rate was about30%. The cisplatin-resistant ofthe tumor contributed to the high mortality rate. Also it is a gargantuan barrierof ovarian cancer during therapy. Therefore,in order to increase the sensitivityof ovarian cancer to platinum and prolong the effective use of platinum, it isurgent to research on the mechanisms of tumor resistance and the study of newdrug in recent years. The very chaperone, named Heat shock Protein90(HSP90) acts an essential role within human body,which regulates cellcycle,the occurrence and development of tumor and cell opoptosis, viainterfering its nearly150kinds of downstream proteins as well as protectingand activating their effectors.Hence,the inhibition of HSP90activity leads tothe failing in regular binding to its client proteins and its substancesdecomposed afterwards. As a result,the development of tumor cells weresuspended because of the inhibition of cell signalling.17-DMAG is a specificinhibitor of the HSP90, exerts an effect of reversal of multidrugresistance.According to previous datas,17-DMAG,at one hand, attenuates theexpression of survivin and livin through debilitating Hsp90;and at the other,itinduces gastric carcinoma cells death by the way of affecting STAT pathwayand p53mutation.Unfortunately, up till now there have been few experimentsworking on the mechanism of17-DMAG function.This study is investigatedthe influence and mechanism of reversing drug resistance of17-DMAG combination with cisplatin on human ovarian Cancer Cells line SKOV3andSKOV3/DDP, offering reference basis for the mechanism of drug resistanceon ovarian cancer.Methods:1The inhibition effect of proliferation was tested by MTT in differentconcentrations of cisplatin(1,2,4,8,16μg/mL),17-DMAG (2,4,8,16,32μmol/L)and17-DMAG combination with cisplatin on human ovarian Cancer Cells lineSKOV3and cisplatin resistance on ovarian cancer cell line SKOV3/DDP, andthe reversing multiple RF of17-DMAG combination with cisplatin wascalculated.2Examining the opoptosis rate of SKOV3and SKOV3/DDP cells withdifferent drug treatment by FCM method.3RT-PCR was used to analyze the expression p53，bcl-2and survivinmRNA in SKOV3and SKOV3/DDP cells treated by cisplatin,17-DMAG and17-DMAG combination with cisplatin.4Western-blot was used to analyze the expression p53，bcl-2andsurvivin protein in SKOV3and SKOV3/DDP cells treated by cisplatin,17-DMAG and17-DMAG combination with cisplatin.Results:117-DMAG (2，4，8，16，32μmol/L) has signifant inhibition effects onproliferation of SKOV3, SKOV3/DDP cells. With the increase of17-DMAGconcentration, the inhibition effects on proliferation of SKOV3, SKOV3/DDPcells increases gradually on dose depending manner, but differentconcentrations of17-DMAG have no signifant inhibition effects onproliferation of SKOV3, SKOV3/DDP cells. Different concentrations ofcisplatin(1，2，4，8,16μg/mL) have signifant inhibition effects on proliferationof SKOV3，SKOV3/DDP cells that were treated48hours on dose dependingmanner, and the inhibition effect on SKOV3is more obvious compared withthat on SKOV3/DDP. The reversing multiple RF of17-DMAG combinationwith cisplatin were2.24,5.52. The results showed that17-DMAGcombination with cisplatin also have obvious inhibition effects on proliferation of SKOV3, SKOV3/DDP, the inhibition effects obviously higherthan single drug of cisplatin and17-DMAG, and can reverse drug resistance ofDDP.2We got the respective inhibition ratio according to FCM methods listedas follows:SKOV3cells blank group,17-DMAG only group(6μmol/L),cis-platinum only group(1.5μg/mL),6μmol/L of17-DMAG and1.5μg/mL ofcis-platinum group; SKOV3/DPP cells, blank group,17-DMAG only group(6μmol/L), cis-platinum only group(3μg/mL),6μmol/L of17-DMAG and3μg/mL of cis-platinum group. In accordance to King’s Formula, the q value ofSKOV3cells was0.941with daul drug pretreatment, and SKOV3/DPP cells0.999, correspondingly.3Detection of RT-PCR gene level, p53，bcl-2and survivin mRNA inSKOV3and SKOV3/DDP cells changes, the results showed that after SKOV3cell treated with48h by cisplatin, the expression of p53，bcl-2and survivinmRNA increaseed with significant difference compared with control group(P<0.05).17-DMAG combined with cisplatin group reducing these resistancegenes of p53，bcl-2and survivin mRNA was obviously higher than that ofsingle drug of17-DMAG groups; After SKOV3cell treated with48h by17-DMAG and17-DMAG combination with cisplatin group drugs, theexpression of p53，bcl-2and survivin mRNA degraded with significantdifference compared with control group (P<0.05). And17-DMAG combinedwith cisplatin group reducing p53，bcl-2and survivin resistance genes wassignificantly better than that of single drug of17-DMAG and cisplatin group.4Detection of Western-blot gene level, p53，bcl-2and surviving proteinin SKOV3and SKOV3/DDP cells changes, the results showed that afterSKOV3cell treated with48h by cisplatin, the expression of p53，bcl-2andsurvivin mRNA increased with significant difference compared with controlgroup (P<0.05). SKOV3cell treated with48h by17-DMAG and17-DMAGcombined with cisplatin the expression of p53，bcl-2and survivin mRNAdecreased with significant difference compared with control group(P<0.05).And17-DMAG combined with cisplatin group reducing these resistance genes of p53，bcl-2and survivin mRNA was obviously higher thanthat of single drug of17-DMAG groups; After SKOV3cell treated with48hby17-DMAG and17-DMAG combination with cisplatin group drugs, theexpression of p53，bcl-2and survivin mRNA degraded with significantdifference compared with control group (P<0.05). And17-DMAG combinedwith cisplatin group reducing p53，bcl-2and survivin resistance genes wassignificantly better than that of single drug of17-DMAG and cisplatin group.Conclusions:117-DMAG combined with cis-platin has signifant inhibition effects onproliferation of SKOV3, SKOV3/DDP cells, the inhibition effects onproliferation of SKOV3, SKOV3/DDP was obviously better than that of singledrug of17-DMAG and cisplatin, and can signifantly reverse drug resistance ofDDP.217-DMAG combined with cis-platin group can signifantly reduce theexpression of resistance genes of p53，bcl-2and survivin mRNA in SKOV3and SKOV3/DDP cells.Hence, we were convinced that17-DMAG mayameliorate its resistance against cis-platin by cutting the level of resistancegene p53within SKOV3/DDP cells. |
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