The Effect Of17-AAG And YM155Acted Separately And Combined On TE-1Esophageal Squamous Cancer Cell Line

Back ground: Heat shock protein90(HSP90) generally exists ineukaryotic and prokaryotic cells, is a group of highly conserved molecularchaperone. Studies confirmed that HSP90had high expression in malignanttumor tissues, however very low in normal tissues. Recently HSP90hasbecome the new targets for cancer treatment and the study of the inhibitorsbecomes very famous in the anti-cancer research.17-AAG is one of the mostfamous HSP90inhibitors which has been entered clinical Ⅲ period.Themechanism of17-AAG act as a anti-cancer agent is it can competeHSP90ATP binding sites from which HSP90endogenous ATP enzymeactivity is inhibited and leads to a lose combine of HSP90peptide protein andfollowing by the loss of stability and degraded by proteinase. The excited invivo research results of the antitumor activity of17-AAG and its lowhepatotoxicity bring the17-AAG a wildly attention. However, the in vitrostudy about the effects of17-AAG in the treatment of esophageal squamouscancer is rarely reported.HSP90has many substrate proteins, many of which play an importantrole in the cell pathway and in tumor occurrence and evolution. HSP90plays aprotective role in keeping the substrate proteins from protease degradation. Innumerous substrate protein of HSP90,Survivin is an imporent one. Survivinthe strongest inhibition of apoptosis protein ever found is one new member ofthe inhibiting apoptosis protein (IAP) family. It is highly expressed in humanembryo and almost all human common malignant tumor tissues. On the otherhand, its expression is very low in healthy adults and terminal differentiationtissue. All of these to give a hint it has a close relationship with the occurrenceof malignant tumors. Survivin has become a new star of anti-tumor targetresearch. Studies have shown that it can effectively destroy the cell survival and development of tumor induce apoptosis degradation. YM155is currentlythe most successful small molecule antagonists of Survivin and has entered thestage of Ⅱ phase clinical trials. YM155inhibits Survivin gene promoteractivity and depress the growth of tumor cell proliferation. Yet about its effecton esophageal squamous cancer cells is rarely reported both at home andabroad.Objective:Our experiment uses17-AAG and YM155separately andcombined to treat TE-1esophageal squamous cancer cells. To observe theproliferation activity, apoptosis and the protein expression of HSP90andsurvivin of TE-1cell. All the data was collected to analysis the relationship ofthe expression of the HSP90and Survivin. Provied the clinical application of17-AAG and YM155acted separately and combined.Methods:1Cell cultureTE-1esophageal squamous cancer cells used in experiments is providedby the fourth hospital, hebei medical university research center, cells toRPMI1640complete culture medium containing10%fetal bovine serum at37℃and in saturated humidity, CO25%incubator in conventional subculture.2To detect the proliferation activity by MTT MethodRespectively,we use different concentrations of17-AAG and YM155act-ed separately and combined on TE-1esophageal squamous cancer cells, toTE-1cells contained no drug as control group. Acted cells after24h,48h,72h,to detecte the inhibition rate of TE-1cells proliferation.3To detect TE-1cells apoptosis rate by flow cytometry (FCM)To detect TE-1cells apoptosis rate by flow cytometry(PI single dyedfluid cytology method), we quantitative analysis the control group and thedifferent concentrations of17-AAG and YM155separated and combined onapplication processing TE-1cells respectively after48h.4Protein immunoblot (Western blot)We use control group and different concentrations of17AAG, YM155separated and combined on TE-1cells after48h later, collect total protein, to detect the HSP90and survivin protein expression level by western blotmethod.5Statistical methodsUsing SPSS16.0software to carry on the single factor analysis of varianceand t test, data with x±s, check with（P <0.05） for significant differences.Results:1The results determined by MTT show that:TE-1cells were treated with a final concentration0.02μM、0.2μM、2μM、20μM and40μM17-AAG and a final concentration of0.01nM,0.1nM,1nM,10nM and100nM of YM155, with the increase of drug concentration in24h,48h,72h inhibition rate increased, the inhibition rate of the difference hasstatistical significance (P <0.05)（Fig.1、2；Table1），and show dependencyof time and dose.Combined use of17-AAG, YM155with separate application, in the roleof esophageal squamous cancer cells for24h,48h,72h of inhibition rate, nam-ely, respectively, to a final concentration of0.2μM17-AAG and the final con-centration of0.1nM,1nM,10nM YM155,2μM17-AAG final concentrait-on and the final concentration of0.1nM,1nM,10nM YM155, finishingconcentration20μM17-AAG and concentration of0.1nM,1nM,10nM YM155, respectively to compare the corresponding single drug groups on TE-1cell inhibition rate, the difference in24h,48h and72h were statistically sig-nificant (P <0.05)（Fig.3-5、Table2-4）. Prompt that combination proli-feration suppression effect is better than alone.2The results of flow cytometry show that:Respectively, TE-1cells were treated with a final concentration of0.2μM,2μM,20μM17-AAG and a final concentration of0.1nM,1nM, the10nMYM155has an effect on TE-1cells after48h, with the increase of drugconcentration in the apoptosis rate increased, the apoptosis rate of thedifference has statistical significance (P <0.05（)Fig.6、7；Table5）, and showdependency of dose. Respectively, TE-1cells were treated with a finalconcentration of0.2μM17-AAG and the final concentration of0.1nM,1nM, 10nM YM155,2μM17-AAG final concentration and the final concentrationof0.1nM,1nM,10nM YM155, finishing concentration20μM17-AAG andconcentration of0.1nM,1nM,10nM YM155, by the joint action ofcombination group corresponding alone were compared for TE-1cellapoptosis rate, apoptosis rate of the difference has statistical significance (P <0.05)（Fig.6-8；Table6）. Prompt combination effect on promoting apoptosisis better than alone. and show dependency of dose.3The results of western blot show that:Respectively, TE-1cells were treated with a final concentration of0μM,0.2μM,2μM and20μM17-AAG and0nM and0.1nM,1nMand10nMYM155after48h, results showed that two kinds of different drugconcentrations HSP90/GADPH gray ratio difference between groups had nostatistical significance (P>0.05)（Fig.9、10、12、13；Table7）. Respectively,TE-1cells were treated with a final concentration of0.2μM17-AAG and thefinal concentration of0.1nM,1nM,10nM YM155,20μM17-AAG finalconcentration and the final concentration of0.1nM,1nM,10nM YM155,finishing concentration20μM17-AAG and concentration of0.1nM,1nM,10nM YM155combined, the result shows that the combination group and thecorresponding single shade of HSP90/GADPH ratio difference betweentreatment group had no statistical significance (P>0.05)（Fig.11、Fig.14-22；Table8）.Respectively, TE-1cells were treated with a final concentration of0nM,0.2μM,2μM and20uM17-AAG and0nM and0.1nM,1nM, the10nMYM155after48h, results showed that two kinds of different drugconcentrations between Survivin/GADPH gray ratio difference was statis-tically significant (P <0.05)（Fig.9、10、12、13；Table.7）, And with theincrease of drug concentration, the gray level ratio gradually reduce, and showdependency of dose. respectively, TE-1cells were treated with a finalconcentration of0.2μM17-AAG and the final concentration of0.1nM,1nM,10nM YM155,2μM17-AAG final concentration and the final concentrationof0.1nM,1nM,10nM YM155, finishing concentration20μM17-AAG and concentration of0.1nM,1nM,10nM YM155combined, the result showsthat the combination group and corresponding alone between survivin/GADPH gray ratio difference was statistically significant (P <0.05)（Fig.11、Fig.14-22；Table9）. The gray level ratio of combination group was obviouslylower alone group. Tip: combined treatment is better than alone could cutsurvivin protein expression.Conclusions:117-AAG, YM155can effectively suppress esophageal squamouscarcinoma cell proliferation activity of TE-1, and both on TE-1cellproliferation activity is in time-and dose-dependent inhibitory effect.217-AAG combined YM155had a stronger inhibitory effect.317-AAG, YM155could promote apoptosis of esophageal squamouscarci-noma cell TE-1and both the cell apoptosis of TE-1were dosedependent.4Combined17-AAG and YM155to TE-1cell has a stronger effect onpromoting apoptosis.517-AAG and YM155single and combined use on TE-1cell HSP90protein expression had no obvious influence.617-AAG, YM155can cut in esophageal squamous cell carcinoma TE-1the expression of survivin protein, and were dose dependent. Combined17-AAG and YM-155cut more expression of survivin protein.