Studies On Expression And Significance Of HSP90α In Esophageal Squamous Cell Carcinoma And The Effection Of17-AAG On Esophageal Squamous Cells

Objectives：Heat shock protein90(HSP90)is a group of ubiquitous highlyconserved molecular chaperone in Prokaryotes and eukaryotes.According totheir different gene-encoding, HSP90is composed of HSP90α and HSP90βsubunits. It has been identified that HSP90is overexpressing in multiplemalignant tumors. HSP90α has demonstrated increasing expressing in contrastto HSP90β with no change in general, suggesting that HSP90α was morerelevant to the malignant tumors and represented a potential therapeutic targetfor malignant tumors.17-AAG interferes the association between clientproteins and HSP90by occupying the ATP binding site of Hsp90as theHSP90inhibitor, inducing Hsp90dissociation from client proteins anddegradation. In addition, the activity of anti-cancer was presented in multiplemalignant tumor cells exposed to17-AAG in vitro.17-AAG was currentlybeing tested in clinical phase Ⅲ trials for anticancer treatment, but studies onthe effect of17-AAG on esophageal squamous cell carcinoma and theexpressing of HSP90α are absence.Hypoxia-inducible factor1(HIF-1) is an oxygen-regulated transcriptionalactivator that plays essential roles in tumor cell adaptation to hypoxia andangiogenesis resulting in the development and metastasis of tumors. HIF-1α isoverexpressed in common cancers and their metastases, including prostate,breast, colon, and lung cancers. It was also reported that HIF-1α wasoverexpressed in ESCC according to recently studies including Takala H's.Moreover, the transcription factors HIF-1α have been identified as being clientproteins of HSP90and HSP90plays an key role in maintain the activity ofHIF-1α. GA and its derivative17-AAG presented an effect on inducingdegradation of HIF-1α in malignant tumors including prostate and hepatic cancer, suppressing the invasion and metastasis. However,the studies onwhether17-AAG induces degradation of HIF-1α protein in ESCC was few.Here, we explored the expessing of HSP90α on tissues and cells of ESCCby immunohistochemisty and immunocytochemical staining respectively.MTT and FCM was required to detect the rate of inhibition of proliferationand apoptosis in TE-1and TE-13cells cultured in vitro. RT-PCR and Westernblot was performanced to manifest whether the effect of17-AAG onexpressing of HIF-1α on the level of mRNA and protein as well theircorrelation.Method:1The expression and clinical significance of heat shock protein90α inesophageal squamous cell carcinomasThe40cases of specimens were obtained from the Fourth Hospital ofHebei Medical University, Thoracic Surgery.The sample were comprise withcancer,mucosa adjacent to cancer (the edge of the tumor is2-5cm) andnormal esophageal mucosa(the edge of the tumor＞5cm and without invasion).All the patients had no radiotherapy or chemotherapy before the operations.The samples of the tumor are all ESCC by the pathology examination. Weapplied S-P immunohistochemical staining to detect the expressions and thequantity of the HSP90α in the ESCC, the mucosa adjacent to cancer and thenormal esophagus mucous membrane. Furthermore, we explore itsrelationship with the length of the tumor, location of the tumor, thepathological grade, the depth of invasion,the clinical stage and the lymph nodemetastasis by statistical analysis of software SPSS13.0.2Cell cultureThe esophageal squamous cell lines TE-1, TE-13and Yes-2Cellsprovided by Hei Bei Medical University. They were cultured in RPMI-1640medium supplemented with10%foetal bovine serum in a37℃incubatorwith humidified atmosphere and5%CO2.3The expression of heat shock protein90α in TE-1, TE-13and Yes-2cells The cells in the stage of exponential phase of growth were selected toparticipate this experiment and the density was modulate to5×105/ml. Theglass coverslip was added to the wells of6-well plates and the cells wereseeded on it with complete culture solution in37℃incubator with humidifiedatmosphere and5%CO2for48h when the cells compeletly attached. Thenterminate the culturing and adherent cells were fixed to microscope slidepreparing for the S-P immunocytochemical staining next to observe theexpressing of HSP90α in esophageal squamous cell lines.4Anti-proliferation effects on the TE-1,TE-13and Yes-2Cell linetreated with17-AAG via MTT assayAnti-proliferation effects on the TE-1, TE-13and Yes-2Cell line wereevaluated by MTT assay.In brief, the TE-1, TE-13and Yes-2Cells wereseeded into96-well plates before modulating the density of cells to1×104/mland allowed to adhere overnight. TE-1,TE-13and Yes-2Cells were thenincubated with17-AAG at various concentrations for24h,48h, or72h, and10ul MTT was added.The precipitated formazan crystals were dissolved in150μl DMSO. Cell viability was assessed at492nm for each well, which630nm as reference wavelength,and calculated as follows:(1-optical densityof experiment sample/optical density of control)×100％.5Assessment of apoptosisApoptosis was measured by staining with annexin V-FITC/PI. TE-1andTE-13cells were incubated in culture medium alone or in culture mediumcontaining0-20μM of17-AAG for48h, harvested, washed twice with PBS,and resuspended in annexin V-FITC and PI.By this method, at least1×104cells per sample should be analyzable on flow cytometry.The degree ofapoptosis was determined as the percentage of cells stained with annexinV-FITC/PI.6The effects of17-AAG on HIF-1α in esophageal squamous cell lineSemi-quantitative reverse transcription (RT-PCT) and Western blotanalysis was performed to explore whether the expressing of HSP90α orHIF-1α at the level of mRNA was in parallel with the level of protein,otherwise, the expressing of HSP90α in parallel with the expressing ofHIF-1α on the same level treated by17-AAG with homo-final concentrationfor48h. The total Protein or mRNA was extracted from TE-1and TE-13cellswhich incubated in culture medium containing0-20uM of17-AAG for48h.Result:1The results showed that the overall positive expression rate of HSP90αin the ESCC, mucosa adjacent to cancer and normal esophagus mucousmembrane was80.0%(32/40),37.5%(15/40),24%(10/40) respectively.Analysis of χ~2test showed that the difference expression rate of HSP90αbetween normal esophageal mucosa and mucosa adjacent to cancer wassignificant(P=0.413＞0.05). The difference expression rate of HSP90α in theESCC between normal esophageal mucosa or mucosa adjacent to cancer waseither significant respectively. The expression of HSP90α in the ESCC casesdid not correlated with depth of invasion, location of tumor and lymph nodemetastasis(P>0.05). There was significant correlation between the expressionof HSP90α and length of tumor and length of tumor.(P=0.000<0.05).2The results displayed that the positive expression rate of HSP90α inTE-1, TE-13and Yes-2Cell line was94.25±2.41％,75.15±2.49％and89.55±1.85％in each high power field respectively. The number of positivecells expressing HSP90α was46.8±4.7,90.4±3.3and78.2±4.57in averagein TE-1, TE-13and Yes-2Cells respectively.3The inhibitory fraction of TE-1Cells exposed to20μmol/L of17-AAGfor24h,48h and72h was39.24%,60.43%and86.58％；As well as2nM,200nM, and20μM of17-AAG for48h was6.25%,40.26%and60.43%,respectively. The inhibitory fraction of TE-13Cells exposed to20μmol/L of17-AAG for24h,48h and72h was45.64％,56.78％and78.81％; As well as2nM,200nM, and20μM of17-AAG for48h was6.58%,41.23%and56.78％,respectively. The effect of17-AAG was dose-time-dependent.4Compared with the rate of control was9.06％and2.54％in TE-13and TE-1respectively, after exposed to the final concentration of0.2μM,2μMand20μM with17-AAG for48h, the rate of apoptosis was12.90±2.7 ％,20.39±1.9％and35.15±2.6％,respectively in TE-13cells,as10.7±3.1％,14.37±2.5％and28.15±3.5％respectively in TE-1cells. A similarpattern was presented in Yes-2cells.5The expressing of HSP90α or HIF-1α at gene level was relevant toprotein level in TE-13cells exposed to increasing17-AAG by linearcorrelation analysis of SPSS. The statistical data showed that the correlationcoefficient of Pearson was0.843,P=0.000and0.907, P=0.000, respectivelyand that were statistically significant.Conclusion:1The total positive expression rate of HSP90α in ESCC was highter thanthoese in the mucosa adjacent to cancer and the normal esophagus mucousmembrane and the difference was no statistic significant between the twolatter. This also indicated that there was no significant correlation between theexpression of HSP90α and location,depth of invasion and lymph nodemetastasis of tumor and it may be associated with the length of the tumor andTNM staging as well as representing a potential marker on evaluation of TNMstaging.2Obviously,comparing with control, the expressing of HSP90α in TE-1,TE-13and Yes-2cells was in consistent with the sample of esophagealsquamous cell cancer in the location of staining and the positive rate, confirmsthat the HSP90α expression either in esophageal carcinoma tissue or cells ispositive.3TE-1and TE-13cells was inhibited by increasing17-AAG that hasshowed a dose-time-dependent manner. Because of the degree of celldifferentiation and the state of growth, the sensitivity to17-AAG was different.The sensitivity of TE-1and TE-13was higher than Yes-2which exposed to17-AAG, prompting17-AAG as a potential target of individualized regimensfor treatment.4Increasing concentrations of17-AAG-induced apoptosis in TE-1andTE-13cells was in a dose-dependent manner.5The expressing of HSP90α or HIF-1α was down-regulated by increasing17-AAG at mRNA level in TE-13cells respectively, and thedown-regulating of HSP90α was in line with HIF-1α that suggested17-AAGcould down-regulate the HIF-1α at RNA level as the inhibitor of HSP90viacertain pathways.6The increasing17-AAG down-regulated the expressing of HSP90α andHIF-1α at protein level, and the degree of down-regulating of HSP90α was inparalleled with HIF-1α in esophageal squamous cells.7The down-regulating of HSP90α or HIF-1α at mRNA level exposed toincreasing17-AAG in TE-13cells was in correlation with protein level.