Expression Of Hsp90α And Cyclin B1 Were Related To Prognosis Of Esophageal Squamous Cell Carcinoma And Keratin Pearl Formation

BackgroundEsophageal carcinoma（ICD-10:C15）is a normal cancer in China.Estimated by the international agency for World Cancer research on cancer incidence and mortality of the 2011 report that,there are 481600 cases of ESCC in the world,259200 cases from China,accounting for 53.82%,the dead cases are 406500 in the world,211100 cases from China,accounting for 51.92%.Surgery,radiotherapy and chemotherapy are the main treatment for esophageal cancer,whereas the chemotherapy is an important means of treatment of esophageal cancer.But the overall prognosis is still poor,with five-year survival rate less than 10%.The main reason is the most current chemotherapy drugs are cytotoxic drugs,which is differential selectivity of tumor cells,with the toxic side effects,it is difficult to meet the current clinical needs,always resulted in drug resistance.Many researchers have suggested that tumor micro environment and interstitial cells play an important role in mediating invasion and metastasis.These malignant tumors always consume a large quantity of oxygen,contain acidosis,growth factor,proteases or proteolytic enzymes and the inflammatory cells.Due to a higher ability to adapt to suffering harsh environment from outside of tumor cells.The effect is often not obvious because of the feature which allows the tumor radiotherapy and chemotherapy treatment,even greater harm to the patients themselves.keratin pearls is a typical structure of squamous cell carcinoma nests central keratosis.Such as skin,lips,mouth,esophagus,larynx,cervix,vagina,penis,etc.which is covered by squamous epithelium of the cancer original site.Observating by microscope,the well differentiated squamous cell carcinoma cancer nests in the middle with a layered keratosis,name cancer beads,around it is equivalent to prickle cell layer,and the outer layer is equivalent to basal cells.Heat shock proteins（heat shock proteins,HSPs）are a group of highly conservative proteins which synthetize in the stimulation such as hyperthermia,radiation and chemical poison.HSPs are widespread in prokaryote and eucaryon.They play roles as molecular chaperone in human.Hsp90（heat shock protein 90）is largely exist in cytoplasm as an important member of HSPs,which participate in various cell progress contain signal transduction,protein folding and degradation.Hsp90 is known as molecular chaperone for regulating substrates protein without changing target protein structure.Until now,there are more than 200 proteins found to be related with Hsp90,most of which are kinase.In addition,the expression of Hsp90 in tumor is 2 to 10 times higher than normal cells.In recent years,Hsp90 becomes the target of cancer treatment and many Hsp90 inhibitors have been used in clinical trials for cancer therapy for its important role in the tumor cell growth or proliferation.CyclinB1 plays a key role to control the cells in G2/M phase,activating and forming compounds with CDKs that means CDC2（cell division cycle）,promote cell to G2/M phase transition.It can lead to uncontrolled cell growth and even being a tumor because of the uncontrolled mechanism of cell cycle regulation.Recent studies had shown that,the abnormal expression of Cyclin B1 in many tumors（such as over expression,location changes）,showing the characteristics of cancer gene,the relationship of the genesis and development of tumors,prognosis,diagnosis,therapeutic is a researching hot pot.Cyclin B1,as a client protein of Hsp90,and also regulating the key cell cycle in G2/M phase,so it can be used as an effect indexes that related with cell cycle regulation,used to elucidate the relationship between Hsp90a and intracellular oxidative stress induced by H₂O₂.Hydrogen peroxide（H₂O₂）is a representative membrane-permeable oxidant and is the most abundant ROS in cells and regulates metabolism,aging,apoptosis and the intensity of growth factor signaling.In addition,it acts as a negative or positive regulator of cell signaling,and is very diffusible within and between cells and so any H₂O₂ might presumably difuse into the cells for removal by catalase,glutathione peroxidases and other H₂O₂-removing systems.Our team experimental results show that there is a reverse relationship of Hsp90 a and Cyclin B1 treated by H₂O₂ in HepG2 cell.Then if there is the same relationship of Hsp90 a and Cyclin B1 in esophageal squamous cell carcinoma?This is the main content of this research.Here,we explored the expression of Hsp90a and Cyclin B1 on tissues and cells of ESCC by immunohistochemisty staining respectively.CCK-8 and FCM was required to detect the rate of inhibition of proliferation and cell cycle in TE-1 cells cultured in vitro.Western Blot was performed to manifest whether the effect of H₂O₂ on expressing of Hsp90a and Cyclin B1 on the level of protein as well as their correlation.Objectives1.To explore the expression of Hsp90a and Cyclin B1 in ESCC,including the level of expression and the cellular localization.Analysis their significance in the progression and prognosis of ESCC;2.To explore the relationship between Hsp90a and Cyclin B1 in ESCC and research the effect of tumor microenvironment on expression of Hsp90 a and Cyclin B1 and formation mechanism of keratin pearls;3.To study whether the treatment with H₂O₂ influences the cell proliferation of TE-1;4.To study whether the treatment with H₂O₂ influences the cell cycle of TE-1;5.Detect the expression of Hsp90a and CyclinB1 of TE-1 cells after treated with H₂O₂;6.In the previous step analysis the effect of H₂O₂ in cell cycle,and in order to further investigate the relationship among oxidative stress induced by H₂O₂,and cell cycle.In addition,investigate the mechanism of cell cycle arrest which induced by and regulating effect mechanism of Hsp90a,then to study the mechanism of ESCC.MethodsImmunohistochemistry1.Immunohistochemistry SP methods were performed to detect the expression of the proteins.In the specimens from 105 ESCC patients(81 stained with Hsp90a antibody by Immunohistochemistry,65 with Cyclin B1 antibody,and among them,41 paired specimens were stained with Hsp90a and Cyclin B1.The results were scored with staining intensity and cell positive rate,as for no immmunoreactivity is 0;weak immunoreactivity is 1;moderate is 2;strong is 3.Cell positive rate:<1%is 0;<10%is 1;<50%is 2;<80%is 3;>80%is 4,and the sum of the both scores is the final result.0-2 for-;3-4 for +;5-6 for++;>7 for +++.Cell experiments1.Established model of oxidative stress in TE-1 cells:The cells were divided into control group,group of oxidative stress 6h,24h group.Oxidative stress factors:500μmol/L H₂O₂ to stimulate oxidative stress for the appropriate time;Use CCK-8 to observe cell survival.Use Flow cytometry observe the cell cycle of different time arrest under H₂O₂ in TE-1 cells.To determine the 24h time point for the H₂O₂ induced G2/M phase arrest;2.Western blot is used to detect content of protein Hsp90a and cyclinB1;To investigate the expression of Hsp90a and its client proteins Cyclin B1 protein level treated with hydrogen peroxide and if is consistent with histological experiments.3.All statistical analysis were carried out with the use of SPSS 16.0 statistical software package.One-way ANOVA test was used in the comparison of multiple sample averages.When the variances are heterogeneous,Dunnett’s T3 test were used for multiple comparisons between groups.Otherwise,LSD test were used;Correlation of non-normal distribution between two variable used Spearman method;Kaplan-Meier method was used for univariate survival analysis,log-rank test was for the significance of differences in survival,P<0.05 was considered statistically significant in all the analysis.RESULTS:Immunohistochemical staining of the proteins in ESCC1.Immunohistochemical staining of Hsp90a in ESSCThe higher intensity of Hsp90a staining signals were observed predominantly in the cancer cells,locate in both of cytoplasm and nucleus.In well differentiated ESCC,which characterized with more keratin pearls,the intensity of immunohistochemical staining of Hsp90aα is lower than the poor differentiated ones.Furthermore,the cells at the edge of well-differentiated tumors demonstrated higher expression of Hsp90a than the other cells.All these indicate Hsp90a might be necessary for the tumor growth and invasion.Among 81 cases of ESSC in surgical specimens,Hsp90a was significantly overexpressed in 78 cases（96.3%）.No association was found between overexpression of Hsp90a in patients with age,gender,tumor site,tumor status,and nodal status.However,significantly increased Hsp90a expression was more frequently observed in later clinical pathological stage than other stages2.Cyclin B1 expression is related to the differentiation of ESSCThe localization of Cyclin B1 was observed by immunoreactivity in the cytoplasm and nucleus of cancer cells,and poor differentiated ESCC was with higher intensity and more nucleus distribution of Cyclin B1.In 65 surgical specimens of ESSC,Cyclin B1 was overexpressed in 55 cases（84.6%）.No association was found between overexpression of Cyclin B1 in patients with age,gender,tumor site.As to TMN stage,Cyclin B1 expression is significantly related to T stage（P=0.002）.Furthermore,the expression of Cyclin B1（the locations of Cyclin B1 in the keratin pearls were excluded）was association with the clinical pathological stages.3.Correlation between Hsp90a and Cyclin B1 expression41 paired specimens were stained with Hsp90a and cyclinB1 antibody respectively,and the matched areas were observed and photographed one by one.The result showed the Hsp90a expression was positively correlated to Cyclin B1 expression（Gamma=0.503,P =0.015）in cancer cells except the center of keratin pearl area.The keratin pearl is a character of well-differentiated ESCC,which is further confirmed by HE staining.Whereas the strong staining signal of Cyclin B1 always appear in the center of the keratin pearls,the Hsp90a expression levels are low and even became negative in the center of them,which indicates the Hsp90a might be involved in the regulation of cyclinB1 expression and degradation.The statistical analysis was done with the 44 matched keratin pearls,and the result showed there was a negative correlation between the expression of Hsp90a and cyclinB1.（Gamma=-0.692,P =0.007）4.Survival analysisKaplan-Meier survival analysis was used to determine survival rate with respect to the expression of Hsp90a and Cyclin B1.The result showed that higher Hsp90a expression were significantly associated with the worse prognosis（P=0.002）and Cyclin B1 showed the same tendency.（P =0.002）.2.Oxidative stress in TE-1 cells model established by H₂O₂1.The result of CCK-8 colorimetric assay cell viability:Under 500pM H₂O₂ concentration,the inhibition of cell proliferation was time dependent（n=3,P<0.05）,With the time prolongation,the inhibition gradually increased（n=3,P<0.05）,（cell viability:control>oxidative stress 6h,24h,48h,P<0.05）;2.Flow cytometry detection of oxidative stress TE-1 cells cycle results:oxidative stress TE-1 24h presented obvious G2/M phase arrest（G2/M phase arrest:control<oxidative stress 6h,24h,48h,P<0.05）;3.Western-blot detection of oxidative stress on the expression of Hsp90a and Cyclin B1 results:oxidative stress lead to increased expression of intracellular Hsp90a at 6h,24h decreased to normal;Cyclin B1 increased with the time prolongation;CONCLUSIONS:1.The expression of Hsp90α and Cyclin B1 was not associated with lymph node metastasis and TNM stage,tumor size and gender.However,significantly increased Hsp90a and Cyclin B1 expression was more frequently observed in later clinical pathological stage than other stages.The survival analysis results showed that higher Hsp90a expression were significantly associated with the worse prognosis and Cyclin B1 showed the same tendency.2.There is a negative correlation between Hsp90a and Cyclin B1 in keratin pearls,but the relationship is opposite around the keratin pearls.The decrease or deletion of Hsp90a expression may be related to the abnormal activation of Cyclin B1 protein,and play an important role in the initiation and the development of ESCC.