Studies On Expression And Significance Of HSP70 In Esophageal Squamous Cell Carcinoma

Esophageal carcinoma (EC) is one of common malignances in China.During past decades, the diagnosis and treatment of EC have been greatlyimproved. However, the prognosis of patients with EC is still relativelypoor, especially those with advanced stages. Long-term survival rate isquite disappointing and determined mainly by the clinical stage. But aninteresting phenomenon has been observed that a few patients with laterclinical stage or low differentiated carcinoma or metastasis of lymph nodehave a longer term survival. However, a few patients with ealier clinicalstage or high differentiated carcinoma or without metastasis of lymphnode have a shorter term survival. Therefore, how to accuratly predictand obviously improve prognosis of EC and increase the long-termsurvival rate of EC patients has been become one of the medical focusesin molecular biology during the past decades.Heat shock protein (HSP) is a ubiquitous highly conservedpolypeptide protein family. It consists of several subfamilies, includingHSP27, HSP60, HSP70 and HSP90 according to their molecular weight.Their main function is to sustain the stability of proteins essential forcellular viability, which is considered as functions on "housekeeping" andcell protection and important role of regulation. Recently, the relationshipof HSP with diseases, especially tumors, has been attracted increasinglymore attention. Studies on roles of HSP in apoptosis of tumor cells and anti-tumor immunity and prognosis of patients with carcinomas havebeen become the hot topics for researchers on oncology and immunology.Among members of HSP, HSP70 have been found to correlate closelywith prognosis of cancer. Researches have demonstrated thatover-expressions of HSP70 in gastric carcinoma, breast cancer andprostate carcinoma correlated inversely with depth of invasion, tumordifferentiation and metastasis of lymph node. Patients with highexpression of HSP70 tended to have a poor prognosis. However, therewere only a few reports on expression of HSP70 in EC. The prognosticsignificance of expression of HSP70 in EC is not completely understood.Esophageal squamous cell carcinoma(ESCC) is the chief histological typeof EC, and about 95 percent. Therefore, we mainly study the relationbetween HSP70 and ESCC.Part oneObjective: To explore the significance of HSP70 and its correlationwith prognosis of ESCC. Methods: 100 cases of tumor tissue embeddedin paraffin with clinical data were got. Immunochemistry were used toexplore the expression of HSP70. Results: The positive rate of HSP70protein in ESCC was 90%, and was higher than that in normal esophagealmucosa(13.3%); a higher expression of HSP70 was observed in ESCCcompared with the normal esophageal mucosa (P＜0.01), indicating thatHSP70 might participate in the carcinogenisis of ESCC. The positive rate of HSP70 protein in ESCC without metastasis of lymph node was 96%,and was lower than that in ESCC with metastasis of lymph node (84%);A higher expression of HSP70 was observed in ESCC with metastasis oflymph node (P＜0.05) and correlated with a lower rate of lymph nodemetastasis. The positive rate of HSP70 protein in ESCC withoutlonger-term survival was 83.3%, and was lower than that in ESCC withlonger-term survival (96.1%); A higher expression of HSP70 wasobserved in ESCC with longer-term survival (P＜0.05) and correlated witha better prognosis. Conclusion: we regarded that HSP70 might serve asan indicator to foretell the prognosis of ESCC.Part TwoObjective: To study the expression and significance of HSP70 in thenormal esophageal mucosa and ESCC. Methods: Fresh samples wereobtained from 33 patients who underwent resection of ESCC. All sampleswere put in liguid nitrogon immediately, which consisted of esophagealcarcinoma tissues and normal epithelia at cuting edge. The whole cellprotein was extracted, then protein quantification and western blottingwere performed for detection of the protein expression of HSP70. TotalRNA were extracted, then the mRNA expression of regulatory genes weredetected by semi-quantitative RT-PCR.β-actin, a house-keeping gene,was the internal standard. All results were analysed using Image Tool 3.0analysis software. The relationship between the expression of HSP70 protein and the parameter of clinical-pathology was analyzed at the sametime. Results: The positive rate of HSP70 mRNA and protein in ESCCwas 100% and 93.9%, and was higher than that in normal esophagealmucosa(69.7% and 45.5%, respectively); The results of detection bysemi-quantitive RT-PCR showed that the ratio of HSP70 toβ-actin inESCC and normal esophageal mucosa was1.35±0.21 and 0.73±0.19,respectively, and with great significance(P＜0.01); The expression ofHSP70 protein did not correlated with patient's age, gender and the depthof tumour infiltration, however, correlated with the status of metastasisof lymphonode. Conclusion: Compared with normal esophageal mucosa,the expression of HSP70 in ESCC was markedly increased and correlatedwith the status of metastasis of lymphonode. That indicated theexpression of HSP70 took part in the carcinogenesis and development ofESCC.Part ThreeObjective: To investigate the role of HSP70 in the proliferation,apoptosis and the ability of infiltration of esophageal carcinoma cells byinhibiting HSP70 expression with HSP70 antisense oligonucleotides.Methods: After esophageal carcinoma cells, Eca-109, were transfectedwith HSP70 antisense oligonucleotides, we analyzed its mRNA andprotein expression by RT-PCR and western blotting respectively; itsproliferation through cell grow curve; kinetics of induction of apoptosisand cell cycle by flow cytometry; and the ability of infiltration by transwell chamber. Results: After transfection with HSP70 antisenseoligonucleotides, the expression of HSPT0 was partially blocked; theproliferation of Eca-109 cells was apparently inhibited; and its inhibitoryrate was increased to 20.4% at 48h and 25.5% at 72h respectively; thecell number in G0/G1 phase was increased, but which in S phase wasdecreased; Apoptosis rate induced by HSP70 antisense oligonucleotideswas higher than those by HSP70 sense oligonucleotides or untransfectedgroup, with significant differences (P＜0.05); compared to two othergroups, the ability of infiltration has no obvious change in HSP70antisense oligonucleotides group(P＞0.05). Conclusion: HSP70 antisenseoligonucleotides could not only inhibit the proliferation but also inducethe apoptosis in esophageal carcinoma cells, and have no influence on theability of infiltration in Eca-109.