The Study Of Heat Shock Protein 27 In Neoplastic Progression Of Benign Breast Lesions And Its Impact On Biological Behaviour And Chemotherapy Sensitivity Of Breast Cancer Cells

PartⅠExpression of HSP27 in proliferative breast lesions of varying histological risk and breast cancer and their significanceObjective To define and compare the level of expression of hormone-related protein HSP27 by RT-PCR in proliferative breast lesions of varying histological risk (hyperplasia of usual type and atypical ductal hyperplasia)and breast cancer tissues, to asssess the function of HSP27 in breast cancer initiation and development,analyze its expression against clinical data in breast cancer.Methods a cohort of 68 patients who had primary breast carcinomas treated by surgery at Qilu hospital between December,2004 and June,2005 were investigated. 27 hyperplasia tissues of usual type,15 atypical hyperplasia tissues in the same period were collected.With reference to the expression ofβ-actin,the expression of HSP27 mRNA was evaluated by reverse transcription-polymerase chain reaction (RT-PCR)in these samples.Results Expression of HSP27 in hyperplasia of usual type,atypical ductal hyperplasia and breast carcinoma was 0.251±0.032,0.611±0.038 and 0.589±0.041 respectively.Compared with normal hyperplasia,HSP27 was highly expressed in breast cancer and atypical hyperplasia(P＜0.05).In adjacent non-neoplastic tissues, HSP27 expressed at 0.377±0.023,which was lower than cancer tissues(P＜0.05), higher than normal hyperplasia tissues but didn't reach the significant point.In breast cancer tissues,the expression of HSP27 was positively related with ER expression(r_s =0.764,P＜0.05),but had no association with menstrucal status,pathologic type, histological stage,tumor size,PR status,HER2 status and axillary node status.Conclusions The differential expression of HSP27 modulates the phenotypic behaviour of morphologically benign epithelial cells.HSP27 overexpression existed at the early stage of initiation of breast cancer and hence may be an important determinant in initiating,or promoting a population of human mammary cancers, especially those with positive ER status.HSP27 may be a new target in the prevention and treatment of breast cancer.PartⅡConstruction and identification of eukaryotic expression vector for human heat shock protein 27(HSP27)Objective To construct the eukaryotic expression plasmid of recombinant human heat shock protein 27 and to analyze its expression in breast cancer cell line MDA-MB-231.Methods Total RNA was isolated from human breast cancer cells MCF-7 in which HSP27 highly expressed,then mRNA was reverse transcribed into cDNA.The full-length coding sequence of HSP27 was constructed by standard PCR method by using a pair of specific primers which was designed according to the CDS sequence of HSP27 eDNA(forward primer 5'-AAGCTTCTGAGCAGACGTCCAGAGCA- 3', dark letter was the target sequence of HindⅢ;reverse primer 5'-GGATCC GCATCCGGGCTAAGGCTTT- 3',dark letter was the target sequence of BamHⅠ). Then the fragment was inserted into the shuttle vector T.Transformation of E.coli DH-5αwith recombinant plasmids and identification of bacterial colonies containing recombinant plasmids by LB-agar plate containing 100μg/ml of ampicillin were conducted,and recombinant plasmids were extracted and purified.All sequences amplified by PCR were confirmed by complete sequencing.Correct sequences were cloned into the pCDNA3.1/myc-His(+)A vector and recombinant pCDNA3.1-HSP27 was acquired,pCDNA3.1-HSP27 was transfected into breast cancer cells MDA-MB-231 which has a low endogenous expression of HSP27.The HSP27 expression in transfected cells was evaluated by RT-PCR and Western blot.Results The amplified sequences were 679bp in length,including the full HSP27 coding sequence.Result of complete sequencing indicated that they were identical with the sequence of HSP27 CDS provided by Genbank(NM001540). Agarose gel electrophoresis indicated fragments of 5.5Kb and 679bp were got by cutting the recombinant plasmid pCDNA3.1-HSP27 with enzyme HindⅢand BamHⅠ. RT-PCR and Western blot demonstrated the expression of HSP27 was up-regulated in transfected MDA-MB-231 cells at both mRNA and protein levels.Recombinant pCDNA3.1-HSP27 was constructed,and it contained the correct recombinant human HSP27 sequences.Conclusions The pCDNA3.1-HSP27 plasmids can express the target gene at high level.The pCDNA3.1-HSP27 plasmid is constructed successfully and will be useful for further research.PartⅢEffects of HSP27 transfection on the biological behaviors of breast epithelial cell HBL-100 and breast cancer cell MDA-MB-231Objective To investigate the effects of HSP27 on the biological behaviors of breast epithelial cell HBL-100 and breast cancer cell MDA-MB-231 by transfection with eukaryotic expression plasmid of recombinant human heat shock protein 27.Methods Both HBL-100 and MDA-MB-231 expressed endogenous HSP27 at low level,pCDNA3.1-HSP27 was transfected into these two kinds of cells by lipofectamine.Cells transfected with pCDNA3.1/myc-His(+)A and those didn't receive transfection served as control.After G418 selection,transfected cells were cloned.The HSP27 expressions before and after transfection were detected by RT-PCR and Western blot to confirm a successful transfection.The morphology was observed by phase contrast microscopy.MTT assay was used to evaluate the cellular proliferating ability.DNA index,S-phase fraction and proliferation index was assessed by flow cytometry.The expression ofcyclinD1,p21^CIP1/WAF1and p27^KIP1was evaluated by RT-PCR.Adhesion,invasion and motility of MDA-MB-231 before and after transfection were analyzed by MTT and Transwell chamber respectively.The expression of CD44v6,MMP-9 and TIMP-1 were tested by flow cytometry.Results Stably transfected HBL-100 and MDA-MB-231 were successfully identified.Protein level of HSP27 in transfected HBL-100 and MDA-MB-231 increased 2.7,3.5 folds respectively compared with those untransfected.The change of morphology was not observed in both kinds of cells.Transfected HBL-100 presented a higher proliferation rate and higher S-phase fractions than those untransfected(P＜0.05).The level of p27^KIP1in transfected HBL-100 decreased significantly(P＜0.05).However,there were no difference in proliferation before and after transfection of MDA-MB-231.DNA index increased significantly after transfection in MDA-MB-231(P＜0.05).For MDA-MB-231,the adhesion ability on Matrigel increased significantly after transfection(P＜0.05).This was partly due to,if not all,the up-regulation of CD44v6 in transfected cells.The capability of invasion increased significantly too.The imbalance between MMP-9 and TIMP-1 induced by HSP27 transfectation may be the reason.In transfected MDA-MB-231,expression of MMP-9 was stimulated,whereas TIMP-1 level was down-regulated.The motility of transfected MDA-MB-231 was inhibited.Transfection of empty plasmid vector pCDNA3.1/myc-His(+)A didn't change the characters of both kinds of cells.Conclusions The HBL-100 and MDA-MB-231 subclones with HSP27 over-expression were identified.Through comparing the biological behavior before and after transfection in two kinds of cells,we concluded;up-regulation of HSP27 significantly increased the S-phase fraction and proliferation rate of HBL-100 which is partly,if not all,due to the downregulation of p27^KIP1.Simultaneously the DNA index of transfected HBL-100 increased.This maybe how HSP27 induce the neoplastic progression of benign breast lesions;MDA-MB-231 after transfection presented a higher DNA index;HSP27 stimulated the expression of CD44v6 and MMP9 and inhibited TIMP-1 expression in MDA-MB-231 which in turn caused its enhanced abilities of adhesion and invasion but limited motility.PartⅣThe study on impact of HSP27 expression on the sensitivity of breast cancer cells to antitumor drugs.Objective Cancer cells with overexpression of heat shock protein 27(HSP27) confer chemoresistance to doxorubicin(Dox).Paclitaxel(Pacl)was reported to suppress HSP27 expression in ovarian and uterine cancer cells.The purpose of this study is to determine whether Pacl inhibits the expression of HSP27 in breast cancer cells,to evaluate whether Pacl can sensitize breast cancer cells with HSP27 overexpression to Dox,and to define the more effective schedule for the combination of Dox with Pacl.Methods The HSP27 high-expressing human breast cancer cell lines MCF-7 and MDA-MB-435 and the HSP27 low-expressing cell line MDA-MB-231 were used in this study.The level of liSP27,topoⅡαandβexpression was assessed by western blot.The cytotoxic activities of Dox,Pacl and combination of these two drugs were evaluated by MTT and flow cytometric assay.Results Pacl(0.1μmol/L)inhibited HSP27 expression by about 2-fold in MCF-7 and MDA-MB-435,and up-regulate the level of topoⅡαandβwhile expression of HSP27 in MDA-MB-231 did not change significantly before and after Pacl.There were synergistic effects in both Pacl-Dox and Dox-Pacl sequence,while the former was stronger in cancer cells with HSP27 overexpression.MCF-7 and MDA-MB-435 treated in Pacl-Dox sequence indicated lower viabilities and a higher apoptotic rate compared with those treated in Dox-Pacl sequence.Conclusions Paclitaxel can significantly decrease the level of HSP27 in breast cancer cells overexpressing this protein.Compared with Dox-Pacl,Pacl-Dox sequence maybe a more effective schedule of chemotherapy to remove breast cancer cells with high HSP27 expression.