HSP70在放线菌素D所致肺腺癌细胞增殖抑制和凋亡中的作用

Roles of HSP 70 in Actinomycin D-Induced Inhibition of Proliferation and Apoptosis in Lung Adenocarcinoma Cell

为探讨热休克蛋白70(heatshockprotein70,HSP70)在肺腺癌中的表达及其作用,首先采用免疫印迹检测经临床支纤镜确诊并行手术切除的肺腺癌组织标本中HSP70的表达,结果显示在癌旁正常组织中HSP70的表达量显著低于其在癌组织中的表达量.其次,采用HSP70反义寡核苷酸阻断A549细胞中HSP70表达后,经MTT和Hoechst33258检测发现,HSP70下调能显著促进放线菌素D(actinomycinD,ActD)所致的A549细胞增殖抑制及凋亡,反义寡核苷酸处理组与正义或随机寡核苷酸处理组比较P值均小于0.05.进一步构建HSP70真核重组质粒并瞬时转染A549细胞后,能显著增加A549细胞中HSP70表达,同时采用MTT和流式细胞术及基因组DNA琼脂糖凝胶电泳检测发现,HSP70过表达能显著抵消ActD所致细胞增殖抑制及凋亡,转染HSP70重组质粒组与转染空载体组相比P值小于0.05.上述结果提示,HSP70在肺腺癌组织中高表达,高表达的HSP70在降低肺腺癌细胞对ActD的敏感性及促进癌细胞增殖与抑制凋亡等方面发挥了重要作用.

It had been proved by many evidences that several heat shock proteins （HSPs） expression is up-regulated in tissue-derived primary lung cancer, and HSPs may play important roles in development, in resistant to drugs and in prognosis of lung cancer. However, there have not still systemic research on which HSPs, especially HSP70 can be or not thought as a new biological target in the therapy to lung cancer. In order to address the expression and roles of heat shock protein 70 （HSP70） in lung adenocarcinoma, immunoblotting was performed to detect the expression of HSP70 in tissue specimens from lung adenocarcinoma which were diagnosed unambiguously by branch fibromicroscopy and were excised. It showed that in the normal lung tissues, the expression of HSP70 was less then that in cancer tissues. After down-regulation of HSP70 protein by HSP70 anti-sense oligonucleotides in A549 cell line, MTT assay showed that the proliferation ofA549 cells was inhibited remarkably after the treatment with HSP70 antisense oligonucleotides and Act D. There had significant differential in HSP70 antisense treatment group followed by Act D treatment and Act D treatment group. Results of Hoechst33258 staining revealed that HSP70 antisense oligonucleotides could promote Act D-mediated apoptosis in A549 cells with a higher percentage of apoptotic cells （26.91±3.73）% than that of Act D-treated group （16.83±3.41）% （P 〈 0.01）. Furthermore, the expression of HSP70 protein was increased remarkably by transfection with a full length HSP70 recombinant plasmid into A549 cells. MTT assay revealed that HSP70 over-expression could block significantly the inhibition of proliferation induced by Act D, there had statistic significance between HSP70 over-expression group and only Act D-treated group （P 〈 0.01）. Furthermore, over-expression of HSP70 could significantly inhibit Act D-mediated apoptosis in A549 cells with a lower percentage of apoptotic cells （4.25±1.48）% in HSP70 transfection group, （12.89±2.03）% in vector transfection group and （14.37±2.56）% in Act D treatment group and disappearance of DNA laddering. These results suggests that increased levels of HSP70 in lung adenocarcinoma tissues decrease the sensitivity of lung adenocarcinoma to Act D, promote proliferation and suppress apoptosis of lung adenocarcinoma cells.