The Role Of HSP70 Regulated LSCC Radiosensitivity And Its Molecular Mechanism

Recent studies have shown that certain intrinsic tumor gene play a key role in the molecular mechanism of radiation resistance,so modulating the biology of SCCHN in combination with a physically targeted agent can impact on radiation therapeutic outcome is a hot topic in the field.Therefore,we have obtained eight patient's gene expression with purificatied LSCC in our previous studies,and results indicated that HSP70 mRNA in tissues of LSCC was up-regulated 7.7 folds than that in the adjacent tissues.However,the effective molecular mechanisms of HSP70 in LSCC radiation resistance remains unknown.HSP70 is the most important member of heat shock protein family and plays an important role in the cells endogenous protection mechanisms.Act as one of the most conserved molecular chaperones,HSP70 is essential for proper folding and assembly of proteins.It has been repoted that HSP70.1 and HSP70.3 gene play a key role in the maintenance and repair of chromosome stability in mouse fibroblast cell after radiation,the radiosensitivity in HSF1 gene knockout mice is higher than that in wild-type mice.These results suggested that HSP70 is an important radiation resistance gene.However,these results came from the non-tumor cell experiment. Based on the above findings,as well as HSP70 cell endogenous protection function, we can initially speculate HSP70 is an imortant radiatherapy resistant gene in laryngeal carcinoma,but its molecular mechanism is unclear.C23,a nonhistone nucelolar RNA binding protein,which plays important role in maintaining the balance between anti-apoptosis and pro-apoptosis,become a hot topic in this field.In our previous studies,we have found that HSP70 could interact with nucleolin(C23) and inhibiting H₂O₂-induced cleavage and down-regulation of C23,thereby inhibiting reactive oxygen species-induced cell apoptosis.Nucleolin is an abundant nucleolar protein located in the DFCs and GCs of nucloli,and plays essential roles in promoting cell proliferation.There are two ways for radiotherapy to destruct tumor cells:(1) X-ray directly broke the cell DNA into fragmentations,leading to cell apoptosis;(2) X-ray released oxygen free radicals to attack tumor cells.Theoretically,it leading to cleavage and down-regulation of C23.Theoretically,we speculated that HSP70 was an important target molecular for LSCC,and down-regulation of HSPT0 was associated with cleavage and degradation of C23,which was an important molecular mechanism for the radiotherapy resistance of LSCC.Firstly,we constructed High-quality tissue microarray(TMA) with fifty tumor samples including different stages of LSCC,and then investigated whether HSP70 was associated with histological grade of layngeal squamous cell carcinomas(LSCC) using immunohistochemistry.Secondly,we screened an effective ASODN,and then the HSP70 antisense and random oligos were injected into laryngeal carcinoma xenografts through intratumoral injection,and the mice were treated with radiation.The treatment effect was observed. The volumes and weights of implantation tumors in the group injected with antisense oligos were less than that in group injected with random oligos.Thirdly,HSP70 expression in each group was detected by western blot and immunohistochemical staining.The results showed that HSP70 antisense oligos could effectively downregulate HSP70 expression in laryngeal carcinoma xenografts.The data showed that the expression of HSP70 in antisense group was lower than that in random group significantly.The levels of cleavage and degradation of C23 in each group were detected by western blot.The results indicated that HSP70 down-regulation was associated with cleavage and degradation of C23.Moreover,the apoptosis cells in each group were detected by tunel.The results showed that the apoptosis cells in group A were more than that in group B.We thus concluded that HSP70 expression was significantly lower in early stage of LSCC than that in late stage of LSCC(P＜0.05),and HSP70 might be a molecular target for LSCC radiotherapy by inhibiting cleavage and degradation of nucleolin.