| Heat shock protein 70(HSP70)has become a new target for anti-cancer therapy.It was involved with the occurrence,development,treatment,prognosis and drug resistance in breast cancer.In this dissertation,the expression of HSP70 was enhanced by heat shock(HS)or hyperthermia(43 ?),while the function of HSP70 was inhibited by HSP70 inhibitor VER-155008.Function of HSP70 in the cell proliferation was studied by MTT method.Meanwhile,the functions of HSP70 in the metabolism connected mitochondrial membrane potential,NADH,FAD and redox ratio(RR)in breast cancer cells were measured by the laser scanning confocal microscopy and the two-photon fluorescence microscopy.Firstly,the contents of HSP70 were measured by BCA protein quantitative kit and human HSP70 ELISA kit before and after HS.Moreover,the proliferations of the breast cells MCF-7,MDA-MB-231 and MCF-10A were measured by MTT method after the sole HS(43 ?)treatment,the sole VER-155008 treatment and the combination treatment.The results showed that there were significant differences of hyperthemia tolerances in three kinds of breast cells,from high to low were MCF-10A,MCF-7 and MDA-MB-231.The effects of the cell pro-proliferation and the proliferation inhibition after HS were related with the subtype of cells and the treatment durations(1h,2h and 3h).The proliferation inhibition was observed after HSP70 inhibition,and the effects of the inhibition presented dosage and treatment duration dependence.Meanwhile,the cell proliferations after the combination treatment were determined by the effects of both treatments.Secondly,the laser scanning confocal microscopy and mitochondrial membrane potential probe JC-1 were applied to visualize and quantify the morphology,membrane potential,content of mitochondria and cell size after sole HS treatment(1h),sole VER-155008 treatment and the combination treatment in MCF-7 and MDA-MB-231 after the treatment beginning 24h,48h and 72h.The results indicated that the mitochondrial networks of cells were intact,extended,and covering the majority of the cells in the control cells and the sole HS treatment cells,while they were both shrinkage,damaged,and fragmented dramatically from long filamentous interconnected tubules into short tubules with the sole VER-155008 treatment and the combination treatment.In addition,there were no obvious discrepancies of the mitochondrial membrane potential,contents of mitochondria and cell sizes between the sole HS cells and the control cells.while mitochondrial membrane potential and cell size were decreased,the mitochondrial contents were reduced in the sole VER-155008 treatment and the combination treatment.The decreases of the combination treatment were less than those of the sole VER-155008 treatment cells.It indicated that HSP70 inhibition induced apoptosis in MCF-7 and MDA-MB-231 breast cancer cells,and its promoting apoptosis effect could be alleviated by HS.Finally,autofluorescence NADH and FAD are important coenzymes in the redox respiratory chain,which can be used as sensitive endogenous oxygen-containing indicators to reflect the metabolic status and cell vitality in breast cancer cells.A metabolism measurement system of living cell was built to monitor the dynamic change of the metabolism related NADH,FAD and redox ratio in MCF-7 and MDA-MB-231 after HS 1h,2h and 3h separately.Moreover,the influences of the HS treatment,the VER-155008 treatment,the tamoxifen treatment and the combination treatment of two of them on NADH,FAD and redox ratio were measured in breast cancer cells.The conclusions revealed that the relationship between the change of metabolism and the effect of the proliferation promotion or proliferation inhibition after HS.In addition,the great significance of targeting HSP70 for anti breast cancer therapy was further highlighted.The redox ratio measurement based on two-photon fluorescence microscopy can be used in the efficacy of anti cancer therapy monitoring,drug screening and the therapy plan optimizing. |
PDF Full Text Request