Role Of HSP70in The Apoptosis Of A549Cells Induced By Actinomycin D

Background:Lung cancer as a primary malignant tumor, the morbidity and mortality is showing an upward trend year after year in china. Therefore, study of the pathogenesis of lung cancer, and the establishment of some anti-tumor therapy for lung cancer, is becoming a hot topic of the life science research.The JNK signaling pathway is a mitogen-activated protein kinase pathway, it is mainly mediated by extracellular signaling to the cell interior, and then initiate apoptosis program, which led to apoptosis. Heat shock proteins Heat shock proteins (HSPs). also known as heat stess protein, is a highly conserved protein which was produced because of the body is stimulated by the various internal and external factors. Its biological functions involved in maintaining the conformation of the intracellular protein, promote protein folding processing and transit, as well as molecular chaperone function etc HSP70is an important member of the heat shock protein family of proteins, the study found that it have an extremely important relationship in tumorigenesis, development, and the occurrence of drug resistance and prognosis. Studies have shown that high expression of HSP70in tumor tissue, involved in the process of tumor development, was closely related to tumor proliferation and the malignant degree. Park, Hee-sae studies have shown that HSP70can inhibit JNK activation, thus affecting the role of c-jun N-terminal kinase apoptosis signal transduction pathways involved in apoptosis.The subject to be through the establishment of an apoptosis induction model, actinomycin D as exposure agent to role in lung adenocarcinoma cell line A549, flow cytometry method was detected apoptosis rate, western blot method was detected HSP70and phosphorylation of JNK expression, through the mechanism study of HSP70in the JNK pathway on the apoptosis of A549cells, provide some experimental and theoretical basis for the treatment of tumors of this program through the blocking of HSP70anti-apoptotic effects.Objective:The high expression of HSP70can or not cause actinomycin D(Act D)-induced apoptosis rate changing, if changed, could explore the possible mechanism of HSP70caused in this change.Materials and Methods:1. Determine the exposure concentration:the MTT assay was detected to different concentrations of Act D exposure of the cells activity in each group. Take the logarithmic growth phase cells, digested and respended cells, adjust the cell concentration5×104/ml, transferred to96well plates, each hole200μl,5%CO2,37℃incubation to which cells covered the bottom of96well plates. Absorb culture fluid, added the final concentration of (0,5,10.20,40,60,80,160) μg/ml actinomycin D respectively(the culture medium as a solvent to prepare of the corresponding concentration of the Act D). one group for the solvent control group (only plus1%DMSO). In each group of five parallel holes, cultured for12h, washed2to3times by PBS, then added to each hole5mg/ml MTT dye20μl into the incubator to continue cultured for3h, discard culture medium, added to each well of DMSO100μl, shock into the shaker for10min at room temperature, the ELISA detected’ absorbance at492nm, and then Calculate the cell death rate according to the formula, determine the exposure dose based on the results.2. Experimental groups:Actinomycin D (Act D) as an apoptosis inducer, the A549cells were divided into four groups:①normal control group;②Act D exposure group;③The JNK inhibitor of SP600125+Act Group D;④eat treatment+Act D group.3. Identification of Apoptosis:Annexin V-FITC and PI anchor double staining to stain cells, when completed, flow cytometry to detect the apoptosis rate of each group.4. HSP70protein and JNK protein expression were detected by western blot: removed each group cells, protein extraction kit to extract total protein each treated group; then transferred to a membrane after SDS-PAGE gel electrophoresis, antibody incubation, ECL coloration, use Gelpro4software to analyze the relative expression levels of the two kinds of proteins, then proceed the correlation analysis.5. Obtained experimental data are express by mean plus or minus standard deviation (x±s), the application SPSS12.0software for statistical analysis. The differences between the groups were compared using analysis of variance, pairwise comparisons using LSD assay, the detection level of α=0.05.Results:1. To detection apoptosis rate of each group:These results show that the apoptosis rate of the Act D group with the control group compared to higher, the difference was statistically significant(P<0.05); Inhibitor group compared with the control group, the difference was no significant(.P>0.05); Heat treatment group compared with the control group, the difference was no significant(P>0.05). Act D group as a control group and compared with the inhibitor group, apoptosis rate decreased, the difference was statistically significant(P<0.05), heat treatment group compared with the Act D group, the apoptosis rate also decreased, the difference was statistically significant (P<0.05). Handle the A549cells with Act D, the apoptosis rate increased.but in the conditions of inhibitors or heat treatment, compared to Act D group, the apoptosis rate reduced(P<0.05).2. western blot detect HSP70and p-JNK protein expression:①For the relative expression levels of HSP70which was obtained were detected by analysis of variance and LSD tests, the results showed that blank group as the control group, the relative expression levels of HSP70increases in Act D treatment group, the difference was statistically significant (P<0.05); Inhibitor group compared with the control group, the expression were increased, the difference was statistically significant (P<0.05); Heat treatment group compared with the control group, expression levels were significantly increased, the difference was statistically significant (P<0.05). Act D group as a control group and compared with the inhibitor group, the difference was statistically significant (P<0.05); the heat treatment group Compare with it, the difference was statistically significant (P<0.05).②For the relative expression levels of obtained p-JNK were detected by analysis of variance and LSD tests, The results showed that the blank group as the control group, Act D treatment group relative expression level of HSP70increased, the difference was statistically significant (P<0.05); Inhibitor group compared with the control group, the expression level decreased, the difference was statistically significant (P<0.05); Heat treatment group compared with the control group, the expression level decreased, the difference was statistically significant (P<0.05). Act D group as a control group, compared with the inhibitor group, the expression levels is lower, the difference was statistically significant (P>0.05); Heat treatment group compare with it, the expression was significantly decreased, the difference was statistically significant (P<0.05).Conclusions:The result showed that high expression of HSP70inhibited Act D induced of apoptosis A549cells, high expression of HSP70inhibited p-JNK protein expression. Thus, The possible mechanism of HSP70inhibition of A549cells apoptosis as follows:The high expression of HSP70inhibiting p-JNK expression, thus inhibiting the JNK apoptotic signal transduction pathway, thereby inhibiting apoptosis of A549cells which induced by Act D.