Heat Shock Protein Inhibitors On Cell Proliferation And Apoptosis In Human Nasopharyngeal Carcinoma

Objective:Different concentrations of human nasopharyngeal carcinoma Hsp90 inhibitor 17-AAG and catechins(EGCG) monotherapy or in combination induced apoptosis when CNE cells to explore new molecular targets for the treatment of nasopharyngeal carcinoma,for Hsp90 inhibitor alone or in combination with catechin(EGCG) treatment of nasopharyngeal carcinoma,enhance its therapeutic effect and provide experimental evidence. Methods:1.MTT assay drug inhibitory effect of different concentrations of 17-AAG,EGCG alone or combined effects of 24 h,48h and 72 h of CNE cells for in vitro growth inhibitory effect.2.Hoechst 33258 fluorescence staining of CNE cells in different concentrations 17-AAG,EGCG monotherapy or in combination for 48 h induced cell morphology.3.By Acridine orange(A0) staining and under the Fluorescence microscope and photographed of nasopharyngeal carcinoma with 7-AAG(0.625μmol / L),EGCG(80mg / L) or both drugs 17-AAG(0.625μmol / L) and EGCG(80mg / L),individual the morphological changes of cells,after the treatment for 48 h.4.By Acridine orange/Ethidium bromide(A0/EB) staining and under the Fluorescence microscope and photographed of nasopharyngeal carcinoma with in different concentrations 17-AAG,EGCG monotherapy or in combination for 48 h induced cell morphology.5.By flow cytometry(FACS) analysis to detect different concentrations of 17-AAG, EGCG alone and in combination 48 h human cell cycle changes of CNE.6.Annexin V- FITC / PI double staining CNE cells change in(0.625μmol/L)、EGCG(40mg/L)alone and combined effects of 48 h, the apoptosis rate.7.By RT-PCR to detect the CNE cells in 17- AAG(0.625 μmol/L),EGCG(80 mg/L) alone and combined 48 h,detection of apoptosis gene Bcl- 2,Bax m RNA expression and apoptosis signal transduction molecules Caspase-3 m RNA expression.8.By Western Blotting detection of human nasopharyngeal carcinoma cells CNE EGCG(60,80,100 mg / L) separately and combined with 17-AAG(0.625μmol / L) role in 48 h,changes in the expression of Bcl-2、Bax protein.9.Statistical analysis.All statistical analysis was performed using SPSS software(version,22..0). P-values < 0.05 were statistically significant.Results:1. MTT assay: 17-AAG(0.315、0.625、1.25、2.5、5μmol/L)inhibited CNE cells within 24h、48h and 72 h. EGCG(40、60、80、100mg/L)also inhibited CNE cells within 24h、48h and 72 h. 17-AAG( 0.625μmol/L)and EGCG(40、60、80、100mg/L)their inhibitory effect were both time dependence and concentration dependence. The inhibitiory effect of 17-AAG on CNE cells was significantly promoted when combined with EGCG,statistically significant difference(P<0.01).2.Optical microscopy, fluorescence microscopy(Hoechst 33258 staining, A0 staining, A0/EB double fluorescent staining) : With the drug concentration increasing, more and more irregular cell morphology, chromatin condensation, nuclear shrinkage, apoptotic bodies, autophagic vacuoles was observed.3. Flow cytometry(FACS): FACS analysis showed that 17-AAG alone arrest CNE cells mainly in the S phase,while EGCG arrest CNE cells mainly in the G2/M phase. When 17-AAG and EGCG were both added, CNE cells was blocked mainly in the G2/M phase and S phase. The resulted showed statistically significant differences(P <0.01).4.Annexin V- FITC / PI double staining: 17-AAG(0.625μmol/L)、EGCG(40mg/L) combination group showed higher CNE apoptotic rate than 17-AAG and EGCG group, and the difference was statistically significant(P<0.01).5.RT-PCR:CNE cells were incubated with EGCG(80mg/L), 17-AAG(0.625μmol/L) alone or combined(17-AAG/ EGCG) for 48 h. The resulted showed that 17-AAG/ EGCG group expressed higher Caspase-3 and Bax m RNA and lower Bcl-2 than 17-AAG and EGCG group.6.Western Blotting Detection:CNE cells were incubated with EGCG(40,60,80,100mg/L), 17-AAG(0.625μmol/L) or 17-AAG/ EGCG for 48 h. The combined administration promoted Bax protein expression significantly and suppressed Bcl-2 protein expression when compared to 17-AAG or EGCG alone.