An Experimental Study On HDE Inhibiting The Proliferation Of MDS Cell Line SKM-1

Section One:HDE Inhibit Proliferation Of MDS Cells Line Skm-1Objective:To observe the inhibition about MDS cells lines Skm-1 induced by HDE,and to explore the mechanism further.Methods:MTT colorimetric assay was used to examine the growth inhibition on Skm-1 cell by HDE;The cell morphological changes were observed using Hoechst33258 staining;The apoptosis rate and Changes in cell cycle were analyzed by flow cytometry;The expression of proteins which Regulate cell cycle and apoptosis were examined by Western blotting.Results:(1)Inhibition rates of Skm-1 cells treated with(0.05-0.125 mg/ml)of HDE for24 h were 3.30%,15.83%,49.53%,67.93%,48 h were 4.10%,11.30%,58.47%,80.60%respectively,The IC50 is 1.8mg/ml,0.8mg/ml.(2)Hoechst33258 staining showed the typical appearance of cell apoptosis after the cells were treated with(0.05-0.1 mg/ml)of HDE for 24h(3)The apoptosis rate were analyzed by Annexin V/PI flow cytometry showed the early-apoptosis rate of 24 hours were 4.49%,7.28%,9.37%,18.1%.G0/G1 phase arrest was also observed through flow cytometry,the G0/G1 phase ratio was 34.89%in Control,while 41.53%after 0.075mg/ml the HDE-treated,and the ratio increased to 48.02%(4)western blotting showed that the cell cycle-related proteins CDK4,CDK6 associated protein,CyclinDl down-regulation after treatment with HDE(5)HDE can increase the apoptosis-related proteins caspase-8,caspase-9,caspase-3 and PRAP expression in a dose-dependent manner significantly(6)HDE can up-regulate cytc,Bak,down-regulate Bcl-2,Bcl-xl,Mcl-1,XIAP1,CIAP1,CIAP-2 significantly,but Bax,Bid expression was not significantly affected.(7)HDE significantly inhibit PI3K,phosphorylated Akt(p-Akt)expression and total Akt,in addition,HDE also inhibited the expression of p65 and protein pp65Conclusions:HDE could significantly inhibit the growth of Skm-1 cells in a time-and concentration-dependent manner within a certain range of concentrations,and HDE also could induces apoptosis and cell arrest in Skm-1 cells,and the mechanisms is affected by Bcl-2 family proteins Bcl-2,Mcl-1,Bcl-xl and inhibit the expression of IAPs family cause mitochondrial damage makes Caspase-9,Caspase-3 activation-induced apoptosis and death receptor pathway may also have certain relationship.(3)HDE induced apoptosis through inhibition of cell cycle.(4)HDE induced apoptos Apoptosis,cell cycle,PI3K/AKT NF-κB inhibition of the PI3K/Akt signaling pathway and NF-κB signaling pathwaySection Two:Heat shock protein 90 inhibitors BIIB021 InhibitProliferation Of MDS Cells Line Skm-1Objective:To observe the inhibition about MDS cells lines Skm-1 induced by Heat shock protein 90 inhibitors BIIB021,and to explore the mechanism further.Methods:MTT colorimetric assay was used to examine the growth inhibition on Skm-1 cell by BIIB021;The cell morphological changes were observed using Hoechst33258 staining;The apoptosis rate and Changes in cell cycle were analyzed by flow cytometry;The expression of proteins which Regulate cell cycle and apoptosis were examined by Western blotting.Results:(1)Inhibition rates of Skm-1 cells treated with(50-400nmol/L)of BIIB021 for24 h were 5.10%、16.20%、38.40%、54.30%,48 h were 14.60%、28.70%、60.00%、79.40%respectively,The IC50 is 355.69nmol/L,168.6nmol/L.(2)Hoechst33258 staining showed the typical appearance of cell apoptosis after the cells were treated with(100-400nmol/L)of BIIB021 for 24h(3)The apoptosis rate were analyzed by Annexin V/PI flow cytometry showed the early-apoptosis rate of 24 hours were 3.95%、9.30%、14.50%、24.30%.G0/G1 phase arrest was also observed through flow cytometry,the G0/G1 phase ratio was 34.94%in Control,while 40.52%after 100nM BIIB021-treated,and the ratio increased to 42.91%after 200nM BIIB021-treated(4)western blotting showed that the cell cycle-related proteins CDK4,CDK6 associated protein,CyclinD1 down-regulation,caspase-8,caspase-3 and PRAP activiated after treated with BIIB021,BIIB021 aslo significantly inhibit PI3K,phosphorylated Akt(p-Akt)expression and total Akt,in addition,BIIB021 also inhibited the expression of p65 and protein pp65.Conclusions:BIIB021 could significantly inhibit the growth of Skm-1 cells in a time-and concentration-dependent manner within a certain range of concentrations,and BIIB021 also could induces apoptosis and cell arrest in Skm-1 cells,and the mechanisms is activated by Caspase-8 Caspase-3,Parp and cell cycle related protein-related arrest,and by affecting the PI3K/Akt and NF-κB pathway.Section Three:HDE Combined with BIIB021 Inhibit Proliferation Of MDS Cells Line Skm-1Objective:To study the synergistic killing effects of HDE combined with BIIB021 in MDS cell line Skm-1,and to explore its mechanisms.Methods:MTT colorimetric assay was used to examine the growth inhibition on Skm-1 cell by HDE combined with BIIB021;The cell morphological changes were observed using Hoechst33258 staining;The apoptosis rate and Changes in cell cycle were analyzed by flow cytometry;The expression of proteins which Regulate cell cycle and apoptosis were examined by Western blotting.Results:(1)BIIB021 combined with low-dose HDE group(0.075mg/ml)on cell proliferation has statistically significant(P<0.05)Compared with the control group.with concentration increased,the inhibitor rate arise,the low-dose HDE group with high-dose group has statistically significant(P<0.05).(2)Hoechst33258 staining showed more significant morphological changes and more apoptosis in combination treatment;(3)The apoptosis rate were analyzed by Annexin V/PI flow cytometry showed that:early-apoptosis rate low-dose HDE group was 8.25%,while control were 4.35%,100 nM BIIB021 group was 13.7%,early-apoptosis rate was 21.1%in combination treatment,G0/G1 phase arrest was also observed through flow cytometry,the G0/G1 phase ratio was 37.8%in Control,while 43.1%after low-dose HDE group or 100 nM BIIB021 group-treated,and the ratio increased to 93.1%with combination treatment.(4)Western blot showed an activation of Caspase-9,-3,PRAP of HDE combined with BIIB021 could down-regulate the expression of CDK4,CDK6,CyclinDl,PI3K,Akt,P-Akt,p65 and pp65.Conclusions:HDE has sensitizing effect for BIIB021 inhibition Skm-1 cell line proliferation,the mechanism may be related to PI3K/Akt and NF-κB signaling pathway activation.