Influlence Of Silencing Survivin Gene In Combination With Heat Shock Protein 90 Inhibitor On Biological Features Of LoVo Cells

Background and AimColorectal cancer is one of the most common malignant tumor in the world,and also is a serious threat to human life and health.In our country,with the improvement of living standards and changes in diet,colorectal cancer morbidity and mortality trends were increasing year by year.Traditional treatment of colorectal cancer,with surgical excision,supplemented by chemotherapy and radiotherapy.However,there are still 20%of the patients lose the opportunity to surgery,and to chemotherapy and radiotherapy is not sensitive.Looking for a new chemotherapy drugs and gene therapy for the treatment of colorectal cancer provides a new direction.Combination therapy for colorectal cancer may provide new opportunities.Hsp90 plays a central role in the cellular stress response,which is consititutively up-regulated in cancer cells to enhance adaptation to enviromental changes.Specifically,Hsp90 is involved in the stabilization and conformational maturation of several proteins belonging to signal transduction pathways,which are deregulated in cancers and contribute to all major components of the malignnt phenotype.The chaperone function of Hsp90 requires the formation of a multichaperone complex,which is dependent on the hydrolysis of ATP and ADP/ATP exchange. Most current inhibitors of Hsp90 act as nucleotide mimetics,which block the intrinsic ATPase activity of this molecular chaperone.The first-in-class inhibitor to enter and complete phaseⅠclinical trials was the geldanamycin analogue,17-allylamino-17-demethoxygeldanamycin.The results of these trials have demonstrated that Hsp90 is a valid drug target.Evidence of clinical activity has been seen in patients with melanoma,breast and prostate cancer.2003,The indentification of Survivin as a new client protein for Hsp90 links the cellular stress response to the dual cell viability/mitotic checkpoint maintained by Survivin.Survivin is a relatively unique member of the inhibitor of apoptosis protein(IAP) family in that it contains a single baculovirus IAP repeat(BIR) domain combined with a COOH terminalα-helix coiled-coil domain instead of the more common zinc-binding RING finger,and play an important role in the generation,development and metastasis of human large intestine cancers。it is the most powerful apoptosis inhibition gene.Because of the repression of apoptosis by Survivin gene carcinoma cells can keep on cleavaging and proliferating,induce carcinoma cells metaptosis.And the patients or cell lines positive expressing Survivin resist chemotherapy and radiotherapy.Related researches find the positive rate of Survivin gene express in colorectal carcinoma is more than 60%,but it has even no express in normal tissue and colorectal benign tumor.Further more than half of the Survivin positive patients has been found metaptosis.Our group pre-test the use of siRNA(small interfering RNAs) and short hairpin RNA(shot hairpin RNA,shRNA) technology in colorectal cancerLoVo cell to silence gene Survivin,the results of moreLoVo cell block in the G0/G1 period,an increase of apoptosis,Survivin gene expression inhibited the decline inLoVo cells.In this study,We will observed the effcets of Hsp90 inhibitor on proliferation,apoptosis and cycles of colorectal cancer LoVo cells and observed if silencing Survivin gene enhance the sensitivity of LoVo cells to Hsp90 inhibitor 17-AAG and to explore its possible mechanism.Method1.The effects of Heat shock protein 90 inhibitor 17-AAG on proliferation,apoptosis and cycles of Lo Vo cells1.1 LoVo cells were treated with 17-AAG.The cell proliferation inhibition rate was evaluated by MTT,assay 50%inhibiting concentration(IC50)1.2 karyomorphology of apoptotic cells was observed by using Hoechst 33258 staining1.3 the apoptotic rate and cell cycle distribution by flow cytometry analysis combined with Annexin V-FITC/PI staining1.4 The cell cycle were detected by flow cytometry with PI staining.1.5 The expression of Hsp90 client protein Survivin was detected by western blot.1.6 Activity of Caspase-3 was evaluated with a commercial spectrophotometric kit.2.The effects of Heat shock protein 90 inhibitor 17-AAG on proliferation and cycles stable transfected Survivin-shRNA-pGenesil-1 plasmid LoVo cells.2.1 Using lipofetion technology to build a pre-test of Surviving-shRNA-pGenesil-1 plasmid transtected into colorectal cancerLo Vo cells,G418 selection,monoclonal cell culture choose to build a stable cell line.2.2 Western blot and ImageAnalyst analysis software were used to survey the proteic level of Survivin;2.3 LoVo cells were treated with 17-AAG.The cell proliferation inhibition rate was evaluated by MTT,assay 50%inhibiting concentration(IC50)2.4 PI staining and flow cytometry analysis were used to examine cell cycle. Results1.17-AAG time-dose-dependently inhibit the proliferation of Lo Vo cells,after 100ng/ml,500ng/ml and 800ng/ml 17-AAG exposure for 24h,the cell proliferation inhibition rate was (21.073±0.147)%,(40.807±0.853)%,(60.343±1.446)%respectively,after exposure for 48h, the cell proliferation inhibition rate was increased to(27.287±0.530)%,(48.170±0.573)%,(80.970±0.747)%respectively,after exposure for 72h,the cell proliferation inhibition rate was to (34.747±0.662)%,(67.810±0.602)%,(88.416±0.533)%;Recording to the cell proliferation inhibition,IC50 of 17-AAG on LoVo cells respectively is IC50(24h):(823.967±48.146)ng/ml,IC50(48h):(356.813±5.045)ng/ml,IC50(72h):(180.245±1.468)ng/ml;72 hours after treated with 17-AAG,the karyomorphological diversity of apoptotic cells was observed in LoVo cells,the apoptosis rate increased,when treated with 500ng/ml 17-AAG,apoptosis rate was (11.800+4.547)%,the apoptosis rate of 100ng/ml and non-teated Lo Vo cells respectively was (6.863±2.535)%and(1.873±0.687)%(P＜0.01);17-AAG arrested cell cycle,When Lo Vo cells exposure to 100ng/ml 17-AAG for 72h,the cell ratio of G0/G1 phase was(61.243±3.539)%,when to 500ng/ml for 72h,cell ratio of G0/G1 phase was increased to(74.050±3.306) %,while LoVo cell did not treated with 17-AAG,ratio of G0/G1 phase was just(48.237±0.895)% (P＜0.01).Hsp90 client protein Survivin was deprived by 17-AAG from Lo Vo cells;when Lo Vo cells treated with 17-AAG for 72h,the activity of Caspase-3 markedly upregulated(P＜0.01).2.Through lipofection Survivin-shRNA-pGenesil-1 plasmid into Lo Vo cells,successfully constructed a stable interference Survivin gene expression in Lo Vo cell line,Western blot results showed that strains of cells transfected with Survivin protein expression was significantly lower than non-transfection group;17-AAG in a time-dose-dependently inhibition Stable transfection cell proliferation,after 100ng/ml,500ng/ml and 800ng/ml 17-AAG exposure for 24h,the cell proliferation inhibition rate was(31.826±0.283)%,(54.500±0.485)%,(76.297±0.628)% respectively,after exposure for 48h,the cell proliferation inhibition rate was increased to(39.380±0.356)%,(73.730±0.671)%,(78.433±0.569)%respectively,after exposure for 72,the cell proliferation inhibition rate was to(45.063±0.215)%,(83.110±0.276)%,(95.777±0.808)%; IC50(24h):(314.793±2.42)ng/ml,IC50(48h):(179.180±0.465)ng/ml,IC50(72h):(83.497±0.505) ng/ml,the results of Cell cycle,17-AAG at 100ng/ml can be caused by(75.880±3.157)%of transfected cells in the G0/G1 phase.Conclusion17-AAG time-dose-dependently inhibited Lo Vo cell proliferation,promote apoptosis by activation of Caspase-3 ways,can block the cell cycle in G0/G1 phase,reducing the level of Survivin protein;to Lo Vo cells which Survivin gene was silenced by shRNA,17-AAG not only can time-dose-dependently inhibited cell proliferation,but also can block the cell cycle in G0/G1 phase at lower concentrations.