Mechanism Of Survivin Upregulation Induced By DNA Damage Reagent Epirubicin

Survivin is the smallest member of eight IAP (inhibitor of apoptosis protein) family: X-IAP,c-IAP-1,c-IAP-2,NIAP,ILP2,ML-IAP/Livin,apollon and survivin ,and is the most attractive for its significance in clinical and basic research. However, survivin was is a unique member of the IAP gene family in that it is expressed in most human cancers, but largely undetectable in most differentiated adult tissues. Another feature of survivin is a cell-cycle dependent expression: survivin was very low in G1 phase, increased in S phase and peaked in G2/M phase. More importantly, in retroclinical research, highly expressed survivin in tumors is an index of cancer rapid development and malignancy, which always means resistance against cancer therapy-induced apoptosis, abbreviated patient survival and high relapse. Otherwise, inhibition of survivin by antisense, RNA interference, ribozyme, dominant negative mutant, small molecular inhibitors can increase sensitivity of cancer to therapy. Hence, survivin is called"pivotal cancer gene"and many projects targeted survivin was in preclinical or clinical stage.Most attractively, in basic cancer-therapy research, DNA damage reagents such as doxorubicin and cisplatin can induce survivin upregulation in many cancer cells lines and tissures, which causes resistance of cancer to chemotherapy. Otherwise, inhibition of survivin can increase sensitivity of cancer cells to DNA damage stress. Howerever, mechanism of resistance to DNA damage caused by survivin was unclear. There is a scientific problem why survivin is upregulated by DNA damage drugs, and so we adopts the common drugs epirubicin and HeLa cells as experimental materials to explore the mechanism of survivin upregulation in DNA damage stress. At last, it may provide rationale for anticancer strategy targeting survivin.Firstly, we make a model of survivin regulation: survivin was detected by immunoblot in HeLa cells treated with epirubicin in different time points and concentrations. Immunoblots analysis showed that survivin was induced upregulation in a time and concentration dependent manner. Furthermore, synchronization of HeLa cells confirmed that survivin was strictly regulated in a cell cycle dependent manner. Survivin was also upregulated by epirubicin in synchronization in the G1 phase, that is to say, responsive upregulation of survivin by epirubicin was not cell cycle dependent. In the counting viable cells, inducible upregulation of survivin in HeLa cells was associated with therapy-resistance: viable cells decreased significantly in survivin-knockdown HeLa cells.Secondly, eukaryotic gene is regulated in multiple level and complex process, and so experiments of different level was designed to seek key step of survivin control in DNA damage response. Activity of survivin proximal promoter was almost not affected by epirubicin in dual luciferase assay; survivin had no change at mRNA level in real-time PCR after stimulated by epirubicin. Howerer, the protein stability of survivin in HeLa cells detected by cycloheximide is increased by epirubicin. At the same time, exogenous survivin controlled by the promoter in the pEGFP-plasmid was induced upregulation, too. Hence, epirubicin had an effects on survivin expression at protein level, and repressed degradation of survivin.It is reported that p53-survivin pathway is important for resistance of cancer to chemotherapy. Considering that p53 is degraded by HPV-E6(an oncoprotein from human papillomavirus,HPV) , exogenous p53 was overexpressed in HeLa cells. Accidentally, activated p53 could represse proximal promoter of survivin, but not affected on survivin expression at protein and mRNA level. However, HeLa cells overexpressing p53 caused much death and weakend delay of S phase. We proposed that although p53 repressed transcription of survivin, other mechanism of survivin regulation neutralized transcriptional repression of p53.Thirdly, immunoflorescence analysis showed that survivin translocated from cytoplasm to nucleus after stimulation by epirubicin. The same results was confirmed in the experiment using GFP- tagged fused protein. Immunoblot analysis in different cell fractions showed survivin was nuclear accumulation: protein of survivin in the nuclei is elevated gradually with the increasingly concentration of epirubicin, while protein of survivin in the cytoplasm was almost unchanged. Counting cells located in cytoplasm or nucleus under the flurescence microscopy at different time points,we found that survivin located in the nuclus in most cells within 3 hours, which was not related to cell cycle. It is reported that CRM-1/survivin complex is required for survivin location. CRM-1 is a transportation protein in the nuclear pore and transport many protein between cytoplasm and nucleus, such as p53, cyclin B, cdc25c. it is wondered if epirubicin had an effects on function of CRM-1.Co-IP analysis using GFP antibody showed that epirubicin had no effects on the binding activity of GFP-survivin and CRM-1. Observation by confocal microscopy showed that quantitative colocalization of survivin and CRM-1 was not affected by epirubicin, but survivin and CRM-1 colocalized in the nucleus. On the contrary, survivin and CRM-1 colocalized in the nuclear envelope in the control cells. Therefore, epirubicin change location of survivin in the cells through the relocalization of CRM-1/survivin complex, and it interfered exportation of survivin into cytoplasm.Forthly, immunofluorescence analysis in situ detergent extraction showed that GFP-survivin and DAPI colocalized in the nucleus, that is to say, epirubicin made survivin binds to chromatin structure. It is accidentally found that epirubicin powerfully prevent HeLa cells into mitosis in hypotonic Chromosome spreads experiment. when survivin was inhibited by RNAi, some HeLa cells could enter mitosis and manifested abnormal chromosomes. During mitosis, survivin binds to Aurora B,INCENP,Borealin to form chromosome passenger complex (CPC), which binds to centromere and contributes to form spindle. Hence, binding of survivin and chromatin may disable formation or location of CPC, which prevents HeLa cells into mitosis and may be in favour of DNA repair. In comet assay, DNA damage in HeLa cells overexpressing wt survivin is in the lower degree. On the contrary, DNA damage in survivin-knockdown HeLa cells is in the higher degree. This results confirmed that survivin can enhance DNA repair, but the mechanism needs to be explored deeply. Transient transfection with wt survivin weakened sensitivity of HeLa cells to epirubicin. Otherwise, Transient transfection with survivin T34A and survivin RNAi enhanced sensitivity to epirubicin. In apoptosis analysis, inhibition of survivin by transient transfection with survivin T34A increased apoptosis by 79.2%. Hence, it is implied that survivin antagonize DNA damage-induced apoptosis, which is at least partly caused by function of survivin in DNA repair.In summary, it is found that upregulation of survivin induced by epirubicin in HeLa cells was associated closely with resistance of cancer to chemotherapy. DNA damage reagent epirubicin can make survivin bind to chromatin fastly, which may prevent or weaken degradation of survivin in the cytoplasm. Therefore, haf-time of survivin is increased ,while survivin was not changed at RNA level. In present study, it is first time that we observed binding of survivin to chromatin in DNA damage response, which may involve in prevention of mitotic enter and DNA damage repair. This detail may provide a clue for exploring mechanism of antagonization to DNA damage by survivin, and contribute to uncover molecular mechanism of antitherapy caused by survivin, which provide scientific instructions for utility of clinical DNA damage drug.