The Role Of Smac In Apoptotic Pathway And Its Antitumor Effect In Vitro And In Vivo

Smac is the second mitochondrial protein, along with cytochrome c,that promotesapoptosis by activating caspases. Smac is a normal mitochondrial protein. Afterreceiving aPoptotic sighals, ndtochondria not only releases cytochrome c to inducethe formation ofcaspase-9 activating aPoptosome, bu aiso releases Sma tocounteract the dribitory activity of IAPs. Mafore Smac binds tO all Anown hUInanIAP family members and relieves thcir inhibition of caspases. The achvated caspasesare nessW for almost all cell aPoptosis pathways. So Smac play an boortant rolein celI apoPtosis.In this stUdy, the hUInan osteosarcoma cell line Saos-2 was used to establish theaPoptosis model. First, the effect of Smac expression on cell growth effect wasstudied. Secondly, if Smac can enhance aPoptosis in various apoptotic pathways wasexPlored. Thirdly,we boer discussed the counteract betWeen smac and otherapoptotic regUlatory protein such as P53 \ Bcl-2 and Bax. Lastly, the anh-boor effectof Smac both in the and vivo was investingated. The datu may provide boerinformation for POssibility of Smac aPPlication in cancer theraPy.To inveshgate the biological effect of Smac exPression on Saos-2 cell, smac andanti-sense smac were tusfected into the Saos-2 cell. The change of cell cycle, cellgrowh curv and the sensihvity to etoposide were then analysed. The resultsdemonstrated that Over-exPressed Smac could Promote the sensihvity of the cells tochemotherapeuhc trig but has little affect on cell cycle or cell growth. habition ofSmac exPression with ani-sense smac could facilitate the cell growth and Gl/G0phase cells would be increased, which the sensihvity to chemotheraPy drug decreased.To investigate the role of Smac in apoptosis, etoPside and M to induce aPoptOsiswere used. The Smac distribution during aPoptosis was detected by westemblodrig.The resultS shOwed tha: Smac is a protein localed on mitochodrial and released to thecytosol M aPoptOsis. Smac increases the cell sensihvity to apoptosiss in bothndtochondrial and the wr aPoptotic pathWays.In order to StUdy the relationship between Smac and othe aPOPtosis-regUlatingprotein includin P53' Bcl-2. Bax and Smac, we examed the distribuhon of thecytosol Smac and the aPOPtohc rate of the various tranSfected Saos-2 cells indUed bydiW Wtohc signals. The results demoMd tha Bcl-2 could block the Smacrelease to the cytOsol in aPoPtOsis process induced by na' P53 andetoposide.Whereas, Bcl-2 had no effect on Ad.smac-indUed aPoptOsis, whichindicated that Smac functions in the down-stream ofbcl-2 in aPoPtohc pathWny. Smacwas needed in P53-induce aPoptosis, Which can be inhibited by BcL2 over-exPressingto reduce Smac releasing into cytosol or thensfection with anti-sense smac to inhbitthe Smac exPression. Bax can also enhance the cell aPOptosis by regUlating Smacrelease. The data boer illustrate the role of Smac in aPOptosis and exPand theknowledge of the apoptosis regulation.Because Smac was involved in almost apoptotic pathways and was able to turn the apoptotic-resisting cells into apoptotic-sensetive ones, it may have potential application in cancer therapy. To explore the anti-tumor effect of Ad.smac in vitro, Replication deficient adenovirus Adsmac was successfully developed. The anti-tumor effects of Smac on various tumor cells were observed after Ad.smac infection. Adsmac can effectively induce the death of Saos-2, HepG2^ PLC/PRF/5, ACC-2 in vitro. Ad.smac treatment alone can inhibit the HepG2 growth in null mice, and when combined with 5-FU, its anti-tumor effects were greatly enhanced which induce complete tumor regression.the results suggest that this new strategy may be useful for the treatment of cancer.