XIAP Inhibitor Embelin Interacts Synergistically With STI-571 To Induce Growth Inhibition And Apoptosis In K562 Cells

STI-571 is a tyrosine kinase inhibitor.STI-571 induces CML(chronic myeloid leukemia) cells death by effectively inhibiting the tyrosine kinase activity of the BCR-ABL onco-protein.STI-571 was approved by FDA for treatment of CML in 2001.However,the emergence of STI-571 resistance in some CML patients initially responsive to this agent and the observation that patients in accelerated phase CML or blast crisis are less likely to respond to STI-571 have prompted the search for additional approaches to the treatment of this disease.One possible approach to this problem involves increase of sensitivity of CML cells to STI-571 treatment.In this study,we tried to find some strategies to increase the sensitivity of CML cells to STI-571.We found that both Embelin,a small molecule XIAP inhibitor,and transfection of the ARF tumor suppressor gene,could increase the sensitivity of K562 cells to STI-571-mediated killing.STI-571,as well as Embelin,inhibited the growth of K562 cells in a dose-dependent manner.Compared with STI-571 or Embelin treatment alone,the combination of STI-571 and Embelin greatly inhibited the proliferation and clonogenicity of K562 cells in vitro.The Calcusyn analysis indicates that Embelin interacts in a highly synergistic way with STI-571 to inhibit K562 cell growth. Co-administration of low concentrations of STI-571(50 nM) and Embelin(20μM) resulted in a marked increase in apoptosis in K562 cell.Flow cytometry analysis showed that STI-571 potentiates the ability of Embelin to induce G1 cell cycle arrest in K562 cells.STI-571 and Embelin co-treatment of K562 cells also resulted in an increase in p21 expression,but exerted no effects on p27 expression.The expression of the anti-apoptotic proteins Mcl-1 and XIAP decreased dramatically in cells exposed to STI-571 and Embelin.Combined treatment with STI-571 and Embelin also resulted in an increase in the phosphorylation of Akt.STI-571 attenuated the expression of Bcl-2 induced by Embelin,but had no effect on the expression of Bcl-xl induced by Embelin.STI-571 or Embelin alone induced slight increases in the expression of the apoptotic protein Bim.Co-administration of STI-571 and Embelin together resulted in marked up-regulation in Bim protein levels.To further study the role of Bim in the synergic growth inhibition induction by STI-571 and Embelin,we generated a subclone of K562 cells(K562/shBim) in which Bim expression was suppressed by retrovirus-mediated RNAi.K562/shBim cells were significantly less sensitive to the STI-571 and Embelin co-treatment than parent K562 cells.Our findings suggest that Bim plays an important role in the enhanced cell death of K562 cells exposed to STI-571 in combination with Embelin.We further developed a STI571-resistant K562 cell line(K562/R) by culturing K562 cells in the presence of increased concentrations of STI-571.Interestingly,we found that Embelin can enhance the sensitivity of K562/R cells to STI-571 killing.Together,these findings indicated that STI-571 and Embelin synergistically induces growth inhibition of K562 cells.Embelin or expression of ARF enhances STI-571-mediated killing of K562 cells.The combination of STI-571 and Embelin may provide a useful strategy for treatment of clinical STI-571 resistant CML. Presently concerned methods of oncotherapy for various types of tumors involve synergetic inhibition of different signal pathways in tumor cells,and our work on the combined effect of STI-571 and Embelin on apoptotic induction and related signal pathways in leukemia cell K562 could be a beneficial and significant exploration for synergetic and targeted therapies for leukemia.