Protein Levels Of Caspase-3 And Bcl-X_L In K562 Cells During The Apoptosis Induced By Arsenic Trioxide And STI571

Chronic myelogenous leukemia (CML) is a disorder of malignant clonal hematopoietic stem cell, distinguished by the presence of the Philadelphia (Ph) chromosome. The Ph chromosome arised as the result of a reciprocal translocation between the long arms of chromosomes 9 and 22, t (9; 22). This fusion gene encodes chimeric protein Bcr-Abl. Bcr-Abl is a constitutively active tyrosine kinase with markedly enhanced tyrosine kinase activity and is critical for its transforming potential. Ectopic or endogenous Bcr-Abl protein expression in HL-60/Bcr-Abl or K562 cells respectively, upregulates several antiapoptotic protein level including BCL-XL. The ectopic and endogenous protein expression of Bcr-Abl was demonstrated to block the mitochondril permeability transition pore and release of cytochrome C, thereby inhibiting the activition of the 'excutioner' caspases and apoptosis. Studies demonstrated that inhibition of the enzymatic activity of Bcr-Abl could be an effective treatment of CML, as Bcr-Abl is present in most patients with CML, and essential for malignant transformation.STI571 was identified as a potent molecule that occupies the kinase pocket of the Bcr-Abl protein, blocking access to ATP and thus preventing phosphorylation of any substrate. Treatment with STI571 has achieved excellent effects in CML at all stages of the disease. However, there are limitations to the successful use of STI571 as a single agent includes the problem of resistance. It is nessesary to improve the quality or used by combination with other drugs. Arsenic trioxide (As₂O₃) was recently demonstrated to be an effective inducer of apoptosis in patients with relapsed acute promyelocytic leukemia(APL) as well as in patients with APL in whom all-trans-retinoic acid and conventional chemotherapy failed. Then the theraputic effect of As₂O₃ on K562 cell line was studied. It is demonstrated that As₂O₃ can induces K562 cell to apoptosis. But the exact machenism is unknow.To determin if combine STI571 with As₂O₃ could enhance the cytotoxic effects on K562 cells, and to explore the machanism of As₂O₃ or STI571 induced apoptosis. The inhibitory effect of As₂O₃ and STI571 used respectively or combinedly on K562 cells were examined. The protein levels of caspase-3 and Bcl-xL after incubation were studied.Methods:K562 cells were maintained in RPMI1640 medium supplemented with 10% PCS. 1 μ mol/L As₂O₃ and 2 μ mol/L STI571 were added to refresh cultured K562 cellrespectively or combinedly. Cellular numbers were accounted to examine the drug's inhibitory effect. The effects of apoptosis were determined by Giema's staining and genomic DNA fragmentation analysis by agarose gel electrophoresis. The Caspase-3 and Bel-XL protein level were detected by Western blot analysis.Result:(1) 1 u mol/L As₂O₃ or 2 μ mol/L ST1571 can inhibit the proliferation and induce apoptosis of K562 cells, (P<0.05). When used the two drugs combinedly the effects were enhanced, (P<0.05). (2) Analyzed by agarose gel electrophoresis, the DNA fragments became evident after incubation of K562 cells with 1 μ mol/L As₂O₃ , 2 μ mol/L STI571 respectively or combinedly for 48 h. (3) Incubation of K562 cells with 1 μ mol/L As₂O₃ or 2μ mol/L STI571 for 36 hours could activate caspase-3 as determined by a Western blot analysis. The activition effect was more significent in 1 u mol/L As₂O₃ + 2 μ mol/L STI571 group, (P<0.01). (4) The protein levels of BCl-XL were downregulated after incubation with 2μ mol/L STI571 for 36 hours, (P<0.01) . Incubation with 1 μ mol/L As₂O₃ also could downregulation the protein level of BCL-XL; but the effect was less significent than that of 2 μ mol/L STI571, (P<0.01). When 1 u mol/L As₂O₃ and 2 μ mol/L ST1571 used combinedly, the downregulation effect was more significant than used each alone, (P<0.01).Conclusions:(1) As₂O₃and STI571 could exert inhibitory effects on the proliferation of K562 cells. (2) STI571 could activate...