The Effect Of Arsenic Trioxide On Imatinib-Resistant Cell Lines K562R And KBM5R

Objective:Chronic myeloid leukemia (CML) is a malignant clone disease induced by multipotent hemopoietic stem cell anomalous proliferation. Marked genetic abnormality is t (9; 22) (q34; q11) translocation, resulting in Bcr-Abl fusion gene. Its encoded chimeric protein Bcr-Abl non-receptor tyrosine kinase (NRTK), with abnormal tyrosine kinase activity, leading to itself tyrosine residues and many important substrate protein phosphorylation, and activation of multiple signal transduction pathway . So that the malignant transformation of cells, excessive proliferation, differentiation and inhibition of apoptosis, thus leading to CML. Imatinib(IM) as a selective tyrosine kinase inhibitor is currently most effective treatment of chronic phase CML. However, using IM in some patients for long time may develop resistance, it is a serious impact on the efficacy of IM. How to effectively overcome IM resistance become the most pressing problem. Arsenic trioxide (ATO) is an ancient drug , and has been re-known because of its unique efficacy to APL, ATO can induce malignant cells apoptosis through a variety of ways .In vitro, ATO has a cytotoxic effect for the majority of blood diseases and solid tumor cells. Our data of trial discovers that ATO not only inhibits the proliferation of IM-sensitive cell lines K562 and KBM5 also inhibits proliferation of IM-resistant cell lines K562R and KBM5R. it's inhibitory effect is more obviously for the IM-resistant cells with T315i mutation. Our test select human IM-sensitive and IM-resistant chronic myeloid leukemia cell lines for study in order to observe the influence of ATO on these cell lines at cell cycle and apoptosis ,and observe the changes of Bcr-Abl gene and protein level and its downstream related proteins P-STAT5(Tyr694)and PI3K-AKT signaling pathway. And wish to provide a theoretical basis for ATO treatment of CML and particular resistant to IM. Methods:(1) Detected the resistance of K562R,KBM5R cells to IM by MTT(2) Detected the influence of ATO on K562, K562R, KBM5, KBM5R cells growth at different concentrations and time points by MTT and draw growth curve;(3) Detected the influence of ATO on apoptosis and cell cycle of K562, K562R, KBM5, KBM5R cells by FCM.(4) Detected the influence of ATO on Bcr-Abl mRNA of the K562, K562R, KBM5, KBM5R cells by real-time RT-PCR.(5) Detected the influence of ATO on Caspase-3, 8,9 protein expression of the K562, K562R, KBM5, KBM5R cells by Western blotting.(6) Detected the influence of ATO on Bcr-Abl and its expression of downstream-related protein P-CRKL(Tyr207),CRKL,P-AKT(Ser473),AKT,P-STAT5(Tyr694),STAT5 of the K562, K562R, KBM5, KBM5R cells by Western blot.(7) Detected the influence of ATO on P21, P27 protein expression of the K562, K562R, KBM5, KBM5R cells by Western blot.Results:(1) K562R, KBM5R has a significant resistance to IM.(2) ATO can inhibit K562, K562R, KBM5, KBM5R cell growth, and with time - effect and the dose - effect dependence.(3) 48h MTT showed that the growth inhibition of ATO on K562R cell was no significant difference compared with the K562 cell line; the growth inhibition of ATO on KBM5R cell was significant difference compared with the KBM5 cell line, ATO has stronger growth inhibition role on KBM5R cells.(4) ATO can induce apoptosis of K562, K562R, KBM5, KBM5R cell lines, and has a dose - effect dependence, the effect of induction apoptosis of ATO on cells KBM5R is more significant compared with KBM5 cells.(5) ATO can make cell lines K562, K562R, KBM5, KBM5R arrest at G2-M phase in a dose - effect dependence.(6) ATO did not affect the volume of Bcr-Abl mRNA of K562, K562R, KBM5, KBM5R cells.(7) ATO can activate Caspase-3, 8,9 protein of K562, K562R, KBM5, KBM5R cells.(8) ATO can down regulate Bcr-Abl and its downstream-related proteins P-CRKL(Tyr207), P-STAT5(Tyr694), P-AKT(Ser473) protein expression of K562, K562R, KBM5, KBM5R cells in a dose - effect dependence.(9) ATO can up regulate P21, P27 protein expression of K562, K562R, KBM5, KBM5R cells in a dose - effect dependence. Conclusion:(1) ATO can inhibit proliferation of CML cell lines K562,K562R,KBM5,KBM5R in vitro.(2) Compared with wild-type, IM-resistant CML cells with T315i mutation is more sensitive to ATO cytotoxicity.(3) ATO can block CML cell lines K562,K562R,KBM5,KBM5R in G2-M stage and perhaps have a relation with the upregalation of p21 and p27 protein.(4) ATO can induce apoptosis of CML cell lines K562,K562R,KBM5,KBM5R by inhibiting downstream related proteins P-STAT5(Tyr694)and PI3K-AKT signaling pathway.