The Inhibitory Effect Of Huaier And Imatinib On Chronic Myeloid Leukemia Resistant Cell K562G01 And The Mechanism

Objective:Imatinib mesylate,a tyrosine kinase inhibitor specifically targeting the BCR-ABL fusion protein,induces hematological remission in many patients with chronic myeloid leukemia(CML).However,nearly 40% of patients with CML after receiving imatinib stopped taking the drug within five years due to primary drug resistance,secondary drug resistance and treatment intolerance.the resistance of leukemia cell to imatinib remains one of the great challenges in the treatment of CML.Huaier,a traditional Chinese medicine,has been generally used in the treatment of cancer.While the anti-proliferation effect of Huaier on imatinib-resistant CML cells and the possible mechanism involved is not clear.Here we study the anti-leukemia effect of Huaier in combination with imatinib in CML resistant cell line K562G01 cells Method:We measured the anti-proliferative effect of different concentration imatinib on K562 and K562G01 by CCK8,and anti-proliferative effect of different concentration huaier on K562G01 was also involved,then we calculated the resistance index and the huaier or imatinib half maximal inhibitory concentration(IC50)to K562G01.The combination index(CI)was calculated through the Compusyn software.The apoptosis rate and protein tyrosine kinase(PTK)activity in each group were assayed by flow cytometry and Elisa.Western blotting was used to assess the levels of proteins associated with apoptosis including Cleaved caspase-3,Bax,Bcl-2 and the possible signaling pathways proteins Lyn,P-Lyn,STAT5,P-STAT5 were involved.Result:The resistance index of K562G01 was about 21.6 times and huaier had anti-proliferative effect on K562G01 in a dose-and time-dependent manner.The IC50 of huaier and imatinib to K562G01 were 8.9 mg/ml and 5.63 uM respectively and the CI was lower than 1,suggesting the combined drugs had a synergistic effect on the growth inhibition of K562G01.The result of flow cytometry showed the apoptosis rate in combination group was more significant(P<0.05)compared with imatinib group.The results of Elisa and Western blot suggested that huaier decreased the PTK activity and the expression of BCR-ABL in K562G01.Furthermore,it also upregulated the expression of Cleaved caspase-3,Bax and downregulated Bcl-2 protein,the combined group was more significant(P<0.05).The total protein expression of Lyn and STAT5 had little change in each group,whereas,huaier decreased the expression of P-Lyn and P-STAT5 and in the combined group the level of P-STAT5 and P-LYN generated more decline compared with the group treated with imatinib(P<0.05).Conclusion:Our studies suggest that huaier have a anti-leukemia effect on imatinib-resistant K562G01 cell by inhibiting cell growth,promoting apoptosis,reducing BCR-ABL expression and PTK activity and it play a more significant role in leukemia in combination with imatinib.The anti-leukemic mechanisms are related to the signaling pathways of STAT5 and Lyn.