The Traditional Chinese Medicine Of Berberine Overcomes Imatinib-resistance In Chronic Myelogenous Leukemia

Chronic myelogenous leukemia(CML) is a clonal myeloprolifetative disease, which is caused by a reciprocal t(9;22) chromosome translocation, resulting in the short Philadelphia chromosome with the BCR-ABL oncogene. BCR-ABL regulates a large array of signal and survival pathways that ultimately lead to a enhanced the malignant behavior of leukemia cells. Imatinib is a tyrosine kinase inhibitor that acts as an ATP-competitve inhibitor of BCR-ABL. The emergence of imatinib for the treatment of CML has the revolutionary significance. Although Imatinib is a very potent and efficient drug in the chronic phase of CML, response rates reduce during disease progression. Drug resistance during Imatinib treatment is mostly related to point mutations occurring within the kinase domain of BCR-ABL,but in ~20% of CML cases resistance mechanisms do not involve altered BCR-ABL function. Among all mutatins of ABL kinase domain, T315 I mutation accounts for 15-20% of mutations of the ABL kinase domain. Berberine is an isoquinolinne alkaloid with multiple pharmacological activties, including anti-inflammatry and anti-diarrhea effect, the induction of apoptosis and anti-cancer effect. However, the mechanism of berberine-affect in the drug resistance of CML cells is not fully understood.To inverstigate the mechanism of berbeine-affected cell viablity, the drug-sensitivity of human CML cell line(K562 and BaF3-P210 cell lines) and drug-resistance cell line(BaF3-P210-T315 I and SFO2 cells lines) were treatd with different concentration of berbeine(1-8μM). The most significant cellular growth arrest was observed in the cells treated with 5μM of berberine for 24 h in both drug-sensitivity cell line and drug-resistance cell line. The drug-sensitivity of human CML cell line(K562 and BaF3-P210 cell lines) and drug-resistance cell line(BaF3-P210-T315 I cells line) were treatd with different concentration of Imatinib and berberine. The experimental results showed that berberine can increase the sensitivity and overcome the resistance to imatinib. Meanwhile, we also measure the ability of cell colony formation after treatment with berberine. The colony number of BaF3-P210-T315 I cell line in berberine group was fewer than in blank control group, but without a significant change in Imatinbi group compared with blank control. The colony number of BaF3-P210 cell line were downregulated in both berberine group and imatinib groups. Further study, our experiment show that berberine reduce BCR-ABL protein expression in the CML-like cell line(BaF3-P210) and drug-resistance cell line(BaF3-P210-T315I), without a sigifiicant difference in the levels of BCR-ABL mRNA after treatment with berberine. To explain the mechanism by which berberine down-regulate BCR-ABL protein, we examined autophagy and ubiuitnation levels, respectively, at the celluar level. The results showed that the pathway of ubiquitination was found after berberine treatment in BaF3-P210 cells, without autophagy. Immunoprecipitation show that berberine down-regulate BCR-ABL oncoprotein through ubiquitination pathway. Furthemore, using software of Discover Studio to docking berberin with BCR-ABL, We found that three regions of ABL, including SH2, protein kinase and F-actin binding regions, could dock with berberine. The Lys, which is the potential point of ubiquitination pathway in protein, was found in these thress regions of ABL.These results show that berberine down-reguate BCR-ABL oncoprotein through ubiquitination pathway and berberine might be a novel BCR-ABL inhibitior with potent anti-CML resistance.