Natural Product Berbamine Enhances The Efficacy Of Imatinib In The Treatment Of CML And Its Mechanisms

Background:Chronic myeloid leukemia (CML) accounts for approximately20%of all adult leukemias and significantly affects people’s health. CML is characterized by the presence of the Philadelphia chromosome (Ph+), which results from a chromosomal translocation between the Bcr gene on chromosome22and the Abl gene on chromosome9. This translocation produces the fusion protein Bcr-Abl that has constitutive kinase activity and actives multiple signaling pathways. These abnormal activations result in incontrollable cell proliferation and apoptosis tolerance, which ultimately induces CML. Accordingly, tyrosine kinase inhibitors (TKIs, such as imatinib, IM) targeting Bcr-Abl has become a series of novel and attractive durgs in clinical setting. However, imatinib is not capable to kill all those cells with Bcr-Abl and simply effective in controlling CML at chronic phase, which partly attribute to TKI-resistance. Evidence indicates leukemia stem cell (LSCs) is one of the core mechanisms in TKI-resistance. Targeting this cluster of cells is a promising method in overcoming TKI-resistance.Evidence indicates that traditional Chinese medicine(TCM) is one of promising drugs for CML treatment. Berbamine(BBM) is a structurally unique bisbenzylisoquinoline isolated from TCM Berberis amurensis, and has been used for treating a variety of diseases from inflammation to tumors for many years. Previous study of our group demonstrated that BBM and its derivatives potently inhibited the growth of imatinib (IM)-resistant CML cells but not normal hematopoietic cells. These results indicate BBM may significantly kill CML cells including LSCs, which will provide a novel alternative way in CML therapy with IM and curing this disease. And in the current study, we will study the feasibility and the advantage of the combination of BBM with IM, and its action mechanism.Objects:1. To compare the effects of natural product BBM, IM and their combination on LSCs or T315I mutant-Bcr-Abl cell clones of CML;2. To compare the effects of BBM, IM and their combination on CML in vivo using mouse model;3. To explore mechanisms by which BBM, IM and their combination inhibit the growth of CML cells.Methods:1. We investigated the effects of BBM, IM and their combination on imatinib (IM)-resistant-K562cells and T315I mutant-Bcr-Abl of CML through MTT in vitroand calculate the CI(combination index).2. We assessed the effects of BBM, IM and their combination on CML leukemic primitive stem/progenitor cells by soft agar colony assay.3. We evaluated the inhibitory effect of BBM, IM and their combination on IM-resistance-cells K562xenografts in nude mice.4. Flow cytometry was used for cell cycle analysis by Annexin-V/PI.5. The protein levels of p210BcR-ABL and CaMKIIy were determined by Western blot analysis.Results:1. BBM and IM have a significant synergism.72h IC50value of BBM and IM on CML cells was10.02±0.064μM,12.538±0.073μM,14.759±0.08μM,14.194±0.032μM and0.065±0.011μ,2.665±0.031μM,14.678±0.077μM,0.065± 0.012μM;2. Combination of BBM with IM also significantly inhibited the proliferation of both TKIs-resistant K562cells and KCL-22M cells with T315I mutant BCR-ABL in vitro.The combination index was less than1.3. Combination of BBM with IM exhibted higher anti-leukemia activity than that of BBM or IM alone in nude mice.4. Combination of BBM with IM greatly promoted apoptosis and cell cycle arrest of CML cells.5. Combination of BBM with IM exhibited more effects in reducing the levels of Bcr-Abl protein and CaMKIIy than that BBM or IM alone.6. Combination of BBM with IM exhibted higher anti-leukemia activity than that of BBM or IM alone in nude mice.lt had better results in terms of reducing tumor weight,promoting tumor regression,futher more,no gross abnormalities of mouse organs were observed.Conclusions:1BBM is a drug with effective anti-tumor effect on both LSCs and T315I cells with BCR-ABL mutation. BBM exerts strong cytotoxicity on CML cells though suppressing CaMKIIy kinase.2BBM and IM displayed synergistic effect in above cells. BBM and IM synergistically inhibites BCR-ABL and its phosphorylation, which indicate a promising way in reversal of imatinib resistance though combination therapy.3Combination of BBM and IM has potent antitumor effect on human CML cells in vivo.4BBM may sensitize the anti-tumor effects of imatinib. On the other hand, berbamine is a drug with high cost-effective and little side-effect. As a consequence, it may paly a potential clinical effects in treatment CML, especially those with imatinib resistance and recurrence.