Effects Of Berbamine On K562-resistant Cells In Vitro And In Vivo

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disorder. Approximately 95% of CML patients carry a characteristic Philadephia chromosome (Ph), a genetic abnormality resulting from a reciprocal translocation between the long arms of chromosomes 9 and 22. The molecular consequence of this translocation is the generation of the fusion gene bcr-abl, which encodes a constitutively active tyrosine kinase. The BCR-ABL kinase signals to multiple downstream survival pathways, including Ras/Raf/MEK/ERK, PI3K/AKT, JAK-STAT, and nuclear factorκ-B(NF-κB), which contribute to the pathogenesis of CML. Imatinib mesylate (STI571, Gleevec or Glivec, Novartis) is a specific inhibitor of BCR-ABL kinase. Imatinib shows high efficiency and low toxicity in the treatment of CML patients. Unfortunately, resistance to imatinib has been observed especially in more advanced phases of the disease. Mutation or amplification of the BCR-ABL, P-glyco-protein (Pgp) overexpression, clonal evolution, BCR-ABL independence and LYN kinase overexpression are known to be involved in imatinib treatment failures.Many different approaches to overcome clinical resistance to imatinib have been reported. Farnesyltransferase inhibitors such as SCH66326 and lonafarnib, dual Src-family kinase/ABL kinase inhibitors dasatinib and bosutinib, homoharringtonine were shown to have growth inhibitory effects on imatinib-resistant leukemias. Though these and other new drugs are known to inhibit the growth of resistant CML cells, obvious side effects or high cost limits their usefulness. Thus it is imperative to design new treatment strategies for CML.Berbamine, a natural compound from berberis, is a bis-benzylisoquinoline alkaloid that has been widely used in China for leukopenia treatment over the past decades. Our previous studies demonstrated that berbamine displayed a selective anti-proliferative activity against leukemia cells and it could inhibit the growth of several cell types including K562 cells, Jurkat cells and NB4 cells in vitro, and has negligible side effects on normal bone marrow cells. To our best knowledge, little research exists on the effects of berbamine on CML K562 resistant cells (K562-r) which are resistant to many drugs including imatinib.Part 1The apoptotic/anti-proliferation effects on K562-r cells induced by berbamine Objective: The aim of this part is to investigate in viro effect of berbamine on K562-r cells.Methods: The growth of K562-r cells was examined using the MTT assay. Morphological analysis and DNA agarose electrophoresis were used to detect apoptosis, the extent of the cells in the sub-G₁ cell cycle phase and the apoptosis rate were measured using flow cytometry. The BCR-ABL kinase domain was amplified by a long PCR method and sequenced consequently. bcr-abl mRNA and mdr-1 mRNA were detected by RT-PCR. BCR-ABL, phospho-BCR-ABL, P-glycoprotein, PARP were detected by Westernblot.Results: MTT tests revealed that berbamine significantly inhibited K562-r cell proliferation in dose- and time-dependent manner. The IC50 values of berbamine were 12.9μg/ml and 8.4μg/ml for 24h and 48h. Moreover, berbamine can increase the chemo-sensitivity of K562-r cells to imatinib. The IC₅₀ of imatinib was decreased to 2.23μM when K562-r cells were treated with a combination of imatinib at different concentration and 4.0μg/ml berbamine (IC₁₀ berbamine to K562-r cells at 48h) for 48h. The IC₅₀ of imatinib was decreased to 0.35μM. Cells with characteristics of apoptosis were confirmed by morphology examination and DNA agarose electrophoresis and percentage of apoptosis were increased after treatment with berbamine. The results also showed that berbamine was able to down-regulate BCR-ABL and phospho-BCR-ABL proteins by affecting bcr-abl mRNA expression. Expression levels of mdr-1 mRNA and P-gp protein were high in untreated K562-r cells and were significantly down-regulated by berbamine treatment. Berbamine-treated K562-r cells also exhibited stimulated proteolytic cleavage of PARP. K562-r cells had no mutations in the ATP-binding region of BCR-ABL.Conclusion: Berbamine could inhibit the proliferation of K562-r cells and induce apoptosis. Berbamine in combination with imatinib restored the chemo-sensitivity of K562-r cells to imatinib. The mechanisms may be related at least in part, to inhibit the expression of bcr-abl and mdr-1 both at the mRNA and protein level. Mutation of BCR-ABL kinase domain did not exist and so was not involved in resistance of K562-r cells to imatinib.Part 2The caner transduction signaling involved in apoptotic effect on K562-r cells inducedby berbamineObjective: The aim of this part is to investigate the caner transduction signalinginvolved in apoptotic effect on K562-r cells induced by berbamine.Methods: To characterize changes in the gene expression profile of K562-r cells inresponse to berbamine, we performed cDNA microarray analysis using the GEArray Qseries Signal Transduction in Cancer Gene Array (HS-044), which contains 128 genesincluding androgen pathway, PI-3K/AKT pathway, Survival/NF-κB pathway, Wntpathway and other related pathways. Westernblot method was used to investigate theresults of cDNA microarray analysis.Results: The cDNA microarray results showed the majority of genes examined showedonly small differences in expression. Serveral genes which changed over 2-fold wererelated with Bcl-2 pathway, Survival/NF-κB pathway, PI-3K/AKT pathway, MAPKpathway. The Westernblot results showed (1) berbamine-treated K562-r cells also exhibited down-regulated expressions of anti-apoptotic proteins Bcl-2 and Bcl-X_l, up-regulated expressions of apoptotic proteins Bax and cytoplasmic cytC. (2)Berbamine was able to decrease expression of nuclear NF-κB, phospho-IκBα, IKKαand Survivin without affecting the expression of IκBα. (3) The effects on other pathways. The effects on PI3K pathway. The expression of PI3K/p110 subunit was down-regulated after treatment with berbamine for 6h, and that of PI3K/p85 subunit, AKT, phospho-AKT(pAKT) were not changed after berbamine treatment for 6h, 12h, but only slightly reduced at 24h. The effects on MAPK pathway. The expressions of ERK1/2, JNK, phospho-JNK(pJNK) were obviously down-regulated after berbamine treatment. Conclusion: Berbamine-induced apoptosis in K562-r cells appeared to occur through several pathways including Bcl-2 family proteins and NF-κB pathway, as well as MAPK pathway. But the relationship between them and which one plays a center role during regulation is needed to be explored in the future.Part 3The effect of berbamine on K562-r cells in vivoObjective: The aim of this part is to investigate the effect of berbamine on K562-r cells in vivo.Methods: BABL/C nu/nu mice (5-to7-week-old) were obtained from Laboratory Animal Center of Shanghai Institute For Biology Science. All mice used in this study were bred and maintained in a specific pathogen-free environment. For this study, 5×10⁷ K562-r cells in 0.2 ml medium were injected subcutaneously in the midline dorsal region of nude mice on day 0. At twenty-four hours post injection, mice were randomly assigned to 2 groups (treated group and control group, 6 mice per group). Berbamine was administered at a dose of 60 mg/kg (in 0.3 ml) twice a day, at 8 am and 4 pm, for 3 weeks. The mice in the control group were given equal volumes of isotonic saline. The experiment was repeated two times. Tumor volumes were measured with calipers every five days. Mice were euthanized on day 30 and peripheral blood, tumors, livers, and spleens from each mouse were harvested.Results: The tumor incidence was 100% in the control group(12/12) within 10 days and66 % (8/12) in the treated group in the same period. Tumor growth was significantlyinhibited when mice were treated with berbamine. The inhibitory rate of treated groupwas 60.43%. There was no weight loss in mice in the treated group and no statisticallysignificant differences in white blood cell counts between two groups. No abnormalhistopathology was noted in liver and spleen at autopsy. Therapeutic assessments inanimal models suggest berbamine has significant antileukemic activity with littletoxicity.Conclusion: In vivo, berbamine can display an anti-leukemic effect without obvious sideeffects.