Anti-proliferation Effect And Mechanism Of Berbamine And Berbamine Derivatives Synthesis On Multiple Myeloma Cells

Multiple myeloma (MM) is a B-cell malignancy characterized by abnormal proliferation of plasma cells in bone marrow secreting monoclonal immunoglobulins. MM accounts for 2% of all cancers and more than 10% of all hematologic cancers. Prior treatment options for MM include chemotherapy, stem cell transplantation and immunotherapy with interferon-gamma. Recently, novel agents such as bortezomib, thalidomide and lenalidomide were approved for MM treatment and have improved the complete remission rate and patient outcome. However, MM remains incurable, and development of drug resistance or intolerable toxicity emerges as a major problem especially for patients with refractory or relapsed MM. Therefore, design, identification and validation of novel chemicals with therapeutic potential are clearly needed for MM treatment.Berbamine (BBM), a natural small molecular compound, is distilled from Chinese traditional medicine Berberis amurensis, which has been shown to improve normal hematopoiesis and immune function of cancer patients over the past decades. Our former studies showed that berbamine down-regulates p210 bcr-abl oncoprotein in K562 cells, and induces apoptosis through a caspase-3-dependent pathway. In addition, berbamine was highly effective in suppressing the growth of imatinib-resistant cell line K562 (K562-r) cells, human acute promyelocytic leukemia cell line NB4 cells and T-cell leukemia cell line Jurkat cells. Therefore, berbamine appears to have broad anti-tumor activities with complex mechanism of actions. These previous studies prompted us to explore whether BBM exist anti-proliferation effect on MM cells and develop novel berbamine derivatives (BBD) designed to treat malignant disease.This is the first report to identify the effects of berbamine on MM cells, and a series of new berbamine derivatives were obtained by synthesis. In this series, high to very high activity has been found. The aim of our study is to investigate the target genes and the transduction signaling involved in the effects on MM cells induced by berbamine and berbamine derivatives. Our studies provide enough rational for considering berbamine worthy of more research and designing chemically synthetic compounds based on berbamine may represent a promising strategy to develop novel anti-myeloma drugs.Part 1 Anti-proliferation Effect and Mechanism of Berbamine on Multiple Myeloma CellsObjective:We sought to investigate the effect of berbamine on the growth of human multiple myeloma cells and elucidate the mechanism of its action.Methods:MTT assay was used to determine the inhibitory effect of berbamine alone or combined with chemotherapeutic drugs. Morphological analysis was used to detect apoptosis. Flow cytometry was performed to characterize cell cycle profile upon berbamine treatment. Changes and subcellular localization of p65 protein were observed by fluorescence microscopy. Western blot was used to measure the protein levels of p65, IκB Kinase a (IKKa), IκBa, p-IκBα, TNFAIP3 (A20), cyclinD1, Bcl-2 family proteins and survivin.Results:Berbamine inhibits the proliferation of KM3, RPMI8226 and U266 cells in a dose-and time-dependent manner, and IC50 of 48 hours were 5.09μg/ml,3.83μg/ml and 4.7μg/ml respectively. A remarkable increased antiproliferative activity on MM cells was found in combination of berbamine with dexamethasone (Dex), doxorubicin (Dox) or arsenic trioxide (ATO). Flow cytometric analysis revealed that KM3 cells were arrested at G1/S phase and apoptotic cells increased from 0.54% to 51.83% for 36 hours. Morphological changes of cells undergoing apoptosis were observed under light microscope. Mechanistically,8μg/ml berbamine treatment led to increased expression of A20, down-regulation of IKKa, p-IκBa, and followed by inhibition of p65 nuclear localization. As a result, the expression of NF-κB downstream targets such as cyclinD1, Bcl-xL, Bid and survivin were down-regulated.Conclusion:Berbamine inhibited the proliferation of MM cells and induced G1/S arrest as well as apoptosis. The mechanism of berbamine on MM cells may be through modulating NF-κB activity. Our results suggest that berbamine is a novel inhibitor of NF-κB activity with remarkable anti-myeloma efficacy.Part 2 Anti-proliferation Effect and Mechanism of Berbamine Derivatives Synthesis on Multiple Myeloma CellsObjective:To explore anti-proliferation effect and mechanism of berbamine derivatives Synthesis on Multiple Myeloma Cells.Methods:A series of new berbamine derivatives were obtained by synthesis and the anti-myeloma activities of them against RPMI8226 cells were evaluated. Compounds 9# showed the best activity and had low cytotoxicity on normal blood cells. MTT assay was used to determine the inhibitory effects of BBD9# and BBD24# on KM3, RPMI8226 and U266 cells individually or combined with conventional anti-myeloma drugs. Flow cytometric analysis was performed to evaluate the effects of BBD9#on cell cycle progression and apoptosis. Changes and subcellular localization of p65 protein were observed by fluorescence microscopy. Western blotting was used to evaluate the protein levels of p65 and its up-stream and down-stream events in the NF-kB activation cascade in response to 1μg/m BBD9# reatment and the same method was used to evaluate the activation of ERK, AKT and JNK pathways.Results:BBD9# and BBD24# displayed higher anti-MM activities against RPMI8226 cells. Both of berbamine derivatives inhibit the proliferation of KM3, RPMI8226 and U266 cells in a dose-and time-dependent manner, and IC50 of 48h were 0.65μg/ml to 0.8μg/ml. Low does BBD9# potentiated the cytotoxicity effecs of other chemotherapeutic agents. It induced cell cycle arrest at Gl/S plase, and then triggered MM cells apoptosis by Flow cytometric analysis. After 24 hours treated withμg/ml BBD9#, the apoptotic cells of KM3, RPMI8226 and U266 cells increased from 0.67%, 1.12% and 1.08% to 36.60%,47.30% and 26.48% respectively. Western blot showed the expressions of IKKa and p-IΚBαwere down-regulated and the expression of NF-κB downstream targets such as cyclinDl and survivin proteins were down-regulated. We also found the expressions of p-JNK and c-Jun were increased in different time, whereas had no effect on the activation of ERK and AKT patyways. JNK inhibitor SP600125 partly blocks BBD9#-induced cell death, suggest that the activation of JNK modulated BBD9#-induced apoptosis on RPMI8226 cells.Conclusion:Both BBD9# and BBD24# can improve its cytotoxicity against MM cells significantly. BBD9#can suppress proliferation and induce apoptosis in RPMI8226 cells mediate suppression of the NF-κB pathway and its effect in part through the up-regulateing JNK signaling pathway. Designing chemically synthetic compounds based on berbamine may represent a promising strategy to develop novel anti-myeloma drugs.