EBB (O-(4-ethoxyl-butyl)-berbamine


Suppression of growth, migration and invasion of highly-metastatic human breast cancer cells by berbamine and its molecular mechanisms of action.
Breast cancer is the second leading cause of cancer related deaths among females worldwide. Berbamine (BER), a kind of bis-benzylisoquinoline alkaloid, has been used to treat clinical patients with inflammation and cancer for many years in China. The purpose of this study is to investigate the activity of BER against highly-metastatic human breast cancer and its molecular mechanisms of action.

In our study, we found that BER inhibits growth of highly-metastatic human breast cancer cell lines MDA-MB-231 and MDA-MB-435S cells dose-dependently and time-dependently. The sera from BER-treated rats suppress the growth of MDA-MB-231 cells. BER shows synergistic effects with some existing anticancer agents such as trichostatin A (TSA, the histone deacetylase inhibitor), celecoxib (the inhibitor of COX-2), and carmofur against the growth of MDA-MB-231 cells. BER also displays the strong activity of inducing apoptosis in both estrogen receptor-negative MDA-MB-231 cells and estrogen receptor-alpha-positive MCF-7 breast cancer cells, but not in normal human mammary epithelial cell line MCF10A. BER down-regulates anti-apoptotic protein Bcl-2 levels and up-regulates pro-apoptotic protein Bax expressions in MDA-MB-231 and MDA-MB-435S cells. BER also has synergistic effects with anticancer agents trichostatin A, celecoxib and/or carmofur on reducing Bcl-2/Bax ratios and VEGF secretions in MDA-MB-231 cells. In addition, BER significantly suppresses cell migration and invasion, as well as decreases pro-MMP-9/pro-MMP-2 activation in breast cancer cells. Furthermore, BER suppresses Akt and nuclear factor kappaB signaling by reducing the phosphorylation of c-Met and Akt, and inhibiting their downstream targets such as nuclear factor kappaB p-65, Bcl-2/Bax, osteopontin, VEGF, MMP-9 and MMP-2 on protein and/or mRNA levels in breast cancer cells.

Our findings have showed that BER suppresses the growth, migration and invasion in highly-metastatic human breast cancer cells by possibly inhibiting Akt and NF-kappaB signaling with their upstream target c-Met and downstream targets Bcl-2/Bax, osteopontin, VEGF, MMP-9 and MMP-2. BER has synergistic effects with anticancer agents trichostatin A, celecoxib and carmofur on inhibiting the growth of MDA-MB-231 cells and reducing the ratio of Bcl-2/Bax and/or VEGF expressions in the cancer cells. These findings suggest that BER may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human breast cancer and other cancers.
Wang S, Liu Q, Zhang Y, et al. Mol Cancer. 2009 Oct 1;8:81.

Reversal of multidrug resistance in drug-resistant human breast cancer cell line MCF-7/ADR by calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine
To investigate the reversal effect of O-(4-ethoxyl-butyl)-berbamine (EBB) on multidrug resistance (MDR) in MCF-7/ADR cell.
Methods: 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the antitumor effect of EBB and determine the reversal effects of different concentrations ( < or = IC20) of EBB on MCF-7/ADR cell. Flow cytometry was applied to observe the intracellular accumulation of Rh123 and cell cycle in the presence of EBB. The expressions of MDR-related genes mdr 1 and topoisomerase II b (top II b) were evaluated by reverse transcription-polymerase chain reaction.
Results: The sensitivity of MCF-7/ADR to adriamycin (ADR) was enhanced up to 50. 40, 89.80, and 14.88 folds after exposure of the cells to 3 micromol/L EBB, 7.5 micromol/L EBB, and 10 micromol/L verapamil (VPL), respectively. After 2 hours of incubation with 6 micromol/L EBB, intracellular Rh123 accumulation in MCF-7/ADR cells was increased to the level comparable to that in MCF-7 cells. When 6 micromol/L EBB was added together with 2 micromol/L ADR, MCF-7/ ADR cells showed to be arrested in the G2/M phase. The declination of mdr 1 gene expression was observed when 6 micromol/L EBB, 12 micromol/L EBB, and 10 micromol/L VPL were added for 48 hours; meanwhile, the expression of top II b mRNA showed no significant change.
Conclusion: EBB has a strong reversal effect on MDR in MCF-7/ ADR cell, which may be achieved by enhancing the arrestment of MCF-7/ADR cells at G2/M phase and increasing intracellular drug concentration.
Cheng YH, Qi J, Xiong DS, Liu JW, Qi SL, Pan B, Yang CZ, Zhu HF. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2006 Apr;28(2):164-8.

Inhibition of Telomerase Activity and bcl-2 Expression in Berbamine-Induced Apoptosis in HL-60 Cells
The present study was aimed to investigate the effect of telomerase activity in berbamine-induced apoptosis and the regulation of B cell leukemia/lymphoma 2 (bcl-2) gene expression in human leukaemia HL-60 cells. Apoptosis of HL-60 cells was induced by berbamine (10 μM) for 3, 6, 12 and 24 h. Apoptosis and bcl-2 were determined by flow cytometry analysis. A polymerase chain reaction-based telomeric repeat amplification protocol assay was used to detect the telomerase activity. Berbamine induced growth arrest and apoptotic cell death in HL-60 cells. The telomerase activity was inhibited in a time-dependent manner during the berbamine-induced apoptosis of HL-60 cells, and the expression of bcl-2 was progressively down-regulated by berbamine. Inhibition of the telomerase activity of HL-60 cells was closely related to the berbamine-induced apoptosis. The present results indicate that inhibition of telomerase and reduced bcl-2 gene expression may play a role in the berbamine-induced apoptosis of HL-60 cells.
Zhao-Ning Ji, Wen-Cai Ye, Guo-Qing Liu, Yu Huang. Pharmacology. Planta Med 2002; 68(7): 596-600. DOI: 10.1055/s-2002-32896

Berbamine exhibits potent antitumor effects on imatinib-resistant CML cells in vitro and in vivo
Cell viability was measured by MTT assay, and apoptotic morphology changes were detected by fluorescence microscopy. The apoptosis rate was measured by flow cytometric assay. mdr-1 mRNA levels were determined by RT-PCR. Bcl-2 family proteins, cytochrome c(cyt C), poly (ADP-ribose) polymerase (PARP), and P-glycoprotein were detected by Western blot. BALB/c nu/nu mice were injected with K562-r cells subcutaneously. Tumor-bearing mice were treated intravenously with berbamine.
Results: MTT tests revealed that berbamine significantly inhibited K562-r cell proliferation and increased the chemo-sensitivity of K562-r cells to imatinib. The apoptosis rate was significantly increased following treatment with 21.2 μmol/L berbamine; formation of typical apoptotic blebs was apparent, as observed by fluorescence microscopy. Expression levels of mdr-1 mRNA and P-gp protein were high in untreated K562-r cells and significantly down-regulated by berbamine treatment. Berbamine-treated K562-r cells also exhibited down-regulated expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, up-regulated expression of the apoptotic proteins Bax and cytoplasmic cyt C, and stimulated proteolytic cleavage of PARP. In addition, berbamine also suppressed the growth of K562-r xenotransplanted tumors in vivo.
Conclusion: Berbamine inhibited proliferation of K562-r cells both in vitro and in vivo. Berbamine-induced apoptosis in K562-r cells appeared to occur through a mechanism involving Bcl-2 family proteins, as well as mdr-1 mRNA and P-gp protein. Berbamine in combination with imatinib restored the chemo-sensitivity of K562-r cells to imatinib. Our findings suggest that berbamine may be useful in treating imatinib-resistant CML patients
Wei Y-l, Lei Xu L, Yun Liang Y, Xu X-h and Xiao-ying Zhao X-y. Acta Pharmacologica Sinica (2009) 30: 451–457; doi: 10.1038/aps.2009.19
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