Bcr-Abl; NF-κB; CaMKII

Following the down-regulation of IKKα, p-IκBα, caused by berbamine treatment, p65 nuclear localization is then inhibited. This results in an increase in the expression of A20. Due to this, cyclin D1, Bcl-xL, Bid, and survivin, which are downstream targets of NF-κB, are downregulated. KM3 cells are treated with Berbamine at different concentrations for 24, 48, and 72 hours, respectively, to see if it has an effect on myeloma cell growth before MTT assays are used to measure cell viability. The growth of KM3 cells is inhibited by berbamine in a dose- and time-dependent manner; the IC50 values are, respectively, 8.17 μg/mL, 5.09 μg/mL, and 3.84 μg/mL for treatment over 24, 48, and 72 h. A normal hematopoietic cell's IC50 value for berbamine is 185.20 μg/mL at 48 hours, in contrast[1].

Berbamine (BBM) is a natural bisbenzylisoquinoline compound, has been used to treat inflammatory and other diseases since it was isolated from the traditional Chinese herb Berberis amurensis.On a xenograft animal model, Berbamine's anti-tumor properties are assessed. Huh7 (epithelial) and SK-Hep-1 (mesenchymal-like), two liver cancer cell lines, are subcutaneously injected into NOD/SCID mice. On the basis of the tumor weight, the oral Berbamine therapy significantly slows the progression of Huh7 xenografted tumors over time.

Berbamine inhibits the growth of liver cancer cells and cancer-initiating cells by targeting Ca²⁺/calmodulin-dependent protein kinase II. The human CAMKIIγ coding sequence with a kozak site was cloned into the retroviral vectors pMSCV-puro (Addgene 24828) and pRetroX-Tight-puro. A MOI of 3–5 was used for retroviral transduction of the liver cancer cells. The retroviral experiments were performed following the manual of Retro-X™ Tet-On® Advanced Inducible Expression System. A lentiviral vector pLKO.1-TRC (Addgene 10878) was used for the knockdown of CAMK2γ. The following targets in the coding sequences were selected for the design of shRNAs: GGATATGTCGACTTCTGAAAC, GGAGCCTATGATTTCCCATCA, GCCACAAACCACTGTGGTACA, GCATCCATGATGCATCGTCAGGA. A MOI of 3 was applied for the infection of the target cells. Puromycin was used to select the cells after lentiviral infection. The stable cells were used for the following animal experiments. Both retroviruses and lentiviruses were packaged in Hek293T cells and titrated with HT1080 cells.[2]

MTT assay is used to measure Berbamine's inhibitory effect on KM3 cell growth. Briefly, for 24, 48, or 72 hours, KM3 cells ((8×103 per well) are incubated with increasing concentrations of Berbamine (1-32 μ/mL), followed by a pulse of 20 μL of 5 mg/mL MTT for the final 4 hours. 200 L DMSO is then added to dissolve the formazan crystals. To determine the inhibitory concentration of 50% (IC50), dye absorbance in viable cells is measured at 570 nm[1].

Mice: The NOD/SCID mice are injected with 5×106 Huh7 cells in 50% Matrigel dissolved in PBS. Each xenograft receives 5×106 SK-Hep-1 cells without the use of Matrigel. After the tumors grow to a diameter of 2 mm, mice are given oral treatments with 100 mg/kg of Berbamine twice daily for 5 days straight. The regimen is carried out once more after two days of withdrawal[2].

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Berbamine (BBM) was dissolved in pure sterile water for animal experiments. 5 × 106 Huh7 cells in 50% Matrigel (BD bioscience, San Jose, CA) dissolved in PBS were inoculated in a NOD/SCID mouse. 5 × 106 SK-Hep-1 cells were applied for each xenograft without Matrigel. 100 mg/kg of BBM was orally treated to mice with a regimen of twice a day for 5 consecutive days after the tumors reached a size of 2 mm in diameter. After 2 days withdraw, the regimen was repeated once. All the procedures followed the National Institutes of Health guidelines for the care and use of laboratory animals.[2]

rat LDLo intraperitoneal 500 mg/kg National Academy of Sciences, National Research Council, Chemical-Biological Coordination Center, Review., 5(26), 1953
mouse LD50 oral 1700 mg/kg Zhongcaoyao. Chinese Traditional and Herbal Medicine., 14(45), 1983
mouse LD50 intraperitoneal 75 mg/kg Chemical and Pharmaceutical Bulletin., 24(2413), 1976 [PMID:1017086]
mouse LD50 intravenous 17430 ug/kg Zhongcaoyao. Chinese Traditional and Herbal Medicine., 14(45), 1983

[1]. Berbamine, a novel nuclear factor kappaB inhibitor, inhibits growth and induces apoptosis in human myeloma cells. Acta Pharmacol Sin. 2009 Dec;30(12):1659-65.

[2]. Berbamine inhibits the growth of liver cancer cells and cancer-initiating cells by targeting Ca2+/calmodulin-dependent protein kinase II. Mol Cancer Ther. 2013 Oct;12(10):2067-77.

Berbamine is a member of isoquinolines and a bisbenzylisoquinoline alkaloid.
Berbamine has been reported in Berberis silva-taroucana, Berberis ferdinandi-coburgii, and other organisms with data available.

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Aim: We sought to investigate the effect of berbamine on the growth of human multiple myeloma cell line KM3 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. Flow cytometry was performed to characterize cell cycle profile in response to berbamine treatment. Western blot was used to measure the protein levels of p65, IkappaB Kinase alpha (IKKalpha), TNFAIP3 (A20), IkappaBalpha, p-IkappaBalpha, cyclinD1, Bcl-2, BAX, Bcl-x(L), Bid, and survivin. Results: Berbamine inhibits the proliferation of KM3 cells in a dose- and time-dependent manner. Combination of berbamine with dexamethasone (Dex), doxorubicin (Dox) or arsenic trioxide (ATO) resulted in enhanced inhibition of cell growth. Flow cytometric analysis revealed that KM3 cells were arrested at G(1) phase and apoptotic cells increased from 0.54% to 51.83% for 36 h. Morphological changes of cells undergoing apoptosis were observed under light microscope. Berbamine treatment led to increased expression of A20, down-regulation of IKKalpha, p-IkappaBalpha, and followed by inhibition of p65 nuclear localization. As a result, NF-kappaB downstream targets such as cyclinD1, Bcl-x(L), Bid and survivin were down-regulated. Conclusion: Berbamine inhibits the growth of KM3 cells by inducing G(1) arrest as well as apoptosis. Berbamine blocks NF-kappaB signaling pathway through up-regulating A20, down-regulating IKKalpha, p-IkappaBalpha, and then inhibiting p65 nuclear translocation, and resulting in decreased expression of the downstream targets of NF-kappaB. Our results suggest that berbamine is a novel inhibitor of NF-kappaB activity with remarkable anti-myeloma efficacy.[1]

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Liver cancer is the third leading cause of cancer deaths worldwide but no effective treatment toward liver cancer is available so far. Therefore, there is an unmet medical need to identify novel therapies to efficiently treat liver cancer and improve the prognosis of this disease. Here, we report that berbamine and one of its derivatives, bbd24, potently suppressed liver cancer cell proliferation and induced cancer cell death by targeting Ca(2+)/calmodulin-dependent protein kinase II (CAMKII). Furthermore, berbamine inhibited the in vivo tumorigenicity of liver cancer cells in NOD/SCID mice and downregulated the self-renewal abilities of liver cancer-initiating cells. Chemical inhibition or short hairpin RNA-mediated knockdown of CAMKII recapitulated the effects of berbamine, whereas overexpression of CAMKII promoted cancer cell proliferation and increased the resistance of liver cancer cells to berbamine treatments. Western blot analyses of human liver cancer specimens showed that CAMKII was hyperphosphorylated in liver tumors compared with the paired peritumor tissues, which supports a role of CAMKII in promoting human liver cancer progression and the potential clinical use of berbamine for liver cancer therapies. Our data suggest that berbamine and its derivatives are promising agents to suppress liver cancer growth by targeting CAMKII. [2]

C₃₇H₄₂CL₂N₂O₆

681.65

680.2419925

C, 65.20; H, 6.21; Cl, 10.40; N, 4.11; O, 14.08

6078-17-7

Berbamine;478-61-5

275182

White to off-white solid

744.4ºC at 760 mmHg

250-253ºC(lit.)

404ºC

7.537

1

8

3

45

963

2

COC1=C(OC2=CC([C@]3([H])CC(C=C4)=CC=C4O5)=C(CCN3C)C=C2OC)C([C@@]6([H])CC7=CC=C(O)C5=C7)=C(CCN6C)C=C1OC.Cl.Cl

USRXDYNDPPUBSG-KKXMJGKMSA-N

InChI=1S/C37H40N2O6.2ClH/c1-38-14-12-24-19-32(41-3)33-21-27(24)28(38)16-22-6-9-26(10-7-22)44-31-18-23(8-11-30(31)40)17-29-35-25(13-15-39(29)2)20-34(42-4)36(43-5)37(35)45-33;;/h6-11,18-21,28-29,40H,12-17H2,1-5H3;2*1H/t28-,29+;;/m0../s1

(1S,14R)-20,21,25-trimethoxy-15,30-dimethyl-7,23-dioxa-15,30-diazaheptacyclo[22.6.2.23,6.18,12.114,18.027,31.022,33]hexatriaconta-3(36),4,6(35),8,10,12(34),18,20,22(33),24,26,31-dodecaen-9-ol;dihydrochloride

Berbamine HCl; Berbamine dihydrochloride; 6078-17-7; Berbamine (dihydrochloride); berbamine hydrochloride; K44OC6U989; Berbenine; (1S,14R)-20,21,25-trimethoxy-15,30-dimethyl-7,23-dioxa-15,30-diazaheptacyclo[22.6.2.23,6.18,12.114,18.027,31.022,33]hexatriaconta-3(36),4,6(35),8,10,12(34),18,20,22(33),24,26,31-dodecaen-9-ol;dihydrochloride; Berbamine 2HCl; Berbamine hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~146.7 mM)
Water: ~50 mg/mL (~73.4 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.05 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)