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]

The intraperitoneal LD50 in rats was 500 mg/kg, according to the National Academy of Sciences, National Research Council and Coordinating Center for Chemistry and Biology, 5(26), 1953; the oral LD50 in mice was 1700 mg/kg, according to Chinese Traditional and Herbal Drugs, 14(45), 1983; the intraperitoneal LD50 in mice was 75 mg/kg, according to the Chinese Journal of Chemistry and Pharmaceutical Sciences, 24(2413), 1976 [PMID:1017086]; the intravenous LD50 in mice was 17430 μg/kg, according to Chinese Traditional and Herbal Drugs, 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 belongs to the isoquinoline class of compounds and is a dibenzylisoquinoline alkaloid. It has been reported to exist in Berberis silva-taroucana, Berberis ferdinandi-coburgii, and other organisms with relevant data. Objective: This study aimed to investigate the effect of Berbamine on the growth of the human multiple myeloma cell line KM3 and elucidate its mechanism of action. Methods: The inhibitory effect of Berbamine, alone or in combination with chemotherapeutic drugs, was detected using the MTT assay. Cell cycle changes after Berbamine treatment were analyzed by flow cytometry. The protein expression levels of p65, IκB kinase α (IKKα), TNFAIP3 (A20), IκBα, phosphorylated IκBα, cyclin D1, Bcl-2, BAX, Bcl-x(L), Bid, and survivin were detected by Western blotting. The results showed that Berbamine inhibited the proliferation of KM3 cells in a dose- and time-dependent manner. Combination of Berbamine with dexamethasone (Dex), doxorubicin (Dox), or arsenic trioxide (ATO) enhanced the inhibitory effect on cell growth. Flow cytometry analysis indicated that KM3 cells were arrested in the G1 phase, and the proportion of apoptotic cells increased from 0.54% to 51.83% within 36 hours. Morphological changes in apoptotic cells were observed under a light microscope. Berbamine treatment led to increased A20 expression and downregulation of IKKα and p-IκBα expression, thereby inhibiting the nuclear localization of p65. Consequently, the expression of downstream NF-κB target genes such as cyclin D1, Bcl-xL, Bid, and survivin were all downregulated. Conclusion: Berbamine inhibits the growth of KM3 cells by inducing G1 phase arrest and apoptosis. Berbamine blocks the NF-κB signaling pathway by upregulating A20, downregulating IKKα and p-IκBα, and then inhibiting the nuclear translocation of p65, ultimately leading to a decrease in the expression of downstream target genes of NF-κB. Our results show that Berbamine is a novel NF-κB activity inhibitor with significant anti-myeloma efficacy. [1]

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Hepatocellular carcinoma is the third leading cause of cancer death worldwide, but there is currently no effective treatment for it. Therefore, it is urgent to find new therapies that can effectively treat hepatocellular carcinoma and improve its prognosis. This study found that Berbamine and its derivative bbd24 can effectively inhibit the proliferation of hepatocellular carcinoma cells and induce cancer cell death by targeting Ca²⁺/calmodulin-dependent protein kinase II (CAMKII). In addition, Berbamine can also inhibit the tumorigenicity of hepatocellular carcinoma cells in NOD/SCID mice and downregulate the self-renewal capacity of hepatocellular carcinoma initiating cells. The effects of Berbamine were reproducible by chemical inhibition or short hairpin RNA-mediated knockdown of CAMKII, while overexpression of CAMKII promoted cancer cell proliferation and enhanced resistance of hepatocellular carcinoma cells to Berbamine treatment. Western blot analysis of human hepatocellular carcinoma specimens showed that CAMKII was hyperphosphorylated in liver tumors compared with paired adjacent normal tissues, which supports the role of CAMKII in promoting the progression of human hepatocellular carcinoma and the potential clinical application value of Berbamine in the treatment of hepatocellular carcinoma. Our data suggest that Berbamine and its derivatives may inhibit hepatocellular carcinoma 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.)