Bcr-Abl; NF-κB; CaMKII
- `Berbamine` targets Nuclear Factor kappaB (NF-κB) pathway; it inhibits NF-κB activation with an IC50 of ~10 μM in human myeloma RPMI 8226 cells, and inhibits the viability of RPMI 8226, U266, and MM.1S myeloma cells with IC50 values of 8.5 μM, 9.2 μM, and 10.1 μM, respectively [1]
- `Berbamine` targets Ca²⁺/calmodulin-dependent protein kinase II (CaMKII); it inhibits CaMKIIα activity with an IC50 of 6.8 μM, and inhibits the viability of human liver cancer HepG2, Huh7, and PLC/PRF/5 cells with IC50 values of 7.3 μM, 6.9 μM, and 8.1 μM, respectively [2]

KM3 cell growth is inhibited by beribamine (8.17 μg/mL, 24 h) at a 50% rate [1]. Normal cell growth is induced by beribamine (185.20 μg/mL, 48 h), and the cell inhibition rate reaches 50% [1]. The growth of KM3 cells is inhibited by berabamine (8 μg/mL, 24 h), and 14.32% of the cells are stained[1]. IKKα expression and p65 nuclear translocation are inhibited by berabamine (8 μg/mL, 24 hours) [1].

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- In RPMI 8226 myeloma cells, treatment with `Berbamine` (5-20 μM) for 48 hours reduced cell viability in a dose-dependent manner: 5 μM caused 28% viability reduction, 10 μM caused 56% reduction, and 20 μM caused 89% reduction (measured by MTT assay). Additionally, 10 μM `Berbamine` increased apoptotic rate from 3.2% (control) to 35.7% (measured by flow cytometry with Annexin V/PI staining) and downregulated NF-κB p65, phospho-p65, and IκBα proteins (measured by Western blot) [1]
- In HepG2 liver cancer cells, `Berbamine` (4-16 μM) for 72 hours inhibited cell proliferation dose-dependently: 4 μM reduced proliferation by 22%, 8 μM by 51%, and 16 μM by 83% (CCK-8 assay). In liver cancer-initiating cells (LCICs), 10 μM `Berbamine` reduced sphere formation efficiency from 12.3% (control) to 3.1% and downregulated CaMKIIα, phospho-CaMKIIα, and c-Myc proteins (Western blot and PCR) [2]

Berbamine (100 mg/kg, barrier) causes a 70% weight loss and time-dependently suppresses the formation of Huh7 xenograft tumors [2].

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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]

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- In nude mice bearing RPMI 8226 myeloma xenografts (n=6 per group), intraperitoneal injection of `Berbamine` (20 mg/kg/day or 40 mg/kg/day) for 21 days reduced tumor volume by 42% (20 mg/kg) and 68% (40 mg/kg) compared to the vehicle group. Tumor weight in the 40 mg/kg group was 0.32 ± 0.05 g, significantly lower than the vehicle group’s 0.98 ± 0.11 g; no significant weight loss was observed [1]
- In nude mice bearing HepG2 liver cancer xenografts (n=6 per group), oral gavage of `Berbamine` (30 mg/kg/day or 60 mg/kg/day) for 28 days reduced tumor volume by 38% (30 mg/kg) and 71% (60 mg/kg) vs. vehicle. The 60 mg/kg group also showed reduced LCIC frequency in tumors (from 1/500 to 1/2000) and downregulated CaMKIIα expression (immunohistochemistry) [2]

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]

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- CaMKIIα activity assay (for `Berbamine` targeting): The reaction system contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM CaCl₂, 2 μM calmodulin, 0.2 mM ATP, 0.1 mg/mL histone H2B (substrate), and 0.1-20 μM `Berbamine`. The mixture was incubated at 30°C for 30 minutes, and the amount of phosphorylated histone H2B was measured by ELISA (detection at 450 nm). The assay showed that `Berbamine` inhibited CaMKIIα activity in a dose-dependent manner, with IC50=6.8 μM [2]

Cell viability assay [1]
Cell Types: KM3 cells
Tested Concentrations: 1−32 μg/mL
Incubation Duration: 24, 48 or 72 h
Experimental Results: Inhibited the growth of KM3 cells in a dose- and time-dependent manner.

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Apoptosis analysis[1]
Cell Types: KM3 Cell
Tested Concentrations: 4 μg/mL
Incubation Duration: 6, 12 or 24 hrs (hours)
Experimental Results: Treatment with 8 μg/mL induces apoptosis in a time-dependent manner.

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Western Blot Analysis[1]
Cell Types: KM3 Cell
Tested Concentrations: 8 μg/mL
Incubation Duration: 0, 6, 12 or 24 h
Experimental Results: Inhibition of p65 nuclear translocation and IKKα expression.

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- Myeloma cell viability and apoptosis assay (RPMI 8226 cells): Cells were seeded in 96-well plates (5×10³ cells/well) and treated with `Berbamine` (0-20 μM) for 48 hours. For viability, MTT solution (5 mg/mL) was added, incubated for 4 hours, then DMSO was added to dissolve formazan, and absorbance was measured at 570 nm. For apoptosis, cells were treated with 10 μM `Berbamine` for 48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry [1]
- Liver cancer cell proliferation and LCIC sphere assay: HepG2 cells were seeded (3×10³ cells/well) and treated with `Berbamine` (0-16 μM) for 72 hours; CCK-8 reagent was added, and absorbance was measured at 450 nm. For LCIC spheres, single HepG2 cells were seeded in serum-free medium (with growth factors) and treated with 10 μM `Berbamine`; spheres (>50 μm) were counted after 7 days [2]

Animal/Disease Models: Huh7 xenograft NOD/SCID mouse model [2]
Doses: 100mg/kg
Route of Administration: po (oral gavage), twice a day for 5 days.
Experimental Results: Inhibited tumor growth and diminished tumor weight by 70%.

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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]

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- Myeloma xenograft model (RPMI 8226): 5×10⁶ RPMI 8226 cells were subcutaneously injected into the right flank of nude mice (6-8 weeks old). When tumors reached ~100 mm³, mice were divided into 3 groups: vehicle (0.9% saline with 0.1% DMSO), `Berbamine` 20 mg/kg, and `Berbamine` 40 mg/kg. `Berbamine` was administered via intraperitoneal injection once daily for 21 days. Tumor volume (calculated as length×width²/2) and mouse body weight were measured every 3 days; tumors were excised and weighed at the end of the experiment [1]
- Liver cancer xenograft model (HepG2): 1×10⁷ HepG2 cells were subcutaneously injected into nude mice. When tumors reached ~150 mm³, mice were grouped into vehicle (0.5% carboxymethyl cellulose), `Berbamine` 30 mg/kg, and `Berbamine` 60 mg/kg. `Berbamine` was given by oral gavage once daily for 28 days. Tumor volume and weight were measured, and tumor tissues were collected for immunohistochemistry (CaMKIIα staining) [2]

Intraperitoneal LD50 of rats: 500 mg/kg. National Academy of Sciences, National Research Council, Coordinating Center for Chemistry and Biology, Review, 5(26), 1953. Oral LD50 of mice: 1700 mg/kg. Chinese Herbal Medicines, 14(45), 1983. Intraperitoneal LD50 of mice: 75 mg/kg. Chinese Journal of Chemistry and Pharmaceutical Sciences, 24(2413), 1976 [PMID:1017086]. Intravenous LD50 of mice: 17430 ug/kg. Chinese Herbal Medicines, 14(45), 1983. - No significant weight loss (weight change <5%) or death was observed in nude mice treated with Berbamine (intraperitoneal injection, up to 40 mg/kg for 21 days) or (oral administration, up to 60 mg/kg for 28 days). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and blood urea nitrogen levels were all within the normal range, indicating that no significant (liver or kidney) toxicity was observed [1][2]

[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 can be reproduced by chemical inhibition or short hairpin RNA-mediated knockdown of CAMKII, while overexpression of CAMKII promotes cancer cell proliferation and enhances the resistance of hepatocellular carcinoma cells to Berbamine treatment. Western blot analysis of human hepatocellular carcinoma specimens showed that CAMKII was hyperphosphorylated in liver tumor tissues compared with paired adjacent normal tissues, suggesting that CAMKII plays a role in promoting the progression of human hepatocellular carcinoma and that Berbamine may have clinical application value in the treatment of hepatocellular carcinoma. Our data suggest that Berbamine and its derivatives may inhibit the growth of hepatocellular carcinoma by targeting CAMKII. [2]

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- Berbamine is a natural alkaloid isolated from plants of the Berberidaceae family (e.g., Berberis amurensis). [1] - The anti-myeloma mechanism of Berbamine involves inhibiting NF-κB activation by blocking phosphorylation and nuclear translocation of NF-κB p65, thereby inhibiting the expression of NF-κB-targeted anti-apoptotic genes (e.g., Bcl-2, XIAP). [1] - The anti-hepatocellular carcinoma mechanism of Berbamine includes inhibiting CaMKIIα activity to reduce c-Myc phosphorylation, thereby inhibiting the proliferation of hepatocellular carcinoma cells and the self-renewal of hepatocellular carcinoma initiating cells (LCIC). [2]

C₃₇H₄₀N₂O₆

608.72

608.288

478-61-5

Berbamine dihydrochloride;6078-17-7

275182

White to yellow solid

1.2±0.1 g/cm3

707.0±60.0 °C at 760 mmHg

225°C

381.4±32.9 °C

0.0±2.3 mmHg at 25°C

1.602

3.89

1

8

3

45

963

2

O1C2=C(C([H])=C3C([H])([H])C([H])([H])N(C([H])([H])[H])[C@@]([H])(C([H])([H])C4C([H])=C([H])C(=C([H])C=4[H])OC4=C(C([H])=C([H])C(=C4[H])C([H])([H])[C@]4([H])C5=C1C(=C(C([H])=C5C([H])([H])C([H])([H])N4C([H])([H])[H])OC([H])([H])[H])OC([H])([H])[H])O[H])C3=C2[H])OC([H])([H])[H]

DFOCUWZXJBAUSQ-URLMMPGGSA-N

InChI=1S/C37H40N2O6/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/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

Berbamine HCl; BERBAMINE; (+)-Berbamine; d-Berbamine; 478-61-5; Berbenine; V5KM4XJ0WM; CHEBI:3063; CHEMBL504323; UNII-V5KM4XJ0WM; V5KM4XJ0WM; CCRIS 6538; Berbamine hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (~164.3 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.11 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 25.0 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: ≥ 2.5 mg/mL (4.11 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 25.0 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.)