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. 2012 Oct;6(5):484-93.
doi: 10.1016/j.molonc.2012.05.002. Epub 2012 Jun 2.

Novel synthetic derivatives of the natural product berbamine inhibit Jak2/Stat3 signaling and induce apoptosis of human melanoma cells

Sangkil Nam  1 , Jun Xie, Angela Perkins, Yuelong Ma, Fan Yang, Jun Wu, Yan Wang, Rong-Zhen Xu, Wendong Huang, David A Horne, Richard Jove
Affiliations

Novel synthetic derivatives of the natural product berbamine inhibit Jak2/Stat3 signaling and induce apoptosis of human melanoma cells

Sangkil Nam et al. Mol Oncol. 2012 Oct.

Abstract

Persistent Jak/Stat3 signal transduction plays a crucial role in tumorigenesis and immune development. Activated Jak/Stat3 signaling has been validated as a promising molecular target for cancer therapeutics discovery and development. Berbamine (BBM), a natural bis-benzylisoquinoline alkaloid, was identified from the traditional Chinese herbal medicine Berberis amurensis used for treatment of cancer patients. While BBM has been shown to have potent antitumor activities with low toxicity in various cancer types, the molecular mechanism of action of BBM remains largely unknown. Here, we determine the antitumor activities of 13 synthetic berbamine derivatives (BBMDs) against human solid tumor cells. BBMD3, which is the most potent in this series of novel BBMDs, exhibits over 6-fold increase in biological activity compared to natural BBM. Moreover, BBMD3, directly inhibits Jak2 autophosphorylation kinase activity in vitro with IC(50)0.69 μM. Autophosphorylation of Jak2 kinase at Tyr1007/1008 sites also was strongly inhibited in the range of 15 μM of BBMD3 in human melanoma cells at 4h after treatment. Following inhibition of autophosphorylation of Jak2, BBMD3 blocked constitutive activation of downstream Stat3 signaling in melanoma cells. BBMD3 also down-regulated expression of the Stat3 target proteins Mcl-1and Bcl-x(L), associated with induction of apoptosis. In sum, our findings demonstrate that the novel berbamine derivative BBMD3 is an inhibitor of the Jak2/Stat3 signaling pathway, providing evidence for a molecular mechanism whereby BBMD3 exerts at least in part the apoptosis of human melanoma cells. In addition, BBMD3 represents a promising lead compound for development of new therapeutics for cancer treatment.

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Figures

Figure 1
Figure 1
(A) Structures of BBMDs. (B) BBMDs inhibit human cancer cell viabilities. Cell viability was determined using MTS assays as described in Section 2. To determine IC50 values of BBMDs against human A2058 melanoma and DU145 prostate cancer cells, cells were treated with BBMDs in a dose‐dependent manner for 48h. Each experiment was performed in quadruplicate. Data are mean.
Figure 2
Figure 2
Effects of BBMD3 on various melanoma cells. Human A2058, A375, G361, SK‐MEL‐28 and SK‐MEL‐5 melanoma (A), and NHDF (B) cells were treated with BBMD3 in a dose‐dependent manner for 48h. Cell viability was determined using MTS assays as described in Section 2. (C) IC50 values of BBMD3 were determined in these cell lines. Each experiment was performed in quadruplicate. Data are mean.
Figure 3
Figure 3
BBMD3 inhibits Jak2/Stat3 signaling in cells. (A) A2058, A375, G361 and SK‐MEL‐5 cells were treated with 10μM of BBMD3 for 4h. Whole‐cell lysates were resolved by SDS‐PAGE and immunoblotted with specific antibodies to p‐Jak2 (Tyr1007/1008), Jak2, p‐Stat3 and Stat3. (B) A2058 melanoma cells were treated with BBMD3 in a dose‐dependent manner for 4h. Whole‐cell lysates were immunoblotted with specific antibodies to p‐Jak2 (Tyr1007/1008), Jak2, p‐src family (Tyr419), Src, p‐Stat3 (Tyr705), Stat3, p‐Tyk2 (Tyr1054/1055), Tyk2, p‐Erk1/2 (Thr202/Tyr204), Erk1/2 and β‐actin as performed in A. (C). A2058 cells were treated with 1μM or 10μM BBMD3 in a time‐dependent manner. Whole‐cell lysates were resolved by SDS‐PAGE and immunoblotted with specific antibodies to p‐Jak2 (Tyr1007/1008), Jak2, p‐Src family (Tyr419), Src, p‐Stat3 (Tyr705) and Stat3.
Figure 4
Figure 4
Inhibition of Jak2/Stat3 signaling by BBMD3 is associated with loss of cell viability. (A) A2058 cells were incubated in RPMI‐1640 medium containing 5% charcoal‐stripped serum overnight. Cells were treated with BBMD3 in a dose‐dependent manner for 4h, followed by IL‐6 stimulation for 10min. Whole‐cell lysates were resolved by SDS‐PAGE and immunoblotted with specific antibodies to p‐Jak2 (Tyr1007/1008), Jak2, p‐Stat3 (Tyr705), Stat3 and β‐actin. B. A2058 cells were treated with 5μM of BBMD3 for 1h. Whole‐cell lysates were resolved by SDS‐PAGE and immunoblotted with specific antibodies to p‐Jak2 (Tyr1007/1008), Jak2, p‐Stat3 (Tyr705) and Stat3. Viabilities of cells treated with 5μM of BBMD3 for 1h were determined using MTS assays. Each experiment was performed in quadruplicate. Data are mean±SD.
Figure 5
Figure 5
BBMD3 inhibits Jak2 autophosphorylation kinase activity in vitro. (A) Jak2 autophosphorylation kinase assay in vitro was performed as described in Section 2. The kinase assay in vitro was carried with non‐activated recombinant Jak2 protein in the absence of substrates. DMSO, BBMD3, AG490 JAK inhibitor, or AZD01 Jak2 inhibitor was preincubated for 10min at room temperature. ATP (20μM) was added to each reaction tube. Reaction mixtures were boiled with SDS‐PAGE sample buffer for 5min and resolved on SDS‐PAGE gels. Then, samples were immunoblotted with specific antibody to p‐Jak2 (Tyr1007/1008) and reblotted with specific antibody to Jak2. Positive immuno‐reactive proteins were detected using the ECL system. (B). To determine the IC50 value of BBMD3 against Jak2 autophosphorylation kinase activity in vitro shown in (A), levels of positive immuno‐reactive proteins were quantified with ImageQuant software (molecular Dynamics). Protein levels of p‐Jak2 were normalized to total Jak2.
Figure 6
Figure 6
BBMD3 down‐regulates Mcl‐1 and Bcl‐xL, associated with induction of apoptosis. (A) A2058 melanoma cells were treated with BBMD3 in a dose‐dependent manner for 24h. Whole‐cell lysates were immunoblotted with specific antibodies to Mcl‐1, Bcl‐xL and β‐actin. (B) BBMD3 induces apoptosis. A2058 melanoma cells were treated with BBMD3 in a dose‐dependent manner for 48h. Cells were labeled with Annexin V‐PE. Cells were analyzed using a FACScan flow cytometer to quantify fluorescence. Apoptotic cells were defined as Annexin V‐PE positive. Each experiment was performed in quadruplicate. Data are mean±SD.
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