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. 2011 Jun 1;71(11):3972-9.
doi: 10.1158/0008-5472.CAN-10-3852. Epub 2011 May 24.

6-Bromoindirubin-3'-oxime inhibits JAK/STAT3 signaling and induces apoptosis of human melanoma cells

Lucy Liu  1 , Sangkil Nam, Yan Tian, Fan Yang, Jun Wu, Yan Wang, Anna Scuto, Panos Polychronopoulos, Prokopios Magiatis, Leandros Skaltsounis, Richard Jove
Affiliations

6-Bromoindirubin-3'-oxime inhibits JAK/STAT3 signaling and induces apoptosis of human melanoma cells

Lucy Liu et al. Cancer Res. .

Abstract

STAT3 is persistently activated and contributes to malignant progression in various cancers. Janus activated kinases (JAK) phosphorylate STAT3 in response to stimulation by cytokines or growth factors. The STAT3 signaling pathway has been validated as a promising target for development of anticancer therapeutics. Small-molecule inhibitors of JAK/STAT3 signaling represent potential molecular-targeted cancer therapeutic agents. In this study, we investigated the role of JAK/STAT3 signaling in 6-bromoindirubin-3'-oxime (6BIO)-mediated growth inhibition of human melanoma cells and assessed 6BIO as a potential anticancer drug candidate. We found that 6BIO is a pan-JAK inhibitor that induces apoptosis of human melanoma cells. 6BIO directly inhibited JAK-family kinase activity, both in vitro and in cancer cells. Apoptosis of human melanoma cells induced by 6BIO was associated with reduced phosphorylation of JAKs and STAT3 in both dose- and time-dependent manners. Consistent with inhibition of STAT3 signaling, expression of the antiapoptotic protein Mcl-1 was downregulated. In contrast to the decreased levels of phosphorylation of JAKs and STAT3, phosphorylation levels of the Akt and mitogen-activated protein kinase (MAPK) signaling proteins were not inhibited in cells treated with 6BIO. Importantly, 6BIO suppressed tumor growth in vivo with low toxicity in a mouse xenograft model of melanoma. Taken together, these results show that 6BIO is a novel pan-JAK inhibitor that can selectively inhibit STAT3 signaling and induces tumor cell apoptosis. Our findings support further development of 6BIO as a potential anticancer therapeutic agent that targets JAK/STAT3 signaling in tumor cells.

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Figures

Figure 1
Figure 1
Effects of 6BIO on human melanoma cell lines. A, MTS cell viability assays were performed in A2058, G361, SK-MEL-5, and SK-MEL-28 human melanoma cells. Cells were treated with 6BIO at various concentrations for 48 h. B, human melanoma cells were treated with 10 μmol/L of 6BIO for 4 h. Cells were lysed for Western blot analysis using antibodies specific to p-Jak2, Jak2, p-Src, Src, p-Stat3, Stat3, and β-Actin.
Figure 2
Figure 2
6BIO inhibits phosphorylation of JAK family kinases in cells and JAK enzymatic activity in vitro. A, A2058 human melanoma cells were treated with 6BIO at various concentrations for 4 h. Cells were lysed for Western blot analysis using antibodies specific to p-Jak1, Jak1, p-Jak2, Jak2, p-Tyk2, Tyk2, and β-Actin . B, A2058 human melanoma cells were treated with 6BIO at various concentrations for 24 h. Cells were lysed for Western blot analysis using antibodies specific to p-Jak1, Jak1, p-Jak2, Jak2, p-Tyk2, Tyk2, and β-Actin. C, A2058 human melanoma cells were treated with 10 μmol/L of 6BIO for various lengths of time. Cells were lysed for Western blot analysis using antibodies specific to p-Jak1, Jak1, p-Jak2, Jak2, p-Tyk2, Tyk2, and β-Actin. D, in vitro kinase assays of JAK1, JAK2 and TYK2 were conducted using recombinant JAK proteins, substrates and 33P-labeled ATP in the presence of 6BIO at various concentrations. Radioactivity was measured for determination of kinase activity.
Figure 3
Figure 3
6BIO selectively inhibits phosphorylation of STAT3 in human melanoma cells. A, A2058 human melanoma cells were treated with 6BIO at various concentrations for 4 h. Cells were lysed for Western blot analysis using antibodies specific to p-Src, Src, p-Stat3, Stat3, p-Akt, Akt, p-Erk1/2, Erk1/2 and β-Actin. B, SK-MEL-5, SK-MEL-28 and G361 human melanoma cells were treated with 10 μmol/L of 6BIO for 4 h. Cells were lysed for Western blot analysis using antibodies specific to p-Akt, Akt, p-Erk1/2, Erk1/2 and β-Actin.
Figure 4
Figure 4
6BIO induces apoptosis associated with down-regulation of anti-apoptotic protein Mcl-1. A, A2058 human melanoma cells were treated with 6BIO at various concentrations for 24 h. Cells were lysed for Western blot analysis using antibodies specific to Mcl-1, Bcl-2, Bcl-xL, Survivin and β-Actin. B, human melanoma SK-MEL-5, SK-MEL-28 and G361 cells were treated with 10 μmol/L of 6BIO for 24 h. Cells were lysed for Western blot analysis using antibodies specific to Mcl-1 and β-Actin. C, human melanoma A2058 cells were treated with 6BIO at various concentrations for 24 h. Cells were lysed for Western blot analysis using antibodies specific to PARP, Caspase-3 and β-Actin. D, A2058 cells were treated with 6BIO at various concentrations for 24 h or 48 h. Apoptosis was analyzed by flow cytometry using Annexin V-FITC staining.
Figure 5
Figure 5
6BIO suppresses melanoma tumor growth in a mouse xenograft model. A, Toxicity study with BALB/c normal mice. 6BIO or vehicle control was administered through oral gavage once daily to each group, at doses of 50, 75 and 100 mg/kg body weight. The maximum tolerated dose (MTD) was determined as 100 mg/kg. B, 6BIO suppressed tumor growth of A2058 human melanoma xenografts in NSG mice. A2058 cells (5×106) were inoculated subcutaneously into the dorsal area of NSG mice. When tumors became palpable, 6BIO or vehicle control was administrated via oral gavage once daily. Points, mean (n = 8); bars, SE. P < 0.001.
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