MLS-2384, a new 6-bromoindirubin derivative with dual JAK/Src kinase inhibitory activity, suppresses growth of diverse cancer cells

Abstract

Janus kinase (JAK) and Src kinase are the two major tyrosine kinase families upstream of signal transducer and activator of transcription (STAT). Among the seven STAT family proteins, STAT3 is constitutively activated in many diverse cancers. Upon activation, JAK and Src kinases phosphorylate STAT3, and thereby promote cell growth and survival. MLS-2384 is a novel 6-bromoindirubin derivative with a bromo-group at the 6-position on one indole ring and a hydrophilic group at the 3'-position on the other indole ring. In this study, we investigated the kinase inhibitory activity and anticancer activity of MLS-2384. Our data from in vitro kinase assays, cell viability analyses, western blotting analyses, and animal model studies, demonstrate that MLS-2384 is a dual JAK/Src kinase inhibitor, and suppresses growth of various human cancer cells, such as prostate, breast, skin, ovarian, lung, and liver. Consistent with the inactivation of JAK and Src kinases, phosphorylation of STAT3 was inhibited in a dose-dependent manner in the cancer cells treated with MLS-2384. STAT3 downstream proteins involved in cell proliferation and survival, such as c-Myc and Mcl-1, are downregulated by MLS-2384 in prostate cancer cells, whereas survivin is downregulated in A2058 cells. In these two cancer cell lines, PARP is cleaved, indicating that MLS-2384 induces apoptosis in human melanoma and prostate cancer cells. Importantly, MLS-2384 suppresses tumor growth with low toxicity in a mouse xenograft model of human melanoma. Taken together, MLS-2384 demonstrates dual JAK/Src inhibitory activity and suppresses tumor cell growth both in vitro and in vivo. Our findings support further development of MLS-2384 as a potential small-molecule therapeutic agent that targets JAK, Src, and STAT3 signaling in multiple human cancer cells.

Keywords: JAK; STAT3; Src; apoptosis; bromoindirubin; cancer cells; indirubin.

Figure 1. MLS-2384 inhibits phosphorylation of JAK2, Src, and STAT3 in various human cancer cells. Human DU145 prostate, MDA-MB-468 breast, A2058 skin, A2780 ovarian, and A 549 lung cancer cells were treated with MLS-2384 at various concentrations 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. MLS-2384 suppresses viability of diverse human cancer cells. Prostate (DU145), breast (MDA-MB-468), skin (A2058), ovarian (A2780), lung (A549), and liver (HepG2) cancer cells were treated with MLS-2384 at various concentrations for 24 h. Viable cells were counted by using a cell viability analyzer. The values of cell viability were calculated as percentages of viable cell numbers from bromoindirubin-treated cells to viable cell numbers from the DMSO-treated cells.

Figure 3. Cleavage of PARP and expression levels of STAT3 downstream proteins in human A2058 melanoma and DU145 prostate cancer cells. A2058 and DU145 cells were treated with MLS-2384 at various concentrations for 24 h. Cells were lysed for western blot analysis using antibodies specific to PARP/cleaved PARP, c-Myc, Mcl-1, survivin, and β-Actin.

Figure 4. Toxicity studies in NHDF cells and BALB/c mice. (A) NHDF cells were treated with MLS-2384 at various concentrations for 24 and 48 h. MTS cell proliferation assays were performed according manufacturer’s instruction. (B) Vehicle and MLS-2384 at doses of 25, 50, 75, and 100 mg/kg were administrated through oral gavage once daily to normal BALB/c mice for 7 d. Mouse body weight was measured every day. Points, mean (n = 4).

Figure 5. MLS-2384 suppresses tumor growth of A2058 human melanoma xenografts in NSG mice. MLS-2384 was administrated to NSG immunodeficient mice through oral gavage twice daily at a dose of 25 mg/kg for 18 d. Mouse tumor volume and body weight were measured for every 3 or 4 d for MLS-2384-or vehicle- treated mice. (A) Tumor volume vs. days of treatments. Points, mean (n = 8); bars, SE; *P < 0.01 vs. control. (B) Body weight vs. days of treatments. Points, mean (n = 8); bars, SE.