A novel 7-bromoindirubin with potent anticancer activity suppresses survival of human melanoma cells associated with inhibition of STAT3 and Akt signaling

Figures & data

Figure 1. MLS-2438 suppresses viability of human melanoma cells and tumor growth in a mouse xenograft model. (A) structure of MLS-2438. Indirubin molecule was derivatized with a bromo-group at the 7-position on one indole ring and a hydrophilic group at the 3′-position on the other indole ring. The synthesis of this 7-bromoindirubin derivative was reported previously.³¹ (B) A2058, A375, G361 and MeWo human melanoma cells were treated with MLS-2438 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. (C) MLS-2438 suppressed tumor growth of MeWo human melanoma xenografts in NSG mice. (D) Body weight change. MLS-2438-or vehicle- treated mice were weighed on the same dates as tumors were measured. Body weight change is shown as percentage to the weight on the starting date. Points, mean (n = 8); bars, SE; *p < 0.01 vs. control.

Figure 2. MLS-2438 induces apoptosis of human melanoma cells. A2058, A375, G361 and MeWo human melanoma cells were treated with MLS-2438 at various concentrations for 24 h. (A) apoptosis was analyzed by flow cytometry by using Annexin V-FITC and DAPI double staining. (B) cells were lysed for Western blot analysis using antibodies specific to PARP, Caspase-3 and β-Actin.

Figure 3. Expression levels of pro- apoptotic and pro-survival proteins in human melanoma cells treated with MLS-2438. A2058, A375, G361 and MeWo human melanoma cells were treated with MLS-2438 at various concentrations for 24 h. Cells were lysed for Western blot analysis using antibodies specific to pro-apoptotic Bcl-2 family proteins such as Bax, Bak, Bad, Bim and Bid and pro-survival proteins such as Mcl-1, Bcl-2 and Survivin. β-Actin was used as a loading control.

Figure 4. MLS-2438 inhibits phosphorylation of JAK2, Src, STAT3 and Akt in human melanoma cells. A2058 human melanoma cells were treated with MLS-2438 at various concentrations for 4 h (A) and 24 h (B). C, A375, G361 and MeWo human melanoma cells were treated with 10 μmol/L of MLS-2438 for 4 h. Cells were lysed for Western blot analysis using antibodies specific to p-JAK2, JAK2, p-Src, Src, p-STAT3, STAT3, p-Akt, Akt, p-Erk1/2, Erk1/2 and β-Actin.

Figure 5. MLS-2438 inhibits Src kinase activity in vitro and phosphorylation of Src, Akt and JAK2 in A2058 cells. (A) In vitro kinase assays of Src, Akt and JAK2 were conducted using recombinant proteins, relevant substrates and ³³P-labeled ATP in the presence of MLS-2438 at various concentrations. Radioactivity was measured for determination of kinase activity. (B) A2058 human melanoma cells were treated with 10 μmol/L of MLS-2438 in a short time course from 5 to 30 min. Cells were lysed for Western blot analysis using antibodies specific to p-Src, Src, p-Akt, Akt, p-JAK2, JAK2 and β-Actin.

Figure 6. Schematic representation of MLS-2438-mediated apoptosis in human melanoma cells. MLS-2438, as a Src inhibitor, inhibits phosphorylation of Src, JAK2, STAT3 and Akt and induces apoptosis in human melanoma cells. The MLS-2438-mediated apoptosis at least in part is associated with inhibition of STAT3 and Akt signaling.

Ferandin Y, Bettayeb K, Kritsanida M, Lozach O, Polychronopoulos P, Magiatis P, et al. 3′-Substituted 7-halogenoindirubins, a new class of cell death inducing agents. J Med Chem 2006; 49:4638 - 49; http://dx.doi.org/10.1021/jm060314i; PMID: 16854069

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