Spirooxindole derivative SOID-8 induces apoptosis associated with inhibition of JAK2/STAT3 signaling in melanoma cells

Abstract

Melanoma is generally refractory to current chemotherapy, thus new treatment strategies are needed. In this study, we synthesized a series of spirooxindole derivatives (SOID-1 to SOID-12) and evaluated their antitumor effects on melanoma. Among the 12 spirooxindole derivatives, SOID-8 showed the strongest antitumor activity by viability screening. SOID-8 inhibited viability of A2058, A375, SK-MEL-5 and SK-MEL-28 human melanoma cells in a dose- and time-dependent manner. SOID-8 also induced apoptosis of these tumor cells, which was confirmed by positive Annexin V staining and an increase of poly(ADP-ribose) polymerase cleavage. The antiapoptotic protein Mcl-1, a member of the Bcl-2 family, was downregulated and correlated with SOID-8 induced apoptosis. In addition, SOID-8 reduced tyrosine phosphorylation of Signal Tansducer and Activator of Transcription 3 (STAT3) in both dose- and time-dependent manners. This inhibition was associated with decreased levels of phosphorylation of Janus-activated kinase-2 (JAK2), an upstream kinase that mediates STAT3 phosphorylation at Tyr705. Accordingly, SOID-8 inhibited IL-6-induced activation of STAT3 and JAK2 in melanoma cells. Finally, SOID-8 suppressed melanoma tumor growth in a mouse xenograft model, accompanied with a decrease of phosphorylation of JAK2 and STAT3. Our results indicate that the antitumor activity of SOID-8 is at least partially due to inhibition of JAK2/STAT3 signaling in melanoma cells. These findings suggest that the spirooxindole derivative SOID-8 is a promising lead compound for further development of new preventive and therapeutic agents for melanoma.

Figure 1. Screening of spirooxindole derivatives for anti-tumor activity on melanoma cells.

(A) Structure of analogues SOID-1 to SOID-12 inspired by natural product Sprotryprostatin B. (B) Effect of spirooxindole derivatives on melanoma cell growth. A2058 and A375 cells were treated with a series of spirooxindole derivatives (30 µM) for 24 h, and cell viability was evaluated by MTS assay. DMSO was used as vehicle control. Data are shown as means ± SEM. Statistical significance between SOID-8 treatment and DMSO control, determined by the two-tailed Student's t test, is indicated by ***, p<0.001.

Figure 2. SOID-8 inhibits melanoma cell growth.

(A) SOID-8 significantly inhibits viability of human melanoma cell lines (A2058, A375, SK-MEL-28 and SK-MEL-5), but has minor effect on normal human cells [NHDFs (normal human dermal fibroblasts) and melanocytes]. Cells were treated with increasing concentrations (2.5, 5, 10, 20 µM) of SOID-8 for 24 (left) and 48 h (right), and cell viability was evaluated by MTS assay. DMSO was used as vehicle control. Data are shown as means ± SEM. Statistical significance between any one of two normal cell lines (NHDF or melanocyte) and any one of four melanoma cell lines (A2058, A375, SK-MEL-28, or SK-MEL-5) was determined by the two-tailed Student's t test. *, p<0.05; **, p<0.005; ***, p<0.001.

Figure 3. Effects of SOID-8 on apoptosis of melanoma cells.

(A) SOID-8 induces apoptosis of A2058 and A375 cells. Cells were treated with SOID-8 at indicated concentrations for 24 (left) and 48 h (right), respectively. Apoptotic cells are represented by propidium iodide and Annexin V-FITC double-positive staining as determined by fluorescence-activated cell sorting. Each experiment was done in triplicate and repeated twice independently. (B) Effects of SOID-8 on apoptosis-related proteins. A2058 and A375 cells were treated with increasing concentrations of SOID-8 for 24 h and the level of PARP, Mcl-1 and Bcl-xL protein was measured by western blot.

Figure 4. SOID-8 represses the JAK2/STAT3 signaling pathway in a dose- and time-dependent manner.

(A) Total proteins were isolated from A2058 (left) and A375 (right) cells incubated with 2.5, 5, 10, or 20 µM SOID-8 for 24 h. Western blot was done with antibodies to total or phosphorylated (p) STAT3, JAK2 and Src using 40 µg total proteins. β-Actin was used as a loading control. (B) Time course of inhibition of STAT3 upstream regulatory proteins JAK2 following SOID-8 treatment. A2058 cells were treated with 10 µM of SOID-8 for indicated times. Total proteins were isolated and the level of total or phosphorylated (p) STAT3, JAK2 and Src was measured by western blot.

Figure 5. SOID-8 inhibits IL-6-induced phosphorylated STAT3 and JAK2 in A2058 (left) and A375 (right) cells.

The cells were serum-starved overnight, then left untreated or were treated with SOID-8 (5–20 µM) for 24 h. The untreated and SOID-8-treated cells were stimulated with IL-6 (10 ng/mL). Cells were then harvested after 30 minutes and the level of total or phosphorylated (p) STAT3 and JAK2 was analyzed by western blot.

Figure 6. SOID-8 inhibits melanoma tumor growth in a mouse xenograft model.

(A) SOID-8 suppressed tumor growth of A2058 melanoma xenografts. A2058 cells (2.5×106) were implanted subcutaneously into the flanks of NSG mice. After tumor development (one week later), SOID-8 or vehicle control was administered by oral gavage twice a day at 50 mg/kg for 17 days. Data are mean ± SEM. Statistical significance, determined by the two-tailed ANOVA, is indicated by *, p<0.01, n = 8 mice/group. (B) SOID-8 inhibits the level of phospho-JAK2 and phospho-STAT3, and Mcl-1 in A2058 tumors, as determined by western blot analysis. (C) SOID-8 inhibits viability of B16 mouse melanoma cells. Cells were treated with increasing concentrations of SOID-8 (1.25, 2.5, 5, 10, or 20 µM) for 24 and 48 h, and cell viability was evaluated by MTS assay. DMSO was used as vehicle control.