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

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

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

<p>(<b>A</b>) 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 (<i>p</i>) 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 (<i>p</i>) STAT3, JAK2 and Src was measured by western blot.</p

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

<p>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 (<i>p</i>) STAT3 and JAK2 was analyzed by western blot.</p

Effects of SOID-8 on apoptosis of melanoma cells.

<p>(<b>A</b>) 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>B</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.</p