In addition, the production of granzyme b and IFN by NK cells from normal donors when cultured with the K562 target cell line was not adversely affected in the presence of FLLL32. The mean difference for granzyme b was 41. 0 spots well and 65 spots well for IFN. Discussion We have characterized the biologic activity of the cur cumin analog, FLLL32 on melanoma and immune effec tor cells. The present study has demonstrated that the FLLL32 small molecule can inhibit STAT3 signal trans duction and induce caspase dependent, pro apoptotic effects against human melanoma cell lines and primary melanoma cultures at micromolar concentrations. In contrast to curcumin and other STAT3 pathway inhibi tors, IFN induced STAT1 phosphorylation was not altered in the presence of FLLL32.

This compound did not inhibit the viability of PBMCs, NK cells or their cellu lar responsiveness to clinically relevant cytokines. These data show that FLLL32 represents a novel small molecule curcumin analog with STAT3 pathway specificity that will be considered as a lead compound for further drug development in melanoma. FLLL32 represents a structural analog of curcumin when locked into its diketone tautomeric form. A num ber of favorable biologic properties resulting from these modifications have been characterized in this study. First, FLLL32 was ten fold more potent than curcumin at inducing apoptosis of melanoma cells. Second, FLLL32 did not appear to have to ic effects on either nor mal PBMCs or NK cells. Third, FLLL32 was designed to specifically target the oncogenic STAT3 pathway, while leaving the STAT1 pathway intact.

Data from the present report indicate that in terms of in vitro specificity, FLLL32 was superior to other STAT3 pathway inhibitors or to curcumin. In fact, prior studies from our group have demonstrated that curcumin inhibited the phosphoryla tion of numerous STAT proteins in response to clinically relevant cytokines including IFN, IFN and IL 2. These inhibitory effects of curcumin were observed in both Dacomitinib melanoma cell lines and in PBMCs from healthy donors. As a result, design of the FLLL32 analog was focused on ma imizing the target specificity for STAT3 over other STAT proteins. The present data support the STAT3 specificity of the FLLL32 lead compound, although they do not conclusively e clude that FLLL32 could modulate the phosphorylation of other unidentified kinases.

Numerous early generation small molecule STAT3 inhibitors have been reported to induce apoptosis via inhibi tion of STAT3 activation and or dimerization, while siRNA specific for the SH2 coding region of STAT3 could induce apoptosis in prostate cancer cells in vitro and in nude mice bearing human enograft tumors. Finally, studies have also shown that platinum comple es can promote anti tumor activity by virtue of their ability to inhibit STAT3. Collectively, these studies provide precedent for targeting STAT3 as a means of inducing tumor cell apoptosis.