Of concern were the effects of FLLL32 on signal transduction in response to IFN, a cytokine that mediates its cellular effects via phosphorylation of STAT1, and a resulting STAT1 STAT1 homodimer. To test these interactions in a biologic system, we investigated the effects of FLLL32 or curcumin pre treatment on IFN induced signaling and gene e pression. Pre treatment of pSTAT3 positive A375 and Hs294T cells with FLLL32 or curcumin led to reduced pSTAT3 versus DMSO treated cells. However, in contrast to curcumin, FLLL32 did not adversely affect IFN induced pSTAT1. A unique advantage of FLLL32 versus other STAT3 pathway inhibitors was its apparent specificity. Despite a similar degree of cytoto icity and the ability to reduce basal pSTAT3 in human melanoma cells, the WP1066, JSI 124, and Stattic compounds also inhibited IFN induced STAT1 phos phorylation.

Pre treatment with FLLL32 also enhanced transcription of the pro apoptotic interferon regulatory factor 1 gene in response to IFN stimulation as determined by Real Time PCR. This IFN responsive gene has been shown to be tran scribed via STAT1 STAT1 homodimers binding to a gamma activated sequence element. Consis tent with our prior studies, IFN stimulated IRF1 transcription was reduced in all cells pre treated with curcumin. The induction of IRF1 was not enhanced in the pSTAT3 negative 1106 MEL cell line, suggesting that cross reactivity of FLLL32 with STAT1 was negligible, and that IFN driven gene transcription can be augmented via STAT3 inhibition.

These data indicated that IFN induced signal transduc tion and gene e pression were not reduced by FLLL32 and that its inhibitory actions were specific for STAT3 and not other homologous STAT proteins that function Cilengitide as tumor suppressors. Effects of FLLL32 on immune effector cells STAT3 function in immune cells can promote tolerance to developing or established tumors. We therefore evalu ated whether FLLL32 would affect the responsiveness of PBMCs to stimulation with clinically relevant cytokines that mediate tumor progression, immunosurveil lance or T and NK cell survival. Pre treatment with increasing doses of FLLL32 reduced basal pSTAT3 in PBMCs from healthy donors and led to reduced IL 6 induced pSTAT3 in PBMCs. FLLL32 pre treatment also did not adversely affect the level of IFN induced pSTAT1 or IRF1 gene e pression in PBMC. The level of IL 2 induced pSTAT5 also was not altered by FLLL32 pre treatment. The FLLL32 compound did not decrease via bility of PBMCs after a 24 hour treatment as compared to treatment with DMSO alone as determined by Anne in V PI staining or PARP cleavage.