The FLLL32 curcumin analog induced apoptosis in human melanoma cells. (A) The molecular structure of curcumin indicates that the molecule exists in two distinct tautomeric forms: 1) a diketone form and 2) a keto-enol form. FLLL32 was designed as a novel structural analog of curcumin that approximates a modified version of the molecule when locked into the keto-form. (B) Annexin V/PI staining of human metastatic melanoma cells following a 48 hour treatment with FLLL32. Error bars show 95% prediction limits based on the model fit at the estimated IC50 from two or more independent experiments. The non-responsive 1106 MEL and 1259 MEL cell lines were pSTAT3-negative. (C) Annexin V/PI staining of representative pSTAT3+ melanoma cells treated with either 20 μM curcumin or 2 μM FLLL32. Data are presented as the mean percentage of apoptotic cells. Error bars represent the standard deviation from at least two individual experiments. (D) Immunoblot analysis (left panel) or immunoprecipitation for total Jak2 protein (right panel; blot with Jak2 or pJak2 antibodies) of pSTAT3-positive A375 and Hs294T cells following 24 hour treatment. (E) FLLL32 treatment reduced pSTAT3, the STAT3-regulated gene, cyclin D1 and induced apoptosis in primary human cells derived from recurrent cutaneous melanoma tumors. These primary melanoma cell cultures have been previously described by our group . Cells were treated for 48 hours with the indicated concentrations of FLLL32 or curcumin (20 mM) as a biologic control and analyzed by immunoblot. Membranes were probed with β actin as a loading control and all blots represent data from at least two independent experiments.