Treatment effects of fenretinide (4-HPR, 5 μmol/L), 2-ME (2 Treatment effects of fenretinide (4-HPR, 5 μmol/L), 2-ME (2.5 μmol/L), and the STAT3 inhibitor LY5 (0.5 μmol/L; ref. 50) on STAT3 phosphorylation and nuclear translocation. Treatment effects of fenretinide (4-HPR, 5 μmol/L), 2-ME (2.5 μmol/L), and the STAT3 inhibitor LY5 (0.5 μmol/L; ref. 50) on STAT3 phosphorylation and nuclear translocation. A, JSCC1 cells; B, JSCC2 cells; C, JSCC3 cells. Cytosolic and nuclear proteins were harvested from sera-deprived JSCC1 and JSCC2 cell lines following 24 hours of treatment (5 μmol/L 4-HPR, 2.5 μmol/L 2-ME, or 0.5 μmol/L LY5) or vehicle (0.1% DMSO) control. Multiple experiments confirmed 5 μmol/L 4-HPR treatment significantly reduced constitutive STAT3 phosphorylation and pSTAT3 nuclear translocation in the STAT3 constitutively active JSCC1 and JSCC2 cells (P < 0.05, Wilcoxon matched pairs signed rank test, n = 7). C, As the JSCC3 cells do not constitutively express pSTAT3, these cells underwent 24 hours stimulation in base medium supplemented with 10 ng/mL of IL6 or 5 ng/mL of TGFα, with or without 4-HPE, 2-ME, or LY5 treatment, followed by harvest. The combination treatment of 2-ME and 4-HPR significantly inhibited IL6 or TGFα-induced pSTAT3 nuclear translocation in JSCC3 cells (n = 3, P < 0.05, Wilcoxon matched pairs signed rank test). D, Effect of single and combination treatments of fenretinide (4-HPR, 5 μmol/L), 2-ME (2.5 μmol/L) and the IL6 receptor inhibitor TOC (1 μg/mL) on STAT3 and pSTAT3 levels. D and E, All cell lines with constitutive pSTAT3 expression (2095sc, JSCC1, and JSCC2) were sera deprived for 24 hours, followed by an additional 24 hours of treatment in sera-free medium that contained: control (0.1% DMSO), 5 μmol/L 4-HPR, 2.5 μmol/L 2-ME, 1 μg/mL TOC (initial agent concentrations same for single and combinations). As the JSCC3 cells do not constitutively express pSTAT3, these cells underwent 24 hours stimulation in base medium supplemented with 10 ng/mL of IL6 and 5 ng/mL of TGFα, with or without 4-HPR, 2-ME, and TOC followed by harvest. Selective treatments induced reduction of both STAT3 and pSTAT3 levels in 2095sc cells, and to a lesser extent JSCC1 and JSCC3 cells. The treatment combinations of TOC + 4-HPR and TOC+4-HPR+2-ME induced significant inhibition of STAT3 phosphorylation [P < 0.05, n = 12 total with n = 3 for every individual cell line (including JSCC3)], Kruskal–Wallis followed by Dunn's multiple comparison post hoc test). Susan R. Mallery et al. Cancer Prev Res 2017;10:76-88 ©2017 by American Association for Cancer Research