N-(4-Hydroxyphenyl) Retinamide Potentiated Anti-Tumor Efficacy of Genistein in Human Ewing’s Sarcoma Xenografts

Abstract

BackgroundEwing’s sarcoma is a pediatric tumor that mainly occurs in soft tissues and bones. New therapeutic strategies are urgently needed for treatment of Ewing’s sarcoma. We examined for the first time the efficacy of N-(4-hydroxyphenyl) retinamide (4-HPR) and genistein (GST) alone and also in combination for controlling growth of human Ewing’s sarcoma SK-N-MC and RD-ES xenografts.MethodsEfficacy of combination therapy was evaluated using histopathological parameters. Molecular mechanisms of combination therapy were detected using Western blotting and immunofluorescence microscopy.ResultsHistopathological examination of tumor sections showed that control group maintained characteristic growth of tumors, 4-HPR alone inhibited proliferation of tumor cells, GST alone induced apoptosis to some extent, and combination of 4-HPR and GST significantly induced apoptosis in both Ewing’s sarcoma xenografts. Time-dependent reductions in body weight, tumor volume, and tumor weight were also found. Combination therapy increased Bax : Bcl-2 ratio to trigger mitochondrial release of Smac/Diablo into the cytosol to downregulate the baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins such as BIRC-2 and BIRC-3 and thereby promote apoptosis. Activation of caspase-3 and mitochondrial release of apoptosis-inducing factor (AIF) occurred in course of apoptosis. Downregulation of the survival factor NF-κB and the angiogenic factors VEGF and FGF2 and increase in caspase-3 activity controlled tumor growth. In situ immunofluorescent labelings showed overexpression of calpain, caspase-12 and caspase-3, and AIF in xenografts, indicating induction of cysteine proteases and AIF for apoptosis.ConclusionsResults revealed that combination of 4-HPR and GST could be highly effective treatment for inhibiting Ewing’s sarcomas in vivo.