Abstract #54: Broccoli isothiocyanates induce cell cycle arrest and apoptosis in association with histone deacetylase inhibition and suppression of oncogenic signaling in human bladder cancer cells

Background: Epidemiologic evidence suggests that cruciferous vegetables, especially broccoli, may reduce bladder cancer risk. Our objectives were to characterize glucosinolates (GLUs) and their metabolites isothiocyanates (ITCs), found in broccoli and broccoli sprouts, and define mechanisms by which they may inhibit human bladder cancer. Methods: Broccoli and broccoli sprout GLUs and ITCs were characterized by HPLC. In vitro effects of non-hydrolyzed (GLUs) and hydrolyzed broccoli and broccoli sprouts (ITCs), and pure ITCs sulforaphane (SFN), erucin (ECN), iberin and allyl ITC, were studied on normal bladder urothelial cells (NBC) and a panel of human bladder cancer cell lines (BCC) representing the spectrum of bladder cancer biology (RT4: non-invasive; J82 and UMUC3: invasive). Cell viability (MTS and SRB assays), apoptosis (caspase-3/7 activity and PARP cleavage), cell cycle analysis (flow cytometry), histone deacetylase (HDAC) activity (Fluor de Lys^TM assay and immunoblotting), and Western blot analysis for several bladder cancer-related molecular markers (EGFR, Her2/neu, thymidylate synthase (TS), and survivin) were performed. Results: We observed a significant dose-dependent decrease in cell viability of bladder cancer cell lines (BCC) by broccoli, broccoli sprout ITCs and pure ITCs. Broccoli sprout ITCs and SFN (IC₅₀ = 2.52 µM) followed by ECN (IC₅₀ = 2.24 µM) were the most potent inhibitors. As compared to BCC, normal bladder urothelial cells showed less growth inhibition by SFN and ECN. Forty-eight hours treatment of BCC with SFN and ECN resulted in a dose-dependent induction of apoptosis and modulation of the cell cycle, in G₂/M and S phases. To elucidate mechanisms by which these biological changes occurred, we studied bladder cancer-related molecular markers and showed a significant dose-dependent decrease in TS, survivin, EGFR and Her2/neu protein expression. Interestingly, HDAC inhibition was also observed in BCC treated with either SFN or ECN, but this seems to vary by cell type. Conclusion: Broccoli and broccoli sprout ITCs inhibit cell viability, induce apoptosis and cell cycle arrest, and modulate different biological pathways in BCC. These effects may in part be related to inhibition of HDACs and ErbB family of receptors, novel targets of ITCs. We will also discuss the relevance of these findings to in vivo rodent studies. These studies suggest different, novel mechanisms of action for broccoli, broccoli sprouts and their components in bladder cancer, supporting continued preclinical and clinical studies. (Support: Shoen Cancer Prevention Research Fund; Center for Functional Foods and Research Entrepreneurship (CAFFRE); OSU CCC P30 CA16058)

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 54.