Background: Sulforaphane (SUL) is an isothiocyanate naturally present in widely consumed vegetables, particularly in broccoli. Epidemiological studies indicate that human exposure to SUL through consumption of cruciferous vegetables may decrease cancer risk. That is, SUL exerts protective effects against potential carcinogens by inducing phase II enzymes that convert them to less toxic and more easily excretable products. Furthermore, SUL has recently been focused due to its inhibitory effects on tumor cell growth by inducing apoptosis and/or cell cycle arrest. Whereas apoptosis is an efficient programmed cell death process, which results in tumor suppression, autophagy is a process of "self-eating" that involves enzymatic digestion and recycling of cellular constituents in response to metabolic stress, which contributes to both cancer cell death and survival. Here, we aimed to investigate the potential ability of SUL to stimulate the autophagic pathway in human colon cancer cells, and the possible contribution of autophagy inhibition in potentiating the pro-apoptotic effect of SUL. Methods: The human colon cancer cell line WiDr was used. The proliferation of cells, treated with SUL at different concentrations, was assessed by the MTS assay. Apoptosis was investigated by flow-cytometry after double-staining with FITC-conjugated Annexin-V and propidium iodine. The formation of acidic vesicular organelles (AVOs) was detected by fluorescence microscopy and flow-cytometry following staining with the lysosomotropic agent acridine orange. Detection of microtubule-associated protein 1 light chain 3 (LC3) levels was investigated by Western blot. Localizations of LC3 and cytochrome c were analyzed by immunocytochemistry. Results: SUL-treatment induced the dose-dependent inhibition of proliferative activity of WiDr, which was mostly dependent on induction of apoptosis. Additionally, exposure of WiDr to lower concentrations of SUL resulted in the detection of several specific features of autophagy, including formation of AVOs. The SUL-induced autophagy was associated with up-regulation, processing, and recruitment to autophagosomes of LC3. Treatment of cells with specific inhibitors of autophagy (Lysosomal lumen alkalizer, Chloroquine diphosphate) increased the localization of LC3 to autophagosomes and cytosolic release of cytochrome c, and potentiated the apoptosis-inducing effect of SUL on colon cancer cells. Conclusion: The present results demonstrate the induction of autophagy represents a defense mechanism against SUL-induced apoptosis in human colon cancer cells. And the inhibition of autophagy potentiated the pro-apoptotic effect of SUL, suggestive that combination of inhibitors of autophagy with pro-apoptotic agents, including chemotherapeutic agents and plant extracts, would be a promising strategy to improve anti-cancer therapy.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 384.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO