Abstract 4841: Inhibition of autophagy potentiates the anti-angiogenic effects of sulforaphane
Authors: Nishikawa Takeshi , Hiyoshi Masaya , Tsuno Nelson H. , Okaji Yurai , Sunami Eiji , Shuno Yasutaka , Sasaki Kazuhito , Hongo Kumiko , Kaneko Manabu , Kawai Kazushige , Kitayama Joji , Takahashi Koki , Nagawa Hirokazu 
Published: February 2011

Background: Sulforaphane (SUL), a kind of isothiocyanate, has been reported to exert strong inhibitory effects on various cancer cells by inducing apoptosis and/or cell cycle arrest. Recently autophagy induction by cancer cells has been focused as a defense mechanism against various proapoptotic agents, which may result in resistance to cancer therapy. Therefore, inhibition of autophagy in cancer cells may be a novel strategy in cancer therapy.

Tumor cells depend on angiogenesis for their growth and metastasis and, therefore, anti-angiogenic therapy is a promising anti-cancer strategy.

In the present study, we aimed to investigate the anti-angiogenic effect of SUL, and the possible induction of autophagy by tumor vascular endothelial cells as a defense mechanism against SUL-induced apoptosis. Additionally, we investigated the role of specific autophagy inhibitor in potentiating the proapoptotic effect of SUL. For these purposes, the human umbilical vein endothelial cells (HUVECs) were used as the in vitro model of tumor endothelium.

Methods: HUVECs were isolated from umbilical cords, obtained from normal pregnants at the delivery, from whom informed consent was obtained. Cells were treated with SUL at different concentrations, and their proliferative activity 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 acridine orange. Detection of light chain 3 (LC3) levels was investigated by Western blot. Localizations of LC3 and cytochrome c were analyzed by immunocytochemistry. Finally, the vascular-like structure formation on Matrigel was investigated. The role of the specific autophagy inhibitor (3-methyladenine (3-MA)) in potentiating the effects of SUL was similarly investigated.

Results: The proapoptotic effect was observed by treatment of cells with relatively high concentrations of SUL for long periods of time. At 16 hour-treatment with lower concentrations of SUL, the features of autophagy, were observed in HUVECs. Treatment of cells with 3-MA potentiated the proapoptotic effect of SUL, which was dependent on the activation of caspases and the release of cytochrome c to the cytosol. SUL dose-dependently inhibited the tube-like formation by HUVECs on Matrigel, an important event in the process of angiogenesis, and treatment of 3-MA potentiated this effect.

Conclusion: The present results demonstrate the induction of autophagy in endothelial cells as a protective reaction against the proapoptotic effect of SUL, and consequently, the potentiation of the proapoptotic effect by the inhibition of autophagy. These findings open premises for the use of autophagy inhibitors in combination with chemotherapeutic agents for the anti-angiogenic agents.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4841.