The inhibition of autophagy potentiates anti-angiogenic effects of sulforaphane by inducing apoptosis.
BACKGROUND
Sulforaphane (SUL), a kind of isothiocyanate, has recently been focused due to its strong pro-apoptotic effect on cancer cells as well as tumor vascular endothelial cells (ECs). And recently, we demonstrated the induction of autophagy by colon cancer cells as a protective mechanism against SUL. In the present study, we aimed to investigate the possible role of autophagy induction by ECs as a defense mechanism against SUL.


METHODS
Human umbilical vein endothelial cells (HUVECs) were used as the in vitro model of angiogenic ECs. The induction of autophagy was evaluated by the detection of acidic vesicular organelles (AVOs) by flow-cytometry, after the staining with acridine orange, as well as the detection of light chain 3(LC3) by Western blot. Finally, the functional implication of autophagy inhibition and SUL treatment in ECs was investigated by their ability to form vascular-like structures on Matrigel.


RESULTS
Treatment of HUVECs with relatively low concentrations of SUL for 16 h resulted in the evident formation of AVOs and the recruitment of LC3 to autophagosomes, the pathognomonic features of autophagy. Co-treatment of cells with the specific autophagy inhibitor (3-methyladenine) potentiated the proapoptotic effect of SUL. And inhibition of autophagy potentiated the inhibitory effect of SUL on the ability of ECs to form capillary-like structures.


CONCLUSIONS
Similar to cancer cells, ECs induced autophagy in response to the pro-apoptotic agent, SUL, and the inhibition of autophagy potentiated the pro-apoptotic effect. These findings open premises for the use of autophagy inhibitors in combination with anti-angiogenic agents.
Nishikawa
Takeshi
T
Department of Surgical Oncology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. takn-tky@umin.ac.jp
Tsuno
Nelson H
NH
Okaji
Yurai
Y
Sunami
Eiji
E
Shuno
Yasutaka
Y
Sasaki
Kazuhito
K
Hongo
Kumiko
K
Kaneko
Manabu
M
Hiyoshi
Masaya
M
Kawai
Kazushige
K
Kitayama
Joji
J
Takahashi
Koki
K
Nagawa
Hirokazu
H
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D003114:Colony-Forming Units Assay; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005434:Flow Cytometry; D006801:Humans; D017879:Isothiocyanates; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D015388:Organelles / Q000187:drug effects / Q000378:metabolism; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology
D020533:Angiogenesis Inhibitors; D017879:Isothiocyanates; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D013454:Sulfoxides; D013861:Thiocyanates; D020169:Caspases; C016766:sulforaphane