Inhibition of autophagy potentiates sulforaphane-induced apoptosis in human colon cancer cells.
BACKGROUND
Sulforaphane (SUL), an isothiocyanate naturally present in widely consumed vegetables, particularly broccoli, has recently attracted attention due to its inhibitory effects on tumor cell growth by inducing apoptosis. We investigated the ability of SUL to induce autophagy in human colon cancer cells and whether inhibition of autophagy could potentiate the proapoptotic effect of SUL.


METHODS
The proliferation of cells treated with SUL was assessed by MTS assay and colony-forming assay. Apoptosis and caspases activity were investigated by flow cytometry. The formation of acidic vesicular organelles (AVOs) was detected in acridine-orange-stained cells by flow cytometry. Western blotting was used for the detection of light chain 3 (LC3). Localizations of LC3 and cytochrome c were analyzed by immunocytochemistry.


RESULTS
The proapoptotic effect was observed by treatment of cells with relatively high concentrations of SUL for long periods of time. After 16 h of treatment, evident formation of AVOs and recruitment of LC3 to autophagosomes, features of autophagy, were observed. Treatment of cells with a specific autophagy inhibitor (3-methyladenine) potentiated the proapoptotic effect of SUL, which was dependent on the activation of caspases and the release of cytochrome c to the cytosol.


CONCLUSIONS
The present results demonstrate induction of autophagy in colon cancer cells as a protective reaction against the proapoptotic effect of SUL, and consequently, the potentiation of the proapoptotic effect by autophagy inhibition. These findings provide a premise for use of autophagy inhibitors in combination with chemotherapeutic agents for treatment of colorectal cancer.
Nishikawa
Takeshi
T
Department of Surgical Oncology, Faculty of Medical Science, The University of Tokyo, Tokyo, Japan. takn-tky@umin.ac.jp
Tsuno
Nelson H
NH
Okaji
Yurai
Y
Shuno
Yasutaka
Y
Sasaki
Kazuhito
K
Hongo
Kumiko
K
Sunami
Eiji
E
Kitayama
Joji
J
Takahashi
Koki
K
Nagawa
Hirokazu
H
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000473:pathology*; D003114:Colony-Forming Units Assay; D045304:Cytochromes c / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*
D016588:Anticarcinogenic Agents; D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates; C025946:3-methyladenine; D045304:Cytochromes c; D020169:Caspases; C016766:sulforaphane; D000225:Adenine