Inhibition of autophagy by 3-MA enhances the effect of 5-FU-induced apoptosis in colon cancer cells.
BACKGROUND
5-fluorouracil-(5-FU)-based adjuvant chemotherapy is widely used for the treatment of colorectal cancer. However, 5-FU resistance in the course of treatment has become more common. Therefore, new therapeutic strategies and/or new adjuvant drugs still need to be explored.


METHODS
Two colon-cancer-derived cell lines, colon26 and HT29, were used to investigate the effect of 5-FU, 3-methyladenine (3-MA, an autophagy inhibitor), or their combination on apoptotic cell death and autophagy. MTT assay, Hochest plus propidium iodide (PI) staining, and DNA fragmentation assay were used to observe apoptosis. Meanwhile, monodansylcadaverine (MDC) was used to detect autophagy. Finally, immunoblotting assay was used to explore the molecular change that occurred.


RESULTS
We observed the apoptosis induced by 5-FU in colon cancer cells. Meanwhile, autophagy was also stimulated. The combination treatment of 3-MA and 5-FU significantly increased the apoptotic cell death. By isolating the subcellular fractions of mitochondria and cytosol, we observed that the release of cytochrome c was increased in combination-treated cells. Cytochrome c resulted in the activation of caspase-3, thus activating PARP. Moreover, the anti-apoptotic protein, Bcl-xL, was significantly downregulated by 3-MA.


CONCLUSIONS
Our results suggest that 5-FU-induced apoptosis in colon cancer cells can be enhanced by the inhibitor of autophagy, 3-MA. Autophagy might play a role as a self-defense mechanism in 5-FU-treated colon cancer cells, and its inhibition could be a promising strategy for the adjuvant chemotherapy of colon cancer.
Li
Jie
J
Department of General Surgical Science (Surgery I), Graduate School of Medicine, Gunma University, Maebashi, Gunma 371-8511, Japan. lijie@med.gunma-u.ac.jp
Hou
Ni
N
Faried
Ahmad
A
Tsutsumi
Soichi
S
Takeuchi
Toshiyuki
T
Kuwano
Hiroyuki
H
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D045304:Cytochromes c / Q000378:metabolism; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans
D000964:Antimetabolites, Antineoplastic; D051017:Apoptosis Regulatory Proteins; C025946:3-methyladenine; D045304:Cytochromes c; D053148:Caspase 3; D000225:Adenine; D005472:Fluorouracil