Glutathione-S-transferase P1-1 protects aberrant crypt foci from apoptosis induced by deoxycholic acid.
OBJECTIVE
Aberrant crypt foci, precursors of colonic adenoma, are frequently positive for glutathione-S-transferase P1-1. Because deoxycholic acid is an apoptosis-inducing xenobiotic in the colon, we examined the possibility that aberrant crypt foci, through the cytoprotecting function of glutathione-S-transferase P1-1, resist deoxycholic acid-induced apoptosis, thereby surviving to become adenomas and subsequently cancer.


METHODS
Glutathione-S-transferase P1-1 or cyclooxygenase-2 expression and the percentage of apoptotic cells in aberrant crypt foci were examined by immunohistochemistry and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, respectively. Glutathione-S-transferase P1-1 was transfected into colon cancer cells (M7609) and human lung fibroblasts, and deoxycholic acid-induced apoptosis was evaluated by a dye-uptake assay and flow cytometry. Binding of deoxycholic acid to glutathione-S-transferase P1-1 was analyzed by circular dichroism and immunoprecipitation. Caspase activities were determined by colorimetric protease assay, and sulindac binding to glutathione-S-transferase P1-1 was determined by inhibition assay of glutathione-S-transferase P1-1 activity.


RESULTS
Aberrant crypt foci showed positive immunostaining for glutathione-S-transferase P1-1 but negative staining for cyclooxygenase-2. The percentage of apoptotic cells in aberrant crypt foci was significantly lower than in healthy epithelium, and the difference became more apparent with deoxycholic acid treatment. The impaired sensitivity of aberrant crypt foci to deoxycholic acid was restored by the glutathione-S-transferase P1-1-specific inhibitor gamma-glutamyl-S-(benzyl)cysteinyl-R-phenylglycine diethylester. By transfection of glutathione-S-transferase P1-1, M7609 cells became more resistant to deoxycholic acid-induced apoptosis than mock transfectants. Direct binding of glutathione-S-transferase P1-1 to deoxycholic acid was proven by circular dichroism and by immunoprecipitation. The aberrant crypt foci in adenoma patients treated with sulindac, which was shown to bind to glutathione-S-transferase P1-1, underwent apoptosis in 4 days and mostly regressed in 2-3 months.


CONCLUSIONS
Glutathione-S-transferase P1-1 protects aberrant crypt foci from deoxycholic acid-induced apoptosis and may play a pivotal role in early colon carcinogenesis.
Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, Japan.
Atsushi
A
Nobuoka
Tetsuji
T
Takayama
Koji
K
Miyanishi
Tsutomu
T
Sato
Kunihiro
K
Takanashi
Tsuyoshi
T
Hayashi
Takehiro
T
Kukitsu
Yasushi
Y
Sato
Minoru
M
Takahashi
Tetsuro
T
Okamoto
Takuya
T
Matsunaga
Junji
J
Kato
Masayuki
M
Oda
Takachika
T
Azuma
Yoshiro
Y
Niitsu
D000236:Adenoma / Q000188:drug therapy* / Q000473:pathology / Q000517:prevention & control; D000970:Antineoplastic Agents / Q000008:administration & dosage; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000473:pathology / Q000517:prevention & control; D051546:Cyclooxygenase 2; D003840:Deoxycholic Acid / Q000378:metabolism / Q000633:toxicity*; D003902:Detergents / Q000378:metabolism / Q000633:toxicity*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000473:pathology; D007527:Isoenzymes / Q000235:genetics; D008168:Lung / Q000166:cytology; D008565:Membrane Proteins; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D013467:Sulindac / Q000008:administration & dosage / Q000031:analogs & derivatives*; D014162:Transfection
D000970:Antineoplastic Agents; D003902:Detergents; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; C096329:gamma-Glu-S-BzCys-PhGly diethyl ester; D003840:Deoxycholic Acid; D013467:Sulindac; C025462:sulindac sulfide; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D005982:Glutathione Transferase; D005978:Glutathione; C025463:sulindac sulfone