Abstract
Background. Enterocolitis is the major toxicity of methotrexate-based cancer chemotherapy, which
limits its clinical applications. Methotrexate induces gut mucosal apoptosis in vivo;
however, little is known about the molecular mechanism involved. The effectors of
apoptosis include the caspase family of proteases, which are selectively activated
in a stimulus-specific and tissue-specific fashion. The aims of this study were (1)
to establish an in vitro model of methotrexate-induced gut apoptosis and (2) to determine
the role of caspases in methotrexate-induced apoptosis in intestinal epithelial cells.
Methods. Rat intestinal epithelial cells (RIE-1) were treated with methotrexate in the absence
or presence of ZVAD-fluoromethyl ketone, a general caspase inhibitor. Apoptosis was
quantified by means of deoxyribonucleic acid (DNA) fragmentation assays and Hoechst
nuclear staining. Caspase activation was measured with the use of fluorogenic substrates.
Results. Methotrexate induced apoptosis and decreased cell number in RIE-1 cells. DNA fragmentation
was preceded by the sequential activation of caspases 9, 2, and 3, whereas caspases
1 and 8 remained inactive. ZVAD-fluoromethyl ketone inhibited methotrexate-induced
caspase activation, DNA fragmentation, and nuclear condensation. Conclusions. These results indicate that methotrexate activates specific caspases and induces
apoptosis in RIE-1 cells. Furthermore, caspases may play an important role in methotrexate-induced
apoptosis in RIE-1 cells and may be potential therapeutic targets to attenuate methotrexate-induced
enterocolitis. (Surgery 2001;859-65.)
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Article info
Publication history
Accepted:
May 18,
2001
Footnotes
*Supported by National Institutes of Health grants F32 DK09867, KO8 CA64191, PO1 DK35608, and EY 08547; American Cancer Society, Texas Division, Inc; Lions Eye Bank Foundation; and Walls Medical Research Foundation.
**Reprint requests: Tien C. Ko, MD, Department of Surgery, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-0542.
Identification
Copyright
© 2001 Mosby, Inc. Published by Elsevier Inc. All rights reserved.
