The role of caspases in methotrexate-induced gastrointestinal toxicity.
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.
Papaconstantinou
H T
HT
Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555-0542, USA.
Xie
C
C
Zhang
W
W
Ansari
N H
NH
Hellmich
M R
MR
Townsend
C M
CM
Ko
T C
TC
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology*; D002452:Cell Count; D002460:Cell Line; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D007413:Intestinal Mucosa / Q000187:drug effects*; D008727:Methotrexate / Q000633:toxicity*; D051381:Rats
D000590:Amino Acid Chloromethyl Ketones; D000964:Antimetabolites, Antineoplastic; D061945:Caspase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D020169:Caspases; D008727:Methotrexate