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.