The role of caspases in methotrexate-induced gastrointestinal toxicity

Background. Enterocolitis is the major toxicity of methotrexate-based cance r chemotherapy, which limits its clinical applications. Methotrexate induce s gut mucosal apoptosis in vivo; however, little is known about the molecul ar mechanism involved. The effectors of apoptosis include the caspase famil y 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 epithelia l cells. Methods. Rat intestinal epithelial cells (RIE-1) were treated with methotre xate in the absence or presence of ZVAD-fluoromethyl ketone, a general casp ase 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 caspa ses 9, 2, and 3, whereas caspases I and 8 remained inactive. ZVAD-fluoromet hyl ketone inhibited methotrexate-induced caspase activation, DNA fragmenta tion, and nuclear condensation. Conclusions. These results indicate that methotrexate activates specific ca spases 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 enteroc olitis.