Original Communications| Volume 130, ISSUE 5, P859-865, November 2001

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. (Surgery 2001;859-65.)
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Surgery
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Meyers PA
        • Gorlick R
        • Heller G
        • Casper E
        • Lane J
        • Huvos AG
        • et al.
        Intensification of preoperative chemotherapy for osteogenic sarcoma: results of the Memorial Sloan-Kettering (T12) protocol.
        J Clin Oncol. 1998; 16: 2452-2458
        • Silverman LB
        • McLean TW
        • Gelber RD
        • Donnelly MJ
        • Gilliland DG
        • Tarbell NJ
        • et al.
        Intensified therapy for infants with acute lymphoblastic leukemia: results from the Dana-Farber Cancer Institute Consortium.
        Cancer. 1997; 80: 2285-2295
        • Williams SF
        • Gilewski T
        • Mick R
        • Bitran JD.
        High-dose consolidation therapy with autologous stem-cell rescue in stage IV breast cancer: follow-up report.
        J Clin Oncol. 1992; 10: 1743-1747
        • Livingston RB.
        High-dose chemotherapy in human solid tumors: rationale and practical use.
        in: Cancer chemotherapy: challenges for the future. Excerpta Medica, Tokyo1986: 130-143
        • Anzai T
        • Wang Y-M
        • Sasaki K
        • Sung A
        • Cangir A
        • Jaffe N
        • et al.
        Clinical pharmacology of high-dose methotrexate in humans.
        in: Methotrexate in cancer therapy. Raven Press, New York1986: 55-68
        • Mitchell EP
        • Schein PS
        Gastrointestinal toxicity of chemotherapeutic agents.
        in: Toxicity of chemotherapy. Grune & Stratton, New York1984: 269-295
        • da Silva CP
        • de Oliveira CR
        • da Conceicao M
        • de Lima P.
        Apoptosis as a mechanism of cell death induced by different chemotherapeutic drugs in human leukemic T-lymphocytes.
        Biochem Pharmacol. 1996; 51: 1331-1340
        • Huschtscha LI
        • Bartier WA
        • Ross CE
        • Tattersall MH.
        Characteristics of cancer cell death after exposure to cytotoxic drugs in vitro.
        Br J Cancer. 1996; 73: 54-60
        • Verburg M
        • Renes IB
        • Meijer HP
        • Taminiau JA
        • Buller HA
        • Einerhand AW
        • et al.
        Selective sparing of goblet cells and paneth cells in the intestine of methotrexate-treated rats.
        Am J Physiol Gastrointest Liver Physiol. 2000; 279: G1037-G1047
        • Chu KU
        • Higashide S
        • Evers BM
        • Rajaraman S
        • Ishizuka J
        • Townsend Jr, CM
        • et al.
        Bombesin improves survival from methotrexate-induced enterocolitis.
        Ann Surg. 1994; 220: 570-576
        • Hetts SW.
        To die or not to die: an overview of apoptosis and its role in disease.
        JAMA. 1998; 279: 300-307
        • Thornberry NA
        • Lazebnik Y.
        Caspases: enemies within.
        Science. 1998; 281: 1312-1316
        • Li P
        • Nijhawan D
        • Budihardjo I
        • Srinivasula SM
        • Ahmad M
        • Alnemri ES
        • et al.
        Cytochrome C and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.
        Cell. 1997; 91: 479-489
        • Blay J
        • Brown KD.
        Characterization of an epithelioid cell line derived from rat small intestine: demonstration of cytokeratin filaments.
        Cell Biol Int Rep. 1969; 8: 551-560
        • Ko TC
        • Bresnahan WA
        • Thompson EA.
        Intestinal cell cycle regulation.
        in: Progress in cell cycle research. Plenum Press, New York1997: 43-52
        • Papaconstantinou HT
        • Hwang KO
        • Rajaraman S
        • Hellmich MR
        • Townsend Jr, CM
        • Ko TC.
        Glutamine deprivation induces apoptosis in intestinal epithelial cells.
        Surgery. 1998; 124: 152-159
        • Zhang W
        • Khanna P
        • Chan LL
        • Campbell G
        • Ansari NH.
        Diabetes-induced apoptosis in rat kidney.
        Biochem Mol Med. 1997; 61: 58-62
        • Papaconstantinou HT
        • Chung DH
        • Zhang W
        • Ansari NH
        • Hellmich MR
        • Townsend Jr, CM
        • et al.
        Prevention of mucosal atrophy: role of glutamine and caspases in apoptosis in intestinal epithelial cells.
        J Gastrointest Surg. 2000; 4: 416-423
        • Sarin A
        • Wu ML
        • Henkart PA.
        Different interleukin-1 beta converting enzyme (ICE) family protease requirements for the apoptotic death of T lymphocytes triggered by diverse stimuli.
        J Exp Med. 1996; 184: 2445-2450
        • Yaoita H
        • Ogawa K
        • Maehara K
        • Maruyama Y.
        Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor.
        Circulation. 1998; 97: 276-281
        • Bossy-Wetzel E
        • Newmeyer DD
        • Green DR.
        Mitochondrial cytochrome C release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization.
        EMBO J. 1998; 17: 37-49
        • Potten CS
        • Wilson JW
        • Booth C.
        Regulation and significance of apoptosis in the stem cells of the gastrointestinal epithelium.
        Stem Cells. 1997; 15: 82-93
        • Keane MM
        • Ettenberg SA
        • Nau MM
        • Russell EK
        • Lipkowitz S.
        Chemotherapy augments TRAIL-induced apoptosis in breast cell lines.
        Cancer Res. 1999; 59: 734-741
        • Krajewska M
        • Wang HG
        • Krajewski S
        • Zapata JM
        • Shabaik A
        • Gascoyne R
        • et al.
        Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease.
        Cancer Res. 1997; 57: 1605-1613