title: Characterization of cell death induced by ethacrynic acid in a human colon cancer cell line DLD-1 and suppression by N-acetyl-L-cysteine.
abstract: Since ethacrynic acid (EA), an SH modifier as well as glutathione S-transferase (GST) inhibitor, has been suggested to induce apoptosis in some cell lines, its effects on a human colon cancer cell line DLD-1 were examined. EA enhanced cell proliferation at 20-40 microM, while it caused cell death at 60-100 microM. Caspase inhibitors did not block cell death and DNA ladder formation was not detected. Poly(ADP-ribose) polymerase, however, was cleaved into an 82-kDa fragment, different from an 85-kDa fragment that is specific for apoptosisis. The 82-kDa fragment was not recognized by antibody against PARP fragment cleaved by caspase 3. N-Acetyl-L-cysteine (NAC) completely inhibited EA-induced cell death, but 3(2)-t-butyl-4-hydroxyanisole or pyrrolidinedithiocarbamate ammonium salt did not. Glutathione (GSH) levels were dose-dependently increased in cells treated with EA and this increase was hardly affected by NAC addition. Mitogen-activated protein kinase (MAPK) kinase (MEK) 1, extracellular signal-regulated kinase (ERK) 1 and GST P1-1 were increased in cells treated with 25-75 microM EA, while c-Jun N-terminal kinase (JNK) 1 and p38 MAPK were markedly decreased by 100 microM EA. NAC repressed EA-induced alterations in these MAPKs and GST P1-1. p38 MAPK inhibitors, SB203580 and FR167653, dose-dependently enhanced EA-induced cell death. An MEK inhibitor, U0126, did not affect EA-induced cell death. These studies revealed that EA induced cell death concomitantly with a novel PARP fragmentation, but without DNA fragmentation. p38 MAPK was suggested to play an inhibitory role in EA-induced cell death.
authors.affiliation: Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki, Aomori 036-8562, Japan. tsuchida@cc.hirosaki-u.ac.jp
authors.forename: Shu
authors.initials: S
authors.lastname: Aizawa
authors.forename: Keizou
authors.initials: K
authors.lastname: Ookawa
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Kudo
authors.forename: Junpei
authors.initials: J
authors.lastname: Asano
authors.forename: Makoto
authors.initials: M
authors.lastname: Hayakari
authors.forename: Shigeki
authors.initials: S
authors.lastname: Tsuchida
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology*; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004976:Ethacrynic Acid / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D005453:Fluorescence; D005978:Glutathione / Q000378:metabolism; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000975:Antioxidants; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D020928:Mitogen-Activated Protein Kinases; D005978:Glutathione; D004976:Ethacrynic Acid; D000111:Acetylcysteine