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. 2005 Aug 19;94(10):886–893. doi: 10.1111/j.1349-7006.2003.tb01371.x

Characterization of cell death induced by ethacrynic acid in a human colon cancer cell line DLD‐1 and suppression by N‐acetyl‐l‐cysteine

Shu Aizawa 1, Keizou Ookawa 1, Toshihiro Kudo 1, Junpei Asano 1, Makoto Hayakari 1, Shigeki Tsuchida 1,✉
PMCID: PMC11160201  PMID: 14556662

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 μM, while it caused cell death at 60–100 μM. 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 μM EA, while c‐Jun N‐terminal kinase (JNK) 1 and p38 MAPK were markedly decreased by 100 μM 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.

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