Kim T I, Jin S H, Kim W H, Kang E H, Choi K Y, Kim H J, Shin S K, Kang J K
Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea.
Int J Colorectal Dis. 2001 Jun;16(3):167-73. doi: 10.1007/s003840100301.
The mechanisms of the antineoplastic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) still are unknown, but the induction of apoptosis is one of the possible mechanisms. We attempted to demonstrate the role of mitogen-activated protein (MAP) kinases, generally considered to be important mediators of proliferative and apoptotic signals, in NSAID-induced colon cancer cell apoptosis. Apoptosis was detected by demonstration of DNA fragmentation in agarose gel electrophoresis. Cell death was assessed by trypan blue dye exclusion method. MAP kinase activation was assessed by Western blot using phosphospecific antibodies to MAP kinases. Kinase assay using activating transcription factor-2 (ATF-2) fusion protein as a substrate was also performed for measuring p38 MAP kinase activity. For the inhibition of p38 MAP kinase, pyridinylimidazole compound (SB203580) was utilized. Caspase-3 activity was measured using the tetrapeptide fluorogenic substrate Ac-DEVD-AMC. Treatment of HT-29 cells with NSAIDs results in time- and dose-dependent induction of apoptosis, accompanied by sustained activation of all three MAP kinase subfamilies. The SB203580, a p38 MAP kinase inhibitor, reduced indomethacin-induced cell death by 43%, while PD098059, a MAPK/ERK kinase (MEK)1 inhibitor, did not affect cell death. p38 MAP kinase and caspase-3 activation were not significantly interlinked in indomethacin-induced apoptosis. From these results, we conclude that NSAIDs can induce prolonged activation of MAP kinases in colon cancer cells and that, of these, p38 MAP kinase may play a partial but significant role in indomethacin-induced apoptosis.
非甾体抗炎药（NSAIDs）抗肿瘤作用的机制尚不清楚，但诱导细胞凋亡是可能的机制之一。我们试图证明丝裂原活化蛋白（MAP）激酶在NSAIDs诱导的结肠癌细胞凋亡中的作用，MAP激酶通常被认为是增殖和凋亡信号的重要介质。通过琼脂糖凝胶电泳显示DNA片段化来检测细胞凋亡。通过台盼蓝拒染法评估细胞死亡情况。使用针对MAP激酶的磷酸特异性抗体通过蛋白质印迹法评估MAP激酶的激活。还使用激活转录因子-2（ATF-2）融合蛋白作为底物进行激酶测定以测量p38 MAP激酶活性。为了抑制p38 MAP激酶，使用了吡啶基咪唑化合物（SB203580）。使用四肽荧光底物Ac-DEVD-AMC测量半胱天冬酶-3的活性。用NSAIDs处理HT-29细胞会导致细胞凋亡的时间和剂量依赖性诱导，并伴随着所有三个MAP激酶亚家族的持续激活。p38 MAP激酶抑制剂SB203580使吲哚美辛诱导的细胞死亡减少了43%，而MAPK/ERK激酶（MEK）1抑制剂PD098059对细胞死亡没有影响。在吲哚美辛诱导的细胞凋亡中，p38 MAP激酶和半胱天冬酶-3的激活没有显著的相互联系。从这些结果中，我们得出结论，NSAIDs可以诱导结肠癌细胞中MAP激酶的长期激活，其中p38 MAP激酶可能在吲哚美辛诱导的细胞凋亡中起部分但重要的作用。
