Denkert Carsten, Fürstenberg Antje, Daniel Peter Ted, Koch Ines, Köbel Martin, Weichert Wilko, Siegert Antje, Hauptmann Steffen
1Institute of Pathology, Charité Hospital, Humboldt University, Campus Mitte, Schumannstr. 20/21, D-10117 Berlin, Germany.
Oncogene. 2003 Nov 27;22(54):8653-61. doi: 10.1038/sj.onc.1206920.
Cyclooxygenases, particularly COX-2, play an important role in tumor development and progression. We have previously shown that COX-2 expression is an independent prognostic factor in human ovarian carcinoma. In this study, we investigated the effects of the inhibition of COX isoforms by the NSAID NS-398 as well as by COX-isoform-specific RNA interference (RNAi) in the human ovarian carcinoma cell lines OVCAR-3 and SKOV-3. OVCAR-3 cells showed a constitutive expression of COX-1 and an induction of high levels of COX-2 and PGE(2) after stimulation with interleukin-1beta. In contrast, SKOV-3 cells were negative for both COX isoforms. In OVCAR-3 cells, PGE(2) production was inhibited by NS-398 in concentrations of 1 microM and by a COX-2-specific silencing RNA (siRNA), while a COX-1-specific siRNA did not have an effect. This suggests that COX-2 is the major source of PGE(2) in this cell line. To dissociate COX-2-specific and non-COX-2-specific effects on cell proliferation, a proliferation assay was performed after incubation of cells with NS-398 and COX siRNAs. NS-398 induced an inhibition of cell proliferation at concentrations of 50-500 microM, which are above the concentrations needed for the inhibition of PGE(2) production. This inhibitory effect was present in the COX-positive cell line OVCAR-3 as well as in the COX-negative cell line SKOV-3 and could not be reverted by addition of exogenous PGE(2). Neither COX-1- nor COX-2-specific siRNAs had an effect on cell proliferation of OVCAR-3 cells. Cell cycle analysis showed an increased accumulation of cells in the G0/G1 phase after treatment with NS-398, but not with COX siRNAs. These experiments suggest that NS-398 reduced cell proliferation in ovarian carcinoma cells by induction of G0/G1 cell cycle arrest independent of COX-2 inhibition. Our study shows that specific inhibition of COX isoforms by RNAi could be used to dissociate effects of NSAIDs. Furthermore, our results suggest that cell cycle arrest is one of the primary mechanisms responsible for the antiproliferative effects of NS-398 on ovarian carcinoma cells.
环氧化酶，尤其是COX-2，在肿瘤的发生和发展中起着重要作用。我们之前已经表明，COX-2的表达是人类卵巢癌的一个独立预后因素。在本研究中，我们调查了非甾体抗炎药NS-398以及COX同工型特异性RNA干扰（RNAi）对人类卵巢癌细胞系OVCAR-3和SKOV-3中COX同工型的抑制作用。OVCAR-3细胞显示出COX-1的组成型表达，在用白细胞介素-1β刺激后诱导高水平的COX-2和前列腺素E2（PGE2）表达。相比之下，SKOV-3细胞两种COX同工型均为阴性。在OVCAR-3细胞中，1 microM浓度的NS-398以及COX-2特异性沉默RNA（siRNA）可抑制PGE2的产生，而COX-1特异性siRNA则没有作用。这表明COX-2是该细胞系中PGE2的主要来源。为了区分COX-2特异性和非COX-2特异性对细胞增殖的影响，在用NS-398和COX siRNAs孵育细胞后进行了增殖试验。NS-398在50 - 500 microM浓度时诱导细胞增殖受到抑制，该浓度高于抑制PGE2产生所需的浓度。这种抑制作用在COX阳性细胞系OVCAR-3以及COX阴性细胞系SKOV-3中均存在，并且不能通过添加外源性PGE2来逆转。COX-1特异性和COX-2特异性siRNAs对OVCAR-3细胞的增殖均无影响。细胞周期分析显示，用NS-398处理后，细胞在G0/G1期的积累增加，但用COX siRNAs处理则不然。这些实验表明，NS-398通过诱导G0/G1细胞周期停滞来降低卵巢癌细胞的增殖，而与COX-2抑制无关。我们的研究表明，RNAi对COX同工型的特异性抑制可用于区分非甾体抗炎药的作用。此外，我们的结果表明，细胞周期停滞是NS-398对卵巢癌细胞抗增殖作用的主要机制之一。
