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尼美舒利抑制肝癌生长（中国药理学报已发表）
AIM: To investigate whether nimesulide could suppress tumor growth and induce apoptosis in implanted hepatoma mice and to explore the molecular mechanisms. METHODS: Male mice received nimesulide 10 mg/kg, 20 mg/kg, and 40 mg/kg ig daily for 21 d. Electron microscopy (EM), flow cytometry (FCM), DNA ladder, radioimmunoassay (RIA), and Western blot analysis were employed to investigate effect of nimesulide on mice hepatoma and the related molecular mechanisms. RESULT: Nimesulide inhibited the growth of hepatoma (from 14 ％ to 62 ％) and elicited typical apoptotic morphologic changes. The DNA ladder of high dose nimesulide was more clearly observed and apoptotic rate was 51.3 %±1.5 %. Nimesulide also decreased cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and Bcl-2 expression, while increased the level of Bax protein. CONCLUSION: Nimesulide suppresses tumor growth and induces apoptosis by inhibiting COX-2 and PGE2 expression, which may be related to overexpression of Bax over Bcl-2.
INTRODUCTION
Cyclooxygenase (COX) is responsible for the conversions of arachidonic acid to prostaglandin (PG). Increased PGE2 synthesis occurs in adenomatous polyps, colonic carcinomas[1] and epithelial ovarian cancers[2]. Significant advances have furthered our understanding of the potential role of PG in cancer biology. Evidences from in vitro and in vivo studies suggest an important role for PG and their synthesizing enzyme COX-2 in carcinogenesis[1,3]. In mammalian cells, the COX enzyme consists of two isozymes encoded by separate genes. The COX-1 gene is constitutively expressed in most tissues, and the protein levels do not fluctuate in response to stimuli such as cytokines or growth factors[4]. The COX-2 gene has been characterized as an immediate early gene associated with cellular growth and differentiation. Recently, elevated levels of COX-2 expression have been found in human carcinomas, including examples in the colon, lung, stomach, and pancreas[3,5-7]. Recent studies have suggested that overexpression of COX-2 might be one of the leading factors in hepatic carcinogenesis[8]. COX-2 can induce angiogenesis via vascular endothelial growth factor (VEGF) and prostaglandin production and can also inhibit apoptosis by inducing the antiapoptotic factor Bcl-2 as well as activating antiapoptotic signalling through Akt/PKB[9]. Therefore, the use of selective inhibitors for the down-regulation of COX-2 activity might be a target for preventing hepatic carcinoma development. Our previous studies have demonstrated that selective COX-2 inhibitors, SC58125 and JTE-522 have exerted cell proliferation inhibition and apoptosis induction on some cancer cell lines in vitro [10,11]. Nimesulide, a sulfonanilide class COX-2 inhibitor which can bind to the large catalytic moiety of COX-2 but not COX-1, preferentially inhibits sheep placenta COX-2 activity in vitro, with an IC50 of 0.07 μmol/L and appears to possess much less adverse on the gastrointestinal tract than non-specific NSAID[12,13]. Studies indicated that nimesulide possessed chemopreventive activity against intestinal polyp development in mice[12], PhIP-induced mammary carcinogesis and CDAA-induced hepatocarcinogenesis in rats[14-16]. Some effort to assess the efficacy of nimesulide as a anticancer agent compared to other potential anticancer compounds, including conventional NSAID and other selective COX-2 inhibitors for liver cancer, in experimental animal models seems worthwhile. So, the present study was designed to determine the effects of nimesulide on hepatoma in vivo.
MATERIALS AND METHODS
Materials Nimesulide was purchased from Sigma Chemical Co (St Louis, MO, USA) and suspended in phosphate-buffered saline (PBS) pH 7.2 solution. Monoclonal anti-mouse antibodies of COX-2, Bcl-2, Bax, C-myc, and β-actin were obtained from Santa Cruz. Final dilutions were 1:300 for bcl-2 and bax antibodies, 1:100 for anti-c-myc antibodies, 1:500 for COX-2 and β-actin antibodies. RIA kit was purchased from Institute of Blood, Suzhou Medical University, China.
Animals and tumor model Male Kunming mice at about 18-22 g were sterilely inoculated subcutaneously in the flank with 1×107 mice hepatoma cell line H22 and were bred on standard mouse chow and water freely under standard conditions. They were randomly separated into four groups (10 mice in each group). The following day they were treated with nimesulide 10 mg/kg, 20 mg/kg, 40 mg/kg, or vehicle, respectively and were ig per day from the 1st day to the 21th day. Throughout the experimentation period, food and water was available to animals ad libitum. After 21 d test period animals were killed by cervical dislocation. Solid tumor was weighed, then fixed or pulverized using a mortar and pestle.
Tumor inhibition rate Tumor growth was evaluated by the inhibition rate as assessed by the formula: IR=(1-T/C) ×100 %. Where IR is the mean inhibition rate, T is the mean tumor weight in the treatment group and C is the mean tumor weight in the control group.
Morphological Analysis of Apoptosis Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by electron microscopy (EM). Solid tumors were fixed with glutaraldehyde 20 mL/L. EM analysis was performed as described previously [17]. Thin sections were viewed in an electron microscope (JEM-100CX 11/T, Japan).
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作者:admin@医学,生命科学 2011-02-27 17:11
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