联合应用选择性环氧合酶-2抑制剂NS-398和脂氧合酶抑制剂NDGA对人肝癌细胞株HepG-2增殖及凋亡的影响
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摘要
肝细胞性肝癌(hepatocellular carcinoma, HCC)是我国常见的恶性肿瘤之一,近二十年来,我国HCC的死亡率增加了41.7%,约占全世界HCC死亡人数的42%。目前,手术切除仍是治疗HCC的最佳手段,但本病起病隐匿,病情发展迅速,大多数患者就诊时已发展到中晚期,失去了手术治疗的机会,患者5年生存率低于10%。环氧合酶(cylooxygenases,COX)和脂氧合酶(lipoxygenase,LOX)是体内催化花生四烯酸(arachidonic acid, AA)代谢的两个关键酶。细胞膜上的磷脂在磷脂酶A_2作用下释放出AA,AA经COX和LOX两条通路代谢产生一系列的生物活性物质,如前列腺素(PG)、血栓烷素A2 (TXA2)、羟基廿碳四烯酸(HETES)和白细胞三烯(LTS)等。通过这些活性物质,COX及LOX影响细胞信号转导及代谢,进而对包括肿瘤,炎症在内的多种疾病产生作用。NS-398是一种选择性COX-2抑制剂,与传统的非甾体类抗炎药(nonsteroidal anti-inflammatory drugs,NSAIDS)不同的是其抑制COX-1的作用非常弱,从而减轻了胃肠道的不良反应。目前其已被发现能够抑制肝癌细胞增殖和诱导细胞周期阻滞。LOX抑制剂NDGA对于人肝癌细胞株HepG2细胞具有明显生长抑制作用,同时还可以诱导HepG_2细胞凋亡,引起细胞周期被阻滞于G1/S期。本研究选用人肝癌细胞株HepG_2作为研究对象,探讨NS-398
和NDGA联合应用对人肝癌细胞的增殖抑制和诱导凋亡作用及对bcl-2mRNA表达的影响,为将NS-398和NDGA联合应用于HCC的治疗提供初步的实验依据。
目的:
探讨单独和联合应用选择性环氧合酶-2抑制剂(cyclooxygenase-2 selective inhibitor) NS-398和脂氧合酶抑制剂(lipoxygenase inhibitor)NDGA对于人肝癌细胞株HepG2的增殖、凋亡和对bcl-2mRNA表达的影响。
方法:
(1)采用四甲基偶氮唑蓝(MTT)比色法检测单独及联合应用NS-398和NDGA对于人肝癌细胞株HepG2的增殖抑制作用并应用金正均法判断两药的相互作用。
(2)采用DNA末端原位标记染色法(TUNEL法)观察药物作用后人肝癌细胞株HepG2的形态学改变并计算凋亡指数(apoptosis index, AI)。
(3)采用流式细胞术检测药物作用后人肝癌细胞株HepG2细胞凋亡率。
(4)采用RT-PCR检测药物作用后人肝癌细胞株HepG2中bcl-2 mRNA的表达。
结果:
(1) MTT结果显示NS-398 (20~160μmol/L)或NDGA (25~200μmol/L)单独应用对人肝癌细胞株HepG2均有增殖抑制作用,药物浓度越高,抑制作用越强,呈现明显的剂量效应关系。系列浓度NS-398或NDGA作用于细胞24h、48h、72h后观察细胞的生长情况,结果显示NS-398或NDGA作用时间越长,对上述细胞的抑制作用越强,呈明显的时间效应关系。依据金正均法NS-398在20~160μmol·L~(-1)浓度范围内与NDGA 25~100μmol·L~(-1)浓度范围内及NS-398 20μmol·L~(-1)与NDGA 200μmol·L~(-1)联合应用显示出协同作用(q >1.15),余浓度联合应用显示相加作用(0.85 (2) TUNEL染色结果显示经NS-398(160μmol/L)和NDGA(200μmol/L)单独和联合应用48h后均可诱导人肝癌细胞株HepG2发生凋亡,凋亡细胞胞核成棕色,核固缩,大小不一;非凋亡细胞细胞核淡蓝紫色,核形态大小较一致。联合用药组较各单独用药组及对照组凋亡指数增高。
(3)流式细胞术结果显示经NS-398(160μmol/L)和NDGA(200μmol/L)单独和联合应用48h后均可诱导人肝癌细胞株HepG2发生凋亡,在DNA直方图上G0/G1期前可见亚G1峰即凋亡峰,NS-398与NDGA联合应用48h后凋亡率明显高于各单独用药组,提示两药合用对诱导人肝癌细胞株HepG2凋亡有协同作用。
(4) RT-PCR检测bcl-2 mRNA的结果显示NS-398(160μmol/L)或NDGA(200μmol/L)单独和联合用药48h后HepG2细胞bcl-2 mRNA的表达均降低,两药联合用药组人肝癌HepG2细胞bcl-2 mRNA表达明显低于NS-398或NDGA单独用药组,差异具有显著性(**P<0.01)。
结论:
(1)单独应用NS-398或NDGA对于人肝癌细胞株HepG2均有增殖抑制作用,且呈剂量和时间效应关系。NS-398和NDGA联合用药对人肝癌细胞株HepG2的增殖抑制具有协同作用。
(2)单独应用NS-398或NDGA对于人肝癌细胞株HepG2均有诱导凋亡作用; NS-398和NDGA联合用药对于人肝癌细胞株HepG2有协同诱导凋亡作用,并可能与抑制bcl-2 mRNA表达的作用增强有关。
Hepatocellular carcinoma (HCC) is one of the most common malignancies in China, which tends to present at an advanced stage before it is detected clinically. It is not amenable to curative therapies and the prognosis of such patients is extremely poor, with a 5-year survival of less than 10%. Cylooxygenases (COX) and lipoxygenase (LOX) are two key enzymes in the metabolism of arachidonic acid (AA). Phospholipid (PHL) in cellular membrane released AA under the effect of phosphatidolipase. AA are catalyzed to a series of biotic activators such as PG, TXA_2, HETES, LTS etc. through COX and LOX. COX and LOX effect on cell signal transduction and metabolism through these biotic activators and play an important role in many kinds of disease including tumor and inflammation. NS-398 is a selective COX-2 inhibitor, different from the traditional nonsteroidal anti-inflammatory drugs (NSAIDS), its inhibition effect on COX-1 is poor, so palliate adverse reaction of gastrointestinal tract. NS-398 can inhibit the proliferation and induce cell life cycle arrest in human hepatoma carcinoma cell. NDGA is a LOX inhibitor, also can inhibit the proliferation, induce apoptosis and cell cycle arrest at the G_1/S phase in human hepatoma carcinoma cell HepG2. The present study was designed to explore the effect of NS-398 in combination with NDGA on the proliferation, apoptosis and expression of bcl-2 mRNA in human hepatocellular carcinoma cell line HepG2, in order to develop an effective combination therapy for HCC.
OBJECTIVE
To explore the effect of NS-398 in combination with NDGA or alone on the proliferation, apoptosis and expression of bcl-2 mRNA in human hepatocellular carcinoma cell line HepG2.
METHODS
(1) The inhibitory effect of NS-398 in combination with NDGA or alone on the proliferation of HepG2 in vitro was measured by MTT assay. The Jin’s formula was used to analyze the synergic inhibitory effect.
(2) Morphological evidence of apoptosis induced by NS-398 in combination with NDGA or alone of HepG2 cells was detected by TUNEL. The apoptosis index (AI) was also calculated.
(3) Apoptosis of HepG2 cells caused by NS-398 in combination with NDGA or alone was detected by flow cytometry (FCM).
(4) The expression of bcl-2 mRNA of HepG2 treated with NS-398 in combination with NDGA or alone was detected by RT-PCR.
RESULTS
(1) The MTT results suggested that NS-398 or NDGA had inhibitory effect on the proliferation of HepG2. The higher the concentration of NS-398 or NDGA was, the stronger the cytotoxic effect reached, which suggested obvious dose-dependent manner of NS-398 or NDGA. And the inhibitory effects were augmented with the prolongation of culture time (24h, 48h to 72h), which had time-dependence. NS-398(20、40、80、 160μmol/L) in combination with NDGA(25、50、100μmol/L) showed synergic effect on the proliferation of human cell line HepG2 (q >1.15), and others were addition(0.85< q <1.15 )effect. There was significant difference between the combined group and the NDGA group alone(*P<0.05,**P<0.01).
(2) Results of TUNEL suggested that treatment with NS-398 (160μmol/L) or NDGA (200μmol/L) or a combination of the two agents for 48h. The AI of combination group was higher than drugs used alone, which demonstrated that NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells.
(3) FCM assay showed that treatment with NS-398 (160μmol/L) or NDGA (200μmol/L) or a combination of the two agents for 48h, the sub-G1 peak appeared before G1 phase, which represents apoptotic cell population, was observed clearly in the HepG2 cells, and the apoptotic rate of combination group was higher than drugs used alone. The results demonstrated that NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells.
(4) The results of RT-PCR showed that the expression of bcl-2 mRNA of HepG2 cells in combined group or alone all decreased. There was significant difference between combined group than alone(**P<0.01).
CONCLUSIONS
(1) NS-398 or NDGA used alone has antiproliferative effect on the human hepatoma cell line HepG2, with an obvious dose- and time-dependent manner. NS-398 in combination with NDGA had synergistic growth-inhibitory effect on the HepG2 cells.
(2) NS-398 or NDGA used alone could induce apoptosis on the human hepatoma cell line HepG2. NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells and correlated with potentialize reducing the expression of bcl-2 mRNA probably.
和NDGA联合应用对人肝癌细胞的增殖抑制和诱导凋亡作用及对bcl-2mRNA表达的影响,为将NS-398和NDGA联合应用于HCC的治疗提供初步的实验依据。
目的:
探讨单独和联合应用选择性环氧合酶-2抑制剂(cyclooxygenase-2 selective inhibitor) NS-398和脂氧合酶抑制剂(lipoxygenase inhibitor)NDGA对于人肝癌细胞株HepG2的增殖、凋亡和对bcl-2mRNA表达的影响。
方法:
(1)采用四甲基偶氮唑蓝(MTT)比色法检测单独及联合应用NS-398和NDGA对于人肝癌细胞株HepG2的增殖抑制作用并应用金正均法判断两药的相互作用。
(2)采用DNA末端原位标记染色法(TUNEL法)观察药物作用后人肝癌细胞株HepG2的形态学改变并计算凋亡指数(apoptosis index, AI)。
(3)采用流式细胞术检测药物作用后人肝癌细胞株HepG2细胞凋亡率。
(4)采用RT-PCR检测药物作用后人肝癌细胞株HepG2中bcl-2 mRNA的表达。
结果:
(1) MTT结果显示NS-398 (20~160μmol/L)或NDGA (25~200μmol/L)单独应用对人肝癌细胞株HepG2均有增殖抑制作用,药物浓度越高,抑制作用越强,呈现明显的剂量效应关系。系列浓度NS-398或NDGA作用于细胞24h、48h、72h后观察细胞的生长情况,结果显示NS-398或NDGA作用时间越长,对上述细胞的抑制作用越强,呈明显的时间效应关系。依据金正均法NS-398在20~160μmol·L~(-1)浓度范围内与NDGA 25~100μmol·L~(-1)浓度范围内及NS-398 20μmol·L~(-1)与NDGA 200μmol·L~(-1)联合应用显示出协同作用(q >1.15),余浓度联合应用显示相加作用(0.85
(3)流式细胞术结果显示经NS-398(160μmol/L)和NDGA(200μmol/L)单独和联合应用48h后均可诱导人肝癌细胞株HepG2发生凋亡,在DNA直方图上G0/G1期前可见亚G1峰即凋亡峰,NS-398与NDGA联合应用48h后凋亡率明显高于各单独用药组,提示两药合用对诱导人肝癌细胞株HepG2凋亡有协同作用。
(4) RT-PCR检测bcl-2 mRNA的结果显示NS-398(160μmol/L)或NDGA(200μmol/L)单独和联合用药48h后HepG2细胞bcl-2 mRNA的表达均降低,两药联合用药组人肝癌HepG2细胞bcl-2 mRNA表达明显低于NS-398或NDGA单独用药组,差异具有显著性(**P<0.01)。
结论:
(1)单独应用NS-398或NDGA对于人肝癌细胞株HepG2均有增殖抑制作用,且呈剂量和时间效应关系。NS-398和NDGA联合用药对人肝癌细胞株HepG2的增殖抑制具有协同作用。
(2)单独应用NS-398或NDGA对于人肝癌细胞株HepG2均有诱导凋亡作用; NS-398和NDGA联合用药对于人肝癌细胞株HepG2有协同诱导凋亡作用,并可能与抑制bcl-2 mRNA表达的作用增强有关。
Hepatocellular carcinoma (HCC) is one of the most common malignancies in China, which tends to present at an advanced stage before it is detected clinically. It is not amenable to curative therapies and the prognosis of such patients is extremely poor, with a 5-year survival of less than 10%. Cylooxygenases (COX) and lipoxygenase (LOX) are two key enzymes in the metabolism of arachidonic acid (AA). Phospholipid (PHL) in cellular membrane released AA under the effect of phosphatidolipase. AA are catalyzed to a series of biotic activators such as PG, TXA_2, HETES, LTS etc. through COX and LOX. COX and LOX effect on cell signal transduction and metabolism through these biotic activators and play an important role in many kinds of disease including tumor and inflammation. NS-398 is a selective COX-2 inhibitor, different from the traditional nonsteroidal anti-inflammatory drugs (NSAIDS), its inhibition effect on COX-1 is poor, so palliate adverse reaction of gastrointestinal tract. NS-398 can inhibit the proliferation and induce cell life cycle arrest in human hepatoma carcinoma cell. NDGA is a LOX inhibitor, also can inhibit the proliferation, induce apoptosis and cell cycle arrest at the G_1/S phase in human hepatoma carcinoma cell HepG2. The present study was designed to explore the effect of NS-398 in combination with NDGA on the proliferation, apoptosis and expression of bcl-2 mRNA in human hepatocellular carcinoma cell line HepG2, in order to develop an effective combination therapy for HCC.
OBJECTIVE
To explore the effect of NS-398 in combination with NDGA or alone on the proliferation, apoptosis and expression of bcl-2 mRNA in human hepatocellular carcinoma cell line HepG2.
METHODS
(1) The inhibitory effect of NS-398 in combination with NDGA or alone on the proliferation of HepG2 in vitro was measured by MTT assay. The Jin’s formula was used to analyze the synergic inhibitory effect.
(2) Morphological evidence of apoptosis induced by NS-398 in combination with NDGA or alone of HepG2 cells was detected by TUNEL. The apoptosis index (AI) was also calculated.
(3) Apoptosis of HepG2 cells caused by NS-398 in combination with NDGA or alone was detected by flow cytometry (FCM).
(4) The expression of bcl-2 mRNA of HepG2 treated with NS-398 in combination with NDGA or alone was detected by RT-PCR.
RESULTS
(1) The MTT results suggested that NS-398 or NDGA had inhibitory effect on the proliferation of HepG2. The higher the concentration of NS-398 or NDGA was, the stronger the cytotoxic effect reached, which suggested obvious dose-dependent manner of NS-398 or NDGA. And the inhibitory effects were augmented with the prolongation of culture time (24h, 48h to 72h), which had time-dependence. NS-398(20、40、80、 160μmol/L) in combination with NDGA(25、50、100μmol/L) showed synergic effect on the proliferation of human cell line HepG2 (q >1.15), and others were addition(0.85< q <1.15 )effect. There was significant difference between the combined group and the NDGA group alone(*P<0.05,**P<0.01).
(2) Results of TUNEL suggested that treatment with NS-398 (160μmol/L) or NDGA (200μmol/L) or a combination of the two agents for 48h. The AI of combination group was higher than drugs used alone, which demonstrated that NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells.
(3) FCM assay showed that treatment with NS-398 (160μmol/L) or NDGA (200μmol/L) or a combination of the two agents for 48h, the sub-G1 peak appeared before G1 phase, which represents apoptotic cell population, was observed clearly in the HepG2 cells, and the apoptotic rate of combination group was higher than drugs used alone. The results demonstrated that NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells.
(4) The results of RT-PCR showed that the expression of bcl-2 mRNA of HepG2 cells in combined group or alone all decreased. There was significant difference between combined group than alone(**P<0.01).
CONCLUSIONS
(1) NS-398 or NDGA used alone has antiproliferative effect on the human hepatoma cell line HepG2, with an obvious dose- and time-dependent manner. NS-398 in combination with NDGA had synergistic growth-inhibitory effect on the HepG2 cells.
(2) NS-398 or NDGA used alone could induce apoptosis on the human hepatoma cell line HepG2. NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells and correlated with potentialize reducing the expression of bcl-2 mRNA probably.
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37.彭黎明,王曾礼主编.细胞凋亡的基础与临床.北京:人民卫生出版社, 2000:153.
38. Kirkin V, Joos S, Z?rnig M. The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta[J]. 2004;1644(2-3):229-49
39. RosséT, Olivier R, Monney L, Rager M, Conus S, Fellay I, Jansen B, Borner C. Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c.. Nature[J]. 1998;391(6666):496-499.
1. McGlynn KA, London WT. Epidemiology and natural history of hepatocellular carcinoma.Best Pract Res Clin Gastroenterol[J].2005;19(1):3-23.
2. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer[J].2001,94(2):153-156.
3. Di Bisceglie AM. Epidemiology and clinical presentation of hepatocellular carcinoma.J Vasc Interv Radiol[J]. 2002;13(9 Pt 2):S169-171.
4. Bruix J, Llovet JM. Prognostic prediction and treatment strategy in hepatocellular carcinoma.Hepatology[J]. 2002;35(3):519-524.
5.陈建国,宋新明.中国肝癌发病水平的估算及分析[J].中国肿瘤,2005;14(1):28-31.
6. Williams CS, Mann M, DuBois RN. The role of cyclooxygenases in inflammation, cancer, and development.Oncogene[J]. 1999;18(55):7908-7916.
7. Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochem J[J]. 1994;302 ( Pt 3):723-727.
8. Hempel SL, Monick MM, Hunninghake GW. Lipopolysaccharide induces prostaglandin H synthase-2 protein and mRNA in human alveolar macrophages and blood monocytes. J Clin Invest[J].1994;93(1):391-396.
9. Fong CY, Pang L, Holland E, Knox AJ. TGF-beta1 stimulates IL-8 release, COX-2 expression, and PGE(2) release in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol[J]. 2000;279(1):L201-L207.
10. Chen CC, Sun YT, Chen JJ, Chang YJ. Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and IkappaB kinase 1/2 in human alveolar epithelial cells. Mol Pharmacol[J]. 2001;59(3):493-500.
11. Laporte JD, Moore PE, Lahiri T, Schwartzman IN, Panettieri RA Jr, Shore SA. p38MAP kinase regulates IL-1 beta responses in cultured airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol[J].2000;279(5):L932-L941.
12. Tazawa R, Xu XM, Wu KK, Wang LH. Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene. Biochem Biophys Res Commun[J]. 1994;203(1):190-199.
13. Lee SH, Soyoola E, Chanmugam P, Hart S, Sun W, Zhong H, Liou S, Simmons D, Hwang D. Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J Biol Chem[J].1992;267(36):25934-25938.
14.Onoe Y, Miyaura C, Kaminakayashiki T, Nagai Y, Noguchi K, Chen QR, Seo H, Ohta H, Nozawa S, Kudo I, Suda T. IL-13 and IL-4 inhibit bone resorption by suppressing cyclooxygenase-2-dependent prostaglandin synthesis in osteoblasts. J Immunol[J]. 1996;156(2):758-764.
15. Niiro H, Otsuka T, Izuhara K, Yamaoka K, Ohshima K, Tanabe T, Hara S, Nemoto Y, Tanaka Y, Nakashima H, Niho Y. Regulation by interleukin-10 and interleukin-4 of cyclooxygenase-2 expression in human neutrophils. Blood[J].1997;89(5):1621-1628.
16. Patrignani P, Tacconelli S, Sciulli MG, Capone ML. New insights into COX-2 biology and inhibition. Brain Res Brain Res Rev[J]. 2005;48(2):352-359.
17. FitzGerald GA. COX-2 and beyond: Approaches to prostaglandin inhibition in human disease. Nat Rev Drug Discov[J]. 2003;2(11):879-890.
18. Qiu DK, Ma X, Peng YS, Chen XY. Significance of cyclooxygenase-2 expression in human primary hepatocellular carcinoma. World J Gastroenterol[J]. 2002;8(5):815-817.
19. Koga H, Sakisaka S, Ohishi M, Kawaguchi T, Taniguchi E, Sasatomi K, Harada M, Kusaba T, Tanaka M, Kimura R, Nakashima Y, Nakashima O, Kojiro M, Kurohiji T, Sata M. Expression of cyclooxygenase-2 in human hepatocellular carcinoma:relevance to tumor dedifferentiation. Hepatology[J]. 1999;29(3):688-96.
20. Kondo M, Yamamoto H, Nagano H, Okami J, Ito Y, Shimizu J, Eguchi H, Miyamoto A, Dono K, Umeshita K, Matsuura N, Wakasa K, Nakamori S, Sakon M, Monden M. Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma. Clin Cancer Res[J]. 1999;5(12):4005-4012.
21.韩少良,唐槐静,林东听,邵永孚.肝细胞癌组织内COX-2表达的临床意义[J].中华肝胆外科杂志2002;8(5):307-308.
22.张必翔,陈孝平,余红平,张万广,朱虹,罗顺峰,王其,吴在德,裘法祖.环氧合酶-2在肝细胞癌中的表达及其临床意义[J].中华实验外科杂志(3). 2004;21:305-306.
23. Leng J, Han C, Demetris AJ, Michalopoulos GK, Wu T. Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth through Akt activation: evidence for Akt inhibition in celecoxib-induced apoptosis. Hepatology[J]. 2003;38(3):756-768.
24. Cheng AS, Chan HL, To KF, Leung WK, Chan KK, Liew CT, Sung JJ. Cyclooxygenase-2 pathway correlates with vascular endothelial growth factor expression and tumor angiogenesis in hepatitis B virus-associated hepatocellular carcinoma. Int J Oncol[J]. 2004;24(4):853-860.
25. Tang TC, Poon RT, Lau CP, Xie D, Fan ST. Tumor cyclooxygenase-2 levels correlate with tumor invasiveness in human hepatocellular carcinoma. World J Gastroenterol[J]. 2005;11(13):1896-1902.
26.张必翔,陈孝平,余红平,张万广,朱虹,罗顺峰,王其,吴在德,裘法祖.环氧合酶-2和血管内皮生长因子共表达与肝细胞癌血管形成的关系[J].中华实验外科杂志2004;21(6):673-675.
27. Rahman MA, Dhar DK, Yamaguchi E, Maruyama S, Sato T, Hayashi H, Ono T, Yamanoi A, Kohno H, Nagasue N. Coexpression of inducible nitric oxide synthase and COX-2 in hepatocellular carcinoma and surrounding liver: possibleinvolvement of COX-2 in the angiogenesis of hepatitis C virus-positive cases. Clin Cancer Res[J]. 2001;7(5):1325-1332.
28.张必翔,陈孝平,张万广,余红平,朱虹,罗顺峰,王其,吴在德,裘法祖.核转录因子-КB对肝细胞癌Hep3B COX-2表达的调节[J].中华实验外科杂志. 2004;21(7):824-825.
29.孙波,吴云林,王升年,张学军,贺恒益,乔敏敏,钟捷.舒林酸诱导人肝细胞癌凋亡及对环氧合酶-2和Bcl-2蛋白表达的影响[J].中华消化杂志. 2002;22(6):338-340.
30. Bae SH, Jung ES, Park YM, Kim BS, Kim BK, Kim DG, Ryu WS. Expression of cyclooxygenase-2 (COX-2) in hepatocellular carcinoma and growth inhibition of hepatoma cell lines by a COX-2 inhibitor, NS-398. Clin Cancer Res[J]. 2001;7(5):1410-1418.
31. Hu KQ, Yu CH, Mineyama Y, McCracken JD, Hillebrand DJ, Hasan M. Inhibited proliferation of cyclooxygenase-2 expressing human hepatoma cells by NS-398, a selective COX-2 inhibitor. Int J Oncol[J]. 2003;22(4):757-763.
32. Rahman MA, Dhar DK, Masunaga R, Yamanoi A, Kohno H, Nagasue N. Sulindac and exisulind exhibit a significant antiproliferative effect and induce apoptosis in human hepatocellular carcinoma cell lines. Cancer Res[J]. 2000;60(8):2085-2089.
33. Tian G, Yu JP, Luo HS, Yu BP, Yue H, Li JY, Mei Q. Effect of nimesulide on proliferation and apoptosis of human hepatoma SMMC-7721 cells. World J Gastroenterol[J]. 2002;8(3):483-487.
34. Denda A, Kitayama W, Murata A, Kishida H, Sasaki Y, Kusuoka O, Tsujiuchi T, Tsutsumi M, Nakae D, Takagi H, Konishi Y. Increased expression of cyclooxygenase-2 protein during rat hepatocarcinogenesis caused by a choline-deficient, L-amino acid-defined diet and chemopreventive efficacy of a specific inhibitor, nimesulide. Carcinogenesis[J]. 2002;23(2):245-256
35. Liu W, Nakamura H, Tsujimura T, Cheng J, Yamamoto T, Iwamoto Y, Imanishi H,Shimomura S, Yamamoto T, Hirasawa T, Inagaki S, Nishiguchi S, Hada T.Chemoprevention of spontaneous development of hepatocellular carcinomas in fatty liver Shionogi mice by a cyclooxygenase-2 inhibitor. Cancer Sci[J]. 2006;97(8):768-773.
36. Kern MA, Sch?neweiss MM, Sahi D, Bahlo M, Haugg AM, Kasper HU, Dienes HP, K?ferstein H, Breuhahn K, Schirmacher P.Cyclooxygenase-2 inhibitors suppress the growth of human hepatocellular carcinoma implants in nude mice. Carcinogenesis[J]. 2004;25(7):1193-1199.
37. Marquez-Rosado L,Trejo-Solis MC,Garcia-Cuellar CM,Villa-Trevino S.Celecoxib, a cyclooxygenase-2 inhibitor, prevents induction of liver preneoplastic lesions in rats. J Hepatol[J]. 2005;43(4):653-660.
38. Abiru S, Nakao K, Ichikawa T, Migita K, Shigeno M, Sakamoto M, Ishikawa H, Hamasaki K, Nakata K, Eguchi K.Aspirin and NS-398 inhibit hepatocyte growth factor-induced invasiveness of human hepatoma cells. Hepatology[J]. 2002;35(5):1117-1124.
2. Di Bisceglie AM. Epidemiology and clinical presentation of hepatocellular carcinoma. J Vasc Interv Radiol[J]. 2002;13(9 Pt 2):S169-171.
3. Bruix J, Llovet JM. Prognostic prediction and treatment strategy in hepatocellular carcinoma. Hepatology[J]. 2002;35(3):519-524.
4. McGlynn KA, London WT. Epidemiology and natural history of hepatocellular carcinoma. Best Pract Res Clin Gastroenterol[J]. 2005; 19(1): 3-23.
5. El-Serag HB. Hepatocellular carcinoma: recent trends in the United States. Gastroenterology[J]. 2004;127(5 Suppl 1): S27-34.
6.陈建国,宋新明.中国肝癌发病水平的估算及分析.中国肿瘤[J]. 2005; 14(1):28-31.
7. Motola-Kuba D, Zamora-Valdés D, Uribe M, Méndez-Sánchez N. Hepatocellular carcinoma. An overview. Ann Hepatol[J]. 2006;5(1):16-24.
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9. Teo EK, Fock KM. Hepatocellular carcinoma: an Asian perspective. Dig Dis[J]. 2001;19(4):263-268.
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11. Schwartz JD, Schwartz M, Mandeli J, Sung M. Neoadjuvant and adjuvant therapy for respectable hepatocellular carcinoma: review of the randomized clinical trials. Lancet Oncol[J]. 2002;3(10):593-603
12. Sun W, Sonnad S, Furth E. Adjuvant chemotherapy in patients with hepatocellularcarcinoma after orthotopic liver transplantation. Proc Am Soc Clin Oncol[J]. 2005, Abstract 4126-4130.
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14. Romano M, Claria J. Cyclooxygenase-2 and 5-lipoxygenase converging functions on cell proliferation and tumor angiogenesis: implications for cancer therapy. FASEB J[J]. 2003;17(14):1986-1995.
15. Clària J, Romano M. Pharmacological intervention of cyclooxygenase-2 and 5-lipoxygenase pathways. Impact on inflammation and cancer. Curr Pharm Des[J]. 2005;11(26):3431-3447.
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17. Shariat SF, Kim JH, Ayala GE, Kho K, Wheeler TM, Lerner SP. Cyclooxygenase-2 is highly expressed in carcinoma in situ and T1 transitional cell carcinoma of the bladder. J Urol[J]. 2003;169(3):938-942
18. Chen WS, Liu JH, Wei SJ, Liu JM, Hong CY, Yang WK. Colon cancer cells with high invasive potential are susceptible to induction of apoptosis by a selective COX-2 inhibitor. Cancer Sci[J]. 2003;94(3):253-258
19. Chen WS, Wei SJ, Liu JM, Hsiao M, Kou-Lin J, Yang WK. Tumor invasiveness and liver metastasis of colon cancer cells correlated with cyclooxygenase-2 (COX-2) expression and inhibited by a COX-2-selective inhibitor, etodolac. Int J Cancer[J]. 2001; 91(6):894-899.
20. Han C, Leng J, Demetris AJ, Wu T. Cyclooxygenase-2promotes human cholangiocarcinoma growth: evidence for cyclooxygenase-2-independent mechanism in celecoxib-mediated induction of p21waf1/cip1 and p27kip1 and cell cycle arrest. Cancer Res[J]. 2004; 64(4):1369-1376.
21. Baek JY, Hur W, Wang JS, Bae SH, Yoon SK. Selective COX-2 inhibitor, NS-398, suppresses cellular proliferation in human hepatocellular carcinoma cell lines via cell cycle arrest. World J Gastroenterol[J]. 2007,13(8):1175-1181.
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35. Miao J, Chen GG, Chun SY, Chak EC, Lai PB. Bid sensitizes apoptosis induced by chemotherapeutic drugs in hepatocellular carcinoma. Int J Oncol[J]. 2004;25(3): 651-659.
36. Cipak L, Paulikova H, Novotny L, Jarosova M, Rauko P. Potentiation of doxorubicin-induced apoptosis and differentiation by indomethacin in K-562 leukemia cells. Neoplasma[J]. 2004;51(3):188-192.
37.彭黎明,王曾礼主编.细胞凋亡的基础与临床.北京:人民卫生出版社, 2000:153.
38. Kirkin V, Joos S, Z?rnig M. The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta[J]. 2004;1644(2-3):229-49
39. RosséT, Olivier R, Monney L, Rager M, Conus S, Fellay I, Jansen B, Borner C. Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c.. Nature[J]. 1998;391(6666):496-499.
1. McGlynn KA, London WT. Epidemiology and natural history of hepatocellular carcinoma.Best Pract Res Clin Gastroenterol[J].2005;19(1):3-23.
2. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer[J].2001,94(2):153-156.
3. Di Bisceglie AM. Epidemiology and clinical presentation of hepatocellular carcinoma.J Vasc Interv Radiol[J]. 2002;13(9 Pt 2):S169-171.
4. Bruix J, Llovet JM. Prognostic prediction and treatment strategy in hepatocellular carcinoma.Hepatology[J]. 2002;35(3):519-524.
5.陈建国,宋新明.中国肝癌发病水平的估算及分析[J].中国肿瘤,2005;14(1):28-31.
6. Williams CS, Mann M, DuBois RN. The role of cyclooxygenases in inflammation, cancer, and development.Oncogene[J]. 1999;18(55):7908-7916.
7. Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochem J[J]. 1994;302 ( Pt 3):723-727.
8. Hempel SL, Monick MM, Hunninghake GW. Lipopolysaccharide induces prostaglandin H synthase-2 protein and mRNA in human alveolar macrophages and blood monocytes. J Clin Invest[J].1994;93(1):391-396.
9. Fong CY, Pang L, Holland E, Knox AJ. TGF-beta1 stimulates IL-8 release, COX-2 expression, and PGE(2) release in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol[J]. 2000;279(1):L201-L207.
10. Chen CC, Sun YT, Chen JJ, Chang YJ. Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and IkappaB kinase 1/2 in human alveolar epithelial cells. Mol Pharmacol[J]. 2001;59(3):493-500.
11. Laporte JD, Moore PE, Lahiri T, Schwartzman IN, Panettieri RA Jr, Shore SA. p38MAP kinase regulates IL-1 beta responses in cultured airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol[J].2000;279(5):L932-L941.
12. Tazawa R, Xu XM, Wu KK, Wang LH. Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene. Biochem Biophys Res Commun[J]. 1994;203(1):190-199.
13. Lee SH, Soyoola E, Chanmugam P, Hart S, Sun W, Zhong H, Liou S, Simmons D, Hwang D. Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J Biol Chem[J].1992;267(36):25934-25938.
14.Onoe Y, Miyaura C, Kaminakayashiki T, Nagai Y, Noguchi K, Chen QR, Seo H, Ohta H, Nozawa S, Kudo I, Suda T. IL-13 and IL-4 inhibit bone resorption by suppressing cyclooxygenase-2-dependent prostaglandin synthesis in osteoblasts. J Immunol[J]. 1996;156(2):758-764.
15. Niiro H, Otsuka T, Izuhara K, Yamaoka K, Ohshima K, Tanabe T, Hara S, Nemoto Y, Tanaka Y, Nakashima H, Niho Y. Regulation by interleukin-10 and interleukin-4 of cyclooxygenase-2 expression in human neutrophils. Blood[J].1997;89(5):1621-1628.
16. Patrignani P, Tacconelli S, Sciulli MG, Capone ML. New insights into COX-2 biology and inhibition. Brain Res Brain Res Rev[J]. 2005;48(2):352-359.
17. FitzGerald GA. COX-2 and beyond: Approaches to prostaglandin inhibition in human disease. Nat Rev Drug Discov[J]. 2003;2(11):879-890.
18. Qiu DK, Ma X, Peng YS, Chen XY. Significance of cyclooxygenase-2 expression in human primary hepatocellular carcinoma. World J Gastroenterol[J]. 2002;8(5):815-817.
19. Koga H, Sakisaka S, Ohishi M, Kawaguchi T, Taniguchi E, Sasatomi K, Harada M, Kusaba T, Tanaka M, Kimura R, Nakashima Y, Nakashima O, Kojiro M, Kurohiji T, Sata M. Expression of cyclooxygenase-2 in human hepatocellular carcinoma:relevance to tumor dedifferentiation. Hepatology[J]. 1999;29(3):688-96.
20. Kondo M, Yamamoto H, Nagano H, Okami J, Ito Y, Shimizu J, Eguchi H, Miyamoto A, Dono K, Umeshita K, Matsuura N, Wakasa K, Nakamori S, Sakon M, Monden M. Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma. Clin Cancer Res[J]. 1999;5(12):4005-4012.
21.韩少良,唐槐静,林东听,邵永孚.肝细胞癌组织内COX-2表达的临床意义[J].中华肝胆外科杂志2002;8(5):307-308.
22.张必翔,陈孝平,余红平,张万广,朱虹,罗顺峰,王其,吴在德,裘法祖.环氧合酶-2在肝细胞癌中的表达及其临床意义[J].中华实验外科杂志(3). 2004;21:305-306.
23. Leng J, Han C, Demetris AJ, Michalopoulos GK, Wu T. Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth through Akt activation: evidence for Akt inhibition in celecoxib-induced apoptosis. Hepatology[J]. 2003;38(3):756-768.
24. Cheng AS, Chan HL, To KF, Leung WK, Chan KK, Liew CT, Sung JJ. Cyclooxygenase-2 pathway correlates with vascular endothelial growth factor expression and tumor angiogenesis in hepatitis B virus-associated hepatocellular carcinoma. Int J Oncol[J]. 2004;24(4):853-860.
25. Tang TC, Poon RT, Lau CP, Xie D, Fan ST. Tumor cyclooxygenase-2 levels correlate with tumor invasiveness in human hepatocellular carcinoma. World J Gastroenterol[J]. 2005;11(13):1896-1902.
26.张必翔,陈孝平,余红平,张万广,朱虹,罗顺峰,王其,吴在德,裘法祖.环氧合酶-2和血管内皮生长因子共表达与肝细胞癌血管形成的关系[J].中华实验外科杂志2004;21(6):673-675.
27. Rahman MA, Dhar DK, Yamaguchi E, Maruyama S, Sato T, Hayashi H, Ono T, Yamanoi A, Kohno H, Nagasue N. Coexpression of inducible nitric oxide synthase and COX-2 in hepatocellular carcinoma and surrounding liver: possibleinvolvement of COX-2 in the angiogenesis of hepatitis C virus-positive cases. Clin Cancer Res[J]. 2001;7(5):1325-1332.
28.张必翔,陈孝平,张万广,余红平,朱虹,罗顺峰,王其,吴在德,裘法祖.核转录因子-КB对肝细胞癌Hep3B COX-2表达的调节[J].中华实验外科杂志. 2004;21(7):824-825.
29.孙波,吴云林,王升年,张学军,贺恒益,乔敏敏,钟捷.舒林酸诱导人肝细胞癌凋亡及对环氧合酶-2和Bcl-2蛋白表达的影响[J].中华消化杂志. 2002;22(6):338-340.
30. Bae SH, Jung ES, Park YM, Kim BS, Kim BK, Kim DG, Ryu WS. Expression of cyclooxygenase-2 (COX-2) in hepatocellular carcinoma and growth inhibition of hepatoma cell lines by a COX-2 inhibitor, NS-398. Clin Cancer Res[J]. 2001;7(5):1410-1418.
31. Hu KQ, Yu CH, Mineyama Y, McCracken JD, Hillebrand DJ, Hasan M. Inhibited proliferation of cyclooxygenase-2 expressing human hepatoma cells by NS-398, a selective COX-2 inhibitor. Int J Oncol[J]. 2003;22(4):757-763.
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