特异性环氧化酶-2抑制剂塞莱西布体外诱导胆囊癌细胞系GBC-SD凋亡及FK506对急性重症胰腺炎大鼠的治疗作用
摘要
目的:本研究旨在研究特异性环氧化酶-2的抑制剂—塞莱西布在体外对人类胆囊癌细胞系GBC-SD的生长抑制、促凋亡作用。通过观察其在不同浓度时抑制胆囊癌细胞生长及促凋亡作用,为临床应用打下基础。方法:1 通过计数法测定胆囊癌细胞的生长曲线,获得该细胞系基本生长特性;2 胆囊癌及细胞用含不同浓度(0,40,80,160和320μmol/L)的塞莱西布的培养基培养,24小时后应用MTT测定生长抑制率,流式细胞技术来观察细胞凋亡情况;3 胆囊癌细胞用含160μmol/L塞莱西布的培养基培养,分别用Hoechst33258试剂以及TUNEL方法染色。结果:1 塞莱西布对胆囊癌细胞具有生长抑制作用,并呈现量-效关系。通过流式细胞技术,可观察到明显的凋亡细胞;2 通过Hoechst33258试剂及TUNEL染色,发现明显存在的凋亡细胞。结论:塞莱西布对胆囊癌细胞具有一定的生长抑制作用及诱导肿瘤细胞出现凋亡的作用,这种作用是剂量依赖性和时间依赖性的。
AIM:The current studies were designed to study the effects of inhibiting proliferation and inducing apoptosis of celecoxib on human gall bladder cancer cells in vitro. After oberserving the inhibitive and inducing effects of celecoxib at various concentrations, we discussed the mechanism behind those phenomena of specific inhibitor of COX-2 in favor of clinical practice primarily.METHODS: 1 Through counting method to set out growth curve of gall bladder cancer cells, and understand its basic characteristic.2 The gall bladder cancer cell were cultured with celecoxib at various concentrations (0, 40, 80,160&320μmol/L). Growth suppression was detected by MTT colorimetric assay, cell apoptotic alterations were evaluated by flow cytometry (FCM). 3 The gall bladder cancer cells were cultured with celecoxib(160μmoL/L), and had been stained by Hoechst33258 and TUNEL method.RESULTS:1 Through counting method to set out growth curve of gall bladder cancer cells.2 The inhibition of proliferation on gall bladder cancer cells can be observed. And the inhibitive effect was dose-dependent and time-dependent. Apoptotic cells were observed by FCM.3 By stain of Hoechst33258 and TUNEL, we observed apoptosis. CONCLUSION:Celecoxib
AIM:The current studies were designed to study the effects of inhibiting proliferation and inducing apoptosis of celecoxib on human gall bladder cancer cells in vitro. After oberserving the inhibitive and inducing effects of celecoxib at various concentrations, we discussed the mechanism behind those phenomena of specific inhibitor of COX-2 in favor of clinical practice primarily.METHODS: 1 Through counting method to set out growth curve of gall bladder cancer cells, and understand its basic characteristic.2 The gall bladder cancer cell were cultured with celecoxib at various concentrations (0, 40, 80,160&320μmol/L). Growth suppression was detected by MTT colorimetric assay, cell apoptotic alterations were evaluated by flow cytometry (FCM). 3 The gall bladder cancer cells were cultured with celecoxib(160μmoL/L), and had been stained by Hoechst33258 and TUNEL method.RESULTS:1 Through counting method to set out growth curve of gall bladder cancer cells.2 The inhibition of proliferation on gall bladder cancer cells can be observed. And the inhibitive effect was dose-dependent and time-dependent. Apoptotic cells were observed by FCM.3 By stain of Hoechst33258 and TUNEL, we observed apoptosis. CONCLUSION:Celecoxib
引文
1 Kune GA, Kune S, Watson LF. Colorectal cancer risk, chronic illnesses, operations, and medications: case control results from the Melbourne Colorectal Cancer Study. Cancer Res, 1988; 48(15): 4399-4404
2 Thun MJ, Namboodiri MM, Calle EE, Flanders WD, Heath CW Jr. Aspirin use and risk of fatal cancer. CancerRes, 1993; 53(6): 1322-1327
3 Steinbach G, Lynch PM, Phillips RK, Wallace MH, Hawk E, Gordon GB, Wakabayashi N, Saunders B, Shen Y, Fujimura T, Su LK, Levin B. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med, 2000; 342 (26): 1946-1952.
4 Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA, 1997; 94(7):3336-3340
5 Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell, 1998; 94(2): 705-716
6 Sawaoka H, Tsuji S, Tsujii M, Gunawan ES, Sasaki Y, Kawano S, Hori M. Cyclooxygenase inhibitors suppress angiogenesis and reduce tumor growth in vivo. Lab Invest, 1999;79(12):1469-1477
7 Stolina M, Sharma S, Lin Y, Dohadwala M, Gardner B, Luo J, Zhu L, Kronenberg M, Miller PW, Portanova J, Lee JC, Dubinett SM. Specific inhibition of cyclooxygenase-2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis. J Immuno, 2000; 164(1): 361-370
8 Castonguay A, Rioux N. Inhibition of lung tumorigenesis by sulindac: comparison of two experimental protocols. Carcinogenesis, 1997; 18(3): 491-496
9 Vane JR, Botting RM. The mechanism of action of aspirin. Thromb Res, 2003; 110(5-6):255-258
10 Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol, 1998; 38: 97-120.
11 王兆钺.环氧化酶-2及其意义.国外医学生理病理科学与临床,1996;16(2):97-99.
12 Inoue H, Yokoyama C, Ham S, Tone Y, Tanabe T. Transcription regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. J Biol Chem, 1995; 270(42): 24965-24971.
13 Marin V, Kaplanski G, Gres S, Famarier C, Bongrand P. Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. Journal of Immunological Methods, 2001; 254(1-2): 183-190.
14 Xie WL, Herschman HR. V-src induces prostaglandin synthasse 2 gene expression by activation of the c-Jun N-terminal Kinase and the c-Jun transcription factor. J Biol Chem, 1995; 270(46): 27622-27628.
15 Hla T, Neilson K. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci USA 1992; 89(16): 7384-7388.
16 陈美若,梁统,周克元.环氧合酶-2表达调控的研究进展.中国药理通讯,2003;20(3):16-17
17 Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med, 1971; 285(21): 1182-1186.
18 Morita I, Schindler M, Regier MK, Otto JC, Hori T, DeWitt DL, Smith WL. Different intracellular location for prostaglandin endoperoxide H synthase-1 and-2. J Biol Chem, 1995; 270(18): 10902-10908.
19 李玲,吴开春,聂永战,吴汉平,王春梅,樊代明.人细胞系环氧合酶的表达、分布及其意义.中华消化杂志,2000;20(6):374-377
20 Nie D, Honn KV. Eicosanoid regulation of angiogenesis in tumors. Semin Thromb Hemost, 2004; 30(1): 119-125.
21 Smith WL, Mamett LJ. Prostaglandin endoperoxide synthase: Structure and catalysis. Biochim Biophys Acta, 1991; 1083(1): 1-17
22 Smith WL. The eicosanoids and their biochemical mechanisms of action. Biochem J, 1989; 259(2): 315-324
23 ApplebySB,RistimakiA,NeilsonK, etal.Structure of the human cyclo-oxygenase-2 gene [J].BiochemJ,1994,302(part3):723-727.
24 AkhtarM,ChengY, MaguoRM,etal.Promoter methylation regulates helicobacter pylori-stirnulated cyclooxygenase-2 expression in gastric epithelial cells[J]. CancerRes,2001,61 (6):2399-2403.
25 NaborsLB,GillespieGY, HarkinsL,etal.HuR,a RNA stability factor, is expressed in malignant brain tumors and binds to adenine-and uridine-rich elements within the 3'untranslated regions of cytokine and angiogenic factor mRNAs[J]. CancerRes,2001,61(5):2154-2161.
26 Kune GA, Kune S, Watson LF. Colorectal cancer risk, chronic illness, operations, and medications: case control results from the Melbourne colorectal cancer study. Cancer Res 1988; 48(15): 4399-4404.
27 Sano H, Kawahito Y, Wilder RL, Hashiramoto A, Mukai S, Asai K, Kimura S, Kato H, Kondo M, Hla T. Expression of cyclooxygenase-1 and-2 in human colorectal cancer. Cancer Res, 1995; 55(17): 3785-3789
28 Eberhart CE, Coffey R J, Radhika A, Giardiello FM, Ferrenbach S, DuBois RN. Up-regulation of cyclooxygenase-2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology, 1994; 107(4): 1183-1188
29 罗玉琴,吴开春,孙安华,潘伯荣,张学庸,樊代明.浅表性胃炎、胃粘膜不典型增生及组织中COX-1,COX-2,iNOS表达的意义.中化消化杂志,2000;20(4):223-226
30 Sawaoka H, Kawano S, Tsuji S, Tsujii M, Murata H, Hori M. Effects of NSAIDs on proliferation of gastric cancer cells in vitro: possible implication of cyclooxygenase-2 in cancer development. J Clin Gastroenterol, 1998; 27 Suppl 1: S47-52
31 Murata H, Kawano S, Tsuji S, Tsuji M, Sawaoka H, Kimura Y, Shiozaki H, Hori M. Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis in human gastric carcinoma. Am J Gastrol, 1999; 94(2): 451-455
32 Sawaoka H, Kawano S, Tsuji S, Tsujii M, Gunawan ES, Takei Y, Nagano K, Hori M. Cyclooxygenase-2 inhibitors suppress the growth of gastric cancer xenografts via induction of apoptosis in nude mice. Am J Physiol, 1998; 274(6 pt 1): G1061-1067
33 O'nell GP, Ford-Hutchinson AW. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett, 1993; 330(2): 156-160
34 Shiota G, Okubo M, Noumi T, Noguchi N, Oyama K, Takano Y, Yashima K, Kishimoto Y, Kawasaki H. Cyclooxygenase-2 expression in hepatocellular carcinoma. Hepatogastroenterology, 1999; 46(25): 407-412
35 Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochemical J, 1994; 302(Pt3): 723-727
36 Wolff H, Saukkonen K, Anttila S, Karjalainen A, Vainio H, Ristimaki A. Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res, 1998; 58(22): 4997-5001
37 Zimmermann KC, Sarbia M, Weber AA, Borchard F, Gabbert HE, Schror K. Cyclooxygenase-2 expression in human esophageal carcinoma. Cancer Res, 1999; 59(1): 198-204
38 Tucker ON, Dannenberg AJ, Yang EK, Zhang F, Teng L, Daly JM, Soslow RA, Masferrer JL, Woerner BM, Koki AT, Fahey TJ 3rd. Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer. Cancer Res, 1999; 59(5): 987-990
39 Hwang D, Scollard D, Byrne J, Levine E. Expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer. J Natl Cancer Inst, 1998; 90(6): 455-460
40 Yoshimura R, Sano H, Masuda C, Kawamura M, Tsubouchi Y, Chargui J, Yoshimura N, Hla T, Wada S. Expression of cyclooxygenase-2 in prostate carcinoma. Cancer, 2000; 89(3): 589-596
41 Uotila P, Valve E, Martikainen P, Nevalainen M, Nurmi M, Harkonen P. Increased expression of cyclooxygenase-2 and nitric oxide synthase-2 in human prostate cancer. Urol Res, 2001; 29(1): 23-28
42 Shirahama T, Sakakura C. Overexpression of cyclooxygenase-2 in squamous cell carcinoma of the urinary bladder. Clin Cancer Res, 2001; 7(3): 558-561
43 Morecki S, Yacovlev L, Slavin S. Effect of indomethacin on tumorigenicity and immunity induction in a murine model of mammary carcinoma. Int J Cancer, 1998; 75(6): 894-899
44 Vainio H, Morgan G Cyclo-oxygenase 2 and breast cancer prevention. Non-steroidal anti-inflammatory agents are worth testing in breast cancer. BMJ, 1998; 317(7162): 828
45 Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, Levine AC. Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol, 2000; 164(3 Pt 1): 820-825
46 Eling TE, Thompson DC, Foureman GL, Curtis JF, Hughes MF. Prostaglandin H synthase and xenobiotic oxidation. Annu Rev Pharmacol Toxicol, 1990; 30: 1-45
47 Sammon AM, Maize meal, non-exterified linoleic acid , and endemic cancer of the esophagus-prelimary findings [J]. Prostaglandins OtherLipid Mediat, 1999; 57(2 - 3):167-171.
48 Reddy BS, Hirose Y, Cohen LA, et al. Preventive potential of what bran fractions against experimental colon carcinogenesis: implications for human colon cancer
2 Thun MJ, Namboodiri MM, Calle EE, Flanders WD, Heath CW Jr. Aspirin use and risk of fatal cancer. CancerRes, 1993; 53(6): 1322-1327
3 Steinbach G, Lynch PM, Phillips RK, Wallace MH, Hawk E, Gordon GB, Wakabayashi N, Saunders B, Shen Y, Fujimura T, Su LK, Levin B. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med, 2000; 342 (26): 1946-1952.
4 Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA, 1997; 94(7):3336-3340
5 Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell, 1998; 94(2): 705-716
6 Sawaoka H, Tsuji S, Tsujii M, Gunawan ES, Sasaki Y, Kawano S, Hori M. Cyclooxygenase inhibitors suppress angiogenesis and reduce tumor growth in vivo. Lab Invest, 1999;79(12):1469-1477
7 Stolina M, Sharma S, Lin Y, Dohadwala M, Gardner B, Luo J, Zhu L, Kronenberg M, Miller PW, Portanova J, Lee JC, Dubinett SM. Specific inhibition of cyclooxygenase-2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis. J Immuno, 2000; 164(1): 361-370
8 Castonguay A, Rioux N. Inhibition of lung tumorigenesis by sulindac: comparison of two experimental protocols. Carcinogenesis, 1997; 18(3): 491-496
9 Vane JR, Botting RM. The mechanism of action of aspirin. Thromb Res, 2003; 110(5-6):255-258
10 Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol, 1998; 38: 97-120.
11 王兆钺.环氧化酶-2及其意义.国外医学生理病理科学与临床,1996;16(2):97-99.
12 Inoue H, Yokoyama C, Ham S, Tone Y, Tanabe T. Transcription regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. J Biol Chem, 1995; 270(42): 24965-24971.
13 Marin V, Kaplanski G, Gres S, Famarier C, Bongrand P. Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. Journal of Immunological Methods, 2001; 254(1-2): 183-190.
14 Xie WL, Herschman HR. V-src induces prostaglandin synthasse 2 gene expression by activation of the c-Jun N-terminal Kinase and the c-Jun transcription factor. J Biol Chem, 1995; 270(46): 27622-27628.
15 Hla T, Neilson K. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci USA 1992; 89(16): 7384-7388.
16 陈美若,梁统,周克元.环氧合酶-2表达调控的研究进展.中国药理通讯,2003;20(3):16-17
17 Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med, 1971; 285(21): 1182-1186.
18 Morita I, Schindler M, Regier MK, Otto JC, Hori T, DeWitt DL, Smith WL. Different intracellular location for prostaglandin endoperoxide H synthase-1 and-2. J Biol Chem, 1995; 270(18): 10902-10908.
19 李玲,吴开春,聂永战,吴汉平,王春梅,樊代明.人细胞系环氧合酶的表达、分布及其意义.中华消化杂志,2000;20(6):374-377
20 Nie D, Honn KV. Eicosanoid regulation of angiogenesis in tumors. Semin Thromb Hemost, 2004; 30(1): 119-125.
21 Smith WL, Mamett LJ. Prostaglandin endoperoxide synthase: Structure and catalysis. Biochim Biophys Acta, 1991; 1083(1): 1-17
22 Smith WL. The eicosanoids and their biochemical mechanisms of action. Biochem J, 1989; 259(2): 315-324
23 ApplebySB,RistimakiA,NeilsonK, etal.Structure of the human cyclo-oxygenase-2 gene [J].BiochemJ,1994,302(part3):723-727.
24 AkhtarM,ChengY, MaguoRM,etal.Promoter methylation regulates helicobacter pylori-stirnulated cyclooxygenase-2 expression in gastric epithelial cells[J]. CancerRes,2001,61 (6):2399-2403.
25 NaborsLB,GillespieGY, HarkinsL,etal.HuR,a RNA stability factor, is expressed in malignant brain tumors and binds to adenine-and uridine-rich elements within the 3'untranslated regions of cytokine and angiogenic factor mRNAs[J]. CancerRes,2001,61(5):2154-2161.
26 Kune GA, Kune S, Watson LF. Colorectal cancer risk, chronic illness, operations, and medications: case control results from the Melbourne colorectal cancer study. Cancer Res 1988; 48(15): 4399-4404.
27 Sano H, Kawahito Y, Wilder RL, Hashiramoto A, Mukai S, Asai K, Kimura S, Kato H, Kondo M, Hla T. Expression of cyclooxygenase-1 and-2 in human colorectal cancer. Cancer Res, 1995; 55(17): 3785-3789
28 Eberhart CE, Coffey R J, Radhika A, Giardiello FM, Ferrenbach S, DuBois RN. Up-regulation of cyclooxygenase-2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology, 1994; 107(4): 1183-1188
29 罗玉琴,吴开春,孙安华,潘伯荣,张学庸,樊代明.浅表性胃炎、胃粘膜不典型增生及组织中COX-1,COX-2,iNOS表达的意义.中化消化杂志,2000;20(4):223-226
30 Sawaoka H, Kawano S, Tsuji S, Tsujii M, Murata H, Hori M. Effects of NSAIDs on proliferation of gastric cancer cells in vitro: possible implication of cyclooxygenase-2 in cancer development. J Clin Gastroenterol, 1998; 27 Suppl 1: S47-52
31 Murata H, Kawano S, Tsuji S, Tsuji M, Sawaoka H, Kimura Y, Shiozaki H, Hori M. Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis in human gastric carcinoma. Am J Gastrol, 1999; 94(2): 451-455
32 Sawaoka H, Kawano S, Tsuji S, Tsujii M, Gunawan ES, Takei Y, Nagano K, Hori M. Cyclooxygenase-2 inhibitors suppress the growth of gastric cancer xenografts via induction of apoptosis in nude mice. Am J Physiol, 1998; 274(6 pt 1): G1061-1067
33 O'nell GP, Ford-Hutchinson AW. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett, 1993; 330(2): 156-160
34 Shiota G, Okubo M, Noumi T, Noguchi N, Oyama K, Takano Y, Yashima K, Kishimoto Y, Kawasaki H. Cyclooxygenase-2 expression in hepatocellular carcinoma. Hepatogastroenterology, 1999; 46(25): 407-412
35 Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochemical J, 1994; 302(Pt3): 723-727
36 Wolff H, Saukkonen K, Anttila S, Karjalainen A, Vainio H, Ristimaki A. Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res, 1998; 58(22): 4997-5001
37 Zimmermann KC, Sarbia M, Weber AA, Borchard F, Gabbert HE, Schror K. Cyclooxygenase-2 expression in human esophageal carcinoma. Cancer Res, 1999; 59(1): 198-204
38 Tucker ON, Dannenberg AJ, Yang EK, Zhang F, Teng L, Daly JM, Soslow RA, Masferrer JL, Woerner BM, Koki AT, Fahey TJ 3rd. Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer. Cancer Res, 1999; 59(5): 987-990
39 Hwang D, Scollard D, Byrne J, Levine E. Expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer. J Natl Cancer Inst, 1998; 90(6): 455-460
40 Yoshimura R, Sano H, Masuda C, Kawamura M, Tsubouchi Y, Chargui J, Yoshimura N, Hla T, Wada S. Expression of cyclooxygenase-2 in prostate carcinoma. Cancer, 2000; 89(3): 589-596
41 Uotila P, Valve E, Martikainen P, Nevalainen M, Nurmi M, Harkonen P. Increased expression of cyclooxygenase-2 and nitric oxide synthase-2 in human prostate cancer. Urol Res, 2001; 29(1): 23-28
42 Shirahama T, Sakakura C. Overexpression of cyclooxygenase-2 in squamous cell carcinoma of the urinary bladder. Clin Cancer Res, 2001; 7(3): 558-561
43 Morecki S, Yacovlev L, Slavin S. Effect of indomethacin on tumorigenicity and immunity induction in a murine model of mammary carcinoma. Int J Cancer, 1998; 75(6): 894-899
44 Vainio H, Morgan G Cyclo-oxygenase 2 and breast cancer prevention. Non-steroidal anti-inflammatory agents are worth testing in breast cancer. BMJ, 1998; 317(7162): 828
45 Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, Levine AC. Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol, 2000; 164(3 Pt 1): 820-825
46 Eling TE, Thompson DC, Foureman GL, Curtis JF, Hughes MF. Prostaglandin H synthase and xenobiotic oxidation. Annu Rev Pharmacol Toxicol, 1990; 30: 1-45
47 Sammon AM, Maize meal, non-exterified linoleic acid , and endemic cancer of the esophagus-prelimary findings [J]. Prostaglandins OtherLipid Mediat, 1999; 57(2 - 3):167-171.
48 Reddy BS, Hirose Y, Cohen LA, et al. Preventive potential of what bran fractions against experimental colon carcinogenesis: implications for human colon cancer