胡桃醌体外抗胰腺癌细胞机制的初步研究
摘要
目的：了解胡桃醌体外抗胰腺癌细胞的机制。
方法：采用MTT法检测胡桃醌对BXPC-3细胞的生长抑制作用,流式细胞仪分析胡桃醌对BXPC-3细胞周期的影响,免疫组化染色法检测不同浓度胡桃醌作用后BXPC-3细胞bcl-2蛋白表达。
结果：MTT法试验结果显示,胡桃醌对体外培养的人胰腺癌BXPC-3细胞具有明显的生长抑制作用,24、48h的IC50值为1.64×10-5、5.8×10-5mol/L。同时胡桃醌能随着浓度的增加,时间的延长,诱导BXPC-3细胞周期停滞于G2期和S期,呈G2期和S期阻滞作用。不同浓度胡桃醌作用于BXPC-3细胞后,免疫组化染色,bcl-2蛋白表达：细胞浆、核膜出现棕黄色颗粒。随着胡桃醌浓度的增加,细胞表达数量减少,色泽变浅,bcl-2蛋白的表达水平呈下降趋势。
结论：胡桃醌对胰腺癌BXPC-3细胞有明显的生长抑制作用,并能阻止其周期,诱导BXPC-3细胞凋亡。
Objective:Understanding of juglone against pancreatic cancer cells in vitro mechanism.
Methods:Juglone assay on BXPC-3 cell growth inhibition by MTT, Analysis juglone on BXPC-3 cell cycle by Flow cytometry.
Results:Juglone inhibited the growth of vitro BXPC-3 human pancreatic cancer cells significantly. The IC50 is respectively 1.64×10-5mol/L,5.8×10-5mol/L after treatment for 24,48h by juglone. As the concentration and time of juglone can increase, induction of BXPC-3 cell cycle arrest in G2 phase and S phase. The role of different concentration of juglone in BXPC-3 cells immunohistochenmistry,bcl-2 protein expression:cytoplasm nuclear membrane appeared brown-yellow granules.With the increasing concentration of juglone,reduce the number of cells,the color lighter,bcl-2 protein expression was decreased
Conclusion:Conclusion Juglone significantly inhibited the growth of BXPC-3 cells,and prevent cell cycle and induction apoptosis.
方法：采用MTT法检测胡桃醌对BXPC-3细胞的生长抑制作用,流式细胞仪分析胡桃醌对BXPC-3细胞周期的影响,免疫组化染色法检测不同浓度胡桃醌作用后BXPC-3细胞bcl-2蛋白表达。
结果：MTT法试验结果显示,胡桃醌对体外培养的人胰腺癌BXPC-3细胞具有明显的生长抑制作用,24、48h的IC50值为1.64×10-5、5.8×10-5mol/L。同时胡桃醌能随着浓度的增加,时间的延长,诱导BXPC-3细胞周期停滞于G2期和S期,呈G2期和S期阻滞作用。不同浓度胡桃醌作用于BXPC-3细胞后,免疫组化染色,bcl-2蛋白表达：细胞浆、核膜出现棕黄色颗粒。随着胡桃醌浓度的增加,细胞表达数量减少,色泽变浅,bcl-2蛋白的表达水平呈下降趋势。
结论：胡桃醌对胰腺癌BXPC-3细胞有明显的生长抑制作用,并能阻止其周期,诱导BXPC-3细胞凋亡。
Objective:Understanding of juglone against pancreatic cancer cells in vitro mechanism.
Methods:Juglone assay on BXPC-3 cell growth inhibition by MTT, Analysis juglone on BXPC-3 cell cycle by Flow cytometry.
Results:Juglone inhibited the growth of vitro BXPC-3 human pancreatic cancer cells significantly. The IC50 is respectively 1.64×10-5mol/L,5.8×10-5mol/L after treatment for 24,48h by juglone. As the concentration and time of juglone can increase, induction of BXPC-3 cell cycle arrest in G2 phase and S phase. The role of different concentration of juglone in BXPC-3 cells immunohistochenmistry,bcl-2 protein expression:cytoplasm nuclear membrane appeared brown-yellow granules.With the increasing concentration of juglone,reduce the number of cells,the color lighter,bcl-2 protein expression was decreased
Conclusion:Conclusion Juglone significantly inhibited the growth of BXPC-3 cells,and prevent cell cycle and induction apoptosis.
引文
[1]孙燕.肿瘤的发生情况与防治趋向[N].中国医学论坛报,2002,28(14)：11
[2]李立明.流行病学[M].人民卫生出版社.第四版2007.218.
[3]Changbaishan Plant8Records. Jilin People Press.Jilin Institute of Traditional Chinese Medicine and Materia Medica. Jilin People Press.1982.203
[4]Gava B, Bhargave UC,Wesfall BA. J Pharm sci,1968,57(10):1674.1674.
[5]陈凤凰,唐文明.核桃树皮化学成分分析及活性研究[J].天然产物研究与开放.2008,20：16-18.
[6]王少东,脱朝伟.核桃楸青果皮抗肿瘤作用的药理研究.辽宁中医杂志,1990,11：37.9
[7]翟梅枝.杨秀萍.刘路.核桃叶抑菌成分的提取及其抑菌活性.西北林学报200316(4)：89～91.
[8]季宇彬,曲中原,邹翔,崔兰,胡国军.青龙衣中胡桃酮对5180肉瘤小鼠的抑瘤作用研究明.中国药学杂志,2009年2月第44卷第3期.
[9]RYO A, L.DU Y C, LU K P. et al. Prolyl isomerase Pin1:acatalyst for on cogenesis and a potential therapeutic target in cancer [J]. CellScj 2003,116 (5):773-783.
[10]SARDRI JE, BOIXADERAMA, SARDIJ. N eoadjuvantchemo-therapy in cervieal Cancer a new trend[J]. CurrOpin Obstet Gynecol 2005,17:43-47.
[11]张红英,杨光华,步宏,等.不同转移潜能的人体横纹肌肉瘤细胞系细胞骨架的研究[J].肿瘤.2002.22(4)291-294.
[12]Miyamoto Y Hosotani R Wada M. Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in pancreatic cancers. Oncology,1999(1)
[13]Yan RLWang JB, Hui HX, Xin XY, Chen BL. Expression and significance of proapoptosis gene bax in epithelial ovarian tu-mour [J].Di-si Junyi Daxue Xuebao (J Fourth Mil Med Univ),1998:19 (3):322-324.
[14]李胜,李占元,张卫东.人胰腺癌中bcl-2基因和蛋白的过度表达[J].胰腺病学,2002,2：166-168.
[15]盖宝东,郑泽霖,王凤军.细胞凋亡相关基因bcl-2和Bax在胰腺癌中的表达及其意义[J].实用肿瘤学杂志,2001,15(2)：102-104.
[16]Adrain C. Martin SJ The mitochondrial apoptosome:a killer unleashed by the cytochrome seas 2001(6)
[17]Vande VC. Cizeau J. Dubik D BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore 2000(15)
[18]HENN IG L,CHRISTENER C, K IP PING M, etal. Selective in-activation of parvulin-like peptidyl-prolylcis/trans isomerases by juglone[J]. Biochem-istry,1998,37 (17):59553-5960.
[19]WU J, KOU W, GAO M T, et al. Preliminary studies on effects of the derivative of D-amine-glucose in inhibiting growth of HepG-2 and inducing its apoptosis [J]. Chin J Cancer Prey Trea,2005,16(12):1223-1227.
[20]孔陵生,邹翔,季宇彬.青龙衣中胡桃醌对A-549细胞周期及凋亡影响哈尔滨商业大学学报(自然科学版)第24卷第6期2008年12月
[21]陈丽,那顺巴雅尔,张建等.胡桃醌对人肝癌BEL-7402细胞亚显微结构的影响南方医科大学学报,2009；29(6)
[22]WU J, KOU W, GAO M T. et al. Preliminary studies on effects of the derivative of D-amine-glucose in inhibiting growth of HepG-2 and inducing its apoptosis[J]. Chin J CancerPreyTrea,2005,16(12):1223-1227.
[23]B.Kiran.Aithal,M.R.Sunil.Kumar,B,Nageshwar Rao,Nayanabhirama Udupa,B.S.Satish Rao.Juglone,a naphthoquinone from walnut,exerts cytotoxic and genotoxic effects against cultured melanoma tumor cells[J].Cell Biology International,2009,(33):1039-1049
[24]Lawrence C.-M. Chiu, T.-S. Ho, Elaine Y-L.Wong, Vincent E. C. Ooi. Ethyl acetate extract of Patrinia scabiosaefolia downregulates anti-apoptotic Bcl-2/Bcl-Xi. expression, and induces apoptosis in human breast carcinoma MCF-2 cells independent of caspase-9 activation[J].Journal of Ethnopharmacology,2006, (105):263-268.
[25]Kamei H. Koide T. Kojima T. et al. Inhibition of cell growth in culture by quinones[J]. Cancer biotherapy & Radiopharmaceuticals,1998,13(3):185-188.
[1]孙燕.肿瘤的发生情况与防治趋向[N].中国医学论坛报,2002,28(14)：11
[2]李立明.流行病学[M].人民卫生出版社.第四版2007.218.
[3]杨小燕,张玉祥,王泽生.胰腺癌BXPC3细胞中Src激酶对Notch-1活化的调节.基础医学与临床,2008年月第28卷第9期--
[4]Chen Q, Lu Q, Wang Y, et al. Cytoskeleton Disorganization during Apoptosis Induced by Curcumin in A549 Lung Adenocarcinoma Cells[J]. Planta Med.2009,5.
[5]张红英,杨光华,步宏,等.不同转移潜能的人体横纹肌肉瘤细胞系细胞骨架的研究[J].肿瘤.2002.22(4)291-294.
[6]Miyamoto Y Hosotani R Wada M. Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in pancreatic cancers. Oncology,1999(1)
[7]Yan RLWang JB, Hui HX, Xin XY, Chen BL. Expression and significance of proapoptosis gene bax in epithelial ovarian tu-mour [J]. Di-si Junyi Daxue Xuebao (J Fourth Mil Med Univ), 1998;19 (3):322-324.
[8]Bukholm IK, Nesland JM. Protein expression of p53, p21 (WAF1/CIP1), bcl-2, Bax, cyclin Dland pRb in human colon carcinomas[J]. Virchows Arch,2000;436 (3):224-228
[9]Kitada S, Andersen J, Akar S, et al. Blood,1998,91(9):3379-3389
[10]KroemerG. NatureMed,1997,3(6):614-620.
[11]李胜,李占元,张卫东.人胰腺癌中bcl-2基因和蛋白的过度表达[J].胰腺病学,2002,2：166-168.
[12]盖宝东,郑泽霖,王凤军.细胞凋亡相关基因bcl-2和Bax在胰腺癌中的表达及其意义[J].实用肿瘤学杂志,2001,15(2)：102-104.
[13]XuH, EI-Gewely MR. Differentially expressed downstream genes in cells with normal or mutated p53.OncolRes,2003,13:429-43
[14]Adrain C.Martin SJ The mitochondrial apoptosome:a killer unleashed by the cytochrome seas 2001(6)
[15]Vande VC. Cizeau J. Dubik D BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore 2000(15)
[16]Petit PX.Zamzami N. Vayssiere JL Implication of mitochondria in apoptosis 1997(1-2)
[17]朱玉山,陈侄.线粒体与细胞凋亡调控.生命科学2008,20(4)：506-515
[18]蔡黔,肖光夏,于晟.线粒体在细胞凋亡中的作用研究进展西北国防医学杂志2002 23(5)
[19]HENN IG L, CHRISTENER C, K IP PING M, etal. Selective in-activation of parvulin-like peptidyl-prolylcis/trans isomerases by juglone[J]. Biochem-istry,1998,37 (17):59553-5960.
[20]WU J, KOU W, GAO M T, et al. Preliminary studies on effects of the derivative of D-amine-glucose in inhibiting growth of HepG-2 and inducing its apoptosis[J]. Chin J Cancer Prey Trea,2005,16(12):1223-1227.
[21]孔陵生,邹翔,季宇彬.青龙衣中胡桃醌对A-549细胞周期及凋亡影响哈尔滨商业大学学报(自然科学版)第24卷第6期2008年12月
[22]朱继红,刘丽萍,童蓓燕,等.肝癌发生与转移过程中E—选择素在裸鼠肝、肺、肾微血管内皮细胞的表达[J].解剖学杂志,2001,24(3)：269-272.
[23]Hanahan D, Weinberg RA. The halhnarks of cancer Cell,2000,100:57-70.
[24]陶景玉,王忠裕金属蛋白酶-9(MMP-9)及其组织抑制物-1(TIMP-1)在胰腺癌中的表达医学与哲学(临床决策论坛版)2009年1月第30卷第1期总第373期
[25]张慧芹,刘阁玲基质金属蛋白酶-9在甲状腺癌中的表达及临床意义[J].医学理论与实践,2005,18：786-788.
[26]Jodele S,Blavier L, Yoon J M. Modifying the soil to affect the seed:role of stromal-derived matrix metalloproteinases in cancer progression[J]. Cancer metastasis rev,2006,25(1):35-43.
[27]Menashi S,Serova M. Regulation of extracellular matrixmetal-loproteinase inducer and matrix metalloproteinase expression by amphiregulllin in transformed human breast epithelial cells[J]. Cancer Rev,2003,63:7575-7580.
[2]李立明.流行病学[M].人民卫生出版社.第四版2007.218.
[3]Changbaishan Plant8Records. Jilin People Press.Jilin Institute of Traditional Chinese Medicine and Materia Medica. Jilin People Press.1982.203
[4]Gava B, Bhargave UC,Wesfall BA. J Pharm sci,1968,57(10):1674.1674.
[5]陈凤凰,唐文明.核桃树皮化学成分分析及活性研究[J].天然产物研究与开放.2008,20：16-18.
[6]王少东,脱朝伟.核桃楸青果皮抗肿瘤作用的药理研究.辽宁中医杂志,1990,11：37.9
[7]翟梅枝.杨秀萍.刘路.核桃叶抑菌成分的提取及其抑菌活性.西北林学报200316(4)：89～91.
[8]季宇彬,曲中原,邹翔,崔兰,胡国军.青龙衣中胡桃酮对5180肉瘤小鼠的抑瘤作用研究明.中国药学杂志,2009年2月第44卷第3期.
[9]RYO A, L.DU Y C, LU K P. et al. Prolyl isomerase Pin1:acatalyst for on cogenesis and a potential therapeutic target in cancer [J]. CellScj 2003,116 (5):773-783.
[10]SARDRI JE, BOIXADERAMA, SARDIJ. N eoadjuvantchemo-therapy in cervieal Cancer a new trend[J]. CurrOpin Obstet Gynecol 2005,17:43-47.
[11]张红英,杨光华,步宏,等.不同转移潜能的人体横纹肌肉瘤细胞系细胞骨架的研究[J].肿瘤.2002.22(4)291-294.
[12]Miyamoto Y Hosotani R Wada M. Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in pancreatic cancers. Oncology,1999(1)
[13]Yan RLWang JB, Hui HX, Xin XY, Chen BL. Expression and significance of proapoptosis gene bax in epithelial ovarian tu-mour [J].Di-si Junyi Daxue Xuebao (J Fourth Mil Med Univ),1998:19 (3):322-324.
[14]李胜,李占元,张卫东.人胰腺癌中bcl-2基因和蛋白的过度表达[J].胰腺病学,2002,2：166-168.
[15]盖宝东,郑泽霖,王凤军.细胞凋亡相关基因bcl-2和Bax在胰腺癌中的表达及其意义[J].实用肿瘤学杂志,2001,15(2)：102-104.
[16]Adrain C. Martin SJ The mitochondrial apoptosome:a killer unleashed by the cytochrome seas 2001(6)
[17]Vande VC. Cizeau J. Dubik D BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore 2000(15)
[18]HENN IG L,CHRISTENER C, K IP PING M, etal. Selective in-activation of parvulin-like peptidyl-prolylcis/trans isomerases by juglone[J]. Biochem-istry,1998,37 (17):59553-5960.
[19]WU J, KOU W, GAO M T, et al. Preliminary studies on effects of the derivative of D-amine-glucose in inhibiting growth of HepG-2 and inducing its apoptosis [J]. Chin J Cancer Prey Trea,2005,16(12):1223-1227.
[20]孔陵生,邹翔,季宇彬.青龙衣中胡桃醌对A-549细胞周期及凋亡影响哈尔滨商业大学学报(自然科学版)第24卷第6期2008年12月
[21]陈丽,那顺巴雅尔,张建等.胡桃醌对人肝癌BEL-7402细胞亚显微结构的影响南方医科大学学报,2009；29(6)
[22]WU J, KOU W, GAO M T. et al. Preliminary studies on effects of the derivative of D-amine-glucose in inhibiting growth of HepG-2 and inducing its apoptosis[J]. Chin J CancerPreyTrea,2005,16(12):1223-1227.
[23]B.Kiran.Aithal,M.R.Sunil.Kumar,B,Nageshwar Rao,Nayanabhirama Udupa,B.S.Satish Rao.Juglone,a naphthoquinone from walnut,exerts cytotoxic and genotoxic effects against cultured melanoma tumor cells[J].Cell Biology International,2009,(33):1039-1049
[24]Lawrence C.-M. Chiu, T.-S. Ho, Elaine Y-L.Wong, Vincent E. C. Ooi. Ethyl acetate extract of Patrinia scabiosaefolia downregulates anti-apoptotic Bcl-2/Bcl-Xi. expression, and induces apoptosis in human breast carcinoma MCF-2 cells independent of caspase-9 activation[J].Journal of Ethnopharmacology,2006, (105):263-268.
[25]Kamei H. Koide T. Kojima T. et al. Inhibition of cell growth in culture by quinones[J]. Cancer biotherapy & Radiopharmaceuticals,1998,13(3):185-188.
[1]孙燕.肿瘤的发生情况与防治趋向[N].中国医学论坛报,2002,28(14)：11
[2]李立明.流行病学[M].人民卫生出版社.第四版2007.218.
[3]杨小燕,张玉祥,王泽生.胰腺癌BXPC3细胞中Src激酶对Notch-1活化的调节.基础医学与临床,2008年月第28卷第9期--
[4]Chen Q, Lu Q, Wang Y, et al. Cytoskeleton Disorganization during Apoptosis Induced by Curcumin in A549 Lung Adenocarcinoma Cells[J]. Planta Med.2009,5.
[5]张红英,杨光华,步宏,等.不同转移潜能的人体横纹肌肉瘤细胞系细胞骨架的研究[J].肿瘤.2002.22(4)291-294.
[6]Miyamoto Y Hosotani R Wada M. Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in pancreatic cancers. Oncology,1999(1)
[7]Yan RLWang JB, Hui HX, Xin XY, Chen BL. Expression and significance of proapoptosis gene bax in epithelial ovarian tu-mour [J]. Di-si Junyi Daxue Xuebao (J Fourth Mil Med Univ), 1998;19 (3):322-324.
[8]Bukholm IK, Nesland JM. Protein expression of p53, p21 (WAF1/CIP1), bcl-2, Bax, cyclin Dland pRb in human colon carcinomas[J]. Virchows Arch,2000;436 (3):224-228
[9]Kitada S, Andersen J, Akar S, et al. Blood,1998,91(9):3379-3389
[10]KroemerG. NatureMed,1997,3(6):614-620.
[11]李胜,李占元,张卫东.人胰腺癌中bcl-2基因和蛋白的过度表达[J].胰腺病学,2002,2：166-168.
[12]盖宝东,郑泽霖,王凤军.细胞凋亡相关基因bcl-2和Bax在胰腺癌中的表达及其意义[J].实用肿瘤学杂志,2001,15(2)：102-104.
[13]XuH, EI-Gewely MR. Differentially expressed downstream genes in cells with normal or mutated p53.OncolRes,2003,13:429-43
[14]Adrain C.Martin SJ The mitochondrial apoptosome:a killer unleashed by the cytochrome seas 2001(6)
[15]Vande VC. Cizeau J. Dubik D BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore 2000(15)
[16]Petit PX.Zamzami N. Vayssiere JL Implication of mitochondria in apoptosis 1997(1-2)
[17]朱玉山,陈侄.线粒体与细胞凋亡调控.生命科学2008,20(4)：506-515
[18]蔡黔,肖光夏,于晟.线粒体在细胞凋亡中的作用研究进展西北国防医学杂志2002 23(5)
[19]HENN IG L, CHRISTENER C, K IP PING M, etal. Selective in-activation of parvulin-like peptidyl-prolylcis/trans isomerases by juglone[J]. Biochem-istry,1998,37 (17):59553-5960.
[20]WU J, KOU W, GAO M T, et al. Preliminary studies on effects of the derivative of D-amine-glucose in inhibiting growth of HepG-2 and inducing its apoptosis[J]. Chin J Cancer Prey Trea,2005,16(12):1223-1227.
[21]孔陵生,邹翔,季宇彬.青龙衣中胡桃醌对A-549细胞周期及凋亡影响哈尔滨商业大学学报(自然科学版)第24卷第6期2008年12月
[22]朱继红,刘丽萍,童蓓燕,等.肝癌发生与转移过程中E—选择素在裸鼠肝、肺、肾微血管内皮细胞的表达[J].解剖学杂志,2001,24(3)：269-272.
[23]Hanahan D, Weinberg RA. The halhnarks of cancer Cell,2000,100:57-70.
[24]陶景玉,王忠裕金属蛋白酶-9(MMP-9)及其组织抑制物-1(TIMP-1)在胰腺癌中的表达医学与哲学(临床决策论坛版)2009年1月第30卷第1期总第373期
[25]张慧芹,刘阁玲基质金属蛋白酶-9在甲状腺癌中的表达及临床意义[J].医学理论与实践,2005,18：786-788.
[26]Jodele S,Blavier L, Yoon J M. Modifying the soil to affect the seed:role of stromal-derived matrix metalloproteinases in cancer progression[J]. Cancer metastasis rev,2006,25(1):35-43.
[27]Menashi S,Serova M. Regulation of extracellular matrixmetal-loproteinase inducer and matrix metalloproteinase expression by amphiregulllin in transformed human breast epithelial cells[J]. Cancer Rev,2003,63:7575-7580.