抗肿瘤药物筛选及白桦酯酸诱导人宫颈癌细胞凋亡机制研究
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摘要
本课题的研究目的是筛选具有抗肿瘤活性的药物,并在此基础上,进一步研究肿瘤细胞生长抑制的分子机制。通过体外生长抑制实验,结晶紫染色法测定细胞死亡率,我们发现从萝藦科(A sclep iadaceae)牛奶菜属(Marsdenia R.Br.)植物乌骨藤[M arsde ia tenacissin a (Roxb) .W ight etAm ]的藤茎中提取出的多种成分均可影响几种肿瘤细胞的生长。实验证明其含有的成分之一白桦酯酸(betulinic acid,BA)可以诱导HeLa细胞凋亡,且具有浓度和时间依赖性。通过以100μg/mLBA作用HeLa细胞48h后,对HeLa细胞总RNA进行提取后,进行RT-PCR反应,经琼脂糖凝胶电泳检测证明,BA可以诱导P21 mRNA和P53 mRNA表达的增高,细胞周期蛋白cyclinA mRNA变化不大,cyclinB1 mRNA表达降低。研究结果显示,白桦酯酸诱导肿瘤细胞凋亡可能与P53,P21基因改变有关,白桦酯酸可能进一步影响细胞周期的进程。
Cell apoptosis is also named cell programmed death, which is one of the newest researches in biology. It is one of considerable vital phenomena in living nature. Apoptosis is closely associated with nemathelminth and high mammalia animal, embryo and adult, pathological and physiological conditions,life and die. It plays an important role in human health. As far as 100 years ago, Carl Vogt found this process. Kerr et al. have named it as“apoptosis”at 1972. Apoptosis is regulatoried by different genes, such as Ced gene family, bcl - 2 gene family, Fas/Apo gene family, P53 gene and ICE gene family etc.
As one of Chinese traditional medicine, M arsdenia tenacissim a (Roxb) .Wight et Arn has been used for curing disease for thousands years in China. The factions and components were isolated from the fruits of M arsdenia tenacissim a (Roxb) .Wight et Arn. In our laboratory, those separate were screen for suppression activity of tumor cells. The result was shown that betulinic acid and betulin induced tumor cell died.
This passage sieves medicine for curing tumor from constituents and chemical compounds are extracted from M arsdenia tenacissim a (Roxb) .Wight et Arn
.This study on the apoptotic molecule mechanism in HeLa cell induced by betulinic acid. Do it with methods of crystal violet dying, RT-PCR were operated. Indicate the growth of tumor cells were significantly inhibited by betulin and betulinic acid in a time- and dose-dependent fashion. The furthers study indicated that betulinic acid induced HeLa cells apoptosis. The expression of P53 mRNA and P21 mRNA was increased and the expression of cyclinB1 mRNA was reduced in HeLa cells by Betulinic acid. But the expression of cyclinA mRNA was no change. The effect of Betulinic acid is mainly associated with apoptosis and cell cycle arrest.
Cell apoptosis is also named cell programmed death, which is one of the newest researches in biology. It is one of considerable vital phenomena in living nature. Apoptosis is closely associated with nemathelminth and high mammalia animal, embryo and adult, pathological and physiological conditions,life and die. It plays an important role in human health. As far as 100 years ago, Carl Vogt found this process. Kerr et al. have named it as“apoptosis”at 1972. Apoptosis is regulatoried by different genes, such as Ced gene family, bcl - 2 gene family, Fas/Apo gene family, P53 gene and ICE gene family etc.
As one of Chinese traditional medicine, M arsdenia tenacissim a (Roxb) .Wight et Arn has been used for curing disease for thousands years in China. The factions and components were isolated from the fruits of M arsdenia tenacissim a (Roxb) .Wight et Arn. In our laboratory, those separate were screen for suppression activity of tumor cells. The result was shown that betulinic acid and betulin induced tumor cell died.
This passage sieves medicine for curing tumor from constituents and chemical compounds are extracted from M arsdenia tenacissim a (Roxb) .Wight et Arn
.This study on the apoptotic molecule mechanism in HeLa cell induced by betulinic acid. Do it with methods of crystal violet dying, RT-PCR were operated. Indicate the growth of tumor cells were significantly inhibited by betulin and betulinic acid in a time- and dose-dependent fashion. The furthers study indicated that betulinic acid induced HeLa cells apoptosis. The expression of P53 mRNA and P21 mRNA was increased and the expression of cyclinB1 mRNA was reduced in HeLa cells by Betulinic acid. But the expression of cyclinA mRNA was no change. The effect of Betulinic acid is mainly associated with apoptosis and cell cycle arrest.
引文
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4. Fang M, Zhang H, Xue S, et al. Intracellular calcium distribution in apoptosis of HL-60 cells induced by harringtonine: intranuclear accumulation and regionalization. Cancer lett, 1998, 127: 113-121.
5. Beroad c , Soussi T. P53 gene mutation :software and database[J ] . Nucleic Acids Res, 1998, 26:200 - 204.
6. El-Deiry WS, Tokino T, Velculescu VE, et al. WAFl, a potential mediator of p53 tumor suppression. Cell, 1993, 75∶817-825.
7. Harper JW, Adami GR, Wei N, et al. The p21 cdk-interacting protein Cip 1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 1993, 75∶805-816.
8. Xiong Y, Hannon GJ, Zhang H, et al. P21 WAFl/Cipl is a universal inhibitor of cyclin kinases. Nature, 1993, 366∶701-704.
9. Macleod KF, Sherry N, Hannon G, et al. p53 dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev, 1995, 9∶935-944.
10. Ohnishi T, Wang X, Ohnishi K, et al. p53-dependent induction of WAFlby heat treatment in human glioblastoma cells. J Biol Chem, 1996, 271∶14510-14513.
11. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl- 2: release of cytochrome c from mitochondria blocked. Science, 1997, 275:1129-1132.
12. Cheng EH, Kirsch DG, Clem RJ, et al. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science, 1997,278:1966-1968.
13. Selzner M,Rüdiger HA , Selzner N,et al . Transgenic mice overexpressing human bcl - 2 are resistant to hepatic ischemia and reperfusion [J] . J Hepatol ,2002 ,36(2) :2181.
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15. Golstein P. Controlling cell death. Science, 1997, 275: 1081 – 1082 .
16. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation . Science,1998, 281:1305-1308.
17. Raff M. Cell suicide for beginners. Nature, 1998,396:119-122.
18. Adams JM, Cory S.The bcl-2 protein family: arbiters of cell survival. Science,1998,281:1322-1326.
19. Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature, 1998,391:96-99.
20. Enari M, Sakahira H, Yokoyama H, et al. A caspases-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature ,1998,391:43-50.
21. Green DR, Reed JC. Mitochondria and apoptosis. Science, 1998, 281:1309-1312.
22. Camilleri Broet S, Vanderwerff H, Caldwell E, et al. Distinct alterationsin mitochondrial mass and function characterize different models of apoptosis.Exp Cell Res,1998,239:277-292.
23. Askenazi A, Dixit VM. Death receptors: signaling and modulation. Science, 1998, 281 (5381): 1305 - 1308.
24. Thornberry NA,Lazebnik Y. Caspases:enemies within. Science, 1998,281:1312-1316.
25. 邢旺兴, 程荣珍, 陈斌, 等. 乌骨藤常见混用品种辨析[ J ]. 现代中药研究与实践, 2004, 18 (1) : 33 - 36.
26. 孙珏, 沈建华, 朱美华, 等. 消癌平对人胃癌细胞治疗作用的实验研究[ J ]. 上海中医药大学学报, 2000, 14 (2) : 41 - 44.
27. 孙珏, 沈建华, 朱美华, 等. 中药消癌平针剂经肝动脉介入治疗转移性肝癌的临床研究[ J ]. 上海中医药杂志, 2000, 34 (1) : 14 - 17.
28. 蒋毅, 罗思齐. 通关藤中新 C21 甾体甙的化学结构研究[ J ]. 中国医药工业杂志, 1996, 27 (9) : 391 - 395.
29. 李茂全, 沈建华, 胥彬, 等. 消癌平对SGC27901胃癌细胞的作用及机制的实验研究[ J ]. 介入放射学杂志, 2001, 10 (4) : 228 - 231.
30. 韩大庆, 刘伟, 王力平, 等. 消癌平口服液抗肿瘤作用研究[ J ]. 中华综合临床医学杂志. 2004, 6 (1) : 20 - 21.
31. 孙珏, 沈建华, 范忠泽, 等. 消癌平对人肝癌细胞治疗作用的实验研究[ J ]. 上海中医药杂志, 2000, 34 (7) : 12 - 14.
32. 张民庆, 龚惠明. 抗癌中药的临床应用[M ]. 北京:人民卫生出版社, 1998, 100 - 101.
33. 邢旺兴, 程荣珍, 陈园昕, 等. 通光藤的药理作用与临床应用[ J ]. 中南药学, 2003, 4 (1) : 229 - 231.
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37. 王志良, 樊青霞, 范魁生, 等. 消癌平临床疗效观察[ J ]. 河南医科大学学报, 1994, 29 (1) : 79 - 80.
38. Pisha E ,Cahi H ,Lee IS , et al . Discovery of betulinic as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nature Med ,1995 ,1 :1046.
39. Fulda S ,Jeremias I ,Steiner HH , et al . Betulinic acid :A new cytotoxic agent against malignant braintumor cells. Int J Cancer , 1999 , 82 :435.
40. Schmidt ML , Kuzmanoff KL ,LingIndeck L , et al . Betulinic acid induces apoptosis in human neuroblastoma cell lines. Eur J Cancer ,1997 ,33 :2007.
41. Zuco V ,Supino R ,Righetti SC , et al . Selective cytotoxicity of betulinic acid on tumor cell lines ,but not normal cells. Cancer Lett ,2002 ,175 :17.
42. Selzer E , Pimentel E ,Wacheck V , et al . Effects of betulinic acid alone and in combination with irradiation in human melanoma cells. J Invest Dermatol ,2000 ,114 :935.
43. Fulda S ,Friesen C ,Los M, et al . Betulinic acid triggers CD95(APO21/ Fas) and p53independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res , 1997 , 57 :4956.
44. Safayhi H ,Sailer ER. Antiinflammatory actions of pentacyclictriterpenes. Planta Med ,1997 ,63 :487.
45. Recio MC , Giner RM,Manez S , et al . Investigations on the steroidal antiinflammatory activity of triterpenoids from Diospyros leucomelas. Planta Med ,1995 ,61 :9.
46. Min B ,Lee GI ,Ha JY, et al . Inhibitory effects of triterpenoids on interleukin8/ CINC1 induction in LPSstimulated rat peritoneal macrophages. Nat Prod Sci ,1996 ,2 :48.
47. Ha JY,Min KR , Kang SH , et al . Suppressiveeffects of triter penoids on CINC21 induction in interleukin1bstimulated ratbroblast NRK49F cells. Arch Pharm Res ,1997 ,20 :234.
48. Huguet AI ,Recio MC ,Manez S , et al . Effect of triterpenoids on the inflammation induced by protein kinase C activators , neuronallyacting irritantsand otheragents. Eur J Pharmacol , 2000 ,410 :69.
49. Dunstan CA ,Liu B ,Welch CJ , et al . Alphitol ,a phenolic substance from Alphitonia zizyphoides which inhibits prost aglan dinbio synthes is in vitro. Phytochemistry ,1998 ,48 :495.
50. Krogh R ,Kroth R ,Berti C , et al . Isolation and identification of compounds with antinociceptive action from Ipomoea pescaprae(L. ) R Br Pharmazie ,1999 ,54 :464.
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52. Chang CW,Wang JP ,Wu PP , et al . Terpenoids from Ocium basilicum. Chin Pharm J ,1999 ,51 :181.
53. Higa M,Ogihara K, Yogi S. Bioactive naphthoquinone derivatives from 田佳川 Diospyros maritime Blume. Chem Pharm Bull ,1998 ,
54. Fulda S ,Jeremias I ,Steiner HH , et al . Betulinic acid :A new cytotoxic agent against malignant braintumor cells. Int J Cancer ,1999 ,82 :435.
55. Wachter GA ,Valcic S ,Flagg ML , et al . Antitubercular activity of pentacyclic triterpenoids from plants of Argentina and Chile. Phytomedicine ,1999 ,6 :341.
56. Kashiwada Y,Hashimoto F ,Cosentino LM,et al .Betulinic acid and dihydrobetulinic acid derivatives as potent antiHIV agents. J Med Chem ,1996 ,39 :1016.
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2. Conradt B, Horvitz H R. The C. elegans protein EGL-1is required forprogrammed cell death and interacts with the bcl-2-like protein CED-9.Cell, 1998, 93: 519-529.
3. 细胞凋亡的生物学特征. 赵卫红,寿好长,闫福岭,等主编. 细胞凋亡.郑州: 河南医科大学出版社,1997.7-11.
4. Fang M, Zhang H, Xue S, et al. Intracellular calcium distribution in apoptosis of HL-60 cells induced by harringtonine: intranuclear accumulation and regionalization. Cancer lett, 1998, 127: 113-121.
5. Beroad c , Soussi T. P53 gene mutation :software and database[J ] . Nucleic Acids Res, 1998, 26:200 - 204.
6. El-Deiry WS, Tokino T, Velculescu VE, et al. WAFl, a potential mediator of p53 tumor suppression. Cell, 1993, 75∶817-825.
7. Harper JW, Adami GR, Wei N, et al. The p21 cdk-interacting protein Cip 1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 1993, 75∶805-816.
8. Xiong Y, Hannon GJ, Zhang H, et al. P21 WAFl/Cipl is a universal inhibitor of cyclin kinases. Nature, 1993, 366∶701-704.
9. Macleod KF, Sherry N, Hannon G, et al. p53 dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev, 1995, 9∶935-944.
10. Ohnishi T, Wang X, Ohnishi K, et al. p53-dependent induction of WAFlby heat treatment in human glioblastoma cells. J Biol Chem, 1996, 271∶14510-14513.
11. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl- 2: release of cytochrome c from mitochondria blocked. Science, 1997, 275:1129-1132.
12. Cheng EH, Kirsch DG, Clem RJ, et al. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science, 1997,278:1966-1968.
13. Selzner M,Rüdiger HA , Selzner N,et al . Transgenic mice overexpressing human bcl - 2 are resistant to hepatic ischemia and reperfusion [J] . J Hepatol ,2002 ,36(2) :2181.
14. Johnson A L. Intracellular mechanisms regulating cell survival in ovarian follicles[J ] . Animal Reprod Sci , 2003 ,78(3) :185.
15. Golstein P. Controlling cell death. Science, 1997, 275: 1081 – 1082 .
16. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation . Science,1998, 281:1305-1308.
17. Raff M. Cell suicide for beginners. Nature, 1998,396:119-122.
18. Adams JM, Cory S.The bcl-2 protein family: arbiters of cell survival. Science,1998,281:1322-1326.
19. Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature, 1998,391:96-99.
20. Enari M, Sakahira H, Yokoyama H, et al. A caspases-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature ,1998,391:43-50.
21. Green DR, Reed JC. Mitochondria and apoptosis. Science, 1998, 281:1309-1312.
22. Camilleri Broet S, Vanderwerff H, Caldwell E, et al. Distinct alterationsin mitochondrial mass and function characterize different models of apoptosis.Exp Cell Res,1998,239:277-292.
23. Askenazi A, Dixit VM. Death receptors: signaling and modulation. Science, 1998, 281 (5381): 1305 - 1308.
24. Thornberry NA,Lazebnik Y. Caspases:enemies within. Science, 1998,281:1312-1316.
25. 邢旺兴, 程荣珍, 陈斌, 等. 乌骨藤常见混用品种辨析[ J ]. 现代中药研究与实践, 2004, 18 (1) : 33 - 36.
26. 孙珏, 沈建华, 朱美华, 等. 消癌平对人胃癌细胞治疗作用的实验研究[ J ]. 上海中医药大学学报, 2000, 14 (2) : 41 - 44.
27. 孙珏, 沈建华, 朱美华, 等. 中药消癌平针剂经肝动脉介入治疗转移性肝癌的临床研究[ J ]. 上海中医药杂志, 2000, 34 (1) : 14 - 17.
28. 蒋毅, 罗思齐. 通关藤中新 C21 甾体甙的化学结构研究[ J ]. 中国医药工业杂志, 1996, 27 (9) : 391 - 395.
29. 李茂全, 沈建华, 胥彬, 等. 消癌平对SGC27901胃癌细胞的作用及机制的实验研究[ J ]. 介入放射学杂志, 2001, 10 (4) : 228 - 231.
30. 韩大庆, 刘伟, 王力平, 等. 消癌平口服液抗肿瘤作用研究[ J ]. 中华综合临床医学杂志. 2004, 6 (1) : 20 - 21.
31. 孙珏, 沈建华, 范忠泽, 等. 消癌平对人肝癌细胞治疗作用的实验研究[ J ]. 上海中医药杂志, 2000, 34 (7) : 12 - 14.
32. 张民庆, 龚惠明. 抗癌中药的临床应用[M ]. 北京:人民卫生出版社, 1998, 100 - 101.
33. 邢旺兴, 程荣珍, 陈园昕, 等. 通光藤的药理作用与临床应用[ J ]. 中南药学, 2003, 4 (1) : 229 - 231.
34. 江苏新医学院. 中药大辞典(下册) [M ]. 上海: 上海科学技术出版社,1999, 1976.
35. 中国药科大学. 中药辞海(第 2 卷) [M ]. 北京: 中国医药科技出版社, 1996, 2360 - 2361.
36. 中国科学院中国植物志编辑委员会. 中国植物志[M ]. 北京: 科学出版社, 1977, (63) : 442 - 465.
37. 王志良, 樊青霞, 范魁生, 等. 消癌平临床疗效观察[ J ]. 河南医科大学学报, 1994, 29 (1) : 79 - 80.
38. Pisha E ,Cahi H ,Lee IS , et al . Discovery of betulinic as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nature Med ,1995 ,1 :1046.
39. Fulda S ,Jeremias I ,Steiner HH , et al . Betulinic acid :A new cytotoxic agent against malignant braintumor cells. Int J Cancer , 1999 , 82 :435.
40. Schmidt ML , Kuzmanoff KL ,LingIndeck L , et al . Betulinic acid induces apoptosis in human neuroblastoma cell lines. Eur J Cancer ,1997 ,33 :2007.
41. Zuco V ,Supino R ,Righetti SC , et al . Selective cytotoxicity of betulinic acid on tumor cell lines ,but not normal cells. Cancer Lett ,2002 ,175 :17.
42. Selzer E , Pimentel E ,Wacheck V , et al . Effects of betulinic acid alone and in combination with irradiation in human melanoma cells. J Invest Dermatol ,2000 ,114 :935.
43. Fulda S ,Friesen C ,Los M, et al . Betulinic acid triggers CD95(APO21/ Fas) and p53independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res , 1997 , 57 :4956.
44. Safayhi H ,Sailer ER. Antiinflammatory actions of pentacyclictriterpenes. Planta Med ,1997 ,63 :487.
45. Recio MC , Giner RM,Manez S , et al . Investigations on the steroidal antiinflammatory activity of triterpenoids from Diospyros leucomelas. Planta Med ,1995 ,61 :9.
46. Min B ,Lee GI ,Ha JY, et al . Inhibitory effects of triterpenoids on interleukin8/ CINC1 induction in LPSstimulated rat peritoneal macrophages. Nat Prod Sci ,1996 ,2 :48.
47. Ha JY,Min KR , Kang SH , et al . Suppressiveeffects of triter penoids on CINC21 induction in interleukin1bstimulated ratbroblast NRK49F cells. Arch Pharm Res ,1997 ,20 :234.
48. Huguet AI ,Recio MC ,Manez S , et al . Effect of triterpenoids on the inflammation induced by protein kinase C activators , neuronallyacting irritantsand otheragents. Eur J Pharmacol , 2000 ,410 :69.
49. Dunstan CA ,Liu B ,Welch CJ , et al . Alphitol ,a phenolic substance from Alphitonia zizyphoides which inhibits prost aglan dinbio synthes is in vitro. Phytochemistry ,1998 ,48 :495.
50. Krogh R ,Kroth R ,Berti C , et al . Isolation and identification of compounds with antinociceptive action from Ipomoea pescaprae(L. ) R Br Pharmazie ,1999 ,54 :464.
51. Ryu SY,Oak MH , Yoon SK, et al . Antiallergic and antin flammatory triterpenes from the herb of Prunella vulgaris.Planta Med ,2000 ,66 :358.
52. Chang CW,Wang JP ,Wu PP , et al . Terpenoids from Ocium basilicum. Chin Pharm J ,1999 ,51 :181.
53. Higa M,Ogihara K, Yogi S. Bioactive naphthoquinone derivatives from 田佳川 Diospyros maritime Blume. Chem Pharm Bull ,1998 ,
54. Fulda S ,Jeremias I ,Steiner HH , et al . Betulinic acid :A new cytotoxic agent against malignant braintumor cells. Int J Cancer ,1999 ,82 :435.
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