Survivin在紫杉醇抗肿瘤机制中作用及其反义核酸干预胃癌形成的研究
摘要
Survivin是近年来发现的凋亡抑制蛋白家族（IAP family）的新成员，能通过与Caspase3、7等的结合抑制Caspase活性而抑制凋亡。研究表明survivin在凋亡的发生中起极重要的调控作用，同时survivin表达又与肿瘤的发生，一些患者的预后不良，生存率降低有关，还有研究认为survivin可能是良好的抗肿瘤治疗靶点。紫杉醇是从红豆杉中提取的抗肿瘤药物，目前的研究表明该药对胃癌等消化系统肿瘤有很好的应用前景，其主要抗肿瘤机理之一是诱导肿瘤细胞凋亡，故紫杉醇极可能会影响到survivin的表达。但紫杉醇能否通过抑制survivin表达来诱导凋亡目前尚无报道，与凋亡密切相关的JNK、p38蛋白激酶通路对survivin表达有无调控亦不清楚，并缺乏以survivin为靶点的体内基因治疗研究。我们研究survivin在紫杉醇抗肿瘤机制中的作用将有助于进一步明确紫杉醇的抗肿瘤机理；采用脂质体介导的体内转染法，研究survivin反义寡核苷酸及联合紫杉醇对实验性大鼠胃癌形成的影响，探讨以survivin为靶点进行体内抗肿瘤基因治疗的效果和可行性，可以为基因治疗早日进入临床应用提供理论和实验基础。
第一部分 survivin在人胃癌中的表达与临床病理及p53、c-erbB2表达的关系
目的 研究人胃癌组织中survivin蛋白表达与临床病理及与p53和c-erbB2表达的关系。方法 采用免疫组化的方法检测56例胃癌患者肿瘤组织及20例慢性胃炎患者胃粘膜中survivin和p53、c-erbB2的表达。结果 20例慢性胃炎患者胃粘膜中survivin的表达阳性1例，阳性率为5％；56例胃癌组织中survivin阳性27例，阳性率48.2%；56例胃癌患者c-erbB2阳性率35.7%（20/56），p53阳性率39.3%（22/56）。survivin表达与患者年龄、肿瘤浸润深度、淋巴结转移、临床分级等无明显相关性，而与肿瘤Lauren组织分型相关。survivin 阳性率在肠型胃癌（20/32，62.5%）中显著高于弥漫型（7/24，29.2% P<0.05）。survivin 在c-erbB2阳性和阴性患者中阳性率分别为80%（16/20）和30.6％（11/36），两者间有显著差异（P<0.01）；survivin 在 p53阳性和阴性患者中阳性率分别为68.2%（15/22）和35.3%（12/34），两者比较亦有显著差异（P<0.05）。结论 胃癌患者survivin的异常表达在胃癌的发生发展中起了重要作用，survivin表达与胃癌中p53和c-erbB2异常表达相关。
第二部分 紫杉醇对胃癌细胞survivin表达的影响及其调控机理
目的 研究紫杉醇诱导胃癌细胞凋亡过程中对survivin表达的影响及其中的调控机理。 方法 采用Western blot的方法检测五株胃癌细胞中survivin表达，用
MTT方法检测紫杉醇对各株细胞的IC50，分析survivin表达与IC50的关系；检测紫杉醇干预后72小时内survivin、JNK和p38激酶表达的变化，并检测分别预先采用curcumin和SB203580抑制JNK和p38激酶通路后survivin表达的改变；用Western blot检测89kDa-PARP的方法和TUNEL法检测抑制剂使用前后凋亡的变化。 结果 紫杉醇对五株胃癌细胞的IC50与细胞的survivin表达正相关；在60ng/ml紫杉醇作用下，五株胃癌细胞中的survivin在24小时内均有不同程度的升高；600ng/ml紫杉醇能导致survivin表达在24小时内有较明显的升高（增幅近70％），而36至72小时内明显下降，而紫杉醇干预后JNK明显升高，一直持续到48小时，p38激酶的磷酸化在1、3小时时明显升高；预先以curcumin抑制JNK激酶，survivin的表达升高更为显著（增幅130％），89kDa-PARP高峰后移， TUNEL示凋亡率下降；而预先以SB203580抑制p38激酶则survivin表达不再明显升高，89kDa-PARP提前出现，TUNEL示凋亡率有所增高。 结论 紫杉醇对胃癌细胞株的IC50与细胞中survivin表达呈正相关；紫杉醇能促进JNK表达和p38磷酸化，JNK介导了凋亡的发生，并能抑制survivin表达，而p38介导凋亡抑制并促进survivin表达；紫杉醇诱导的凋亡与其激活JNK最终导致survivin下降有一定的关系，而p38介导的凋亡抑制作用与其使survivin表达升高有关。
第三部分 survivin反义寡核苷酸的抗肿瘤作用和与紫杉醇的协同作用
目的 研究survivin反义寡核苷酸及联合紫杉醇对胃癌细胞生长增殖的影响。 方法 以脂质体包裹survivin反义或正义寡核苷酸瞬时转染胃癌BGC-823细胞，以MTT法检测寡核苷酸转染后及联合紫杉醇时对细胞生长增殖的抑制作用。 结果 survivin正、反义寡核苷酸均能顺利转入BGC-823细胞，在胞核和胞浆均见到绿色荧光；survivin反义寡核苷酸能明显抑制survivin蛋白的表达，并抑制BGC-823的生长，其IC50约350nM左右，survivin正义寡核苷酸对BGC-823的生长影响不大； 紫杉醇与survivin反义寡核苷酸联合能显著降低其IC50值，起到协同抗肿瘤作用。 结论 survivin反义寡核苷酸能抑制BGC-823细胞生长，和紫杉醇有协同抗肿瘤作用。
第四部分 survivin在实验性大鼠胃癌中的表达及survivin反义寡核苷酸联合化疗药物对胃癌的干预作用
目的 研究在胃癌发生过程中survivin表达的变化及survivin反义寡核苷酸联合化疗药物对胃癌形成的干预作用。 方法 121只六周龄的雄性Wistar大鼠随机分为正常组和模型组，正常对照组16只不作任何处理；模型组105只，给予含
Survivin , a new member of IAP family, can inhibit apoptosis by combining with Caspase3 and Caspase7. Sufficient data show that survivin plays an important role in apoptosis pathway and that its' expression is highly related with cancer and associated with an unfavorable prognosis. Some study indicated that survivin may be a promising target of anticancer therapy. Taxol is a compound extracted from Yew and clinical data has proved that taxol is a promising drug for the chemotherapy of gastrointestinal cancer. Inducing apoptosis is an important antitumor mechanism of Taxol, hence Taxol may have an influence on the expression of survivin. Studying the role of survivin in Taxol's effects will contribute to clarify the antitumor mechanism of Taxol. With the transfection mediated by Lipofect Amine, we studied effects of survivin antisense and it's combination with Taxol on the development of experimental gastric cancer in order to evaluate the effect of survivin targeted gene therapy in vivo.
Part 1: The expression of survivin in gastric carcinoma, and it's relationship with the expression of p53 and c-erbB2
Objective: To study the expression of survivin in gastric carcinomas and its relationship with the expression of p53, c-erbB2.
Method: Using immunohistochemical staining method, we examined the expression of survivin, p53 and c-erbB2 genes in 20 cases of chronic gastritis and 56 cases of gastric carcinomas.
Results: Survivin expressed in 27 of 56(48.2%) cases of gastric carcinoma tissues and 1 of 20(5%) cases of chronic gastritis. Over expression of survivin had no relation with age, tumor depth, tumor size, and disease stage, but was significantly related to histological type. The expression of survivin significantly segregated with intestinal type cases as compared with diffuse type cases. Survivin positive cases were significantly associated with p53 expression(15/22,68.2% versus 12/34,35.3%,P<0.05) and c-erbB2 expression(16/20,80% versus 11/36,30.6%,P<0.01).
Conclusions: These data indicated that survivin play a important role in the onset of gastric cacinoma and suggested potential correlation between survivin and c-erbB2, and between survivin and p53 as well.
Part2: Regulation on survivin expression in gastric cancer cells by Taxol and it's mechanism
Objective: To study the regulation of Taxol on the expression of survivin in gastric cancer cells and it's mechanism.
Method: Survivin expression of five gastric cancer cell lines were examined by western blot, and the IC50 of Taxol to each cell line was detected by MTT. We also examined survivin expression of each cell line after treated with 60ng/ml Taxol in 24 hours, and examined survivin expression in BGC-823 cell line after treat with 600ng/ml Taxol in 72 hours. The expression of JNK kinase, phosphorylation of JNK kinase and phosphorylation of p38 kinase were detected simultaneously. In addition, SB203580 and curcumin were used to inhibit p38 and JNK pathway respectively before treatment with Taxol for 72 hours, then survivin expression were examined by western blot, and cell apoptosis were detected by TUNEL and 89kDa-PARP.
Result: The IC50 of Taxol to these five gastric cell lines were correlated with survivin expression. Survivin expression of these five cell lines increased to some extend respectively during the 24 hours after 60ng/ml Taxol treatment. When BGC-823 cells were treated with 600ng/ml Taxol, the expression of survivin increased 7 fold at 24 hour and then decreased. JNK kinase expression increased after Taxol treatment and remain at a high level for about 48 hours, and phosphorylation of p38 kinase increased from 1 to 3 hour after Taxol treatment. Pretreated BGC-823 with curcumin 10uM for 1 hour before Taxol treatment, survivin expression increased 1.3 time in 24 hour, the peak of 89kDa-PARP delayed, and apoptosis index examined by TUNEL decreased. When pretreated with SB203580 10uM for 1 hour, no increase of survivin expression was detected during 24 hour, the presence of 89kDa-PARP was brought forward,
第一部分 survivin在人胃癌中的表达与临床病理及p53、c-erbB2表达的关系
目的 研究人胃癌组织中survivin蛋白表达与临床病理及与p53和c-erbB2表达的关系。方法 采用免疫组化的方法检测56例胃癌患者肿瘤组织及20例慢性胃炎患者胃粘膜中survivin和p53、c-erbB2的表达。结果 20例慢性胃炎患者胃粘膜中survivin的表达阳性1例，阳性率为5％；56例胃癌组织中survivin阳性27例，阳性率48.2%；56例胃癌患者c-erbB2阳性率35.7%（20/56），p53阳性率39.3%（22/56）。survivin表达与患者年龄、肿瘤浸润深度、淋巴结转移、临床分级等无明显相关性，而与肿瘤Lauren组织分型相关。survivin 阳性率在肠型胃癌（20/32，62.5%）中显著高于弥漫型（7/24，29.2% P<0.05）。survivin 在c-erbB2阳性和阴性患者中阳性率分别为80%（16/20）和30.6％（11/36），两者间有显著差异（P<0.01）；survivin 在 p53阳性和阴性患者中阳性率分别为68.2%（15/22）和35.3%（12/34），两者比较亦有显著差异（P<0.05）。结论 胃癌患者survivin的异常表达在胃癌的发生发展中起了重要作用，survivin表达与胃癌中p53和c-erbB2异常表达相关。
第二部分 紫杉醇对胃癌细胞survivin表达的影响及其调控机理
目的 研究紫杉醇诱导胃癌细胞凋亡过程中对survivin表达的影响及其中的调控机理。 方法 采用Western blot的方法检测五株胃癌细胞中survivin表达，用
MTT方法检测紫杉醇对各株细胞的IC50，分析survivin表达与IC50的关系；检测紫杉醇干预后72小时内survivin、JNK和p38激酶表达的变化，并检测分别预先采用curcumin和SB203580抑制JNK和p38激酶通路后survivin表达的改变；用Western blot检测89kDa-PARP的方法和TUNEL法检测抑制剂使用前后凋亡的变化。 结果 紫杉醇对五株胃癌细胞的IC50与细胞的survivin表达正相关；在60ng/ml紫杉醇作用下，五株胃癌细胞中的survivin在24小时内均有不同程度的升高；600ng/ml紫杉醇能导致survivin表达在24小时内有较明显的升高（增幅近70％），而36至72小时内明显下降，而紫杉醇干预后JNK明显升高，一直持续到48小时，p38激酶的磷酸化在1、3小时时明显升高；预先以curcumin抑制JNK激酶，survivin的表达升高更为显著（增幅130％），89kDa-PARP高峰后移， TUNEL示凋亡率下降；而预先以SB203580抑制p38激酶则survivin表达不再明显升高，89kDa-PARP提前出现，TUNEL示凋亡率有所增高。 结论 紫杉醇对胃癌细胞株的IC50与细胞中survivin表达呈正相关；紫杉醇能促进JNK表达和p38磷酸化，JNK介导了凋亡的发生，并能抑制survivin表达，而p38介导凋亡抑制并促进survivin表达；紫杉醇诱导的凋亡与其激活JNK最终导致survivin下降有一定的关系，而p38介导的凋亡抑制作用与其使survivin表达升高有关。
第三部分 survivin反义寡核苷酸的抗肿瘤作用和与紫杉醇的协同作用
目的 研究survivin反义寡核苷酸及联合紫杉醇对胃癌细胞生长增殖的影响。 方法 以脂质体包裹survivin反义或正义寡核苷酸瞬时转染胃癌BGC-823细胞，以MTT法检测寡核苷酸转染后及联合紫杉醇时对细胞生长增殖的抑制作用。 结果 survivin正、反义寡核苷酸均能顺利转入BGC-823细胞，在胞核和胞浆均见到绿色荧光；survivin反义寡核苷酸能明显抑制survivin蛋白的表达，并抑制BGC-823的生长，其IC50约350nM左右，survivin正义寡核苷酸对BGC-823的生长影响不大； 紫杉醇与survivin反义寡核苷酸联合能显著降低其IC50值，起到协同抗肿瘤作用。 结论 survivin反义寡核苷酸能抑制BGC-823细胞生长，和紫杉醇有协同抗肿瘤作用。
第四部分 survivin在实验性大鼠胃癌中的表达及survivin反义寡核苷酸联合化疗药物对胃癌的干预作用
目的 研究在胃癌发生过程中survivin表达的变化及survivin反义寡核苷酸联合化疗药物对胃癌形成的干预作用。 方法 121只六周龄的雄性Wistar大鼠随机分为正常组和模型组，正常对照组16只不作任何处理；模型组105只，给予含
Survivin , a new member of IAP family, can inhibit apoptosis by combining with Caspase3 and Caspase7. Sufficient data show that survivin plays an important role in apoptosis pathway and that its' expression is highly related with cancer and associated with an unfavorable prognosis. Some study indicated that survivin may be a promising target of anticancer therapy. Taxol is a compound extracted from Yew and clinical data has proved that taxol is a promising drug for the chemotherapy of gastrointestinal cancer. Inducing apoptosis is an important antitumor mechanism of Taxol, hence Taxol may have an influence on the expression of survivin. Studying the role of survivin in Taxol's effects will contribute to clarify the antitumor mechanism of Taxol. With the transfection mediated by Lipofect Amine, we studied effects of survivin antisense and it's combination with Taxol on the development of experimental gastric cancer in order to evaluate the effect of survivin targeted gene therapy in vivo.
Part 1: The expression of survivin in gastric carcinoma, and it's relationship with the expression of p53 and c-erbB2
Objective: To study the expression of survivin in gastric carcinomas and its relationship with the expression of p53, c-erbB2.
Method: Using immunohistochemical staining method, we examined the expression of survivin, p53 and c-erbB2 genes in 20 cases of chronic gastritis and 56 cases of gastric carcinomas.
Results: Survivin expressed in 27 of 56(48.2%) cases of gastric carcinoma tissues and 1 of 20(5%) cases of chronic gastritis. Over expression of survivin had no relation with age, tumor depth, tumor size, and disease stage, but was significantly related to histological type. The expression of survivin significantly segregated with intestinal type cases as compared with diffuse type cases. Survivin positive cases were significantly associated with p53 expression(15/22,68.2% versus 12/34,35.3%,P<0.05) and c-erbB2 expression(16/20,80% versus 11/36,30.6%,P<0.01).
Conclusions: These data indicated that survivin play a important role in the onset of gastric cacinoma and suggested potential correlation between survivin and c-erbB2, and between survivin and p53 as well.
Part2: Regulation on survivin expression in gastric cancer cells by Taxol and it's mechanism
Objective: To study the regulation of Taxol on the expression of survivin in gastric cancer cells and it's mechanism.
Method: Survivin expression of five gastric cancer cell lines were examined by western blot, and the IC50 of Taxol to each cell line was detected by MTT. We also examined survivin expression of each cell line after treated with 60ng/ml Taxol in 24 hours, and examined survivin expression in BGC-823 cell line after treat with 600ng/ml Taxol in 72 hours. The expression of JNK kinase, phosphorylation of JNK kinase and phosphorylation of p38 kinase were detected simultaneously. In addition, SB203580 and curcumin were used to inhibit p38 and JNK pathway respectively before treatment with Taxol for 72 hours, then survivin expression were examined by western blot, and cell apoptosis were detected by TUNEL and 89kDa-PARP.
Result: The IC50 of Taxol to these five gastric cell lines were correlated with survivin expression. Survivin expression of these five cell lines increased to some extend respectively during the 24 hours after 60ng/ml Taxol treatment. When BGC-823 cells were treated with 600ng/ml Taxol, the expression of survivin increased 7 fold at 24 hour and then decreased. JNK kinase expression increased after Taxol treatment and remain at a high level for about 48 hours, and phosphorylation of p38 kinase increased from 1 to 3 hour after Taxol treatment. Pretreated BGC-823 with curcumin 10uM for 1 hour before Taxol treatment, survivin expression increased 1.3 time in 24 hour, the peak of 89kDa-PARP delayed, and apoptosis index examined by TUNEL decreased. When pretreated with SB203580 10uM for 1 hour, no increase of survivin expression was detected during 24 hour, the presence of 89kDa-PARP was brought forward,
引文
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24. 张佩范 进展期胃癌的病理 见：张文范，张荫昌，陈峻青，主编， 胃癌， 上海： 上海科技技术出版社，第二版 2001：109-110
25. Leung WK, Sung JJ. Review article: intestinal metaplasia and gastric carcinogenesis. Aliment Pharmacol Ther 2002,16(7):1209-16
26. Olie RA, Simoes-Wust AP, Baumann B,et al. A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res 2000,60:2805
27. Crown J, O'Leary M. The taxanes: an update. Lancet 2000,355(9210):1176-8
28. Weiner LM. Paclitaxel in the treatment of esophageal cancer. Semin Oncol 1999,26(1 Suppl 2):106-8
Gadgeel SM, Shields AF, Heilbrun LK. et al. Phase II study of paclitaxel and
29. carboplatin in patients with advanced gastric cancer. Am J Clin Oncol 2003,26(1):37-41
30. Gotaskie GE, Andreassi BF. Paclitaxel: a new antimitotic chemotherapeutic agent. Cancer Pract 1994,2(1):27-33
31. Horwitz SB. Taxol (paclitaxel): mechanisms of action. Ann Oncol 1994;5 Suppl 6:S3-6
32. Herceg Z, Wang ZQ. Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat Res 2001,477(1-2):97-110
33. Oliver FJ, de la Rubia G, Rolli V, et al. Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. J Biol Chem 1998 Dec 11;273(50):33533-9
34. Barrachina M, Secades J, Lozano R, et al. Citicoline increases glutathione redox ratio and reduces caspase-3 activation and cell death in staurosporine-treated SH-SY5Y human neuroblastoma cells. Brain Res 2002,957(1):84-90
35. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002,298(5600):1911-2
36. Schaeffer HJ, Weber MJ. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 1999 Apr;19(4):2435-44
37. Whitmarsh AJ, Yang SH, Su MS, et al. Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors. Mol Cell Biol 1997,17(5):2360-71
38. Wang XZ, Ron D. Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase. Science 1996,272(5266):1347-9
39. Dunn C, Wiltshire C, MacLaren A, et al. Molecular mechanism and biological functions of c-Jun N-terminal kinase signalling via the c-Jun transcription factor. Cell Signal 2002,14(7):585-93
40. Gupta S, Campbell D, Derijard B, et al. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 1995,267(5196):389-93
41. Cuenda A, Rouse J, Doza YN, et al. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 1995,364(2):229-33
42. Takenaka K, Moriguchi T, Nishida E. Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest. Science 1998,280(5363):599-602
Chen YR, Tan TH. Inhibition of the c-Jun N-terminal kinase (JNK) signaling
43. pathway by curcumin. Oncogene 1998,17(2):173-8
44. Li D, Zimmerman TL, Thevananther S, et al. Interleukin-1 beta-mediated suppression of RXR:RAR transactivation of the Ntcp promoter is JNK-dependent. J Biol Chem 2002,277(35):31416-22
45. Moreno-Manzano V, Lucio-Cazana J, Konta T, et al. Enhancement of TNF-alpha-induced apoptosis by immobilized arginine-glycine-aspartate: involvement of a tyrosine kinase-dependent, MAP kinase-independent mechanism. Biochem Biophys Res Commun 2000,277(2):293-8
46. Lee LF, Li G, Templeton DJ, et al. Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK). J Biol Chem 1998,273(43):28253-60
47. Okano J, Rustgi AK. Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery. J Biol Chem 2001,276(22):19555-64
48. Yamamoto K, Ichijo H, Korsmeyer SJ. BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol Cell Biol 1999,19(12):8469-78
49. Figueroa-Masot XA, Hetman M, Higgins MJ, et al. Taxol induces apoptosis in cortical neurons by a mechanism independent of Bcl-2 phosphorylation. J Neurosci 2001,21(13):4657-67
50. Stone AA, Chambers TC. Microtubule inhibitors elicit differential effects on MAP kinase (JNK, ERK, and p38) signaling pathways in human KB-3 carcinoma cells. Exp Cell Res 2000,254(1):110-9
51. Yamaki K, Hong J, Hiraizumi K, et al. Participation of various kinases in staurosporine induced apoptosis of RAW 264.7 cells. J Pharm Pharmacol 2002,54(11):1535-44
52. Tadlock L, Yamagiwa Y, Marienfeld C, et al. Double stranded RNA activates a p38 Mitogen Activated Protein Kinase dependent Cell Survival Program in Biliary Epithelia. Am J Physiol Gastrointest Liver Physiol 2003 Jan 22
53. Grossman D, Kim PJ, Schechner JS, et al. Inhibition of melanoma tumor growth in vivo by survivin targeting. Proc Natl Acad Sci U S A 2001,98(2):635-40
54. Zaffaroni N, Daidone MG. Survivin expression and resistance to anticancer treatments: perspectives for new therapeutic interventions. Drug Resist Updat 2002,(2):65-72
Capaccioli S, Di Pasquale G, Mini E, et al. Cationic lipids improve antisense
55. oligonucleotide uptake and prevent degradation in cultured cells and in human serum. Biochem Biophys Res Commun 1993,197(2):818-25
56. Haller H, Maasch C, Dragun D, et al. Antisense oligodesoxynucleotide strategies in renal and cardiovascular disease. Kidney Int 1998,53(6):1550-8
57. Merdan T, Kopecek J, Kissel T. Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Adv Drug Deliv Rev 2002,54(5):715-58
58. Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 1998,58(9):1808-1812
59. Grossman D, McNiff JM, Li F, Altieri DC. Expression of the apoptosis inhibitor, survivin, in nonmelanoma skin cancer and gene targeting in a keratinocyte cell line. Lab Invest 1999,79(9):1121-1126
60. Kawasaki H, Toyoda M, Shinohara H, et al. Expression of survivin correlates with apoptosis, proliferation, and angiogenesis during human colorectal tumorigenesis. Cancer 2001,91(11):2026-32
61. 全国胃癌防治研究协作组编著. 胃及十二指肠粘膜活检病理. 121－128, 沈阳：辽宁人民出版社，1992年
62. Parker SB, Dobrian AD, Wade SS, et al. AT(1) receptor inhibition does not reduce arterial wall hypertrophy or PDGF-A expression in renal hypertension. Am J Physiol Heart Circ Physiol 2000,278(2):H613-22
63. Birner P, Ritzi M, Musahl C, et al. Immunohistochemical detection of cell growth fraction in formalin-fixed and paraffin-embedded murine tissue. Am J Pathol 2001,158(6):1991-6
64. Sugie S, Okamoto K, Watanabe T, et al. Suppressive effect of irsogladine maleate on N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-initiated and glyoxal-promoted gastric carcinogenesis in rats. Toxicology 2001,166(1-2):53-61
65. Iishi H, Tatsuta M, Baba M, et al. Low-protein diet promotes sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats. Cancer Lett 1999,141(1-2):117-22
66. Takahashi M, Nishikawa A, Furukawa F, et al. Dose-dependent promoting effects of sodium chloride (NaCl) on rat glandular stomach carcinogenesis initiated with N-methyl-N'-nitro-N-nitrosoguanidine. Carcinogenesis 1994,15(7):1429-32
Tanakamaru Z, Nishikawa A, Furukawa F,et al. Failure of dietary alpha-difluoromethylornithine to inhibit gastric carcinogenesis in rats after 8 weeks of
67. treatment with N-methyl-N'-nitro-N-nitrosoguanidine and sodium chloride. Cancer Lett 1997,120(1):95-100
68. Dolbeare F. Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridization. Histochem J 1996,28(8):531-75
69. Niklinska W, Chyczewski L, Niklinski J. New molecular approaches to lung cancer: biological and clinical implications of P53, P16 and K-RAS studies. Folia Histochem Cytobiol 2001,39(2):99-103
70. Wistuba II, Gazdar AF, Minna JD. Molecular genetics of small cell lung carcinoma. Semin Oncol 2001,28(2 Suppl 4):3-13
71. Greco O, Scott SD, Marples B, et al. Cancer gene therapy: 'delivery, delivery, delivery' . Front Biosci 2002,7:d1516-24
72. Yazawa K, Fisher WE, Brunicardi FC. Current progress in suicide gene therapy for cancer. World J Surg 2002,26(7):783-9
2. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 1997;3:917-21.
3. Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res 1998,58(23):5315-20
4. Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998,396(6711):580-4
5. Kawasaki H, Altieri DC, Lu CD, et al. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res 1998,58(22):5071-4
6. Yamaguchi K, Tada M, Horikoshi N, et al. Phase II study of paclitaxel with 3-h infusion in patients with advanced gastric cancer. Gastric Cancer 2002;5(2):90-5
7. Constenla M, Garcia-Arroyo R, Lorenzo I, et al. Docetaxel, 5-fluorouracil, and leucovorin as treatment for advanced gastric cancer: results of a phase II study. Gastric Cancer 2002;5(3):142-7
8. Diaz JF, Strobe R, Engelborghs Y. et al. Molecular recognition of taxol by microtubules. Kinetics and thermodynamics of binding of fluorescent taxol derivatives to an exposed site. J Biol Chem 2000,275(34):26265-76
9. Moos PJ, Fitzpatrick FA. Taxanes propagate apoptosis via two cell populations with distinctive cytological and molecular traits. Cell Growth Differ 1998,9(8):687-97
10. Chantalat L, Skoufias DA, Kleman JP, et al. Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions. Mol Cell 2000,6:183
11. Eskes R, Desagher S, Antonsson B, et al. Bid induces the oligomerization and insertion of Bax into the outermitochondrial membrane. Mol Cell Biol, 2000,20(3):929-935
12. Ashkenazi A , Dixit VM. Death receptors: signaling and modulation. Science, 1998, 281(5381): 1305-1308
13. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science, 1995,267(5203):1456-1462
14. Yang E,Korsmeyer S.J. Molecular thanatopsis:a discourse on the BCL2 family. Blood,1996,88:386-401
15. Kappler M, Kotzsch M, Bartel F, et al. Elevated expression level of survivin protein in soft-tissue sarcomas is a strong independent predictor of survival. Clin Cancer Res 2003,9(3):1098-104
16. Ikeguchi M, Kaibara N. survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma. Br J Cancer 2002,87(8):883-7
17. Kato J, Kuwabara Y, Mitani M, et al. Expression of survivin in esophageal cancer: correlation with the prognosis and response to chemotherapy. Int J Cancer 2001,95(2):92-5
18. Tamura G. Genetic and epigenetic alterations of tumor suppressor and tumor-related genes in gastric cancer. Histol Histopathol 2002,17(1):323-9
19. Wu MS, Shun CT, Sheu JC, et al. Overexpression of mutant p53 and c-erbB-2 proteins and mutations of the p15 and p16 genes in human gastric carcinoma: with respect to histological subtypes and stages. J Gastroenterol Hepatol 1998,13(3):305-10
20. Wu MS, Shun CT, Wang HP, et al. Genetic alterations in gastric cancer: relation to histological subtypes, tumor stage, and Helicobacter pylori infection. Gastroenterology 1997,112(5):1457-65
21. 陈波，王舒宝 胃癌的临床表现与分期 见：张文范，张荫昌，陈峻青，主编， 胃癌， 上海： 上海科技技术出版社，第二版 2001：249-255
22. Hoffman WH, Biade S, Zilfou JT, et al. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 2002,277:3247
23. Mirza A, McGuirk M, Hockenberry TN, et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene 2002,21:2613
24. 张佩范 进展期胃癌的病理 见：张文范，张荫昌，陈峻青，主编， 胃癌， 上海： 上海科技技术出版社，第二版 2001：109-110
25. Leung WK, Sung JJ. Review article: intestinal metaplasia and gastric carcinogenesis. Aliment Pharmacol Ther 2002,16(7):1209-16
26. Olie RA, Simoes-Wust AP, Baumann B,et al. A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res 2000,60:2805
27. Crown J, O'Leary M. The taxanes: an update. Lancet 2000,355(9210):1176-8
28. Weiner LM. Paclitaxel in the treatment of esophageal cancer. Semin Oncol 1999,26(1 Suppl 2):106-8
Gadgeel SM, Shields AF, Heilbrun LK. et al. Phase II study of paclitaxel and
29. carboplatin in patients with advanced gastric cancer. Am J Clin Oncol 2003,26(1):37-41
30. Gotaskie GE, Andreassi BF. Paclitaxel: a new antimitotic chemotherapeutic agent. Cancer Pract 1994,2(1):27-33
31. Horwitz SB. Taxol (paclitaxel): mechanisms of action. Ann Oncol 1994;5 Suppl 6:S3-6
32. Herceg Z, Wang ZQ. Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat Res 2001,477(1-2):97-110
33. Oliver FJ, de la Rubia G, Rolli V, et al. Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. J Biol Chem 1998 Dec 11;273(50):33533-9
34. Barrachina M, Secades J, Lozano R, et al. Citicoline increases glutathione redox ratio and reduces caspase-3 activation and cell death in staurosporine-treated SH-SY5Y human neuroblastoma cells. Brain Res 2002,957(1):84-90
35. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002,298(5600):1911-2
36. Schaeffer HJ, Weber MJ. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 1999 Apr;19(4):2435-44
37. Whitmarsh AJ, Yang SH, Su MS, et al. Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors. Mol Cell Biol 1997,17(5):2360-71
38. Wang XZ, Ron D. Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase. Science 1996,272(5266):1347-9
39. Dunn C, Wiltshire C, MacLaren A, et al. Molecular mechanism and biological functions of c-Jun N-terminal kinase signalling via the c-Jun transcription factor. Cell Signal 2002,14(7):585-93
40. Gupta S, Campbell D, Derijard B, et al. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 1995,267(5196):389-93
41. Cuenda A, Rouse J, Doza YN, et al. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 1995,364(2):229-33
42. Takenaka K, Moriguchi T, Nishida E. Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest. Science 1998,280(5363):599-602
Chen YR, Tan TH. Inhibition of the c-Jun N-terminal kinase (JNK) signaling
43. pathway by curcumin. Oncogene 1998,17(2):173-8
44. Li D, Zimmerman TL, Thevananther S, et al. Interleukin-1 beta-mediated suppression of RXR:RAR transactivation of the Ntcp promoter is JNK-dependent. J Biol Chem 2002,277(35):31416-22
45. Moreno-Manzano V, Lucio-Cazana J, Konta T, et al. Enhancement of TNF-alpha-induced apoptosis by immobilized arginine-glycine-aspartate: involvement of a tyrosine kinase-dependent, MAP kinase-independent mechanism. Biochem Biophys Res Commun 2000,277(2):293-8
46. Lee LF, Li G, Templeton DJ, et al. Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK). J Biol Chem 1998,273(43):28253-60
47. Okano J, Rustgi AK. Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery. J Biol Chem 2001,276(22):19555-64
48. Yamamoto K, Ichijo H, Korsmeyer SJ. BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol Cell Biol 1999,19(12):8469-78
49. Figueroa-Masot XA, Hetman M, Higgins MJ, et al. Taxol induces apoptosis in cortical neurons by a mechanism independent of Bcl-2 phosphorylation. J Neurosci 2001,21(13):4657-67
50. Stone AA, Chambers TC. Microtubule inhibitors elicit differential effects on MAP kinase (JNK, ERK, and p38) signaling pathways in human KB-3 carcinoma cells. Exp Cell Res 2000,254(1):110-9
51. Yamaki K, Hong J, Hiraizumi K, et al. Participation of various kinases in staurosporine induced apoptosis of RAW 264.7 cells. J Pharm Pharmacol 2002,54(11):1535-44
52. Tadlock L, Yamagiwa Y, Marienfeld C, et al. Double stranded RNA activates a p38 Mitogen Activated Protein Kinase dependent Cell Survival Program in Biliary Epithelia. Am J Physiol Gastrointest Liver Physiol 2003 Jan 22
53. Grossman D, Kim PJ, Schechner JS, et al. Inhibition of melanoma tumor growth in vivo by survivin targeting. Proc Natl Acad Sci U S A 2001,98(2):635-40
54. Zaffaroni N, Daidone MG. Survivin expression and resistance to anticancer treatments: perspectives for new therapeutic interventions. Drug Resist Updat 2002,(2):65-72
Capaccioli S, Di Pasquale G, Mini E, et al. Cationic lipids improve antisense
55. oligonucleotide uptake and prevent degradation in cultured cells and in human serum. Biochem Biophys Res Commun 1993,197(2):818-25
56. Haller H, Maasch C, Dragun D, et al. Antisense oligodesoxynucleotide strategies in renal and cardiovascular disease. Kidney Int 1998,53(6):1550-8
57. Merdan T, Kopecek J, Kissel T. Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Adv Drug Deliv Rev 2002,54(5):715-58
58. Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 1998,58(9):1808-1812
59. Grossman D, McNiff JM, Li F, Altieri DC. Expression of the apoptosis inhibitor, survivin, in nonmelanoma skin cancer and gene targeting in a keratinocyte cell line. Lab Invest 1999,79(9):1121-1126
60. Kawasaki H, Toyoda M, Shinohara H, et al. Expression of survivin correlates with apoptosis, proliferation, and angiogenesis during human colorectal tumorigenesis. Cancer 2001,91(11):2026-32
61. 全国胃癌防治研究协作组编著. 胃及十二指肠粘膜活检病理. 121－128, 沈阳：辽宁人民出版社，1992年
62. Parker SB, Dobrian AD, Wade SS, et al. AT(1) receptor inhibition does not reduce arterial wall hypertrophy or PDGF-A expression in renal hypertension. Am J Physiol Heart Circ Physiol 2000,278(2):H613-22
63. Birner P, Ritzi M, Musahl C, et al. Immunohistochemical detection of cell growth fraction in formalin-fixed and paraffin-embedded murine tissue. Am J Pathol 2001,158(6):1991-6
64. Sugie S, Okamoto K, Watanabe T, et al. Suppressive effect of irsogladine maleate on N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-initiated and glyoxal-promoted gastric carcinogenesis in rats. Toxicology 2001,166(1-2):53-61
65. Iishi H, Tatsuta M, Baba M, et al. Low-protein diet promotes sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats. Cancer Lett 1999,141(1-2):117-22
66. Takahashi M, Nishikawa A, Furukawa F, et al. Dose-dependent promoting effects of sodium chloride (NaCl) on rat glandular stomach carcinogenesis initiated with N-methyl-N'-nitro-N-nitrosoguanidine. Carcinogenesis 1994,15(7):1429-32
Tanakamaru Z, Nishikawa A, Furukawa F,et al. Failure of dietary alpha-difluoromethylornithine to inhibit gastric carcinogenesis in rats after 8 weeks of
67. treatment with N-methyl-N'-nitro-N-nitrosoguanidine and sodium chloride. Cancer Lett 1997,120(1):95-100
68. Dolbeare F. Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridization. Histochem J 1996,28(8):531-75
69. Niklinska W, Chyczewski L, Niklinski J. New molecular approaches to lung cancer: biological and clinical implications of P53, P16 and K-RAS studies. Folia Histochem Cytobiol 2001,39(2):99-103
70. Wistuba II, Gazdar AF, Minna JD. Molecular genetics of small cell lung carcinoma. Semin Oncol 2001,28(2 Suppl 4):3-13
71. Greco O, Scott SD, Marples B, et al. Cancer gene therapy: 'delivery, delivery, delivery' . Front Biosci 2002,7:d1516-24
72. Yazawa K, Fisher WE, Brunicardi FC. Current progress in suicide gene therapy for cancer. World J Surg 2002,26(7):783-9