姜黄素对亚硝胺诱导的大鼠肝癌的化学防护作用研究
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摘要
背景/目的：
人类许多肿瘤的形成和发展是一个长期的多阶段的过程，化学预防可作用于其
中的任何阶段。从低风险的正常人群到因环境、生活方式、基因易感性和癌前病
变等因素造成的中等风险人群，再到曾患过肿瘤而易再发肿瘤的高风险人群，都
可以采用适当的化学预防措施降低肿瘤的发病率和病死率。因而，人们希望能找
到安全、高效的肿瘤化学预防药物。食物来源的天然药物因其具备较低的毒副作
用、较高的安全性和较高的依从性，可被各类人群尤其是低风险人群长期服用，
而倍受关注。姜黄素是天然植物姜黄的主要活性成分，生活中可作为调料和食物
染色剂。姜黄素的药理作用广泛，具有抗炎、抗氧化、降血脂等作用。近年来，
其对肿瘤的化学预防作用受到重视，具有抗癌谱广，毒副作用小等特点。尽管已
对姜黄素的作用机制进行了初步阐明，但对其作用的分子靶点依然不清。基因芯
片能同时检测几千个甚至上万个基因的表达，使系统的研究姜黄素作用的分子机
制成为可能。本研究旨在探讨以下问题：
1）观察姜黄根粉对亚硝胺诱导的实验性大鼠肝癌的化学预防作用，评价姜黄素
对肝癌的化学预防效果；
2）寻找姜黄素对肝癌预防作用评价的中间生物指标，并利用中间生物指标建立
姜黄素预防效果的早期判别函数；
3）利用基因芯片技术探讨姜黄素对肝癌化学防护作用的分子机理，分析姜黄素
98级博士学位论义
可能作用的基因功能组。
方 法：
1）利用二乙基亚硝胺和 Nitrosomorpholine诱导 Wistar大鼠原发性肝癌模型。
预防组A于诱癌前7天喂食含2％姜黄根粉饲料，预防组B于诱癌24周后加用
2％姜黄根粉饲料。分别于 16、20。24周宰杀部分大鼠，观察大鼠肿瘤发生情
况。32周时，宰杀所有大鼠，统计各组大鼠的肿瘤发生率、淋巴结转移率和
肺转移率。HE染色进行形态学观察和病理定性分析；
2）利用细胞增殖核抗原和细胞原位末端标记技术分别检测各组肝组织的细胞增
殖指数和细胞凋亡指数，并用这两个指标作为中间生物标记评价姜黄素对肝
癌的化学预防作用，建立以它们为参数的判别函数。
3）分别取诱癌早期（20周）和诱癌晚期（32周）组织和对应的姜黄素化学预防
组织，进行基因芯片检测。用异硫氰酸肌一步法抽提组织RNA，比色检测纯度，
电泳检测RNA的完整性，用oligodT亲和柱过柱纯化，得到纯mRNA。逆转录
为 CDNA，分别用荧光染料CY3和 CYS标记成探针，与大鼠基因芯片杂交，42
OC孵育15l7hr，洗片，扫描，进行结果分析。
结 果：
1）大鼠经 32周二乙基亚硝胺和 Nitrosomorpholine处理，肝癌的发生率在诱癌
组为100％（12／12），肿瘤在肝脏形成多发癌灶（22．3土6．0个），最多达31
个，同时肺转移率为75．0％（9／12），淋巴结转移率为66．7％（S／12）。而姜黄素
早期预防组 A肝癌的发生率仅为 16．7见 肝癌结节明显小而少，平均为 2．0土
1．4，未发现肺转移和淋巴结转移；而在诱癌的中晚期枯4周）给予姜黄素，肝
癌的发生率为100％，肝癌结节达9．8士2．2，肺转移率和淋巴结转移率均为
16．7叭2／l人形态学上，诱癌早期，诱癌组呈典型肝硬化表现，形成假小叶。
而预防组则仅见少量气泡样变性和点状坏死：诱癌晚期，诱癌组可见肿瘤形
南方医院全军消化内科研究所 第5贞
98级博士学位论文
成，而预防组仅见轻度肝硬化形成。同时，诱癌组可见许多肺转移结节，镜
下见肿瘤细胞呈灶性生长；而预防组则少见。
2）与诱癌组相比，预防组增殖指数在 16，20，24和 32周均较同期诱癌组明显
降低而凋亡指数明显增高，差异显著。提示姜黄素对肝癌的化学预防作用与
抑制增殖和诱导凋亡有关。利用增殖指数和凋亡指数为参数，建立起判别函
数，以期在早期判定姜黄素的预防效果。
3）应用基因芯片检测了诱癌早期和诱癌晚期姜黄素可能的作用靶点，发现在早
期调节 217个基因的表达，而晚期调节 106个基因的表达，与多个功能基因
组有关。
结 论：
1）姜黄素对亚硝胺诱发的大鼠肝癌具有明显的化学预防作用，其早期化学预防
作用良好，明显降低肝癌的发生率和多结节性，降低肿瘤自发转移率；晚期
给予姜黄素也有一定的防护作用；能延缓疾病进展，降低肝癌的转移率。
2）细胞增殖指数和细胞凋亡指数可作为评价姜黄素化学预防作用的中间生物指
标，可在诱癌的早中期反映姜黄素的化学预防效果，对今后的临床实践有借
鉴作用。
3）姜黄素对肝癌的化学预防作用与调节肝脏代谢功能，抑制增殖，诱导凋亡，
抗细胞骨架，增强机体兔疫力和桔抗亚硝胺的基因调节作用有关。
Backgroud/Objectives:
The development of human cancer is a long-time and multiphase process,
the scope of chemoprevention encompasses cohorts at all phases of this
process梖rom healthy subjects at normal risk, to populations at intermediate
risk resulting from environmental and lifestyle factors, genetic predisposition,
and precancerous lesions, and then to the patients at high risk for second
malignancy. So they expect to get chemopreventive drugs with safety and
efficiency. Diet-derived chemopreventive agents are highly interested because
of their safety and compliance. Curcumin is dominating active components of
Curcuma longa and can be used as spice and edible pigment. Simultaneously,
curcumin possess widespread pharmacological effect, such as
antiinflammation, antioxidation, anticoagulation et al. Recently, curcumin is
attached importance on account of its anticancer properties. Understanding
possible molecular targets is very important to elucidate mechanisms for
curoumin chemoprevention. Gene chip allows monitoring the expression of
thousands or tens of thousands genes simultaneously in one hybridization
experiment. Employing this technique, detection of differentially expressed
genes regulated by curcumin is greatly facilitated. The thesis aimed to observe
the inhibition effects of curcumin on hepatocarcinogenesis induced by
nitrosamine on Wistar rats and to evaluate chemopreventive effect of curcumin
on hepatocarcingenesis, and to investigate mechanisms for chemoprevention
by curcumin with possible molecular targets by gene chip. It is focused on the
questions as follows.
1) To observe the inhibition effects of diet containing 2% turmeric powder on
hepatocarcinogenesis induced by nitrosamine on Wistar rats.
2) To investigate the intermediate biomarker that evaluates the
chemopreventive effects of curcumin on hepatocarcinogenesis in order to
be used in clinical research.
3) To discover the possible molecular targets by gene chip in order to
elucidate the mechanisms of chemoprevention by curcumin.
Methods
1) Wistar rats were given a single i.p. injection of 40mg/kg body weight
N-diethylnitrosamine(DEN) at age 6 weeks. After 4 weeks, rats received
l2Oppm N-nitrosomorpholine in drinking water for 28 weeks. The curcumin
A group started eating 2% turmeric powder-containing diet 7 days before
DEN injection until execution. The rats in curcumin B group were given 2%
turmeric powder-containing diet until hepatocellular carcinoma had been
induced at 24th week. Then, the rats in experimental group and curcumin
A group were sacrificed at 16th, 20th and 24th week to examine the
development of hepatocellular carcinoma. At the 32nd week, all rats were
executed to compare the curcumin group with the experimental group on
incidence, multiplicity and metastasis of hepatocarcinoma. The changes of
hepatic tissues in morphology were observed through hematoxylin-eosin
(HE) staining..
2) Proliferation index displayed by PCNA through immunohistochemistry and
apoptotic index revealed by TUNEL are used to evaluate the
chemopreveritive effects on curcumin for every stage of
hepatocarcinogenesis induced by nitrosamine, and to set up a discriminant
function to develop discriminant analysis in order to determine the
preventive effects using intermedial biomarker at early-middle stage.
3) The hepatic tissue specimens come from
人类许多肿瘤的形成和发展是一个长期的多阶段的过程，化学预防可作用于其
中的任何阶段。从低风险的正常人群到因环境、生活方式、基因易感性和癌前病
变等因素造成的中等风险人群，再到曾患过肿瘤而易再发肿瘤的高风险人群，都
可以采用适当的化学预防措施降低肿瘤的发病率和病死率。因而，人们希望能找
到安全、高效的肿瘤化学预防药物。食物来源的天然药物因其具备较低的毒副作
用、较高的安全性和较高的依从性，可被各类人群尤其是低风险人群长期服用，
而倍受关注。姜黄素是天然植物姜黄的主要活性成分，生活中可作为调料和食物
染色剂。姜黄素的药理作用广泛，具有抗炎、抗氧化、降血脂等作用。近年来，
其对肿瘤的化学预防作用受到重视，具有抗癌谱广，毒副作用小等特点。尽管已
对姜黄素的作用机制进行了初步阐明，但对其作用的分子靶点依然不清。基因芯
片能同时检测几千个甚至上万个基因的表达，使系统的研究姜黄素作用的分子机
制成为可能。本研究旨在探讨以下问题：
1）观察姜黄根粉对亚硝胺诱导的实验性大鼠肝癌的化学预防作用，评价姜黄素
对肝癌的化学预防效果；
2）寻找姜黄素对肝癌预防作用评价的中间生物指标，并利用中间生物指标建立
姜黄素预防效果的早期判别函数；
3）利用基因芯片技术探讨姜黄素对肝癌化学防护作用的分子机理，分析姜黄素
98级博士学位论义
可能作用的基因功能组。
方 法：
1）利用二乙基亚硝胺和 Nitrosomorpholine诱导 Wistar大鼠原发性肝癌模型。
预防组A于诱癌前7天喂食含2％姜黄根粉饲料，预防组B于诱癌24周后加用
2％姜黄根粉饲料。分别于 16、20。24周宰杀部分大鼠，观察大鼠肿瘤发生情
况。32周时，宰杀所有大鼠，统计各组大鼠的肿瘤发生率、淋巴结转移率和
肺转移率。HE染色进行形态学观察和病理定性分析；
2）利用细胞增殖核抗原和细胞原位末端标记技术分别检测各组肝组织的细胞增
殖指数和细胞凋亡指数，并用这两个指标作为中间生物标记评价姜黄素对肝
癌的化学预防作用，建立以它们为参数的判别函数。
3）分别取诱癌早期（20周）和诱癌晚期（32周）组织和对应的姜黄素化学预防
组织，进行基因芯片检测。用异硫氰酸肌一步法抽提组织RNA，比色检测纯度，
电泳检测RNA的完整性，用oligodT亲和柱过柱纯化，得到纯mRNA。逆转录
为 CDNA，分别用荧光染料CY3和 CYS标记成探针，与大鼠基因芯片杂交，42
OC孵育15l7hr，洗片，扫描，进行结果分析。
结 果：
1）大鼠经 32周二乙基亚硝胺和 Nitrosomorpholine处理，肝癌的发生率在诱癌
组为100％（12／12），肿瘤在肝脏形成多发癌灶（22．3土6．0个），最多达31
个，同时肺转移率为75．0％（9／12），淋巴结转移率为66．7％（S／12）。而姜黄素
早期预防组 A肝癌的发生率仅为 16．7见 肝癌结节明显小而少，平均为 2．0土
1．4，未发现肺转移和淋巴结转移；而在诱癌的中晚期枯4周）给予姜黄素，肝
癌的发生率为100％，肝癌结节达9．8士2．2，肺转移率和淋巴结转移率均为
16．7叭2／l人形态学上，诱癌早期，诱癌组呈典型肝硬化表现，形成假小叶。
而预防组则仅见少量气泡样变性和点状坏死：诱癌晚期，诱癌组可见肿瘤形
南方医院全军消化内科研究所 第5贞
98级博士学位论文
成，而预防组仅见轻度肝硬化形成。同时，诱癌组可见许多肺转移结节，镜
下见肿瘤细胞呈灶性生长；而预防组则少见。
2）与诱癌组相比，预防组增殖指数在 16，20，24和 32周均较同期诱癌组明显
降低而凋亡指数明显增高，差异显著。提示姜黄素对肝癌的化学预防作用与
抑制增殖和诱导凋亡有关。利用增殖指数和凋亡指数为参数，建立起判别函
数，以期在早期判定姜黄素的预防效果。
3）应用基因芯片检测了诱癌早期和诱癌晚期姜黄素可能的作用靶点，发现在早
期调节 217个基因的表达，而晚期调节 106个基因的表达，与多个功能基因
组有关。
结 论：
1）姜黄素对亚硝胺诱发的大鼠肝癌具有明显的化学预防作用，其早期化学预防
作用良好，明显降低肝癌的发生率和多结节性，降低肿瘤自发转移率；晚期
给予姜黄素也有一定的防护作用；能延缓疾病进展，降低肝癌的转移率。
2）细胞增殖指数和细胞凋亡指数可作为评价姜黄素化学预防作用的中间生物指
标，可在诱癌的早中期反映姜黄素的化学预防效果，对今后的临床实践有借
鉴作用。
3）姜黄素对肝癌的化学预防作用与调节肝脏代谢功能，抑制增殖，诱导凋亡，
抗细胞骨架，增强机体兔疫力和桔抗亚硝胺的基因调节作用有关。
Backgroud/Objectives:
The development of human cancer is a long-time and multiphase process,
the scope of chemoprevention encompasses cohorts at all phases of this
process梖rom healthy subjects at normal risk, to populations at intermediate
risk resulting from environmental and lifestyle factors, genetic predisposition,
and precancerous lesions, and then to the patients at high risk for second
malignancy. So they expect to get chemopreventive drugs with safety and
efficiency. Diet-derived chemopreventive agents are highly interested because
of their safety and compliance. Curcumin is dominating active components of
Curcuma longa and can be used as spice and edible pigment. Simultaneously,
curcumin possess widespread pharmacological effect, such as
antiinflammation, antioxidation, anticoagulation et al. Recently, curcumin is
attached importance on account of its anticancer properties. Understanding
possible molecular targets is very important to elucidate mechanisms for
curoumin chemoprevention. Gene chip allows monitoring the expression of
thousands or tens of thousands genes simultaneously in one hybridization
experiment. Employing this technique, detection of differentially expressed
genes regulated by curcumin is greatly facilitated. The thesis aimed to observe
the inhibition effects of curcumin on hepatocarcinogenesis induced by
nitrosamine on Wistar rats and to evaluate chemopreventive effect of curcumin
on hepatocarcingenesis, and to investigate mechanisms for chemoprevention
by curcumin with possible molecular targets by gene chip. It is focused on the
questions as follows.
1) To observe the inhibition effects of diet containing 2% turmeric powder on
hepatocarcinogenesis induced by nitrosamine on Wistar rats.
2) To investigate the intermediate biomarker that evaluates the
chemopreventive effects of curcumin on hepatocarcinogenesis in order to
be used in clinical research.
3) To discover the possible molecular targets by gene chip in order to
elucidate the mechanisms of chemoprevention by curcumin.
Methods
1) Wistar rats were given a single i.p. injection of 40mg/kg body weight
N-diethylnitrosamine(DEN) at age 6 weeks. After 4 weeks, rats received
l2Oppm N-nitrosomorpholine in drinking water for 28 weeks. The curcumin
A group started eating 2% turmeric powder-containing diet 7 days before
DEN injection until execution. The rats in curcumin B group were given 2%
turmeric powder-containing diet until hepatocellular carcinoma had been
induced at 24th week. Then, the rats in experimental group and curcumin
A group were sacrificed at 16th, 20th and 24th week to examine the
development of hepatocellular carcinoma. At the 32nd week, all rats were
executed to compare the curcumin group with the experimental group on
incidence, multiplicity and metastasis of hepatocarcinoma. The changes of
hepatic tissues in morphology were observed through hematoxylin-eosin
(HE) staining..
2) Proliferation index displayed by PCNA through immunohistochemistry and
apoptotic index revealed by TUNEL are used to evaluate the
chemopreveritive effects on curcumin for every stage of
hepatocarcinogenesis induced by nitrosamine, and to set up a discriminant
function to develop discriminant analysis in order to determine the
preventive effects using intermedial biomarker at early-middle stage.
3) The hepatic tissue specimens come from
引文
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4. Kelloff GJ, Hawk ET, Karp JE, et al. Progress in clinical chemoprevention. Semi Onco, 1997; 24:241-252
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7. Masui T; Nakanishi H; Inada K; et al. Highly metastatic hepatocellolar carcinomas induced in male F344 rats treated with N-nitrosomorpholine in combination with other hepatocarcinogens show a high incidence of P53 gene mutatons along with altered mRNA expressed of tumor-related genes. Cancer lett, 1997; 112:33-45
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20. Samaha HS, Kelloff G J, Steele V, et al. Modulation of apoptosis by Sulindac, Curcumin, Phenylethyl-3-methylcaffeate, and 6-Phenylhexyl isothiocyanate: Apoptotic index as a biomarker in colom cancer chemoprevention and promotion. Cancer Research, 1997; 57:1301-1305
21. Churchill M, Chadburn A, Bilinski RT, et al. Inhibition of intestinal tumors by curcumin is associated with changes in the intestinal immune cell profile. J Surg Res. 2000; 89(2): 169-75
22. Arbiser JL, Klauber N, Rohan R,et al. Curcumin is an in vivo inhibitor of angiogenesis. Mol Med. 1998; 4(6): 376-83
23．沃兴德，洪行球，高承贤．姜黄素最大耐受量试验，浙江中医学院学报 2000；24(2)：55
24．沃兴德，洪行球，高承贤．姜黄素长期毒性试验，浙江中医学院学报 2000；24(1)：61-65
25. Boone CW, Kelloff G J, Malone WE, et al. Identification of candidate cancer chemopreventive agents and their evaluation in animal models and human clinical trials: a review. Cancer Res, 1990,50(1):2-9
26. Greenwald P, Witkin KM, Malone WF, et al. The study of markers of biological effect in cancer prevention research trials. Int J Cancer. 1992; 52(2): 189-96
27. Debouck C, Goodfellow PN. DNA microarrays in drug discovery and
28. Braxton S, Bedilion T. The integration of microarray information in the drug development process. Curr Opin Biotechnol. 1998; 9(6): 643-9
29. Afshari CA, Nuwaysir EF, Barrett JC Application of complementary DNA microarray technology to carcinogen identification, toxicology, and drug safety evaluation. Cancer Res. 1999; 59(19): 4759-60
30．刘鹏，邢婉丽，程京，生物芯片技术-21 世纪革命性的技术 生理通讯 2000；19(2)：29-31
31．沈忠英，细胞凋亡研究进展．中华病理学杂志．2000；29(1)：63-65
32. Loeffier M, Kroemer G. The mitochondrion in cell death control: certainties and incognita. Exp Cell Res. 2000; 256(1): 19-26
33. Ning XH, Zhang J, Liu J, et al. Signaling and expression for mitochondrial membrane proteins during left ventricular remodeling and contractile failure after myocardial infarction. J Am Coll Cardiol. 2000; 36(1): 282-7
34. Kaukonen J, Juselius JK, Tiranti V, et al. Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science. 2000; 289(5480): 782-5
35. Halestrap AP. The mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury. Biochem Soc Symp. 1999; 66:181-203
36. Crompton M, Virji S, Doyle V, et al. The mitochondrial permeability transition pore. Biochem Soc Symp. 1999; 66:167-79
37. Bernardi P. Mitochondria in muscle cell death. Ital J Neurol Sci. 1999 Dec; 20(6): 395-400
38. Halestrap AP, Doran E, Gillespie JP, et al. Mitochondria and cell death. Biochem Soc Trans. 2000; 28(2): 170-7
39. Yuan-ZQ, Sun-M, Feldman-RI,et al. Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer. Oncogene. 2000; 19(19): 2324-30
40. Page C, Lin H J, Jin Y, et al. Overexpression of Akt/AKT can modulate chemotherapy-induced apoptosis. Anticancer-Res. 2000; 20(1A): 407-16
41. Daugas E, Nochy D, Ravagnan L, et al. Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett. 2000; 476(3): 118-23
42. Lorenzo HK, Susin SA, Penninger J, et al. Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ. 1999; 6(6): 516-24
43. Morin D, Barthelemy S, Zini R, et al. Curcumin induces the mitochondrial pereability transition pore mediated by membrane protein thioloxidation. FEBS Lett. 2001,495:131-136
44. Pan MH, Chang WL, Lin S ,et al. Induction of apoptosis by garcinol and curcumin through cytochrome C release and activation of caspases in human leukemia H1-60 cells. J Agric Food Chem. 2001; 49(3):1464-1474.
45． Yamazaki Y, Tsuruga M, Zhou D, et al. Cytoskeletal disruption accelerates caspase-3 activation and alters the intracellular membrane reorganization in DNA damage-induced apoptosis. Exp Cell Res. 2000 25; 259(1) : 64-78
46． Marcellus RC, Chan H, Paquette D,et al.Induction of p53-independent apoptosis by the adenovirus E4orf4 protein requires binding to the Balpha subunit of protein phosphatase 2A. J Virol. 2000; 74(17) : 7869-77
47． Bokoch GM. Regulation of cell function by Rho family GTPases. Immunol Res. 2000; 21(2-3) : 139-48
48． Mimura S, Masuda T, Matsui T, et al. Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts. Genes Cells. 2000 ; 5(6) : 439-52
49． Mizushima T, Takahashi N, Stillman B . Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro. Genes Dev. 2000; 14(13) : 1631-41
50． Madine MA, Swietlik M, Pelizon C, et al. The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells. J Struct Biol. 2000; 129(2-3) : 198-210
51． Wahl MC, Huber R, Marinkovic S, et al. Structural investigations of the highly flexible recombinant ribosomal protein L12 from Thermotoga maritima. Biol Chem. 2000; 381(3) : 221-9
52． Chenuil A, Solignac M, Bernard M. Evolution of the large-subunit ribosomal RNA binding site for protein L23/25． Mol Biol Evol. 1997 ; 14(5) : 578-88
53． Maguire BA, Wild DG . The roles of proteins L28 and L33 in the assembly and function of Escherichia coli ribosomes in vivo. Mol Microbiol. 1997; 23(2) : 237-45
54． Maguire BA, Wild DG. The effects of mutations in the rpmB,G operon of Escherichia coli on ribosome assembly and ribosomal protein synthesis. Biochim Biophys Acta. 1997; 1353(2) : 137-47
55． Spence J, Gali RR, Dittmar G, et al. Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell. 2000; 102(1) : 67-76
56． Miilward TA, Zolnierowicz S, Hemmings BA. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci. 1999 ; 24(5) : 186-91
57． Abraham D, Podar K, Pacher M, et al. Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation. J Biol Chem. 2000; 275(29) : 22300-4
58． King AJ, Sun H, Diaz B,et al. The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338． Nature. 1998; 396(6707) : 180-3
59． Sun H, King AJ, Diaz HB, et al. Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak. Curr-Biol. 2000; 10(5) : 281-4
60． Radcliffe PA, Garcia MA, Toda T .The cofactor-dependent pathways for alpha-and beta-tubulins in microtubule biogenesis are functionally different in fission yeast. Genetics. 2000; 156(1) : 93-103
61． Ngan VK, Bellman K, Panda D, et al. Novel actions of the antitumor drugs vinflunine and vinorelbine on microtubules. Cancer Res. 2000; 60(18) : 5045-51
62． Goode BL, Drubin DG, Barnes G .Functional cooperation between the microtubule and actin cytoskeletons. Curr Opin Cell Biol. 2000; 12(1) : 63-71
63． de Hostos EL . The coronin family of actin-associated proteins. Trends Cell Biol. 1999; 9(9) : 345-50
64． Fukui-Y, Engler-S, Inoue-S, et al. Architectural dynamics and gene replacement of coronin suggest its role in cytokinesis. Cell Motil Cytoskeleton. 1999; 42(3) : 204-17
65． Hynes GM, Willison KR. Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of beta-actin. J Biol Chem. 2000; 275(25) : 18985-94
66． Sander EE, ten Klooster JP, van Delft S, et al. Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J Cell Biol. 1999; 147(5) : 1009-22
67． Akiyama TE, Ward JM, Gonzalez FJ. Regulation of the liver fatty acid-binding protein gene by hepatocyte nuclear factor 1 alpha (HNF1alpha). Alterations in fatty acid homeostasis in HNF1 alpha-deficient mice. J Biol Chem. 2000; 275(35) : 27117-22
68． Storch J, Thumser AE. The fatty acid transport function of fatty acid-binding proteins. Biochim Biophys Acta. 2000 26; 1486(1) : 28-44
69． Yang X, Bhaumik M, Bhattacharyya R, et al. New evidence for an extra-hepatic role of N-acetylglucosaminyltransferase Ⅲ in the progression of diethylnitrosamine-induced liver tumors in mice. Cancer Res. 2000 15; 60(12) : 3313-9
70． Bishayee A, Roy S, Chatterjee M. Characterization of selective induction and alteration of xenobiotic biotransforming enzymes by vanadium during diethylnitrosamine-induced chemical rat liver carcinogenesis. Oncol Res. 1999; 11(1) : 41-53
71． Henderson CJ, Sahraouei A, Wolf CR . Cytochrome P450s and chemoprevention. Biochem Soc Trans. 2000 Feb; 28(2) : 42-6
72． Antony S, Kuttan R, Kuttan G. et al. Imrnunomodulatory activity of curcumin. Immunol Invest. 1999; 28(5-6) : 291-303
73． T Rovira P, Mascarell L, Truffa Bachi-P. The impact of immunosuppressive drugs on the analysis of T cell activation. Curr Med Chem. 2000; 7(7) : 673-92
74． Rusnak F, Mertz P.Calcineurin: form and function. Physiol Rev. 2000; 80(4) : 1483-521
75． Bhattacharjee A, Lappi VR, Rutherford MS, et al. Molecular dissection of dimethylnitrosamine (DMN)-induced hepatotoxicity by mRNA differential display. Toxicol Appl Pharmacol. 1998 ; 150(1) : 186-95
76． Cavaggioni A, Mucignat C, Tirindelli R . Pheromone signalling in the mouse: role
of urinary proteins and vomeronasal organ. Arch Ital Biol. 1999; 137(2-3) : 193-200
77． Novotny MV, Ma W, Wiesler D, et al. Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein. Proc-R-Soc-Lond-B-Biol-Sci. 1999; 266(1432) : 2017-22
78． Bech Obschir D, Kraft R, Huang X et al. COP9 signalosome-specific phosphorlation target P53 to degration by the ubiquitin system. EMBO J. 2001,20(7) :1630-1639
2. Kelloff GJ; Crowell JA; Steele VE; et al. Progress in cancer chemoprevention. Ann N Y Acad Sci. 1999; 889:1-13
3. Kelloff GJ; Boone CW; Malone WF; et al. Chemoprevention clinical trials. Mutat Res. 1992; 267(2): 291-5
4. Kelloff GJ, Hawk ET, Karp JE, et al. Progress in clinical chemoprevention. Semi Onco, 1997; 24:241-252
5. Kelloff GJ; Hawk ET; Crowell JA; et al. Strategies for identification and clinic evalutation of promising chemopreventive agents. Oncology, 1996; 10:1471-1484
6. Coller HA; Grandori C; Tamayo P; et al. Expression analysis with oligonucletide microarrays reveals that MYC regulates gene involves in growth, cell cycle, signaling, and adhesion. Proc Natl Acad Sci USA, 2000,97:3260-3265
7. Masui T; Nakanishi H; Inada K; et al. Highly metastatic hepatocellolar carcinomas induced in male F344 rats treated with N-nitrosomorpholine in combination with other hepatocarcinogens show a high incidence of P53 gene mutatons along with altered mRNA expressed of tumor-related genes. Cancer lett, 1997; 112:33-45
8．陈宏、张振书、张亚历等，姜黄素抗癌作用与诱导肿瘤细胞凋亡．中华肿瘤杂志，1999，21(2)：118
9. Chen H, Zhang ZS, Zhang YL et al. Curcumin inhibits cell proliferation by interfering with the cell cycle and inducing apoptosis in colon carcinoma cells. Anticancer Research, 1999; 19:3675-3680
10．李连弟、鲁凤珠、张思维等，中国恶性肿瘤死亡率20年变化趋势和近期预测分析．中华肿瘤杂志，1997，19(1)：3～9
11. Rao CV, Simi B, Reddy BS. Inhibition by dietary curcumin of azoxymethane-induced ornithine decarboxylase, tyrosine protein kinase, arachidonic acid metabolism, and aberrant crypt foci formation in the rat colon. Carcinogenesis, 1993; 14:2219-2225
12. Azuine MA, Bhide SV. Chemopreventive effect of turmeric against stomach and skin tumors induced by chemical carcinogens in Swiss mice. Nurr Cancer, 1992; 17:77-83
13. Chuang SE, Kuo MI, Hsu CH, et al. Curcumin-containing diet inhibits diethylnitrosamine-induced murine hepatocarcinogenesis. Carcinogenesis, 2000; 21(2): 331-335
14. Krishnaswamy K, Goud VK, Sesikeran B, et al. Retardation of experimental tumorigenesis and reduction in DNA adducts by turmeric and curcumin. Nutr Cancer. 1998; 30(2): 163-6
15. Nakamura Y, Ohto Y, Murakami A. Inhibitory effects of curcumin and tetrahydrocurcuminoids on the tumor promoter-induced reactive oxygen species generation in leukocytes in vitro and in vivo. Jpn J Cancer Res. 1998; 89(4): 361-70
16. Piper JT, Singhal SS, Salameh MS, et al. Mechanisms of anticarcinogenic properties of curcumin: the effect of curcumin on glutathione linked detoxification enzymes in rat liver. Int J Biochem Cell Biol. 1998; 30(4): 445-56
17. Conney AH, Lysz T, Ferraro T, et al. Inhibitory effect of curcumin and some related dietary compounds on tumor promotion and arachidonic acid metabolism in mouse skin. Adv Enzyme Regul, 1991; 31:385-96
18. Kuo ML, Huang TS, Lin JK. Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells. Biochim Biophys Acta. 1996 15; 1317(2): 95-100
19. Anto RJ, Maliekal TT, Karunagaran D. L-929 cells harboring ectopically expressed RelA resist curcumin-induced apoptosis. J Biol Chem. 2000 26; 275(21): 15601-4
20. Samaha HS, Kelloff G J, Steele V, et al. Modulation of apoptosis by Sulindac, Curcumin, Phenylethyl-3-methylcaffeate, and 6-Phenylhexyl isothiocyanate: Apoptotic index as a biomarker in colom cancer chemoprevention and promotion. Cancer Research, 1997; 57:1301-1305
21. Churchill M, Chadburn A, Bilinski RT, et al. Inhibition of intestinal tumors by curcumin is associated with changes in the intestinal immune cell profile. J Surg Res. 2000; 89(2): 169-75
22. Arbiser JL, Klauber N, Rohan R,et al. Curcumin is an in vivo inhibitor of angiogenesis. Mol Med. 1998; 4(6): 376-83
23．沃兴德，洪行球，高承贤．姜黄素最大耐受量试验，浙江中医学院学报 2000；24(2)：55
24．沃兴德，洪行球，高承贤．姜黄素长期毒性试验，浙江中医学院学报 2000；24(1)：61-65
25. Boone CW, Kelloff G J, Malone WE, et al. Identification of candidate cancer chemopreventive agents and their evaluation in animal models and human clinical trials: a review. Cancer Res, 1990,50(1):2-9
26. Greenwald P, Witkin KM, Malone WF, et al. The study of markers of biological effect in cancer prevention research trials. Int J Cancer. 1992; 52(2): 189-96
27. Debouck C, Goodfellow PN. DNA microarrays in drug discovery and
28. Braxton S, Bedilion T. The integration of microarray information in the drug development process. Curr Opin Biotechnol. 1998; 9(6): 643-9
29. Afshari CA, Nuwaysir EF, Barrett JC Application of complementary DNA microarray technology to carcinogen identification, toxicology, and drug safety evaluation. Cancer Res. 1999; 59(19): 4759-60
30．刘鹏，邢婉丽，程京，生物芯片技术-21 世纪革命性的技术 生理通讯 2000；19(2)：29-31
31．沈忠英，细胞凋亡研究进展．中华病理学杂志．2000；29(1)：63-65
32. Loeffier M, Kroemer G. The mitochondrion in cell death control: certainties and incognita. Exp Cell Res. 2000; 256(1): 19-26
33. Ning XH, Zhang J, Liu J, et al. Signaling and expression for mitochondrial membrane proteins during left ventricular remodeling and contractile failure after myocardial infarction. J Am Coll Cardiol. 2000; 36(1): 282-7
34. Kaukonen J, Juselius JK, Tiranti V, et al. Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science. 2000; 289(5480): 782-5
35. Halestrap AP. The mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury. Biochem Soc Symp. 1999; 66:181-203
36. Crompton M, Virji S, Doyle V, et al. The mitochondrial permeability transition pore. Biochem Soc Symp. 1999; 66:167-79
37. Bernardi P. Mitochondria in muscle cell death. Ital J Neurol Sci. 1999 Dec; 20(6): 395-400
38. Halestrap AP, Doran E, Gillespie JP, et al. Mitochondria and cell death. Biochem Soc Trans. 2000; 28(2): 170-7
39. Yuan-ZQ, Sun-M, Feldman-RI,et al. Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer. Oncogene. 2000; 19(19): 2324-30
40. Page C, Lin H J, Jin Y, et al. Overexpression of Akt/AKT can modulate chemotherapy-induced apoptosis. Anticancer-Res. 2000; 20(1A): 407-16
41. Daugas E, Nochy D, Ravagnan L, et al. Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett. 2000; 476(3): 118-23
42. Lorenzo HK, Susin SA, Penninger J, et al. Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ. 1999; 6(6): 516-24
43. Morin D, Barthelemy S, Zini R, et al. Curcumin induces the mitochondrial pereability transition pore mediated by membrane protein thioloxidation. FEBS Lett. 2001,495:131-136
44. Pan MH, Chang WL, Lin S ,et al. Induction of apoptosis by garcinol and curcumin through cytochrome C release and activation of caspases in human leukemia H1-60 cells. J Agric Food Chem. 2001; 49(3):1464-1474.
45． Yamazaki Y, Tsuruga M, Zhou D, et al. Cytoskeletal disruption accelerates caspase-3 activation and alters the intracellular membrane reorganization in DNA damage-induced apoptosis. Exp Cell Res. 2000 25; 259(1) : 64-78
46． Marcellus RC, Chan H, Paquette D,et al.Induction of p53-independent apoptosis by the adenovirus E4orf4 protein requires binding to the Balpha subunit of protein phosphatase 2A. J Virol. 2000; 74(17) : 7869-77
47． Bokoch GM. Regulation of cell function by Rho family GTPases. Immunol Res. 2000; 21(2-3) : 139-48
48． Mimura S, Masuda T, Matsui T, et al. Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts. Genes Cells. 2000 ; 5(6) : 439-52
49． Mizushima T, Takahashi N, Stillman B . Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro. Genes Dev. 2000; 14(13) : 1631-41
50． Madine MA, Swietlik M, Pelizon C, et al. The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells. J Struct Biol. 2000; 129(2-3) : 198-210
51． Wahl MC, Huber R, Marinkovic S, et al. Structural investigations of the highly flexible recombinant ribosomal protein L12 from Thermotoga maritima. Biol Chem. 2000; 381(3) : 221-9
52． Chenuil A, Solignac M, Bernard M. Evolution of the large-subunit ribosomal RNA binding site for protein L23/25． Mol Biol Evol. 1997 ; 14(5) : 578-88
53． Maguire BA, Wild DG . The roles of proteins L28 and L33 in the assembly and function of Escherichia coli ribosomes in vivo. Mol Microbiol. 1997; 23(2) : 237-45
54． Maguire BA, Wild DG. The effects of mutations in the rpmB,G operon of Escherichia coli on ribosome assembly and ribosomal protein synthesis. Biochim Biophys Acta. 1997; 1353(2) : 137-47
55． Spence J, Gali RR, Dittmar G, et al. Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell. 2000; 102(1) : 67-76
56． Miilward TA, Zolnierowicz S, Hemmings BA. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci. 1999 ; 24(5) : 186-91
57． Abraham D, Podar K, Pacher M, et al. Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation. J Biol Chem. 2000; 275(29) : 22300-4
58． King AJ, Sun H, Diaz B,et al. The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338． Nature. 1998; 396(6707) : 180-3
59． Sun H, King AJ, Diaz HB, et al. Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak. Curr-Biol. 2000; 10(5) : 281-4
60． Radcliffe PA, Garcia MA, Toda T .The cofactor-dependent pathways for alpha-and beta-tubulins in microtubule biogenesis are functionally different in fission yeast. Genetics. 2000; 156(1) : 93-103
61． Ngan VK, Bellman K, Panda D, et al. Novel actions of the antitumor drugs vinflunine and vinorelbine on microtubules. Cancer Res. 2000; 60(18) : 5045-51
62． Goode BL, Drubin DG, Barnes G .Functional cooperation between the microtubule and actin cytoskeletons. Curr Opin Cell Biol. 2000; 12(1) : 63-71
63． de Hostos EL . The coronin family of actin-associated proteins. Trends Cell Biol. 1999; 9(9) : 345-50
64． Fukui-Y, Engler-S, Inoue-S, et al. Architectural dynamics and gene replacement of coronin suggest its role in cytokinesis. Cell Motil Cytoskeleton. 1999; 42(3) : 204-17
65． Hynes GM, Willison KR. Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of beta-actin. J Biol Chem. 2000; 275(25) : 18985-94
66． Sander EE, ten Klooster JP, van Delft S, et al. Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J Cell Biol. 1999; 147(5) : 1009-22
67． Akiyama TE, Ward JM, Gonzalez FJ. Regulation of the liver fatty acid-binding protein gene by hepatocyte nuclear factor 1 alpha (HNF1alpha). Alterations in fatty acid homeostasis in HNF1 alpha-deficient mice. J Biol Chem. 2000; 275(35) : 27117-22
68． Storch J, Thumser AE. The fatty acid transport function of fatty acid-binding proteins. Biochim Biophys Acta. 2000 26; 1486(1) : 28-44
69． Yang X, Bhaumik M, Bhattacharyya R, et al. New evidence for an extra-hepatic role of N-acetylglucosaminyltransferase Ⅲ in the progression of diethylnitrosamine-induced liver tumors in mice. Cancer Res. 2000 15; 60(12) : 3313-9
70． Bishayee A, Roy S, Chatterjee M. Characterization of selective induction and alteration of xenobiotic biotransforming enzymes by vanadium during diethylnitrosamine-induced chemical rat liver carcinogenesis. Oncol Res. 1999; 11(1) : 41-53
71． Henderson CJ, Sahraouei A, Wolf CR . Cytochrome P450s and chemoprevention. Biochem Soc Trans. 2000 Feb; 28(2) : 42-6
72． Antony S, Kuttan R, Kuttan G. et al. Imrnunomodulatory activity of curcumin. Immunol Invest. 1999; 28(5-6) : 291-303
73． T Rovira P, Mascarell L, Truffa Bachi-P. The impact of immunosuppressive drugs on the analysis of T cell activation. Curr Med Chem. 2000; 7(7) : 673-92
74． Rusnak F, Mertz P.Calcineurin: form and function. Physiol Rev. 2000; 80(4) : 1483-521
75． Bhattacharjee A, Lappi VR, Rutherford MS, et al. Molecular dissection of dimethylnitrosamine (DMN)-induced hepatotoxicity by mRNA differential display. Toxicol Appl Pharmacol. 1998 ; 150(1) : 186-95
76． Cavaggioni A, Mucignat C, Tirindelli R . Pheromone signalling in the mouse: role
of urinary proteins and vomeronasal organ. Arch Ital Biol. 1999; 137(2-3) : 193-200
77． Novotny MV, Ma W, Wiesler D, et al. Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein. Proc-R-Soc-Lond-B-Biol-Sci. 1999; 266(1432) : 2017-22
78． Bech Obschir D, Kraft R, Huang X et al. COP9 signalosome-specific phosphorlation target P53 to degration by the ubiquitin system. EMBO J. 2001,20(7) :1630-1639