在我們之前的研究中發現綠茶中所含有的唲茶素，尤其是
EGCG，能夠抑制3T3-L1 前脂肪細胞的生長或是誘發其凋亡，然而
這些現象可能導因於EGCG 改變了細胞週期的動態變化。因此，我
們利用pTargeTTM 載體建構出Cdk2 過度表達和顯性抑制(將Cdk2 基
因序列上Asp145 突變為Asn145)的穩定表達的鼠類3T3-L1 前脂肪細胞
株作為研究素材，且更進一步地實驗確認Cdk2 是否在EGCG 所調控
的3T3-L1 前脂肪細胞的生長或凋亡上，扮演一必要性之角色。在實
驗中，我們發現在沒有EGCG 處理的環境下，Cdk2 過度表達的細胞
數較Cdk2 顯性抑制型的細胞數約增加了50%，且降低了細胞週期
G1 期約20%，增加了磷酸化Histone H1 的活性；另外，Cdk2 過度表
達的細胞的生長速度也較Cdk2 顯性抑制型、Lac Z 轉殖型的細胞來
得快。在不同轉殖的細胞中處理20~100μM 的四種綠茶唲茶素後，
只有EGCG 可以降低野生型和Lac Z 轉殖型的細胞數，而對Cdk2 過
度表達型的細胞影響不大；但所有的綠茶唲茶素皆對Cdk2 顯性抑制
型的細胞沒有產生任何作用。同樣地，EGCG 會使野生型的細胞週期
延滯約10%，對於Cdk2 過度表達的細胞週期影響不大，但卻不會改
變Cdk2 顯性抑制的細胞週期動態。Cdk2 顯性抑制型的細胞不論在
EGCG 有無的環境下，皆會有細胞凋亡而產生DNA 梯度、Sub-G1 期
的現象。利用免疫沉澱將有無處理EGCG 之不同轉殖細胞的Cdk2 沉
澱下來，再用西方點墨法觀察細胞週期抑制蛋白p18、p21、p27 與
Cdk2 的結合量，結果並沒有很大的差異。因此，我們斷定EGCG 在
抑制3T3-L1 前脂肪細胞的生長及誘發其凋亡上，是透過一Cdk2 途
徑，且有別於其他的綠茶唲茶素；其作用機制則細胞週期抑制蛋白與
Cdk2 的結合無關。

Previously we have found that green tea catechins, particularly EGCG, inhibit
growth and induce apoptosis of 3T3-L1 preadipocytes and such effects result from its
altering different phases of cell cycle. Using 3T3-L1 preadipose cell lines
transfected with pTargeTTM plasmid containing either wild type of cyclin-dependent
kinase 2 (CDK2) or CDK2-dominant negative (CDK2-dn) gene mutated from Asp145
of CDK2 to Asn145, we further examined whether CDK2 was essential in the EGCG
modulation of preadipocyte growth and apoptosis. As indicated by 50% increases in
cell number, 20% lower in G1 phase of cell cycle, and higher activity in
phosphorylating Histone H1, CDK2-overexpressed cells in the absence of EGCG
treatment grew faster than vector-transfected cells or CDK2-dn cells. At 20-100 µM,
EGCG, but not structurally-related EC, ECG, or EGC, reduced cell number by 30-50%
in wild type of cells and vector-transfected cells, and, to a lesser extent, in
CDK2-overexpressed cells; however, all catechins had no effect on CDK2-dn cells.
Also, EGCG arrested normal cells by 10% and, to a much lesser extent,
CDK2-overexpressed cells in G1 phase, but did not alter the G1 percentage of
CDK2-dn cell cycle. CDK2-dn cells displayed the similar extent of apoptosis, as
indicated by the appearance of DNA ladder and sub-G1 phase of cell cycle, when they
were treated with or without EGCG. CDK2 immunoprecipitates from normal,
vector-transfected, CDK2-transfected, or CDK2-dn cells treated with or without
EGCG contained the corresponding amounts of CDK2 inhibitors such as p18, p21, or
p27. We conclude that EGCG mediate growth and apoptosis of preadipocytes
through CDK2-dependent pathway and such pathway of EGCG action seems different
from other green tea catechins and may not rely on the binding between CDK2 and
CDK2 inhibitors.

第六章參考文獻
Ahmad, N., Adhami, V. M., Gupta, S., Cheng, P., and Mukhtar, H. (2002). Role of the
retinoblastoma (pRb)-E2F/DP pathway in cancer chemopreventive effects of
green tea polyphenol epigallocatechin-3-gallate, Arch Biochem Biophys 398
Ahmad, N., Cheng, P., and Mukhtar, H. (2000). Cell cycle dysregulation by green tea
polyphenol epigallocatechin-3-gallate, Biochem Biophys Res Commun 275
34.
Allison, D. B., and Fontaine, K. R. (1999). Annual deaths attribuatable to obesity in
the Unite States, JAMA 282 1530-1538.
Bradfield, A. E., and Bata-Smith, E. C. (1950). Chromatographic behavior and
chemical structure:the tea catechins, Biochem Biophys Acta 4 441-444.
Bray, G. A., and Tartaglia, L. A. (2000). Medicinal strategies in the treatment of
obesity, Nature 404 72-677.
Chen, L., Lee, M. J., Li, H., and Yang, C. S. (1997). Absorption, distribution,
elimination of tea polyphenols in rats, Drug Metab Dispos 25 1045-50.
Chen, W. J., Chang, C. Y., Lin, J.K. (2003). Induction of G1 phase arrest in MCF
human breast cancer cells by pentagalloyl glucose through the down-regulation of
CDK4 and CDK2 activities and up-regulation of the CDK inhibitors p27(Kip) and
p21(Cip), Biochem Pharmacol. 65 1777-85.
Cheng, M., Olivier, P., Diehl, J. Fero, M., Roussel, M., JM, R., and Sherr, C. (1999).
The p21(Cip1) and p27(Kip1) CDK ''inhibitors'' are essential activators of cyclin
D-dependent kinases in murine fibroblasts, EMBO J. 18 1571-83.
Chung, L. Y., Cheung, T. C., Kong, S. K., Fung, K. P., Choy, Y. M., Chan, Z. Y., and
Kwok, T. T. (2001). Induction of apoptosis by green tea catechins in human
prostate cancer DU145 cells, Life Sci 681 207-14.
Deng, J., Hua, K., and Lesser, S. S. (2000). Activation of signal transducer and
activator of transcription-3 during proliferative of 3T3-L1 adipogenesis,
Endocrinology 142 370-2376.
Dobashi, Y., Kudoh, T., Matsumine, A., Toyoshimas, K., and Akiyama, T. (1995).
Constitutive overexpession of cdk2 inhibits differentiation of rat
pheochromocytoma PC12 cells, J. Biol. Chem. 270 23031-23037.Dulloo, A., G,
and Duret, C. (1999). Efficacy of a green tea extract rich in catechin polyphenols
and caffeine in increasing 24-h expenditure and fat oxidation in humans, Am J
Clin Nutr 70 1040-1045.
Dvorakova, K., Dorr, R. T., Valcic, S., Timmermann, B., and Alberts, D. S. (1999).
Pharmacokinetics of the green tea derivative, EGCG, by the topical route of
administration in mouse and human skin, Cancer Chemother Pharmaco l43 331-5.
Graham, H. N. (1992). Green tea composition,consumption,and polyphenol chemistry,
Prev Med 21 334-350.
Gupta, S., Ahmad, N., Nieminen, A. L., and Mukhtar, H. (2000). Growth inhibition,
cell-cycle dysregulation, and induction of apoptosis by green tea constituent
(-)-epigallocatechin-3-gallate in androgen-sensitive and androgen-insensitive
human prostate carcinoma cells, Toxicol Appl Pharmacol 64 2-90.
Harmon, A. W., and Harp, J. B. (2001). Differential effects of flavonoids on 3T3-L1
adipogenesis and lipolysis, Am J Physiol Cell Physiol 280 C807-13.
Harrls, T. E., Albrecht, J. H., Nakanishi, M., and Darlington, G. J. (2001).
CCAAT/Enhancer-binding Protein- Cooperates with p21 to Inhibit
Cyclin-dependent Kinase-2 Activity and Induces Growth Arrest Independent of
DNA Binding, J. Biol. Chem. 276 29200-29209.
Hayakawa, S., Saeki, K., Sazuka, M., Suzuki, Y., Shoji, Y., Ohta, T., Kaji, K., Yuo, A.,
and Isemura, M. (2001). Apoptosis induction by epigallocatechin gallate involves
its binding to Fas, Biochem Biophys Res Commun 285 1102-6.
Heuvel, S. V. D., and Harlow, E. (1993). Distinct roles for cyclin-dependent kinases in
cell cycle control, A.A.C . 262 2050-2054.
Hu, B., Jayashree, M., Heuvel, S. V. D., and Enders, G. H. (2001). S and G2 phase
roles for cdk2 revealed by inducible expression of a dominant negative mutant in
human cells, Mol. Cell. Biol. 21 2755-2766.
Islam, S., Islam, N., Kermode, T., Johnstone, B., Mukhtar, H., Moskowitz, R. W.,
Goldberg, V. M., Malemud, C. J., and Haqqi, T. M. (2000). Involvement of
caspase-3 in epigallocatechin-3-gallate-mediated apoptosis of human
chondrosarcoma cells, Biochem Biophys Res Commun 270 793-7.
Jung, Y. D., Kim, M. S., Shin, B. A., Chay, K. O., Ahn, B. W., Liu, W., Bucana, C. D.,
Gallick, G. E., and Ellis, L. M. (2001). EGCG, a major component of green tea,
inhibits tumour growth by inhibiting VEGF induction in human colon carcinoma
cells, Br J Cancer 84 844-50.
Kao, Y. H., Hiipakka, R. A., and Liao, S. (2000). Modulation of endocrine systems
and food intake by green tea epigallocatechin gallate, Endocrinology 141 980-7.
Katdare, M., Osborne, M., and Telang, N. (2002). Soy isoflavone genistein modulates
cell cycle progression and induces apoptosis in HER-2/neu oncogene expressing
human breast epithelial cells, Int J Oncol. 21 809-15.
Kavanagh, K. T., Hafer, L. J., Kim, D. W., Mann, K. K., Sherr, D. H., Rogers, A. E.,
and Sonenshein, G. E. (2001). Green tea extracts decrease carcinogen-induced
mammary tumor burden in rats and rate of breast cancer cell proliferation in
culture, J Cell Biochem 82 387-98.
Kelly, G., and Husband, A. (2003). Flavonoid compounds in the prevention and
treatment of prostate cancer, Methods Mol Med. 81 377-94.
Kopelman, P. G. (2000). Obesity as a medical problem, Nature 404 635-643.
Lea Currie, Y. R., Monroe, D., and McIntosh, M. K. (1999). Dehydroepiandrosterone
and related steroids alter 3T3-L1 preadipocyte proliferation and differentiation,
Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 123 17-25.
Liang, Y. C., Lin Shiau, S. Y., Chen, C. F., and Lin, J. K. (1999). Inhibition of
cyclin-dependent kinases 2 and 4 activities as well as induction of Cdk inhibitors
p21 and p27 during growth arrest of human breast carcinoma cells by
(-)-epigallocatechin-3-gallate, J Cell Biochem 75 1-12.
Liang, Y. C., Lin, S. S. Y., Chen, C. F., and Lin, J. K. (1999). Inhibition of
cyclin-dependent kinases 2 and 4 activities as well as induction of cdk inhibitors
p21 and p27 during growth arrerst of human breast carcinoma cells by
(-)-epigallocatechin-3-gallate, J.Cell.Biochem. 75 1-12.
Liberto, M., and Cobrinik, D. (2000). Growth factor-dependent induction of p21(CIP1)
by the green tea polyphenol, epigallocatechin gallate, Cancer Lett 154 151-61.
Lopez-Lazaro, M., and Akiyama, M. (2002). Flavonoids as anticancer agents:
structure-activity relationship study, Curr Med Chem Anti-Canc Agents. 2
691-714.
Manthey, J., and Guthrie, N. (2002). Anti proliferative activities of citrus flavonoids
against six human cancer cell lines, J Agric Food Chem. 50 5837-43.
Morgan, D. O. (1995). Principles of cdk regulation, Nature 374 131-134.
Morrison, R. F., and Farmer, S. R. (1999). Role of PPARgamma in regulating a
cascade expression of cyclin-dependent kinase inhibitors, p18(INK4c) and
p21(Waf1/Cip1), during adipogenesis, J Biol Chem 274 17088-97.
Must, A., Field, A. E., and Dietz, W. H. (1999). the disease burden association with
overweight and obesity, JAMA 282 1523-1529.
Nakagawa, K., and Miyazawa, T. (1997). Chemiluminescence-high-performance
liquid chromatographic determination of tea catechin, (-)-epigallocatechin
3-gallate, at picomole levels in rat and human plasma, Anal Biochem 248 41-9.
Nihal, A., Cheng, P., and Hasan, M. (2000). Cell cycle dysregulation by green tea
polyphenol epigallocathchin-3-gallate, Biochem. Biophys. Res. Commun. 275
328-334.
Opare Kennedy, D., Kojima, A., Hasuma, T., Yano, Y., Otani, S., and Matsui Yuasa, I.
(2001). Growth inhibitory effect of green tea extract and (-)-epigallocatechin in
Ehrlich ascites tumor cells involves a cellular thiol-dependent activation of
mitogenic-activated protein kinases, Chem Biol Interact 134 113-33.
Paschka, A. G., Butler, R., and Young, C. Y. (1998). Induction of apoptosis in prostate
cancer cell lines by the green tea component, (-)-epigallocatechin-3-gallate,
Cancer Lett 130 1-7.
Phelps, D. E., and Xiong, Y. (1998). Regulation of cyclin-dependent kinase 4 during
adipogenesis involves switching of cyclin D subunits and concurrent binding of
p18INK4c and p27Kip1, Cell Growth Differ 9 595-610.
Rao, S., Lowe, M., Herliczek, T., and Keyomarsi, K. (1998). Lovastatin mediated G1
arrest in normal and tumor breast cells is through inhibition of CDK2 activity and
redistribution of p21 and p27, independent of p53, Oncogene. 17 2393-402.
Reichert, M., and Eick, D. (1999). Analysis of cell cycle arrest in adipocyte
differentiation, Oncogene 18 459-66.
Richon, V. M., Lyle, R. E., and McGehee, R. E., Jr. (1997). Regulation and expression
of retinoblastoma proteins p107 and p130 during 3T3-L1 adipocyte differentiation,
J Biol Chem 272 10117-24.
Saeki, K., Hayakawa, S., Isemura, M., and Miyase, T. (2000). Importance of a
pyrogallol-type structure in catechin compounds for apoptosis-inducing activity,
Phytochemistry 53 391-4.
Sanjay, G., Tajamul, H., and Hasan, M. (2003). Molecular pathway for
(-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human
prostate carcinoma cells, A.B.B. 410 177-185.
Shime, H., Kariya, M., Orii, A., Momma, C., Kanamori, T., Fukuhara, K., Kusakari,
T., Tsuruta, Y., Takakura, K., Nikaido, T., and Fujii, S. (2002). Tranilast inhibits
the proliferation of uterine leiomyoma cells in vitro through G1 arrest associated
with the induction of p21(waf1) and p53, J Clin Endocrinol Metab. 87 5610-7.
Tai, T. Y., Chuang, L. M., and Wu, H. P. (1992). Association of body bulid with
non-insulin-dependent diabets mellitus and hypertension among Chinese adults,
IntJEpidemiol 21 511-517.
Tan, X., Hu, D., Li, S., Han, Y., Zhang, Y., and Zhou, D. (2000). Differences of four
catechins in cell cycle arrest and induction of apoptosis in LoVo cells, Cancer Lett
158 1-6.
Watanabe, J., and Niki, R. (1998). isolation and identification of acetyl-coA
carboxylase inhibitors from green tea, Biosci Biophys Biochem 62 532-534.
William, M., and Schlegel, R. (1996). Suppression of apoptosis by dominant negative
mutants of cyclin-dependent protein kinases, J. Biol. Chem. 271 10205-10209.
Yamamoto, T., and Juneja, L. R. (1997). Chemistry and application of green tea, CRC
Press,Boca Raton,Florida.
Yang, C. S., Chen, L., Lee, M. J., Balentine, D., Kuo, M. C., and Schantz, S. P.
(1998a). Blood and urine levels of tea catechins after ingestion of different
amounts of green tea by human volunteers, Cancer Epidemiol Biomarkers Prev 7
351-4.
Yang, G. Y., Liao, J., Kim, K., Yurkow, E. J., and Yang, C. S. (1998b). Inhibition of
growth and induction of apoptosis in human cancer cell lines by tea polyphenols,
Carcinogenesis 19 611-6.
Yu, Z., Zhang, L., and Wu, D. (2003). Genistein induced apoptosis in MCF-7 and
T47D cells, Wei Sheng Yan Jiu 32 125-7.