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系統識別號 U0026-0812200913431519
論文名稱(中文) 穿心蓮內酯於體內及體外抗癌活性之探討
論文名稱(英文) In vivo and in vitro anti-cancer activities of andrographolide derived from Andrographis paniculata
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 95
學期 2
出版年 96
研究生(中文) 蔡含卿
研究生(英文) Han-Ching Tsai
學號 s2694105
學位類別 碩士
語文別 英文
論文頁數 52頁
口試委員 指導教授-李益謙
口試委員-鄭瑞棠
口試委員-洪正路
中文關鍵字 穿心蓮內酯  轉移  癌症  細胞週期 
英文關鍵字 andrographolide  cancer  metastasis  cell cycle 
學科別分類
中文摘要 穿心蓮(Andrographis paniculata)為爵床科植物,以往被用為抗菌、消炎解熱及免疫促進之傳統治療藥物。其中被認為最主要的活性成分為雙萜類的穿心蓮內酯。近年來陸續有研究發現穿心蓮內酯亦表現出抑癌效果。因此,我們欲進一步證實並探討其抗癌活性。首先,利用多種人類及小鼠癌細胞株進行毒殺試驗、DAPI細胞核染色法及DNA片段分析,發現穿心蓮內酯的確對於癌細胞具有抑制增生並導致細胞凋亡的作用。接下來以流式細胞儀(FACS)分析細胞週期的變化,可觀察到癌細胞在處理藥物後,其細胞週期停滯在G0/G1期。為釐清其中所牽涉的細胞週期調控蛋白,我們藉由西方墨點法(Western blotting)觀察到穿心蓮內酯可提升癌細胞中細胞週期抑制因子(cyclin dependent kinase inhibitor, CDKI),p21的表現量並降低細胞週期素依賴性激酶(cyclin dependent kinase, CDK),CDK4的含量。此外,於小鼠原位癌模式中發現穿心蓮內酯在活體內亦有抑制腫瘤細胞生長的能力;另外發現在給予藥物後,腫瘤中血管新生情形相對於控制組有減少的現象。於是進一步利用大鼠主動脈環實驗觀察到穿心蓮內酯具有抑制主動脈微脈管形成的作用。而後,由酶譜(gelatin zymography) 以及細胞移行分析(cell migration assay)亦發現穿心蓮內酯可降低癌細胞移行能力並抑制與腫瘤轉移極具相關性的物質,基質金屬蛋白酶(Matrix Metalloproteinase, MMP)-2及-9,的分泌。最後,藉由小鼠黑色素瘤肺轉移模式證實穿心蓮內酯在動物體內具有抑制癌症轉移的效果。以上結果顯示穿心蓮內酯在活體內及活體外皆具有抗癌活性。然而,在抗癌治療策略走向多元化的現今,穿心蓮內酯在詳細抗癌機制以及在臨床所發揮的抗癌功效上,還有很大的空間值得去深究探討。
英文摘要 Andrographolide (AG) is a bicyclic diterpenoid lactone isolated from the leaves of Andrographis paniculata, an herbal medicine used traditionally as an anti-inflammatory drug for the treatments of laryngitis, diarrhea and rheumatoid arthritis. The present investigation aims at making a systemic study, both in vitro and in vivo, of the anti-cancer cell proliferation, cell cycle arrest, anti-angiogenesis, anti-metastasis and anti-tumor activities of AG. In vitro, AG shows anti-proliferative activity toward a variety of human cancer cell lines. FACS and Western blot analyses indicate that AG induces cells to undergo cell cycle arrest at the G0/G1 phase by inducing the expression of p21, a cyclin dependent kinase inhibitor (CDKI), and a concomitant decrease of cyclin dependent kinase 4 (CDK4). AG also exhibits anti-angiogenesis activity in both the immunostaining of CD31 and rat aortic ring assay. In a gelatin zymography assay for matrix metalloproteinase (MMP), AG inhibits the secretion of MMP-2 and -9 from A549 cells, suggesting AG is involved in suppressing metastasis. The notion is strengthened by the finding that AG inhibits the migrations of A549 and B16-F10 cells by either the Boyden chamber assay or the wound healing assay. The anti-metastasis of AG is finally supported in vivo by the reduction of number of metastasized lung nodules after a tail vein injection of B16-F10 melanoma cells into C57BL/6J mice. AG also significantly suppresses the primary implanted Lewis lung carcinoma tumor in the same mouse strain. Our data suggest that AG is an interesting pharmacophore with anti-cancer activities in vitro and in vivo. Its molecular mechanism of action deserves further elucidation in the future studies.
論文目次 口試合格證明
Abstract.........................................I
摘要.............................................II
Contents.........................................III
Index............................................VI
Abbreviation.....................................VIII
Introduction
I Overview of cancer..........................1
II Cell apoptosis..............................2
III Cell cycle..................................4
IV Angiogenesis and cancer.....................6
V Metastasis and cancer.......................7
VII Review of andrographolide...................8
VI The aims of this study......................10
Materials and Methods
I Materials
1. Reagents.................................11
2. Solutions................................13
II Methods
1. Cell lines...............................17
2. Cell culture.............................17
3. Cell viability assay.....................17
4. Detection of apoptosis...................18
5. DNA fragmentation assay..................18
6. Cell cycle analysis......................18
7. Rat aortic ring assay....................18
8. Gelatin zymography.......................19
9. In vitro wound healing assay.............19
10. Boyden chamber cell-migration assay......19
11. Western analysis.........................19
12. Immunohistochemistry.....................20
13. In vivo anti-tumor model.................21
14. In vivo metastatic model.................21
Results
I. Growth inhibitory and apoptosis-inducing
effects of andrographolide on multiple
cancer cell lines..........................22
II. Andrographolide induced G0/G1 cell cycle
arrests in A549 cells and altered cell
cycle regulatory proteins..................22
III. Andrographolide suppressed the tumor growth
and angiogenesis in LLC-bearing mice.......23
IV. Andrographolide inhibits microvessel
formation in the ex vivo rat aortic
ring angiogenesis assay....................24
V. Andrographlide inhibits migration and
the secreted matrix metalloproteinase
(MMP) activity in A549 cells...............25
VI. Andrographolide inhibits B16-F10 melanoma
metastasis in C57BL/6 mice.................26
Discussion.......................................27
References.......................................31
Curriculum Vitae.................................52
Index
Tab. 1 The IC50 values of andrographolide
effects on various cancer cell lines....39

Fig. 1 Cell cycle control in mammalian cells...40
Fig. 2 Schematic representation of the
cell cycle..............................41
Fig. 3 Andrographolide inhibits proliferation
of multiple tumor cell types............42
Fig. 4 Andrographolide induces apoptosis.......43
Fig. 5 Andrographolide arrests at G0/G1 phase
of cell cycle progress inA549 cells.....44
Fig. 6 Cell cycle regulatory proteins
expression is altered byandrographolide.45
Fig. 7 Andrographolide inhibits tumor growth
in mice inoculated with LLC cells. .....46
Fig. 8 Andrographolide decreased expression of
CD31 in the LLCtumor from C57BL/6 mice..47
Fig. 9 Andrographolide exhibits dose-dependent
anti-angiogenic activity on rat
aortic rings............................48
Fig. 10 Andrographlide inhibits the secreted
matrix metalloproteinase (MMP) activity
and migration in A549 cells............49
Fig. 11 Andrographolide inhibited of B16-F10
melanoma metastasis in vivo and
in vitro...............................50
Fig. 12 Adrographolide effects of on the
pathology and the expression of lung
metastatic nodules.....................51
參考文獻 1.Andres, V. Control of vascular cell proliferation
and migration by cyclin-dependent kinase signalling:
new perspectives and therapeutic potential. Cardiovasc.
Res. 63, 11-21(2004).
2.Armstrong, J.S. Mitochondria: a target for cancer
therapy. Br. J. Pharmacol. 147, 239-248 (2006).
3.Ausprunk, D.H., Knighton, D.R., Folkman, J.
Differentiation of vascular endothelium in the chick
chorioallantois: a structural and autoradiographic
study. Dev. Biol. 38, 237-248 (1974).
4.Ausprunk, D.H., Folkman, J. Migration and proliferation
of endothelial cells in preformed and newly formed
blood vessels during tumor angiogenesis. Microvasc.
Res. 14, 53-65 (1977).
5.Bergsland, E.K. Vascular endothelial growth factor
as a therapeutic target in cancer. Am. J. Health
Syst. Pharm. 61, S4–S11 (2004).
6.Blagosklonny, M.V. Antiangiogenic therapy and tumor
progression. Cancer Cell 5, 13-17 (2004)
7.Bookstein, R., MacGrogan, D., Hilsenbeck, S.G.,
Sharkey, F., Allred, D.C. p53 is mutated in a subset
of advanced-stage prostate cancers. Cancer Res. 53,
3369-3373 (1993).
8.Burgos, R.A., Seguel, K., Perez, M., Meneses, A.,
Ortega, M., Guarda, M.I., Loaiza, A., Hancke, J.L.
Andrographolide inhibits IFN-gamma and IL-2 cytokine
production and protects against cell apoptosis.
Planta Med. 71, 429-434 (2005).
9.Bussolino, F., Mantovani, A., Persico, G. Molecular
mechanisms of blood vessel formation. Trends. Biochem.
Sci. 22, 251-256 (1997).
10.Carmeliet, P., Jain, R.K. Angiogenesis in cancer and
other diseases. Nature 407, 249-257 (2000).
11.Cavallaro, U., Christofori, G.. Cell adhesion and
signalling by cadherins and Ig-CAMs in cancer.
Nat. Rev. Cancer 4, 118-132 (2004).
12.Chambers, A.F., Groom, A.C., MacDonald, I.C.
Dissemination and growth of cancer cells in metastatic
sites. Nat. Rev. Cancer 2, 563-572 (2002).
13.Chen, W., Fu, J., Liu, Q., Ruan, C., Xiao, S.
Retroviral endostatin gene transfer inhibits human
colon cancer cell growth in vivo. Chin. Med. J. (Engl).
116, 1582-1584 (2003).
14.Chen, J.H., Hsiao, G., Lee, A.R., Wu, C.C., Yen, M.H.
Andrographolide suppresses endothelial cell apoptosis
via activation of phosphatidyl inositol-3-kinase/Akt
pathway. Biochem. Pharmacol. 67, 1337-1345 (2004).
15.Cheung, H.Y., Cheung, S.H., Li, J., Cheung, C.S.,
Lai, W.P., Fong, W.F., Leung, F.M. Andrographolide
isolated from Andrographis paniculata induces cell
cycle arrest and mitochondrial-mediated apoptosis in
human leukemic HL-60 cells. Planta Med. 71, 1106-1111
(2005).
16.Curran, S., Murray, G.I. Matrix metalloproteinases:
molecular aspects of their roles in tumour invasion
and metastasis. Eur. J. Cancer 36, 1621-1630 (2000).
17.Datto, M.B., Li, Y., Panus, J.F., Howe, D.J., Xiong,
Y., Wang, X.F. Transforming growth factor beta induces
the cyclin-dependent kinase inhibitor p21 through a
p53-independent mechanism. Proc. Natl. Acad. Sci.U.S.A.
92, 5545-5549 (1995).
18.Dupont, J., Le Roith, D. Insulin-like growth factor 1
and oestradiol promote cell proliferation of MCF-7
breast cancer cells: new insights into their
synergistic effects. Mol. Pathol. 54, 149-154 (2001).
19.Englert, C., Maheswaran, S., Garvin, A.J., Kreidberg,
J., Haber, D.A. Induction of p21 by the Wilms’ tumor
suppressor gene WT1. Cancer Res. 57, 1429-1434 (1997).
20.Ferrara, N. Role of vascular endothelial growth factor
in the regulation of angiogenesis. Kidney Int. 56, 794-
814 (1999).
21.Fidler, I.J. The pathogenesis of cancer metastasis:
the 'seed and soil' hypothesis revisited. Nat. Rev.
Cancer 3, 453-458 (2003).
22.Fidler, I.J., Ellis, L.M. The implications of
angiogenesis for the biology and therapy of cancer
metastasis. Cell 79, 185-188 (1994).
23.Folkman, J., Merler, E., Abernathy, C., Williams, G.
Isolation of a tumor factor responsible on
angiogenesis. J. Exp. Med. 133, 275-288 (1971).
24.Folkman, J. What is the evidence that tumors are
angiogenesis dependent? J. Natl.Cancer Inst. 82, 4-6
(1990).
25.Folkman, J. Seminars in medicine of the Beth Israel
Hospital, Boston. Clinical applications of research on
angiogenesis. N. Engl. J. Med. 333, 1757-1763 (1995).
26.Fong, T.A., Shawver, L.K., Sun, L., Tang, C., App, H.,
Powell, T.J., Kim, Y.H., Schreck, R., Wang, X., Risau,
W., Ullrich, A., Hirth, K.P., McMahon, G. SU5416 is a
potent and selective inhibitor of the vascular
endothelial growth factor receptor (Flk-1/KDR) that
inhibits tyrosine kinase catalysis, tumor
vascularization, and growth of multiple tumor types.
Cancer Res. 59, 99-106 (1999).
27.Friedl, P., Wolf, K. Tumour-cell invasion and
migration: diversity and escape mechanisms. Nat. Rev.
Cancer 3, 362-374 (2003).
28.Gale, N.W., Yancopoulos, G.D. Growth factors acting via
endothelial cell-specific receptor tyrosine kinases:
VEGFs, angiopoietins and ephrins in vascular
development. Genes Dev. 13, 1055-1066 (1999).
29.Gartel, A.L., Radhakrishnan, S.K. Lost in
transcription: p21 repression, mechanisms, and
consequences. Cancer Res. 15, 3980-3985 (2005).
30.Green, D.R., Reed, J.C. Mitochondria and apoptosis.
Science 281, 1309-1312 (1998).
31.Itoh, T., Tanioka, M., Yoshida, H., Yoshioka, T.,
Nishimoto, H., Itohara, S. Reduced angiogenesis and
tumor progression in gelatinase A-deficient mice.
Cancer Res. 58, 1048-1051 (1998).
32.Itoh, T., Tanioka, M., Matsuda, H., Nishimoto, H.,
Yoshioka, T., Suzuki, R., Uehira, M. Experimental
metastasis is suppressed in MMP-9-deficient mice. Clin.
Exp. Metastasis 17, 177-181 (1999).
33.Kapil, A., Koul, I.B., Banerjee, S.K., Gupta, B.D.
Antihepatotoxic effects of major diterpenoid
constituents of Andrographis paniculata. Biochem.
Pharmacol. 46, 182-185 (1993).
34.Kim, L.S., Huang, S., Lu, W., Lev, D.C., Price, J.E.
Vascular endothelial growth factor expression promotes
the growth of breast cancer brain metastases in nude
mice. Clin. Exp. Metastasis 21, 107-118 (2004).
35.Kim, Y.S., Milner, J.A. Targets for indole-3-carbinol
in cancer prevention. J. Nutr. Biochem. 16, 65-73
(2005).
36.Kluck, R.M., Bossy-Wetzel, E., Green, D.R., Newmeyer,
D.D. The release of cytochrome c from mitochondria: a
primary site for Bcl-2 regulation of apoptosis. Science
275, 1132-1136 (1997).
37.Korsmeyer, S.J., Wei, M.C., Saito, M., Weiler, S., Oh,
K.J., Schlesinger, P.H. Pro-apoptotic cascade activates
BID, which oligomerizes BAK or BAX into pores that
result in the release of cytochrome c. Cell Death
Differ. 7, 1166-1173 (2000).
38.Kumar, R.A., Sridevi, K., Kumar, N.V., Nanduri, S.,
Rajagopal, S. Anticancer and immunostimulatory
compounds from Andrographis paniculata. J.
Ethnopharmacol. 92, 291-295 (2004).
39.Li, C.Y., Shan, S., Cao, Y., Dewhirst, M.W. Role of
incipient angiogenesis in cancer metastasis. Cancer
Metastasis Rev. 19, 7-11 (2000).
40.Li, J., Cheung, H.Y., Zhang, Z., Chan, G.K., Fong, W.F.
Andrographolide induces cell cycle arrest at G2/M phase
and cell death in HepG2 cells via alteration of
reactive oxygen species. Eur. J. Pharmacol. (2007).
41.Liu, S., Bishop, W.R., Liu, M. Differential effects of
cell cycle regulatory protein p21WAF/Cip1 on apoptosis
and sensitivity to cancer chemotherapy. Drug Resist
Update 6, 183-195 (2003).
42.Locksley, R.M., Killeen, N., Lenardo, M.J. The TNF and
TNF receptor superfamilies: integrating mammalian
biology. Cell 104, 487-501 (2001).
43.Mehlen, P., Puisieux, A. Metastasis: a question of life
or death. Nat. Rev. Cancer 6, 449-458 (2006).
44.Nakagawa, T., Yuan, J. Cross-talk between two cysteine
protease families. Activation of caspase-12 by calpain
in apoptosis. J. Cell Biol. 150, 887-894 (2000).
45.Ortega, S., Malumbres, M., Barbacid, M. Cyclin D-
dependent kinases, INK4 inhibitors and cancer. Biochim.
Biophys. Acta. 1602, 73-87 (2002).
46.Pardal, R., Clarke, M.F., Morrison, S.J. Applying the
principles of stem-cell biology to cancer. Nat. Rev.
Cancer 3, 895-902 (2003).
47.Rajagopal, S., Kumar, R.A., Deevi, D.S., Satyanarayana,
C., Rajagopalan, R. Andrographolide, a potential cancer
therapeutic agent isolated from Andrographis
paniculata. J. Exp. Ther. Oncol. 3, 147-158 (2003).
48.Rao, R.V., Hermel, E., Castro-Obregon, S., del Rio, G.,
Ellerby, L.M., Ellerby, H.M., Bredesen, D.E. Coupling
endoplasmic reticulum stress to the cell death program.
Mechanism of caspase activation. J. Biol. Chem. 276,
33869-33874 (2001).
49.Sandler, A.B., Johnson, D.H., Herbst, R.S. Anti-
vascular endothelial growth factor monoclonals in non-
small cell lung cancer. Clin. Cancer Res. 10, 4258s-
4262s (2004).
50.Satyanarayana, C., Deevi, D.S., Rajagopalan, R.,
Srinivas, N., Rajagopal, S. DRF 3188 a novel semi-
synthetic analog of andrographolide: cellular response
to MCF 7 breast cancer cells. BMC Cancer. 4 (2004).
51.Scorrano, L., Korsmeyer, S.J. Mechanisms of cytochrome
c release by proapoptotic BCL-2 family members.
Biochem. Biophys. Res. Commun. 304, 437-44 (2003).
52.Scorrano, L., Oakes, S.A., Opferman, J.T., Cheng, E.H.,
Sorcinelli, M.D., Pozzan, T., Korsmeyer, S.J. BAX and
BAK regulation of endoplasmic reticulum Ca2+: a control
point for apoptosis. Science 300, 135-139 (2003).
53.Sherr, C.J. D-type cyclins. Trends. Biochem. Sci. 20,
187-190 (1995).
54.Sherr, C.J., Roberts, J.M. CDK inhibitors: positive and
negative regulators of G1-phase progression. Genes Dev.
13, 1501-1512 (1999).
55.Singha, P.K., Roy, S., Dey, S. Protective activity of
andrographolide and arabinogalactan proteins from
Andrographis paniculata Nees. against ethanol-induced
toxicity in mice. J. Ethnopharmacol. 111, 13-21 (2007).
56.Somasundaram, K., Zhang, H., Zeng, Y.X., Houvras, Y.,
Peng, Y., Zhang, H., Wu, G.S., Licht, J.D., Weber,
B.L., El-Deiry, W.S. Arrest of the cell cycle by the
tumour-suppressor BRCA1 requires the CDK-inhibitor
p21/Waf1/Cip1. Nature 389, 187-190 (1997).
57.Steeg, P.S., Zhou, Q. Cyclins and breast cancer. Breast
Cancer Res. Treat. 52, 17-28 (1998).
58.Tanaka, S., Sugimachi, K., Yamashita, Y., Shirabe, K.,
Shimada, M., Wands, J.R., Sugimachi, K. Angiogenic
switch as a molecular target of malignant tumors. J
Gastroenterol. 38 Suppl 15, 93-97 (2003).
59.Thisoda, P., Rangkadilok, N., Pholphana, N.,
Worasuttayangkurn, L., Ruchirawat, S., Satayavivad, J.
Inhibitory effect of Andrographis paniculata extract
and its active diterpenoids on platelet aggregation.
Eur. J. Pharmacol. 553, 39-45 (2006).
60.Tsai, H.R., Yang, L.M., Tsai, W.J., Chiou, W.F.
Andrographolide acts through inhibition of ERK1/2 and
Akt phosphorylation to suppress chemotactic migration.
Eur. J. Pharmacol. 498, 45-52 (2004).
61.Visen, P.K., Shukla, B., Patnaik, G.K., Dhawan, B.N.
Andrographolide protects rat hepatocytes against
paracetamol-induced damage. J. Ethnopharmacol. 40, 131-
136 (1993).
62.Wang, H., Iakova, P., Wilde, M., Welm, A., Goode, T.,
Roesler, W.J., Timchenko, N.A. C/EBPalpha arrests cell
proliferation through direct inhibition of Cdk2 and
Cdk4. Mol. Cell. 8, 817-828 (2001).
63.Wang, H., Goode, T., Iakova, P., Albrecht, J.H.,
Timchenko, N.A. C/EBPalpha triggers proteasome-
dependent degradation of cdk4 during growth arrest.
Embo J. 21, 930-941 (2002).
64.Wolfel, T., Hauer, M., Schneider, J., Serrano, M.,
Wolfel, C., Klehmann-Hieb, E., De Plaen, E., Hankeln,
T., Meyerzum Buschenfelde, K.H., Beach, D. A p16INK4a-
insensitive CDK4 mutant targeted by cytolytic T
lymphocytes in a human melanoma. Science 269, 1281-
1284 (1995).
65.Yanase, T., Tamura, M., Fujita, K., Kodama, S., Tanaka,
K. Inhibitory effect of angiogenesis inhibitor TNP-470
on tumor growth and metastasis of human cell lines in
vitro and in vivo. Cancer Res. 53, 2566-2570 (1993).
66.Yancopoulos, G.D., Klagsbrun, M., Folkman, J.
Vasculogenesis, angiogenesis and growth factors:
ephrins enter the fray at the border. Cell 93, 661-664
(1998).
67.Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M.,
Cai, J., Peng, T.I., Jones DP, Wang X. Prevention of
apoptosis by Bcl-2: release of cytochrome c from
mitochondria blocked. Science 275, 1129-1132 (1997).
68.Yuan, J.Y., Horvitz, H.R. The Caenorhabditis elegans
genes ced-3 and ced-4 act cell autonomously to cause
programmed cell death. Dev. Biol. 138, 33-41 (1990).
69.Zhou, J., Zhang, S., Ong, C.N., Shen, H.M. Critical
role of pro-apoptotic Bcl-2 family members in
andrographolide-induced apoptosis in human cancer
cells. Biochem. Pharmacol. 7, 136-144 (2006).
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