臺灣博碩士論文加值系統

在腫瘤生長及轉移的過程中需要新的血管由周邊發展出來，供給腫瘤細胞所需的氧氣、養份及生長因子。在腫瘤生長的過程中，由於生長速度過快造成內部血管來不及長好，而形成一種缺氧的環境。細胞在缺氧環境下會開啟缺氧誘發因子 (hypoxia-inducible factor 1, HIF-1) 的調控路徑，促進腫瘤生長及血管新生。HIF-1是一種由 HIF-1α及HIF-1β組成的異形二聚物 (heterodimer) 。當HIF-1α 與下游基因調控子中的缺氧反應元件 (hypoxia-response element, HRE) 結合後，啟動下游的缺氧反應基因轉錄，例如: 血管內皮細胞生長因子 (vascular endothelial growth factor, VEGF)。血管內皮細胞生長因子為一個血管新生因子，幫助血管新生有助於腫瘤細胞的生長，因此透過減少血管內皮細胞生長因子的分泌，能有效抑制血管新生及抗腫瘤生長。半枝蓮 (Scutellaria barbata, S. barbata) 為一中草藥，被用於抗發炎及抗腫瘤的治療上。在過去的研究中指出半枝蓮可以抑制腫瘤生長及誘發癌細胞發生細胞凋亡 (apoptosis)。在我們的研究中，試圖建立一個由帶有缺氧反應元件 (Hypoxia response element) 調控子調控的冷光酶 (luciferase) 基因 (6xHRE-Luc) 當作篩選具抑制血管新生功能的中草藥平台。我們利用人類肺腺癌細胞CL1-5在多種中草藥中做篩選，發現半枝蓮萃取液可以透過減少HIF-1 α蛋白量，抑制由缺氧誘發升高的HIF-1轉錄活性。我們進一步證實給予半枝蓮的人類肺腺癌細胞其分泌血管內皮細胞生長因子的能力被抑制。由於半枝蓮減少癌細胞分泌的血管內皮細胞生長因子，因此我們利用博登細胞爬行分析 (Boyden chamber) 證明，半枝蓮可抑制人類臍帶微血管的內皮細胞 (human microvascular endothelial cell line-1, HMEC-1) 的爬行及增生。同時我們也證實半枝蓮可透過抑制Akt磷酸化路徑，而減少HIF-1α蛋白表現。此研究中，我們證實半枝蓮具有抑制肺腺癌的效果，進一步提供中草藥未來在臨床運用的機會。

Angiogenesis is the formation of new blood vessels from preexisting ones and is required for supplying oxygen, nutrient, and growth factors to tumor growth and metastasis. In the processes of tumor growth, a rapidly growing tumor mass quickly outstrips its vasculature and lacks oxygen and nutrients, thus making a hypoxia environment. One way that cells respond to hypoxia is through hypoxia-inducible factor 1 (HIF-1), leading to promoting tumor growth and angiogenesis. HIF-1 is a heterodimer that consists of the hypoxic response factor HIF-1α and HIF-1β. HIF-1α binds to hypoxia-response elements (HREs), thereby activating the expression of numerous hypoxia-response genes, such as vascular endothelial growth factor (VEGF). VEGF is an angiogenic factor, which regulates neoangiogenesis to promote tumor growth. Therefore, anti-angiogenesis is a promising approach for cancer therapy. Scutellaria barbata is a traditional Chinese medicine and has been used as anti-inflammatory and antitumor agents. Previous studies have shown that S. barbata inhibits tumor growth and induces cancer cell apoptosis. In this study, we constructed a reporter vector in which a luciferase reporter gene was driven by the CMV minimal promoter conjugated with HREs as a selective platform to screen herbal medicines as anticancer drugs. In several herbal medicines, we found that the extract of S. barbata could decrease the transcriptional activity of HIF-1 under hypoxic condition in CL1-5 lung adenocarcinoma cells through the reduction of HIF-1α protein level. The production of VEGF in CL1-5 cells was reduced after S. barbata treatment. S. barbata inhibited migration and proliferation of human microvascular endothelial cells (HMEC-1), as demonstrated by the Boyden chamber assay. Furthermore, S. barbata decreased HIF-1α expression through inhibiting the phosphorylation of Akt. Taken together, this study demonstrated therapeutic efficacy of S. barbata for lung adenocarcinoma. Furthermore, this strategy may have implications for clinical trials on this cancer.

中文摘要.....................................................................................................1
英文摘要.....................................................................................................3
附圖目錄.....................................................................................................9
縮寫...........................................................................................................10
緒論...........................................................................................................12
一. 肺癌 (Lung cancer) ....................................................................12
二. 血管新生與轉移 (Angiogenesis and metastasis)......................13
三. 缺氧 (Hypoxia) 及缺氧誘發因子-1 (Hypoxia inducible
factor-1, HIF-1)..................................................................................14
四. 半枝蓮 (Scutellaria barbata) 的抑癌作用..............................15
五. 研究目的與實驗策略.................................................................16
材料與方法...............................................................................................17
一. 材料.............................................................................................17
1. 質體 (Plasmid) ......................................................................17
2. 細胞株....................................................................................17
3. 寡去氧核苷酸 (Oligodeoxynucleotides, ODN)...................18
4. 抗體 (Antibody)....................................................................19
二. 方法.............................................................................................19
1. 慢病毒系統質體之構築 (pWPXL/6xHRE-CMVmini-Luc )
7
.....................................................................................................19
2. 慢病毒的生產 (Manipulation of lentivirus vector)..............20
3. 穩定表現冷光酶細胞株 (stable clone) 的選殖..................21
4. 半枝蓮粗萃取物製備............................................................22
5. 半枝蓮對細胞半抑制率濃度分析 (50% inhibitory
concentration, IC50) .....................................................................22
6. 細胞HIF-1 轉錄活性分析....................................................23
7. 反轉錄酶-聚合酶連鎖反應 (Reverse
transcriptase-polymerase chain reaction, RT-PCR)....................24
7-1. 反轉錄作用.................................................................24
7-2. 聚合酶連鎖反應.........................................................24
8. 測細胞上清液中血管內皮細胞生長因子 (vascular
endothelial growth factor, VEGF) 蛋白量.................................25
9. 細胞爬行分析 (Migration assay) .........................................25
10. 西方墨點法 (Western blot) ................................................26
11. 癌細胞轉移模擬實驗.........................................................27
結果...........................................................................................................29
一. 構築帶有pWPXL/6xHRE-CMVmini-Luc 的慢病毒載體......29
二. 測量藥物對正常人類胚胎HEL299 肺細胞及人類肺癌細胞的
細胞毒性............................................................................................29
三. 利用CL1-5/6xHRE-CMVmini-Luc 細胞篩選中草藥.............30
8
四.半枝蓮專一抑制肺癌細胞中HIF-1 的轉錄活性.......................31
五. 半枝蓮抑制肺癌細胞產生血管內皮細胞生長因子................31
六. 半枝蓮抑制內皮細胞的移動及增生........................................32
七. Luteolin 抑制肺癌細胞分泌血管內皮細胞生長因子及減少內
皮細胞的增生....................................................................................33
八. 半枝蓮及luteolin 對小鼠細胞LL2 的抑制作用.....................34
九. 半枝蓮透過Akt 路徑抑制HIF-1α 蛋白................................35
討論...........................................................................................................38
附圖...........................................................................................................46

參考文獻
Brahimi-Horn, C., and Pouyssegur, J. (2006). The role of the
hypoxia-inducible factor in tumor metabolism growth and invasion. Bull
Cancer 93, E73-80.
Brugarolas, J. B., Vazquez, F., Reddy, A., Sellers, W. R., and Kaelin,
W. G., Jr. (2003). TSC2 regulates VEGF through mTOR-dependent and
-independent pathways. Cancer Cell 4, 147-158.
Chen, Z., and Han, Z. C. (2008). STAT3: a critical transcription
activator in angiogenesis. Med Res Rev 28, 185-200.
Chiang, C. T., Way, T. D., and Lin, J. K. (2007). Sensitizing
HER2-overexpressing cancer cells to luteolin-induced apoptosis through
suppressing p21(WAF1/CIP1) expression with rapamycin. Mol Cancer
Ther 6, 2127-2138.
Chiu, F. L., and Lin, J. K. (2008). Downregulation of androgen
receptor expression by luteolin causes inhibition of cell proliferation and
induction of apoptosis in human prostate cancer cells and xenografts.
Prostate 68, 61-71.
Chu, Y. W., Yang, P. C., Yang, S. C., Shyu, Y. C., Hendrix, M. J., Wu,
R., and Wu, C. W. (1997). Selection of invasive and metastatic
subpopulations from a human lung adenocarcinoma cell line. Am J Respir
Cell Mol Biol 17, 353-360.
Daghini, E., Zhu, X. Y., Versari, D., Bentley, M. D., Napoli, C.,
Lerman, A., and Lerman, L. O. (2007). Antioxidant vitamins induce
angiogenesis in the normal pig kidney. Am J Physiol Renal Physiol 293,
F371-381.
Eichhorn, M. E., Kleespies, A., Angele, M. K., Jauch, K. W., and
Bruns, C. J. (2007). Angiogenesis in cancer: molecular mechanisms,
clinical impact. Langenbecks Arch Surg 392, 371-379.
Huang, Y. T., Hwang, J. J., Lee, P. P., Ke, F. C., Huang, J. H., Huang,
44
C. J., Kandaswami, C., Middleton, E., Jr., and Lee, M. T. (1999). Effects
of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and
metastasis-associated properties in A431 cells overexpressing epidermal
growth factor receptor. Br J Pharmacol 128, 999-1010.
Jiang, B. H., and Liu, L. Z. (2008). PI3K/PTEN signaling in
tumorigenesis and angiogenesis. Biochim Biophys Acta 1784, 150-158.
Kim, D. I., Lee, T. K., Lim, I. S., Kim, H., Lee, Y. C., and Kim, C. H.
(2005). Regulation of IGF-I production and proliferation of human
leiomyomal smooth muscle cells by Scutellaria barbata D. Don in vitro:
isolation of flavonoids of apigenin and luteolin as acting compounds.
Toxicol Appl Pharmacol 205, 213-224.
Kim, J. H., Lee, E. O., Lee, H. J., Ku, J. S., Lee, M. H., Yang, D. C.,
and Kim, S. H. (2006). Caspase activation and extracellular
signal-regulated kinase/Akt inhibition were involved in luteolin-induced
apoptosis in Lewis lung carcinoma cells. Ann N Y Acad Sci 1090,
147-160.
Lee, C. H., Wu, C. L., and Shiau, A. L. (2007). Hypoxia-induced
cytosine deaminase gene expression for cancer therapy. Hum Gene Ther
18, 27-38.
Liu, Y. V., Baek, J. H., Zhang, H., Diez, R., Cole, R. N., and Semenza,
G. L. (2007). RACK1 competes with HSP90 for binding to HIF-1alpha
and is required for O(2)-independent and HSP90 inhibitor-induced
degradation of HIF-1alpha. Mol Cell 25, 207-217.
Mirzoeva, S., Kim, N. D., Chiu, K., Franzen, C. A., Bergan, R. C., and
Pelling, J. C. (2008). Inhibition of HIF-1 alpha and VEGF expression by
the chemopreventive bioflavonoid apigenin is accompanied by Akt
inhibition in human prostate carcinoma PC3-M cells. Mol Carcinog.
Pore, N., Jiang, Z., Shu, H. K., Bernhard, E., Kao, G. D., and Maity, A.
(2006). Akt1 activation can augment hypoxia-inducible factor-1alpha
expression by increasing protein translation through a mammalian target
of rapamycin-independent pathway. Mol Cancer Res 4, 471-479.
45
Skinner, H. D., Zheng, J. Z., Fang, J., Agani, F., and Jiang, B. H.
(2004). Vascular endothelial growth factor transcriptional activation is
mediated by hypoxia-inducible factor 1alpha, HDM2, and p70S6K1 in
response to phosphatidylinositol 3-kinase/AKT signaling. J Biol Chem
279, 45643-45651.
Vaupel, P. (2004). Tumor microenvironmental physiology and its
implications for radiation oncology. Semin Radiat Oncol 14, 198-206.
Wiznerowicz, M., and Trono, D. (2003). Conditional suppression of
cellular genes: lentivirus vector-mediated drug-inducible RNA
interference. J Virol 77, 8957-8961.
Yang, S. F., Yang, W. E., Chang, H. R., Chu, S. C., and Hsieh, Y. S.
(2008). Luteolin induces apoptosis in oral squamous cancer cells. J Dent
Res 87, 401-406.
Zhang, Q., Zhao, X. H., and Wang, Z. J. (2008). Flavones and
flavonols exert cytotoxic effects on a human oesophageal
adenocarcinoma cell line (OE33) by causing G2/M arrest and inducing
apoptosis. Food Chem Toxicol 46, 2042-2053.
王瑋祥 (2007). Evaluation of the therapeutic effects of interleukin
-8-dependent oncolytic adenovirus on orthotopic lung adenocarcinoma by
in vivo non-invasive imaging. Master's Thesis, National Cheng Kung
University, Tainan, Taiwan.