臺灣博碩士論文加值系統

從天然物中尋找並發展成抗癌藥物是研究治療癌症一項重要的方法。先前實驗室發現自台灣杉科植物台灣杉 (Taiwania cryptomerioides Hayata) 心材所分離出的化合物176對腫瘤細胞株有很強的毒殺作用，進一步研究其結構衍生物時，發現化合物462-5對於肝癌細胞株Hep G2/A2與Hep 3B/T2有不同的毒性表現。
本論文係以人類肝癌細胞株 Hep G2/A2 與 Hep 3B/T2 為模式系統，比較在體外培養條件下，化合物462-5對這兩種細胞株引發不同程度的細胞毒性效果。化合物462-5處理 Hep 3B/T2 細胞兩天後會造成細胞內部液泡的增加，並使細胞逐漸變得破碎及不完整，在處理四天後即有顯著的細胞毒殺作用。而且其對Hep G2/A2與Hep 3B/T2處理六天後以MTT分析法得到毒殺細胞之EC50值分別為大於20 M與5 M。此外將化合物462-5處理細胞四天後移除，再trypsinize與replating細胞並將之種於新的培養盤中，Hep 3B/T2細胞的生長能力仍會持續地降低，以20 M化合物462-5處理四天後，Hep 3B/T2細胞在無血清環境下，存活率約為65 %，而在含血清環境中，亦僅有75 %，顯示化合物462-5對癌細胞的毒殺作用是不可逆的。
利用抗-tubulin的抗體做螢光染色觀察細胞內tubulin分佈，並以Hoechst dye染色觀察細胞內染色體分佈，顯示化合物462-5不會對細胞tubulin與染色體的分佈有影響。又利用流式細胞分析儀分析細胞DNA分佈情形與細胞週期，發現化合物462-5不會影響細胞的細胞週期分佈。而兩種細胞株若是以化合物462-5處理二或四天後再以低細胞密度replating回培養盤中，或是進行anchorage-independent growth將細胞種殖在soft agar中，Hep G2/A2細胞株只有在濃度20 M的化合物462-5處理後，在兩項實驗中細胞叢的數目會略為減少。而Hep 3B/T2細胞經過2或20 M化合物462-5處理二或四天後，其細胞叢生成數目均會顯著地減少。

Cancer is a serious health problem in human. Development of anticancer drugs from natural products is one of the important chemotherapy. A compound 176 was isolated from the heartwood of Taiwania cryptomerioides Hayata, and it showed strong cytotoxicity to several tumor cells. Continuation of screening cytotoxic compounds against human hepatoma cells from structure derivatives of 176, we found compound 462-5 shown cytotoxicity in human hepatoma cells, Hep 3B/T2 but not in Hep G2/A2.
In this thesis, in vitro, I compared the cytotoxic effect of compound 462-5 between human hepatoma cells Hep G2/A2 and Hep 3B/T2. It provoked the formation of vacuoles in cells and cells broken gradually on compound 462-5-treated Hep 3B/T2 cells for two days. Compound 462-5 showed cytotoxicity on Hep 3B/T2 cells for four days treatment. The EC50 value of the cytotoxicity on Hep G2/A2 and Hep 3B/T2 cells were out of 20 M and 5 M, respectively, for six days treatment of compound 462-5. It is predicted that the cytotoxic effect of compound 462-5 is irreversible because the ability of cell growth is still declining shown on removing of drug and reseeding experiments.
We didn’t find the influence of compound 462-5 on tubulin and DNA distribution on Hep 3B/T2 and Hep G2/A2 cells using -tubulin immunofluorescence and Hoechst dye staining methods. Also from cell cycle analysis, compound 462-5 didn’t show any difference with controlled cells on both cell lines. Furthermore, cells treated with compound 462-5 for two or four days, and then reseeding cells on culture dishes in low cell density or seeding cells in soft agar for anchorage-independent growth test. We found numbers of both types of colony formation on Hep 3B/T2 cells less than on Hep G2/A2 cells obviously.

目錄
中文摘要..........................................................................1
英文摘要..........................................................................2
一、緒論..........................................................................4
二、材料及方法..............................................................8
三、結果........................................................................14
四、討論........................................................................20
五、參考文獻................................................................25
六、圖............................................................................30
七、表格........................................................................56
八、附表.........................................................................59
九、附圖.......................................................................

Alley M. C., Scudiero D. A., Monks A., Hursey M. L., Czerwinski M. J., Fine D. L., Abbott B. J., Mayo J. G., Shoemaker R. H., and Boyd M. R. (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48: 589-601.
Bojean K. A.P.M., Pauw-Gillet M.-C. A.J. DE, Quetin-Leclercq J., Angenot L., and Bassleer R. J.B. (1996) In vitro cytotoxic activity of two potential anticancer drugs isolated from Strychnos: Strychnopentamine and Usambarensine. Anticancer Res. 16: 1129-1138.
Burden D.A., Kingma P. S., Froelich-Ammon S. J., Bjornsti M. -A., Patchan M. W., Thompson R. B. and Osheroff N. (1996) Topoisomerase II etoposide interaction direct the formation od drug-induced enzyme-DNA cleavage complex. J. Biol. Chem. 46: 29238-29244.
Butel J. S., Dudley J. P., and Medina D. (1977) Comparison of the growth properties in vitro and transplantability of continuous mouse mammary tumor cell lines and clonal derivatives. Cancer Res. 37: 1892-1900.
Carstens C.-P., Kramer A., and Fahl W. E. (1996) Adhesion-dependent control of cyclin E/cdk2 activity and cell cycle progression in normal cells but not in Ha-ras transformed NRK cells. Exp. Cell. Res. 229: 86-92.
Guadagno T. M., Ohtsubo M., Robert J. M., and Assoian R. K. (1993) A link between cyclin A expression and adhesion-dependent cell cycle progression. Science 262: 1572-1575.
Cragg G. and Suffness M. (1988) Metabolism of plant-derived anticancer agents. Pharmacol. Ther. 37: 425.
Daley L., Guminski Y., Demerseman P., Kruczynski A., Etievant C., Imbert T., Hill B. T., and Monneret C. (1998) Synthesis and antitumor activity of new glycosides of epipodophyllotoxin, analogues of etoposide, and NK 611. J. Med. Chem. 41: 4475-4485.
Damayanthi Y. and Lown J. W. (1998) Podophyllotoxins: current status and recent developments. Curr. Med. Chem. 5: 205-252.
Fang F., Orend G., Watanabe N., Huntre T., and Ruoslahti E. (1996) Dependence of cyclin E-CDK2 kinase activity on cell anchorage. Science 271: 499-502.
Fukazawa H., Mizuno S., and Uehara Y. (1995) A microplate assay for quantitation of anchorage-independent growth of transformed cells. Anal. Biochem. 228: 83-90.
Horwitz S. B. (1992) Mechanism of action of taxol. Trends Pharmacol. Sci. 13: 134-136.
Kang J.-S., and Krauss R. S. (1996) Ras induces anchorage-independent growth by subverting multiple adhesion-regulated cell cycle events. Mol. Cell. Biol. 16: 3370-3380.
Kingston D.G.I. (1994) Taxol: the chemistry and structure-activity relationships of a novel anticancer agent. Trends Biotechnol. 12: 222-225.
Korytynski E. A., Kelloff G. J., Suk W. A., Sharma S. and Elmore E. (1996) The development of an anchorage-independence assay using human lung tumor cells to screen potential chemopreventive agents. Anticancer Res. 16: 1091-1094.
Macpherson I. (1964) Agar suspension culture for quantitation of transformed cells. Virology 23: 291-294.
Macrae W. D. and Towers G. H. N. (1984) Biological activities of lignans. Phytochem. 23 (6): 1207-1220.
Minocha A. and Long B. (1984) Inhibition of the DNA catenation activity of type II topoisomerase by VP-16-213 and VM-26. Biochem. Biophys. Res. Commun. 122: 165-171.
Miyashita K. and Kakunaga T. (1975) Isolation of heat- and cold-sensitive mutants of Chinese hamster lung cells affected in their abolity to express the transformed state. Cell 5: 131-138.
Moore S. M., Rintoul R. C., Walker T. R., Chilvers E. R., Haslett C., and Sethi T. (1998) The presence of a constitutively active phosphoinositide 3-kinase in small cell lung cancer cells mediates anchorage-independent proliferation via a protein kinase B and p70s6k-dependent pathway. Cancer Res. 58: 5239-5247.
Nederman T., Norling B., Glimelius B., Carlsson J., and Brunk U. (1984) Demonstration of an extracellular matrix in multicellular tumor spheroids. Cancer Res. 44: 3090-3097.
Rao C. B. S. (1978) In: Chemistry of lignans. Andhra university press, India. pp. 1-42.
Reasor M. J. (1989) A review of the biology and toxicologic implications of the induction of lysosomal lamellar bodies by drugs. Toxicol. Appl. Pharm. 97: 47-56.
Saulnier R., Bhardwaj B., Klassen J., Leopold D., Rahimi N., Tremblay E., Moscher D., and Elliott B. (1996) Fibronectin fibrils and growth factors stimulate anchorage-independent growth of a murine mammary carcinoma. Exp. Cell Res. 222: 360-369.
Schulze A., Zerfass-Thome K., Berges J., Middendorf S., Jansen-Durr P., and Henglin B. (1996) Anchorage-dependent transcription of the cyclin A gene. Mol. Cell. Biol. 26: 4632-4638.
Seckl M. J., Seufferlein T., and Rozengurt E. (1994) Lysophosphatidic acid-depleted serum, hepatocyte growth factor and stem cell growth factor stimulate colony growth of small cell lung cancer cells through a calcium-independent pathway. Cancer Res. 54: 6143-6147.
Shin S., Freedman V. H., Risser R., and Pollack R. (1975) Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc. Natl. Acad. Sci. USA 73: 4435-4439.
Steele V. E., Morrissey R. E., Elmore E. L., Gurganus-Rocha D., Wilkinson B. P., Curren R. D., Schmetter B. S., Louie A. T., Lamb J. C., and Yang L.-L. (1988) Evaluation of two in vitro assays to screen for potential developmental toxicants. Fund. App. Toxicol. 11: 673-684.
Tada H., Shiho O., Kuroshima K., Koyama M., and Tsukamato K. (1986) An improved colorimetric assay for interleukin 2. J. Immunol. Meth. 93: 157-165.
Wozniak A. J. and Ross W. E. (1983) DNA damage as a basis for 4’-demethyl-epipodophyllotoxin-9-(4,6-O-ethylidene--D-glucopyranoside) (Etoposide) cytotoxicity. Cancer Res. 43: 120-124.
Yang J.-J., Kang J.-S., and Krauss R. S. (1998) Ras signals to the cell cycle machinery via multiple pathways to induce anchorage-independent growth. Mol. Cell. Biol. 18 (5): 2586-2595.