臺灣博碩士論文加值系統

葉下珠一名珍珠草，屬於大戟科葉下珠屬的植物，它是民間已長期使用的中草藥。在亞洲，葉下珠廣泛的運用於防治B型肝炎。在美洲，葉下珠多用於拮抗平滑肌的收縮，用於治療尿路結石，膽道結石。止痛方面則運用於神經性疼痛及風濕性疼痛。對於腫瘤的治療，葉下珠屬的植物可降低原發性肝癌發生的機率，並保護肝臟細胞。體外研究則顯示葉下珠屬植物的根亦可抑制鼠類血癌細胞及黑色素腫瘤細胞株的生長，但其中相關的機制並未有詳細的探討。有關於葉下珠本身運用於抗癌的實驗研究，近來僅有一報告指出和降低Bcl-2及Telomerase的活性有關，但僅限於細胞株的實驗研究。今我們以葉下珠的全草，萃取冷凍乾燥後，加藥於各種不同人類腫瘤細胞株，觀察其是否有抑制細胞生長的作用，並探索其相關之分子機轉，同時我們也建立動物模型，探討葉下珠在活體實驗是否也有抑制腫瘤生長的作用，並進一步分析其可能的相關機制。本論文以四個實驗設計來分別探討上述問題。
實驗設計一：探討葉下珠水抽出物對各種不同人類腫瘤細胞株的抗癌效應，研究葉下珠是否會誘發腫瘤細胞株產生凋亡。在本實驗中我們觀察細胞形態的變化及進行去氧核醣核酸片段化分析。對於正常細胞如人類臍帶血管內皮細胞及人類正常肝細胞則未出現細胞毒性
，因此本實驗證明葉下珠水抽出物對於各種不同人類腫瘤細胞株有誘發細胞凋亡的特異性，但對正常細胞則沒有任何毒性副作用。
實驗設計二：探討葉下珠水抽出物對於人類急性髓性白血病細胞株（HL-60）的抗癌效應，並探索其相關之分子機制。葉下珠加藥於HL-60細胞後，觀察其細胞形態的變化，分析去氧核醣核酸片段化的表現及caspase-3活性，均隨著藥物濃度及時間的增加而明顯增加，證明葉下珠會誘發HL-60產生細胞凋亡。HL-60細胞的凋亡伴隨著Bax基因表現增加及Bcl-2基因表現的調降，同時也增加Fas receptor / ligand基因的表現，但卻不影響P53的表現。fumonicin B1為ceramide合成酉每之抑制劑，HL-60細胞先以fumonicin B1處理過後，發現其可以幾乎完全阻斷葉下珠所誘發之細胞凋亡。另外以葉下珠餵食小鼠二週後，抽取其血清，亦發現有抑制HL-60細胞生長的作用。本實驗證明葉下珠抑制HL-60細胞生長乃經由凋亡的分子機制。
實驗設計三：探討葉下珠水抽出物對於小鼠Lewis肺癌細胞株（LLC）的抗癌效應並探索其相關之分子機制。葉下珠加藥於LLC細胞株，以MTT測試及細胞流式儀檢測細胞的存活率，發現LLC細胞隨著加藥的濃度及時間的增加，細胞存活率隨之下降，以去氧核醣核酸片段化分析及Caspase-3活性證明其細胞死亡的模式乃經由細胞凋亡。而凋亡的路徑乃經由Bcl-2的基因調降，但不影響Bax，P53及P21的表現。cyclosporin A是一種粒線體膜孔轉移抑制劑，LLC經cyclosporin A前處理後，發現其有部份抑制LLC細胞凋亡的作用，因此本實驗證明葉下珠誘發LLC細胞凋亡乃與調降Bcl-2基因表現及與經由活化粒線體的內在徑路有關。
實驗設計四：探討葉下珠水抽出物對於C57B/L6小鼠腫瘤生長及對血管新生的抑制作用。服用葉下珠組的小鼠腫瘤明顯小於控制組，兩組腫瘤重量分別為0.89±0.83及2.32±1.67g。控制組17隻小鼠2周內全部長出腫瘤，而實驗組長出腫瘤的時間則較慢，且有2隻直到犧牲仍未產生腫塊。對於兩組間，各主要器官如心、肝、脾、肺、腎、以HE染色法並未有明顯差異及腫瘤細胞轉移現象。而經服用葉下珠的小鼠腫塊，以免疫組織化學染色法（C D31）及trichrome染色發現腫瘤內血管的分佈密度有明顯的減少。同時以人類臍帶內皮細胞檢測其葉下珠有抑制細胞移行及MMP活性的作用，但對內皮細胞並未有明顯的毒副作用。本實驗證明葉下珠抑制腫瘤生長及減少腫瘤內血管增生，可能機制與抑制血管內皮細胞的移行與MMP活性有關。因此葉下珠水抽出物可能為一安全而能抗腫瘤血管增生的物質。
本論文所得結論如下，葉下珠水抽出物可以導致各種人類腫瘤細胞株的死亡，但不影響正常細胞，而其誘發腫瘤細胞的死亡途徑乃經由細胞凋亡，對於HL-60細胞乃經由調降Bcl-2基因表現，活化Bax，Fas receptor / ligand等基因表現，但不影響P53，而fumonicin B1。可以阻斷葉下珠所誘發之凋亡，推論其凋亡的路徑和ceramide有關。對於LLC細胞乃經由Bcl-2的調降作用，而cyclosporin A有部分抑制葉下珠所誘發細胞凋亡的作用，推論其凋亡的路徑可能與粒線體內在徑路有關。在動物活體實驗則發現葉下珠有抑制小鼠腫瘤生長及延緩腫瘤形成的作用，葉下珠它會抑制腫瘤內血管增生，同時對於體外內皮細胞的移行和MMP活性有抑制作用。因此葉下珠水抽出物有可能成為臨床有用的抗癌藥物之一，在未來仍須更進一步研究分析其分子機轉及分離鑑定出有效的分子成份。

Phyllanthus urinaria (P. urinaria), one of the herbal plants belonging to the genus Phyllanthus (Euphobiaceae), is widely distributed in China, Southern India and America. It has long been used in folk medicine for the treatment of several diseases. In Asia, P. urinaria has been shown to treat the chronic B hepatitis. It also has been proved to be effective to antagonist the contraction of the smooth muscle and to relax the nephrolithiasis and gall stone. The neurological and rheumatological pain can be alleviated by the use of P. urinaria in South America. The anticancer effect of the genus Phyllanthus has been reported in a few papers. Phyllanthus amarus protected the liver from hepatocarcinogenesis by N-nitrodiethylamine in animal models. Root of the Phyllanthus acuminatus has been shown to inhibit the growth of murine P-388 lymphocytic leukemia and B-16 melanoma cell lines. However, the real mechanism is still not clear so far. The ethyl fraction of P. urinaria was shown recently to exhibit anticancer activity by inducing apoptosis through the inhibition of Bcl-2 expression and telomerase activity in cell line experiments. In this thesis study, the whole plant of P. urinaria was extracted in boiling water and various concentrations of P. urinaria were use to treat various kinds of human cancer cell lines. We have studied the inhibitory effect of P. urinaria on cell growth and investigated the correlative molecular mechanism. We also established the animal model to examine the inhibition of tumor growth in vivo by P. urinaria and study the possible underlying mechanism further. There were four experimental designs in this thesis which were listed in the followings.
The experimental design I was to investigate the anticancer effect of aqueous extract prepared from P. urinaria by analyzing its potential to induce apoptosis in human cancer cells. We showed that the aqueous extract of P. urinaria could reduce the viability by inducing the apoptosis in human cancer cells derived from several different origins as demonstrated by morphological changes and DNA fragmentation. Yet, P. urinaria extract exhibited no cytotoxic effect on normal human cells including vascular endothelial cells and liver cells under the same condition. It suggests that the aqueous extract of P. urinaria is substantially useful in treating various kinds of human cancer cells without toxic side effect on normal cells.
The experimental design II was to investigate the water extract of P. urinaria on inducing the apoptosis of HL-60 cells as demonstrated by morphological change, DNA fragmentation and increased caspase-3 activity. However, normal human peripheral mononuclear cells remained viable under the same treatment. The P. urinaria-induced apoptosis of HL-60 cells was associated with the increased Bax gene expression and decreased Bcl-2 gene expression. In addition, the gene expressions of Fas receptor and Fas ligand, but not p53, were also induced in HL-60 cells dose- and time-dependently. The inhibitor of ceramide synthase, fumonisin B1, completely suppressed the apoptosis induced by P. urinaria. It indicated that the activity of ceramide synthase is critical for the P. urinaria-induced apoptosis in HL-60 cells. It suggests that P. urinaria may be useful in treating myeloid leukemia without toxic side effect on normal cells.
The experimental design III was to investigate the effect of water extract of P. urinaria on decreasing the number of Lewis lung carcinoma cells in a dose- and time-dependent manner as determined by MTT assay. However, the water extract of P. urinaria did not exert any cytotoxic effect on normal cells such as endothelial cells and liver cells. Result from flow cytometry revealed a dose-dependent increase of dead cells 24 hours after treating Lewis lung carcinoma cells with P. urinaria extract. The anticancer activity of P. urinaria extract was due to the apoptosis induced in Lewis lung carcinoma cells, which was demonstrated by DNA fragmentation analysis and increased caspase-3 activity. The apoptosis triggered by P. urinaria extract in Lewis lung carcinoma cells was associated with the down-regulation of Bcl-2 gene expression, but not with p53, p21 and Bax. Furthermore, the partial inhibition of P. urinaria-induced apoptosis in Lewis lung carcinoma cells by pretreatment with cyclosporin A, a mitochondria permeability transition pore inhibitor, suggesting that P. urinaria extract induced the apoptosis of Lewis lung carcinoma cells, at least in part, through a mitochondria-associated intrinsic pathway.
The experimental design IV was to investigate the anti-cancer effect of P. urinaria in vivo. The anti-tumor activity of the water extract of P. urinaria was evaluated by their effect on the onset of tumor, tumor size, and angiogenesis in tumor mass in vivo. The effect of P. urinaria on the migration and MMP activity of endothelial cells were studied in vitro. The reduction of tumor mass was observed in the P. urinaria-treated group (0.98±0.83), comparing with those in the control group (2.32±1.67). Mice in the control group all developed tumor in shorter time period, where mice treated with P. urinaria developed tumor later and the incidence was less than 100 % as in the control group. By analyzing the tumor histology with immunohistochemical method and trichrome stain, we found that tumor in the P. urinaria-treated group had obviously less vascularization, indicating the potential anti-angiogenesis effect of P. urinaria. As we expected, there were no cytotoxic effects in vital organs including heart, lung, liver, spleen, and kidney as revealed by HE stain. The dose-dependent inhibitory effect of P. urinaria on the migration of HUVECs was demonstrated by transwell migration assay. The observation that P. urinaria could also decrease the MMPs activities of HUVECs, again suggesting that water extract of P. urinaria could be used as a safe anti-angiogenesis agent.
In conclusion, P. urinaria considerably exhibited the inhibitory effect on cell growth of various cancer cells through inducing apoptosis pathway and had no toxic side effect on normal cells. The P. urinaria-induced apoptosis of HL-60 was associated with down regulation of Bcl-2 gene expression, activation of Bax, Fas receptor/ ligand, but not P53. Fumonisin B1 completely suppressed the apoptosis induced by P. urinaria, demonstrating the critical role of ceramide synthesis in this pathway. The anticancer effect of P. urinaria on Lewis lung carcinoma cells was mediated by apoptosis with down regulation of Bcl-2 gene expression but not with Bax, P53 and P21. Result from the use of cyclosporin A pretreatment demonstrated a partial inhibition of P. urinaria-induced apoptosis, indicating the possible molecular mechanism through a mitochondria-associated intrinsic pathway. In our in vivo study, P. urinaria exhibited the significant inhibitory effect on tumor mass growth and delayed the initial tumor formation. The demonstration of reduced tumor vascularization and inhibited migration and MMPs activities in endothelial cells strongly suggests the potential role of P. urinaria in anti-angiogenesis. In conclusion, with continuous pharmacological analysis and study, P. urinaria could become one of the potential clinical anti-cancer drugs.

第一章 簡介----------------------------------------------------1
第二章 假說與目的---------------------------------------------19
第三章 葉下珠水抽出物誘發多種人類腫瘤細胞株的凋亡-------------23
第四章 葉下珠誘發HL-60血癌細胞株Fas receptor / ligand表現
及藉由ceramide所引起的細胞凋亡-------------------------35
第五章 葉下珠引發Lewis lung carcinoma細胞株凋亡及調降…Bcl-2
基因的表現---------------------------------------------55
第六章 葉下珠水抽出物抑制小鼠腫瘤生長及腫瘤內血管新生---------71
第七章 結論與展望---------------------------------------------90
參考文獻------------------------------------------------------95
附錄---------------------------------------------------------107

1. 中藥大辭典
2. Yao QQ, Zuo CX. Chemical studies on the constituents of Phyllanthus nrinaria L. Axta pharmaceutica Sinica 1993; 28: 829-35.
3. Calixto, J.B., A.R. Santos, V. Cechinel Filho and R.A. Yunes. A review of the plants of the genus Phyllanthus: their chemistry, pharmacology, and therapeutic potential. Medicinal Research Reviews 1998; 18: 225-58.
4. Thyagarajan SP, Subramanian S, Thirunalasundari T, Venkateswaran PS, Blumberg BS. Effect of Phyllanthus amarus on chronic carriers of hepatitisB virus. Lancet 1988; 2: 764-6.
5. Wang M, Cheng H, Li Y, Meng L, Zhao G, Mai K. Herbs of the genus Phyllanthus in the treatment of chronic hepatitis B: observations with three preparations from different geographic sites. Journal of Laboratory & Clinical Medecine 1995; 126: 350-2.
6. Ji XH, Qin YZ, Wang WY, Zhu JY, Liu XT. Effects of extracts from Phyllanthus urinaria L. on HbsAg production in PLC/PRF/5 cell line. Zhongguo Zhong Yao Za Zhi 1993; 18: 496-8, 511.
7. Ott M, Thyagarajan SP, Gupta S. Phyllanthus amarus suppresseshepatitis B virus by interrupting interactions between HBV enhancer I and cellular transcrition factors. European Journal of Clinical Investigation 1997; 27: 908-15.
8. Lee CD, Ott M, Thyagarajan SP, Shafritz DA, Burk RD, Gupta S. Phyllanthus amarus down-regulates hepatitis B virus mRNA transcription and replication. European Journal of Clinical Investigation 1996; 26: 1069-76.
9. Zhou S, Xu C, Zhou N, Huang Y, Huang L, Chen X, Hu Y, Liao Y. Mechanism of protective action of Phyllanthus urinaria L. against injuries of liver cells. Zhongguo Zhong Yao Za Zhi 1997; 22: 109-11.
10. Ogata T, Higuchi H, Mochida S, Matsumoto H, Kato A, Endo T, Kaji A, Kaji H. HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri. AIDS Research & Human Retroviruses 1992; 8: 1937-44.
11. Suthienkul O, Miyazaki O, Chulasiri M, Kositanont U, Oishi K. Retroviral reverse transcriptase inhibitory activity in Thai herbs and spices: screening with Moloney murine leukemia viral enzyme. Southeast Asian J Trop Med Public Health 1993; 24: 751-5.
12. Qian-Cutrone J, Huang S, Trimble J, Li H, Lin PF, Alam M, Klohr SE, Kadow KF. Niruriside, a new HIV REV/RRE binding inhibitor from Phyllanthus niruri. Journal of Natural Products 1996; 59: 196-99.
13. Higashino H, Suzuki A, Tanaka Y, Pootakham K. Hypoglycemic effects of Siamese Momordica charantia and Phyllanthus urinaria extracts in streptozotocin-induced diabetic rats. Nippon Yakurigaku Zasshi 1992; 100: 415-21.
14. Srividya N. Periwal S. Diuretic, hypotensive and hypoglycaemiaeffect of Phyllanthus amarus. Indian Journal of Experimental Biology 1995; 33: 861-4.
15. Dias MA, Campos AH, Cechinel-Filho V, Yunes RA., Calixto JB. Analysis of the mechanisms underlying the contractile responses induced by the hydroalcoholic extract of Phyllanthus urinaria in the guinea pig urinary bladder in-vitro. Journal of Pharmacy & Pharmacology 1995; 47: 846-51.
16. Paulino N, Cechinel-Filho V, Yunes RA, Calixto JB. The relaxant effect of extract of Phyllanthus urinaria in the guinea-pig isolated trachea. Evidence for involvement of ATP-sensitive potassium channels. Journal of Pharmacy & Pharmacology 1996; 48: 1158-63.
17. Paulino N, Cechinel-Filho V, Pizzolatti MG, Yunes RA, Calixto JB. Mechanisms involved in the contractile responses induced by the hydroalcoholic extract of Phyllanthus urinaria on the guinea pig isolated trachea: evidence for participation of tachykinins and influx of extracellular Ca2+ sensitive to ruthenium red. General Pharmacology 1996; 27: 795-802.
18. Santos AR, Cechinel-Filho V, Yunes RA, Calixto JB. Analysis of the mechanisms underlying the antinociceptive effect of the extracts of plants from the genus Phyllanthus. General Pharmacology 1995; 26: 1499-506.
19. Santos AR, De Campos RO, Miguel OG, Cechinel-Filho V, Yunes RA, Calixto JB. The involvement of K+ channels and Gi/o protein in the antinociceptive action of the gallic acid ethyl ester. European Journal of Pharmacology1999; 379: 7-17.
20. Yeh SF, Hong CY, Huang YL, Liu TY, Choo KB, Chou CK. Effect of an extract from Phyllanthus amarus on hepatitis B surface antigen gene expression in huma hepatoma cells. Antiviral Research 1993; 20: 185-92.
21. Blumberg BS, Millman I, Venkateswaran PS, Thyagarajan SP. Hepatitis B virus and primary hepatocellular carcinoma: treatment of HBV carries with Phyllanthus amarus. Vaccine 1990; 8 suppl: S86-92.
22. Blumberg BS, Millman I, Venkateswaran PS, Thyagarajan SP. Hepatitis B virus and hepatocellular carcinoma —treatment of HBV carries with Phyllanthus amarus. Cancer Detection and prevention 1989; 14: 195-201.
23. Jeena KJ, Joy KL., Kuttan R. Effect of Emblica officinalis, Phyllanthus amarus and Picrorrhiza Kurroa on N-nitrosodiethylamine induced hepatocarcinogenesis. Cancer Letter 1999; 136: 11-6.
24. Rajeshkumar NV, Kuttan R. Phyllanthus amarus extract administration increases the life span of rats with hepatocellular carcinoma. Journal of Ethnopharmacology 2000; 73: 215-9.
25. Evan G, Littlewood T. A matter of life and cell death. Science 1998; 281: 1317-22.
26. Williams, G.T. Programmed cell death: apoptosis and oncogenesis. Cell 1991; 65: 1097-8.
27. Nagata S. Apoptosis by death factor. Cell 1997; 88: 355-65.
28. Hale AJ, Smith CA, Sutherland LC, Stoneman VEA, Longthorne VL, Culhane AC, Williams GT. Apoptosis: molecular regulation of cell death. European Journal of Biochemistry 1996; 236: 1-26.
29. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312-6.
30. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281: 1309-12.
31. Cheng EH, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K. Hardwick JM. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science 1997; 278: 1966-8.
32. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 1997; 275: 1132-6.
33. Chinnaiyan AM, O''Rourke K, Lane BR, Dixit VM. Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death. Science 1997; 275: 1122-6.
34. Green DR. A Myc-induced apoptosis pathway surfaces. Science 1997; 278: 1246-7.
35. Derry WB, Putzke AP, Rothman JH. Caenorhabditis elegans p53: role in apoptosis, meiosis, and stress resistance. Science 2001; 294: 591-5.
36. Soengas MS, Alarcon RM, Yoshida H, Giaccia AJ, Hakem R, Mak TW, Lowe SW. Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition. Science 1999; 284: 156-9.
37. Gupta S. Molecular steps of death receptor and mitochondria pathways of apoptosis. Life Sciences 2001; 69: 2957-64.
38. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998; 281: 1305-8.
39. Cappello P, Novelli F, Forni G, Giovarelli M. Death receptor ligands in tumors. Journal of Immunotherapy 2002; 25: 1-15.
40. Baetu TM, Hiscott J. On the TRAIL to apoptosis. Cytokine & Growth Factor Reviews 2002; 13: 199-207.
41. Yonehara S. Death receptor Fas and autoimmune disease: from the original generation to therapeutic application of agonistic anti-Fas monoclonal antibody. Cytokine & Growth Factor Reviews 2002; 13: 393-402.
42. Wajant H. The Fas signaling pathway: more than a paradigm. Science 2002; 296: 1635-6.
43. Beg AA. Baltimore D. An essential role for NF-kappaB in preventing TNF-alpha-induced cell death. Science 1996; 274: 782-784.
44. Bernardi P, Petronilli V, Di Lisa F, Forte M. A mitochondrial perspective on cell death. Trends in Biochemical Sciences 2001; 26: 112-7.
45. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nature Reviews. Cancer 2002; 2: 647-56.
46. Plas DR, Thompson CB. Cell metabolism in the regulation of programmed cell death. Trends in Endocrinology & Metabolism 2002; 13: 75-8.
47. Maceyka M, Payne SG, Milstien S, Spiegel S. Sphingosine kinase, sphingosine-1-phosphate, and apoptosis. Biochimica et Biophysica Acta 2002; 1585: 193-201.
48. English D. Kovala AT. Welch Z. Harvey KA. Siddiqui RA. Brindley DN. Garcia JG. Induction of endothelial cell chemotaxis by sphingosine 1-phosphate and stabilization of endothelial monolayer barrier function by lysophosphatidic acid, potential mediators of hematopoietic angiogenesis. Journal of Hematotherapy & Stem Cell Research 1999; 8: 627-34.
49. Pettus BJ, Chalfant CE, Hannun YA. Ceramide in apoptosis: an overview and current perspective. Biochimica et Biophysica Acta 2002; 1585: 114-25.
50. Andrieu-Abadie N, Levade T. Sphingomyelin hydrolysis during apoptosis. Biochimica et Biophysica Acta 2002; 1585: 126-34.
51. Hanada K. Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism. Biochimica et Biophysica Acta 2003: 1632: 16-30.
52. Levade T, Malagarie-Cazenave S, Gouaze V, Segui B, Tardy C, Betito S, Andrieu-Abadie N, Cuvillier O. Ceramide in apoptosis: a revisited role. Neurochemical Research 2002; 27: 601-7.
53. Chen M, Wang J. Initiator caspases in apoptosis signaling pathways. Apoptosis 2002; 7: 313-9.
54. Scholz SR, Korn C, Gimadutdinow O, Knoblauch M, Pingoud A, Meiss G. The effect of ICAD-S on the formation and intracellular distribution of a nucleolytically active caspase-activated DNase. Nucleic Acids Research 2002; 30: 3045-51.
55. Sakahira H, Nagata S. Co-translational folding of caspase-activated DNase with Hsp70, Hsp40, and inhibitor of caspase-activated DNase. Journal of Biological Chemistry 2002; 277: 3364-70.
56. Lynch CC, Matrisian LM. Matrix metalloproteinases in tumor-host cell communication. Differentiation 2002; 70: 561-73.
57. Silletti S, Kessler T, Goldberg J, Boger DI, Cheresh DA. Disruption of matrix metalloproteinase 2 binding to intergrin alpha vbeta by an organic molecule inhibits angiogenesis and tumor growth in vivo. Proceedings of the National Academy of Sciences of the United States of America 2001; 98: 119-24.
58. Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nature Cell Biology 2000; 2: 737-44.
59. Hashimoto G, Inoki I, Fujii Y, Aoki T, Ikeda E, Okada Y. Matrix metalloproteinases cleave connective tissue growth factor and reactivate angiogenic activity of vascular endothelial growth factor 165. Journal of Biological Chemistry 2002; 277: 36288-95.
60. Patteeson BC, Sang QA. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelastinase type IV collagenase (MMP-9). Journal of Biological Chemistry 1997; 272: 28823-5.
61. Cornelius LA, Nehring LC, Harding E, Bolanowski M, Welgis HG, Kobayashi DK, Pierce RA, Shapiro SD. Matrix metalloproteinases generate angiostatin: effects on neovasculization. Journal of Immunology 1998; 161: 6845-52.
62. Iivanainen E, Kahari VM, Heino J, Elenius K. Endothelial cell-matrix interactions. Microscopy Research & Technique 2003; 60: 13-22.
63. Ferrara N. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Seminars in Oncology 2002; 29: 10-4.
64. Distler O, Neidhart M, Gay RE, Gay S. The molecular control of angiogenesis. International Reviews of Immunology 2002; 21: 33-49.
65. Williams GT. Programmed cell death: apoptosis and oncogenesis. Cell 1991; 65: 1097-8.
66. Hale AJ, Smith CA, Sutherland LC, Stoneman VEA, Longthorne VL, Culhane AC, Williams GT. Apoptosis: molecular regulation of cell death. European Journal of Biochemistry 1996; 236: 1-26.
67. Pettit GR, Schaufelberger DE, Nieman RA, Dufresne C, Saenz-Renauld JA. Antineoplastic agents, 177. Isolation and structure of phyllanthostatin 6. Journal of Natural Products 1990; 53: 1406-13.
68. Petti GR, Schaufelberger DE. Isolation and structure of the cytostatic lignan glycoside phyllanthostatin A. Journal of Natural Products 1988; 51: 1104-12.
69. Powis G, Moore DJ. High-performance liquid chromatographic assay for the antitumor glycoside phllanthoside and its stability in plasma of several species. Journal of Chromatography 1985; 342: 129-34.
70. Sanchez-Lamar AM, Fiore E, Cundari R, Ricordy R, De Salvia R. Phyllanthus orbicularis aqueous extract: cytotoxic, genotoxic, and antimutagenic effects in the CHO cell line. Toxicology and Applied Pharmacology 1999; 161: 231-9.
71. Huang ST, Yang RC, Yang LJ, Lee PN, Pang JHS. Phyllanthus urinaria triggers the apoptosis and Bcl-2 down-regulation in Lewis lung carcinoma cells. Life Sciences 2003; 72: 1705-16.
72. Li, H. L. Flora of Taiwan (2nd. Ed.). Taipei: Editorial Committee of the Flora of Taiwan Publications, 1993, pp. 498.
73. Du YH, Ho PC. Arsenic compounds induce cytotoxicity and apoptosis in cisplatin-sensitive and -resistant gynecological cancer cell lines. Cancer Chemotherapy and Pharmacology 2001; 47: 481-90.
74. Ishiko O, Sumi T, Yoshida H, Ogita S, Yamada R. Expression of apoptosis regulatory proteins in advanced cancer of the uterine cervix after cyclic balloon-occluded arterial infusion chemotherapy. International Journal of Oncology 2001; 18: 1151-5.
75. Bishay K, Ory K, Olivier MF, Lebeau J, Levalois C, Chevillard S. DNA damage-related RNA expression to assess individual sensitivity to ionizing radiation. Carcinogenesis 2001; 22: 1179-83.
76. Naumann U, Kugler S, Wolburg H, Wick W, Rascher G, Schulz JB, Conseiller E, Bahr M, Weller M. Chimeric tumor suppressor 1, a p53-derived chimeric tumor suppressor gene, kills p53 mutant and p53 wild-type glioma cells in synergy with irradiation and CD95 ligand. Cancer Research 2001; 61: 5833-42.
77. Kondo T, Matsuda T, Kitano T, Takahashi A, Tashima M, Ishikura H, Umehara H, Domae N, Uchiyama T, Okazaki T. Role of c-jun expression increased by heat shock- and ceramide-activated caspase-3 in HL-60 cell apoptosis. Possible involvement of ceramide in heat shock-induced apoptosis. Journal of Biological Chemistry 2000; 275: 7668-76.
78. Feng H, Zeng Y, Whitesell L, Katsanis E. Stressed apoptotic tumor cells express heat shock proteins and elicit tumor-specific immunity. Blood 2001; 97: 3505-12.
79. Vouldoukis I, Sivan V, Vozenin MC, Kamate C, Calenda A, Mazier D, Dugas B. Fc-receptor-mediated intracellular delivery of Cu/Zn-superoxide dismutase (SOD1) protects against redox-induced apoptosis through a nitric oxide dependent mechanism. Molecular Medicine 2000; 6: 1042-53.
80. Mansat V, Bettaieb A, Levade T, Laurent G, Jaffrezou JP. Serine protease inhibitors block neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin. FASEB Journal 1997; 11: 695-702.
81. Giridharan P, Somasundaram ST, Perumal K, Vishwakarma RA, Karthikeyan NP, Velmurugan R, Balakrishnan A. Novel substituted methylenedioxy ligan suppresses proliferation of cancer cells by inhibiting telomerase and activation of c-myc and caspases leading to apoptosis. British Journal of Cancer 2002; 87:98-105.
82. Riordan FA, Bravery CA, Mengubas K, Ray N, Borthwick NJ, Akbar AN, Hart SM, Hoffbrand AV, Mehta AB, Wickremasinghe RG. Herbimycin A accelerates the induction of apoptosis following etoposide treatment or gamma-irradiation of bcr/abl-positive leukaemia cells. Oncogene 1998; 16: 1533-42.
83. Jamieson L, Carpenter L, Biden TJ, Fields AP. Protein kinase Ciota activity is necessary for Bcr-Abl-mediated resistance to drug-induced apoptosis. Journal of Biological Chemistry 1999; 274: 3927-30.
84. Talanian RV, Quinlan C, Trautz S, Hackett MC, Mankovich JA, Banach D, Ghayur T, Brady KD, Wong WW. Substrate specificities of caspase family proteases. Journal of Biological Chemistry 1997; 272: 9677-82.
85. Chomczynski P, Sacchi N. Single-step method of RNA isolated by acid guanidium thiocyanate-phenol-chloroform extraction. Ann Biochem 1987; 162: 156-9.
86. Johnson N, Ng TTC, Parkin JM. Camptothecin causes cell cycle perturbations within T-lymphoblastoid cells followed by dose dependent induction of apoptosis. Leukemia Research 1997; 21: 961-72.
87. Narayanan BA, Geoffroy O, Willingham MC, Re GG, Nixon DW. p53/p21 (WAF1/CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells. Cancer Letters 1999; 136: 215-21.
88. Senderowicz AM. Decelopment of cyclin-dependent kinase modulators as novel therapeutic approaches for hematological malignancies. Leukemia 2001; 15: 1-9.
89. Krammer PH, Dhein J, Walczak H, Behrmann I, Mariani S, Matiba B, Fath M, Daniel RT, Knipping E, Westendorp MO. The role of APO-1-mediated apoptosis in the immune system. Immunological Reviews 1994; 142: 175-91.
90. Michael JM, Lavin MF, Watters DJ. Resistance to radiation-induced apoptosis in Burkitt''s lymphoma cells is associated with defective ceramide signaling. Cancer Research 1997; 57: 3600-05.
91. Takeda Y, Tashima M, Takahashi A, Uchiyama T, Okazaki T. Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3. Journal of Biological Chemistry 1999; 274: 10654-60.
92. Kondo T, Matsuda T, Tashima M, Umehara H, Domae N, Yokoyama K, Uchiyama T, Okazaki T. Suppression of heat shock protein-70 by ceramide in heat shock-induced HL-60 cell apoptosis. Journal of Biological Chemistry 2000; 275: 8872-9.
93. Grullich C, Sullards MC, Fuks Z, Merrill AH, §Jr, Kolesnick R. CD95 (Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis. Journal of Biological Chemistry 2000; 275: 8650-6.
94. Furuke K, Bloom ET. Redox-sensitive events in Fas-induced apoptosis in human NK cells include ceramide generation and protein tyrosine dephosphorylation. International Immunology 1998; 10: 1261-72.
95. Herr I, Wilhelm D, Bohler T, Angel P, Debatin KM. Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis. EMBO Journal 1997; 16: 6200-8.
96. VonHaefen C, Wieder T, Gillissen B, Stark L, Graupner V, Dorken B, Daniel PT. Ceramide induces mitochondria activation and apoptosis via a Bax-dependent pathway in human carcinoma. Oncogene 2002; 21: 4009-19.
97. Kim HJ, Mun JY, Chun YJ, Choi KH, Kim MY. Bax-dependent apoptosis induced by ceramide in HL-60 cells. FEBS Letters 2001; 505: 264-8.
98. Muller M, Wilder S, Bannasch D, Israeli D, Lehlbach K, Li-Weber M, Friedman SL, Galle PR, Stremmel W, Oren M, Krammer PH. P53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs. Journal of Experimental Medicine 1998; 188: 2033-45.
99. Fukazawa T, Fujiwara T, Morimoto Y, Shao J, Nishizaki M, Kadowaki Y, Hizuta A, Owen-Schaub LB, Roth JA, Tanaka N. Differential involvement of the CD95 (Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in human cancer cells. Oncogene 1999; 18: 2189-99.
100. Wilson WH, Teruya-Feldstein J, Fest T, Harris C, Steinberg SM, Jaffe ES, Raffeld M. Relationship of p53, bcl-2, and tumor proliferation to clinical drug resistance in non-Hodgkin''s lymphomas. Blood 1997; 89: 601-9.
101. Wattel E, Preudhomme C, Hecquet B, Vanrumbeke M, Quesnel B, Dervite I, Morel P, Fenaux P. p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies. Blood 1994; 84: 3148-57.
102. Rouby SE, Thomas A, Costin D, Rosenberg CR, Potmesil M, Silber R, Newcomb EW. p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression. Blood 1993; 82: 3452-9.
103. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998; 281: 1322-26.
104. Basu A, Haldar S. The relationship between Bcl2, bax and P53: consequences for cell cycle progression and cell death. Molecular Human Production 1998; 4: 1099-109.
105. Furuno T, Kanno T, Arita K, Asami M, Utsumi T, Doi Y, Inoue M, Utsumi K. Roles of long chain fatty acids and carnitine in mitochondrial membrane permeability transition. Biochemical Pharmacology 2001; 62: 1037-46.
106. Shiraishi J, Tatsumi T, Keira N, Akashi K, Mano A, Yamanaka S, Matoba S, Asayama J, Yaoi T, Fushiki S, Fliss H, Nakagawa M. Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis. American Journal of Physiology 2001; 281: H1637-47.
107. Fujimoto K, Hosotani R, Doi R, Wada M, Lee JU, Koshiba T, Miyamoto Y, Tsuji S, Nakajima S, Imamura M. Induction of cell-cycle arrest and apoptosis by a novel retinobenzoic-acid derivative, TAC-101, in human pancreatic-cancer cells. International Journal of Cancer 1999; 81: 637-44.
108. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312-16.
109. Dales JP, Palmerini F, Devilard E, Hassoun J, Birg F, Xerri L. Caspases: conductors of the cell death machinery in lymphoma cells. Leukemia and Lymphoma 2001; 41: 247-53.
110. Budd RC. Activation-induced cell death. Current Opinion in Immunology 2001; 13: 356-62.
111. Isenberg JS, Klaunig JE. Role of the mitochondrial membrane permeability transition (MPT) in rotenone-induced apoptosis in liver cells. Toxicological Sciences 2000; 53: 340-51.
112. Kwon KB, Yang JY, Ryu DG, Rho HW, Kim JS, Park JW, Kim HR, Park BH. Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells. Journal of Biological Chemistry 2001; 276: 47518-23.
113. Belzacq AS, Jacotot E, Vieira HL, Mistro D, Granville DJ, Xie Z, Reed JC, Kroemer G, Brenner C. Apoptosis induction by the photosensitizer verteporfin: identification of mitochondrial adenine nucleotide translocator as a critical target. Cancer Rearch 2001; 61: 1260-04.
114. Lehmann S, Bengtzen S, Paul A, Christensson B, Paul C. Effects of arsenic trioxide (As2O3) on leukemic cells from patients with non-M3 acute myelogenous leukemia: studies of cytotoxicity, apoptosis and the pattern of resistance. European Journal of Haematology 2001; 66: 357-64.
115. Devireddy LR, Teodoro JG, Richard FA, Green MR. Induction of apoptosis by a secreted lipocalin that is transcriptionally regulated by IL-3 deprivation. Science 2001; 293: 829-34.
116. Geller HM, Cheng KY, Goldsmith NK, Romero AA, Zhang AL, Morris EJ, Grandison L. Oxidative stress mediates neuronal DNA damage and apoptosis in response to cytosine arabinoside. Journal of Neurochemistry 2001; 78: 265-75.
117. Seo WG, Pae ho, Oh GS, Chai KY, Yun YG, Chung HT, Jang KK, Kwon TO. Ethyl acetate extract of the stem bark of Cudrania tricuspidata induces apoptosis in human leukemia HL-60 cells. American Journal of Chinese Medicine 2001; 29: 313-20.
118. Bussing A, Stein GM, Herterich-Akinpelu I, Pfuller U. Apoptosis-associated generation of reactive oxygen intermediates and release of pro-inflammatory cytokines in human lymphocytes and granulocytes by extracts from the seeds of Acalypha wilkesiana. Journal of Ethnopharmacology 1999; 66: 301-9.
119. Yoon Y, Kim YO, Jeon WK, Park HJ, Sung HJ. Tanshinone IIA isolated from Salvia miltiorrhiza BUNGE induced apoptosis in HL60 human premyelocytic leukemia cell line. Journal of Ethnopharmacology 1999; 68: 121-7.
120. Adachi H, Preston G, Harvat B, Dawson MI, Jetten AM. Inhibition of cell proliferation and induction of apoptosis by the retinoid AHPN in human lung carcinoma cells. American Journal of Respiratory Cell and Molecular Biology 1998; 18: 323-33.
121. Sirzen F, Zhivotovsky B, Nilsson A, Bergh J, Lewensohn R. Higher spontaneous apoptotic index in small cell compared with non-small cell lung carcinoma cell lines; lack of correlation with Bcl-2/Bax. Lung Cancer 1998; 22: 1-13.
122. Korsmeyer SJ, Shutter JR, Veis DJ, Merry DE, Oltvai ZN. Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell death. Seminars in Cancer Biology 1993; 4: 327-32.
123. Egan B, Beilharz T, George R, Isenmann S, Gratzer S, Wattenberg B, Lithgow T. Targeting of tail-anchored proteins to yeast mitochondria in vivo. FEBS Letters 1999; 451: 243-8.
124. Priault M, Chaudhuri B, Clow A, Camougrand N, Manon S. Investigation of bax-induced release of cytochrome c from yeast mitochondria permeability of mitochondrial membranes, role of VDAC and ATP requirement. European Journal of Biochemistry 1999; 260: 684-91.
125. Kamphaus GD, Colorado PC, Panka DJ, Hopfer H, Ramchandran R, Torre A, Maeshima Y, Mier JW, mSukhatme VP, Kalluri R. Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth. Journal of Biological Chemistry 2000; 275: 1209-15.
126. Cao Y, O’Reilly MS, Marshall B, Flynn E, Ji RW, Folkman J. Expression of angiostatin cDNA in a murine fibrosarcoma supressesprimary tumor growth and produce long-term dormacy of metastases. Journal of Clinical Investigation 1998; 101: 1055-63.
127. Cao R, Farnebo J, Kurimoto M, Cao Y. Interleukin-18 acts as an angiogenesis and tumor suppressor. FASEB Journal 1999; 13: 2195-202.
128. Silvestre JS, Mallat Z, TamaratR, Duriez M, Tedgui A, Levy BI. Regulation of matrix metalloproteinase activity in ischemic tissue by interleukin-10. Circulation Research 2001; 89: 259-64.
129. Yoshiji H, Kuriyama S, Hicklin DJ, Huber J, Yoshi J, Miyamoto Y, Kawata M, Ikenaka Y, Nakatani T, Tsujinoue H, Fukui H. KDH/Flk-1 is a major regulator of vascular endothelial growth factor-induced tumor development and angiogenesis in murine hepatocellular carcinoma cells. Hepatology 1999; 30: 1179-86.
130. Reiher FK, Volpert OV, Jimenez B, Crawford SE, Dinney CP, Henkin J, Haviv F, Bouck NP, Campbell ST. Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics. International Journal of Cancer 2002; 98: 682-9.
131. Burns CJ, Liu W, Davis DW, Shaheen RM, McConkey DJ, Wilson MR, Bucana CD, Hicklin DJ, Ellis LM. Vascular endothelial growth factor is an in vivo survival factor for tumor endothelial in a murine model of colorectal carcinoma liver metastases. Cancer 2000; 89: 488-99.
132. Fathallah-Shaykh HM, Zhao LJ, Kafrouni AI, Smith GM, Forman J. Gene transfer of INF-r into established brain tumors represses growth by antiangiogenesis. Journal of Immunology 2000; 164: 217-22.
133. Mesri M, Morales-Ruiz M, Ackermann EJ, Bennett CF, Pober JS, Sessa WC, Altieri DC. Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting. American Journal of Pathology 2001; 158: 1757-65
134. English D, Garcia JG, Briendley DN. Platelet-released phospholipids link haemostasis and angiogenesis. Cardiovascular Research 2001; 49: 588-99.
135. Riu. A study of staining blood film (Romanowsky system). J Niigata Med Assoc 1956; 70: 635-43.
136. Fox SB, Leek RD, Weekes MP, Whitehouse RM, Gatter KCH. Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, chalkley count, and computer image analysis. Journal of Pathology 1995; 177: 275-83.
137. Chalkley H. Method for the quantitative morphological analysis of tissues. J Natl Cancer Inst 1943; 4: 47-53.
138. Sim BRL, O’Reilly MS, Liang H, Fortier AH, HE W, Madsen JW, Lapcevich R, Nacy CA. A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer. Cancer Research 1997; 57: 1329-34.
139. Seed MP, Brown JR, Freemantle CN, Papworth JL, Colville-Nash PR, Willis D, Somerville KW, Asculai S, Willoughby DA. The inhibition of colon-26 adenocarcinoma development and angiogenesis by topical diclofenac in 2.5% hyaluronan. Cancer Research 1997; 57: 1625-9.
140. Albini A, Morini M, D’Agostini F, Ferrari N, Compelli F, Arena G, Noonan DM, Pesce C, Flora SD. Inhibition of angiogenesis-driven Kaposi’s sarcoma tumor growth on nude mice by oral N-Acetylcysteine. Cancer Research 2001; 61: 8171-8.
141. Maeshima Y, Manfredi M, Reimer C, Holthaus KA, Hopfer H, Chandamuri BR, Kharbanda S, Kalluri R. Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin. Journal of Biological Chemistry 2001; 276: 15240-8.
142. Zhang L, Yu D, Hicklin DJ, Hannay JAF, Ellis LM, Pollock RE. Combined anti-fetal liver kinase 1 monoclonal antibody and continuous low-dose doxorubicin inhibits angiogenesis and growth of human soft tissue sarcoma xerografts by induction of endothelial cell apoptosis. Cancer Research 2002; 62: 2034-42.
143. Yu J, Moon A, Kim HR. Both platelet-derived growth factor receptor (PDGFR)-alpha and PDGFR-beta promote murine fibroblast cell migration. Biochemical and Biophysical Research Communications 2001; 282: 697-700.
144. Dunn IF, Heese O, Black PM. Growth factors in glioma angiogenesis: FGFs, PDGF, EGF, and TGFs. Journal of Neuro-oncology 2000; 50(1-2): 121-37.
145. Nakamura T, Mochizuki Y, Kanetake H, Kanda S. Signals via FGF receptor 2 regulate migration of endothelial cells. Biochemical and Biophysical Research Communications 2001; 289: 801-6.
146. Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends in Pharmacological Sciences 2001; 22: 201-7.
147. Poole TJ, Finkelstein EB, Cox CM. The role of FGF and VEGF in angioblast induction and migration during vascular development. Developmental Dynamics 2001; 220: 1-17.