肝癌在全球癌症致死率排名第二，而肝內轉移途徑是造成肝癌復發與高死亡率的主要原因之一。但肝癌轉移機制仍不清楚。三環氧化物triptolide的衍生物teroxirone為一合成的抗癌試劑，過去研究發現teroxirone會使人類非小細胞肺癌細胞經內源性途徑細胞凋亡。本篇使用常見的肝癌細胞株Huh7 (具有突變p53)、HepG2 (野生型p53)與Hep3B (無p53)進行實驗，實驗顯示teroxirone會使肝癌細胞株Huh7產生外源性途徑細胞凋亡。進一步使用caspase-3抑制劑Z-DEVD-FMK來確認teroxirone所誘導Huh7產生細胞凋亡是否透過外源性途徑，發現細胞在加藥前經過caspase-3抑制劑處理，外源性細胞凋亡相關的蛋白質表現降低。另外，透過傷口癒合檢測 (wound-healing assay)與細胞遷移與侵入實驗 (transwell migration and invasion assay)都顯示藥物濃度低於IC50的teroxirone處理可以抑制Huh7產生細胞遷移與侵入。而利用酶譜分析 (zymography)與西方墨點法 (western blot)之結果可看出在低濃度的teroxirone處理後促進細胞轉移相關的蛋白基因表現皆有受到抑制。此外，證明了teroxirone會去降低Huh7 stem-like cells增生與其幹細胞指標。在動物模式中，異體移植的Huh7腫瘤teroxirone處理，會使腫瘤透過細胞凋亡的方式達到抑制腫瘤生長的目的。結論，teroxirone會透過活化外源性細胞凋亡途徑抑制人類肝癌細胞的生長，且低濃度的藥物處理能抑制肝癌細胞的遷移與轉移。另外，teroxirone能降低肝癌類幹細胞的幹細胞特性，並抑制動物模式中異體移植之腫瘤生長，顯示teroxirone在治療肝癌具有發展潛力。

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality, worldwide. The poor prognosis and high mortality rate is mainly the result of intra-hepatic metastases. The mechanisms underlying the metastasis of HCC remain unclear. The triepoxide derivative, teroxirone, is a synthetic antitumor agent. Previously, teroxirone was shown causing apoptotic cell death in human non-small cell lung cancer cells and the effects related to intrinsic pathway. In this study, human HCC cell lines HepG2 (wild type p53) , Huh7 (mutated p53), and Hep3B (p53 null) were used to study the effect of teroxirone on liver cancer cells. Teroxirone was shown inducing of apoptotic cell death of Huh7 cells through extrinsic pathway. Caspase-3 inhibitor, Z-DEVD-FMK, was used to confirm that teroxirone induced Huh7 apoptosis by inducing extrinsic pathway characters. Protein levels of extrinsic apoptosis pathway markers were decreased as a result caspase-3 inhibitor pretreatment. The data of wound-healing assay, transwell migration assay and transwell invasion assay, showed that low concentrations of teroxirone suppressed migration and invasion determination of Huh7 cells. The zymographic and western blot analysis indicated that the expression of metastasis promoting genes MMP-2, MMP-9, and VEGF were reduced after low concentrations teroxirone treatment. Furthermore, we also proved that teroxirone attenuated the cell viability and the stem cell markers intensity of Huh7 stem-like cells. In an in vivo model, subcutaneous injection of teroxirone suppressed the growth of xenograft Huh7 tumor cells by apoptosis. In conclusion, this research elucidates the effects and molecular mechanism for teroxirone on HCC cells. Thus, teroxirone is a new candidate in eradicating liver cancer cells.

1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, et al. (2015) Global cancer statistics, 2012. Ca-A Cancer Journal for Clinicians 65: 87-108.

2. Fan JY, Huang TJ, Jane SW, Chen MY (2015) Prevention of Liver Cancer Through the Early Detection of Risk-related Behavior Among Hepatitis B or C Carriers. Cancer Nursing 2014/05/20 ed. pp. 169-176.

3. Gopalan B, Narayanan K, Ke Z, Lu T, Zhang Y, et al. (2014) Therapeutic effect of a multi-targeted imidazolium compound in hepatocellular carcinoma. Biomaterials 35: 7479-7487.

4. Mokdad AA, Singal AG, Yopp AC (2015) JAMA PATIENT PAGE. Liver Cancer. Jama 314: 2701.

5. Lee DC, Kang YK, Kim WH, Jang YJ, Kim DJ, et al. (2008) Functional and clinical evidence for NDRG2 as a candidate suppressor of liver cancer metastasis. Cancer Research 68: 4210-4220.

6. Truant S, Sequier C, Leteurtre E, Boleslawski E, Elamrani M, et al. (2015) Tumour biology of colorectal liver metastasis is a more important factor in survival than surgical margin clearance in the era of modern chemotherapy regimens. The Official Journal of the International Hepato Pancreato Biliary Association 17: 176-184.

7. Deryugina EI, Quigley JP (2006) Matrix metalloproteinases and tumor metastasis. Cancer and Metastasis Reviews 25: 9-34.

8. Papetti M, Herman IM (2002) Mechanisms of normal and tumor-derived angiogenesis. American Journal of Physiology: Cell Physiology 282: C947-970.

9. Zetter BR (1998) Angiogenesis and tumor metastasis. Annual Review of Medicine 49: 407-424.

10. Wu H, Zhao W, Liu S, Zheng J, Ji G, et al. (2015) Pure transcatheter arterial chemoembolization therapy for intrahepatic tumors causes a shrink in pulmonary metastases of hepatocellular carcinoma. International Journal of Clinical and Experimental Medicine 8: 1035-1042.

11. Tang ZY (2001) Hepatocellular carcinoma--cause, treatment and metastasis. World Journal of Gastroenterology 7: 445-454.

12. Yakkundi A, Bennett R, Hernandez-Negrete I, Delalande JM, Hanna M, et al. (2015) FKBPL is a critical antiangiogenic regulator of developmental and pathological angiogenesis. Arteriosclerosis, Thrombosis, and Vascular Biology 35: 845-854.

13. Folkman J, Kalluri R (2004) Cancer without disease. Nature 427: 787.

14. Saharinen P, Eklund L, Pulkki K, Bono P, Alitalo K (2011) VEGF and angiopoietin signaling in tumor angiogenesis and metastasis. Trends in Molecular Medicine 17: 347-362.

15. Zheng H, Takahashi H, Murai Y, Cui Z, Nomoto K, et al. (2006) Expressions of MMP-2, MMP-9 and VEGF are closely linked to growth, invasion, metastasis and angiogenesis of gastric carcinoma. Anticancer Research 26: 3579-3583.

16. Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nature Medicine 9: 669-676.

17. Adams RH, Alitalo K (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nature Reviews Molecular Cell Biology 8: 464-478.

18. Maatta M, Soini Y, Liakka A, Autio-Harmainen H (2000) Differential expression of matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP in hepatocellular and pancreatic adenocarcinoma: implications for tumor progression and clinical prognosis. Clinical Cancer Research 6: 2726-2734.

19. Chetty C, Lakka SS, Bhoopathi P, Rao JS (2010) MMP-2 alters VEGF expression via alphaVbeta3 integrin-mediated PI3K/AKT signaling in A549 lung cancer cells. International Journal of Cancer 127: 1081-1095.

20. Shamon LA, Pezzuto JM, Graves JM, Mehta RR, Wangcharoentrakul S, et al. (1997) Evaluation of the mutagenic, cytotoxic, and antitumor potential of triptolide, a highly oxygenated diterpene isolated from Tripterygium wilfordii. Cancer Letters 112: 113-117.

21. Ames MM, Kovach JS, Rubin J (1984) Pharmacological characterization of teroxirone, a triepoxide antitumor agent, in rats, rabbits, and humans. Cancer Research 44: 4151-4156.

22. Neidhart JA, Derocher D, Grever MR, Kraut EH, Malspeis L (1984) Phase I trial of teroxirone. Cancer Treatment Reports 68: 1115-1119.

23. Atassi G, Spreafico F, Dumont P, Nayer P, Klastersky J (1980) Antitumoral effect in mice of a new triepoxyde derivative: 1, 3, 5-triglycidyl-s-triazinetrione (NSC 296934). European Journal of Cancer 16: 1561-1567.

24. Wang JP, Lin KH, Liu CY, Yu YC, Wu PT, et al. (2013) Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53. Toxicology and Applied Pharmacology 273: 110-120.

25. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicologic Pathology 35: 495-516.

26. Zhao X, Liu X, Su L (2014) Parthenolide induces apoptosis via TNFRSF10B and PMAIP1 pathways in human lung cancer cells. Journal of Experimental & Clinical Cancer Research 33: 3.

27. DiPietrantonio AM, Hsieh T, Wu JM (1999) Activation of caspase 3 in HL-60 cells exposed to hydrogen peroxide. Biochemical and Biophysical Research Communications 255: 477-482.

28. Hara H, Friedlander RM, Gagliardini V, Ayata C, Fink K, et al. (1997) Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proceedings of the National Academy of Sciences of the United States of America 94: 2007-2012.

29. Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, et al. (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37-43.

30. Longthorne VL, Williams GT (1997) Caspase activity is required for commitment to Fas-mediated apoptosis. EMBO Journal 16: 3805-3812.

31. Boldin MP, Goncharov TM, Goltsev YV, Wallach D (1996) Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85: 803-815.

32. Knoblich JA (2008) Mechanisms of asymmetric stem cell division. Cell 132: 583-597.

33. Bishop AE, Buttery LD, Polak JM (2002) Embryonic stem cells. The Journal of Pathology 197: 424-429.

34. Yu Z, Pestell TG, Lisanti MP, Pestell RG (2012) Cancer stem cells. International Journal of Biochemistry & Cell Biology 44: 2144-2151.

35. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, et al. (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367: 645-648.

36. Li F, Tiede B, Massague J, Kang Y (2007) Beyond tumorigenesis: cancer stem cells in metastasis. Cell Research 17: 3-14.

37. Dean M, Fojo T, Bates S (2005) Tumour stem cells and drug resistance. Nature Reviews Cancer 5: 275-284.

38. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J (1982) Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cell Research 42: 3858-3863.

39. Knowles BB, Howe CC, Aden DP (1980) Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209: 497-499.

40. Han MS, Barrett T, Brehm MA, Davis RJ (2016) Inflammation Mediated by JNK in Myeloid Cells Promotes the Development of Hepatitis and Hepatocellular Carcinoma. Cell Reports 15: 19-26.

41. Muzio M, Stockwell BR, Stennicke HR, Salvesen GS, Dixit VM (1998) An induced proximity model for caspase-8 activation. Journal of Biological Chemistry 273: 2926-2930.

42. LaVallee TM, Zhan XH, Johnson MS, Herbstritt CJ, Swartz G, et al. (2003) 2-methoxyestradiol up-regulates death receptor 5 and induces apoptosis through activation of the extrinsic pathway. Cancer Research 63: 468-475.

43. Elrod HA, Lin YD, Yue P, Wang X, Lonial S, et al. (2007) The alkylphospholipid perifosine induces apoptosis of human lung cancer cells requiring inhibition of Akt and activation of the extrinsic apoptotic pathway. Molecular Cancer Therapeutics 6: 2029-2038.

44. Li Q, Liu Z, Xu M, Xue Y, Yao B, et al. (2016) PCAF inhibits hepatocellular carcinoma metastasis by inhibition of epithelial-mesenchymal transition by targeting Gli-1. Cancer Letters 375: 190-198.

45. Xu LB, Liu C (2014) Role of liver stem cells in hepatocarcinogenesis. World journal of stem cells 6: 579-590.
46. Yamashita T, Wang XW (2013) Cancer stem cells in the development of liver cancer. Journal of Clinical Investigation 123: 1911-1918.

47. Douville J, Beaulieu R, Balicki D (2009) ALDH1 as a functional marker of cancer stem and progenitor cells. Stem Cells and Development 18: 17-25.

48. Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, et al. (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Genetics 38: 431-440.

49. Ku CY, Wang YR, Lin HY, Lu SC, Lin JY (2015) Corosolic Acid Inhibits Hepatocellular Carcinoma Cell Migration by Targeting the VEGFR2/Src/FAK Pathway. PLoS One 10: e0126725.

50. Shivji MK, Podust VN, Hubscher U, Wood RD (1995) Nucleotide excision repair DNA synthesis by DNA polymerase epsilon in the presence of PCNA, RFC, and RPA. Biochemistry 34: 5011-5017.

51. Kurki P, Vanderlaan M, Dolbeare F, Gray J, Tan EM (1986) Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle. Experimental Cell Research 166: 209-219.

52. Bravo R, Frank R, Blundell PA, Macdonald-Bravo H (1987) Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature 326: 515-517.

53. Nagata S (2000) Apoptotic DNA fragmentation. Experimental Cell Research 256: 12-18.

54. Ihara T, Yamamoto T, Sugamata M, Okumura H, Ueno Y (1998) The process of ultrastructural changes from nuclei to apoptotic body. MedSci Entry for Virchows Archiv 433: 443-447.

55. Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. Journal of Cell Biology 119: 493-501.