Title

Withaferin A與TRAIL引起人類口腔癌細胞株細胞 凋亡協力作用之研究

Translated Titles

Withaferin A synergizes TRAIL-induced apoptosis in human oral cancer cells

Authors

林澤宇

Key Words

細胞凋亡 ; 醉茄素A ; 口腔癌 ; apoptosis ; Withaferin A ; oral cancer

PublicationName

臺灣大學動物學研究所學位論文

Volume or Term/Year and Month of Publication

2013年

Academic Degree Category

碩士

Advisor

郭光雄

Content Language

繁體中文

Chinese Abstract

Withaferin A是純化自一種印度傳統草藥 withania somnifera 的withanolide類化合物。它具有增強免疫、抗菌、抗發炎、抗血管新生的效果。 在台灣,口腔癌占男性癌症死亡的第四名,雖然化療和放療的技術被廣泛使用在治療口腔癌,但病患的五年存活率沒有明顯的改善。 Withaferin A 被發現能誘導許多癌細胞株細胞凋亡,如:乳癌、腎癌、肺癌、子宮頸癌等,因此有成為抗癌藥的潛力。 我們的研究發現,Withaferin A 可明顯抑制口腔癌細胞株 SAS和Ca9-22生長,且濃度愈高或作用時間愈長,抑制效應就愈明顯 (SAS與Ca9-22 之24 小時 IC50 分別為1.1μM及1.2μM)。 藉由流式細胞儀分析細胞之細胞週期結果顯示,以 1.1μM與1.2μM的 Withaferin A 處理 SAS 及 Ca9-22 細胞株 分別在12及24小時後,會明顯增加sub-G1 期細胞的百分比,可見得 Withaferin A 也可引起人類口腔癌細胞的細胞凋亡。西方墨點法和caspase抑制劑的實驗結果指出Withaferin A誘導Fas、DR4和DR5的表現,造成caspase 8(膜受體路徑)和caspase 9(粒線體路徑)活化,最後導致細胞凋亡。次毒性濃度(0.6μM)的 Withaferin A與TRAIL(5ng/ml)對口腔癌細胞株細胞凋亡有協力的效果。對於Withaferin A是否可當作口腔癌抗癌藥物則需要再以更精確的動物實驗及臨床試驗作更深入的評估其抗癌的功效。

English Abstract

Withanolide-like compound, Withaferin A, is purified from an Indian traditional herb, withania somnifera. Withanolide is one of steroidal lactones, it is antioxidant and has clinical medical activities, such as immunity increasing, anti-bacteria, anti-inflammatory, anti-angiogenesis, and antitumor. The last is the most striking activity. In Taiwan, oral cancer is the fourth cancer causing dead, although the technologies in radiotherapy and chemotherapy have been promoted, the five-years-survival rate of patients was no significant improvement. Withaferin A has been found to induce apoptosis in many kinds of cancer cell lines and regarded as a potential anti-cancer drug, ex: breast cancer’ kidney cancer’ lung cancer’ cervical cancer. In this study, we showed the Withaferin A inhibited strongly the growths of oral cancer cell lines, SAS and Ca9-22 with dose-dependent and time-dependent manners(IC50 value 1.1μM and 1.2μM respectively). Withaferin A increased the percentage of sub-G0/G1 dramatically (1.1μM and 1.2μM respectively). With the results of Western blot and trial of caspase inhibitor treatments, the results showed Withaferin A induced Fas’ DR4 and DR5 expressions, then activated caspase 8 (death receptor pathway) and also caspase 9 (mitochondrial pathway) , and final induce cell death. Withaferin A in sub-toxic concentration (0.6μM) could facilitate the sensitivity of oral cancer cell line to TRAIL (5ng/ml) and let to apoptosis. In conclusion, Withaferin A can be used as oral cancer drugs but needs to further research to confirm it efficacy.

Topic Category 生命科學院 > 動物學研究所
生物農學 > 動物學
Reference
  1. Akopova OV, Kolchinskaya LI, Nosar VI, Bouryi VA, Mankovska IN, Sagach VF.(2012) Cytochrome C as an amplifier of ROS release in mitochondria.Fiziol Zh. 2012;58(1):3-12
    連結:
  2. Ashkenazi A. Targeting the extrinsic apoptosis pathway in cancer. Cytokine Growth Factor Rev. 2008 Jun-Aug;19(3-4):325-31.
    連結:
  3. Avi Ashkenazi (2002) “Targeting death and decoy receptors of the tumour-necrosis factor superfamily” Nature Reviews Cancer 2, 420-430
    連結:
  4. Bai, L. Y., Chiu, C. F., Pan, S. L., Sargeant, A. M., Shieh, T. M., Wang, Y. C., & Weng, J. R. (2011). Antitumor activity of a novel histone deacetylase inhibitor (S)-HDAC42 in oral squamous cell carcinoma. Oral Oncol, 47(12), 1127-1133.
    連結:
  5. Bossi G, Sacchi A. “Restoration of wild-type p53 function in human cancer: Relevance for tumor therapy” Head Neck. 2007 Mar;29(3):272-84.
    連結:
  6. Chen, S. C. (2012). Life experiences of Taiwanese oral cancer patients during the postoperative period. Scand J Caring Sci, 26(1), 98-103
    連結:
  7. Chen, Y. W., Kao, S. Y., & Yang, M. H. (2012). Analysis of p16(INK4A) expression of oral squamous cell carcinomas in Taiwan: Prognostic correlation without relevance to betel quid consumption. J Surg Oncol, 106(2), 149-154.
    連結:
  8. Choi, M. J., E. J. Park, et al. (2011). "Endoplasmic reticulum stress mediates Withaferin A-induced apoptosis in human renal carcinoma cells." Toxicol In Vitro 25(3): 692-698.
    連結:
  9. Choudhary, M. I., S. Hussain, et al. (2010). "Chlorinated and diepoxy withanolides from Withania somnifera and their cytotoxic effects against human lung cancer cell line." Phytochemistry 71(17-18): 2205-2209.
    連結:
  10. Franchitto, A., A. Torrice, et al. (2010). "Prostate apoptosis response-4 is expressed in normal cholangiocytes, is down-regulated in human cholangiocarcinoma, and promotes apoptosis of neoplastic cholangiocytes when induced pharmacologically." Am J Pathol 177(4): 1779-1790.
    連結:
  11. Garcia-Perez C, Roy SS, Naghdi S, Lin X, Davies E, Hajnoczky G. (2012)Bid-induced mitochondrial membrane permeabilization waves propagated by local reactive oxygen species (ROS) signaling. Proc Natl Acad Sci U S A. ;109(12):4497-502
    連結:
  12. Gerspach, J., Pfizenmaier, K., & Wajant, H. (2011). Therapeutic targeting of CD95 and the TRAIL death receptors. Recent Pat Anticancer Drug Discov, 6(3), 294-310.
    連結:
  13. Grover, A., A. Shandilya, et al. (2011). "Hsp90/Cdc37 chaperone/co-chaperone complex, a novel junction anticancer target elucidated by the mode of action of herbal drug Withaferin A." BMC Bioinformatics 12 Suppl 1: S30.
    連結:
  14. Grover, A., V. Agrawal, et al. (2011). "Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug Withaferin A." BMC Bioinformatics 12 Suppl 13: S22.
    連結:
  15. Hahm, E. R., J. Lee, et al. (2011). "Withaferin A suppresses estrogen receptor-alpha expression in human breast cancer cells." Mol Carcinog 50(8): 614-624.
    連結:
  16. Hahm, E. R., M. B. Moura, et al. (2011). "Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species." PLoS One 6(8): e23354.
    連結:
  17. Huang, S. F., Cheng, S. D., Chuang, W. Y., Chen, I. H., Liao, C. T., Wang, H. M., & Hsieh, L. L. (2012). Cyclin D1 overexpression and poor clinical outcomes in Taiwanese oral cavity squamous cell carcinoma. World J Surg Oncol, 10, 40
    連結:
  18. Juanita C. Sharpe1, Damien Arnoult, Richard J. Youle(2003) “Control of mitochondrial permeability by Bcl-2 family members” Molecular Cell Research Volume 1644, Issues 2–3, 1, 107–113
    連結:
  19. Kalthur, G. and U. D. Pathirissery (2010). "Enhancement of the response of B16F1 melanoma to fractionated radiotherapy and prolongation of survival by Withaferin A and/or hyperthermia." Integr Cancer Ther 9(4): 370-377.
    連結:
  20. Kerr JF, Wyllie AH, Currie AR. (1972) “Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics” Br J Cancer.
    連結:
  21. Koduru, S., R. Kumar, et al. (2010). "Notch-1 inhibition by Withaferin-A: a therapeutic target against colon carcinogenesis." Mol Cancer Ther 9(1): 202-210.
    連結:
  22. Kumar, S. P., G. Thippeswamy, et al. (2008). "WITHDRAWN: Anti-tumor and proapoptotic effect of Withaferin A is mediated by up-regulation of Bax and inhibition NF-kappaB in Ehrlich ascites tumor cells." Biochim Biophys Acta.
    連結:
  23. Lee, J., E. R. Hahm, et al. (2010). "Withaferin A inhibits activation of signal transducer and activator of transcription 3 in human breast cancer cells." Carcinogenesis 31(11): 1991-1998.
    連結:
  24. Lee, T. J., H. J. Um, et al. (2009). "Withaferin A sensitizes TRAIL-induced apoptosis through reactive oxygen species-mediated up-regulation of death receptor 5 and down-regulation of c-FLIP." Free Radic Biol Med 46(12): 1639-1649.
    連結:
  25. Lee, Y. R., Wu, W. C., Ji, W. T., Chen, J. Y., Cheng, Y. P., Chiang, M. K., & Chen, H. R. (2012). Reversine suppresses oral squamous cell carcinoma via cell cycle arrest and concomitantly apoptosis and autophagy. J Biomed Sci, 19, 9.
    連結:
  26. Lin, Y. S., Chen, S. F., Liu, C. L., & Nieh, S. (2012). The chemoadjuvant potential of grape seed procyanidins on p53-related cell death in oral cancer cells. J Oral Pathol Med, 41(4), 322-331
    連結:
  27. Liu, X., W. Qi, et al. (2011). "An analog of Withaferin A activates the MAPK and glutathione "stress" pathways and inhibits pancreatic cancer cell proliferation." Cancer Invest 29(10): 668-675.
    連結:
  28. Malara, N., D. Foca, et al. (2008). "Simultaneous inhibition of the constitutively activated nuclear factor kappaB and of the interleukin-6 pathways is necessary and sufficient to completely overcome apoptosis resistance of human U266 myeloma cells." Cell Cycle 7(20): 3235-3245.
    連結:
  29. Malik, F., A. Kumar, et al. (2007). "Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound Withaferin A with concomitant protection by N-acetyl cysteine." Apoptosis 12(11): 2115-2133.
    連結:
  30. Mandal, C., A. Dutta, et al. (2008). "Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade." Apoptosis 13(12): 1450-1464.
    連結:
  31. Manoharan, S., K. Panjamurthy, et al. (2009). "Circadian time-dependent chemopreventive potential of withaferin-A in 7,12-dimethylbenz[a]anthracene-induced oral carcinogenesis." Pharmacol Rep 61(4): 719-726.
    連結:
  32. Manoharan, S., K. Panjamurthy, et al. (2009). "Protective effect of Withaferin-A on tumour formation in 7,12-dimethylbenz[a]anthracene induced oral carcinogenesis in hamsters." Indian J Exp Biol 47(1): 16-23.
    連結:
  33. Martinez-Lostao, L., Marzo, I., Anel, A., & Naval, J. (2012). Targeting the Apo2L/TRAIL system for the therapy of autoimmune diseases and cancer. Biochem Pharmacol, 83(11), 1475-1483.
    連結:
  34. Mayola, E., C. Gallerne, et al. (2011). "Withaferin A induces apoptosis in human melanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2." Apoptosis 16(10): 1014-1027.
    連結:
  35. Mehrotra, A., D. Kaul, et al. (2011). "LXR-alpha selectively reprogrammes cancer cells to enter into apoptosis." Mol Cell Biochem 349(1-2): 41-55.
    連結:
  36. Min, K. J., K. Choi, et al. (2011). "Withaferin A down-regulates lipopolysaccharide-induced cyclooxygenase-2 expression and PGE2 production through the inhibition of STAT1/3 activation in microglial cells." Int Immunopharmacol 11(8): 1137-1142.
    連結:
  37. Mirjalili, M. H., E. Moyano, et al. (2009). "Steroidal lactones from Withania somnifera, an ancient plant for novel medicine." Molecules 14(7): 2373-2393.
    連結:
  38. Mirzajani, F., A. Ghassempour, et al. (2010). "Optimisation of a microwave-assisted method for extracting Withaferin A from Withania somnifera Dunal. using central composite design." Phytochem Anal 21(6): 544-549.
    連結:
  39. Nakajima, H., Y. Wakabayashi, et al. (2011). "An Extract of Withania somnifera Attenuates Endothelin-1-stimulated Pigmentation in Human Epidermal Equivalents through the Interruption of PKC Activity Within Melanocytes." Phytother Res.
    連結:
  40. Ndlovu, M. N., C. Van Lint, et al. (2009). "Hyperactivated NF-{kappa}B and AP-1 transcription factors promote highly accessible chromatin and constitutive transcription across the interleukin-6 gene promoter in metastatic breast cancer cells." Mol Cell Biol 29(20): 5488-5504.
    連結:
  41. Oh, J. H. and T. K. Kwon (2009). "Withaferin A inhibits tumor necrosis factor-alpha-induced expression of cell adhesion molecules by inactivation of Akt and NF-kappaB in human pulmonary epithelial cells." Int Immunopharmacol 9(5): 614-619.
    連結:
  42. Oh, J. H., T. J. Lee, et al. (2008). "Induction of apoptosis by Withaferin A in human leukemia U937 cells through down-regulation of Akt phosphorylation." Apoptosis 13(12): 1494-1504.
    連結:
  43. Pai, P. C., Chuang, C. C., Tseng, C. K., Tsang, N. M., Chang, K. P., Yen, T. C., . . . Chang, J. T. (2012). Impact of pretreatment body mass index on patients with head-and-neck cancer treated with radiation. Int J Radiat Oncol Biol Phys, 83(1), e93-e100.
    連結:
  44. Panjamurthy, K., S. Manoharan, et al. (2008). "Protective effect of Withaferin-A on micronucleus frequency and detoxication agents during experimental oral carcinogenesis." Afr J Tradit Complement Altern Med 6(1): 1-8.
    連結:
  45. Panjamurthy, K., S. Manoharan, et al. (2009). "Protective role of Withaferin-A on immunoexpression of p53 and bcl-2 in 7,12-dimethylbenz(a)anthracene-induced experimental oral carcinogenesis." Invest New Drugs 27(5): 447-452.
    連結:
  46. Perego, P., Zuco, V., Gatti, L., & Zunino, F. (2012). Sensitization of tumor cells by targeting histone deacetylases. Biochem Pharmacol, 83(8), 987-994.
    連結:
  47. Pretorius, E., H. M. Oberholzer, et al. (2009). "Comparing the cytotoxic potential of Withania somnifera water and methanol extracts." Afr J Tradit Complement Altern Med 6(3): 275-280.
    連結:
  48. Pugh, T. J., & Lee, A. K. (2011). Role of radiation therapy for the treatment of lymph nodes in urologic malignancies. Urol Clin North Am, 38(4), 497-506, vii.
    連結:
  49. Samadi, A. K., X. Tong, et al. (2010). "Withaferin A, a cytotoxic steroid from Vassobia breviflora, induces apoptosis in human head and neck squamous cell carcinoma." J Nat Prod 73(9): 1476-1481.
    連結:
  50. Santagata, S., Y. M. Xu, et al. (2012). "Using the heat-shock response to discover anticancer compounds that target protein homeostasis." ACS Chem Biol 7(2): 340-349.
    連結:
  51. Sharmila Shankar, Rakesh K Srivastava(2004) “Enhancement of therapeutic potential of TRAIL by cancer chemotherapy and irradiation: mechanisms and clinical implications”. Drug Resistance Updates Volume 7, Issue 2, April 2004, 139–156
    連結:
  52. Shirley, S., Morizot, A., & Micheau, O. (2011). Regulating TRAIL receptor-induced cell death at the membrane : a deadly discussion. Recent Pat Anticancer Drug Discov, 6(3), 311-323.
    連結:
  53. Srinivasan, S., R. S. Ranga, et al. (2007). "Par-4-dependent apoptosis by the dietary compound Withaferin A in prostate cancer cells." Cancer Res 67(1): 246-253.
    連結:
  54. Stan, S. D., E. R. Hahm, et al. (2008). "Withaferin A causes FOXO3a- and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo." Cancer Res 68(18): 7661-7669.
    連結:
  55. Stan, S. D., Y. Zeng, et al. (2008). "Ayurvedic medicine constituent Withaferin A causes G2 and M phase cell cycle arrest in human breast cancer cells." Nutr Cancer 60 Suppl 1: 51-60.
    連結:
  56. Suzanne L Iverson, Sten Orrenius(2003) “The cardiolipin–cytochrome c interaction and the mitochondrial regulation of apoptosis” Archives of Biochemistry and Biophysics Volume 423, Issue 1, 1 March 2004, 37–46
    連結:
  57. Takimoto R, El-Deiry WS. Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site Oncogene. 2000 Mar 30;19(14):1735-43.
    連結:
  58. Thaiparambil, J. T., L. Bender, et al. (2011). "Withaferin A inhibits breast cancer invasion and metastasis at sub-cytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation." Int J Cancer 129(11): 2744-2755.
    連結:
  59. Voelkel-Johnson, C. (2011). TRAIL-mediated signaling in prostate, bladder and renal cancer. Nat Rev Urol, 8(8), 417-427
    連結:
  60. Widodo, N., D. Priyandoko, et al. (2010). "Selective killing of cancer cells by Ashwagandha leaf extract and its component Withanone involves ROS signaling." PLoS One 5(10): e13536.
    連結:
  61. Yang, E. S., M. J. Choi, et al. (2011). "Combination of Withaferin A and X-ray irradiation enhances apoptosis in U937 cells." Toxicol In Vitro 25(8): 1803-1810.
    連結:
  62. Yang, H., G. Shi, et al. (2007). "The tumor proteasome is a primary target for the natural anticancer compound Withaferin A isolated from "Indian winter cherry"." Mol Pharmacol 71(2): 426-437.
    連結:
  63. Yen, C. Y., Chiu, C. C., Haung, R. W., Yeh, C. C., Huang, K. J., Chang, K. F., . . . Wu, Y. C. (2012). Antiproliferative effects of goniothalamin on Ca9-22 oral cancer cells through apoptosis, DNA damage and ROS induction. Mutat Res, 747(2), 253-258
    連結:
  64. Yerbes, R., Palacios, C., & Lopez-Rivas, A. (2011). The therapeutic potential of TRAIL receptor signalling in cancer cells. Clin Transl Oncol, 13(12), 839-847.
    連結:
  65. Stuart S Martin, Kristiina Vuori(2004) “Regulation of Bcl-2 proteins during anoikis and amorphosis” Biochimica et Biophysica Acta (BBA) - Molecular Cell Research Volume 1692, Issues 2–3, 145–157
  66. Uma Devi, P., H. Utsumiz, et al. (2008). "Enhancement of radiation induced cell death in chicken B lymphocytes by Withaferin A." Indian J Exp Biol 46(6): 437-442