跳到主要內容

臺灣博碩士論文加值系統

(3.215.79.68) 您好!臺灣時間:2022/07/04 04:16
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:吳孟珈
研究生(外文):Meng-Chia Wu
論文名稱:樟芝菌絲體活性成分標靶頭頸癌腫瘤起始細胞之驗證與探討
論文名稱(外文):Identification and Characterization of 4-Acetylantroquinonol B of Antrodia Cinnamomea Mycelia on Targeting Head and Neck Cancer Initiating Cells
指導教授:羅正汎
指導教授(外文):Jeng-Fan Lo
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:67
中文關鍵詞:樟芝菌絲體頭頸癌癌症起始細胞高醋醛脫氫酶
外文關鍵詞:4-Acetylantroquinonol BCancer Initiating CellsHead and Neck CancerAntrodia CinnamomeaMyceliaALDHAldehyde dehydrogenaseALDEFLUOR kitreactive oxygen species
相關次數:
  • 被引用被引用:1
  • 點閱點閱:422
  • 評分評分:
  • 下載下載:6
  • 收藏至我的研究室書目清單書目收藏:0
摘要


癌症起始細胞(Cancer Initiating Cells, CICs)為一群具有幹細胞特性及生成腫瘤能力的亞群癌細胞。癌起始細胞被認為能夠抵抗傳統化療與放射線療法,進而造成腫瘤復發及腫瘤轉移。藥物開發以標靶癌症起始細胞,有助於未來癌症病患之治療。
依據近年來的研究發現高醋醛脫氫酶 (aldehyde dehydrogenase, ALDH)的活性為癌起始細胞之特性。故我們以ALDEFLUOR kit分析細胞內高醋醛脫氫酵素活性自樟芝菌絲體(Antrodia cinnamomea Mycelia, ACM)萃取物中篩選出能標靶頭頸癌症起始細胞的純物質。我們發現樟芝菌絲體萃取物中分離出之純物質4-Acetylantroquinonol B (4-AAQN B)能有效降低細胞內高醋醛脫氫酶活性。在本研究中我將探討4-AAQN B是否能標靶癌起始細胞及探討其對於此癌症起始細胞的影響。4-AAQN B 能夠抑制OECM1與SAS的高醋醛脫氫酶活性以及降低癌症起始幹細胞生物標記的表現如:CD44、uPAR及CCN1。我的研究並發現4-AAQN B 處理下能影響細胞內reactive oxygen species (ROS) 之含量; 而細胞內ROS 之含量亦與癌症起始幹細胞之特性有關。,因此4-AAQN B可做為標靶有活性的癌症起始幹細胞的藥物,可進而結合傳統的化療藥物作為未來癌症治療之方法。

Abstract


Cancer initiating cells (CICs), a subtype of cancer cells, have the enhanced stemness properties and tumorigenic abilities. CICs are also considered to render resistance of chemotherapy or radiotherapy, therefore; it results in recurrence and metastasis. Development of drugs that can specifically target cancer initiating cells, as a therapeutic method to against the CICs, would benefit for future treatment of cancer patients.
Recently, others’ and our discoveries have shown that the elevated activity of aldehyde dehydrogenase (ALDH) is closely related to CICs. Herein, we first used the ALDH assay to screen for purified compounds of the myceluim from Antrodia Cinnamomea (ACM) with inhibitory activity of ALDH in cancer cells. Then, we find out that treatment of 4-acetylantroquinonol B (4-AAQN B), which was isolated from ACM, significantly down regulated the ALDH activity of cancer cells. Further, the anticancer activity of 4-AAQN B by targeting CICs had been characterized.
Our results showed that the ALDH activity and expression of stemness biomarkers including CD44, uPAR and CCN1 of cancer cells were significantly decreased after 4-AAQN B administration. We also observed the reduced cellular viability in 4-AAQN B treated cancer cells. Of note, 4-AQQN B disturbed the intracellular content of reactive oxygen species (ROS) of cancer cell where the subpopulations of cancer cells with differential ROS content have been related to CICs.
The anticancer activity of 4-AAQN B has been reported before. However, we are the first group to confirm that 4-AAQN B can effectively target CICs. The abovementioned findings provide an alternative therapeutics for future cancer treatment in combination with conventional chemotherapy.

Contents
【Signature Page】 I
【Thesis Approval Form】 II
【Acknowledgements】 III
【English Abstract】 IV
【Chinese Abstract】 VI
【Table of Contents】 VIII
【List of Figures】 XI
【List of Figures】 XIII
【Introduction】 1
1.1 Head and neck squamous cell carcinoma (HNSCC) 2
1.2 Head and Neck Cancer initiating cells (HN-CICs) 2
1.3 Identification and characterization of cancer initiating cells 3
1.4 Antrodia Cinnanomea 4
1.5 4-Acetylantroquinonol B (4AAQN B) 5
【Hypothesis and Specific Aims】 5
【Materials and methods】 9
2.1 Cell lines and culture condition 9
2.2 4-Acetylantroquinonol B 10
2.3 Flow cytometry 10
2.4 ALDH assay 12
2.5 Reactive oxygen species (ROS) staining 13
2.6 AnnexinV/PI kit 13
2.7 Cell viability 14
2.8 Western blot 15
2.9 Migration 16
2.10 Anchorage-independent growth 17
2.11 Double stain 18
【Results】 20
3.1 ALDH+ activity, stemness marker, decreased after the treatment of 24 hours 4-Acetylantroqinonol B. 20
3.2 Cell morphology changed after treated 4-Acetylantroquinonol B. 21
3.3 4-Acetylantroquinonol B treatment for 24 hours decreased Cell viability. 21
3.4 CD44 is known as a stemness marker also decreased after 24hr 4-Acetylantroquinonol B treatments in HNSCC cell lines. 22
3.5 Parental cells detected AnnexinV/PI assay after 24 hours 4-Acetylantroquinonol B administrations. 23
3.6 Sphere cells treated 4-Acetylantroquinonol B changed morphology thinly. 23
3.7 4-Acetylantroquinonol B treated 24 hours not decreased cell viability in sphere cells. 24
3.8 Urokinase-type plasminogen activator receptor (uPAR), stemness markers, curtailed after the 24 hours 4-Acetylantroquinonol B administrations in sphere cells. 24
3.9 Stemness markers of CCN1 curtailed after the 24 hours 4-Acetylantroquinonol B administrations in OECM1-Sphere cells. 25
3.10 The cells with high intracellular level of reactive oxygen species (ROSHigh) were shifted toward to another ROS population after 24hr 4-Acetylantroquinonol B treatments in sphere cells. 26
3.11 Anchorage-independent growth was decreased for 24 hours 4-Acetylantroquinonol B treatment of sphere cells. 27
3.12 Sphere cells for 24 hours 4-Acetylantroquinonol B administration curtailed the migratory activity. 27
3.13 AnnexinV/PI assay not increased after 24 hours 4-Acetylantroquinonol B administrations in sphere cells. 28
3.14 The Propidium Iodide (PI+) cells were raised after 24hr 4-Acetylantroquinonol B treatments in sphere cells. 28
3.15 To confirm the stemness biomarkers and differentiation marker in SAS-Sphere cell line of western blotting. 29
3.16 To confirm the ROS expression compared with parental cells and sphere cells. 30
3.17 To confirm the ROS and stemness marker in SAS-Sphere cells. 30
【Discussion】 31

【Reference】 35






【List of Figures】 XI
Figure. 1 The stemness marker ALDH+ activity decreased after the treatment of 4-Acetylantroqinonol B in HNSCC cell lines. 40
Figure. 2 24hr 4-Acetylantroquinonol B treated in HNSCC cell lines cause cell morphology changed. 42
Figure. 3 The Cell viability was decreased after 24 hours and 48 hours 4-Acetylantroquinonol B treatments in HNSCC cell lines. 43
Figure. 4 Stemness biomarker CD44 was decreased after 24hr 4-Acetylantroquinonol B treated in HNSCC cell lines. 44
Figure. 5 The apoptosis cell death detected after 24 hours 4-Acetylantroquinonol B treated in HNSCC cell lines. 45
Figure. 6 24hr 4-Acetylantroquinonol B treated in Sphere cells would not change cell morphology. 46
Figure. 7 After 24 hours 4-Acetylantroquinonol B treated in sphere cells were not influenced the cell viability. 47
Figure. 8 Stemness biomarker uPAR were curtailed after the 24 hours 4-Acetylantroquinonol B administration in sphere cells. 48
Figure. 9 Stemness biomarker CCN1 were curtailed after the 24 hours 4-Acetylantroquinonol B administrated in OECM1-Sphere cells. 49
Figure. 10 The cells with high amount of intracellular reactive oxygen species (ROSHigh) diminished after 24hr 4-Acetylantroquinonol B treatments in sphere cells. 50
Figure. 11 The anchorage-independent growth was decreased after treated 24 hours 4-Acetylantroquinonol B in sphere cells. 51
Figure. 12 The migration capability was decreased after 24 hours hours 4-Acetylantroquinonol B treated in sphere cells. 52
Figure. 13 AnnexinV/PI stained of apoptosis cells death was not increased for 24 hours 4-Acetylantroquinonol B treatments in sphere cells. 53
Figure. 14 24 hours 4-Acetylantroquinonol B administration in sphere cells were increased Propidium Iodide positive (PI+) cells. 54
Figure. 15 Stemness biomarkers Oct4 and Nanog were reduced slightly and differentiation marker Involucrin was raised after the 24 hours 4-Acetylantroquinonol B administrations in SAS-Sphere cell line of western blotting. 55
Figure. 16 Measured reactive oxygen species (ROS) population in contrast to parental cells and sphere cells. 56
Figure. 17 Reactive oxygen species and stemness biomarker double stained in SAS-Sphere cells. 57










【List of Tables】 XIII
Table. 1 Cancer is top one major factor for death in Taiwan and oral cancer is the fifth common cancer. 58
Table. 2 Head and neck cancer congaing several organs. 59
Table. 3 Cancer stem cells as a therapeutic target. 60
Table. 4 Microarray of stemness markers CD44, uPAR, and CCN1 compared parental cells and sphere cells in our lab. 61
Table. 5 Reactive oxygen species (ROS) expressed in cancer stem cells. There were ROSLow and ROSHigh populations. 62
Table. 6 uPAR interacted with integrin and down regulate several signaling pathways. 63
Table. 7 CCN1 interacted with integrin and down regulate several signaling pathways. 64
Table. 8 memGrp78 expression in SAS-Parental cells, OECM1-Parental cells and SAS-Sphere cells. 65
Table. 9 HSC cell viability decreased to baseline under 4-Acetylantroquinonol B treated 40 hours. 66
Table. 10 Conclusion of all experiments under 4-Acetylantroquinonol B administration. 67




References:
1. Vokes EE, Weichselbaum RR, Lippman SM, Hong WK. Head and neck cancer. The New England journal of medicine. 1993;328:184-94.
2. Lewin F, Norell SE, Johansson H, Gustavsson P, Wennerberg J, Biorklund A, et al. Smoking tobacco, oral snuff, and alcohol in the etiology of squamous cell carcinoma of the head and neck: a population-based case-referent study in Sweden. Cancer. 1998;82:1367-75.
3. McKaig RG, Baric RS, Olshan AF. Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head & neck. 1998;20:250-65.
4. Marur S, D'Souza G, Westra WH, Forastiere AA. HPV-associated head and neck cancer: a virus-related cancer epidemic. The lancet oncology. 2010;11:781-9.
5. Chen YJ, Chang JT, Liao CT, Wang HM, Yen TC, Chiu CC, et al. Head and neck cancer in the betel quid chewing area: recent advances in molecular carcinogenesis. Cancer science. 2008;99:1507-14.
6. Brennan JA, Boyle JO, Koch WM, Goodman SN, Hruban RH, Eby YJ, et al. Association between cigarette smoking and mutation of the p53 gene in squamous-cell carcinoma of the head and neck. The New England journal of medicine. 1995;332:712-7.
7. Oksuz DC, Prestwich RJ, Carey B, Wilson S, Senocak MS, Choudhury A, et al. Recurrence patterns of locally advanced head and neck squamous cell carcinoma after 3D conformal (chemo)-radiotherapy. Radiation oncology. 2011;6:54.
8. Schwartz GJ, Mehta RH, Wenig BL, Shaligram C, Portugal LG. Salvage treatment for recurrent squamous cell carcinoma of the oral cavity. Head & neck. 2000;22:34-41.
9. Jerjes W, Upile T, Petrie A, Riskalla A, Hamdoon Z, Vourvachis M, et al. Clinicopathological parameters, recurrence, locoregional and distant metastasis in 115 T1-T2 oral squamous cell carcinoma patients. Head & neck oncology. 2010;2:9.
10. Jordan CT, Guzman ML, Noble M. Cancer stem cells. The New England journal of medicine. 2006;355:1253-61.
11. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646-74.
12. Frank NY, Schatton T, Frank MH. The therapeutic promise of the cancer stem cell concept. The Journal of clinical investigation. 2010;120:41-50.
13. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell stem cell. 2007;1:555-67.
14. Chen YC, Chen YW, Hsu HS, Tseng LM, Huang PI, Lu KH, et al. Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer. Biochemical and biophysical research communications. 2009;385:307-13.
15. Chiou SH, Yu CC, Huang CY, Lin SC, Liu CJ, Tsai TH, et al. Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008;14:4085-95.
16. Pyke C, Kristensen P, Ralfkiaer E, Grondahl-Hansen J, Eriksen J, Blasi F, et al. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas. The American journal of pathology. 1991;138:1059-67.
17. Chen YS, Huang WL, Chang SH, Chang KW, Kao SY, Lo JF, et al. Enhanced filopodium formation and stem-like phenotypes in a novel metastatic head and neck cancer cell model. Oncology reports. 2013;30:2829-37.
18. Jo M, Eastman BM, Webb DL, Stoletov K, Klemke R, Gonias SL. Cell signaling by urokinase-type plasminogen activator receptor induces stem cell-like properties in breast cancer cells. Cancer research. 2010;70:8948-58.
19. Gutova M, Najbauer J, Gevorgyan A, Metz MZ, Weng Y, Shih CC, et al. Identification of uPAR-positive chemoresistant cells in small cell lung cancer. PloS one. 2007;2:e243.
20. Haque I, Mehta S, Majumder M, Dhar K, De A, McGregor D, et al. Cyr61/CCN1 signaling is critical for epithelial-mesenchymal transition and stemness and promotes pancreatic carcinogenesis. Molecular cancer. 2011;10:8.
21. Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature. 2009;458:780-3.
22. Ogasawara MA, Zhang H. Redox regulation and its emerging roles in stem cells and stem-like cancer cells. Antioxidants & redox signaling. 2009;11:1107-22.
23. Phillips TM, McBride WH, Pajonk F. The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. Journal of the National Cancer Institute. 2006;98:1777-85.
24. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:3983-8.
25. Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ. Prospective identification of tumorigenic prostate cancer stem cells. Cancer research. 2005;65:10946-51.
26. Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, Dick JE. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nature medicine. 2006;12:1167-74.
27. Okamoto A, Chikamatsu K, Sakakura K, Hatsushika K, Takahashi G, Masuyama K. Expansion and characterization of cancer stem-like cells in squamous cell carcinoma of the head and neck. Oral oncology. 2009;45:633-9.
28. Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:973-8.
29. Dey-Guha I, Wolfer A, Yeh AC, J GA, Darp R, Leon E, et al. Asymmetric cancer cell division regulated by AKT. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:12845-50.
30. Le Belle JE, Orozco NM, Paucar AA, Saxe JP, Mottahedeh J, Pyle AD, et al. Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt-dependant manner. Cell stem cell. 2011;8:59-71.
31. Song TY, Yen GC. Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCl4-induced hepatic toxicity in rats. Journal of agricultural and food chemistry. 2003;51:1571-7.
32. Lee IH, Huang RL, Chen CT, Chen HC, Hsu WC, Lu MK. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS microbiology letters. 2002;209:63-7.
33. Hseu YC, Chang WC, Hseu YT, Lee CY, Yech YJ, Chen PC, et al. Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life sciences. 2002;71:469-82.
34. Liu JJ, Huang TS, Hsu ML, Chen CC, Lin WS, Lu FJ, et al. Antitumor effects of the partially purified polysaccharides from Antrodia camphorata and the mechanism of its action. Toxicology and applied pharmacology. 2004;201:186-93.
35. Hsu YL, Kuo PL, Cho CY, Ni WC, Tzeng TF, Ng LT, et al. Antrodia cinnamomea fruiting bodies extract suppresses the invasive potential of human liver cancer cell line PLC/PRF/5 through inhibition of nuclear factor kappaB pathway. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association. 2007;45:1249-57.
36. Yang HL, Chen CS, Chang WH, Lu FJ, Lai YC, Chen CC, et al. Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata. Cancer letters. 2006;231:215-27.
37. Hseu YC, Chen SC, Tsai PC, Chen CS, Lu FJ, Chang NW, et al. Inhibition of cyclooxygenase-2 and induction of apoptosis in estrogen-nonresponsive breast cancer cells by Antrodia camphorata. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association. 2007;45:1107-15.
38. Yeh CT, Rao YK, Yao CJ, Yeh CF, Li CH, Chuang SE, et al. Cytotoxic triterpenes from Antrodia camphorata and their mode of action in HT-29 human colon cancer cells. Cancer letters. 2009;285:73-9.
39. Liu YM, Liu YK, Lan KL, Lee YW, Tsai TH, Chen YJ. Medicinal Fungus Antrodia cinnamomea Inhibits Growth and Cancer Stem Cell Characteristics of Hepatocellular Carcinoma. Evidence-based complementary and alternative medicine : eCAM. 2013;2013:569737.
40. Chang CW, Chen CC, Wu MJ, Chen YS, Chen CC, Sheu SJ, et al. Active Component of Antrodia cinnamomea Mycelia Targeting Head and Neck Cancer Initiating Cells through Exaggerated Autophagic Cell Death. Evidence-based complementary and alternative medicine : eCAM. 2013;2013:946451.
41. Geethangili M, Tzeng YM. Review of Pharmacological Effects of Antrodia camphorata and Its Bioactive Compounds. Evidence-based complementary and alternative medicine : eCAM. 2011;2011:212641.
42. Yang SS, Wang GJ, Wang SY, Lin YY, Kuo YH, Lee TH. New constituents with iNOS inhibitory activity from mycelium of Antrodia camphorata. Planta medica. 2009;75:512-6.
43. Lin YW, Pan JH, Liu RH, Kuo YH, Sheen LY, Chiang BH. The 4-acetylantroquinonol B isolated from mycelium of Antrodia cinnamomea inhibits proliferation of hepatoma cells. Journal of the science of food and agriculture. 2010;90:1739-44.
44. Lin YW, Chiang BH. 4-acetylantroquinonol B isolated from Antrodia cinnamomea arrests proliferation of human hepatocellular carcinoma HepG2 cell by affecting p53, p21 and p27 levels. Journal of agricultural and food chemistry. 2011;59:8625-31.
45. Chen YY, Liu FC, Chou PY, Chien YC, Chang WS, Huang GJ, et al. Ethanol extracts of fruiting bodies of Antrodia cinnamomea suppress CL1-5 human lung adenocarcinoma cells migration by inhibiting matrix metalloproteinase-2/9 through ERK, JNK, p38, and PI3K/Akt signaling pathways. Evidence-based complementary and alternative medicine : eCAM. 2012;2012:378415.
46. Chiou SH, Wang ML, Chou YT, Chen CJ, Hong CF, Hsieh WJ, et al. Coexpression of Oct4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiation. Cancer research. 2010;70:10433-44.
47. Jeter CR, Liu B, Liu X, Chen X, Liu C, Calhoun-Davis T, et al. NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene. 2011;30:3833-45.
48. Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, et al. Transcriptional regulation of nanog by OCT4 and SOX2. The Journal of biological chemistry. 2005;280:24731-7.
49. Said JW, Nash G, Sassoon AF, Shintaku IP, Banks-Schlegel S. Involucrin in lung tumors. A specific marker for squamous differentiation. Laboratory investigation; a journal of technical methods and pathology. 1983;49:563-8.
50. Watt FM. Involucrin and other markers of keratinocyte terminal differentiation. The Journal of investigative dermatology. 1983;81:100s-3s.
51. Blasi F, Carmeliet P. uPAR: a versatile signalling orchestrator. Nature reviews Molecular cell biology. 2002;3:932-43.
52. Jun JI, Lau LF. Taking aim at the extracellular matrix: CCN proteins as emerging therapeutic targets. Nature reviews Drug discovery. 2011;10:945-63.
53. Wu MJ, Jan CI, Tsay YG, Yu YH, Huang CY, Lin SC, et al. Elimination of head and neck cancer initiating cells through targeting glucose regulated protein78 signaling. Molecular cancer. 2010;9:283.

連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊