臺灣博碩士論文加值系統

前言：鼠尾草酸(Carnosic acid, CA)是迷迭香中一種主要的多酚類雙萜，已經被發現有許多功能，包括抗癌活性。

方法：本研究中使用人類乳癌細胞株(MCF-7, ZR-75-1, MDA-MB-231, MDA-MB-453, and BT-474)、纖維母細胞株(NIH-3T3)與黑色素瘤細胞株(B16F10)來測試鼠尾草酸的抗癌作用。在乳癌的部分，利用MTT法、傷痕癒合測試與聚落形成實驗來檢測細胞的生長與增殖，以流式細胞儀測定細胞週期，COX4、GAPDH、BAX、Bcl-2、Bcl-xL、β-actin、cytochrome C (Cyt.C)、endonuclease G (EndoG)與apoptosis inducing factor (AIF)的表現量則以西方墨點法與定量即時聚合酶鏈鎖反應進行測定，ATP生產量的測定使用Mitochondrial ToxGlo assay完成。而在黑色素瘤的部分，除了MTT法、傷痕癒合測試與聚落形成實驗，BrdU法亦被使用來測試黑色素瘤細胞B16F10的細胞生長與增殖。細胞週期仍以流式細胞儀測定，而p21與p27的表現量則以西方墨點法檢測。研究中亦建立黑色素瘤細胞移植動物模型，並以鼠尾草酸、卡莫司汀(carmustine，BCNU)或洛莫司汀(lomustine，CCNU)進行治療與評估。

結果：在乳癌細胞的測試結果中，鼠尾草酸的作用並不會受到動情激素受體陽性或陰性的影響，鼠尾草酸可以調節乳癌細胞的BAX、Bcl-2與Bcl-xL的表現量，啟動BAX的訊息傳導並導致凋亡因子釋放。此外，鼠尾草酸與順鉑(cisplatin)的合併療法對MCF-7 與MDA-MB-231細胞具有提升人類乳癌細胞對順鉑的敏感度的傾向。另一方面，本篇研究亦探討鼠尾草酸對黑色素瘤的作用，發現鼠尾草酸顯著抑制黑色素瘤細胞B16F10的生長，且使其細胞週期停滯，鼠尾草酸可使細胞週期滯留在G0/G1 期並增加p21的表現。再者，鼠尾草酸可增強卡莫司汀與洛莫司汀在B16F10細胞中引發的細胞毒性與細胞週期停滯。在體內測試中，鼠尾草酸抑制腫瘤生長同時也降低麩胺酸苯醋酸轉氨基酵素(aspartate aminotransferase，AST)與血清麩胺酸丙酮酸轉氨基酵素(alanine aminotransferase，ALT)的數值。

結論：鼠尾草酸或許可以作為一種安全且有效的新化療藥物。

Background: Carnosic acid (CA), a major polyphenolic diterpene in Rosmarinus officinalis, has been reported to have multiple functions, including anti-tumor activity.

Methods: To evaluate the anti-cancer effects of CA, the human breast cancer cell lines (MCF-7, ZR-75-1, MDA-MB-231, MDA-MB-453, and BT-474), fibroblast cell line (NIH-3T3) and melanoma cell line (B16F10) were used and tested. For the breast cancer cells, MTT assay, wound healing and colony formation were utilized for the detection of cell growth and proliferation. The cell cycle was determined by flow cytometry. COX4, GAPDH, BAX, Bcl-2, Bcl-xL, β-actin, cytochrome c (Cyt.C), endonuclease G (EndoG) and apoptosis inducing factor (AIF) expression were detected by western blotting and quantitative RT-PCR. The ATP production detection was done by Mitochondrial ToxGlo assay. For the melanoma cells, BrdU incorporation was used to detect melanoma cell growth and proliferation in addition to MTT assay, wound healing and colony formation. Flow cytometry was also conducted to detect cell cycle, and western blotting was conducted to detect p21 and p27 expressions. Melanoma cell xenograft model was also established, and the treatment potential of CA, carmustine (BCNU), or lomustine (CCNU) was evaluated in this present study.

Results: According to the results of the breast cancer cells experiment, the effect of CA was not affected by estrogen receptor (ER) either positively or negatively. CA was able to regulate the expression of BAX, Bcl-2 and Bcl-xL, trigger BAX translocation which lead to release of apoptotic factors in breast cancer cells. Moreover, CA was found to have a tendency to sensitize ER-positive and -negative human breast cancer cells to cisplatin by combined treatment of CA and cisplatin on MCF-7 and MDA-MB-231 cells. On the other hand, the investigation of the effect of CA on melanoma found that CA exhibited significant growth inhibition and cell cycle arrest in melanoma B16F10 cells. We also found that CA triggered cell cycle arrest at G0/G1 phase and enhanced p21 expression. Moreover, CA could enhance BCNU- and CCNU-mediated cytotoxicity and cell cycle arrest in B16F10 cells. Finally, we found that CA inhibited tumor growth, and reduced the values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in vivo.

Conclusion: CA may be safe and useful as a novel chemotherapeutic agent.

論文口試委員會審定書 I
謝誌 II
中文摘要 III
英文摘要 V
目錄 VII
表目錄 X
圖目錄 XI
1. Introduction 1
2. Materials and Methods 4
2.1 Cell line and reagents 4
2.2 Cytotoxicity detection 4
2.3 Cell proliferation assay 5
2.4 Wound healing assay 6
2.5 Soft agar colony formation assay 6
2.6 Cell cycle detection 6
2.7 Western blotting 7
2.8 Apoptosis detection 7
2.9 Mitochondrial and cytosolic fractions isolation 8
2.10 Quantitative RT-PCR 8
2.11 ATP production detection 9
2.12 Ethics approval and consent to participate 9
2.13 Mouse Xenograft Model 9
2.14 Statistical Analysis 10
3. Results and discussion 11
3.1 The effect of CA on breast cancer cells. 11
3.1.1 CA inhibited cell growth and induced apoptosis in ER-positive and -negative human breast cancer cells. 11
3.1.2 CA regulated pro- and anti-apoptotic protein expression. 12
3.1.3 CA-triggered BAX translocation led to apoptotic factor release and mitochondrial dysfunction. 13
3.1.4 CA sensitized MCF-7 and MDA-MB-231 cells to cisplatin. 15
3.2 The effect of CA on melanoma cells. 16
3.2.1 CA inhibited cell growth in melanoma cells. 16
3.2.2 CA edhanced the cytotoxic effects of BCNU and CCNU in B16F10 cells 17
3.2.3 CA regulated p21 and p27 protein expression of B16F10. 17
3.2.4 CA inhibited B16F10 tumor growth and enhanced anti-tumor effects of BCNU and CCNU. 18
4. Conclusions 21
Abbreviations: 22
References 23
Tables 31
Figures 38
自述 (個人履歷介紹，附個人照片擋) 62

1.Poeckel D, Greiner C, Verhoff M, Rau O, Tausch L, Hornig C, et al. Carnosic acid and carnosol potently inhibit human 5-lipoxygenase and suppress pro-inflammatory responses of stimulated human polymorphonuclear leukocytes. Biochemical Pharmacology. 2008; 76: 91-7
2.Yesil-Celiktas O, Sevimli C, Bedir E, Vardar-Sukan F. Inhibitory effects of rosemary extracts, carnosic acid and rosmarinic acid on the growth of various human cancer cell lines. Plant Foods for Human Nutrition. 2010; 66: 158-63.
3.Shin HB, Choi MS, Ryu B, Lee NR, Kim HI, Choi HE, et al. Antiviral activity of carnosic acid against respiratory syncytial virus. Virology Journal. 2013; 10: 303.
4.Tsai CW, Lin CY, Lin HH,Chen JH. Carnosic acid, A rosemary phenolic compound, induces apoptosis through reactive oxygen species-mediated p38 activation in human neuroblastoma IMR-32 cells. Neurochemical Research. 2011; 36: 2442-51.
5.Barni MV, Carlini MJ, Cafferata EG, Puricelli L, Moreno S. Carnosic acid inhibits the proliferation and migration capacity of human colorectal cancer cells. Oncology. 2012; 27: 1041-48.
6.Park SY, Song H, Sung MK, Kang YH, Lee KW, Park JH. Carnosic acid inhibits the epithelial-mesenchymal transition in B16F10 melanoma cells: a possible mechanism for the inhibition of cell migration. International Journal of Molecular Sciences. 2014; 15: 12698-713.
7.Rajasekaran D, Manoharan S, Silvan S, Vasudevan K, Baskaran N, Palanimuthu D. Proapoptotic, anti-cell proliferative, anti-inflammatory and anti-angiogenic potential of carnosic acid during 7,12 dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. African Journal of Traditional, Complementary and Alternative medicines. 2012; 10: 102-12.
8.Steiner M, Priel I, Giat J, Levy J, Sharoni Y, Danilenko M. Carnosic acid inhibits proliferation and augments differentiation of human leukemic cells induced by 1,25-dihydroxyvitamin D3 and retinoic acid. Nutrition and Cancer. 2001; 41: 135-44.
9.Visanji JM, Thompson DG, Padfield PJ. Induction of G2/M phase cell cycle arrest by carnosol and carnosic acid is associated with alteration of cyclin A and cyclin B1 levels. Cancer Letter. 2006; 237: 130-36.
10.Pesakhov S, Khanin M, Studzinski GP, Danilenko M. Distinct combinatorial effects of the plant polyphenols curcumin, carnosic acid, and silibinin on proliferation and apoptosis in acute myeloid leukemia cells. Nutrition and Cancer. 2010; 62: 811-24.
11.Kar S, Palit S, Ball WB, Das PK. Carnosic acid modulates Akt/IKK/NF-kB signaling by PP2A and induces intrinsic and extrinsic pathway mediated apoptosis in human prostate carcinoma PC-3 cells. Apoptosis. 2012; 17: 735-47.
12.Jung KJ, Min KJ, Bae JH, Kwon TK. Carnosic acid sensitized TRAIL-mediated apoptosis through down-regulation of c-FLIP and Bcl-2 expression at the post translational levels and CHOP-dependent up-regulation of DR5, Bim, and PUMA expression in human carcinoma caki cells. Oncotarget. 2015; 6: 1556-68.
13.Li, C.; Zhang, G.; Zhao, L.; Ma, Z.; Chen, H. Metabolic reprogramming in cancer cells: glycolysis, glutaminolysis, and Bcl-2 proteins as novel therapeutic targets for cancer. World J. Surg. Oncol. 2016; 14: 15.
14.Garrison, J.B.; Ge, C.; Che, L.; Pullum, D.A.; Peng, G.; Khan, S.; Ben-Jonathan, N.; Wang, J.; Du, C. Knockdown of the Inhibitor of Apoptosis BRUCE Sensitizes Resistant Breast Cancer Cells to Chemotherapeutic Agents. J. Cancer Sci. Ther. 2015; 7: 121-126.
15.Youle, R.J.; Strasser, A. The BCL-2 protein family: Opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59.
16.Cheng, E.H.; Wei, M.C.; Weiler, S.; Flavell, R.A.; Mak, T.W.; Lindsten, T.; Korsmeyer, S.J. BCL-2, BCL-X (L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell. 2001; 8: 705-711.
17.Ghate, N.B.; Das, A.; Chaudhuri, D.; Panja, S.; Mandal, N. Sundew plane, a potential source of anti-inflammatory agents, selectively induces G2/M arrest and apoptosis in MCF-7 cells through upregulation of p53 and Bax’Bcl-2 ratio. Cell Death Discov. 2016; 18: 15062.
18.Cain, K. Chemical-induced apoptosis: formation of the Apaf-1 apoptosome. Drug Metab. Rev. 2003; 35: 337-363.
19.Kitagawa, k.; Niikura, Y. Caspase-independent mitotic death (CIMD). Cell cycle 2008; 7: 1001-1005.
20.Kim, D.H.; Park, K.W.; Chae, I.G.; Kundu, J.; Kim, E.H.; Kundu, J.K.; Chun, K.S. Carnosic acid inhibits STAT3 signaling and induces apoptosis through generation of ROS in human colon cancer HCT116 cells. Mol. Carcinog. 2016; 55: 1096-1110.
21.Bursch, W.; Karwan, A.; Mayer, M.; Dornetshuber, J.; Fröhwein, U.; Schulte-Hermann, R.; Fazi, B.; Di Sano, F.; Piredda, L.; Piacentini, M.; et al. Cell death and autophagy: cytokines, drugs, and nutritional factors. Toxicology 2008; 254: 147-157.
22.Fazi, B.; Bursch, W.; Fimia, G.M.; Nardacci, R..; Piacentini, M.; Di Sano, F.; Piredda, L. Fenretinide induces autophagic cell death in caspase-defective breast cancer cells. Autophagy 2008; 4: 435-441.
23.Einbond L.S.; Wu, H.A.; Kashiwazaki, R.; He, K.; Roller, M.; Su, T.; Wang, X.; Goldsberry, S. Carnosic acid inhibits the growth of ER-negative human breast cancer cells and synergizes with curcumin. Fitoterapia 2012; 83: 1160-168.
24.Ziegler, A.; Luedke, G.H.; Fabbro, D.; Altmann K,H.; Stahel, R.A.; Zangemeister-Wittke, U. Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence. J. Natl. Cancer Inst. 1997; 16: 1027-1036.
25.Zangemeister-Wittke, U.; Schenker, T.; Luedke, G.H.; Stahel, R.A. Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines. Br. J. Cancer. 1998; 78: 1035-1042.
26.Leech, S.H.; Olie, R.A.; Gautschi, O.; Simoes-Wust, A.P.; Tschopp, S.; Haner, R.; Hall, J.; Stahel, R.A.; Zangemeister-Wittke, U. Induction of apoptosis in lung-cancer cells following bcl-xL anti-sense treatment. Int. J. Cancer 2000; 15: 570-576.
27.Poliseno, L.; Mariani, L.; Collecchi, P.; Piras, A.; Zaccaro, L.; Rainaldi, G. Cancer Chemother. Pharmacol. 2002; 50: 127-130.
28.Vanhoefer U, Yin MB, Harstrick A, Seeber S, Rustum YM. Carbamoylation of glutathione reductase by N,N-bis(2-chloroethyl)-N- nitrosourea associated with inhibition of multidrug resistance protein (MRP) function. Biochemical Pharmacology. 1997; 53: 801-809.
29.Stahl W, Krauth-Siegel RL, Schirmer RH, Eisenbrand G. A method to determine the carbamoylating potential of 1-(2-chloroethyl)-1-nitrosoureas. IARC Scientific Publications. 1987; 84:191-193.
30.McGirt MJ, Than KD, Weingart JD, Chaichana KL, Attenello FJ, Olivi A, et al. Gliadel (BCNU) wafer plus concomitant temozolomide therapy after primary resection of glioblastoma multiforme. Journal of Neurosurgery. 2009; 110: 583-8.
31.Panigrahi M, Das PK, Parikh PM. Brain tumor and Gliadel wafer treatment. Indian Journal of Cancer. 2011; 48: 11-7.
32.van den Bent MJ, Brandes AA, Taphoorn MJ, Kros JM, Kouwenhoven MC, Delattre JY, et al. et al. Adjuvant procarbazine, lomustine, and vincristine chemotherapy in newly diagnosed anaplastic oligodendroglioma: long-term follow-up of EORTC Brain Tumor Group study 26951. Journal of Clinical Oncology. 2013; 31: 344-50.
33.van den Bent MJ, Chinot O, Boogerd W, Bravo Marques J, Taphoorn MJ, Kros JM, et al. Second line chemotherapy with temozolomide in recurrent oligodendroglioma after PCV (procarbazine, lomustine and vincristine) chemotherapy: EORTC Brain Tumor Group phase II study 26972. Annals of Oncology. 2003; 14: 599-602.
34.Green MR, Dillman RO, Horton C. Procarbazine, vincristine, CCNU, and cyclophosphamide (POCC) in the treatment of metastatic malignant melanoma. Cancer Treatment Reports. 1980; 64: 139-142.
35.Reithmeier T, Graf E, Piroth T, Trippel M, Pinsker MO, Nikkhah G. BCNU for recurrent glioblastoma multiforme: efficacy, toxicity and prognostic factors. BMC Cancer. 2010; 10: 30.
36.Tai J, Cheung S, Wu M, Hasman D. Antiproliferation effect of Rosemary (Rosmarinus officinalis) on human ovarian cancer cells in vitro. Phytomedicine. 2012; 19: 436-43.
37.Jung K, Mildner D, Jacob B, Scholz D, Precht K. On the pyridoxal-59-phosphate stimulation of AST and ALT in serum and erythrocytes of patients undergoing chronic hemodialysis and with kidney transplants. Clinica Chimica Acta. 1981; 115; 105-10.
38.Wroblewski F. The clinical significance of alterations in transaminase activities of serum and other body fluids. Advances in Clinical Chemistry. 1958; 1: 313-51.
39.Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff LB. Diagnosis and monitoring of hepatic injury. I. Performance characteristics of laboratory tests. Clinical Chemistry. 2000; 46: 2027-49.
40.Doumas BT, Peters T. Serum and urine albumin: a progress report on their measurement and clinical significance. Clinica Chimica Acta. 1997; 258: 3-20.
41.Bonis PA, Tong PA, Tong MJ, Blatt LM, Conrad A, Griffith JL. A predictive model for the development of hepatocellular carcinoma, liver failure, or liver transplantation for patients presenting to clinic with chronic hepatitis C. The American Journal of Gastroenterology. 1999; 94: 1605-12.
42.Cortese K, Daga A, Monticone M, Tavella S, Stefanelli A, Aiello C, et al. Carnosic acid induces proteasomal degradation of Cyclin B1, RB and SOX2 along with cell growth arrest and apoptosis in GBM cells. Phytomedicine. 2016; 23: 679-85.
43.Karjalainen JM, Eskelinen MJ, Kellokoski JK, Reinikainen M, Alhava EM, Kosma VM. p21(WAF1/CIP1) expression in stage I cutaneous malignant melanoma: its relationship with p53, cell proliferation and survival. British Journal of Cancer. 1999; 79: 895-902.
44.Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes & Development. 1999; 13: 1501-12.
45.Gulbis JM, Kelman Z, Hurwitz J, O’Donnell M, Kuriyan J. Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA. Cell. 1996; 87: 297-306.
46.Roy S, Singh RP, Agarwal C, Siriwardana S, Sclafani R, Agarwal R. Downregulation of both p21/Cip1 and p27/Kip1 produces a more aggressive prostate cancer phenotype. Cell Cycle. 2008; 7: 1828-35.
47.Reed SI, Bailly E, Dulic V, Hengst L, Resnitzky D, Slingerland J. G1 control in mammalian cells. Journal of Cell Science. Supplement. 1994; 18: 69-73.
48.Grana X, Reddy EP. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene. 1995; 11: 211-19.
49.Bei D, Meng J, Youan1 BC. Engineering nanomedicines for improved melanoma therapy: progress and promises. Nanomedicine (Lond). 2010; 5: 1385-99.
50.Pyrhönen S, Hahka-Kemppainen M, Muhonen T. A promising interferon plus four-drug chemotherapy regimen for metastatic melanoma. Journal of Clinical Oncology. 1992; 10: 1919-26.
51.Park SY, Song H, Sung MK, Kang YH, Lee KW, Park JHY. Carnosic Acid Inhibits the Epithelial-Mesenchymal Transition in B16F10 Melanoma Cells: A Possible Mechanism for the Inhibition of Cell Migration. Int J Mol Sci. 2014; 15: 12698–12713.
52.Egeblad M., Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat. Rev. Cancer. 2002; 2: 161–174.
53.Chang C., Werb Z. The many faces of metalloproteases: cell growth, invasion, angiogenesis and metastasis. Trends Cell Biol. 2001; 11: S37–S43.
54.Basset P., Okada A., Chenard M.P., Kannan R., Stoll I., Anglard P., Bellocq J.P., Rio M.C. Matrix metalloproteinases as stromal effectors of human carcinoma progression: therapeutic implications. Matrix Biol. 1997; 15: 535–541.