Rhein(大黃酸)，是一種由大黃根萃取出來的傳統中草藥，在中國已經廣泛被用於做瀉劑，雖然有許多研究指出大黃酸會誘導癌細胞死亡，但大黃酸對乳癌細胞的影響仍未知。在本研究中為了確認大黃酸對於乳癌細胞是否具有毒殺性效果以及探討其毒殺機制，我們利用蛋白質體學技術以2D-DIGE結合MS分析乳癌細胞在經過大黃酸作用之下，不同蛋白表現量對於癌細胞的影響為何，結果發現大黃酸治療乳癌細胞研究結果中總共分析出有差異(>1.5-fold changes)的蛋白總共73個以及有差異(>1.3-fold changes)的巰基改變的蛋白9個。綜合蛋白質體學結果分析顯示大黃酸誘導乳癌細胞的毒殺效果是透過細胞骨架喪失調控功能，蛋白質無法正常摺疊及氧化還原喪失正常調節功能等等，並且進一步的實驗結果也發現大黃酸會促進蛋白質不正常摺疊以及細胞內氧化還原失去調控而導致乳癌細胞內質網壓力(ER stress)上升。我們的研究顯示出透過蛋白質體學的技術可提供一個快速的方式得知大黃酸誘導乳癌細胞的毒殺機制，而這些分析出來的目標蛋白未來可能做為在乳癌治療中的潛力目標蛋白。

Rhein, a traditional Chinese herbal derived from the rhizome of Rheum palmatum L., is one of the most important active component among the anthraquinones. It has been reported that rhein shows antitumor activity on many types of cancer. However, the mechanism of rhein-induced breast cancer cells death is still unknown. To investigate the cytotoxic effect of rhein on breast cancer cells, we perform lysine-labeling and cysteine-labeling 2D-DIGE combined MALDI-TOF MS to analyze the differentially expressed proteins on rhein-treated breast cancer cells. The multiplex proteomics studies demonstrate that 73 and 9 protein features are significantly changed in protein expression level and thiol reacticity, respectively, in either MCF-7 or MDA-MB-231 cells. Most of the differentially expression proteins involved in protein folding, cytoskeleton regulation and glycolysis. Further study also indicates that rhein may interfere with proper folding of proteins as well as unbalancing of cellular redox status leading to ER-stress. To sum up, our preliminary approach demonstrates that the combined proteomic strategy offers a rapid strategy to study the molecular mechanisms of rhein-induced cytotoxicity in breast cancer cells. The identified target proteins might be useful for further assessment as potential targets in breast cancer therapy.

TABLE OF CONTENTS
中文摘要 i
Abstract ii
誌謝 iii
TABLE OF CONTENTS v
LIST OF FIGURES AND TABLES vii
ABBREVIATIONS ix
Chapter1 Introduction 1
1.1 Breast cancer 1
1.1.1 The risk factors of breast cancer 1
1.1.2 Stages of breast cancer 3
1.1.3 Treatments of breast cancer 4
1.2 Rhein 6
1.2.1 Anti-tumor properties of rhein 6
1.2.2 Rhein treatment of other diseases 7
1.3　ER stress 8
1.4　Proteomics 10
1.5 Aim of this study 16
Chapter 2 Materials and Methods 17
2.1 Chemicals and reagents 17
2.2 Cell culture 18
2.3 Cell viability 18
2.4　Immunoblotting 19
2.5　Enzyme-linked immunosorbent assay (ELISA) analysis 19
2.6　Sample preparation for proteomic analysis 20
2.7 2D-DIGE and gel image analysis 21
2.8 Protein staining 22
2.9 In-gel digestion 22
2.10 Protein identification by MALDI-TOF MS and MS/MS 23
2.11　Flow cytometry analysis 24
2.12　Assay for endogenous reactive oxygen species using DCFH-DA 24
2.13　Assay for the effect of N-acetylcysteine on rhein-induced cell death 25
2.14 Validation of thiol reactivity changes by immunoprecipitation coupled to immunoblotting 25
Chapter 3 Results 26
3.1 Rhein induces cell death and cell apoptosis in MCF-10A cells, MCF-7 cells 26
and MDA-MB-231 cells 26
3.2 Rhein induces ROS generation in breast cancer cell 31
3.3 2D-DIGE and MALDI-TOF MS analysis of rhein-induced proteomic alterations in MCF-7 cells and MDA-MB-231 cells 34
3.4 Validation of rhein-induced differential protein expression in breast cancer 42
cells by immunoblotting, immunofluorescence and enzyme-linked immunosorbent assay 42
3.5 Rhein-increase ER-stress in MCF-10A cells, MCF-7 cells and MDA-MB-231 cells 45
3.6 Redox 2D-DIGE analysis of rhein-induced differential cysteine- modification in MCF-7 cells and MDA-MB-231 cells. 47
3.7 Validation of the thiol reactive protein, peroxiredoxin-2, identified through 50
redox-proteomic study in MCF-7 cells after rhein treatment by IP-WB. 50
Chapter 4 Discussion 51
Chapter 5 Conclusion 58
Chapter 6 REFERENCE 59
APPENDIX 64
Supplementary table 1. 65
Supplementary table 2. 74

1. Ferlay, J., et al., Breast and cervical cancer in 187 countries between 1980 and 2010. Lancet, 2012. 379(9824): p. 1390-1.
2. Eccles, S.A., et al., Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer. Breast Cancer Res, 2013. 15(5): p. R92.
3. Zhang, M.H., et al., Estrogen receptor-positive breast cancer molecular signatures and therapeutic potentials (Review). Biomed Rep, 2014. 2(1): p. 41-52.
4. Jordan, V.C., Tamoxifen (ICI46,474) as a targeted therapy to treat and prevent breast cancer. Br J Pharmacol, 2006. 147 Suppl 1: p. S269-76.
5. Rubin, I. and Y. Yarden, The basic biology of HER2. Ann Oncol, 2001. 12 Suppl 1: p. S3-8.
6. Livingston, R.B. and F.J. Esteva, Chemotherapy and herceptin for HER2(+) metastatic breast cancer: the best drug? Oncologist, 2001. 6(4): p. 315-6.
7. Konkimalla, V.B. and T. Efferth, Evidence-based Chinese medicine for cancer therapy. J Ethnopharmacol, 2008. 116(2): p. 207-10.
8. Santos Araujo Mdo, C., et al., Uncaria tomentosa-Adjuvant Treatment for Breast Cancer: Clinical Trial. Evid Based Complement Alternat Med, 2012. 2012: p. 676984.
9. Liao, H.F., et al., Effects of herbal medicinal formulas on suppressing viral replication and modulating immune responses. Am J Chin Med, 2010. 38(1): p. 173-90.
10. Castiglione, S., et al., Rhein inhibits glucose uptake in Ehrlich ascites tumor cells by alteration of membrane-associated functions. Anticancer Drugs, 1993. 4(3): p. 407-14.
11. Iosi, F., M.T. Santini, and W. Malorni, Membrane and cytoskeleton are intracellular targets of rhein in A431 cells. Anticancer Res, 1993. 13(2): p. 545-54.
12. Chang, C.Y., et al., Rhein induces apoptosis in human breast cancer cells. Evid Based Complement Alternat Med, 2012. 2012: p. 952504.
13. Lin, M.L., et al., Rhein induces apoptosis through induction of endoplasmic reticulum stress and Ca2+-dependent mitochondrial death pathway in human nasopharyngeal carcinoma cells. Anticancer Res, 2007. 27(5A): p. 3313-22.
14. Du, Q., et al., Role of mitochondrial permeability transition in human hepatocellular carcinoma Hep-G2 cell death induced by rhein. Fitoterapia, 2013. 91: p. 68-73.
15. Li, Y., et al., Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway. Braz J Med Biol Res, 2012. 45(11): p. 1052-9.
16. Hsia, T.C., et al., The roles of endoplasmic reticulum stress and Ca2+ on rhein-induced apoptosis in A-549 human lung cancer cells. Anticancer Res, 2009. 29(1): p. 309-18.
17. Shi, P., Z. Huang, and G. Chen, Rhein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma BEL-7402 cells. Am J Chin Med, 2008. 36(4): p. 805-13.
18. Lin, M.L., et al., Rhein inhibits invasion and migration of human nasopharyngeal carcinoma cells in vitro by down-regulation of matrix metalloproteinases-9 and vascular endothelial growth factor. Oral Oncol, 2009. 45(6): p. 531-7.
19. Lin, Y.J., et al., Rhein lysinate induced S-phase arrest and increased the anti-tumor activity of 5-FU in HeLa cells. Am J Chin Med, 2011. 39(4): p. 817-25.
20. Lin, Y.J., et al., [Rhein lysinate induces apoptosis in breast cancer SK-Br-3 cells by inhibiting HER-2 signal pathway]. Yao Xue Xue Bao, 2008. 43(11): p. 1099-105.
21. Jia, Z.H., et al., Combined therapy of rhein and benazepril on the treatment of diabetic nephropathy in db/db mice. Exp Clin Endocrinol Diabetes, 2007. 115(9): p. 571-6.
22. Gao, Q., et al., Rhein improves renal lesion and ameliorates dyslipidemia in db/db mice with diabetic nephropathy. Planta Med, 2010. 76(1): p. 27-33.
23. Sheng, X., et al., Rhein protects against obesity and related metabolic disorders through liver X receptor-mediated uncoupling protein 1 upregulation in brown adipose tissue. Int J Biol Sci, 2012. 8(10): p. 1375-84.
24. Zhang, Y., et al., Rhein Reduces Fat Weight in db/db Mouse and Prevents Diet-Induced Obesity in C57Bl/6 Mouse through the Inhibition of PPARgamma Signaling. PPAR Res, 2012. 2012: p. 374936.
25. He, Z.H., et al., Anti-angiogenic effect and mechanism of rhein from Rhizoma Rhei. Phytomedicine, 2011. 18(6): p. 470-8.
26. Joung, D.K., et al., Synergistic effect of rhein in combination with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus. Exp Ther Med, 2012. 3(4): p. 608-612.
27. Lin, Y.J., et al., Effects of Rhein lysinate on H2O2-induced cellular senescence of human umbilical vascular endothelial cells. Acta Pharmacol Sin, 2011. 32(10): p. 1246-52.
28. Zhou, H. and R. Liu, ER stress and hepatic lipid metabolism. Front Genet, 2014. 5: p. 112.
29. Fewell, S.W., et al., The action of molecular chaperones in the early secretory pathway. Annu Rev Genet, 2001. 35: p. 149-91.
30. Papanikou, E. and B.S. Glick, Golgi compartmentation and identity. Curr Opin Cell Biol, 2014. 29C: p. 74-81.
31. Kozutsumi, Y., et al., The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature, 1988. 332(6163): p. 462-4.
32. Sugeno, N., et al., Serine 129 phosphorylation of alpha-synuclein induces unfolded protein response-mediated cell death. J Biol Chem, 2008. 283(34): p. 23179-88.
33. Gonzalez-Guerrero, C., et al., Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-kappaB-mediated inflammatory responses in kidney tubular cells. Toxicol Appl Pharmacol, 2013. 272(3): p. 825-41.
34. Zong, W.X., et al., Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J Cell Biol, 2003. 162(1): p. 59-69.
35. Urano, F., A. Bertolotti, and D. Ron, IRE1 and efferent signaling from the endoplasmic reticulum. J Cell Sci, 2000. 113 Pt 21: p. 3697-702.
36. Orrenius, S., B. Zhivotovsky, and P. Nicotera, Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol, 2003. 4(7): p. 552-65.
37. Patterson, S.D. and R.H. Aebersold, Proteomics: the first decade and beyond. Nat Genet, 2003. 33 Suppl: p. 311-23.
38. Unlu, M., M.E. Morgan, and J.S. Minden, Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis, 1997. 18(11): p. 2071-7.
39. Marouga, R., S. David, and E. Hawkins, The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal Bioanal Chem, 2005. 382(3): p. 669-78.
40. Van den Bergh, G. and L. Arckens, Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics. Curr Opin Biotechnol, 2004. 15(1): p. 38-43.
41. Chan, H.L., et al., Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis. Proteomics, 2005. 5(11): p. 2908-26.
42. Aebersold, R. and M. Mann, Mass spectrometry-based proteomics. Nature, 2003. 422(6928): p. 198-207.
43. Hardouin, J., Protein sequence information by matrix-assisted laser desorption/ionization in-source decay mass spectrometry. Mass Spectrom Rev, 2007. 26(5): p. 672-82.
44. Lin, S., M. Fujii, and D.X. Hou, Rhein induces apoptosis in HL-60 cells via reactive oxygen species-independent mitochondrial death pathway. Arch Biochem Biophys, 2003. 418(2): p. 99-107.
45. Timms, J.F. and R. Cramer, Difference gel electrophoresis. Proteomics, 2008. 8(23-24): p. 4886-97.
46. Zhou, J., et al., Differential expression of the early lung cancer detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP-A2/B1) in normal breast and neoplastic breast cancer. Breast Cancer Res Treat, 2001. 66(3): p. 217-24.
47. Yan-Sanders, Y., G.J. Hammons, and B.D. Lyn-Cook, Increased expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP) in pancreatic tissue from smokers and pancreatic tumor cells. Cancer Lett, 2002. 183(2): p. 215-20.
48. Zhou, J., et al., Expression of early lung cancer detection marker: hnRNP-A2/B1 and its relation to microsatellite alteration in non-small cell lung cancer. Lung Cancer, 2001. 34(3): p. 341-50.
49. Goto, Y., et al., Significance of heterogeneous nuclear ribonucleoprotein B1 as a new early detection marker for oral squamous cell carcinoma. Jpn J Cancer Res, 1999. 90(12): p. 1358-63.
50. Chen, C.Y., et al., The antitumor agent PBT-1 directly targets HSP90 and hnRNP A2/B1 and inhibits lung adenocarcinoma growth and metastasis. J Med Chem, 2014. 57(3): p. 677-85.
51. Rubinstein, N., et al., Targeted inhibition of galectin-1 gene expression in tumor cells results in heightened T cell-mediated rejection; A potential mechanism of tumor-immune privilege. Cancer Cell, 2004. 5(3): p. 241-51.
52. Zheng, D., et al., Downregulation of galectin-3 causes a decrease in uPAR levels and inhibits the proliferation, migration and invasion of hepatocellular carcinoma cells. Oncol Rep, 2014. 32(1): p. 411-8.
53. Oka, N., et al., Galectin-3 inhibits tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by activating Akt in human bladder carcinoma cells. Cancer Res, 2005. 65(17): p. 7546-53.
54. Qian, Z., et al., Downregulation of cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9. BMC Cancer, 2012. 12: p. 442.
55. Bonfils, C., et al., Cyclophilin A as negative regulator of apoptosis by sequestering cytochrome c. Biochem Biophys Res Commun, 2010. 393(2): p. 325-30.
56. Yamaguchi, H. and J. Condeelis, Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochim Biophys Acta, 2007. 1773(5): p. 642-52.
57. Polachini, G.M., et al., Proteomic approaches identify members of cofilin pathway involved in oral tumorigenesis. PLoS One, 2012. 7(12): p. e50517.
58. Shenton, D. and C.M. Grant, Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae. Biochem J, 2003. 374(Pt 2): p. 513-9.
59. Weeks, M.E., et al., A parallel proteomic and metabolomic analysis of the hydrogen peroxide- and Sty1p-dependent stress response in Schizosaccharomyces pombe. Proteomics, 2006. 6(9): p. 2772-96.
60. Silva, V.C. and L. Cassimeris, Stathmin and microtubules regulate mitotic entry in HeLa cells by controlling activation of both Aurora kinase A and Plk1. Mol Biol Cell, 2013. 24(24): p. 3819-31.
61. Lee, M.J., et al., Pro-oncogenic potential of NM23-H2 in hepatocellular carcinoma. Exp Mol Med, 2012. 44(3): p. 214-24.
62. Anzinger, J., et al., Secretion of a nucleoside diphosphate kinase (Nm23-H2) by cells from human breast, colon, pancreas and lung tumors. Proc West Pharmacol Soc, 2001. 44: p. 61-3.
63. Chen, L.C., et al., The antiapoptotic protein, FLIP, is regulated by heterogeneous nuclear ribonucleoprotein K and correlates with poor overall survival of nasopharyngeal carcinoma patients. Cell Death Differ, 2010. 17(9): p. 1463-73.
64. Chunhua, L., et al., Apigenin up-regulates transgelin and inhibits invasion and migration of colorectal cancer through decreased phosphorylation of AKT. J Nutr Biochem, 2013. 24(10): p. 1766-75.
65. Godlewski, M.M., et al., Colocalization of BAX with BID and VDAC-1 in nimesulide-induced apoptosis of human colon adenocarcinoma COLO 205 cells. Anticancer Drugs, 2002. 13(10): p. 1017-29.