臺灣博碩士論文加值系統

三陰性乳癌在乳癌病患中占約10-20%。這類乳癌細胞通常較惡性且較具侵襲性，其預後的效果也較差。由於這類乳癌細胞缺乏賀爾蒙及Her2/neu的受體，所以賀爾蒙治療與Her2/neu的標靶治療並不適合他們。三陰性乳癌病患在臨床上的治療通常為手術，輻射線治療及化療，更適合這類病患的治療策略還在研發當中。在本篇研究中，我們發現三陰性乳癌細胞對於萃取自柴胡的丙酮層(BS-AE)的異柴胡內酯(K8)是敏感的。異柴胡內酯(K8)已經被證明在肺癌及攝護腺癌細胞中具有抗腫瘤細胞的活性，然而此抗腫瘤活性的分子機制尚不清楚。我們利用兩種合成消旋構型的異柴胡內酯(E-K8及Z-K8)，以偵測細胞增生率的方式比較此兩種構型的效果，發現人類三陰性乳癌細胞對於Z-K8相較於E-K8是更敏感的。此外，我們在MDA-MB-468人類三陰性乳癌細胞中發現Z-K8藉由活化G2/M細胞週期的檢查點(cdc2/cyclin B複合體)造成G2/M細胞週期停滯及細胞凋亡。磷酸化cdc-2的表現量明顯增加會使細胞週期停滯在G2/M時期。而Z-K8也會誘發DNA損害反應並活化ATR/BRCA1/Chk1訊號路徑。綜合本篇實驗結果，Z-K8會藉由誘發DNA損害反應並造成G2/M細胞週期停滯及細胞凋亡，在未來臨床上應用於三陰性乳癌病患是具有潛力的。

Triple-negative breast cancers (TNBCs) constitute about 10-20% of breast cancers. They are characterized with higher grade, more aggressive biology, and poor prognosis. Since TNBC lack hormone and Her2/neu receptors, hormone therapy and target therapy against Her2/neu are not appropriate for them. Treatments of TNBC patients are usually surgery, irradiation, and chemotherapy; moreover, better therapeutic strategies for them are still developing. In this study, we found that TNBC cells are sensitive to isochaihulactone (K8), which is an acetone extract from root of Bupleurumscorzonerifolium (BS-AE). K8 has been demonstrated to possess anti-tumor activity against lung and prostate cancer cells. However, the molecular mechanism of K8 in anti-tumor activity is still unclear. Here, we tested two synthetic racemic forms (Z-K8 and E-K8) in breast cancer cells and found that TNBC cells were more sensitive to Z-K8 than E-K8 in cell proliferation assay. In addition, we showed that Z-K8 can cause G2/M cell cycle arrest and apoptosis in human MDA-MB-468 TNBC by activating its checkpoint (cdc2/cyclin B complex). Moreover, the expression of cell cycle regulatory proteins including phospho-cdc2 dramatically increased to block cell cycle progression at G2/M boundary. Furthermore, Z-K8 also induced DNA damage response by activating ATR/Chk1/BRCA1 signaling pathway. Taken together, our results revealed that Z-K8 can induce DNA damage response and cause cell cycle arrest at G2/M phase resulting in cell apoptosis and has the potential for future clinical applications on TNBC patients.

致謝 03
摘要 06
Abstract 07
Abbreviations 08
Chapter 1. Introduction 12
1.1. Characteristics of triple-negative breast cancers (TNBCs) 13
1.2. Therapeutic strategies of TNBC 16
1.3. Traditional Chinese medicine (TCM) in anti-cancer drug development 19
1.4. Chemical compositions and targets in anti-cancer drugs developed from TCM 19
1.5. Origin of Isochaihulactone (K8) and its reported functions 26
1.6. Isochaihulactone (K8), a lignan compound isolated from root of Nan-Chai-Hu 27
1.7. Rationale of this study 28
Chapter 2. Material and methods 29
2.1. Cell-lines 30
2.2. Cell proliferation assay 30
2.3. Antibodies 30
2.4. Cell apoptosis assay 31
2.5. TUNEL assay 31
2.6. Cell cycle analysis 31
2.7. Transfection 32
2.8. Infection 32
2.9. Statistical analysis 32
Chapter 3. Results 33
3.1. Z-K8 inhibited TNBCs’ cell proliferation by inducing its cell apoptosis 35
3.2.Z-K8 arrested TNBC cells at G2/M phase by altering cell cycle regulator proteins 42
3.3. Z-K8 induced TNBC cells’ DNA damage and activated DNA damage response 50
Chapter 4. Summary 54
Chapter 5. Discussions 56
References 61

1.Siegel, R., et al., Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin, 2011. 61(4): p. 212-36.
2.Brenton, J.D., et al., Molecular classification and molecular forecasting of breast cancer: ready for clinical application? J Clin Oncol, 2005. 23(29): p. 7350-60.
3.Tan, D.S., et al., Triple negative breast cancer: molecular profiling and prognostic impact in adjuvant anthracycline-treated patients. Breast Cancer Res Treat, 2008. 111(1): p. 27-44.
4.Schneider, B.P., et al., Triple-negative breast cancer: risk factors to potential targets. Clin Cancer Res, 2008. 14(24): p. 8010-8.
5.Foulkes, W.D., I.E. Smith, and J.S. Reis-Filho, Triple-negative breast cancer. N Engl J Med, 2010. 363(20): p. 1938-48.
6.Sandhu, R., et al., Microarray-Based Gene Expression Profiling for Molecular Classification of Breast Cancer and Identification of New Targets for Therapy. Laboratory Medicine, 2010. 41(6): p. 364-372.
7.Rakha, E.A., et al., Triple-negative breast cancer: distinguishing between basal and nonbasal subtypes. Clin Cancer Res, 2009. 15(7): p. 2302-10.
8.Berrada, N., S. Delaloge, and F. Andre, Treatment of triple-negative metastatic breast cancer: toward individualized targeted treatments or chemosensitization? Ann Oncol, 2010. 21 Suppl 7: p. vii30-5.
9.Rodriguez, A.A., et al., DNA repair signature is associated with anthracycline response in triple negative breast cancer patients. Breast Cancer Res Treat, 2010. 123(1): p. 189-96.
10.Luo, X., et al., Expression and clinical significance of androgen receptor in triple negative breast cancer. Chin J Cancer, 2010. 29(6): p. 585-90.
11.Tang, D., et al., The expression and clinical significance of the androgen receptor and E-cadherin in triple-negative breast cancer. Med Oncol, 2012. 29(2): p. 526-33.
12.Hudis, C.A. and L. Gianni, Triple-negative breast cancer: an unmet medical need. Oncologist, 2011. 16 Suppl 1: p. 1-11.
13.Liu, N., X. Wang, and X. Sheng, ''Triple negative'' epithelial ovarian cancer and pathologic markers for prognosis. Curr Opin Obstet Gynecol, 2011. 23(1): p. 19-23.
14.Ueno, N.T. and D. Zhang, Targeting EGFR in Triple Negative Breast Cancer. J Cancer, 2011. 2: p. 324-8.
15.Inoue, S., et al., Nanobiopolymer for direct targeting and inhibition of EGFR expression in triple negative breast cancer. PLoS One, 2012. 7(2): p. e31070.
16.Litzenburger, B.C., et al., High IGF-IR activity in triple-negative breast cancer cell lines and tumorgrafts correlates with sensitivity to anti-IGF-IR therapy. Clin Cancer Res, 2011. 17(8): p. 2314-27.
17.Behbod, F. and J.M. Rosen, Will cancer stem cells provide new therapeutic targets? Carcinogenesis, 2005. 26(4): p. 703-11.
18.Harrison, H., et al., Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res, 2010. 70(2): p. 709-18.
19.Pannuti, A., et al., Targeting Notch to target cancer stem cells. Clin Cancer Res, 2010. 16(12): p. 3141-52.
20.Linderholm, B.K., et al., Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple-negative breast cancer. Ann Oncol, 2009. 20(10): p. 1639-46.
21.Dent, S.F., The role of VEGF in triple-negative breast cancer: where do we go from here? Ann Oncol, 2009. 20(10): p. 1615-7.
22.Amos, K.D., B. Adamo, and C.K. Anders, Triple-negative breast cancer: an update on neoadjuvant clinical trials. Int J Breast Cancer, 2012. 2012: p. 385978.
23.Man, S., et al., Anticancer Drugs from Traditional Toxic Chinese Medicines. Phytother Res, 2012.
24.Lee, T.F., Y.L. Lin, and Y.T. Huang, Protective effects of kaerophyllin against liver fibrogenesis in rats. Eur J Clin Invest, 2012. 42(6): p. 607-16.
25.Dai, J., et al., Crude polysaccharide from an anti-UVB cell clone of Bupleurum scorzonerifolium protect HaCaT cells against UVB-induced oxidative stress. Cytotechnology, 2011. 63(6): p. 599-607.
26.Yu, S.L., et al., Isochaihulactone protects PC12 cell against H(2)O(2) induced oxidative stress and exerts the potent anti-aging effects in D-galactose aging mouse model. Acta Pharmacol Sin, 2010. 31(12): p. 1532-40.
27.Chang, W.L., et al., Immunosuppressive flavones and lignans from Bupleurum scorzonerifolium. Phytochemistry, 2003. 64(8): p. 1375-9.
28.Dai, Y., et al., Metabolomics study on the anti-depression effect of xiaoyaosan on rat model of chronic unpredictable mild stress. J Ethnopharmacol, 2010. 128(2): p. 482-9.
29.Cheng, Y.-L., et al., Acetone extract of Bupleurum scorzonerifolium inhibits proliferation of A549 human lung cancer cells via inducing apoptosis and suppressing telomerase activity. Life Sciences, 2003. 73(18): p. 2383-2394.
30.Chen, Y.L., et al., In vitro and in vivo studies of a novel potential anticancer agent of isochaihulactone on human lung cancer A549 cells. Biochem Pharmacol, 2006. 72(3): p. 308-19.
31.Chen, Y.L., et al., Activation of nonsteroidal anti-inflammatory drug-activated gene-1 via extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase revealed a isochaihulactone-triggered apoptotic pathway in human lung cancer A549 cells. J Pharmacol Exp Ther, 2007. 323(2): p. 746-56.
32.Chiu, S.C., et al., Activation of NAG-1 via JNK signaling revealed an isochaihulactone-triggered cell death in human LNCaP prostate cancer cells. BMC Cancer, 2011. 11: p. 146.
33.Yu, Y.L., et al., Synergistic anti-tumor activity of isochaihulactone and paclitaxel on human lung cancer cells. J Cell Physiol, 2012. 227(1): p. 213-22.
34.Chang, W.-L., et al., Immunosuppressive flavones and lignans from Bupleurum scorzonerifolium. Phytochemistry, 2003. 64(8): p. 1375-1379.
35.Lignans with Anticancer Activity. J. Fac. Pharm. Ankara, 1994. 23, 1-2 (1994).
36.Martin, M.E., et al., Interactions between phytoestrogens and human sex steroid binding protein. Life Sci, 1996. 58(5): p. 429-36.
37.Schwartz, G.K. and M.A. Shah, Targeting the cell cycle: a new approach to cancer therapy. J Clin Oncol, 2005. 23(36): p. 9408-21.
38.Gladden, A.B. and J.A. Diehl, Cell cycle progression without cyclin E/CDK2: breaking down the walls of dogma. Cancer Cell, 2003. 4(3): p. 160-2.
39.Donzelli, M. and G.F. Draetta, Regulating mammalian checkpoints through Cdc25 inactivation. EMBO Rep, 2003. 4(7): p. 671-7.
40.Johnson, D.G. and C.L. Walker, Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol, 1999. 39: p. 295-312.
41.Prigent, C. and S. Dimitrov, Phosphorylation of serine 10 in histone H3, what for? J Cell Sci, 2003. 116(Pt 18): p. 3677-85.
42.Lu, Z. and T. Hunter, Ubiquitylation and proteasomal degradation of the p21(Cip1), p27(Kip1) and p57(Kip2) CDK inhibitors. Cell Cycle, 2010. 9(12): p. 2342-52.
43.Starostina, N.G. and E.T. Kipreos, Multiple degradation pathways regulate versatile CIP/KIP CDK inhibitors. Trends Cell Biol, 2012. 22(1): p. 33-41.
44.Perry, J.A. and S. Kornbluth, Cdc25 and Wee1: analogous opposites? Cell Div, 2007. 2: p. 12.
45.Potapova, T.A., et al., Fine tuning the cell cycle: activation of the Cdk1 inhibitory phosphorylation pathway during mitotic exit. Mol Biol Cell, 2009. 20(6): p. 1737-48.
46.Ruiz, E.J., M. Vilar, and A.R. Nebreda, A two-step inactivation mechanism of Myt1 ensures CDK1/cyclin B activation and meiosis I entry. Curr Biol, 2010. 20(8): p. 717-23.
47.Boutros, R., C. Dozier, and B. Ducommun, The when and wheres of CDC25 phosphatases. Curr Opin Cell Biol, 2006. 18(2): p. 185-91.
48.Hermeking, H., The 14-3-3 cancer connection. Nat Rev Cancer, 2003. 3(12): p. 931-43.
49.Wang, Y., et al., Centrosome-associated regulators of the G(2)/M checkpoint as targets for cancer therapy. Mol Cancer, 2009. 8: p. 8.
50.Foley, J., et al., EGFR signaling in breast cancer: bad to the bone. Semin Cell Dev Biol, 2010. 21(9): p. 951-60.
51.Helleday, T., et al., DNA repair pathways as targets for cancer therapy. Nat Rev Cancer, 2008. 8(3): p. 193-204.
52.Bohgaki, T., M. Bohgaki, and R. Hakem, DNA double-strand break signaling and human disorders. Genome Integr, 2010. 1(1): p. 15.
53.Summers, K.C., et al., Phosphorylation: the molecular switch of double-strand break repair. Int J Proteomics, 2011. 2011: p. 373816.
54.Decatris, M.P., S. Sundar, and K.J. O’Byrne, Platinum-based chemotherapy in metastatic breast cancer: current status. Cancer Treatment Reviews, 2004. 30(1): p. 53-81.
55.Pegram, M.D., et al., Rational Combinations of Trastuzumab With Chemotherapeutic Drugs Used in the Treatment of Breast Cancer. JNCI Journal of the National Cancer Institute, 2004. 96(10): p. 739-749.
56.McGrogan, B.T., et al., Taxanes, microtubules and chemoresistant breast cancer. Biochim Biophys Acta, 2008. 1785(2): p. 96-132.