臺灣博碩士論文加值系統

肺癌是現在全球最嚴重的癌症之一，在全球無論是男性或女性都具有高發生率與高死率。而在台灣也具有第一位的死亡率，已經成為台灣嚴重的公共衛生議題。非小細胞肺癌占了肺癌罹病人數的85%，其特色為對於化療藥物不具有敏感性，這造成了肺癌預後不佳的主要原因。因此尋求效果更佳的化療用藥是目前肺癌研究的目標之一。大黃素(Emodin)，是由大黃分離出來的天然蒽醌衍生物，是中國傳統中藥的有效成分之一，以前的研究已經發現大黃素對於多種癌症以及訊息傳遞路徑有抑制的效果，進而造成細胞的生長抑制或細胞凋亡(apoptosis)。而細胞的生長抑制與細胞週期的進行有很大的關係。在2008年Yu CX.等人指出，大黃素會透過p53-p21路徑造成攝護腺癌細胞凋亡，暗示著細胞週期抑制蛋白(Cyclin-dependent kinase inhibitor, CKI)有牽涉在其中。本篇論文探討大黃素如何影響肺癌細胞株A549的細胞增生。實驗結果顯示，大黃素能夠改變肺癌細胞A549細胞的型態並抑制肺癌細胞株A549增生。在蛋白層面，發現大黃素可以增加p21與p27的蛋白表現。利用定量聚合酶連鎖反應測定p21與p27的mRNA表現有顯著的上升，進一步以核質分離與免疫細胞染色法觀察到，p21的上游p53與p27的上游FOXO3a在細胞核中的表現量有顯著增加。p21與p27的蛋白功能方面結果顯示，大黃素會促進p21與p27在細胞核中的蛋白量。進一步利用免疫共沉澱法觀察p21與p27的目標蛋白CDK2(cyclin-dependent kinase 2, CDK2)的物理性結合有顯著的上升。最後利用激酶活性測定，可觀察到大黃素能夠有效的降低CDK2的激酶活性，並造成細胞週期停滯在G1。總結以上結果，本論文發現在肺癌細胞中，大黃素能透過p21與p27的增加抑制CDK2活性，進而抑制細胞的增生。

Lung cancer is a leading cause of cancer death in both men and women in the world. More than 85% lung cancer patients is non-small cell lung cancer (NSCLC). The 5-year survival rate for individuals with lung cancer is only about 15%. Previous studies have demonstrated that Emodin inhibits cell growth in several type of tumor cells. Cell cycle plays an important role in lung cancer cell growth. A previous report shows that Emodin induces apoptosis in human prostate cancer cell LNCaP through p53-p21 pathway. Moreover the cell cycle inhibitory proteins (Cyclin-dependent kinase inhibitor, CKI) have been involved in Emodin-inhibited cell proliferation. This thesis attempted to investigate how Emodin suppress cell proliferation of A549 cells. The results showed that Emodin could increase the protein expressions of p21 and p27. Data from quantitative polymerase chain reaction assay indicated that p21 and p27 mRNA expression significantly increased upon emodin treatment. With further experiments may confirm p53 and FOXO3a, transcription factor of p21 and p27, were increased in cell nucleus by Emodin treatment. Moreover, I observed that Emodin could increase p21 and p27 protein in the nucleus. The biochemical interaction of p21/CDK2(cyclin-dependent kinase 2)and p27/CDK2 both are increased in A549 cells; however, Emodin could decrease CDK2 kinase activity by enhancing p21/CDK2 and p27/CDK2 interaction.
Finally, the thesis suggested that Emodin can inhibit CDK2 activity by enhanceing biochemical interacton of p21/CDK2 and p27/CDK2, and decrease cell proliferation in lung cancer cells.

中文摘要 i
英文摘要 ii
第一章、前言 1
一、背景 1
(一)、肺部組織與肺癌介紹 1
(二)、細胞週期介紹 3
(三)、癌症與細胞週期 5
(四)、細胞週期抑制劑(cyclin-dependent kinase inhibitor, CKI) 7
(五)、p21介紹 8
(六)、p27介紹 10
(七)、Emodin介紹 11
二、研究動機與目的 14
第二章、材料與方法 15
一、人類肺癌細胞培養 (Cell Culture) 15
二、蛋白質萃取 (Protein Extraction) 16
三、細胞核質分離 (Nuclear/Cytosol Fractionation) 17
四、免疫沉澱法 (Immunoprecipitation) 18
五、西方墨點法 (Western Blotting) 18
(一)、SDS-PAGE膠體電泳法 (SDS-PAGE Electrophoresis) 18
(二)、蛋白質轉漬法 (Immunoblotting, IB) 19
(三)、抗體偵測 (Detection) 19
六、細胞生長分析 (MTT assay) 19
七、mRNA表現分析 19
(一)、Total RNA純化萃取 (Total RNA Purification) 21
(二)、RT-PCT (Reverse-transcription Polymerase Chain Reaction) 22
(三)、qRT-PCR (Quantitative Real-Time Polymerase Chain Reaction) 23
八、免疫細胞化學染色法 (Immunocytochemistry, ICC) 23
九、激酶活性測定法(kinase assay) 24
十、細胞週期分析(Cell cycle analysis) 25
十一、量化分析 (Quantify) 26
十二、統計分析 (Statistics) 26
第三章、實驗結果 27
一、Emodin影響肺癌細胞株A549細胞型態 27
二、Emodin抑制肺癌細胞株A549生長 27
三、 Emodin增加肺癌細胞株A549之p21與p27蛋白表現 28
四、Emodin增加肺癌細胞株A549、H460、H520、H292、CL1-0
與CL1-5的p27¬蛋白量 29


五、 Emodin可能促進肺癌細胞株 A549的p21與p27蛋白質
分解 29
六、Emodin增加肺癌細胞株 A549的p21與p27¬的
mRNA量 30
七、Emodin使肺癌細胞株 A549細胞核中的的p21與p53蛋白
量增加 31
八、Emodin使肺癌細胞株 A549細胞核中的的p27與FOXO3a
蛋白量增加 31
九 、 Emodin使肺癌細胞株 A549細胞核中的的p27、FOXO3a、p21與p53蛋白量增加 32
十 、Emodin增加肺癌細胞株 A549細胞中的p27、p21與
CDK2蛋白結合 33
十一、Emodin降低肺癌細胞株 A549細胞中的CDK2激酶活性
33
十二、 Emodin使肺癌細胞株A549的細胞週期停滯在G1 期 34



第四章、討論 35
一、 Emodin 對於細胞週期的影響 35
二、 Emodin 對於p21與p27的調控 35
三、 Emodin 劑量的探討 36
四、 更深入探討Emodin對於細胞週期的調控 37

第五章、結論 38
第六章、參考文獻 39
第七章、實驗結果圖 45

1.Siegel, R. L., Miller, K. D., and Jemal, A. (2015) Cancer statistics, 2015. CA: a cancer journal for clinicians 65, 5-29
2.Brody, H. (2014) Lung cancer. Nature 513, S1-S1
3.Chang, G. C., Chen, K. C., Yang, T. Y., Yin, M. C., Lin, C. P., Kuo, B. I., and Hsu, J. Y. (2005) Activity of gefitinib in advanced non-small-cell lung cancer with very poor performance status. Investigational new drugs 23, 73-77
4.Norbury, C., and Nurse, P. (1992) Animal cell cycles and their control. Annual review of biochemistry 61, 441-470
5.Nigg, E. A. (1995) Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. BioEssays : news and reviews in molecular, cellular and developmental biology 17, 471-480
6.Vermeulen, K., Van Bockstaele, D. R., and Berneman, Z. N. (2003) The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell proliferation 36, 131-149
7.Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D., and Hunt, T. (1983) Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 33, 389-396
8.Parker, B. W., Kaur, G., Nieves-Neira, W., Taimi, M., Kohlhagen, G., Shimizu, T., Losiewicz, M. D., Pommier, Y., Sausville, E. A., and Senderowicz, A. M. (1998) Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 91, 458-465
9.Morgan, D. O. (1995) Principles of CDK regulation. Nature 374, 131-134
10.Pines, J. (1995) Cyclins and cyclin-dependent kinases: theme and variations. Advances in cancer research 66, 181-212
11.Lapenna, S., and Giordano, A. (2009) Cell cycle kinases as therapeutic targets for cancer. Nature reviews. Drug discovery 8, 547-566
12.Sherr, C. J., and Roberts, J. M. (1995) Inhibitors of mammalian G1 cyclin-dependent kinases. Genes & development 9, 1149-1163
13.Chang, Y. T., Gray, N. S., Rosania, G. R., Sutherlin, D. P., Kwon, S., Norman, T. C., Sarohia, R., Leost, M., Meijer, L., and Schultz, P. G. (1999) Synthesis and application of functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors. Chemistry & biology 6, 361-375
14.Polyak, K., Lee, M. H., Erdjument-Bromage, H., Koff, A., Roberts, J. M., Tempst, P., and Massague, J. (1994) Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78, 59-66
15.Harper, J. W., Elledge, S. J., Keyomarsi, K., Dynlacht, B., Tsai, L. H., Zhang, P., Dobrowolski, S., Bai, C., Connell-Crowley, L., Swindell, E., and et al. (1995) Inhibition of cyclin-dependent kinases by p21. Molecular biology of the cell 6, 387-400
16.Lee, M. H., Reynisdottir, I., and Massague, J. (1995) Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes & development 9, 639-649
17.Hengst, L., and Reed, S. I. (1998) Inhibitors of the Cip/Kip family. Current topics in microbiology and immunology 227, 25-41
18.Wu, S., Bao, Y., Ma, D., Zi, Y., Yang, C., Yang, M., Xing, M., and Yang, W. (2014) Sodium selenite inhibits leukemia HL-60 cell proliferation and induces cell apoptosis by enhancing the phosphorylation of JNK1 and increasing the expression of p21 and p27. International journal of molecular medicine 34, 1175-1179
19.Bates, S., Ryan, K. M., Phillips, A. C., and Vousden, K. H. (1998) Cell cycle arrest and DNA endoreduplication following p21Waf1/Cip1 expression. Oncogene 17, 1691-1703
20.Niculescu, A. B., 3rd, Chen, X., Smeets, M., Hengst, L., Prives, C., and Reed, S. I. (1998) Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Molecular and cellular biology 18, 629-643
21.Chang, B. D., Watanabe, K., Broude, E. V., Fang, J., Poole, J. C., Kalinichenko, T. V., and Roninson, I. B. (2000) Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases. Proceedings of the National Academy of Sciences of the United States of America 97, 4291-4296
22.Wang, W., Nacusi, L., Sheaff, R. J., and Liu, X. (2005) Ubiquitination of p21Cip1/WAF1 by SCFSkp2: substrate requirement and ubiquitination site selection. Biochemistry 44, 14553-14564
23.Sarmento, L. M., Huang, H., Limon, A., Gordon, W., Fernandes, J., Tavares, M. J., Miele, L., Cardoso, A. A., Classon, M., and Carlesso, N. (2005) Notch1 modulates timing of G1-S progression by inducing SKP2 transcription and p27 Kip1 degradation. The Journal of experimental medicine 202, 157-168
24.Yu, Z. K., Gervais, J. L., and Zhang, H. (1998) Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins. Proceedings of the National Academy of Sciences of the United States of America 95, 11324-11329
25.Bornstein, G., Bloom, J., Sitry-Shevah, D., Nakayama, K., Pagano, M., and Hershko, A. (2003) Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase. The Journal of biological chemistry 278, 25752-25757
26.Shats, I., Milyavsky, M., Tang, X., Stambolsky, P., Erez, N., Brosh, R., Kogan, I., Braunstein, I., Tzukerman, M., Ginsberg, D., and Rotter, V. (2004) p53-dependent down-regulation of telomerase is mediated by p21waf1. The Journal of biological chemistry 279, 50976-50985
27.Lohr, K., Moritz, C., Contente, A., and Dobbelstein, M. (2003) p21/CDKN1A mediates negative regulation of transcription by p53. The Journal of biological chemistry 278, 32507-32516
28.Moldovan, G. L., Pfander, B., and Jentsch, S. (2007) PCNA, the maestro of the replication fork. Cell 129, 665-679
29.Delavaine, L., and La Thangue, N. B. (1999) Control of E2F activity by p21Waf1/Cip1. Oncogene 18, 5381-5392
30.Ghanem, L., and Steinman, R. (2005) A proapoptotic function of p21 in differentiating granulocytes. Leukemia research 29, 1315-1323
31.Dotto, G. P. (2000) p21(WAF1/Cip1): more than a break to the cell cycle? Biochimica et biophysica acta 1471, M43-56
32.Roninson, I. B. (2002) Oncogenic functions of tumour suppressor p21(Waf1/Cip1/Sdi1): association with cell senescence and tumour-promoting activities of stromal fibroblasts. Cancer letters 179, 1-14
33.Rossig, L., Jadidi, A. S., Urbich, C., Badorff, C., Zeiher, A. M., and Dimmeler, S. (2001) Akt-dependent phosphorylation of p21(Cip1) regulates PCNA binding and proliferation of endothelial cells. Molecular and cellular biology 21, 5644-5657
34.Li, Y., Dowbenko, D., and Lasky, L. A. (2002) AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. The Journal of biological chemistry 277, 11352-11361
35.Xia, W., Chen, J. S., Zhou, X., Sun, P. R., Lee, D. F., Liao, Y., Zhou, B. P., and Hung, M. C. (2004) Phosphorylation/cytoplasmic localization of p21Cip1/WAF1 is associated with HER2/neu overexpression and provides a novel combination predictor for poor prognosis in breast cancer patients. Clinical cancer research : an official journal of the American Association for Cancer Research 10, 3815-3824
36.Winters, Z. E., Leek, R. D., Bradburn, M. J., Norbury, C. J., and Harris, A. L. (2003) Cytoplasmic p21WAF1/CIP1 expression is correlated with HER-2/ neu in breast cancer and is an independent predictor of prognosis. Breast cancer research : BCR 5, R242-249
37.Zhou, B. P., Liao, Y., Xia, W., Spohn, B., Lee, M. H., and Hung, M. C. (2001) Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nature cell biology 3, 245-252
38.Ping, B., He, X., Xia, W., Lee, D. F., Wei, Y., Yu, D., Mills, G., Shi, D., and Hung, M. C. (2006) Cytoplasmic expression of p21CIP1/WAF1 is correlated with IKKbeta overexpression in human breast cancers. International journal of oncology 29, 1103-1110
39.Abbas, T., and Dutta, A. (2009) p21 in cancer: intricate networks and multiple activities. Nature reviews. Cancer 9, 400-414
40.Sherr, C. J., and Roberts, J. M. (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes & development 13, 1501-1512
41.Nakayama, K. I., and Nakayama, K. (2006) Ubiquitin ligases: cell-cycle control and cancer. Nature reviews. Cancer 6, 369-381
42.Sekimoto, T., Fukumoto, M., and Yoneda, Y. (2004) 14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1). The EMBO journal 23, 1934-1942
43.Shin, I., Rotty, J., Wu, F. Y., and Arteaga, C. L. (2005) Phosphorylation of p27Kip1 at Thr-157 interferes with its association with importin alpha during G1 and prevents nuclear re-entry. The Journal of biological chemistry 280, 6055-6063
44.Liang, J., Zubovitz, J., Petrocelli, T., Kotchetkov, R., Connor, M. K., Han, K., Lee, J. H., Ciarallo, S., Catzavelos, C., Beniston, R., Franssen, E., and Slingerland, J. M. (2002) PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nature medicine 8, 1153-1160
45.Fujita, N., Sato, S., and Tsuruo, T. (2003) Phosphorylation of p27Kip1 at threonine 198 by p90 ribosomal protein S6 kinases promotes its binding to 14-3-3 and cytoplasmic localization. The Journal of biological chemistry 278, 49254-49260
46.Wu, F. Y., Wang, S. E., Sanders, M. E., Shin, I., Rojo, F., Baselga, J., and Arteaga, C. L. (2006) Reduction of cytosolic p27(Kip1) inhibits cancer cell motility, survival, and tumorigenicity. Cancer research 66, 2162-2172
47.Chu, I. M., Hengst, L., and Slingerland, J. M. (2008) The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nature reviews. Cancer 8, 253-267
48.Shrimali, D., Shanmugam, M. K., Kumar, A. P., Zhang, J., Tan, B. K., Ahn, K. S., and Sethi, G. (2013) Targeted abrogation of diverse signal transduction cascades by emodin for the treatment of inflammatory disorders and cancer. Cancer letters 341, 139-149
49.Tan, W., Lu, J., Huang, M., Li, Y., Chen, M., Wu, G., Gong, J., Zhong, Z., Xu, Z., Dang, Y., Guo, J., Chen, X., and Wang, Y. (2011) Anti-cancer natural products isolated from chinese medicinal herbs. Chinese medicine 6, 27
50.Huang, P. H., Huang, C. Y., Chen, M. C., Lee, Y. T., Yue, C. H., Wang, H. Y., and Lin, H. (2013) Emodin and Aloe-Emodin Suppress Breast Cancer Cell Proliferation through ER alpha Inhibition. Evidence-based complementary and alternative medicine : eCAM 2013, 376123
51.Liu, J. B., Gao, X. G., Lian, T., Zhao, A. Z., and Li, K. Z. (2003) [Apoptosis of human hepatoma HepG2 cells induced by emodin in vitro]. Ai zheng = Aizheng = Chinese journal of cancer 22, 1280-1283
52.Yu, C. X., Zhang, X. Q., Kang, L. D., Zhang, P. J., Chen, W. W., Liu, W. W., Liu, Q. W., and Zhang, J. Y. (2008) Emodin induces apoptosis in human prostate cancer cell LNCaP. Asian journal of andrology 10, 625-634
53.He, L., Bi, J. J., Guo, Q., Yu, Y., and Ye, X. F. (2012) Effects of emodin extracted from Chinese herbs on proliferation of non-small cell lung cancer and underlying mechanisms. Asian Pacific journal of cancer prevention : APJCP 13, 1505-1510
54.Zhou, X., Song, B., Jin, L., Hu, D., Diao, C., Xu, G., Zou, Z., and Yang, S. (2006) Isolation and inhibitory activity against ERK phosphorylation of hydroxyanthraquinones from rhubarb. Bioorganic & medicinal chemistry letters 16, 563-568
55.Cha, T. L., Qiu, L., Chen, C. T., Wen, Y., and Hung, M. C. (2005) Emodin down-regulates androgen receptor and inhibits prostate cancer cell growth. Cancer research 65, 2287-2295
56.Su, Y. T., Chang, H. L., Shyue, S. K., and Hsu, S. L. (2005) Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway. Biochemical pharmacology 70, 229-241
57.Kwak, H. J., Park, M. J., Park, C. M., Moon, S. I., Yoo, D. H., Lee, H. C., Lee, S. H., Kim, M. S., Lee, H. W., Shin, W. S., Park, I. C., Rhee, C. H., and Hong, S. I. (2006) Emodin inhibits vascular endothelial growth factor-A-induced angiogenesis by blocking receptor-2 (KDR/Flk-1) phosphorylation. International journal of cancer 118, 2711-2720
58.Dong, X., Fu, J., Yin, X., Cao, S., Li, X., Lin, L., and Ni, J. (2016) Emodin: A Review of its Pharmacology, Toxicity and Pharmacokinetics. Phytotherapy research : PTR 30, 1207-1218
59.Wang, Y., Yu, H., Zhang, J., Ge, X., Gao, J., Zhang, Y., and Lou, G. (2015) Anti-tumor effect of emodin on gynecological cancer cells. Cellular oncology (Dordrecht) 38, 353-363
60.Park, S. J., Jin, M. L., An, H. K., Kim, K. S., Ko, M. J., Kim, C. M., Choi, Y. W., and Lee, Y. C. (2015) Emodin induces neurite outgrowth through PI3K/Akt/GSK-3beta-mediated signaling pathways in Neuro2a cells. Neuroscience letters 588, 101-107
61.Li, C. J., Chang, J. K., Chou, C. H., Wang, G. J., and Ho, M. L. (2010) The PI3K/Akt/FOXO3a/p27Kip1 signaling contributes to anti-inflammatory drug-suppressed proliferation of human osteoblasts. Biochemical pharmacology 79, 926-937
62.Hsu, C. M., Hsu, Y. A., Tsai, Y., Shieh, F. K., Huang, S. H., Wan, L., and Tsai, F. J. (2010) Emodin inhibits the growth of hepatoma cells: finding the common anti-cancer pathway using Huh7, Hep3B, and HepG2 cells. Biochemical and biophysical research communications 392, 473-478
63.Jascur, T., Brickner, H., Salles-Passador, I., Barbier, V., El Khissiin, A., Smith, B., Fotedar, R., and Fotedar, A. (2005) Regulation of p21(WAF1/CIP1) stability by WISp39, a Hsp90 binding TPR protein. Molecular cell 17, 237-249
64.Berlett, B. S., and Stadtman, E. R. (1997) Protein oxidation in aging, disease, and oxidative stress. The Journal of biological chemistry 272, 20313-20316
65. 民國102年癌症登記年報