娃兒藤生物鹼(Tylophorine)是天然存在的菲駢類吲哚生物鹼(phenanthroindolizidine alkaloid)，為印度藥草植物(Tylophorine indica)的主要有效成分。由娃兒藤生物鹼所衍生的化合物具有多種生物學上的功能，包含:抗發炎、抗癌及抗病毒等生物活性。在實驗室先前的研究中，我們報導過在癌細胞中，娃兒藤生物鹼可藉由結合含有caprin-1蛋白質、G3BP1蛋白質及c-Myc信使核醣核酸(mRNA)的核糖核蛋白複合體(ribonucleoprotein complex)，從而防止其相應信使核醣核酸的轉譯(translation)；而在急性的發炎系統[Raw264.7細胞株以脂多醣(LPS)及干擾素伽瑪(IFN-gamma)刺激]中，娃兒藤生物鹼則會透過提高Akt蛋白質的活性，來降低c-Jun蛋白質的表現量，進而達到抑制AP1蛋白質的效果。然而，在Raw264.7細胞株中，其娃兒藤生物鹼所作用的標的物仍舊未知。在蛋白質體外結合實驗(pull-down experiment)中，藉由使用接上生物素(biotin)的娃兒藤生物鹼，我們發現在Raw264.7細胞株中，娃兒藤生物鹼和在癌細胞中所報導的一樣，皆會結合含有caprin-1蛋白質、G3BP1蛋白質及c-Myc信使核醣核酸的核糖核蛋白複合體，並且阻斷c-Myc蛋白質及其下游所調控和瓦氏效應(Warburg effect)相關的基因表現，包含：ASS1、GLS1、Glut1、Glut3、iNOS以及COX II。另一方面，藉由調控RhoC蛋白質的表現量，娃兒藤生物鹼可將活化的Raw264.7細胞株的細胞型態，從特定的樹突狀(巨噬細胞樣)轉變回圓形的樣貌(單核細胞樣)，除此之外，娃兒藤生物鹼及其衍生物dibenzoquinoline-33b也隨使用劑量的增加而大幅抑制細胞激素(cytokine) 的產生，其包含nitric oxide、TNF-alpha及IL-6。因此，我們的研究結果詳細描述出娃兒藤生物鹼的抗發炎機制，也提供一個全新的細胞標靶用以發展抗發炎藥物。

Tylophorine is a naturally occurring phenanthroindolizidine alkaloid and the major active component of herb Tylophorine indica. Tylophorine based compounds exhibit multiple biological functions including activities of anti-inflammatory, anti-cancer and anti-coronaviruses. Previously, we reported that 1) in carcinoma cells, tylophorine compounds target the ribonucleoprotein complex that contains caprin-1, G3BP1, and c-Myc mRNA, and thus preventing the translation of the corresponding mRNA transcripts; 2) In an acute inflammation system (Raw264.7 cells stimulated by LPS/IFN-gamma), tylophorine compounds augment the Akt activation, which leads to the AP1 inhibition through diminishing c-Jun expression. However, in Raw264.7 cells, the cellular targets of tylophorine compounds remain unknown. In the pull-down experiment, using biotinylated tylophorine, we found that tylophorine targeted the ribonucleoprotein complex containing caprin1 and G3BP1, and c-Myc mRNA in LPS/IFN-gamma stimulated Raw264.7 cells as that in carcinoma cells, thus blocking protein expressions of c-Myc and its downstream target genes related to Warburg effect including argininosuccinate synthase 1 (ASS1), kidney-type glutaminase (GLS1), glucose transporter 1 (Glut1), glucose transporter 3 (Glut3), inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX) II. Also, the morphology of activated Raw264.7 cells switched from the specific dendritic shape (macrophage-like) to the generic round shape (monocyte-like) upon tylophorine treatment, which was thought to be mediated by RhoC expression. In addition, tylophorine and its derivative dibenzoquinoline-33b (DBQ 33b) also significantly inhibited cytokine production of nitric oxide, TNF-alpha, and IL-6 in a dose-dependent manner. Therefore, our results delineates the principal mechanism of how tylophorine compounds exhibit their anti-inflammatory activities and provide a novel cellular target for the development of anti-inflammatory drugs.

Abstract (English)………………………………………………………i
Abstract (Chinese)……………………………………………………..ii
Acknowledgement……………………………………………………..iii
List of Figures………………………………………………………….iv
List of Appendix Figures……………………………………………..vii
Abbreviation…………………………………………………………...ix

1. Introduction………………………………………………………….1

1.1. Studies of Tylophora indica / Tylophora ovata and their
related compounds…………1

1.2. The differentiation from monocyte (MO) to M1/M2
macrophage………...2
1.3. The role of c-Myc in metabolism………………………………………….4
1.4. Rho GTPases and their distinct functions………………………………...5

2. Materials and Methods………………………………………………………....18

2.1. Cell cultures and chemicals……………………………………………...18
2.2. Pull-down assay……………………………………………………….....18
2.3. Western blot analysis…………………………………………………….19
2.4. Transfection……………………………………………………………...20
2.5. Reverse transcription polymerase chain reaction (RT-PCR)…………....20
2.6. Active Rho/Rac1/Cdc42 detection assay………………………………...21
2.7. c-Myc gene silencing…………………………………………………….22
2.8. RhoC gene silencing…………………………………………………......22
2.9. RhoA gene silencing……………………………………………………...23
2.10. Griess assay……………………………………………………………...23
2.11. Mouse TNF-α/IL-6 enzyme-linked immunosorbent assay…………….....24

3. Results…………………………………………………………………………...25

3.1. Tylophorine targets the RNP complex containing caprin-1, G3BP1, and mRNAs of c-Myc and Hif1-α…...25
3.2. Tylophorine suppresses not only c-Myc expression but also several gene expression related to Warburg effect in activated Raw264.7 cells……26
3.3. Tylophorine inhibits metabolic switch through suppressing c-Myc ……..27
and its downstream gene expression in activated Raw264.7 cells
3.4. Tylophorine inhibits morphological change of activated Raw264.7 cells from specific dendritic shape to generic round shape through suppressing RhoC expression and its activity…….28
3.5. Tylophorine and DBQ 33b significantly inhibit the cytokine
production of nitric oxide, TNF-alpha, and IL-6 in activated
Raw264.7 cells……………30

4. Discussion………………………………………………………………………..32

Reference…………………………………………………………………………….61

Appendix…………………………………………………………………………….65

1. Gupta, M., Singh, M., Mukhatr, H. M., and Ahmad, S. (2010) HPTLC Fingerprinting of different leaf extracts of Tylophora indica (Burm F.) Merill. Phcog J. 2, 381-385
2. Gopal, K. C., Shankarnarayan, D., Kameswaran, L., and Natarajan, S. (1979) Pharmacological investigation of Tylophorine, Indian. J. Med. Res. 69, 513-520
3. Faisal, M. and Anis, M. (2005). In vitro regeneration and plant establishment of Tylophora indica: Petiole callus culture. In vitro Cell. Dev. Biol. Plant. 41, 511-515
4. Qiu, Y. Q., Yang, C. W., Lee, Y. Z., Yang, R. B., Lee, C. H., Hsu, H. Y., Chang, C. C., and Lee, S. J. (2015) Targeting a ribonucleoprotein complex containing the caprin-1 protein and the c-Myc mRNA suppresses tumor growth in mice: an identification of a noveloncotarget. Oncotarget. 6, 2148–2163
5. Lee, Y. Z., Huang, C. W., Yang, C. W., Hsu, H. Y., Kang, I. J., Chao, Y. S., Chen, I. S., Chang, H. Y., and Lee, S. J. (2011) Isolation and biological activities of phenanthroindolizidine and septicine alkaloids from the formosan tylophora ovata. Planta Med. 77, 1932
6. Lee, Y. Z., Yang, C. W., Hsu, H. Y., Qiu, Y. Q., Yeh, T. K., Chang, H. Y., Chao, Y. S., and Lee, S.-J. (2012) Synthesis and biological evaluation of tylophorine-derived dibenzoquinolines as orally active agents: exploration of the role of tylophorine e ring on biological activity. J. Med. Chem. 55, 10363–10377
7. Yang, C. W., Chen, W. L., Wu, P. L., Tseng, H. Y., and Lee, S. J. (2006) Anti-inflammatory mechanisms of phenanthroindolizidine alkaloids. Mol. Pharmacol. 69, 749−758.
8. Steinman, R. M. and Idoyaga, J. (2010) Features of the dendritic cell lineage. Immunol Rev. 234, 5-17
9. MacMicking, J., Xie, Q. W., and Nathan, C. (1997) Nitric oxide and macrophage function. Annu Rev Immunol. 15, 323-50
10. Guillermo, A. D. and Albert, B. (2014) Macrophage Cytokines: Involvement in Immunity and Infectious Diseases. Front Immunol 5, 491
11. O’Neill, L. A. (2015) A broken krebs cycle in macrophages. Immunity 42, 293-4
12. Netea, M. G., Quintin, J., and van der Meer, J. W. (2011) Trained immunity: A memory for innate host defense. Cell Host Microbe 9, 355–361
13. Ma, A., Moroy, T., Collum, R., Weintraub, H., Alt, F. W., and Blackwell, T. K. (1993). DNA binding by N- and L-Myc proteins. Oncogene 8, 1093-1098
14. Blackwood, E. M., and Eisenman, R. N. (1991) Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 251, 1211-217
15. Blackwood, E. M., Kretzner, L., and Eisenman, R. N. (1992) Myc and Max function as a nucleoprotein complex. Curr. Opin. Genet. Dev. 2, 227-235
16. Luscher, B. and Vervoorts, J. (2012) Regulation of gene transcription by the oncoprotein MYC. Gene 494, 145-160
17. Dang, C. V. (1999) c-Myc Target Genes Involved in Cell Growth, Apoptosis, and Metabolism. Mol. Cell. Biol. 19, 1-11
18. Wahlstrom, T. and Henriksson, M. A. (2015) Impact of MYC in regulation of tumor cell metabolism. Biochim Biophys Acta. 1849, 563-9
19. Tsai, W. B., Aiba, I., Long, Y., Lin, H. K., Feun, L., Savaraj, N., and Kuo, M. T. (2012) Activation of Ras/PI3K/ERK pathway induces c-Myc stabilization to upregulate argininosuccinate synthetase, leading to arginine deiminase resistance in melanoma cells. Cancer Res. 72, 2622-33
20. Gao, P., Tchernyshyov, I., Chang, T. C., Lee, Y. S., Kita, K., Ochi, T., Zeller, K. I., De Marzo, A. M., Van Eyk, J. E., Mendell, J. T., and Dang, C. V. (2009) c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature. 458, 762-5
21. Kim, J. W., Gao, P., Liu, Y. C., Semenza, G. L., and Dang, C. V. (2007) Hypoxia-inducible factor 1 and dysregulated c-Myc cooperatively induce vascular endothelial growth factor and metabolic switches hexokinase 2 and pyruvate dehydrogenase kinase 1. Mol Cell Biol. 27, 7381-93
22. Shim, H., Dolde, C. Lewis, B. C., Wu, C. S., Dang, G., Jungmann, R.A., Dalla-Favera, R., and Dang, C.V. (1997) c-Myc transactivation of LDH-A: implications for tumor metabolism and growth. Proc Natl Acad Sci U S A. 94, 6658-63
23. Osthus, R. C., Shim, H., Kim, S., Li. Q., Reddy, R., Mukherjee, M., Xu, Y., Wonsey, D., Lee, L. A., and Dang, C. V. (2000) Deregulation of glucose transporter 1 and glycolytic gene expression by c-Myc. J Biol Chem. 275, 21797-800
24. Yim, H. C., Li, J. C., Pong, J. C., and Lau, A. S. (2011) A role for c-Myc in regulating anti-mycobacterial responses. Proc Natl Acad Sci U S A. 108, 17749-54
25. Ting, L., Yu, Z., Kwang, S. K., and Heping, Y. (2015) Interactions between Myc and Mediators of Inflammation in Chronic Liver Diseases. Mediators Inflamm. 2015, 276850
26. Vega, F. M., and Ridley, A. J. (2008) Rho GTPases in cancer cell biology. FEBS Lett. 582, 2093-101
27. Jaffe, A. B. and Hall, A. (2005) Rho GTPases: biochemistry and biology. Annu Rev. Cell Dev Biol. 21, 247–269
28. Bos, J. L., Rehmann, H., and Wittinghofer, A. (2007) GEFs and GAPs: critical elements in the control of small G proteins. Cell 129, 865–877
29. Dovas, A. and Couchman, J. R. (2005) RhoGDI: multiple functions in the regulation of Rho family GTPase activities. Biochem. J. 390, 1–9.
30. Wheeler, A. P. and Ridley, A. J. (2004) Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp Cell Res. 301, 43-9
31. Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy, G., Parsons, J. T., and Horwitz, A. R. (2003) Cell migration: integrating signals from front to back. Science 302, 1704-09
32. Sahai, E. and Marshall, C. J. (2002) RHO-GTPases and cancer. Nat Rev Cancer. 2, 133-42
33. Karlsson, R., Pedersen, E. D., Wang, Z., and Brakebusch, C. (2009) Rho GTPase function in tumorigenesis. Biochim Biophys Acta. 1796, 91-8
34. Huang, M. and Prendergast, G. C. (2006) RhoB in cancer suppression. Histol Histopathol. 21, 213-8
35. Clark, E. A., Golub, T.R., Lander, E.S., and Hynes, R.O. (2000) Genomic analysis of metastasis reveals an essential role for RhoC. Nature 406, 532-35
36. Hakem, A., Sanchez-Sweatman, O., You-Ten, A., Duncan, G., Wakeham, A., Khokha, R., and Mak, T.W. (2005) RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis. Genes Dev. 19, 1974-79
37. Bellovin, D.I., Simpson, K.J., Danilov, T., Maynard, E., Rimm, D.L., Oettgen, P., and Mercurio, A.M. (2006) Reciprocal regulation of RhoA and RhoC characterizes the EMT and identifies RhoC as a prognostic marker of colon carcinoma. Oncogene 25, 6959-67
38. Pillé, J. Y., Denoyelle, C., Varet, J., Bertrand, J. R., Soria, J., Opolon, P., Lu, H., Pritchard, L. L., Vannier, J. P., Malvy, C., Soria, C., and Li, H. (2005) Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo. Mol. Ther. 11, 267-74
39. Simpson, K.J., Dugan, A.S., and Mercurio, A.M. (2004) Functional analysis of the contribution of RhoA and RhoC GTPases to invasive breast carcinoma. Cancer Res. 64, 8694-8701
40. Bravo-Cordero, J. J., Hodgson, L., and Condeelis, J. S. (2014) Spatial regulation of tumor cell protrusions by RhoC. Cell Adh Migr. 8, 263-7
41. Yang, C. W., Lee, Y. Z., Kang, I. J., Barnard, D. L., Jan, J. T., Lin, D., Huang, C. W., Yeh, T. K., Chao, Y. S., and Lee, S. J. (2010) Identification of phenanthroindolizines and phenanthroquinolizidines as novel potent anti-coronaviral agents for porcine enteropathogenic coronavirus transmissible gastroenteritis virus and human severe acute respiratory syndrome coronavirus. Antiviral Research. 88, 160-168
42. Giuliani, C., Napolitano, G., Bucci, I., Montani, V., and Monaco, F. (2001) Nf-kB transcription factor: role in the pathogenesis of inflammatory, autoimmune, and neoplastic diseases and therapy implications. Clin Ter. 152, 249-53