臺灣博碩士論文加值系統

穿心蓮內酯 (andrographolide, ANDRO) 是二萜類結構的天然化合物，它是中草藥穿心蓮(Andrographis paniculata)葉片中的主要成分。先前傳醫所傅淑玲老師實驗室將癌細胞處理穿心蓮內酯後，以蛋白質體學方法鑑定到熱休克蛋白90 (Heat shock protein, Hsp90) 斷裂的片段並且利用西方點墨法發現Bcr-Abl的降解。由於過去文獻中的蛋白體學研究資料指出Hsp90是穿心蓮內酯之標靶蛋白，且Bcr-Abl是Hsp90的受質蛋白，我們推測穿心蓮內酯可能透過化學反應與Hsp90產生鍵結而抑制其穩定受質蛋白之能力，再進而導致Bcr-Abl的降解使癌細胞無法存活。為了詳細去探討穿心蓮內酯與Hsp90之間的作用機制，實驗室與台大羅禮強老師合作設計帶有螢光基團的穿心蓮內酯衍生物 (ANDRO-NBD) 以協助找尋穿心蓮內酯的反應對象蛋白或其上的對象胺基酸。首先我的實驗先確認了ANDRO-NBD的確可以與穿心蓮內酯的已知標靶蛋白p50以及Hsp90進行化學鍵結，並且此鍵結在蛋白質結構遭受破壞後受到抑制。除此之外我們更發現ANDRO-NBD比穿心蓮內酯對於標靶蛋白有更好的反應性。之前文獻指出穿心蓮內酯可以藉由與p50的半胱胺酸 (cysteine) 形成共價鍵來抑制NF-κB的活性。我的實驗也確認了ANDRO-NBD同樣地專一性地作用在標靶蛋白之半胱胺酸。利用液相串聯質譜儀 (LC-MS/MS) 鑑定到穿心蓮內酯所鍵結到的含Cys572之Hsp90胜肽片段以及ANDRO-NBD所鍵結到的Cys529、Cys572的Hsp90胜肽片段，不只如此我們也找到了ANDRO-NBD同時標的到兩段分別含有Cys572、Cys420的胜肽複合物。結果顯示穿心蓮內酯以及ANDRO-NBD除了可以透過鍵結一個半胱胺酸來抑制Hsp90的活性外，ANDRO-NBD還會透過二次反應，把Hsp90不同位置的半胱胺酸鍵結在一起。本研究也觀察到在慢性骨髓白血病K562細胞表現突變型Hsp90有些微地降低穿心蓮內酯誘導Bcr-Abl降解的效果，但是目前這部分實驗結果還無法下定論，未來還需深入去探討。總結以上結果，此研究發現了穿心蓮內酯對Hsp90作用的位置，以及ANDRO-NBD對Hsp90還可以進行二次反應，這可能限制了Hsp90的構型以及阻礙Hsp90與受質蛋白的交互作用，提供了ANDRO-NBD新的抗癌作用機轉，另外這些結果也解釋了ANDRO-NBD比穿心蓮內酯對於標靶蛋白有更好的反應性及更好的抗癌效果。未來建議對穿心蓮內酯進行化學結構上的修飾，讓衍生物更專一性作用於Hsp90 Cys572上，提高抑制Hsp90活性的效果。

Andrographolide (ANDRO) is a natural product of diterpenoid lactone, which is isolated from the leaves of Andrographis paniculata. Professor Shu-Ling Fu (NYMU) previously reported that a cleaved fragment of Hsp90 was identified in ANDRO-treated cells by a proteomics approach. In addition, Fu’s lab also reported that ANDRO induced Hsp90 cleavage and decreased levels of Bcr-Abl in K562 cell, a cell line derived from patients with chronic myelogenous leukemia. A previous proteomics research previously reported that Hsp90 was a targeting protein of ANDRO. We speculated that ANDRO promoted the Bcr-Abl degradation and hence suppressed cancer cell proliferation via forming chemical bonds with Hsp90. In order to identify the target proteins of ANDRO, we have collaborated with Professor Lee-Chiang Lo (NTU) to design a fluorescence-based derivative of ANDRO (ANDRO-NBD), which possesses an acetoxy group at C-14 and a nitrobenzoxadiazole (NBD) fluorophore at C-19 hydroxyl group. The fluorescent signal of ANDRO-NBD was detected at the position of p50 on SDS-PAGE gel, and confirmed that ANDRO-NBD covalently labels with a known protein target of ANDRO. In addition, the heating-mediated decrease of fluorescent signal of ANDRO-NBD on target proteins was due to the loss of protein conformation. Furthermore, we confirmed that Hsp90 is a target protein of ANDRO-NBD, and ANDRO-NBD exhibits a stronger activity of protein targeting than ANDRO. Previous studies indicated that ANDRO affects NF-κB function through direct labeling with cysteine residue of p50. I also confirmed that ANDRO-NBD labels cysteine as ANDRO in target proteins. The ANDRO-labeled peptide fragment containing Cys572 of Hsp90 was identified by LC-MS/MS. In addition, the ANDRO-NBD-labeled peptide fragments containing Cys572 and Cys529 were also identified, and I further found that ANDRO-NBD induces crosslinking of two Hsp90 peptides, TKFENLC572K and DYC481TR. Our results suggested that ANDRO/ANDRO-NBD inhibited Hsp90 activity by binding to a cysteine residue. Furthermore, ANDRO-NBD can induce crosslinking of different hsp90 cysteines via 2nd labeling. Moreover, we found that ANDRO-induced Bcr-Abl downregulation was slightly recovered in the cell constitutively expressing Hsp90 C572S in K562 cells. Therefore, this finding cannot jump to a conclusion. In summary, this thesis identified the targeting site(s) of Hsp90 inhibition by ANDRO and ANDRO-NBD. In addition, ANDRO-NBD also induces crosslinking of different cysteines, which may result in conformational change of Hsp90 and the disruption of interaction between Hsp90 and its client proteins. These findings provide a possible mechanism explaining why ANDRO-NBD exhibits better anti-cancer effect than ANDRO. In the future, development of ANDRO analogs, with higher labeling to Cys572 of Hsp90 protein, may exhibit higher inhibitory effect on Hsp90 and merit further development.

中文摘要 i
Abstract ii
目錄 iii
圖目錄 v
第一章、緒論(背景) 1
1. 癌症 1
1.2 癌症共同特徵 1
1.3 黑色素瘤 1
1.4 慢性骨髓性白血病 (Chronic Myelogenous Leukemia, CML) 1
2. 熱休克蛋白90 (Heat shock protein 90, Hsp90) 3
2.1 Hsp90的結構與構型週期 4
2.2 Hsp90與癌症共同特徵的關係 4
2.3 Hsp90小分子抑制劑 4
2.4 Hsp90小分子抑制劑針對慢性骨髓白血病 (CML) 的治療 5
3. 穿心蓮內酯 5
3.1 穿心蓮內的酯標靶蛋白以及可能的作用機制之研究 5
3.2 穿心蓮內酯誘導細胞週期停滯 5
3.3 穿心蓮內酯影響細胞凋亡 6
3.4 穿心蓮內酯對免疫的影響 6
3.5 穿心蓮內酯引發抗發炎以及抗血管生成 6
研究動機與目標 7
第二章、實驗材料與方法 8
1. 實驗材料 8
1.1 試劑與抗體 8
1.2 緩衝液配方 8
1.3 質體 (plasmids)與引物 (primer) 9
1.4 實驗細胞株 9
2. 實驗方法 10
2.1 細胞株培養及收集細胞沉澱物 10
2.2 質體轉染 (transfection) 10
2.3 細胞分離 (Cell Fractionation) 10
2.4 重組蛋白質之選殖與製備 11
2.5 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 12
2.6 西方點墨法 (Western blot) 12
2.7 SDS-PAGE蛋白膠內酶解 (In-gel digestion) 13
2.8 穿心蓮內酯螢光探針 (ANDRO-NBD) 實驗 13
第三章、實驗結果 14
1. 確認穿心蓮內酯衍生物 (ANDRO-NBD) 與穿心蓮內酯標靶蛋白之間的作用 14
1.1 穿心蓮內酯衍生物 (ANDRO-NBD) 能相同地作用於穿心蓮內酯已知的標靶蛋白p50. 14
1.2 ANDRO-NBD直接作用於重組性蛋白Hsp90 14
1.3 ANDRO-NBD可作用於眾多未知的目標蛋白 14
1.4 ANDRO-NBD作用於有穩定結構的標靶蛋白 15
1.5 ANDRO-NBD專一性地作用於標靶蛋白的半胱胺酸 15
1.6 ANDRO-NBD具有比穿心蓮內酯更好的蛋白質反應活性 15
2. 探討穿心蓮內酯及ANDRO-NBD標定Hsp90蛋白的反應機制 16
2.1 穿心蓮內酯標定於Hsp90的Cys572位置 16
2.2 ANDRO-NBD標定於Hsp90的 Cys529、Cys572位置 16
2.3 ANDRO-NBD具有交鏈 (cross link)兩個半胱胺酸的能力 16
2.4 ANDRO-NBD具有交鏈Hsp90的兩段不同半胱胺酸胜肽的能力 17
3. 比較突變型以及野生型的重組蛋白Hsp90對ANDRO-NBD的結合能力 17
4. 穿心蓮內酯與其衍生物可促進Bcr-Abl的降解 17
5. Hsp90調控穿心蓮內酯誘導的Bcr-Abl降解 18
第四章、討論 19
第五章、參考文獻 24
第六章、圖表 29


Figure 1. Chemical structures of andrographolide (ANDRO) and its derivative (ANDRO-NBD). 29
Figure 2. 10% SDS-PAGE analysis of recombinant p50-WT during expression and purification 30
Figure 3. ANDRO-NBD directly labels the recombinant p50 in vitro 31
Figure 4. 10% SDS-PAGE analysis of recombinant His-Hsp90-WT during expression and purification 32
Figure 5. ANDRO-NBD directly labels the recombinant Hsp90 in vitro 33
Figure 6. Binding assay of ANDRO-NBD and NBD in cell lysate of B16F10 34
Figure 7. ANDRO-NBD labels the B16F10 cell lysate proteins from different cell fractions 35
Figure 8 . The heating-mediated decrease of fluorescent signal of ANDRO-NBD on target proteins 36
Figure 9 . Labeling of ANDRO-NBD with protein targets in cell lysates can be inhibited by heating 37
Figure 10. Iodoacetamide (IAA) reduces the labeling of p50 with ANDRO-NBD in vitro 38
Figure 11. Iodoacetamide (IAA) reduces the labeling of Hsp90-WT with ANDRO-NBD in vitro 39
Figure 12. Iodoacetamide (IAA) reduces the labeling of Hsp90 middle-domain with ANDRO-NBD in vitro 40
Figure 13. Iodoacetamide (IAA) reduces the labeling of B16F10 cell lysate proteins with ANDRO-NBD in vitro 41
Figure 14. ANDRO-NBD has better labeling ability with target proteins than ANDRO in vitro 42
Figure 15. High-dose ANDRO reduces the labeling of Hsp90 with ANDRO-NBD 43
Figure 16. MS/MS spectrum of ANDRO-labeled peptide TKFENLC572K of Hsp90α 44
Figure 17. LC-MS/MS spectrum of ANDRO-NBD labeled peptide TKFENLC572K of Hsp90α 45
Figure 18. LC-MS/MS spectrum of ANDRO-NBD labeled peptide HGLEVIYMIEPIDEYC529VQQLK of Hsp90α 46
Figure 19. A schematic diagram of chemical reaction to label the cysteine of proteins by ANDRO 47
Figure 20. A schematic diagram of chemical reaction to label the cysteine by ANDRO-NBD(a.) and cleavage of the ester bond of ANDRO-NBD occurred in LC-MS/MS (b.) 48
Figure 21. N-Acetyl-L-cysteine (NAC) can form two covalent bonds with ANDRO-NBD 49
Figure 22. ANDRO-NBD induces crosslinking two Hsp90 peptides, TKFENLC572K and DYC481TR 50
Figure 23. Construction map of plasmid pET23a-Hsp90-C529S/C572S-His 51
Figure 24. 10% SDS-PAGE analysis of recombinant His-tagged Hsp90 mutants during expression and purification 52
Figure 25. ANDRO-NBD-induced fluorescent signal of Hsp90-C529S and Hsp90-C529S/C572S are not significantly decrease in vitro 53
Figure 26. ANDRO-NBD-induced fluorescent signal of Hsp90-C572S and Hsp90-C529S/C572S are not significantly decrease in vitro 54
Figure 27. Bcr-Abl downregulation was induced by andrographolide (ANDRO) in K562 cells 55
Figure 28. Bcr-Abl downregulation was induced by ANDRO-NBD in K562 cells 56
Figure 29. ANDRO-induced Bcr-Abl downregulation was slightly recovered in the expressing Hsp90 C572S of K562 cells 57

1. Negrini, S., Gorgoulis, V. G., and Halazonetis, T. D. (2010) Genomic instability--an evolving hallmark of cancer. Nat Rev Mol Cell Biol 11, 220-228
2. Hanahan, D., and Weinberg, R. A. (2011) Hallmarks of cancer: the next generation. Cell 144, 646-674
3. Fidler, I. J. (2003) The pathogenesis of cancer metastasis: the ‘seed and soil’hypothesis revisited. Nat Rev Cancer 3, 1
4. Chambers, A. F., Groom, A. C., and MacDonald, I. C. (2002) Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2, 563-572
5. Melo, J. V., and Barnes, D. J. (2007) Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat Rev Cancer 7, 441-453
6. Melo, J. V. (1996) The molecular biology of chronic myeloid leukemia. Leukemia 10, 751-756
7. Pérez-Caro, M., and Sánchez-Garcia, I. (2007) BCR-ABL and Human Cancer. in Apoptosis, Cell Signaling, and Human Diseases: Molecular Mechanisms (Srivastava, R. ed.), Humana Press, Totowa, NJ. pp 3-34
8. Shahrabi, S., Azizidoost, S., Shahjahani, M., Rahim, F., Ahmadzadeh, A., and Saki, N. (2014) New insights in cellular and molecular aspects of BM niche in chronic myelogenous leukemia. Tumor Biology 35, 10627-10633
9. Cilloni, D., and Saglio, G. (2012) Molecular pathways: BCR-ABL. Clin Cancer Res 18, 930-937
10. Melo, J. V., and Deininger, M. W. (2004) Biology of chronic myelogenous leukemia--signaling pathways of initiation and transformation. Hematol Oncol Clin North Am 18, 545-568, vii-viii
11. Zhang, X., Subrahmanyam, R., Wong, R., Gross, A. W., and Ren, R. (2001) The NH(2)-terminal coiled-coil domain and tyrosine 177 play important roles in induction of a myeloproliferative disease in mice by Bcr-Abl. Mol Cell Biol 21, 840-853
12. Kurzrock, R., Gutterman, J., and Talpaz, M. (1988) The molecular genetics of Philadelphia chromosome-positive leukemias. N. Engl. J. Med. 319, 990-998
13. Carlesso, N., Frank, D. A., and Griffin, J. D. (1996) Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. J Exp Med 183, 811-820
14. Talpaz, M., Hehlmann, R., Quintas-Cardama, A., Mercer, J., and Cortes, J. (2013) Re-emergence of interferon-alpha in the treatment of chronic myeloid leukemia. Leukemia 27, 803-812
15. Sacha, T. (2014) Imatinib in chronic myeloid leukemia: an overview. Mediterr J Hematol Infect Dis 6, e2014007
16. Modi, H., McDonald, T., Chu, S., Yee, J. K., Forman, S. J., and Bhatia, R. (2007) Role of BCR/ABL gene-expression levels in determining the phenotype and imatinib sensitivity of transformed human hematopoietic cells. Blood 109, 5411-5421
17. Jabbour, E. J., Cortes, J. E., and Kantarjian, H. M. (2013) Resistance to tyrosine kinase inhibition therapy for chronic myelogenous leukemia: a clinical perspective and emerging treatment options. Clin Lymphoma Myeloma Leuk 13, 515-529
18. Abruzzese, E., Breccia, M., and Latagliata, R. (2014) Second-Generation Tyrosine Kinase Inhibitors in First-Line Treatment of Chronic Myeloid Leukaemia (CML). BioDrugs 28, 17-26
19. Chen, B., Zhong, D., and Monteiro, A. (2006) Comparative genomics and evolution of the HSP90 family of genes across all kingdoms of organisms. BMC Genomics 7, 156
20. Li, J., and Buchner, J. (2013) Structure, function and regulation of the hsp90 machinery. Biomed J 36, 106-117
21. Trepel, J., Mollapour, M., Giaccone, G., and Neckers, L. (2010) Targeting the dynamic HSP90 complex in cancer. Nat Rev Cancer 10, 537-549
22. K., S., and E., P. (2014) HSP90 Inhibitors: Current Development and Potential in Cancer Therapy. Recent Pat Anticancer Drug Discov 9, 1-20
23. Shrestha, L., Bolaender, A., Patel, H. J., and Taldone, T. (2016) Heat Shock Protein (HSP) Drug Discovery and Development: Targeting Heat Shock Proteins in Disease. Curr Top Med Chem 16, 2753-2764
24. Jilani, K., Qadri, S. M., and Lang, F. (2013) Geldanamycin-induced phosphatidylserine translocation in the erythrocyte membrane. Cell Physiol Biochem 32, 1600-1609
25. Jego, G., Hazoume, A., Seigneuric, R., and Garrido, C. (2013) Targeting heat shock proteins in cancer. Cancer letters 332, 275-285
26. Heath, E. I., Hillman, D. W., Vaishampayan, U., Sheng, S., Sarkar, F., Harper, F., Gaskins, M., Pitot, H. C., Tan, W., Ivy, S. P., Pili, R., Carducci, M. A., Erlichman, C., and Liu, G. (2008) A Phase II Trial of 17-Allylamino-17-Demethoxygeldanamycin in Patients with Hormone-Refractory Metastatic Prostate Cancer. Clinical Cancer Research 14, 7940-7946
27. Solit, D. B., Osman, I., Polsky, D., Panageas, K. S., Daud, A., Goydos, J. S., Teitcher, J., Wolchok, J. D., Germino, F. J., Krown, S. E., Coit, D., Rosen, N., and Chapman, P. B. (2008) Phase II trial of 17-allylamino-17-demethoxygeldanamycin in patients with metastatic melanoma. Clin Cancer Res 14, 8302-8307
28. Ronnen, E. A., Kondagunta, G. V., Ishill, N., Sweeney, S. M., DeLuca, J. K., Schwartz, L., Bacik, J., and Motzer, R. J. (2006) A phase II trial of 17-(Allylamino)-17-demethoxygeldanamycin in patients with papillary and clear cell renal cell carcinoma. Investigational New Drugs 24, 543-546
29. Taldone, T., Gozman, A., Maharaj, R., and Chiosis, G. (2008) Targeting Hsp90: small-molecule inhibitors and their clinical development. Curr Opin Pharmacol 8, 370-374
30. Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M., and Neckers, L. M. (2000) The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone. The Journal of biological chemistry 275, 37181-37186
31. Liu, S., Yuan, Y., Okumura, Y., Shinkai, N., and Yamauchi, H. (2010) Camptothecin disrupts androgen receptor signaling and suppresses prostate cancer cell growth. Biochemical and biophysical research communications 394, 297-302
32. Plescia, J., Salz, W., Xia, F., Pennati, M., Zaffaroni, N., Daidone, M. G., Meli, M., Dohi, T., Fortugno, P., Nefedova, Y., Gabrilovich, D. I., Colombo, G., and Altieri, D. C. (2005) Rational design of shepherdin, a novel anticancer agent. Cancer cell 7, 457-468
33. Zackova, M., Mouckova, D., Lopotova, T., Ondrackova, Z., Klamova, H., and Moravcova, J. (2013) Hsp90 - a potential prognostic marker in CML. Blood Cells Mol Dis 50, 184-189
34. Wu, L. X., Xu, J. H., Zhang, K. Z., Lin, Q., Huang, X. W., Wen, C. X., and Chen, Y. Z. (2008) Disruption of the Bcr-Abl/Hsp90 protein complex: a possible mechanism to inhibit Bcr-Abl-positive human leukemic blasts by novobiocin. Leukemia 22, 1402-1409
35. Xia, Y. F., Ye, B. Q., Li, Y. D., Wang, J. G., He, X. J., Lin, X., Yao, X., Ma, D., Slungaard, A., Hebbel, R. P., Key, N. S., and Geng, J. G. (2004) Andrographolide Attenuates Inflammation by Inhibition of NF- B Activation through Covalent Modification of Reduced Cysteine 62 of p50. The Journal of Immunology 173, 4207-4217
36. Hsieh, C. Y., Hsu, M. J., Hsiao, G., Wang, Y. H., Huang, C. W., Chen, S. W., Jayakumar, T., Chiu, P. T., Chiu, Y. H., and Sheu, J. R. (2011) Andrographolide enhances nuclear factor-kappaB subunit p65 Ser536 dephosphorylation through activation of protein phosphatase 2A in vascular smooth muscle cells. The Journal of biological chemistry 286, 5942-5955
37. Wang, J., Tan, X. F., Nguyen, V. S., Yang, P., Zhou, J., Gao, M., Li, Z., Lim, T. K., He, Y., Ong, C. S., Lay, Y., Zhang, J., Zhu, G., Lai, S. L., Ghosh, D., Mok, Y. K., Shen, H. M., and Lin, Q. (2014) A quantitative chemical proteomics approach to profile the specific cellular targets of andrographolide, a promising anticancer agent that suppresses tumor metastasis. Molecular & cellular proteomics : MCP 13, 876-886
38. Hsu, Y. H., Hsu, Y. L., Liu, S. H., Liao, H. C., Lee, P. X., Lin, C. H., Lo, L. C., and Fu, S. L. (2016) Development of a Bifunctional Andrographolide-Based Chemical Probe for Pharmacological Study. PLoS One 11, e0152770
39. Li, L., Wijaya, H., Samanta, S., Lam, Y., and Yao, S. Q. (2015) In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound. Scientific reports 5, 11522
40. Cheung, H. Y., Cheung, S. H., Li, J., Cheung, C. S., Lai, W. P., Fong, W. F., and Leung, F. M. (2005) Andrographolide isolated from Andrographis paniculata induces cell cycle arrest and mitochondrial-mediated apoptosis in human leukemic HL-60 cells. Planta Med 71, 1106-1111
41. Zhou, J., Zhang, S., Ong, C. N., and Shen, H. M. (2006) Critical role of pro-apoptotic Bcl-2 family members in andrographolide-induced apoptosis in human cancer cells. Biochem Pharmacol 72, 132-144
42. Zhou, J., Lu, G. D., Ong, C. S., Ong, C. N., and Shen, H. M. (2008) Andrographolide sensitizes cancer cells to TRAIL-induced apoptosis via p53-mediated death receptor 4 up-regulation. Mol Cancer Ther 7, 2170-2180
43. Kumar, R. A., Sridevi, K., Kumar, N. V., Nanduri, S., and Rajagopal, S. (2004) Anticancer and immunostimulatory compounds from Andrographis paniculata. J Ethnopharmacol 92, 291-295
44. Hidalgo, M. A., Romero, A., Figueroa, J., Cortes, P., Concha, II, Hancke, J. L., and Burgos, R. A. (2005) Andrographolide interferes with binding of nuclear factor-kappaB to DNA in HL-60-derived neutrophilic cells. Br J Pharmacol 144, 680-686
45. Sheeja, K., Guruvayoorappan, C., and Kuttan, G. (2007) Antiangiogenic activity of Andrographis paniculata extract and andrographolide. Int Immunopharmacol 7, 211-221
46. Liu, S.-H., Lin, C.-H., Liang, F.-P., Chen, P.-F., Kuo, C.-D., Alam, M. M., Maiti, B., Hung, S.-K., Chi, C.-W., Sun, C.-M., and Fu, S.-L. (2014) Andrographolide downregulates the v-Src and Bcr-Abl oncoproteins and induces Hsp90 cleavage in the ROS-dependent suppression of cancer malignancy. Biochemical Pharmacology 87, 229-242
47. Dai, L., Wang, G., and Pan, W. (2017) Andrographolide Inhibits Proliferation and Metastasis of SGC7901 Gastric Cancer Cells. Biomed Res Int 2017, 6242103
48. Zhang, M., Xue, E., and Shao, W. (2016) Andrographolide promotes vincristine-induced SK-NEP-1 tumor cell death via PI3K-AKT-p53 signaling pathway. Drug Des Devel Ther 10, 3143-3152
49. Yang, T., Yao, S., Zhang, X., and Guo, Y. (2016) Andrographolide inhibits growth of human T-cell acute lymphoblastic leukemia Jurkat cells by downregulation of PI3K/AKT and upregulation of p38 MAPK pathways. Drug Des Devel Ther 10, 1389-1397
50. Zhang, H. T., Yang, J., Liang, G. H., Gao, X. J., Sang, Y., Gui, T., Liang, Z. J., Tam, M. S., and Zha, Z. G. (2017) Andrographolide Induces Cell Cycle Arrest and Apoptosis of Chondrosarcoma by Targeting TCF-1/SOX9 Axis. J Cell Biochem
51. Luo, X., Luo, W., Lin, C., Zhang, L., and Li, Y. (2014) Andrographolide inhibits proliferation of human lung cancer cells and the related mechanisms. Int J Clin Exp Med 7, 4220-4225
52. Preet, R., Chakraborty, B., Siddharth, S., Mohapatra, P., Das, D., Satapathy, S. R., Das, S., Maiti, N. C., Maulik, P. R., Kundu, C. N., and Chowdhury, C. (2014) Synthesis and biological evaluation of andrographolide analogues as anti-cancer agents. Eur J Med Chem 85, 95-106
53. Sirion, U., Kasemsook, S., Suksen, K., Piyachaturawat, P., Suksamrarn, A., and Saeeng, R. (2012) New substituted C-19-andrographolide analogues with potent cytotoxic activities. Bioorg Med Chem Lett 22, 49-52
54. Beck, R., Verrax, J., Gonze, T., Zappone, M., Pedrosa, R. C., Taper, H., Feron, O., and Calderon, P. B. (2009) Hsp90 cleavage by an oxidative stress leads to its client proteins degradation and cancer cell death. Biochem Pharmacol 77, 375-383
55. Park, S., Park, J. A., Kim, Y. E., Song, S., Kwon, H. J., and Lee, Y. (2015) Suberoylanilide hydroxamic acid induces ROS-mediated cleavage of HSP90 in leukemia cells. Cell Stress Chaperones 20, 149-157
56. Beck, R., Dejeans, N., Glorieux, C., Creton, M., Delaive, E., Dieu, M., Raes, M., Leveque, P., Gallez, B., Depuydt, M., Collet, J. F., Calderon, P. B., and Verrax, J. (2012) Hsp90 is cleaved by reactive oxygen species at a highly conserved N-terminal amino acid motif. PLoS One 7, e40795
57. Chen, H., Xia, Y., Fang, D., Hawke, D., and Lu, Z. (2009) Caspase-10-mediated heat shock protein 90 beta cleavage promotes UVB irradiation-induced cell apoptosis. Mol Cell Biol 29, 3657-3664
58. Fritsch, J., Fickers, R., Klawitter, J., Sarchen, V., Zingler, P., Adam, D., Janssen, O., Krause, E., and Schutze, S. (2016) TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death. Oncotarget 7, 75774-75789
59. Zhang, Y., Dayalan Naidu, S., Samarasinghe, K., Van Hecke, G. C., Pheely, A., Boronina, T. N., Cole, R. N., Benjamin, I. J., Cole, P. A., Ahn, Y. H., and Dinkova-Kostova, A. T. (2014) Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation. Br J Cancer 110, 71-82
60. Moulick, K., Ahn, J. H., Zong, H., Rodina, A., Cerchietti, L., Gomes DaGama, E. M., Caldas-Lopes, E., Beebe, K., Perna, F., Hatzi, K., Vu, L. P., Zhao, X., Zatorska, D., Taldone, T., Smith-Jones, P., Alpaugh, M., Gross, S. S., Pillarsetty, N., Ku, T., Lewis, J. S., Larson, S. M., Levine, R., Erdjument-Bromage, H., Guzman, M. L., Nimer, S. D., Melnick, A., Neckers, L., and Chiosis, G. (2011) Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90. Nat Chem Biol 7, 818-826