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研究生(外文):Shih-Chia Chiu
論文名稱:探討由Sargassum hemiphyllum透析純化之褐藻醣膠對非小細胞肺癌生理活性的影響
論文名稱(外文):Effects of Sargassum hemiphyllum dialyzed fucoidan on anti-cancer biological properties of non-small cell lung cancer
指導教授(外文):Hsien-Yeh Hsu
外文關鍵詞:FucoidanSargassum hemiphyllumDialysisTransforming growth factor receptorNon-small-cell lung cancerSlug
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褐藻醣膠 (Fucoidan) 為利用褐藻 (Laminaria japonica、Sargassum hemiphyllum、Fucus vesiculosus) 所萃取出之天然硫化多醣,現今研究已知褐藻醣膠具有免疫調節作用和抗癌的效果。目前有許多對於褐藻醣膠治療癌症的研究,但對於褐藻醣膠在抗癌治療上的機制及對癌細胞之活性影響並未被廣泛研究。另外於半葉馬尾藻 (Sargassum hemiphyllum) 所萃取出之褐藻醣膠成分中發現含有大量的游離醣類:山梨糖醇 (sorbitol) 和甘露醇 (mannitol),人體攝取過多糖醇類易導致腹痛脹氣、消化不良及電解質失調等副作用。在本篇研究中,我們利用半葉馬尾藻 S. hemiphyllum所萃取之褐藻醣膠水透析純化四天,分析其成分發現透析純化後能有效去除褐藻醣膠中的游離醣類並增加其有效成分岩藻醣 (fucose),經透析後的褐藻醣膠能提高非小細胞肺癌株H1975及 CL1-5的生理活性,阻斷TGF-β signaling並降解Slug蛋白表現,促進誘導細胞凋亡Apoptosis路徑caspase 3及 PARP的活化,且促使細胞內活性氧(ROS) 累積增加,在結合化療藥物Cisplatin後能更有效抑制H1975細胞活性及促進癌細胞apoptosis。綜合上述結果,純化後之褐藻醣膠可避免病人攝取過多糖醇降低可能之副作用,能夠有效抑制TGF-β路徑進而降解Slug表現,增強在非小細胞肺癌株的抗癌效果。
Fucoidan is a sulfated polysaccharide extracted from the brown algae such as Laminaria japonica, Sargassum hemiphyllum and Fucus vesiculosus. Recent studies know that fucoidan exhibits immunomodulatory and anti-cancer activities. A large amount of free form sugars including sorbitol and mannitol were found in the fucoidan extracted from Sargassum hemiphyllum. Ingestion of excess sorbitol and mannitol may cause abdominal pain, indigestion, and electrolyte imbalance. In this study, we use water dialysis purification to remove the free form sugars from S. hemiphyllum. After analysis, our results suggest that the dialysis method effectively remove free form sugar, increase purity and anti-non-small-cell lung cancer (NSCLC) ability, compare to non-dialyzed fucoidan. In addition, the dialyzed fucoidan from S. hemiphyllum improve the anti-tumor efficiency in H1975 and CL1-5 cells, including of inhibit the transforming growth factor receptor (TGFR) and suppression of Slug expression. We further reveal that dialyzed fucoidan enhance apoptosis pathway through caspase 3 as well as PARP activation and induce intracellular reactive oxygen species (ROS) generation in NSCLCs. In summary, use water dialysis method to avoid possible side effects for patient and enhance the anti-tumor ability of NSCLC cells. Dialyzed fucoidan overcome H1975 cells by inhibiting TGF-β pathway to degrade Slug expression and promote tumor cell apoptosis. Simultaneous treatment with dialysis fucoidan and cisplatin are more efficient to enhance apoptosis in H1975 and CL1-5 cells.
Materials and Methods---4
Cell lines and cell culture---4
Reagents and antibodies---4
Sulfated polysccharides extraction---5
Determination of polysaccharides---5
Western blot analysis---5
Determination of cell morphology---6
Cell viability assay---6
Cell migration assay---6
Cell cycle assay---7
Apoptosis analysis---7
Cycloheximide assay---7
Detection of intracellular ROS---7
Preparation the molecular weight of fucoidan to 50-3 kDa---8
Statistical analysis---8
Use water dialysis purification to remove free form monosaccharides.---9
Water dialysis removes the sorbitol and mannitol from the fucoidan.---9
Dialyzed fucoidan inhibits the viability and migration of H1975 and CL1-5 NSCLCs.---10
Dialyzed fucoidan enhances cell apoptosis through activating caspase-3 and PARP.---10
Dialyzed fucoidan downregulates TGFRs and inhibits p-ERK/AKT expression in NSCLCs.---11
Dialyzed fucoidan suppresses EMT via downregulating TGF-β signaling and Slug protein levels in NSCLCs.---12
D4 dialyzed fucoidan inhibits cell viability and promotes proliferation proteins p21/ p27/ p53.---12
D4 dialyzed fucoidan downregulates TGFRs via proteasomal degradation.---13
D4 dialyzed fucoidan induces proteasome-mediated degradation of Slug.---14
D4 dialyzed fucoidan induces intracellular reactive oxygen species ROS generation in NSCLCs.---14
D4 dialyzed fucoidan enhances cisplatin inhibited-cell viability and apoptosis in NSCLCs.---15
Analyzed 50-3 kDa monosaccharide components in D4 dialysis fucoidan.---15
Figure legends---26
Figure 1. Water dialysis mothed to extract sulfated polysaccharides.---26
Figure 2. The composition of polysaccharides in different dialysis day of S. hemiphyllum.---27
Figure 3. Dialyzed fucoidan inhibits the viability and motility of NSCLCs.---29
Figure 4. Dialyzed fucoidan enhances apoptosis signaling and cell cycle-regulatory proteins.---38
Figure 5. Dialyzed fucoidan downregulates TGFRs and inhibits p-ERK & AKT expression.---43
Figure 6. Dialyzed fucoidan supresses EMT markers and Slug protein levels in NSCLCs.---46
Figure 7. Dialyzed fucoidan inhibits cell viability and promotes proliferation protein in dose-dependent.---49
Figure 8. Dialyzed fucoidan downregulates TGFRs via proteasomal degradation.---51
Figure 9. Dialyzed fucoidan induces proteasome-mediated degradation of Slug.---54
Figure 10. Dialyzed fucoidan induces intracellular ROS generation in NSCLCs.---57
Figure 11. Dialyzed fucoidan enhances cisplatin inhibited-cell viability and apoptosis.---59
Table 1. The polysaccharide composition of 50-3 kDa in D4 dialysis fucoidan.---61
Ale, M. T. and A. S. Meyer (2013). "Fucoidans from brown seaweeds: an update on structures, extraction techniques and use of enzymes as tools for structural elucidation." RSC Adv. 3(22): 8131-8141.

Arora, A. and E. M. Scholar (2005). "Role of tyrosine kinase inhibitors in cancer therapy." J Pharmacol Exp Ther 315(3): 971-979.

Atashrazm, F., R. M. Lowenthal, G. M. Woods, A. F. Holloway and J. L. Dickinson (2015). "Fucoidan and cancer: a multifunctional molecule with anti-tumor potential." Mar Drugs 13(4): 2327-2346.

Blandin Knight, S., P. A. Crosbie, H. Balata, J. Chudziak, T. Hussell and C. Dive (2017). "Progress and prospects of early detection in lung cancer." Open Biol 7(9): 170070.

Chaitanya, G. V., A. J. Steven and P. P. Babu (2010). "PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration." Cell Commun Signal 8: 31.

Chen, S., Y. Zhao, Y. Zhang and D. Zhang (2014). "Fucoidan induces cancer cell apoptosis by modulating the endoplasmic reticulum stress cascades." PLoS One 9(9): e108157.

Duan, L. and R. E. Perez (2014). "Increasing cisplatin sensitivity by schedule-dependent inhibition of AKT and Chk1." Cancer Biology 15(12): 1600-1612.

Feng, J., D. Song, S. Jiang, X. Yang, T. Ding, H. Zhang, J. Luo, J. Liao and Q. Yin (2018). "Quercetin restrains TGF-beta1-induced epithelial-mesenchymal transition by inhibiting Twist1 and regulating E-cadherin expression." Biochem Biophys Res Commun 498(1): 132-138.

Florea, A. M. and D. Busselberg (2011). "Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects." Cancers (Basel) 3(1): 1351-1371.

Han, M. H., D. S. Lee, J. W. Jeong, S. H. Hong, I. W. Choi, H. J. Cha, S. Kim, H. S. Kim, C. Park, G. Y. Kim and Y. Hyun Choi (2017). "Fucoidan Induces ROS-Dependent Apoptosis in 5637 Human Bladder Cancer Cells by Downregulating Telomerase Activity via Inactivation of the PI3K/Akt Signaling Pathway." Drug Dev Res 78(1): 37-48.

Hanahan, D. and R. A. Weinberg (2011). "Hallmarks of cancer: the next generation." Cell 144(5): 646-674.
Hombach-Klonisch, S. (2018). "Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells." Molecular Cell Research 18(65): 749-768.

Hong, S. W., K. H. Jung, H. S. Lee, H. M. Zheng, M. J. Choi, C. Lee and S. S. Hong (2011). "Suppression by fucoidan of liver fibrogenesis via the TGF-beta/Smad pathway in protecting against oxidative stress." Biosci Biotechnol Biochem 75(5): 833-840.

Hong, Y.-s. (2014). "Hsp90 inhibition by WK88-1 potently suppresses the growth of gefitinib-resistant H1975 cells harboring the T790M mutation in EGFR." Oncology Reports 31(1): 2619-2624.

Hsu, H. Y., T. Y. Lin, P. A. Hwang, L. M. Tseng, R. H. Chen, S. M. Tsao and J. Hsu (2013). "Fucoidan induces changes in the epithelial to mesenchymal transition and decreases metastasis by enhancing ubiquitin-dependent TGFbeta receptor degradation in breast cancer." Carcinogenesis 34(4): 874-884.

Hsu, H. Y., T. Y. Lin, M. K. Lu, P. J. Leng, S. M. Tsao and Y. C. Wu (2014). "Fucoidan inhibition of lung cancer in vivo and in vitro: role of the Smurf2-dependent ubiquitin proteasome pathway in TGFβ receptor degradation." Oncotarget 5(17): 7870-7885.

Hsu, H. Y., T. Y. Lin, M. K. Lu, P. J. Leng, S. M. Tsao and Y. C. Wu (2017). "Fucoidan induces Toll-like receptor 4-regulated reactive oxygen species and promotes endoplasmic reticulum stress-mediated apoptosis in lung cancer." Sci Rep 7: 44990.

Huang, T.-H. (2015). "Prophylactic Administration of Fucoidan Represses Cancer Metastasis by Inhibiting Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloproteinases (MMPs) in Lewis Tumor-Bearing Mice." Mar Drugs 13: 1882-1900.

Jakowlew, S. B. (2006). "Transforming growth factor-beta in cancer and metastasis." Cancer Metastasis Rev 25(3): 435-457.

Jung-Chien Cheng, Hsun-Ming Chang and P. C. K. Leung (2013). "Transforming Growth Factor- 1 Inhibits Trophoblast Cell Invasion by Inducing Snail-mediated Down-regulation of Vascular Endothelial-cadherin Protein." Journal of Biological Chemistry 288(46): 33181-33192.

Karimian (2016). "Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage." DNA Repair (Amst) 42: 63-71.

Kim, T. H., E. K. Lee, M. J. Lee, J. H. Kim and W. S. Yang (2013). "Fucoidan inhibits activation and receptor binding of transforming growth factor-beta1." Biochem Biophys Res Commun 432(1): 163-168.

Lee, A.-F. (2017). "Reverse epithelial-mesenchymal transition contributes to the regain of drug sensitivity in tyrosine kinase inhibitor-resistant non-small cell lung cancer cells." PLoS One 12(7): e0180383.

Lee, H. E., E. S. Choi, J. A. Shin, S. O. Lee, K. S. Park, N. P. Cho and S. D. Cho (2014). "Fucoidan induces caspase-dependent apoptosis in MC3 human mucoepidermoid carcinoma cells." Exp Ther Med 7(1): 228-232.

Li, B., F. Lu, X. Wei and R. Zhao (2008). "Fucoidan: Structure and Bioactivity." Molecules 13(8): 1671-1695.

Lowe, S. W. (2000). "Apoptosis in cancer." Carcinogenesis 21(3): 485-495.

Mishkin, D. and Leon (1997). "Fructose and Sorbitol Malabsorption in Ambulatory Patients with Functional Dyspepsia." Digestive Diseases and Sciences 42(12): 2591-2598.

Nagaraj, N. S. and P. K. Datta (2010). "Targeting the transforming growth factor-beta signaling pathway in human cancer." Expert Opin Investig Drugs 19(1): 77-91.

O'Brien, T. D., P. Jia, N. E. Caporaso, M. T. Landi and Z. Zhao (2018). "Weak sharing of genetic association signals in three lung cancer subtypes: evidence at the SNP, gene, regulation, and pathway levels." Genome Med 10(1): 16.

Padua, D. and J. Massague (2009). "Roles of TGFbeta in metastasis." Cell Res 19(1): 89-102.

Palma-Nicolas, J. P. and A. M. Lopez-Colome (2013). "Thrombin induces slug-mediated E-cadherin transcriptional repression and the parallel up-regulation of N-cadherin by a transcription-independent mechanism in RPE cells." J Cell Physiol 228(3): 581-589.

Pelicano, H., D. Carney and P. Huang (2004). "ROS stress in cancer cells and therapeutic implications." Drug Resist Updat 7(2): 97-110.

Qin, L. F. (2002). "Induction of apoptosis by cisplatin and its effect on cell cycle-related proteins and cell cycle changes in hepatoma cells." Cancer Letter 175(1): 28-38.

Regales, L., Y. Gong, R. Shen, E. de Stanchina, I. Vivanco, A. Goel, J. A. Koutcher, M. Spassova, O. Ouerfelli, I. K. Mellinghoff, M. F. Zakowski, K. A. Politi and W. Pao (2009). "Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer." J Clin Invest 119(10): 3000-3010.

Schieber, M. and N. S. Chandel (2014). "ROS function in redox signaling and oxidative stress." Curr Biol 24(10): R453-462.

Senthilkumar, K., P. Manivasagan, J. Venkatesan and S. K. Kim (2013). "Brown seaweed fucoidan: biological activity and apoptosis, growth signaling mechanism in cancer." Int J Biol Macromol 60: 366-374.

Shih, J. Y. and P. C. Yang (2011). "The EMT regulator slug and lung carcinogenesis." Carcinogenesis 32(9): 1299-1304.

Silchenko, A. S., M. I. Kusaykin, V. V. Kurilenko, A. M. Zakharenko, V. V. Isakov, T. S. Zaporozhets, A. K. Gazha and T. N. Zvyagintseva (2013). "Hydrolysis of fucoidan by fucoidanase isolated from the marine bacterium, Formosa algae." Mar Drugs 11(7): 2413-2430.

Tao Fan, L. C. (2016). "Autophagy decreases alveolar macrophage apoptosis by attenuating endoplasmic reticulum stress and oxidative stress." Oncotarget 7(52): 87206-87218.

Van Schaeybroeck, S., J. Kyula, D. M. Kelly, A. Karaiskou-McCaul, S. A. Stokesberry, E. Van Cutsem, D. B. Longley and P. G. Johnston (2006). "Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells." Mol Cancer Ther 5(5): 1154-1165.

Vanneman, D. (2012). "Combining immunotherapy and targeted therapies in cancer treatment." Nat Rev Cancer 12(4): 237-251.

Virve Koljonen, E. T. (2006). "Cell Cycle Control by p21, p27 and p53 in Merkel Cell Carcinoma." Anticancer Research 26(1): 2209-2212.

Wang, S., S. Cang and D. Liu (2016). "Third-generation inhibitors targeting EGFR T790M mutation in advanced non-small cell lung cancer." J Hematol Oncol 9: 34.

Xue, M. (2017). "Caspase-dependent and caspase-independent induction of apoptosis in breast cancer by fucoidan via the PI3K/AKT/GSK3b pathway in vivo and in vitro." Biomedicine & Pharmacotherapy 94(1): 898-908.
Yang, L., P. Wang, H. Wang, Q. Li, H. Teng, Z. Liu, W. Yang, L. Hou and X. Zou (2013). "Fucoidan derived from Undaria pinnatifida induces apoptosis in human hepatocellular carcinoma SMMC-7721 cells via the ROS-mediated mitochondrial pathway." Mar Drugs 11(6): 1961-1976.

Yokoyama, H. and Y. Ikehara (2006). "Molecular basis for sensitivity and acquired resistance to gefitinib in HER2-overexpressing human gastric cancer cell lines derived from liver metastasis." British Journal of Cancer 95(1): 1504-1513.

Yu, M., Y. Ji, Z. Qi, D. Cui, G. Xin, B. Wang, Y. Cao and D. Wang (2017). "Anti-tumor activity of sulfated polysaccharides from Sargassum fusiforme." Saudi Pharm J 25(4): 464-468.

Zhang, C. and X. Zhang (2017). "TGF-β2 initiates autophagy via Smad and non-Smad pathway to promote glioma cell invasion." Clinical Cancer Research 36(162): 15.
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