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Title:	Kesan apoptotik dan antioksidatif gama-tokotrienol terhadap sel neuroblastoma manusia sh-sy5y
Authors:	Tan Jen Kit (P59459)
Supervisor:	Wan Zurinah Wan Ngah, Prof. Dato' Dr.
Keywords:	Gama-tokotrienol Sel neuroblastoma Dissertations, Academic -- Malaysia
Issue Date:	31-Mar-2015
Description:	Penentuan sasaran molekul yang dikawal-atur oleh gama-tokotrienol (γT3) untuk mengaruhkan apoptosis dalam sel kanser adalah penting dari segi translasi klinikal. Sebagai antioksidan, keberkesanan γT3 sebagai agen terapeutik mungkin dipengaruhi oleh status redoks dalam sel kanser. Kajian ini bertujuan untuk menentukan mekanisme γT3 sebagai perencat Bcl-2 melalui pengikatannya pada Bcl-2 untuk mengaruhkan apoptosis dalam sel neuroblastoma manusia SH-SY5Y. Berbanding dengan sel tanpa perlakuan, perlakuan γT3 pada 2.5-20 μM selama 16-24 jam secara signifikan (P<0.05) menurunkan viabiliti sel bersandarkan kepada dos, meningkatkan sitotoksisiti 3.6 kali ganda dan apoptosis 1.6 kali ganda, melalui penyahkutuban potensi membran mitokondria, menyebabkan pembebasan sitokrom c ke sitosol, dan meningkatkan aktiviti kaspase-9 sebanyak 1.9 kali ganda dan aktiviti kaspase-3 sebanyak 3.6 kali ganda. γT3 berikat pada alur hidrofobik Bcl-2 melalui analisis perisian pengedokan in siliko YASARA dan asai pengikatan saingan ANS. Perlakuan bersama 20 μM γT3 + 10 μM all-trans-retinoic acid (ATRA) selama 24 jam tidak menurunkan viabiliti berbanding dengan perlakuan γT3 sahaja. Yang menariknya, perlakuan 10 μM ATRA selama 24 jam meningkatkan proliferasi sel sebanyak 7 % (P<0.05) berbanding dengan sel tanpa perlakuan. Berbanding dengan sel tanpa perlakuan, perlakuan 10 μM ATRA selama 6 dan 12 jam meningkatkan ekspresi N-myc, manakala perlakuan 20 μM γT3 selama 24 jam menurunkan ekspresi N-myc. Seterusnya, kesan γT3 terhadap sel SH-SY5Y yang diaruh dengan tekanan oksidatif ditentukan. Perlakuan bersama 0.1 μM γT3 + 10 μM buthionine sulfoximine (BSO) selama 48 jam meningkatkan viabiliti sebanyak 34 %, dan menyekat sepenuhnya sitotoksisiti dan apoptosis apabila berbanding dengan perlakuan BSO sahaja. Perlakuan 10 μM BSO selama 72 jam menurunkan glutation terturun (GSH) berbanding dengan sel tanpa perlakuan, manakala GSH tidak dipengaruhi oleh perlakuan bersama 0.1 μM γT3 + 10 μM BSO berbanding dengan perlakuan BSO sahaja. Perlakuan bersama 0.1 μM γT3 + 10 μM BSO selama 72 jam menurunkan spesies oksigen reaktif berbanding dengan perlakuan BSO sahaja. Berbanding dengan sel tanpa perlakuan, tahap fosforilasi p38 MAPK didapati tidak berubah dalam perlakuan bersama 0.1 μM γT3 + 10 μM BSO selama 48 dan 72 jam. Namun, penambahan perencat p38 MAPK (SB 202190) pada 1, 5 and 10 μM selama 48 jam menurunkan viabiliti sebanyak 21-27 % dalam perlakuan bersama 0.1 μM γT3 + 10 μM BSO apabila berbanding dengan perlakuan tanpa SB 202190. Secara kesimpulannya, γT3 bertindak sebagai perencat Bcl-2 dan menurunkan ekspresi N-myc untuk mengaruh apoptosis dalam sel SH-SY5Y. Kesan pro-kemandirian ATRA terhadap sel SH-SY5Y mungkin kerana ATRA meningkatkan ekspresi N-myc. γT3 melindungi sel SH-SY5Y daripada tekanan oksidatif yang diaruhkan oleh BSO melalui tindakannya sebagai peragut radikal bebas dan pengawal-aturan yang melibatkan pengaktifan p38 MAPK. γT3 berpotensi dikembangkan sebagai perencat Bcl-2 yang lebih spesifik dan perekaan terbitan γT3 yang tidak mempunyai sifat antioksidan mungkin meningkatkan keberkesanan γT3 sebagai agen terapeutik.,Identification of molecular targets that are mediated by gamma-tocotrienol (γT3) to induce apoptosis in cancer cells has a major implication in the perspective of clinical translation. As an antioxidant, the efficacy of γT3 as therapeutic agent may be affected by the redox status of cancer cells. This study was aimed to determine the mechanism of γT3 as a Bcl-2 inhibitor by binding onto Bcl-2 to induce apoptosis in human neuroblastoma SH-SY5Y cells. As compared with untreated cells, exposure of γT3 at 2.5-20 μM for 16-24 hours significantly (P<0.05) reduced the viability of cells in a concentration dependent-manner, increased cytotoxicity by 3.6 fold and apoptosis by 1.6 fold, by depolarizing mitochondria membrane potential, resulting in release of cytochrome c to cytosol, thus increasing the activities of caspase-9 by 1.9 fold and of caspase-3 by 3.6 fold. γT3 binds to the hydrophobic groove of Bcl-2 via the analysis of in silico docking by using YASARA software and ANS competitive binding assay. The co-treatment of 20 μM γT3 + 10 μM all-trans-retinoic acid (ATRA) for 24 hours did not further reduce the viability of cells as compared with treatment of γT3 alone. Interestingly, treatment of 10 μM ATRA alone for 24 hours increased the proliferation of cells by 7 % (P<0.05) as compared with untreated cells. As compared with untreated cells, treatment of 10 μM ATRA for 6 and 12 hours increased the expression of N-myc, while treatment of 20 μM γT3 for 24 hours decreased the N-myc expression. Next, the effect of γT3 on oxidative stress-induced SH-SY5Y cells was determined. Co-treatment of 0.1 μM γT3 + 10 μM buthionine sulfoximine (BSO) for 48 hours increased the viability of cells by 34 %, and completely blocked cytotoxicity and apoptosis as compared with treatment of BSO alone. Treatment of 10 μM BSO for 72 hours decreased reduced glutathione (GSH) as compared with untreated cells, while GSH remaining unaffected by co-treatment of 0.1 μM γT3 + 10 μM BSO as compared with treatment of BSO alone. Co-treatment of 0.1 μM γT3 + 10 μM BSO for 72 hours reduced the reactive oxygen species as compared with treatment of BSO alone. As compared with untreated cells, level of phosphorylated p38 MAPK remained unchanged in cells co-treated with 0.1 μM γT3 + 10 μM BSO for 48 and 72 hours. However, addition of p38 MAPK inhibitor (SB 202190) at 1, 5 and 10 μM for 48 hours decreased the viability of cells by 21-27 % in cells co-treated with 0.1 μM γT3 + 10 μM BSO when compared with treatment without SB 202190. In conclusion, γT3 acted as a Bcl-2 inhibitor and decreased N-myc expression to induce apoptosis in SH-SY5Y cells. The pro-survival effect of ATRA on SH-SY5Y cells may be due to the increase of N-myc expression by ATRA. γT3 protected SH-SY5Y cells from BSO-induced oxidative stress by scavenging free radicals and modulating the activation of p38 MAPK. γT3 has good potential to develop into a more specific Bcl-2 inhibitor and the design of γT3 derivative with a redox-silent property may enhance its efficacy as a potential therapeutic agent.,PhD
Pages:	249
Publisher:	UKM, Kuala Lumpur
Appears in Collections:	Faculty of Medicine / Fakulti Perubatan

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