活性成分抑制黑色素生成、抗氧化及誘導皮膚癌細胞凋亡之機制探討

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Title:	活性成分抑制黑色素生成、抗氧化及誘導皮膚癌細胞凋亡之機制探討 The Mechanism of Active Compund on Anti-Melanogenesis, Antioxidation and Cell Apoptosis in Skin Cancers Cells
Authors:	霍竹芸
Contributors:	化粧品應用與管理系梁家華
Keywords:	抗氧化抗黑色素生成細胞凋亡 Antioxidation Anti-Melanogenesis Apoptosis
Date:	2013
Issue Date:	2015-10-26 20:26:43 (UTC+8)
Abstract:	1.本論文使用活性成分E、H、K和O進行抑制黑色素生成之機制評估。我們進行活性成分E、H、K和O的細胞毒性、蘑菇酪胺酸?活性、細胞內酪胺酸?活性、多巴氧化?和黑色素含量測定，發現活性成分K 8:2抑制細胞內酪胺酸?活性，抑制多巴氧化?活性，及抑制黑色素之功效最佳。而在黑色素生成的路徑中，活性成分K 8:2調控cyclic adenosine monophosphate (cAMP)、p38 MAPK (mitogen-activated protein kinase)、Jun N-terminal kinase (JNK)、extracellular signal-regulated kinase (ERK)、melanocortin-1 receptor (MC1R)、cAMP response element binding protein (CREB)、microphthalmia-associated transcription factor (MITF)、Tyrosinase、tyrosinase-related protein-2 (TRP-2)和TRP-1之基因和蛋白質表現。此外，我們也發現活性成分K 8:2具有抑制發炎反應相關酵素LOX-1活性的能力。因此，活性成分K 8:2具有抗發炎和抑制黑色素生成之潛力。2.本實驗探討活性成分M-X之抗氧化能力及誘導皮膚癌細胞凋亡之機制。以清除DPPH自由基試驗、清除ABTS陽離子自由基試驗、還原力、清除超氧陰離子和抑制酯質過氧化等實驗證實活性成分 M-X具抗氧化效能。於細胞存活度試驗中發現，活性成分P對BCC和SCC25細胞最具細胞毒殺性，且對於正常細胞之毒性較低。活性成分P作用於人類皮膚角質株化細胞，能夠減少氧化反應活性氧(reactive oxygen species, ROS)的產生，增加穀胱甘?(glutathione,GSH)的表現。然而將活性成分P作用於BCC和SCC25細胞，其會增加癌細胞ROS，降低GSH的產生，造成癌細胞損傷。將活性成分P作用於BCC和SCC25細胞後，發現其具抑制細胞菌落的生長。利用annexin V-propidium iodide雙染試驗，發現活性成分P藉由凋亡方式促使癌細胞死亡。在細胞週期實驗中發現，活性成分P使細胞週期停滯於停滯於S和G2/M期，調控細胞週期相關基因與蛋白質之表現，促使Cyclin A、Cdk2、Cyclin B1、Cdk1表現降低；使G0/G1降低，使sub-G1增加，進而促使癌細胞凋亡。利用LPS誘導RAW264.7細胞增加TNF-α表現，經由活性成分P作用能夠降低TNF-α含量。活性成分P調控BCC和SCC25細胞中(a)粒線體途徑、(b) TNF、TRAIL和Fas死亡受體途徑、(c) Caspase凋亡相關基因及蛋白質表現，進而促使癌細胞凋亡。 1.This study aimed to elucidate the anti-melnogenesis of active compound E, H, K, O. We investigated the cytotoxic, mushroom tyrosinase activity, cellular tyrosinse activity, dopa oxidase, and melanin content of that K with differing in ratio surfactants. We found that the ratio of active compound K 8:2 had the excellent ability on inhibiting of tyrosinase acitivity, dopa-oxidase activity and melanin formation. active compound K 8:2 regulated the protein and gene expression of mlanogenesis mechanism including cyclic adenosine monophosphate (cAMP), p38 MAPK (mitogen-activated protein kinase), Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), melanocortin-1 receptor (MC1R), cAMP response element binding protein (CREB), microphthalmia-associated transcription factor (MITF), Tyrosinase, tyrosinase-related protein-2 (TRP-2) and TRP-1. Besides, we also found that active compound K inhibited LOX-1 acitivity which the enzyme of inflammatory. Thus, active compound K 8:2 had a potential capability on anti-melanogenesis and anti-inflammatory activities.2.In this study, active compound M-X has been used on anti-oxidant and anti-tumor activities. Active compound M-X were tested the antioxidant capacity, such as DPPH free radical scavenging activity, ABTS free radical scavenging activity, reducing power, superoxide scavenging activity, and inhibition of lipid peroxidant assay. We further evaluated the cell viability in skin cells by MTT assay. The cytotoxic of active compound P in skin cancer cell (BCC and SCC25) is higher than in skin normal cell (HaCaT and Hs68) and it also inhibited cancer cell colony formation. active compound P was found to deplete the intracellular-reduced GSH (glutathione) and increase ROS (reactive oxygen species) generation in BCC and SCC25 cells. Treatment of the both cells with active compound P increased formations of fragmented DNA and apoptotic body by morphology alteration and annexin V-propidium iodide from a flow-cytometric analysis.And cell cycle indicates that active compound P sensitized both cells in S-G2/M phases with a concomitant significantly increase of sub-G1 population. Active compound P was found to deplete the intracellular-reduced GSH and increase ROS generation and lead to the p53and p21 expression, prevent the Cyclin A, Cdk2, Cyclin B1 and Cdk1 activation. The results herein suggest active compound P may cross-link between the pathways of apoptosis via TNF, TRAIL and Fas death receptor and mitochondria-mediated caspases signaling pathway.
Relation:	學年度：102，108頁
Appears in Collections:	[Dept. of Cosmetic Science and institute of cosmetic science] Dissertations and Theses

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