I. (1)誘發人類皮膚癌細胞之凋亡機制
II.牡丹皮萃取之化合物抑制黑色素生成及抗氧化能力之探討

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Title:	I. (1)誘發人類皮膚癌細胞之凋亡機制 II.牡丹皮萃取之化合物抑制黑色素生成及抗氧化能力之探討 I.The mechanism of (1) on cell apoptosis in human skin cancer II.The antimelanogenesis and antioxidation properties of the compounds from Moutan Cortex
Authors:	曾雅萍
Contributors:	嘉南藥理科技大學：化妝品科技研究所梁家華
Keywords:	抗氧化黑色素生成細胞凋亡皮膚癌 antimelanogenesis antioxidation apoptosis skin cancer
Date:	2011
Issue Date:	2011-10-25 13:28:13 (UTC+8)
Abstract:	I. (1)誘發人類皮膚癌細胞之凋亡機制皮膚癌是全世界常見的惡性腫瘤之一，而皮膚鱗狀細胞癌(squamous cell carcinoma, SCC)亦是常見的非黑色素皮膚癌。本研究利用一新合成化合物(1)作用於人類皮膚癌細胞，探討其抗腫瘤機制。細胞毒性試驗證實化合物(1)對人類皮膚鱗狀上皮癌細胞株(SCC9和SCC25)皆具毒殺作用，其IC50濃度分別為2.3和1.8 μM，毒殺效果顯著。由光學顯微鏡觀察(1)作用於兩株癌細胞後，發現細胞產生皺縮、變圓、染色質濃縮和空泡化等凋亡特徵。利用annexin V—propidium iodide雙染試驗，發現(1)以凋亡方式促使兩株細胞死亡。化合物(1)會造成SCC9和SCC25之細胞週期G0/G1期下降，sub-G1及G2/M期之比例增加。除此之外，(1)在低濃度及短時間作用下會使SCC25細胞停滯於G0/G1期。使用RT-PCR技術、免疫螢光染色、流式細胞儀及西方墨點法探討經(1)作用於兩株癌細胞後與調控細胞凋亡相關之基因與蛋白質表現。結果發現，經(1)作用於兩株癌細胞後reactive oxygen species (ROS)提升、glutathione (GSH)下降，影響p53及p21表現增加，進而抑制CDC2與cyclin B1結合，使細胞週期停滯於G2/M期；另外(1)亦調控tumor necrosis factor (TNF) family，包含FasL/Fas、TNF-α/TNFRs及TNF-related apoptosis-inducing ligand (TRAIL)/TRAILRs表現量增加，活化下游Fas-associated death domain (FADD)、TNFR-1-associated death domain (TRADD)、caspase-8；而p53之表現進而活化下游粒線體途徑cytochrome c、bax、caspase-9及caspase-3表現增加，而抗凋亡bcl-2表現減少。綜合結果證實(1)以誘導人類皮膚鱗狀細胞癌凋亡機制促使癌細胞死亡，推測其具有發展為治療皮膚癌之用藥。 II.牡丹皮萃取之化合物抑制黑色素生成及抗氧化能力之探討本研究以牡丹皮(Moutan Cortex)萃取分離出的化合物進行抑制黑色素生成及抗氧化的活性評估。利用老鼠黑色素瘤細胞(B16)、老鼠肝臟細胞(BNLCL2)、人類角質株化細胞(HaCaT)及人類纖維母細胞(Hs68)，進行細胞毒性試驗。挑選對細胞不具毒性的化合物進行細胞內酪胺酸酶活性試驗，初步篩選後，選擇具抑制酪胺酸酶活性之化合物(M1、M2、M3及M4)進行分析與黑色素生成相關基因及蛋白質之表現。B16細胞經此4種化合物處理後，發現M1、M2、M3及M4分別具抑制接受器(melanocortin-1 receptor, MC1R)和MITF (microphthalmia-associated transcription factor)，並影響酪胺酸酶(tyrosinase)、TRP-1 (tyrosinase-related proteins-1)和TRP-2的基因與蛋白質表現，進而抑制黑色素生成。而由牡丹皮萃取之化合物以DPPH試驗進行抗氧化能力篩選，顯示M5和M6 此兩種化合物具有良好的自由基清除能力，進一步研究清除ABTS自由基、還原力、螯合金屬離子及抑制脂質過氧化能力試驗，評估其抗氧化能力。在細胞內試驗，以老鼠肝細胞BNLCL2，經過H2O2刺激後，測試 E和F對活性氧(reactive oxygen species, ROS)抑制率及榖胱甘肽(reduced glutathione, GSH)生成量。結果證實由牡丹皮中獲得之化合物可抑制黑色素生成及清除自由基功效，具發展成為美白與抗氧化保養品或保健食品添加劑之潛力。 I.The mechanism of (1) on cell apoptosis in human skin cancer Skin cancer is one of the most common human malignancies in the world. Cutaneous squamous cell carcinoma (SCC) is one of most common human non-melanoma skin malignancies. In this study, a new synthesis compound (1) has been investigated the mechanism on anti-human skin cancer cells. The cell viability in human skin squamous cell carcinoma (SCC9 and SCC25) cells has been demonstrated by MTT assay. After treatment with (1), the half-inhibitory concentrations (IC50) for SCC9 and SCC25 cells were 2.3 and 1.8 μM. Treatment with both cells with (1) increased DNA fragmentation and apoptotic body by morphology alteration, and annexin V-propidium iodide from a flow-cytometric analysis. Cell-cycle distribution indicates that (1) sensitized both cells in S-G2/M phases with a concomitant significantly increase of sub-G1 population. Additionally, (1) sensitized SCC25 cells in G0/G1 phase at short time with low concentration of (1). Further, we investigated the expression of apoptosis-associated factors by RT-PCR, immunofluorescence, flow cytometry and western blotting. (1) was found to reduce the intracellular-reduced GSH and increase ROS generation, upregulation p53 and p21 expression, downregulation CDC2 and cyclin b1 activation. This apoptosis process was accompanied by death receptor-operated TNF-α/TNFRs, FasL/Fas, and downstream adaptors TRADD, FADD, and caspase-8 expression. Moreover, p53 expression activated the mitochondria-operated pathway, including upexpression of cytochrome c, Bax, caspase-9 and caspase-3 and downexpression of bcl-2. Hence, these results herein suggest (1) may cross-link between the pathways of apoptosis via both receptor and mitochondria-mediated caspases signaling pathways, and may be effectively exploited in future human skin cancer clinical trials. II.The antimelanogenesis and antioxidation properties of the compounds from Moutan Cortex This study evaluated the antimelanogenic and antioxidant properties of compounds from Moutan Cortex. M1, M2, M3, M4, M5 and M6 which isolated from Moutan Cortex were been demonstrated its safety concentration in skin fibroblast (Hs68 cells), keratinocytes (HaCaT cells), melanoma (B16 cells) and mouse liver cells (BNLCL2 cells). After initial screening, we choose M1, M2, M3 and M4, demonstrated these four compounds suppress tyrosinase activities and DOPA oxidase activities, and reduce melanin synthesis. Moreover, after treatment of B16 cells with M1, M2, M3 and M4, downregulation the gene and protein expressions of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related proteins-2 (TRP-2) and TRP-1 have been demonstrated by RT-PCR and western blot analysis. Additionally, the antioxidant activities of M5 and M6 were evaluated by measuring DPPH and ABTS free-radical scavenging activities, reducing power, and inhibition of lipid peroxidation. Determination the reactive oxygen species (ROS) content and reduced glutathione (GSH) formation in H2O2-treated BNLCL2 cells by resacetophenone and 2,5-dihydroxy-4-methylacetophenone. This result demonstrated that M5 and M6, isolated from Moutan Cortex, exhibits antimelanogenesis and antioxidant activities. Thus, these can be used as a whitening source and food additive.
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