砷和UVB對培養正常人類角質細胞之細胞凋亡影響 —光保護效應

Apoptotic Effects of Arsenic and UVB on Normal Human Cultured Keratinocytes—Effect of Photoprotection

陳柏宏(Po-Hung Chen)

指導教授 ： 陳國熏

高雄醫學大學/醫學院/醫學研究所/碩士(2004年)

摘要

無機砷不僅是一廣為人知之環境毒物，也是一會導致人類癌症之致癌物質。在動物實驗中，砷可做為一共同突變原(co-mutagen)，並可加強紫外線B對於老鼠之致癌效應。細胞凋亡是一個受到良好調控之細胞死亡過程，其對於維持正常發育及組織之恆定(homeostasis)相當重要。一但細胞凋亡產生調控異常將會導致各種病理狀態，例如癌症。本實驗之目的在研究低劑量砷與紫外線B及在其交互作用下對於培養之角質細胞所產生之細胞凋亡效應。培養之角質細胞以砷化鈉１微莫耳及紫外線Ｂ５０mJ/cm2做各類處理，包括控制組，砷單獨處理組(As組)，紫外線Ｂ單獨照射組(UVB組)，先經砷前處理後照射紫外線Ｂ組(UVB-As組)，先經紫外線B照射再加上砷處理組(UVB-As組)。實驗結果顯示在以低濃度砷化鈉單獨刺激下並不會對培養之角質細胞產生細胞凋亡。另在以紫外線B單獨照射之角質細胞組中不但可見其caspase-8,-9,-3活性有明顯上升，且在TUNEL試驗中亦可見其細胞凋亡率強烈增加。而在先經紫外線B照射再加上砷處理之角質細胞組亦可見類似之促細胞凋亡效應。但相對的，與控制組相較下在經砷前處理後照射紫外線Ｂ組中角質細胞之型態與存活率只見輕微改變。而進一步以西方墨點法及caspase活性試驗法檢測caspase-8,-9,-3之結果也證實細胞凋亡的兩大訊息傳導途徑-Fas接受器途徑與粒線體途徑皆無活化。因此吾人認為經紫外線照射之角質細胞可消除因砷處理所致之增殖怍用，此外經砷前處理似可減少因照射紫外線Ｂ所引起之促細胞凋亡作用。

關鍵字

細胞凋亡 ； 砷 ； 紫外線B

並列摘要

Inorganic arsenic is not only a well-known environmental toxin but also a human carcinogen. Being a co-mutagen, arsenic enhances the carcinogenesis of ultraviolet irradiation on mouse skin. Apoptosis, a well regulated cell death process, is essential for normal development and tissue homeostasis. Dysregulation of apoptosis will lead to various kinds of pathological conditions, such as cancers. The purpose of this study is to investigate the apoptotic effect induced by the interaction of arsenic and UVB in cultured human keratinocytes. Cultured keratinocytes were treated with sodium arsenite (1μM) and/or UVB 50mJ/cm2 exposure in different combinations, including arsenic alone (As group), UVB alone (UVB group), arsenic then UVB (As-UVB group), and UVB then As (UVB-As group) treatments. The results revealed that low concentration of sodium arsenite did not stimulate keratinocytes apoptosis. The keratinocytes treated with UVB irradiation showed obvious elevation of caspase-8, -9, and -3 activities and strong induction of apoptotic rate by TUNEL assay. Similar pro-apoptotic effects were observed in keratinocytes pretreated with UVB then incubated with sodium arsenite. In contrast, only subtle changes of cell morphology and survival rate were noticed in keratinocytes pretreated with arsenite followed by UVB irradiation as compared with control group. The results of Western blot and activity assay of caspase-8, -9, and -3 also revealed neither receptor nor mitochondrial apoptotic signaling pathway activation. Therefore, we conclude that the pretreatment of keratinocytes with UVB abolishes the proliferative effect of low concentration of sodium arsenite. In addition, pretreatment with sodium arsenite decreases the pro-apoptotic effects induced by UVB.

並列關鍵字

apoptosis ； UVB ； arsenic

參考文獻

Adams JM (2003): Ways of dying: multiple pathways to apoptosis. Genes Dev 17:2481-95.

Alessi DR, Deak M, Casamayor A, Caudwell FB, Morrice N, Norman DG, Gaffney P, Reese CB, MacDougall CN, Harbison D, Ashworth A, Bownes M (1997a): 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol 7:776-89.

Alessi DR, James SR, Downes CP, Holmes AB, Gaffney PR, Reese CB, Cohen P (1997b): Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7:261-9.

Aragane Y, Kulms D, Metze D, Wilkes G, Poppelmann B, Luger TA, Schwarz T (1998): Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L. J Cell Biol 140:171-82.

Ashkenazi A (2002): Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2:420-30.