Vad Nikhil M, Yount Garret, Moridani Majid Y
Department of Pharmaceutical Sciences, School of Pharmacy, School of Medicine, Texas Tech University Health Sciences Center, Amarillo, Texas, USA.
Melanoma Res. 2008 Dec;18(6):386-99. doi: 10.1097/CMR.0b013e3283107df7.
In the current work, we investigated the biochemical toxicity of acetylsalicylic acid (ASA; Aspirin) in human melanoma cell lines using tyrosinase enzyme as a molecular cancer therapeutic target. At 2 h, ASA was oxidized 88% by tyrosinase. Ascorbic acid and NADH, quinone reducing agents, were significantly depleted during the enzymatic oxidation of ASA by tyrosinase to quinone. The 50% inhibitory concentration (48 h) of ASA and salicylic acid toward SK-MEL-28 cells were 100 micromol/l and 5.2 mmol/l, respectively. ASA at 100 micromol/l was selectively toxic toward human melanocytic SK-MEL-28, MeWo, and SK-MEL-5 and murine melanocytic B16-F0 and B16-F10 melanoma cell lines. However, ASA was not significantly toxic to human amelanotic C32 melanoma cell line, which does not express tyrosinase enzyme, and human nonmelanoma BJ, SW-620, Saos, and PC-3 cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased ASA toxicity toward SK-MEL-28 cells indicating quinone formation and intracellular GSH depletion played important mechanistic roles in ASA-induced melanoma toxicity. Ascorbic acid, a quinone reducing agent, and GSH, an antioxidant and quinone trap substrate, prevented ASA cell toxicity. Trifluoperazine, inhibitor of permeability transition pore in mitochondria, prevented ASA toxicity. ASA led to significant intracellular GSH depletion in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ASA also led to significant reactive oxygen species (ROS) formation in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ROS formation was exacerbated by dicoumarol and 1-bromoheptane in SK-MEL-28. Our investigation suggests that quinone species, intracellular GSH depletion, ROS formation, and mitochondrial toxicity significantly contributed toward ASA selective toxicity in melanocytic SK-MEL-28 melanoma cells.
在当前研究中，我们以酪氨酸酶作为分子癌症治疗靶点，研究了乙酰水杨酸（ASA；阿司匹林）对人黑色素瘤细胞系的生化毒性。在2小时时，ASA被酪氨酸酶氧化了88%。在酪氨酸酶将ASA酶促氧化为醌的过程中，醌还原剂抗坏血酸和NADH显著消耗。ASA和水杨酸对SK-MEL-28细胞的50%抑制浓度（48小时）分别为100微摩尔/升和5.2毫摩尔/升。100微摩尔/升的ASA对人黑素细胞SK-MEL-28、MeWo和SK-MEL-5以及小鼠黑素细胞B16-F0和B16-F10黑色素瘤细胞系具有选择性毒性。然而，ASA对不表达酪氨酸酶的人无黑色素C32黑色素瘤细胞系以及人非黑色素瘤BJ、SW-620、Saos和PC-3细胞无明显毒性。双香豆素（一种黄递酶抑制剂）和1-溴庚烷（一种消耗谷胱甘肽的试剂）增加了ASA对SK-MEL-28细胞的毒性，表明醌的形成和细胞内谷胱甘肽的消耗在ASA诱导的黑色素瘤毒性中起重要机制作用。抗坏血酸（一种醌还原剂）和谷胱甘肽（一种抗氧化剂和醌捕获底物）可防止ASA的细胞毒性。三氟拉嗪（线粒体通透性转换孔抑制剂）可防止ASA毒性。ASA导致黑素细胞SK-MEL-28黑色素瘤细胞内谷胱甘肽显著消耗，但对无黑色素C32黑色素瘤细胞无此作用。ASA还导致黑素细胞SK-MEL-28黑色素瘤细胞内活性氧（ROS）显著形成，但对无黑色素C32黑色素瘤细胞无此作用。双香豆素和1-溴庚烷在SK-MEL-28中加剧了ROS的形成。我们的研究表明，醌类物质、细胞内谷胱甘肽消耗、ROS形成和线粒体毒性在黑素细胞SK-MEL-28黑色素瘤细胞中对ASA的选择性毒性有显著贡献。
