Institute of Medicine, School of Medicine, Chung Shan Medical University, Taichung, Taiwan.
J Dermatol Sci. 2011 Dec;64(3):191-8. doi: 10.1016/j.jdermsci.2011.09.001. Epub 2011 Sep 24.
Glycolic acid (GA) has been widely used in cosmetic agents and superficial chemical peeling in recent years. It has long been concerned that UV irradiation would enhance the photosensitivity of GA on human skin. Therefore, it is mandatory to explore the biologic effects of concomitant exposure of GA and UV irradiation in human keratinocytes.
The aim of the study is to explore the effects of concomitant exposure of GA and UVB in a human keratinocyte cell line (HaCaT).
We used HaCaT to investigate the effects of GA (5mM), UVB (50mJ/cm(2)), and co-treatment with GA and UVB (GA+UVB) in human keratinocytes. We used a phase contrast microscope to observe morphological changes of the cells, and employed flow cytometry to detect cell viability, cell cycle, and mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) levels. Cell damage was detected by DAPI stain, and Western blot was used to detect the activities of apoptosis- related and cycle checkpoint-related proteins such as Bax, Bcl-2, caspases-3, -4, -9, Endo G, AIF, and p21, p27, p53, cdk2, cyclin E, cyclin A.
We found that either GA or UVB alone had inhibitory effect on cell proliferation, and co-treatment with GA and UVB had synergistic anti-proliferative effect. GA alone did not affect the cell cycle, and UVB induced HaCaT cells accumulated at S phase, and co-treatment with GA and UVB arrested cells at S phase more prominently. Moreover, all the treatment with GA, UVB, and GA+UVB in HaCaT cells induced apoptosis. We further demonstrated that GA had synergistic apoptotic effect in human keratinocytes. GA and UVB both had effects on the decline of MMP and increase of ROS release, and GA had synergistic increase in the level of ROS in UVB-treated HaCaT cells. Besides, co-treatment with GA and UVB had synergistic effect on apoptosis through the over-expressions of Bax, p21, p53, caspases-3, -4, -9, Endo G and AIF, and confocal microscopy disclosed translocation of AIF and Endo G from cytoplasm to the nucleus. Therefore, apoptosis induced by co-treatment by GA+UVB was initiated and executed by multiple pathways including mitochondria- and ER-dependent, and caspase-dependent and caspase-independent pathways.
We demonstrated that GA, UVB, GA+UVB inhibited proliferation and induced apoptosis in HaCaT cells. The mechanisms of apoptosis induced by co-treatment of GA and UVB involve multiple pathways. The synergistic photo-toxicity may be related to cell cycle arrest and apoptosis in UVB-treated HaCaT cells. These results highlight the potential adverse effects of GA-containing cosmetic agents on human skin.
近年来，乙醇酸（GA）已广泛应用于美容制剂和浅层化学焕肤中。人们长期以来一直担心，紫外线照射会增强 GA 对人体皮肤的光敏性。因此，有必要探讨 GA 和中波紫外线（UVB）同时暴露对人角质形成细胞的生物学效应。
本研究旨在探讨 GA 和 UVB 同时暴露对人角质形成细胞系（HaCaT）的影响。
我们采用 HaCaT 细胞，观察 GA（5mM）、UVB（50mJ/cm²）以及 GA 和 UVB 共同处理（GA+UVB）对人角质形成细胞的影响。采用相差显微镜观察细胞形态学变化，流式细胞术检测细胞活力、细胞周期和线粒体膜电位（MMP）及细胞内活性氧（ROS）水平。DAPI 染色检测细胞损伤，Western blot 检测凋亡相关和周期检验点相关蛋白如 Bax、Bcl-2、caspases-3、-4、-9、Endo G、AIF 和 p21、p27、p53、cdk2、cyclin E、cyclin A 的活性。
我们发现，GA 或 UVB 单独处理均对细胞增殖有抑制作用，GA 和 UVB 共同处理具有协同的抗增殖作用。GA 单独处理不影响细胞周期，UVB 诱导 HaCaT 细胞在 S 期蓄积，而 GA 和 UVB 共同处理使细胞周期阻滞更为明显。此外，GA、UVB 和 GA+UVB 处理 HaCaT 细胞均诱导细胞凋亡。我们进一步证明，GA 在人角质形成细胞中具有协同的促凋亡作用。GA 和 UVB 均降低 MMP 并增加 ROS 释放，GA 协同增加 UVB 处理的 HaCaT 细胞中 ROS 水平。此外，GA 和 UVB 共同处理通过 Bax、p21、p53、caspases-3、-4、-9、Endo G 和 AIF 的过度表达对细胞凋亡产生协同作用，共聚焦显微镜显示 AIF 和 Endo G 从细胞质易位到细胞核。因此，GA+UVB 共同处理诱导的细胞凋亡是通过线粒体和内质网依赖性、caspase 依赖性和 caspase 非依赖性途径启动和执行的。
我们证明 GA、UVB 和 GA+UVB 抑制 HaCaT 细胞增殖并诱导细胞凋亡。GA 和 UVB 共同处理诱导细胞凋亡的机制涉及多种途径。GA 联合 UVB 的光毒性协同作用可能与 UVB 处理的 HaCaT 细胞中的细胞周期阻滞和凋亡有关。这些结果突出了含 GA 的美容制剂对人体皮肤的潜在不良影响。
