Dose-dependent autophagic effect of titanium dioxide nanoparticles in human HaCaT cells at non-cytotoxic levels.
Dose-dependent autophagic effect of titanium dioxide nanoparticles in human HaCaT cells at non-cytotoxic levels.
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DOI：
10.1186/s12951-016-0174-0
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发表时间：
2016-03-22
影响因子：
10.2
通讯作者：
Cristobal S
中科院分区：
文献类型：
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作者：
Lopes VR;Loitto V;Audinot JN;Bayat N;Gutleb AC;Cristobal S
纳米颗粒和细胞之间的相互作用现在是一个快速发展的研究领域的焦点。虽然许多纳米颗粒与细胞相互作用而没有任何急性毒性反应，但金属氧化物纳米颗粒（包括由二氧化钛（TiO 2-NPs）组成的纳米颗粒）可能会破坏细胞内的巨自噬过程。自噬在人类健康和疾病中起着关键作用，特别是在癌症和神经退行性疾病中。我们在此研究了TiO 2-NPs（18 nm）在非细胞毒性水平对人角质形成细胞（HaCaT）细胞自噬的体外生物学效应。通过透射电子显微镜（TEM）和动态光散射技术对TiO 2-NPs进行表征。通过TEM和NanoSIMS评估的细胞摄取揭示了NP内化导致自噬体的形成。TiO 2-NPs处理没有降低HaCaT细胞的细胞活力，也没有增加氧化应激。通过使用eGFP-LC 3角质形成细胞的共聚焦显微镜、自噬标志物LC 3 I/II的蛋白质印迹、p62和NBR 1蛋白的免疫检测以及通过RT-qPCR的LC 3 II、p62、NBR 1、beclin 1和ATG 5的基因表达，另外评价细胞自噬。我们还证实了在具有LC 3-II上调的NP处理的细胞中自噬体的形成和积累。基于缺乏p62和NBR 1蛋白的降解，高剂量（25.0 μg/ml）的自噬体积累是由于阻断，而低剂量（0.16 μg/ml）促进自噬。细胞活力在两种情况下都没有受到影响。在非细胞毒性条件下，TiO 2-NPs的摄取导致自噬效应的剂量依赖性增加。我们的研究结果表明，随着时间的推移，剂量依赖性自噬效应作为对TiO 2-NP的细胞反应。最重要的是，这些发现表明，简单的毒性数据不足以理解TiO 2-NP的全部影响及其对细胞通路或功能的影响。
Interactions between nanoparticles and cells are now the focus of a fast-growing area of research. Though many nanoparticles interact with cells without any acute toxic responses, metal oxide nanoparticles including those composed of titanium dioxide (TiO2-NPs) may disrupt the intracellular process of macroautophagy. Autophagy plays a key role in human health and disease, particularly in cancer and neurodegenerative diseases. We herein investigated the in vitro biological effects of TiO2-NPs (18 nm) on autophagy in human keratinocytes (HaCaT) cells at non-cytotoxic levels. TiO2-NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering techniques. Cellular uptake, as evaluated by TEM and NanoSIMS revealed that NPs internalization led to the formation of autophagosomes. TiO2-NPs treatment did not reduce cell viability of HaCaT cells nor increased oxidative stress. Cellular autophagy was additionally evaluated by confocal microscopy using eGFP-LC3 keratinocytes, western blotting of autophagy marker LC3I/II, immunodetection of p62 and NBR1 proteins, and gene expression of LC3II, p62, NBR1, beclin1 and ATG5 by RT-qPCR. We also confirmed the formation and accumulation of autophagosomes in NPs treated cells with LC3-II upregulation. Based on the lack of degradation of p62 and NBR1 proteins, autophagosomes accumulation at a high dose (25.0 μg/ml) is due to blockage while a low dose (0.16 μg/ml) promoted autophagy. Cellular viability was not affected in either case. The uptake of TiO2-NPs led to a dose-dependent increase in autophagic effect under non-cytotoxic conditions. Our results suggest dose-dependent autophagic effect over time as a cellular response to TiO2-NPs. Most importantly, these findings suggest that simple toxicity data are not enough to understand the full impact of TiO2-NPs and their effects on cellular pathways or function.