Full length articleGraphene oxide induces p62/SQSTM-dependent apoptosis through the impairment of autophagic flux and lysosomal dysfunction in PC12 cells
Graphical abstract
Introduction
Carbon-based nanomaterials have been widely used because of their unique physical, chemical, and biological properties [1], [2], [3]. Among these materials, graphene oxide (GO), as a novel two-dimensional (2D) carbon nanosheet, has attracted considerable interest in recent years [4], [5], [6]. Compared with pristine graphene, GO possesses larger specific surface area, abundant hydrophilic groups (hydroxyl, carboxylic and epoxide groups) and high stability in aqueous dispersion [7], which have enabled GO to be used in a growing number of applications in biomedical fields, such as drug carriers [8], cellular imaging [9], biosensors [10], [11], and antibacterial agents [12], [13]. Simultaneously, it should be noted that the growing uses of GO have opened various opportunities for these nanomaterials to be released into the environment and the human body. Considering the unknown biological activity of these nanomaterials, it is urgently required to establish a paradigm for accurately evaluating their adverse effects in biological systems.
In recent times, the potential toxic effects of GO on the environment and human health have attracted considerable attention for both toxicologists and engineers. After exposure, many reports suggested that GO can transfer throughout the body, be retained in target organs over an extended period of time [14], [15] and further cause injurious responses, such as thrombus formation [16], inflammatory cell infiltration [17], edema and granuloma formation [18], and even delay development in offspring [19]. Additionally, in vitro data have also demonstrated the cellular toxicity of GO, including the excessive generation of oxidative stress [20], [21], DNA damage [22], cell cycle arrest and apoptosis [23]. Such studies provide deep concerns on the interactions between GO and various biological systems both in vitro and in vivo. The most widely investigated mechanisms of nanomaterial-induced toxicity are oxidative stress, inflammatory response and mitochondrial damage, but the underlying mechanisms are still largely obscure [24]. Note that autophagy has been considered as a novel mechanism underlying GO-caused toxicity [25], [26]. It was reported that GO exposure might induce autophagic response [27] or autophagic cell death [28] both in vitro and in vivo [29], [30]. However, the detailed regulatory mechanism of autophagy in GO-induced toxic effect is still poorly defined.
Autophagy, referring to macroautophagy, is considered to begin as a phagophore followed by interactions with Atg5-Atg12 conjugation. LC3 later inserts into the extending phagophore membrane to facilitate the engulfment of cellular components (including aberrant organelles, proteins, and nanomaterials), which develop into double membrane vesicles, named autophagosomes. During the process of autophagosome assembly, LC3 is converted from its cytosolic form (LC3 I) into an active membrane-bound form (LC3 II) [30]. After that step, autophagosomes are transported to lysosomes to form autolysosomes, allowing intracellular content degradation and recycling for cell viability [31], [32]. Appropriate regulation of autophagy is essential for human health, and autophagy dysfunctions may be related with various human pathologies, such as cancer, abnormal immune responses, neurodegeneration and premature ageing [32]. Furthermore, the integrity of lysosomes is a basic factor for complete the autophagic process [33]. The dynamic process of autophagosomes formation, fusion between autophagosomes and lysosomes, and degradation of intracellular content are determined as the autophagic flux. The proper acid environment and hydrolase activity inside lysosomes is crucial for intracellular content degradation. Under stimulus exposure (such as nanomaterial exposure), the dysfunctions of lysosomes may lead to incomplete autophagy and even initiate cell programmed death [26], [34]. Previous studies reported that graphene-family nanomaterials could cause apoptosis through different signal pathways, including mitochondrial-dependent cascades and direct interactions with death-receptor proteins [35], [36], [37].
Notably, in addition to a set of core autophagy-related (Atg) proteins, p62 has recently become an interesting subject in autophagic studies due to its ability to crosstalk with both autophagy and apoptosis pathways [38]. The protein of p62, also known as sequestosome 1 (SQSTM1), is a well-known substrate for autophagy. The impairment of autophagic flux, such as defects in fusion between autophagosome and lysosome, can lead to an increased level of p62. It is both interesting and important to explore whether p62 is functionally involved in programmed cell death (including apoptosis) and the underlying molecular mechanisms of autophagy activation in response to GO exposure.
In this study, we systematically explored the impact of GO on cell viability at different doses and time points. We further observed that GO could induce autophagy and the impairment of autophagic flux, which resulted in an elevated level of autoghagic substrate p62. Moreover, the relationship between p62 accumulation and caspase-9 mediated apoptosis was also determined using both chemical and genetic methods. These findings reveal that GO treatment may shift the effect on autophagy from induction to blockage and further initiate cell apoptosis via caspase-mediated pathways.
Section snippets
Characterization of GO materials
GO used in the study was purchased from Sigma-Aldrich (Lot # MKDCC0556, St Louis, USA). The microstructure was examined by scanning electron microscope (SEM) (Hitachi Scientific Instruments, Japan), atomic force microscope (AFM) (Agilent Technologies, Inc., USA) and Raman spectroscopy (Renishaw, UK). Additionally, the chemical composition was evaluated by Fourier transform infrared (FTIR) spectroscopy (Thermo Fisher Scientific Inc., USA) and X-ray photoelectron spectroscopy (XPS). Finally, the
Characterization of GO nanosheets
As demonstrated in our previous review, the physicochemical properties of graphene-family nanomaterials, including lateral size, thickness, charge and surface structure, are important factors that mediate their interactions with cells [40]. Thus, it is strongly suggested to acquire detailed information of nanomaterial characterization before its biocompatibility evaluation. The GO materials used in this study were a type of commercial available nanosheet (Sigma) and thoroughly characterized
Conclusions
In this study, we thoroughly explored the toxic effect of GO nanosheets in PC12 cells. We found that cell viability was decreased in a dose- and time-dependent manner following GO exposure. Additionally, GO triggered an increased autophagic response and the impairment of autophagic flux in PC12 cells, which was determined to be associated with defects in lysosome degradation capability. Importantly, the elevated level of p62 protein was functionally involved in cell apoptosis via the caspase-9
Acknowledgements
This work was supported by the National Key Research and Development Program of China (2016YFC1102605) and the National Natural Science Foundation of China (2016A030313673).
Disclosures
The authors declare that they have no competing interests.
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These authors contributed equally to this work.