Graphene oxide induces p62/SQSTM-dependent apoptosis through the impairment of autophagic flux and lysosomal dysfunction in PC12 cells.

Graphene oxide (GO), as a two-dimensional carbon nanosheet, has been extensively studied for potential biomedical applications due to its notable properties. Although a growing number of studies have investigated the adverse effects of GO nanosheets, the available toxicity data concerning GO's effect on the neuronal cells remain highly limited. In this work, we systematically investigated the toxic responses of commercially available GO on a rat pheochromocytoma-derived PC12 cell line, which was an ideal in vitro model to study the neurotoxicity of GO. GO exerted a significant toxic effect on PC12 cells in a dose- and time-dependent manner. GO treatments under doses of 40, 50, and 60 μg/mL triggered an autophagic response and the blockade of autophagic flux via disrupting lysosome degradation capability. Caspase 9-mediated apoptosis was also observed in GO-treated cells. Moreover, GO-induced apoptosis was relevant to the aberrant accumulation of autophagy substrate p62/SQSTM. Inhibitionofthe accumulation of autophagic substrate alleviated GO-caused apoptotic cell death. Our findings raise a concern for the putative biomedical applications of GO in the form of diagnostic and therapeutic tools, where its systematic biocompatibility should be thoroughly explored. STATEMENT OF SIGNIFICANCE: Graphene oxide (GO) has attracted considerable interests in biomedical fields, which also resulted in numerous safety risks to human bodies. It is urgently required to establish a paradigm for accurately evaluating their adverse effects in biological systems. This study thoroughly explored the neurotoxicity of GO in PC12 cells. We found GO triggered an increased autophagic response and the impairment of autophagic flux, which was functionally involved in cell apoptosis. Inhibitionofexcessive accumulation of autophagic cargo attenuated apoptotic cell death. Our findings highlight deep considerations on the regulation mechanism of autophagy-lysosomes-apotosis-axis, which will contribute to a better understanding of the neurotoxicity of graphene-family nanomaterials, and provide a new insight in the treatment of cancer cells at nanoscale levels.