p53 Mediates Colistin-Induced Autophagy and Apoptosis in PC-12 Cells

Abstract

Colistin is used as a last-line treatment option against multidrug-resistant (MDR) Gram-negative bacteria, which can cause life-threatening infections (1-3). However, its clinical use is limited by potential nephrotoxicity and neurotoxicity (4, 5), the mechanisms of which are still unknown. It has been discovered in a mouse model and neuroblastoma 2a cells that autophagy is involved in colistin-induced nephrotoxicity (6, 7). Apoptosis and autophagy are two common forms of cell death (8, 9). Apoptosis is a prevalent form of programmed cell death (PCD) in multicellular organisms, which is the culmination of coordinately regulated intrinsic and extrinsic pathways involving major protein families, including the Bcl-2 family and caspases (10). Autophagy is a catabolic process of degradation and recycling of dysfunctional cellular components by lysosomal systems (11-13). Autophagy participates in organelle turnover and in the bioenergetic management of starvation stresses, pathogen infection, and hypoxia in order to maintain cellular homeostasis (14,15). A number of stimuli can induce autophagy, apoptosis, or both, and recent studies suggest that autophagy delays or promotes apoptosis under certain conditions (16,17). For example, treatment of cells with pemetrexed and simvastatin promoted autophagy and inhibited apoptosis (16), while oridonin phosphate induced autophagy and enhanced apoptotic cell death (17). However, the precise mechanisms that determine autophagy, apoptosis, and their interaction remain to be elucidated.A number of studies in tumor cells (e.g., hepatocellular carcinoma and OVCAR-3 cancer cells) have shown that the p53 tumor suppressor protein, which is an important cellular stress sensor, can trigger cell cycle arrest and apoptosis and also regulate autophagy (18)(19)(20). Activation of p53 in response to a death stimulus leads to the transcription of genes involved in apoptosis, including PUMA (p53 upregulated modulator of apoptosis), AMPK (AMPactivated protein kinase), and Bax in the nucleus. These in turn activate the intrinsic mitochondrial apoptotic pathway in the cytoplasm via inhibition of the antiapoptotic proteins Bcl-2 and Bcl-X L (19,20). In addition, p53 appears to play a dual role in the control of autophagy. At basal levels, p53 has an inhibitory effect, and its activation initiates the autophagic process (21,22). Thus, p53-induced autophagy may either constitute a physiological cellular defense response (23) or contribute to cell death (24). The cellular localization of p53 appears to determine whether a cell will undergo autophagy or apoptosis. Nuclear p53 induces and regulates autophagy, while cytoplasmic p53 inhibits autophagy (22,25).

Quantitative assessment of cell fate decision between autophagy and apoptosis

“…We further validated our model using two additional datasets generated in rat kidney proximal tubular (RPTC) cells 34 and rat adrenal medulla PC-12 cells 35 , respectively. Figure 3c shows the corresponding model predictions ( dashed curves ), which quantitatively reproduce the experimentally observed ( dots ) time courses of autophagy and apoptosis of RPTC cells in response to 20 μM cisplatin (ER stress-inducer).…”

A Dual Role of P53 in Regulating Colistin-Induced Autophagy in PC-12 Cells

“…However, autophagy inhibition by p53 is more obvious when the nuclear localization sequence of p53 is deleted, leading to a merely cytoplasmic p53 localization (Ryan, 2011 ). In our present study, it is clear from the results that autophagy-related genes and protein were inhibited in colistin-treated PC-12-sip53 cells at 12 h, but opposite results occurred at 24 h, indicating that p53 down-regulated autophagy in PC-12-sip53 cells at 12 h and up-regulated autophagy at 24 h. Our previous study showed that colistin induced the highest expression levels of nuclear p53 after 12 h, but it induced the highest expression of cytoplasmic p53 at 24 h (Zhang et al, 2016 ). Thus, we inferred that the neurotoxicity of colistin acted as a stress/stimulation in PC-12 cells to be a signal of p53 translocation into the nucleus, as p53 expression levels was reduced by siRNA, leading to the lower levels of p53-translocated into nucleus, thereby silencing of p53 down-regulated colistin-induced autophagy in PC-12 cells for 12 h. Thereafter, cytoplasmic p53 plays a leading role in down-regulation of autophagy, while the relatively decreased cytoplasmic p53 level resulted in activation of autophagy in PC-12-sip53 cells at 24 h.…”

“…Therefore, understanding the complex functionality of autophagy induction is highly important. In our previous study, we proved that colistin (125 μg/mL) induces a high level of autophagy at 12 h and that autophagy can protect against the colistin-induced apoptosis (Jiang et al, 2014 ), and Zhang et al reported that colistin increased the expression level of p53, and p53 involved in colistin-induced autophagy and apoptosis (Zhang et al, 2016 ). In this follow-up mechanistic study, we are interested in exploring whether p53-mediated autophagy plays a cell survival role in colistin-treated PC-12 cells and p53 could act as a target gene to protect against colistin-induced neurotoxicity.…”

“…They were washed with PBS twice and post-fixed in 1% osmium tetroxide at 4°C for 1 h. Next, the cells were dehydrated by ethanol series and 100% acetone, embedded in epoxy resins. The ultrathin sections were stained with uranyl acetate and lead citrate (Zhang et al, 2016 ), and then observed under a GEM-1200ES transmission electron microscope (JEOL Ltd., Tokyo, Japan).…”

“…Our colleagues studied that colistin-induced neurotoxicity triggered autophagy and apoptosis in PC-12 cells, and that apoptosis was affected by the control of autophagy (Jiang et al, 2014 ; Zhang et al, 2015 ). Importantly, our previous study showed that p53 participates in autophagy and apoptosis in colistin-treated PC-12 cells (Zhang et al, 2016 ). The PC-12 cell line is derived from pheochromocytoma of the rat adrenal medulla and it is commonly used as a neural differentiation and neurosecretion model (Sasaki et al, 2002 ; Chen et al, 2013 ).…”