Perturbation of Cellular Redox Homeostasis Dictates Divergent Effects of Polybutyl Cyanoacrylate (PBCA) Nanoparticles on Autophagy

Abstract

Nanoparticles (NPs) are used in our everyday life, including as drug delivery vehicles. However, the effects of NPs at the cellular level and their impacts on autophagy are poorly understood. Here, we demonstrate that the NP drug delivery vehicle poly(butyl cyanoacrylate) (PBCA) perturbs redox homeostasis in human epithelial cells, and that the degree of redox perturbation dictates divergent effects of PBCA on autophagy. Specifically, PBCA promoted functional autophagy at low concentrations, whereas it inhibited autophagy at high concentrations. Both effects were completely abolished by the antioxidant N-acetyl cysteine (NAC). High concentrations of PBCA inhibited MAP1LC3B/GABARAP lipidation and LC3 flux, and blocked bulk autophagic cargo flux induced by mTOR inhibition. These effects were mimicked by the redox regulator H2O2. In contrast, low concentrations of PBCA enhanced bulk autophagic cargo flux in a Vps34-, ULK1/2- and ATG13-dependent manner, yet interestingly, without an accompanying increase in LC3 lipidation or flux. PBCA activated MAP kinase signaling cascades in a redox-dependent manner, and interference with individual signaling components revealed that the autophagy-stimulating effect of PBCA required the action of the JNK and p38–MK2 pathways, whose activities converged on the pro-autophagic protein Beclin-1. Collectively, our results reveal that PBCA exerts a dual effect on autophagy depending on the severity of the NP insult and the resulting perturbation of redox homeostasis. Such a dual autophagy-modifying effect may be of general relevance for redox-perturbing NPs and have important implications in nanomedicine.

To eat or not to eat: a critical review on the role of autophagy in prostate carcinogenesis and prostate cancer therapeutics

“…Such measurements are frequently used as proxies for autophagic cargo flux. However, there are a number of important limitations and potential confounding factors in the use of autophagic markers like LC3 and p62 in assessing autophagic activity ( Klionsky et al, 2021 ) including potential influences from transcriptional and translational regulation of LC3 and p62 ( Engedal et al, 2013 ; Klionsky et al, 2021 ), conjugation of LC3 to other membrane compartments than phagophores/autophagosomes ( Malhotra, 2013 ; Pimentel-Muiños and Boada-Romero, 2014 ; Durgan et al, 2021 ), conjugation of LC3 to proteins (termed “protein ATG8ylation”) ( Carosi et al, 2021 ), puncta formation due to LC3/p62 aggregation in the absence of phagophores ( Szeto et al, 2006 ; Kuma et al, 2007 ; Runwal et al, 2019 ), a high degree of uncertainty in the correlation between LC3/p62 marker flux versus actual cargo flux, and that LC3 and p62 have many other cellular functions than autophagy ( Moscat et al, 2007 ; Baisamy et al, 2009 ; Hanson et al, 2010 ; Chung et al, 2012 ; Moscat et al, 2016 ; Ramkumar et al, 2017 ; Lindner et al, 2020 ; Kumar et al, 2021 ; Nieto-Torres et al, 2021 ; Yoon et al, 2024 ) and are not always necessary for autophagy ( Sala et al, 2014 ; Szalai et al, 2015 ; Xu et al, 2015 ; Nguyen et al, 2016 ; Vaites et al, 2018 ; Luhr et al, 2019 ; Sønstevold et al, 2021 ). Therefore, the use of autophagic markers alone is insufficient for drawing solid conclusions about autophagic activity ( Klionsky et al, 2021 ).…”

“…Therefore, the use of autophagic markers alone is insufficient for drawing solid conclusions about autophagic activity ( Klionsky et al, 2021 ). Indeed, upon direct comparison of cargo-based versus LC3-based assays, we have identified conditions where LC3 flux measurements show a complete lack of positive correlation with actual autophagic cargo flux in mammalian cells ( Szalai et al, 2015 ; Sønstevold et al, 2021 ).…”

“…The cleaved forms (termed mATG8-I, for instance LC3-I or GABARAP-I) can then be conjugated to phosphatidylethanolamine (PE) ( Kabeya et al, 2004 ; Sou et al, 2006 ) (termed “lipidation”) by sequential ubiquitylation-like reactions catalysed by ATG7 (E1-like enzyme), ATG3 (E2-like enzyme), and the ATG5/ATG12-ATG16L1 complex to generate a membrane-bound form of mATG8-I, referred to as mATG8-II (for instance LC3-II or GABARAP-II) ( Kabeya et al, 2000 ; Tanida et al, 2001 ; Tanida et al, 2002 ; Tanida et al, 2004 ). Initially, both the LC3- and GABARAP subfamilies of the mATG8 proteins were thought to be essential for autophagosome formation ( Weidberg et al, 2010 ), however, recent studies by us and others indicate that the GABARAPs play a dominant role, and that the LC3s are frequently not required for the autophagic pathway to go to completion ( Szalai et al, 2015 ; Nguyen et al, 2016 ; Vaites et al, 2018 ; Luhr et al, 2019 ; Sønstevold et al, 2021 ). An important other function of the mAGT8s is to recruit various types of autophagy receptors to the inner phagophore membrane via interaction with specific motifs/regions in the receptors, the best described being the so-called LC3/ATG8-interacting region/motif (LIR/AIM).…”

Effects of polymer carriers on the occurrence and development of autophagy in drug delivery

“…4). 55 Polydopamine (PDA), as a pH-sensitive material, could control the drug release under pH-stimulus. 56 PDA-coated mesoporous silica nanoparticles could increase LC3-II and p62 levels and decrease beclin1 levels via the inhibition of the AKT-mTOR-p70S6K signaling pathway.…”