Gold Nanoparticles Induced Size Dependent Cytotoxicity on Human Alveolar Adenocarcinoma Cells by Inhibiting the Ubiquitin Proteasome System

Abstract

Gold nanoparticles (AuNPs) are widely used in biomedicine due to their remarkable therapeutic applications. However, little is known about their cytotoxic effects on the ubiquitin proteasome system (UPS). Herein, the cytotoxicity of different sizes of AuNPs (5, 10, and 80 nm) on the UPS was investigated with a particular focus on deubiquitinating enzymes (DUBs) such as ubiquitin-specific proteases (USP) and ubiquitin carboxyl-terminal hydrolases (UCHL-1) in human alveolar epithelial adenocarcinoma (A549). It was found that all sizes of AuNPs reduced the percentage of viable A549 cells and increased lactate dehydrogenase (LDH) release, measured using the MTT and LDH assays, respectively. Furthermore, the 5 nm AuNPs were found to exhibit greater cytotoxicity than the 10 and 80 nm AuNPs. In addition, apoptosis and necrosis were activated through reactive oxygen species (ROS) generation due to AuNPs exposure. The internalisation of AuNPs in A549 cells increased with increasing particle size (80 > 10 > 5 nm). Interestingly, the expression of USP7, USP8, USP10, and UCHL-1 was significantly (p < 0.001) downregulated upon treatment with 5–30 µg/mL of all the AuNPs sizes compared to control cells. Moreover, the inhibition of these proteins triggered mitochondrial-related apoptosis through the upregulation of poly (ADP-ribose) polymerase (PARP), caspase-3, and caspase-9. Collectively, these results indicate that AuNPs suppress the proliferation of A549 cells and can potentially be used as novel inhibitors of the proteasome.

The Ubiquitin–Proteasome System in Tumor Metabolism

“…In prokaryotes, ClpP and Lon are the major proteasomes that participate in the proximal ATP-dependent steps of substrate degradation [ 10 ]. UPS participates in intracellular protein proteasome-dependent degradation, cell metabolism, cell cycle progression, chromosome separation, kinase activation, apoptosis, DNA repair, and so on [ 11 , 12 , 13 ]. Ubiquitination modification is a primary mode of intracellular protein degradation in eukaryotes mediated by ubiquitin; it is initiated by enzyme E1 (ubiquitin-activating enzyme), which generates thioester bonds between its Cys residue and the ubiquitin C terminus using ATP-dependent hydrolysis.…”

Functionalized Gold Nanoparticles Suppress the Proliferation of Human Lung Alveolar Adenocarcinoma Cells by Deubiquitinating Enzymes Inhibition

“…This is the first attempt to study the effects of functionalized AuNPs on the ubiquitin proteasome system enzymes of A549 cells, and other studies are urgently needed to confirm the evidence. However, our previous study showed that AuNPs (5, 10, and 80 nm) were able to induce size-dependent cytotoxicity to A549 cells by inhibiting DUBs such as USP7, USP8, USP10, and UCHL-1 . Other studies have also reported that small molecules like P5091, HBX19818, P22077, b-AP15,, and WP1130 can attenuate the proliferation of different cell lines through the inhibition of ubiquitin-specific proteases such as USP7, − USP8, − USP10, − USP14, , and Ubiquitin C-terminal hydrolases such as UCHL5 .…”

“…In addition, when cells were exposed to a high concentration of functionalized AuNPs for 24 and 48 h, the proliferation of A549 cells relatively decreased. The decrease in cell proliferation at higher concentrations of AuNPs could be mediated by the insufficient nutrient provided in the CCM and more available number of AuNPs to interact with cellular components such as proteins, fatty acids, nucleic acid, and carbohydrates. ,, Based on the cell viability results, the capacity of A549 cells to recover from the detrimental effects caused by AuNPs was studied. Results on cell recovery demonstrated that A549 cells have the capacity to return to normal proliferation at low AuNP concentrations.…”

“…However, there are no reports on the effects of different functionalized AuNPs on deubiquitinating enzymes (DUBs) and their utility in understanding anticancer pathways. DUBs are an important group of enzymes that play a significant role in cancer pathogenesis, development, and proliferation by removing attached ubiquitin from the substrate. , They are classified into ubiquitin c-terminal hydrolases (UCHs), ubiquitin specific proteases (USPs), ovarian tumor proteases (OTUs), machado Joephin domain proteases (MJDs), monocyte chemotactic protein induced proteins (MCPIPs), and JAB1/MPN/Mov34 metalloenzyme (JAMM). − Among these DUBs, USPs, and UCHs are the most well-studied as they are highly overexpressed in human cancers, suggestive of their roles in tumor progression. , It has been found that cell survival in liver, breast, and colorectal cancers was promoted by an elevated level of USPs through simultaneously inhibiting apoptotic proteins and promoting tumor metastasis. , UCHL-1 overexpression has also been positively correlated with development, invasiveness, and chemotherapy resistance in some cancers including pancreatic, myeloma, neuroblastoma, nonsmall cell lung cancer (NSCLC), prostate, and lymphoma. , The overarching aim of this study is to understand the effects of tannate, citrate, and PVP functionalized 5 nm AuNPs on DUBs with a particular focus on USPs and UCHL-1 in A549 cells proliferation and toxicity. The study further hypothesized if the inhibition of these DUBs could lead to the downregulation of PI3K/AKT/mTOR, Wnt signaling pathway related proteins, and activation of mitochondrial apoptosis.…”

Modulation of Heat Shock Protein Expression in Alveolar Adenocarcinoma Cells through Gold Nanoparticles and Cisplatin Treatment

“…In this study, the effects of combined therapy of citrate functionalised AuNPs (10 nm) with the anticancer drug CDDP were evaluated. Monodispersed AuNPs with an average size range of 8-14 nm, zeta potential of −19 ± 1.29 mV, and polydispersity index of 0.301 ± 0.01, which were characterised previously [ 45 ], were used in the current study. The use of transmission electron microscopy showed that the AuNPs were spherical with homogeneous size distribution ( Figure S1 in Supplementary Materials ).…”