Increased DNMT1 Involvement in the Activation of LO2 Cell Death Induced by Silver Nanoparticles via Promoting TFEB-Dependent Autophagy

Abstract

The accumulation of exogenous silver nanoparticles (AgNPs) will terminally bring about liver injury, including cell death, where DNA methylation tends to be a crucial epigenetic modulator. The change in the cell autophagy level verified to be closely associated with hepatocyte death has been followed with wide interest. But the molecular toxicological mechanisms of AgNPs in relation to DNA methylation, autophagy, and cell death remain inconclusive. To address the issue above, in LO2 cells treated with increasing concentrations of AgNPs (0, 5, 10, and 20 μg/mL), a cell cytotoxicity assay was performed to analyze the level of cell death, which also helped to choose an optimal concentration for next experiments. An immunofluorescence assay was used to determine the autophagic flux as well as TFEB translocation, with qRT-PCR and western blot being used to analyze the expression level of autophagy-related genes and proteins. According to our findings, in the determination of cell viability, 20 μg/mL (AgNPs) was adopted as the best working concentration. LO2 cell death, autophagy, and TFEB nuclear translocation were induced by AgNPs, which could be inhibited by lysosome inhibitor chloroquine (CQ) or siRNA specific for TFEB. Moreover, AgNP exposure led to DNA hypermethylation, with DNMT1 taking part mainly, which could be obviously prevented by 5-Aza-2'-deoxycytidine (5-AzaC) or trichostatin A (TSA) treatment or DNMT1 knockout in LO2 cells. Our studies suggest that through TFEB-dependent cell autophagy, increased DNMT1 may facilitate cell death induced by AgNPs.

Keywords: DNMT1; autophagy; cell death; hepatocyte injury; silver nanoparticles.

Figure 1

Toxicity of AgNPs to LO2 cells. Cell viability and LDH release level of LO2 cells after (A,C) 24 h of AgNP (0, 5, 10, 20 μg/mL) treatments and (B,D) different time courses of AgNP (20 μg/mL) treatments, respectively. (* p < 0.05 vs. the 0 μg/mL or 0 h AgNPs group.) AgNPs, silver nanoparticles; LDH, lactic dehydrogenase.

Figure 2

Induction of autophagy was found in LO2 cells treated with AgNPs. (A,B) qRT-PCR was used to detect the mRNA expression levels of LC3 and Beclin1 in LO2 cells dealt with 24 h AgNPs (0, 5, 10, 20 μg/mL). An immunoblot assay was used to measure the protein expression level of (C,D) p62 and LC3 after 24 h of AgNP (0, 5, 10, 20 μg/mL) treatments, as well as (E,F) LC3 upon AgNP-only treatment or AgNP-CQ co-treatment. (G,H) Punctate LC3 signals of LO2 cells treated with 24 h of AgNP (20 μg/mL) were measured via IFC analysis. (* p < 0.05 vs. the 0 μg/mL AgNP group or the control group; ^# p < 0.05 vs. the AgNPs treatment group.) AgNPs, silver nanoparticles; CQ, chloroquine; IFC, immunofluorescence.

Figure 3

AgNPs induced TFEB-dependent cell autophagy. (A,B) LO2 cells were treated with AgNPs (20 μg/mL) for 24 h and immunostained for TFEB (red). The nuclei were stained with DAPI (blue). (C,D) LysoTracker dyes (red) were used after the treatment of AgNPs (20 μg/mL) for 24 h. (E,F) Cell viability and the LDH release level of LO2 cells upon AgNP-only treatment or AgNP-CQ co-treatment. (G,H) Cell viability and the LDH release level were analyzed in LO2 cells transfected with siRNA specific for TFEB (si TFEB) and treated with AgNPs (20 μg/mL) for an additional 24 h. (* p < 0.05 vs. the control group; ^# p < 0.05 vs. the AgNPs treatment group.) AgNPs, silver nanoparticles; TFEB, transcription factor EB; DAPI, 4′,6-diamidino-2-phenylindole; LDH, lactic dehydrogenase; CQ, chloroquine.

Figure 4

DNMTs were involved in autophagy after AgNP exposure. (A–C) qRT-PCR was used to detect the mRNA expression levels of DNMTs, including DNMT1, DNMT3a, and DNMT3b (the same below), in LO2 cells dealt with 24 h of AgNP (0, 5, 10, 20 μg/mL). (D,E) The protein levels of DNMTs in LO2 cells after 24 h of AgNP (0, 5, 10, 20 μg/mL) treatments. A western blot was applied to detect the expression of (F,G) DNMTs, as well as (H,I) p62 and LC3, upon AgNP-5-AZA or AgNP-TSA co-treatment. (J,K) Punctate LC3 signals of LO2 cells upon AgNP-5-AZA or AgNP-TSA co-treatment were measured via IFC analysis. (L,M) LO2 cells were treated with AgNPs (20 μg/mL), as well as 5-AZA or TSA, for 24 h and immunostained for TFEB (red). The nuclei were stained with DAPI (blue). (* p < 0.05 vs. the control group). DNMTs, DNA methyltransferases; AgNPs, silver nanoparticles; 5-AZA, 5-Aza-2′-Deoxycytidine; TSA, Trichostatin A; IFC, immunofluorescence; TFEB, transcription factor EB; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5

DNMT1 knockout inhibits TFEB-dependent autophagic cell death after AgNP exposure. (A–C) Western blot and qRT-PCR were used to detect the protein expression levels of DNMT1, caspase 3, LC3 A/B and the mRNA level of Beclin1 in AgNP (20 μg/mL)-treated LO2 cells with or without DNMT1 knockout. (D,E) Punctate LC3 signals of LO2 cells upon AgNP (20 μg/mL) treatment with or without DNMT1 knockout were measured via IFC analysis. (F,G) LO2 cells with or without DNMT1 knockout were treated with AgNPs (20 μg/mL) for 24 h and immunostained for TFEB (red). The nuclei were stained with DAPI (blue). (H,I) LysoTracker dyes (red) were used after the treatment of AgNPs (20 μg/mL) for 24 h. (* p < 0.05 vs. the KO-NC-0 group; ^# p < 0.05 vs. the KO-NC-20 group.) TFEB, transcription factor EB; AgNPs, silver nanoparticles; IFC, immunofluorescence; DAPI, 4′,6-diamidino-2-phenylindole; KO-NC-0 group, LO2 cells dealt without AgNPs; KO-NC-20 group, LO2 cells dealt with AgNPs (20 μg/mL); KO-DNMT1-0 group, DNMT1 knocking out LO2 cells dealt without AgNPs; KO-DNMT1-20 group, DNMT1 knocking out LO2 cells dealt with AgNPs (20 μg/mL).