ML385 Attenuates Malignant Progression of Silica-induced Lung Adenocarcinoma Cells through the ROS/NRF2-autophagy Axis Pathway

Summary

This study employs animal and cell models to investigate whether NRF2 inhibitor ML385 attenuates silica-induced malignant progression of lung adenocarcinoma through the ROS/NRF2-autophagy axis pathway.

Abstract

Silica exposure is associated with an increased risk of lung adenocarcinoma, but its molecular mechanism remains unclear. This study aims to establish a repeatable experimental protocol to explore how the nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor ML385 inhibits silica-induced malignant progression of lung adenocarcinoma by regulating the ROS/NRF2-autophagy axis pathway. Firstly, a silicosis model was established by intranasal perfusion of silica suspension in C57BL/6 mice. The degree of pulmonary fibrosis was evaluated by Masson staining, the infiltration of immune cells was analyzed by immunofluorescence, and the expressions of NRF2, cyclin-dependent kinase 1 (CDK1), and voltage-dependent anion channel 1 (VDAC1) in lung tissue were detected by immunohistochemistry. Subsequently, in in vitro experiments, the RAW264.7 macrophage cell line was treated with silica. Autophagic flux and oxidative stress levels were evaluated by LC3-lysosome co-localization and ROS probes, and intervention was carried out with the NRF2 inhibitor ML385. Finally, the effects of ML38 on migration, invasion, and apoptosis of lung adenocarcinoma cells were analyzed by scratch assay, cells passing through pores of a specific size assay, and flow cytometry. The results showed that silica could induce pulmonary fibrosis, immune cell infiltration, and upregulation of NRF2, CDK1, and VDAC1 in mice (p < 0.001), and inhibit the autophagic flux of macrophages and reduce ROS levels. ML385 can reverse these effects (p < 0.05). This protocol provides a complete experimental process from in vivo model construction to in vitro mechanism research, offering an operational technical path for studying the molecular mechanism of silica-related lung adenocarcinoma and therapeutic strategies targeting NRF2.

Introduction

Lung adenocarcinoma is the leading cause of cancer-related deaths worldwide, with an estimated 2 million new cases and 1.76 million deaths each year¹. In recent years, the incidence of lung adenocarcinoma among people who have never smoked has risen, which may be related to environmental factors such as air pollution². Silica is a common environmental pollutant and has been identified as a potential pathogenic factor for human lung adenocarcinoma³. Silica can cause persistent chronic inflammation, leading to infiltration of macrophages, lymphocytes, and neutrophils, and the release of reactive oxy....

Protocol

All donor blocks were obtained from archival pathological specimens collected between 2016 and 2018 at the Affiliated Huai'an NO.1 People's Hospital of Nanjing Medical University. The samples were deidentified prior to use and processed in compliance with approved protocols. The ethics committee of the Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University, KY-2024-250-01. The breeding, disposal, and application of experimental mice strictly follow the Regulations on the Administration of Laboratory Animals and international guidelines.

Construction of a silica-induced mouse model
Prep....

Results

Silica mouse model
In Figure 1, the mice were administered silica intranasally at a dosage of 30 mg/mL and 70 µL for 40 consecutive days, which ensured that no death events occurred in the mice and the silica mouse model could be successfully established. Meanwhile, mice gavage with PBS were used as negative controls. The aim was to eliminate the influence of the operation and the solvent itself and ensure that the phenotype was specifically induced by silica. The succes.......

Discussion

Lung adenocarcinoma is one of the main causes of cancer-related deaths worldwide, and its incidence and mortality rates are increasing year by year¹⁷. Silica is classified as a class 1 human carcinogen by the International Agency for Research on Cancer, and the risk of lung adenocarcinoma increases with cumulative occupational exposure^18,19,20,21. Previous studies have p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BeyoGold Transwell	Beyotime Biotechnology	FTW067-48lns	Transwell
CD3e	BD Bioscience	561827	FITC Hamster Anti-Mouse CD3e(145-2C11)
CD86	BD Bioscience	105013	CD86
CD8a	BD Bioscience	100713	CD8a
CDK1	Abcam	ab133327	Anti-CDK1
Cell apoptosis detection kit	Beyotime Biotechnology	C1062L	apoptosis detection
DAPI	Beyotime Biotechnology	P0131-25ml	DAPI
Embedding machine	P.S.J MEDICAL	BM450A	Embedding machine
F4/80	BD Bioscience	123109	F4/80
Fully automatic tissue dehydrator	Leica Biosystems	ASP3005	Fully automatic tissue dehydrator
Glass microscope slides	Citotest	250124A1	Glass microscope slides
H&E dye	Beyotime Biotechnology	C0105M	H&E dye
IHC Kit	Absin Biotechnology	abs996-5ml	IHC Kit
LC3 probe	Beyotime Biotechnology	C3018M	LC3 probe
Low Profile Microtome Blades	Thermo Fisher	3052835	Low Profile Microtome Blades
lysosome probe	Beyotime Biotechnology	C1046	lysosome probe
Marker pen	Deli	SK109	Marker pen
Masson dye	Beyotime Biotechnology	C0189M	Masson dye
Matrix-Gel	Beyotime Biotechnology	C0371-5ml	Matrix adhesive
Microtome	Leica Biosystems	HistoCore BIOCUT	Microtome
ML385	Abcam	ab287109	NRF2 inhibitor
NK1.1	BD Bioscience	561117	NK1.1
NRF2	Abcam	ab1809Y	Anti-NRF2
p62	Abcam	ab20735	Anti-p62
Paraffin wax	Solarbio	YA0012	Paraffin wax
Reactive oxygen species Assay kit	Beyotime Biotechnology	S0033M	ROS probe
Silica	Sigma Aldrich	S5631	Crystalline silica
VDAC1	Abcam	ab34726	Anti-VDAC1