Oxidative damage and malignant migration of hepatocellular carcinoma cells LM3 induced by 14 weeks exposure to sodium arsenite

doi:10.3969/j.issn.1674-8115.2022.12.004

Abstract

Abstract:

Objective ·To investigate the effects of different doses of sodium arsenite exposure on human hepatocellular carcinoma cell line LM3, and analyze the underlying potential molecular mechanisms of sodium arsenite-mediated promotion of malignant migration of LM3 cells. Methods ·The model of sodium arsenite exposure was established by continuously culturing LM3 cells in high DMEM glucose medium containing 0, 1 and 10 μmol/L sodium arsenite for 14 weeks. After exposure, the sodium arsenite-containing cell culture medium was discarded, and the cells were allowed to recover in sodium arsenite-free culture medium for one week. Cells from the same batch without sodium arsenite treatment were used as the control group. Subsequently, CCK-8 proliferation assay, Transwell migration assay, and DCFH-DA reactive oxygen species fluorescence probe experiments were performed to verify the effect of chronic sodium arsenite exposure on LM3 cell proliferation, migration, and oxidative stress. Real-time quantitative PCR and Western blotting were performed to elucidate the effect of chronic sodium arsenite exposure on the expression of mRNA and protein related to tumor metastasis in LM3 cells. Results ·Compared with the control group, the sodium arsenite exposure groups (1 μmol/L and 10 μmol/L) had significantly higher reactive oxygen species (ROS) levels (both P>0.05), and sodium arsenite exposure promoted the malignant migration of LM3 cells in a dose-dependent manner (both P>0.05), but sodium arsenite had no significant effect on the proliferation of LM3 cells. Sodium arsenite exposure up-regulated the gene expression levels of vascular endothelial growth factor (VEGF), nicotinamide adenine dinucleotide phosphate oxidases 2 (NOX2), and mitogen-activated protein kinase 8 (MAPK8),and up-regulated the protein expression level of NOX2 and MAPK8. Conclusions ·Sodium arsenite exposure can promote malignant migration and oxidative stress level of LM3 cells, and up-regulate the expression levels of VEGF, NOX2 and MAPK8 genes, suggesting that the mechanism of sodium arsenite promoting malignant migration of LM3 cells may be related to oxidative damage and up-regulation of these genes.

Key words: sodium arsenite, LM3 cells, proliferation, oxidative stress, malignant migration

CLC Number:

SUN Jinli, SONG Weiwei, XU Ming, LI Jingquan. Oxidative damage and malignant migration of hepatocellular carcinoma cells LM3 induced by 14 weeks exposure to sodium arsenite[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(12): 1677-1684.

Figures/Tables 6

Tab 1 Primer sequences for qRT-PCR

Gene	Forward sequence （5'→3'）	Reverse sequence （5'→3'）
VEGF	AGGGCAGAATCATCACGAAGT	AGGGTCTCGATTGGATGGCA
NOX2	TGCCAGTCTGTCGAAATCTGC	ACTCGGGCATTCACACACC
MAPK8	TGTGTGGAATCAAGCACCTTC	AGGCGTCATCATAAAACTCGTTC
TGFβ	GGCCAGATCCTGTCCAAGC	GTGGGTTTCCACCATTAGCAC
GAPDH	GGCCTCCAAGGAGTAAGACC	AGGGGAGATTCAGTGTGGTG

Fig 1 Effects of chronic sodium arsenite exposure on proliferation of LM3 cells

Fig 2 Effects of chronic sodium arsenite exposure on migration of LM3 cells

Fig 3 Intracellular ROS levels detected by DCFH-DA ROS fluorescence probe

Fig 4 Detection of mRNA expression levels of tumor metastasis-related genes by qRT-PCR

Fig 5 Detection of protein expression levels of NOX2 and MAPK8 by Western blotting

TrendMD

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