Gene expression and pathway analysis of human hepatocellular carcinoma cells treated with cadmium

Abstract

Cadmium (Cd) is a toxic and carcinogenic metal naturally occurring in the Earth's crust. A common route of human exposure is via diet and cadmium accumulates in the liver. The effects of Cd exposure on gene expression in human hepatocellular carcinoma (HepG2) cells were examined in this study. HepG2 cells were acutely-treated with 0.1, 0.5, or 1.0 μM Cd for 24h; or chronically-treated with 0.01, 0.05, or 0.1 μM Cd for three weeks and gene expression analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST Arrays. Acute and chronic exposures significantly altered the expression of 333 and 181 genes, respectively. The genes most upregulated by acute exposure included several metallothioneins. Downregulated genes included the monooxygenase CYP3A7, involved in drug and lipid metabolism. In contrast, CYP3A7 was upregulated by chronic Cd exposure, as was DNAJB9, an anti-apoptotic J protein. Genes downregulated following chronic exposure included the transcriptional regulator early growth response protein 1. Ingenuity Pathway Analysis revealed that the top networks altered by acute exposure were lipid metabolism, small molecule biosynthesis, cell morphology, organization, and development; while top networks altered by chronic exposure were organ morphology, cell cycle, cell signaling, and renal and urological diseases/cancer. Many of the dysregulated genes play important roles in cellular growth, proliferation, and apoptosis, and may be involved in carcinogenesis. In addition to gene expression changes, HepG2 cells treated with cadmium for 24h indicated a reduction in global levels of histone methylation and acetylation that persisted 72 h post-treatment.

Keywords: Affymetrix; Cadmium; Epigenetics; Gene expression; HepG2 cells; Histone modifications.

Figure 1

Heatmaps of differentially expressed genes due to acute (left) and chronic (right) cadmium treatment in HepG2 cells (over 1.25-fold change, p>0.05). Red indicates upregulation while blue indicates downregulation of gene expression relative to control, untreated cells.

Figure 2

Top networks altered by acute exposure to 0.5μM cadmium in HepG2 cells were lipid metabolism, small molecule biochemistry, and vitamin and mineral metabolism (left), and cellular morphology, assembly, organization, and development (right). Red – upregulation; green - downregulation.

Figure 3

Top networks altered by chronic exposure to 0.1μM cadmium in HepG2 cells were organ morphology, cell cycle, and cancer (left), and developmental disorder, renal and urological diseases and cancer (right). Red – upregulation; green – downregulation

Figure 4

Western blot images (A) and graphical representations (B) of relative intensities calculated by ImageJ for cadmium treatment in HepG2 cells. Cells were treated for 24 hours with varying doses of cadmium and cells were lysed post-treatment. Cells treated with 5 μM cadmium were allowed to recover for 72 hours after treatment before lysis (washout). Blots and graphs are representative of two independent experiments. WO; washout

Figure 4

Western blot images (A) and graphical representations (B) of relative intensities calculated by ImageJ for cadmium treatment in HepG2 cells. Cells were treated for 24 hours with varying doses of cadmium and cells were lysed post-treatment. Cells treated with 5 μM cadmium were allowed to recover for 72 hours after treatment before lysis (washout). Blots and graphs are representative of two independent experiments. WO; washout