Mercury Chloride but Not Lead Acetate Causes Apoptotic Cell Death in Human Lung Fibroblast MRC5 Cells via Regulation of Cell Cycle Progression

Abstract

Heavy metals are important for various biological systems, but, in excess, they pose a serious risk to human health. Heavy metals are commonly used in consumer and industrial products. Despite the increasing evidence on the adverse effects of heavy metals, the detailed mechanisms underlying their action on lung cancer progression are still poorly understood. In the present study, we investigated whether heavy metals (mercury chloride and lead acetate) affect cell viability, cell cycle, and apoptotic cell death in human lung fibroblast MRC5 cells. The results showed that mercury chloride arrested the sub-G₁ and G₂/M phases by inducing cyclin B1 expression. In addition, the exposure to mercury chloride increased apoptosis through the activation of caspase-3. However, lead had no cytotoxic effects on human lung fibroblast MRC5 cells at low concentration. These findings demonstrated that mercury chloride affects the cytotoxicity of MRC5 cells by increasing cell cycle progression and apoptotic cell death.

Keywords: HgCl2; MRC5; PbAc; apoptosis; cell cycle arrest; heavy metal; human lung fibroblast cell.

Figure 1

Effect of heavy metals on cell viability and morphology of MRC5 cells. (A) MRC5 cell viability was measured using the MTS assay. Cells were treated with 6.25–50 μM of HgCl₂ and 20–100 μM PbAc for 24 or 48 h. Data are represented as the percentage of the values obtained for the EtOH-treated cells used as the control. Error bars show mean ± SEM. p values obtained by Student’s t-test. ** p value < 0.05. (B) The morphology of MRC5 cells was observed by phase-contrast microscopy after treatment with indicated concentration of HgCl₂ and PbAc for 24 or 48 h. (C) MRC5 cells were stained with calcein-AM (green) and ethidium homodimer (red) by the live/dead assay. EtOH were used as the negative control. Images are representative of three independent experiments. Scale bars represent 200 μM.

Figure 2

Heavy metals treatment reduced the proliferation of MRC5 cells. (A) The heavy metals-treated MRC5 cells were immunostained with anti-Ki-67 antibodies. The cells were counted by FACS analysis. (B) The percentages of Ki-67-positive population are represented as the mean ± S.E.M. of three independent experiments (n = 6), each performed in triplicate. Error bars show mean ± S.E.M. p values obtained by Student’s t-test. *** p value < 0.001.

Figure 3

Cell cycle progression in heavy metals treatment in MRC5 cells. (A) The heavy metals-treated MRC5 cells were stained with propidium iodide (PI), and the cell cycle was measured using FACS analysis. (B) The percentages of population in the sub-G₁, G₀/G₁, S, and G₂/M phases are represented as the mean ± S.E.M. of three independent experiments (n = 6), each performed in triplicate.

Figure 4

The effect of heavy metals on cyclin B1 expression in MRC5 cells. (A) Cells were treated with indicated concentration of heavy metals for 24 or 48 h. Cells were fixed with 1% PFA and stained with anti-cyclin B1 antibody. The cyclin B1 expression was analyzed by FACS analysis. (B) The percentages of cyclin B1-positive cells are represented as the mean ± S.E.M. of three independent experiments (n = 6), each performed in triplicate. EtOH was used as the negative control.

Figure 5

The effect of heavy metals on cyclin D1 expression in MRC5 cells. (A) Cells were treated with indicated concentration of heavy metals for 24 or 48 h. Cells were fixed with 1% PFA and stained with anti-cyclin D1 antibody. The cyclin D1 expression was analyzed by FACS analysis. (B) The percentages of cyclin D1-positive cells are represented as the mean ± S.E.M. of three independent experiments (n = 6), each performed in triplicate. EtOH was used as the negative control.

Figure 6

Effect of apoptotic cell death upon heavy metals treatment in MRC5 cells. (A) Cells were treated with indicated concentration of heavy metals for 24 or 48 h, and were double-stained with annexin V-FITC and PI. The proportion of apoptotic cells was assessed by FACS analysis. The scatter plots represent PI (Y-axis) and annexin V-FITC (X-axis). (B) The percentages of cells in the live, early- and late-apoptotic, and necrotic stages are expressed as the mean ± S.E.M. of three independent experiments (n = 6), each performed in triplicate.

Figure 7

The effect of caspase-3 activity in heavy metals-treated MRC5 cells. (A) Cells were treated with indicated concentration of heavy metals for 24 or 48 h. Cells were fixed with 1% PFA and stained with anti-cleaved caspase-3 antibody. Expression levels of cleaved caspase-3 were analyzed by FACS analysis. (B) The percentages of cleaved caspase-3-positive cells are represented as the mean ± S.E.M. of three independent experiments (n = 6), each performed in triplicate. EtOH was used as the negative control. Error bars show mean ± SEM. p values obtained by Student’s t-test. *** p value < 0.001.