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. Author manuscript; available in PMC: 2015 Sep 21.
Published in final edited form as: Met Ions Biol Med. 2011;11:242–246.

Lead-Induced Cell Cycle Arrest in Human Liver Carcinoma (HepG2) Cells: Involvement of oxidative stress, p53 and Cyclin A

Clement G Yedjou 1,*, Paul B Tchounwou 1
PMCID: PMC4577017  NIHMSID: NIHMS310486  PMID: 26401103

Abstract

Background

Recent studies in our laboratory have demonstrated that lead is cytotoxic to human liver carcinoma (HepG2) cells, showing a 48 h-LD50 of 35.5 ± 9.2ug/mL. However, its molecular mechanisms of toxicity are still largely unknown. Hence, the aim of the present study was to use HepG2 cells as a test model to investigate the molecular mechanisms of lead-induced oxidative stress and modulation of cellular response proteins.

Methods

To achieve this goal, we performed lipid peroxidation assay for malondialdehyde (MDA) determination, western blot and densitometric analyses for genes and related proteins expression in human liver carcinoma cells.

Results

Data obtained from the lipid peroxidation assay demonstrated a significant increase (p ≤ 0.05) of MDA levels in lead-treated HepG2 cells compared to control cells. Western Blot analysis showed a strong dose-response relationship with regard to p53 expression, and a significant repression in cyclin A in lead-treated cells.

Conclusions

Findings from this research indicate that lead is able to cause oxidative stress, cell cycle arrest through activation of the 53-kDa tumor suppressor protein and down regulation of the cyclin A protein in human liver carcinoma (HepG2) cells.

Keywords: Lead, HepG2 cells, p53, cyclin A, lipid peroxidation

INTRODUCTION

A series of recent studies in our laboratory demonstrated that lead induced toxicity and apoptosis in human cancer cells involved several cellular and molecular processes including, induction of cell death and oxidative stress [1], transcriptional activation of stress genes [2], DNA damage [3], externalization of phosphatidylserine, and activation of caspase-3 [4]. Even though lead has been intensively studied for many years, the molecular mechanisms of lead-induced oxidative stress and expression of cellular response proteins in tumor cells remain to be elucidated. Therefore, the present study was designed to determine whether oxidative stress plays a role in lead nitrate-induced toxicity, and to determine whether lead nitrate exposure modulates p53 and cyclin A expression in human liver carcinoma (HepG2) cells.

MATERIAL AND METHODS

Chemicals and media

Reference solution (1000 ± 10 ppm) of lead nitrate (CAS No. 10099-74-8, Lot No. 981735-24) with a purity of 100% was purchased from Fisher Scientific in Fair Lawn, New Jersey. Dulbecco’s Modified Eagle’s Minimal Essential Medium (DMEM, Lot. 1016511) was purchased from Life Technologies in Grand Island, New York.

Lipid Peroxidation Assay

Aldehydes such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA) are formed during lipid peroxidation. The concentration of MDA was measured by using a lipid peroxidation assay kit (Calbiochem-Novabiochem, San Diego, CA). Briefly, 2 × 106 HepG2 cells/mL untreated as a control and treated with different lead nitrate concentrations were cultured in a total volume of 10 ml growth medium for 48 hours. After the incubation period, cells were collected in 15 mL tube, followed by low-speed centrifugation. The cell pellets were re-suspended in 0.5 ml of Tris-HCl, pH 7.4, and lysed using a sonicator (W-220; Ultrasonic, Farmingdale, NY) under the conditions of duty cycle 25% and output control 40% for 5 sec on ice. The protein concentration of the cell suspension was determined using a protein assay kit (BioRad, Hercules, C.A.). A 200μl aliquot of the culture medium or 2 mg of cell lysate protein was assayed for MDA according to the lipid peroxide assay kit protocol (Calbiochem-Novabiochem, San Diego, CA). The absorbance of the sample was monitored at 586 nm, and the concentration of MDA was determined from a standard curve.

Western Blot Analysis for Identification of Specific Cellular Proteins

The Western Blot analysis was conducted to determine specific cellular response gene proteins (p53 and Cyclin A) at 48 hours of lead nitrate exposure. HepG2 cells were grown in 96 well polystyrene tissue culture plates until confluence. Briefly, 200 μL cells (5 × 105 cells/mL) were added to each well of 96 tissue culture plates treated with (10, 20, 30, and 40 μg/mL) lead nitrate for 48 hours. Control well plates were also made without lead nitrate. After incubation period, cells were collected by trypsinazation following by centrifugation at 2000 rpm for 5 minutes, supernatant were carefully aspirated, and cells were washed twice with PBS. The total protein was measured by the Bradford method [5] at 600 nm using a microtiter plate reader (Bio-Tek Instruments Inc). 20uL of native sample buffer (0.2 mol/L Tris, pH 6.8, 1% SDS, 30% glycerol, 7.5% mercaptoethanol, 0.1% bromophenol blue) was added to each well plate and cells were mechanically collected into micro-centrifuge tubes. Cellular protein lysates (15μg/mL) from human liver carcinoma (HepG2) cells containing an equal volume of sample buffer were heated at 100°C for 10 minutes. Appropriate amounts of total cellular protein were loaded onto 10% SDS-polyacrylamide gels and electrophoresed at 100 V constant voltages for one hour. Samples were transferred onto a nitrocellulose membrane on ice and the membrane blocked (Tris buffer saline with 5% nonfat dry milk, 0.1 Tween 20) overnight at 4°C. Detection of membrane-bound proteins was carried out using specific primary antibodies for the proteins of interest (p53 1:1000 and Cyclin A 1:500). Subsequently, the reaction was probed with a 1:750 dilution of alkaline conjugated anti-mouse IgG secondary antibody. NBT/BCIP color substrate was incorporated to develop protein bands. Immunoblot 1-D protein bands were assessed for relative abundance by Total Lab-Image computer software (Nonlinear USA Inc. Durham, NC) [6].

Statistical analysis

Experiments were performed in triplicates. Data were presented as means ± SDs. Where appropriate, one-way ANOVA or Student paired t-test was performed using SAS Software available in the Biostatistics Core Laboratory at Jackson State University. P-values less than 0.05 were considered statistically significant.

RESULTS

Malondialdehyde (MDA) Determination

The effect of lead nitrate on MDA production in HepG2 cells is represented in Figure 1. Data presented in this figure demonstrated that lead nitrate treatment resulted in a significant increase in MDA level, an indicator of lipid peroxidation. Upon 48 hours of exposure, the MDA levels were computed to be 1 ± 0, 2.25 ± 0.35, 3.75 ± 0.35, 8.25 ± 0.35, and 11.75 ± 1.05μM in 0, 10, 20, 30, and 40 μg/mL of lead nitrate respectively.

Fig 1.

Fig 1

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Effects of different concentrations of lead nitrate on MDA production in HepG2 cells. HepG2 cells were cultured with various concentrations of lead nitrate for 48 h and the lipid peroxidation level was determined as described in Materials and Methods. *Significantly different from the control by ANOVA; p < 0.05.

Modulation of p53 and cyclin A Proteins

The expression and abundance levels of p53 in human liver carcinoma (HepG2) cells exposed to lead nitrate are represented in (Fig 2A). The result of Western Blot and the densitometric analyses demonstrated a strong dose-relationship with regard to p53 expression in lead nitrate-treated HepG2 cells. From these data, we found that lead nitrate is able to cause oxidative stress along with cell injury, and cell cycle arrest through activation of the 53-kDa tumor suppressor protein.

Fig 2.

Fig 2

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Expression and relative abundance of p53 (Fig, 2A) and Cyclin A (Fig, 2B) in human liver carcinoma (HepG2) cells exposed to lead nitrate. HepG2 cells were treated with different doses of lead nitrate, and Western blot and densitometric analyses of p53 and cyclin A expression were performed as indicated in the Materials and Methods. Inset shows representative Western Blot analysis. Bars represent p53 and cyclin A abundance. Each point represents the mean value and the standard deviation of three experiments. * Significantly different from control (0μg/mL), p < 0.05.

The expression and abundance levels of cycin A in human liver carcinoma (HepG2) cells exposed to lead nitrate are represented in (Figure 2B). Western Blot and the densitometric analyses demonstrated a down-regulation of Cyclin A in lead nitrate-treated HepG2 cells, meaning lead nitrate plays a key role in cellular arrest at the G2 checkpoint of the cell cycle.

DISCUSSION

Metal-induced lipid peroxidation is mostly attributed to increased production of free radicals [7, 8]. Because heavy metals such as arsenic, cadmium, and lead are powerful catalysts of lipid peroxidation processes [7], the production of MDA was determined following exposure of HepG2 cells to lead nitrate. The treatment of HepG2 cells with lead nitrate resulted in an increase of MDA concentration levels, an indicator of lipid peroxidation. Upon 48 hours of exposure, the MDA concentration level was computed to be 11.75 ± 1.05 μM at highest dose tested. These data are consistent with those of previous literature reporting a similar increase in lipid peroxidation products (MDA) when plants were treated with copper [9–11]. A series of recent studies have also shown that rats exposed to lead had an elevation of blood pressure accompanied by a marked increase of lipid peroxidation product (MDA) in the plasma and tissue and a substantial reduction in urinary excretion of stable NO metabolites (NOx) [12–14].

Western Blot analysis showed a gradual increase of p53 expression in HepG2 cells, with increase of lead nitrate concentration. This result showed that activation of p53 in HepG2 cells may be indicative of a cellular response to DNA damage, along with oxidative damage response [15]. An increase expression of p53 was observed from low to high dose tested, thus indicating the translocation of p53 from the cytoplasm into the nucleus in response to DNA damage.

Cyclin A is one of the first cyclins to be identified and is believed to function between that of cyclin E and cyclin B. Our data of western blot analysis demonstrated a down-regulation of cyclin A in lead nitrate-HepG2 cells at all the tested concentrations (Fig 2). These data indicate that lead nitrate inhibits the activity of cyclin A at the G2 chekpoint of the cell cycle. The mechanisms likely to activate the oncogenic properties of the cyclins include chromosomal translocations, gene amplification and aberrant protein overexpression [16]. Abnormalities of several cyclins have been reported in different tumor types, implicating, in particular, cyclin A, cyclin E and cyclin D [16, 17]. The down regulation of Cyclin A in our study indicates a potential cell cycle arrest at the G2 checkpoint of the cell cycle as a result of lead exposure.

CONCLUSIONS

Data generated from the present study indicate that lead nitrate induces cell cycle in human liver carcinoma (HepG2) cells at least part through p53 up-regulation and cyclin A down-regulation. Study results of lipid peroxidation assay showed a significant increase of malondialdehyde (MDA), an end product of lipid peroxidation in HepG2 cells exposed to lead nitrate. Western Blot along with the densitometry analyses demonstrated a strong dose-response relationship with regard to p53 expression within the dose range of 0–40μg/mL. The up-regulation of p53 observed in lead nitrate-treated cells may be indicative of cell cycle arrest at the G1 checkpoint. Cyclin A expression was significantly suppressed (p < 0.05) in lead nitrate-treated HepG2 cells compared to the control cells, meaning that lead nitrate is blocking the activity of cyclin A at the G2 checkpoint of the cell cycle. Taken together, data obtained from these studies indicate that lead nitrate is able to cause oxidative stress along with cell injury, and cell cycle arrest through activation of the 53-kDa tumor suppressor protein, and down regulation of the cyclin A protein.

Acknowledgments

This research was financially supported by a grant from the National Institutes of Health (Grant No. 5G12RR013459), through the RCMI-Center for Environmental Health at Jackson State University.

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