Stimulation of the proliferation of human normal esophageal epithelial cells by fumonisin B1 and its mechanism

Previous epidemiological studies have demonstrated a correlation between fumonisin B1 (FB1) and human esophageal cancer in China, Iran and South Africa. The purpose of this study was to investigate the effects of FB1 on the proliferation, cell-cycle and apoptosis of normal human esophageal epithelial cells (HEECs) and to explore the molecular mechanisms of these effects. The proliferation of HEECs treated with FB1 was assessed using a colorimetric assay, while analyses of the cell cycle and apoptosis were performed using flow cytometry and the measurement of the protein expressions of genes associated with the cell cycle was conducted using western blotting. The results showed that FB1 stimulated the proliferation of HEECs, decreased the percentage of cells in the G0/G1 phase and reduced apoptosis. The western blotting results showed that FB1 significantly increased the protein expression of cyclin D1 and significantly decreased the protein expression of cyclin E, p21 and p27. The results indicated that FB1 stimulated the proliferation of HEECs by affecting the cell cycle and apoptosis. This mechanism was associated with changes in cyclin D1, cyclin E, p21 and p27 expression.


Introduction
Fumonisins are toxic metabolites produced predominantly by Fusarium verticillioides, one of the most common molds present globally on maize and other agricultural products (1)(2)(3). Areas of low and high esophageal cancer incidence in the former Transkei region of South Africa have been shown to correlate with low and high levels of fumonisin-contaminated home-grown maize, respectively (4). Alizadeh et al (5) detected high levels of fumonisin B 1 (FB 1 ) contamination in corn and rice samples from the Golestan province of Iran, with a significant positive correlation between the FB 1 contamination in rice and the risk of esophageal cancer (5). The high contamination rates of FB 1 , the most common fumonisin, which are noted in food from Huaian where incidences of esophageal cancer are amongst the highest in China, suggest a possible contributing role of FB 1 in human esophageal carcinogenesis (6). It has been proposed that FB 1 may exhibit carcinogenicity in humans and FB 1 is, at present, considered to be a possible carcinogen to humans. A number of studies have investigated the toxicity of FB 1 in human cell lines to provide more information concerning the effects of FB 1 in humans (7)(8)(9)(10). FB 1 was shown to inhibit clonal expansion in human keratinocytes and proliferation in human fibroblasts (7), in addition to causing DNA damage of an apoptotic type in human fibroblasts (8). Furthermore, FB 1 has been demonstrated to induce apoptosis in human proximal tubule-derived cells (IHKE cells) (9) and to cause oxidative stress in the human intestinal cell line Caco-2 (10).
Although the toxic effects of FB 1 on mammalian cells have been studied extensively, the carcinogenicity of FB 1 on normal human esophageal epithelial cells (HEECs), and the possible mechanism underlying the effects, have yet to be elucidated. In recent years, numerous studies have shown that FB 1 exerts significant effects on the cell cycle in certain cells (11,12). Cell cycle progression is controlled by cyclin-dependent kinases (CDKs) and cyclins (13,14). Cyclins D1 and E are necessary for entry into the S phase. CDK inhibitors, such as p16, p21 and p27, bind CDK-cyclin complexes and inhibit CDK activity (15,16).
In this study the effects of FB 1 on the proliferation, cell cycle and apoptosis of HEECs were investigated, in addition to the expression of molecular markers of the cell cycle genes cyclins D1 and E, p16, p21 and p27 in HEECs. Furthermore, the potential esophageal carcinogenicity of FB 1 in humans was examined.

Materials and methods
Materials. FB 1 , propidium iodide (PI), dimethylsulfoxide (DMSO), ethidium bromide (EB) and diethyl pyrocarbonate (DEPC) were purchased from Sigma (St. Louis, MO, USA), SHAO-KANG WANG 1,2 , TING-TING WANG 1,2 , GUI-LING HUANG 1,2 ,  RUO-FU SHI 1,2 , LI-GANG YANG 1,2 and GUI-JU SUN 1,2 while RPMI-1640 and trypsin were obtained from Gibco (Grand Island, NY, USA). A stock solution of FB 1 for cellular assays was prepared in phosphate-buffered saline (PBS) and then diluted in the optimal medium (≤10 µl/ml Harvesting cells. HEECs in the logarithmic growth phase were plated at a density of 1x10 5 cells/ml in 50-cm 2 culture flasks and allowed to grow in 4 ml culture medium. Following cell attachment, the culture medium was poured away and the cells were treated with FB 1 (5, 10, 20 and 40 µmol/l) for 72 h. The cells were then trypsinized and collected for cell cycle and apoptosis analyses, or washed twice with ice-cold PBS and removed from the surface of the flask using a rubber scraper for western blot analysis.

Stimulation of the proliferation of human normal esophageal epithelial cells by fumonisin B 1 and its mechanism
Cell cycle and apoptosis analyses. The cell cycle phase and apoptosis were examined using a Becton-Dickinson FACSCalibur Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The cells were stained with Vindelov's reagent (40 mM Tris, pH 7.6; 100 mM NaCl; 10 mg RNase A/ml; 7.5% PI and 0.1% Nonidet P-40), and data from 10,000 cells were collected for each data file. The experiment and assay were repeated three times. The supernatant was collected following centrifugation for 20 min at 10,000 x g, 4˚C. The protein concentration was assessed using a Pierce ® bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) and lysates (30 µl total protein) were separated using SDS-polyacrylamide gel electrophoresis (PAGE). Following electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes. The membranes were then blocked in Tris-buffered saline with 0.1% Tween-20 (TBST), containing 5% non-fat dry milk, for 1 h at room temperature and incubated with primary antibody at 4˚C overnight. The membranes were then incubated at room temperature with horseradish peroxidase (HRP)-conjugated mouse or rabbit immunoglobulin G (IgG). The antibodies used in the western blotting and their dilutions are shown in Table I. After washing with TBST, incubation with West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA) and detection using Kodak In-Vivo Imaging systems (Carestream Health Inc., Rochester, NY, USA) enabled the visualization of the proteins, which were then quantified by strip densitometry. Actin was used as an internal control.
Statistical analysis. The data are expressed as the mean ± standard deviation. Statistical analysis of the data was performed by one-way analysis of variance (ANOVA) with the SPSS statistical software package (version 13.0; SPSS, Inc., Chicago, IL, USA). Differences among the groups were evaluated using the parametric Least Significant Difference (LSD) test and differences between the experimental and the negative control groups were evaluated using a Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

Results
Effect of FB 1 on the proliferation of HEECs. As shown in Fig. 1, the proliferation of the HEECs was increased compared with that in the control group following treatment with FB 1 for

Effect of FB 1 on the cell cycle and apoptosis of HEECs.
In order to explore the mechanism by which FB 1 affected the proliferation of the HEECs, the percentages of HEECs in the different phases of the cell cycle and cell apoptosis were assessed by flow cytometry. As shown in Fig. 2, the cell cycle progression of the HEECs was blocked in the S phase by treatment with FB 1 in a dose-dependent manner, and was blocked in the G2/M phase by treatment with 40 µmol/l FB 1 . The percentages of HEECs in the G0/G1 phase were 67.1±4.7 and 49.8±5.9%, respectively, following treatment with 20 and 40 µmol/l FB 1 (Fig. 2B). Compared with the control, the percentage of HEECs undergoing apoptosis was significantly decreased by treatment with 40 µmol/l FB 1 (Fig. 2C).

Effect of FB 1 on the protein expression of genes involved in the cell-cycle in HEECs.
The protein expression levels of cyclins D1 and E, p21 and p27 in the HEECs were significantly changed by treatment with FB 1 for 72 h compared with the levels in the control (Fig. 3). However, the HEECs treated with FB 1 and the control cells were negative for the protein expression of p16 (data not shown). Compared with the control, the protein expression of cyclin D1 was significantly increased by treatment with 10, 20 and 40 µmol/l FB 1 , and the protein expression levels of cyclin E, p21 and p27 were significantly decreased by treatment with 5, 10, 20 and 40 µmol/l FB 1 (Fig. 3B). These results showed that FB 1 significantly increased the protein expression level of cyclin D1 and significantly decreased the protein expression levels of cyclin E, p21 and p27 in the HEECs.

Discussion
Mycotoxins pose a health hazard to animals and humans through commonly contaminated staple food grains. FB 1 is a cytotoxic and carcinogenic mycotoxin produced by Fusarium verticillioides, which causes porcine pulmonary edema and equine leukoencephalomalacia and has been implicated in the etiology of esophageal cancer in the Transkei, South Africa (17). Previous epidemiological surveys in China have revealed that FB 1 is associated with the occurrence of human esophageal cancer (6,18). The International Agency for Research on Cancer has classified FB 1 as a class 2B carcinogen, a probable human carcinogen (19). In order to investigate the effect of FB 1 on the human esophagus, HEECs derived from a normal human esophagus were tested in the present study.
The results demonstrated that cell proliferation was stimulated in HEECs treated with FB 1 . Similarly, lower concentrations of mycotoxins (aflatoxin B1, FB 1 , deoxynivalenol and nivalenol) have been shown to enhance cellular proliferation, with the effect being more pronounced in human than in porcine lymphocytes (20). In another study, the stimuli for cell proliferation were introduced 7 days subsequent to the start of the administration of FB 1 , by gavage with different daily doses ranging from 0.14 to 3.5 mg FB 1 /100 g body weight, while cancer initiation was effected over a period of 14 days of FB 1 treatment in the rat liver (21). The results of the present study demonstrated that the maximum proliferation of the HEECs was induced by treatment with 40 µmol/l FB 1 for 96 h (137.3±2.0%). Furthermore, the proliferation of the HEECs induced by treatment with 5, 10, 20 and 40 µmol/l FB 1 for 72 h was increased significantly to 105.0±3.3, 109.0±2.9, 115.4±2.2 and 123.4±3.4%, respectively. However, antiproliferative effects of FB 1 have been observed in human hepatoma cells (22) and in swine peripheral blood mononuclear cells (23). In addition, Fornelli et al (24) showed an inhibition of proliferation in SF-9 cells treated with FB 1 of ~20%. The varying effects of the FB 1 on cell proliferation are dependent on the dose of the mycotoxins and the types of cells.
Compared with the control, treatment with 40 µmol/l FB 1 for 72 h decreased the percentage of cells in the G0/G1 phase and increased the percentage of cells in the S and G2/M phases, in addition to significantly decreasing the percentage of HEECs undergoing apoptosis. In a previous study, flow cytometric and morphological analyses showed that FB 1 lowered the marked apoptosis induced by prostaglandins, particularly prostaglandin A2, in esophageal carcinoma (WHCO3) cells. However, in combination with arachidonic acid and prostaglandins E2 and A2, FB 1 increased the number of G2/M cells (25). In a different study, the results showed that the number of C6 glioma cells in the S phase decreased significantly compared with the control, from 18.7±2.5 to 8.1±1.1%

A B
for 9 µmol/l FB 1 , and the number of cells in the G2/M phase increased significantly compared with the control, from 45.7±0.4 to 54.8±1.1% for 9 µmol/l FB 1 . However, no change occurred in the number of cells in G0/G1 phase (26). It has been demonstrated that FB 1 interferes with the G1/S phase checkpoint, which leads to changes in the cell cycle in animal experiments (27). The percentage of cells blocked in the G0/G1 phase of the cell cycle has been shown to be increased by FB 1 in swine peripheral blood mononuclear cells (23). Thus, there are different effects on the cell cycle and apoptosis in different types of cell following treatment with FB 1 . The stimuli for cell proliferation in HEECs are a common result of the FB 1 -induced changes in the cell cycle and apoptosis. To further investigate the pathogenesis of the effects of FB 1 in the present study, the protein expression of genes involved in the cell cycle in HEECs were analyzed using western blotting.
In eukaryotes, the cell cycle is tightly regulated by a number of protein kinases composed of CDKs, with corresponding regulatory cyclins and CDK inhibitors (28). The activity of the CDK/cyclin complexes is regulated by proliferating cell nuclear antigen, which binds to cyclin D1; cyclin E promotes the progression through G1 phase into S phase. The activity of the CDK/cyclin complexes is negatively regulated by binding to CDK inhibitors. CDK inhibitors are grouped into two distinct families (29): the INK4 family, including p15INK4b, p16INK4a, p18INK4c and p19INK4d (30), and the CIP/KIP family, including p21WAF1/CIP1, p27KIP1 and p57KIP2 (31). In the present study, the increased expression of cyclin D1 and decreased expression of CDK inhibitors, including p21 and p27, strongly suggest that FB1 induced the low expression of the members of KIP/CIP family, which sequentially stimulated the activities of the CDK/cyclin complexes.
In conclusion, the present in vitro study demonstrated that FB 1 stimulated cell proliferation in HEECs, most likely by decreasing the percentage of cells in the G0/G1 phase of the cell cycle, increasing the percentage of cells in S phase, increasing the percentage of cells in G2/M phase and arresting cell apoptosis. The changes in the cell cycle may have been mediated by stimulation of cyclin D1 and inhibition of p21 and p27 expression, thereby accelerating the passing of cells through the G1-S checkpoint (32,33). The inhibition of cyclin E was offset by the role of these genes. It has been shown that the expression of cyclin D1, p21 and p27 is involved in the occurrence of esophageal cancer (34), and p21 and p27 have been proposed as candidate tumor suppressor genes (35). In a study by Huang et al (36), cyclin D1 was shown to be overexpressed in esophageal cancer in southern China (36). Furthermore, it has been demonstrated that the overexpression of cyclin D1, rather than cyclin E, is involved in the pathogenesis of esophageal cancer (37). However, additional studies, particularly in vivo experiments, are required to further demonstrate the effect of FB 1 in normal human esophageal epithelium and to elucidate the correlation between FB 1 and human esophageal cancer.