Regulation of oncogenes and gap junction intercellular communication during the proliferative response of zearalenone in TM3 cells

Abstract

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced by Fusarium species. The exposure risk to humans and animals is the consumption of contaminated food and animal feeds. The aim of this study was to investigate ZEA-induced effects and its tumorigenic mechanism in TM3 cells (mouse Leydig cells). Cell proliferation, apoptosis, and gap junction intercellular communication (GJIC) were assessed in this study. Results showed that low concentrations of ZEA could significantly promote the growth of TM3 cells. The percentage of cell distribution was decreased significantly in G1/G0 phase and was increased significantly in S phase with 10 and 20 μg/L of ZEA for 72 h (p < 0.05, p < 0.01). The expressions of cyclin D1 and Cdk4 were significantly increased in the exposure groups compared with the control group (p < 0.05, p < 0.01). Compared with the control group, the apoptosis was significantly decreased in 10 and 20 μg/L groups (p < 0.01), and the ratio of Bax/Bcl-2 protein level was significantly decreased in a dose-dependent manner. The protein levels of proto-oncogene c-Myc, c-Jun, and c-Fos were significantly elevated and the protein levels of anti-oncogene p53 and phosphatase and tensin homolog (PTEN) were decreased obviously compared with the control group (p < 0.05, p < 0.01). ZEA affected the expressions of connexins and inhibited the activity of GJIC. These results demonstrated that ZEA can disturb the dynamic balance between proliferation and apoptosis and causes abnormal regulation of oncogenes, GJIC, and connexins in TM3 cells, which may easily induce the translation of normal cells into tumor cells.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin, previously known as F-2 toxin, mainly produced by fungi belonging to Fusarium species, which was detected in maize, corn, and milk worldwide. ¹ ZEA is very stable and hard to be degraded during general process of food handling, so it can be found in several ordinary foods like bread and beer. ^2,3 The carbonyl group of ZEA can be reduced by hydroxysteroid dehydrogenase and form two isomeric metabolites α-zearalenol and β-zearalenol. ZEA is genotoxic, immunotoxic, and tumorigenic, and it affects reproductive and developmental processes. It decreases fertility, reduces litter size, alters the weight of adrenal, thyroid, and pituitary glands in offspring, and changes the progesterone and estradiol levels. ZEA can bind estrogen receptors (ERs) and then causes structural and functional changes in reproductive organs. ⁴ ZEA caused testicular germ cell depletion, altered testis morphological parameters, reduced serum testosterone concentrations, and disturbed fertility. ⁵

Several research studies have suggested that ZEA and its metabolites play an important role in increasing the risk of hormone-dependent tumors. ⁶ Serum collected from heifers following 1 month of α-zearalanol implantation stimulated MCF-7 breast cancer cell growth. ⁷ Moreover, in vitro studies have showed that ZEA could obviously enhance the proliferation ability of human breast tumor cells, through estrogen-mediated pathways and by activation of gene profiles similar to those activated by natural estradiol. ⁸ In addition, exposure of female rats to environmentally relevant doses of ZEA resulted in long-term changes in mammary gland development, which may associate with increased risk of mammary tumors. ^9,10

It is well known that Leydig cells play a crucial role in regulating the process of spermatogenesis and synthesizing testosterone. Leydig cells secrete testosterone, which is essential in the maintenance of spermatogenesis and male fertility. Alteration of Leydig cell function can lead to adverse effects on testicular functions. ¹¹ Studies in various animals (e.g. rodents, pigs, and monkeys) have shown that ZEA and its metabolites exhibit estrogenic and anabolic activities. Leydig cells contain not only androgen receptor but also ERα and ERβ. ¹² There are two subtypes of ER including ERα and ERβ, which are members of nuclear receptor superfamily. Studies in various animals (e.g. rodents, pigs, and monkeys) have shown that ZEA and its metabolites exhibit estrogenic and anabolic activities. ¹³ The strong estrogenic effects exhibited by ZEA are due to its competition with 17-β-estradiol for cytosolic ERs present in the uterus, mammary gland, hypothalamus, and pituitary gland. ¹⁴ Yang and colleagues proved that ZEA reduces testosterone secretion both in vitro and in vivo. ¹⁵ ZEA activates the nuclear ER-signaling pathway to limit Nur77 expression, indirectly disturbs the transcription of steroidogenic enzymes. ¹⁶

However, the precise mechanisms of ZEN toxicity in Leydig cells remain incompletely understood and require further investigation. The aim of this study was to investigate ZEA-induced effects and its tumorigenic mechanism in TM3 cells. Cell proliferation, apoptosis, and gap junction intercellular communication (GJIC) were assessed in this study.

Materials and methods

Materials

ZEA was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Dulbecco’s modified eagle medium/nutrient mixture F-12 (DMEM/F-12) medium, fetal bovine serum (FBS), and horse serum (HRS) were obtained from GIBCO BRL (Grand Island, New York, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was provided by Amresco (Solon, Ohio, USA). p53, phosphatase and tensin homolog deleted on chromosome ten (PTEN), c-Myc, c-Fos, c-Jun, Bax, Bcl-2, Cx43, Cx32, and β-actin mAb were purchased from Cell Signaling Technology (Boston, Massachusetts, USA). Enhanced chemiluminescence (ECL) solution was obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Goat anti-rabbit immunoglobulin G-conjugated fluorescein isothiocyanate (IgG-FITC) was obtained from Bioworld Technology (St. Louis, Minnesota, USA).

Cell culture and exposure to ZEA

Experiments were carried out with TM3 cells (mouse Leydig cells) provided by the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). TM3 cells were cultured in DMEM/F-12 medium supplemented with 2.5% FBS, 5% HRS, 150 mg/L of l-glutamine, 1.5 g/L of sodium bicarbonate, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin at 37°C with 5% carbon dioxide in humidified air. Cells were then divided into four groups and treated with ethanol as a control, with various concentrations (5, 10, and 20 μg/L) of ZEA for 72 h.

Cellular proliferation assay

The MTT assay was used to assess TM3 cell viability. The cells were plated in 96-well plates at a density of 5 × 10³ cells/well and treated with different concentrations of ZEA (0, 5, 10, 20, and 50 μg/L) for 72 h. After 20 µL of MTT solution (5 mg/mL) was added to each well containing cells, the 96-well plate was incubated at 37°C for 4 h. Formazan crystals were dissolved by dimethyl sulfoxide, and absorbance was measured by a microplate reader at 490 nm. At last, the results were recorded and analyzed.

Cell cycle determination

The cell cycle was detected by flow cytometry. After treated with different concentrations of ZEA (0, 5, 10, and 20 μg/L), the cells were fixed with 70% ethanol at 4°C for 24 h and washed twice with PBS, dyed with straining solution (20 μg/mL propidium iodide (PI), 50 μg/mL RNaseA, PBS) at room temperature for 30 min. Samples were filtrated using 200 mesh nylon filter, measured by a flow cytometry (FACS Calibur; Becton–Dickinson, Franklin Lakes, New Jersey, USA).

Apoptosis determination

After treated with ZEA, the cells were collected and washed with PBS at 4°C, then dyed with straining solution (5 μL Annexin V-FITC, 5 μL PI, 100 μL binding buffer) for 15 min without light at room temperature. At last, samples were filtrated using 200 mesh nylon filter and determined by flow cytometry (FACS Calibur; Becton–Dickinson, Franklin Lakes, New Jersey, USA).

Western blot analysis

Western blot analysis was used to determine the levels of Bcl-2 family members, proto-oncogenes c-Myc, c-Fos, c-Jun, anti-oncogenes p53 and PTEN and connexins Cx32 and Cx43. After the treatment with ZEA (0, 5, 10, and 20 μg/L) for 72 h, the cells were collected, and then washed with PBS. The total cellular proteins were extracted by radioimmunoprecipitation lysis buffer and ultrasonic cracking apparatus. After 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis electrophoresis, proteins were transferred to nitrocellulose membranes, blocked with 5% nonfat milk in Tris-buffered saline at room temperature for 2 h. The membranes were incubated with specific antibodies overnight at 4°C and incubated with secondary antibodies at room temperature for 2 h. At last, the result was detected by ECL reagents and analyzed by Image J, version 1.47.

Immunofluorescence assay

TM3 cells were plated in 6-well plates and treated with 0, 5, 10, and 20 μg/L of ZEA for 72 h. The cells were washed twice with PBS, fixed in 4% paraformaldehyde solution for 30 min at 4°C, then dealt with 0.5% Triton X-100 and blocked with 5% bovine serum albumin (BSA) for 15 min at room temperature. The cells were incubated with Cx43 antibody (1:200) in 5% BSA solution for 12 h at 4°C and then incubated with goat anti-rabbit IgG FITC (1:200) at room temperature for 1 h. After washed with PBS, TM3 cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. At last, changes were observed under a fluorescence microscope (Leica 2500; Leica Corporation, Germany).

GJIC function detection

Gap junction activity was analyzed using the scrape-loading/dye-transfer method (SL/DT) as reported previously. ¹⁷ Briefly, following treatment the cells were cut with a surgical scalpel in the presence of Lucifer yellow (LY; 0.5 mg/mL) and rhodamine dextran (RD; 2.5 mg/mL), followed by incubation for 3 min at room temperature, then fixed with 4% paraformaldehyde. The level of gap junction activity was quantified as the average distance traveled (μm) by the LY dye from the designated cut to the neighboring cells from six different sites in each sample, measured using a fluorescent microscope (Leica DMI 3000B, Solms, Germany).

Statistical analysis

Data were analyzed using SPSS for Windows 13.0 Software (SPSS Inc., Chicago, Illinois, USA) and presented as means with standard deviation (mean ± SD). Differences between the control and the treatment groups were determined using a one-way analysis of variance followed by the least significant difference multiple range test. A p < 0.05 was considered statistically significant.

Results

ZEA-induced proliferation in TM3 cells

The effect of ZEA (0, 5, 10, 20, and 50 μg/L) on the viability of TM3 cells was assessed by the MTT assay. As shown in Figure 1(a), low-dose concentration of ZEA promoted the growth of TM3 cells. The cell viability for the treatment group 20 μg/L was obviously higher than that in the control group (p < 0.05). In the present research, cell viability with 50 μg/L of ZEA was lower than that with 20 μg/L of ZEA (p < 0.05). Therefore, 0, 5, 10, and 20 μg/L of ZEA were selected as the experimental concentrations.

Figure 1.

The effects of ZEA on the proliferation in TM3 cells. (A) TM3 cells were treated with ZEA (0, 5, 10, 20, and 50 μg/L) and after 72 h viability was detected by the MTT assay. (b and c) TM3 cells were treated with ZEA for 72 h. Cell cycle distribution was measured by flow cytometry. Results were expressed as mean ± SD of three independent experiments. *p < 0.05; **p < 0.01 versus the control group. (d and e) The expressions of cyclin D1 and Cdk4 were measured by Western blot assay after treated with ZEA. Values are presented as mean ± SD. Asterisks indicate a statistically significant difference: *p < 0.05; **p < 0.01. ZEA: zearalenone.

Effects of ZEA on the cell cycle

The results of flow cytometry showed that the percentage of cell distribution was decreased significantly in G1/G0 phase and was increased significantly in S phase with 10 and 20 μg/L of ZEA for 72 h (p < 0.05 and p < 0.01, respectively; Figure 1(b) and (c)). As shown in Figure 1(d) and (e), the expressions of cyclin D1 and Cdk4 were significantly increased in the exposure groups compared with the control group (p < 0.05 and p < 0.01, respectively).

ZEA produced effect on apoptosis in TM3 cells

Annexin V-FITC/PI double staining method was used to detect apoptosis in TM3 cells. Scatter diagram can be divided into four quadrants (Q1: mechanical damage to cells, Q2: late apoptotic cells, Q3: normal cells, Q4: early apoptotic cells), and cell apoptosis rate was Q2 value plus Q4 value. After TM3 cells were exposed to ZEA for 72 h, the result showed that the apoptosis rate of TM3 cells increased obviously with 0–10 μg/L of ZEA (Figure 2(a) and (b)). Comparing with the control group, the apoptosis was significantly decreased with 10 and 20 μg/L of ZEA (p < 0.01).

Figure 2.

ZEA can delay apoptosis in TM3 cells. (a and b) Apoptosis was confirmed by flow cytometry using Annexin V/PI dual staining when TM3 cells were treated with different concentrations of ZEA. (c and d) The expressions of Bax and Bcl-2 were measured by Western blot assay after treated with ZEA. Values are presented as mean ± SD. Asterisks indicate a statistically significant difference: *p < 0.05; **p < 0.01. ZEA: zearalenone; PI: propidium iodide.

ZEA regulated Bcl-2 family members

The results of Western blot showed that the expression of BAX was decreased and the level of Bcl-2 was increased in TM3 cells after treated with ZEA for 72 h. The ratio of Bax/Bcl-2 protein level was significantly decreased in a dose-dependent manner (Figure 2(c) and (d)).

ZEA upregulated c-Myc, c-Fos and c-Jun and downregulated p53 and PTEN

We investigated some key proto-oncogenes and anti-oncogenes to confirm whether the observed changes were associated with the proliferation in TM3 cells. The results from Western blot indicated that the protein levels of proto-oncogene c-Myc, c-Jun, and c-Fos were significantly elevated compared with the control group (p < 0.05, p < 0.01; Figure 3(a) and (b)). Moreover, the protein levels of anti-oncogene p53 and PTEN were decreased obviously (p < 0.01; Figure 3(c) and (d)).

Figure 3.

The expressions of proto-oncogenes and anti-oncogenes were detected in TM3 cells. (a and b) Western blot analysis of C-fos, c-jun, and c-myc after ZEA treatment. (c and d) Western blot analysis of P53 and PTEN after ZEA treatment. Values are presented as mean ± SD. Asterisks indicate a statistically significant difference: *p < 0.05; **p < 0.01. ZEA: zearalenone.

ZEA affected the expressions of connexins and inhibited the activity of GJIC

SL/DT assay was performed to assess GJIC activity in TM3 cells treated with 0, 5, 10, and 20 μg/L of ZEA for 72 h. The results showed that the diffused distance of LY between cells was significantly decreased with 10 and 20 μg/L of ZEA compared to the control group (p < 0.01; Figure 4(a) and (b)). As is shown in Figure 4(d) and (e), the protein levels of Cx32 and Cx43 significantly decreased with 10 and 20 μg/L of ZEA (p < 0.05, p < 0.01). Through the immunofluorescence we found that the distribution of Cx43 was mostly in the intracellular region rather than cell membrane after being exposed to ZEA (Figure 4(c)).

Figure 4.

ZEA induces GJIC inhibition in TM3 cells. (a) ZEA-induced downregulation of GJIC in TM3 cells. LY (green) transferred to adjacent cells via open gap junctions. (b) The average distance of LY spread from the side of the scraped edge from six different sites in each sample was obtained for quantification. (c) The distribution of Cx43 was observed by immunofluorescence in TM3 cells (red arrows represented Cx43). (d and e) The protein expressions of Cx32 and Cx43 were analyzed by Western blot after the treatment with 0, 5, 10, and 20 μg/L of ZEA for 72 h. Values are presented as mean ± SD. Asterisks indicate a statistically significant difference: *p < 0.05; **p < 0.01. ZEA: zearalenone; GJIC: gap junction intercellular communication; LY: Lucifer yellow.

Discussion

Previous studies have proved that ZEA is toxic for human and animal health. Several research studies showed that high concentrations of ZEA can inhibit the proliferation of Leydig cells and cause autophagy. ¹⁷ The other research has showed low concentrations of ZEA mediate the proliferation of SK-N-SH cells and promote the development of hepatocellular adenomas and pituitary adenomas. ¹⁸ The detailed molecular and cellular mechanisms of ZEA toxicity have not yet been completely explained. In the current study, we evaluated the effects of ZEA on TM3cells. The results demonstrated that the ZEA can promote the proliferation of TM3 cells, which means high and low doses of ZEA have different influences on Leydig cells. The mechanisms of ZEA inducing cellular proliferation may contribute to interacting with the ERα and ERβ of Leydig cells. Several studies in various animals (e.g. rodents, pigs, and monkeys) have shown that ZEA and its metabolites exhibit estrogenic activities. ¹³ The strong estrogenic effects exhibited by ZEA are due to its competition with 17-β-estradiol for cytosolic ERs. ¹⁴

Regulation of cell cycle is critical for normal development of multicellular organisms, and loss of control ultimately leads to cancer. ¹⁹ Defects in this balance are thought to contribute to the development of cancer and other pathological conditions. ²⁰ In this study, analysis of cell cycle distribution by flow cytometry indicated that after treatment with ZEA, the percentage of cell distribution was decreased significantly in G1/G0 phase and was increased significantly in S phase and the expressions of cyclin D1 and Cdk4 were significantly increased. G0 phase is a resting phase where the cell has left the cycle and has stopped dividing. S phase is a duplicating DNA phase. Cyclin protein and cyclin-dependent kinases (CDK) are two major components in cell cycle regulation, which can combine into the activated cyclin–CDK kinase complex to promote cell cycle transport. They interact and form maturation promoting factor, which in turn promotes one cell dividing into two cells. ²¹ These results suggested that ZEA can stimulate the TM3 cells division.

Apoptosis or programmed cell death is a highly regulated process of cell deletion; it promotes the turnover of normal cells and the clearance of infected or transformed cells. Apoptosis occurs in a well-choreographed sequence of morphological events via extrinsic and intrinsic pathways. ²² Both pathways have a final common pathway that involves the activation of a cascade of proteases and culminates in the death of cells. ²³ In this study, after exposed to ZEA, the apoptosis rate of the apoptosis was significantly decreased. The ratio of Bax/Bcl-2 protein level was significantly decreased in a dose-dependent manner. These results indicated that ZEA can promote the cells proliferation via downregulating apoptosis.

C-fos and c-jun are two important members of the proto-oncogene family. Activation of these genes would result in the production of related proteins that could form the dimeric transcription factor AP-1. AP-1 has been speculated to be involved in the induction of tumorigenesis. C-myc gene is the best-studied member of Myc oncogene family, which can encode a transcriptional regulator involved in carcinogenesis. ²⁴ PTEN is a tumor suppressor that has been found to be mutated in many types of cancers. P53 is known to interact and regulate PTEN at the transcription as well as protein level. P53 has an effect on the expression of splicing variants of PTEN and thus may serve as an added regulatory mechanism. ²⁵ In our study, after treatment with ZEA, the protein expression levels of proto-oncogene c-Myc, c-Jun, and c-Fos were significantly elevated and the expressions of anti-oncogene p53 and PTEN were decreased obviously. These results indicated that ZEA has potential tumor-promoting activity.

Gap junctions provide a way for the exchange of material and information in adjacent cells, which have been found in testis tissue. GJIC is considered as a key mechanism in the regulation of physiological homeostasis and an important factor of tumorigenesis. Cx43 proteins are transported from trans-Golgi network along microtubules to cytomembrane. ²⁶ In this study, after the treatment of ZEA, the Cx43 protein was almost distributed in the intracellular region rather than cytomembrane. The molecular mechanism of anomalous GJIC has been elucidated, including connexins abnormal localization and downregulation of connexins expression. These results suggested that ZEA can suppress the function of GJIC.

In summary, this study showed that ZEA can disturb the dynamic balance between proliferation and apoptosis and causes abnormal regulation of oncogenes, GJIC, and connexins in TM3 cells. These effects may be associated with the male-specific reproductive toxicity of ZEA. Further studies are warranted to elucidate the specific molecular mechanism of these effects.