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Carcinogenesis Advance Access originally published online on July 5, 2007
Carcinogenesis 2007 28(11):2291-2297; doi:10.1093/carcin/bgm149
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Folate receptor and human reduced folate carrier expression in HepG2 cell line exposed to fumonisin B1 and folate deficiency

Afif M. Abdel Nour1, Diana Ringot1, Jean-Louis Guéant2 and Abalo Chango1,2,*

1 Laboratory of Nutritional Genomics, Institut Polytechnique LaSalle Beauvais—Agrohealth EGEAL, 19 rue Pierre Waguet, F-60026 Beauvais cedex, France
2 INSERM U724, Cellular and Molecular Pathology in Nutrition, Vandoeuvre-les-Nancy, 54505 France

* To whom correspondence should be addressed. Tel: + 33 3 4406 38 67 Fax: +33 3 44 06 25 26; Email: abalo.chango{at}lasalle-beauvais.fr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Fumonisin B1 (FB1) induces apoptosis and decreases the cellular uptake of 5-methyltetrahydrofolate. Two folate transporters (folate receptor, FR, and Reduced Folate Carrier, hRFC1) are involved in the cell uptake of folate. We aimed to study whether FB1 modifies the expression of the FR and the hRFC1 and whether its apoptotic effect is influenced by folate. Incubation of HepG2 cells with FB1 induced apoptosis in concentration and time-dependent manner in complete medium (experimental control medium, ECM), as well as in folate-depleted medium (FDM). FDM increased the toxicity of FB1 as the cells developed apoptosis within 24 h at 1 µM of FB1 instead of 100 µM in ECM. Whereas FR protein expression in cells grown in ECM was significantly inhibited after apoptosis event, protein expression of the hRFC1 was rather increased. The hrfc1 transcription was decreased in the treated cells. Under folate-deficient conditions, dramatic changes were observed on both transcriptional and post-transcriptional expression of the two transporters. FDM alone reduced FR protein expression by 12 ± 2% and 43 ± 1% at 48 and 72 h, respectively. The 5-methytetrahydrofolate attenuates apoptosis in a greater extent than the folic acid. However, its effects in preventing decrease of both folate transporters have not been observed. In conclusion, this study shows that the changes in the expression of FR after FB1 addition are probably a consequence of the FB1 toxicity. The response to FB1 by HepG2 cell lines is influenced by folate status and by folate form. 5-Methyltetrahydrofolate appears to be more effective in preventing apoptosis than folic acid.

Abbreviations: CB, cell buffer; ECM, experimental control medium; FB1, Fumonisin B1; FDM, folate-depleted medium; FR, folate receptor; GPI, glycosylphosphatidylinositol; MEM, Minimum Essential Medium Eagle's; MSM, MTHF-supplemented medium


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
In those relatively rare occurrences of chronic human toxicity due to food contaminants, the underlying mechanisms usually remain unresolved. Among the major contaminants of concern for the food industry are fumonisins, which are a family of several structurally related toxins that were initially isolated in 1988 (1). Fumonisins are mycotoxins produced by Fusarium species, mainly Fusarium verticillioides. Fumonisin B1 (FB1) is the most significant of the fumonisins in terms of toxicity and occurrence. FB1 has been associated with several animal diseases and other adverse consequences of exposure. FB1 causes equine leukoencephalomalacia, porcine pulmonary edema, body weight decrease in poultry, rabbits' brain haemorrhages, atherosclerosis in monkeys, atherogenic effects in the vervet monkey, nephrotoxicity in rats and hepatocellular carcinomas (1,2). Additionally, FB1 have been implicated in the development of human esophageal cancers in Southern Africa (3) and China (4). The International Agency for Research on Cancer has classified FB1 as a class 2B carcinogen, a class of molecules that are possibly carcinogenic to humans (5).

Due to its structural similarity with sphinganine, the mechanism of action of FB1 is believed to primarily occur via disruption of sphingolipid metabolism (6). According to Riley et al. (7), this mechanism of action may initiate the following cascade of events: (i) inhibition of ceramide biosynthesis by blocking the enzyme ceramide synthase, (ii) increases of free sphinganine and sphingosine and (iii) increase in sphingoid base degradation of dietary sphingolipids. Ceramide synthase is a key enzyme responsible for the acylation of sphinganine in the de novo synthesis of sphingolipids and the re-acylation of sphingosine (8). The inhibition of sphingolipid biosynthesis reduces the formation of sphingomyelin, which is a major component of the plasma membrane and is required for the proper function of glycosylphosphatidylinositol (GPI)-anchored proteins, such as the folate receptor (FR) or the folate-binding protein (9).

In mammalian cells, folate is transported by two major mechanisms. One involves the FR, with high affinity for folic acid and reduced folates (10). The other uses a transmembrane transporter protein called the reduced folate carrier (hRFC1, SLC19A1), which is an anion exchanger that has the properties of a classical facilitative carrier with high affinity for reduced folates (11). Folate plays an essential role in carrying the methyl groups within cells. These act as either donors or receivers of one-carbon moieties in a variety of reactions involved in the syntheses of purines, deoxynucleosides, remethylation of homocysteine to methionine, and in the methylation of cytosine in DNA (12). Interest in one-carbon metabolism has been growing this last decade, in part because of reports that link inadequate folate status or intake to increased risk of diseases such as cardiovascular diseases, neural tube defects, neurological and neuropsychiatric disorders, preeclampsia and early pregnancy loss (13–16). Folate deficiency is also associated with an increased risk of certain types of cancer (17,18). In addition, FB1 inhibits the FR and consequently blocks folate internalization in Caco-2, a human colon adenocarcinoma cell line (19). The effect of FB1 on folate transport in hepatocarcinoma has not been yet investigated. A carcinogenetic effect of FB1 has been recently reported in the liver of female B6C3F1 mice fed for 2 years with FB1 (20). One of the pathomechanisms that underlies this effect could be related with an influence of FB1 on the expression of folate transporters in hepatocytes.

To address this hypothesis, we have therefore conducted an in vitro study in the HepG2 human hepatocarcinoma cell line and we studied whether (i) FB1 modifies the expression of the FR and the hRFC1 at the transcriptional and translational levels and (ii) the apoptotic effect of FB1 is influenced by folate.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Reagents and chemicals
FB1 and the 5-methyl-5,6,7,8-tetrahydropteroyl-L-glutamic acid disodium salt (5-MTHF) (Cat#: M0132) were from Sigma–Aldrich (Lyon, France). Minimum Essential Medium Eagle's (MEM) either lacking or containing folic acid (2.27 µM) and phosphate-buffered saline Ca2+ and Mg2+ free (PBS), L-glutamine, streptomycin, penicillin, fetal bovine serum and Calcein-AM was purchased from Invitrogen (Cergy Pontoise, France). To minimize exogenous folate sources, fetal bovine serum was replaced with dialyzed fetal bovine serum (Invitrogen). Annexin V–biotin apoptosis detection kit was obtained from VWR (Fontenay sous Bois, France). Fluorolink Cy5-streptavidin was purchased from Amersham Biosciences (Orsay, France). Cell Fluorescence LabChip Kit and 2100 bioanalyzer are from Agilent Technologies (Waldbronn, Germany). Primary antibody anti-FR (ref. AB2107) and the secondary antibody (ref. AB6566-100) were purchased from AbCam (Paris, France). Antibody anti-human RFC1 (ref. RFC11-A) and the secondary antibody (ref. RFC IgG #1) were from Biovalley (Conches, France). HepG2 cell lines (human hepatocellular carcinoma cells) were ordered from ECACC (No. 85011430).

Cell line culture and exposure conditions
HepG2 cells were routinely grown in MEM, supplemented with 20% heat-inactivated fetal bovine serum (56°C; 30 min), 1% L-glutamine and 1% antibiotics (penicillin/streptomycin) at 37°C in a 5% CO2-humidified incubator. For the experiment, confluent HepG2 cells at 60–80% were first trypsinized, counted and then re-plated into 25-cm2 culture flasks (4 x 105 cells/flask) in the complete medium MEM for 24 h. Cells were counted in using a Beckman Coulter Z1 (Beckman Coulter, Villepinte, France) by diluting 0.5 ml of cell suspension in 10 ml of Z-PakTM Isotron II dilient (Beckman Coulter). After 24 h, the medium was removed, adherent cells were washed gently by PBS and then a new experimental medium, complete MEM hereafter designed as experimental control medium (ECM) or MEM medium without folic acid hereafter designed as folate-depleted medium (FDM), was added, such that the cells were maintained under these conditions for the incubation period. For the treatment with 5-MTHF, the medium was an FDM supplemented with 2.27 µM of 5-MTHF hereafter designed as MTHF-supplemented medium (MSM). The incubation times were 24, 48, or 72 h. FB1 was diluted in PBS and added to the flasks to obtain final FB1 concentrations of 0.5, 1, 10 or 100 µM. Total PBS volume containing FB1 or not corresponded to 1% of volume in the flask. For all experiments, control flasks were systematically included. Controls were cells cultivated in ECM with PBS at 1% (v/v) and incubated for the same periods. All media were supplemented with 3% dialyzed fetal bovine serum and 1% L-glutamine.

Cellular parameters measurement
We used the 2100 bioanalyzer (Agilent Technologies) for measuring cellular fluorescent-labeled cells based on simple flow cytometric analysis. Typical cell applications for this instrument are apoptosis detection, as well as monitoring protein expression by antibody staining.

Apoptosis assay by annexin V
Treated cells were harvested by trypsinization and re-suspended in MEM medium to a cell density of 1.106 cells/ml. Hundred microliters of the cell suspension were mixed with 2 µl of 200 µg/ml annexin V–biotin (Annexin V Biotin Apoptosis Detection Kit) in a microcentrifuge tube and incubated for 10 min at room temperature. The medium was centrifuged (500 g, 2 min) and the supernatant was aspirated. The cells were re-suspended by gentle vortexing in 100 µl of 1x Binding Buffer (Annexin V Biotin Apoptosis Detection Kit) containing 1 µg/ml Fluorolink Cy5-streptavidin and 1 µM Calcein-AM. Calcein was used as an indicator for cells with an intact cell membrane. After incubation for 10 min at room temperature, centrifugation and medium aspiration, cells were re-suspended in 50 µl of cell buffer (CB) by gentle pipetting. Ten microliters of cell suspension (20 000 cells) were added directly to the wells of a cell chip without further treatment or washes. The prepared chip was then loaded on the Agilent 2100 bioanalyzer as described by the manufacturer for analysis. The percentage of apoptosis was calculated by the bioanalyzer system. Results shown are the difference in percentage of apoptosis between treated and control cells.

FR and hRFC1 protein expression assay
Treated cells were harvested and washed with PBS and cell density was adjusted to 3.106 cells/ml in the CB. For FR detection, 10 µl of cell suspension was incubated with 2 µl Calcein-AM (1:50 in CB), 1 µl of primary anti-FR antibody (prediluted 1:500 in CB) and 1 µl of secondary Cy5 marked antibody (optimization of antibody concentration not shown). For hRFC1 detection, 10 µl of cell suspension was incubated with 2 µl Calcein-AM (1:50 in CB), 1 µl of primary anti hRFC antibody (prediluted 1:500 in CB) and 1 µl of secondary Cy5 marked antibody (prediluted 1:500 in CB). Stained cell samples were re-suspended in CB at 2.106 cells/ml and loaded onto the chips as described in the reagent kit guide. Data acquisition was performed using the intuitive software package supplied with the Cell Assay Extension with no requirement to manually set instrument-specific parameters. The FR or hRFC1 protein expressions were calculated as the difference in percentage between treated cells and controls.

RNA isolation and absolute quantification of FR and hRFC1 mRNA expression
Medium was first removed from flasks containing treated and untreated cells, then cells were lysed with Trizol® reagent (Life Technologies, Carlsbad, California) and total RNA was extracted following the manufacturer's instruction. Total RNA samples were stored at –80°C until analysis. One microgram of total RNA was converted to cDNA using Quantitect reverse transcription kit (Qiagen, Courtaboeuf, France). The FR transcript (fr) and the hRFC1 transcript (hrfc1) levels in samples were quantified by quantitative polymerase chain reaction (PCR) using the Applied Biosystems 7300 sequence detection system. For absolute quantification, we used a method developed in our laboratory. Primers and fluorogenic probes (supplementary table 1 is available at Carcinogenesis Online) were designed with Primers Express Software version 2 (Applied Biosystems, Les Ulysses, France), and obtained from the same company. In order to avoid amplification of the target gene in genomic DNA, the probes spanned the junction between two exons, covered by the forward and reverse primers. Quantitative PCR was performed with 100 ng of cDNA, 12.5 µl of 2x Taqman PCR master mix (Applied Biosystems), 300 nmol of each primer and a 200 nmol probe in a final volume of 25 µl. Thermal cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C followed by 45 repeats of 15 s at 95°C and 1 min at 60°C. A negative control containing distilled water was always included (see supplementary table 1 is available at Carcinogenesis Online for details of primers and probes). For each sample, measurements of hrfc1 and fr gene expression were performed in triplicates, and the means of these values were represented. Standard curves for quantification of each transcript were realized using Bacterial Artificial Chromosome (BAC) dilutions ranging from 102 to 107 copies and allowed the determination of copy number for each transcript. In addition, we considered ß-actin as a reference gene and we measured its expression level with quantitative PCR in the samples.

Statistical analysis
Data for all response variables were reported as mean of the triplicate assays conducted in the same experiment ± standard error to the mean. Results were adjusted to the value of the control ECM without FB1, which is the baseline at each time point. Means were compared with the initial values of controls. A one-way analysis of variance was used to test for significant differences. When significant differences were obtained (P < 0.05), a comparison of means was conducted (Student's t-test, P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
FB1-induced apoptosis in HepG2 grown in the ECM
Figure 1 shows that, compared with control condition without FB1, there was no significant apoptotic effect at 24, 48, or 72 h of 0.5 µM of FB1 exposure. At an FB1 concentration of 1 µM, apoptosis was seen to increase by 30 ± 1% at 72 h of exposure, a degree that was significantly higher than the apoptotic degree observed in the controls. When the concentration of FB1 was increased to 10 µM, apoptosis was observed in 14 ± 0% and 32 ± 1% of the cells at 48 and 72 h of exposure, respectively. The apoptotic degree was significantly higher in cells treated with 100 µM FB1 at 24, 48, and 72 h, with respective values of 25 ± 1%, 33 ± 1%, and 49 ± 1% (Figure 1).


Figure 1
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  		Fig. 1. Bioanalyzer analysis of apoptosis in HepG2 cells cultivated in ECM. Effect of different doses of FB1 on the apoptotic cells in HepG2 cells after 24, 48, and 72 h are presented. Error bars represent standard error to the mean. Statistical analysis is performed between treated cells and controls. *P < 0.05 versus control.

 
Effects of FB1 on FR and hRFC1 proteins expression and mRNA levels in the ECM
The Bioanalyzer 2100 results showed a significant decrease in FR protein expression after a 48-h exposure following exposure to 100 µM of FB1 (Figure 2A). Compared with the controls, protein expression levels in the FB1-treated cells were significantly decreased to –9 ± 1% and –10 ± 1% at 48 and 72 h, respectively. For the hRFC1 protein expression (Figure 2B), we observed a significant increase in the protein expression at 24 and 48 h following exposure to 10 and 100 µM of FB1, but not at 72 h. Concerning mRNA levels, Figure 2C showed that FR transcript (fr) copy number was significantly higher at 24 h following exposure to all FB1 concentrations (1–100 µM) when compared with controls. The copy number of fr decreased significantly at 48 and 72 h for the same concentrations. There was a significant change in fr expression only after 72 h of exposure at the 0.5 µM concentration of FB1. Similarly, the exposure to 0.5 µM of FB1 significantly decreased the levels of hrfc1 at 72 h, compared with controls (Figure 2D). All FB1 concentrations tested produced a decrease of hfc1 at 48 and 72 h. However, it is worth noting that a significant increase in hrfc1 expression was observed at 24 h for 10 and 100 µM of FB1. We found no significant difference for cycle threshold values of ß-actin gene quantitation for different conditions in FB1 concentration. The cycle threshold was 25.9 ± 0.2 for control, and varied between 25.1 ± 0.4 to 26.4 ± 0.9 independent of increasing concentrations of FB1 (from 0.5 up to 100 µM).


Figure 2
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  		Fig. 2. Effect of different doses of FB1 on the folate transporters expression in HepG2 cells after 24, 48, and 72 h. Bioanalyzer analysis of FR protein expression (A); hRFC1 protein expression (B) and quantitative reverse transcriptase–PCR for fr (C) and hrfc1 (D) transcript copy number. Data were reported as mean of triplicate assays performed in the same experiment. Error bars represent standard error to the mean. Statistical analysis is performed between treated cells and controls. *P < 0.05 versus control.

 
Folate depletion combined with FB1-induced apoptosis in HepG2 grown in the FDM
The degree of apoptosis in FDM was not significantly higher than that observed in the control, at 24 h (7 ± 3%) (Figure 3). However, we observed a significant increase in the number of apoptotic cells after 48 h of exposure, with apoptosis rates of 11 ± 1% and 25 ± 3% at 48 and 72 h, respectively. When the FB1 was added to the FDM, an increase in the degree of apoptosis was observed at 24 h for 1 µM of FB1, to reach 34 ± 0% when the highest (100 µM) FB1 concentration was used. The absence of folate increased apoptosis in the presence of FB1. The highest apoptosis rate (61 ± 1%) was obtained with 100 µM FB1 in FDM at 72 h, a value that was 1.7-fold higher than that for cells cultivated in ECM with 100 µM of FB1 (36 ± 2%).


Figure 3
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  		Fig. 3. Bioanalyzer analysis of apoptosis in HepG2 cells cultivated en FDM. Effect of folate deficiency and different doses of FB1 on cells in HepG2 cells after 24, 48, and 72 h of treatment. Error bars represent standard error to the mean from independent experiments. Statistical analysis is performed between treated cells and controls. *P < 0.05 versus controls.

 
Effects of FB1 and folate depletion on FR and hRFC1 proteins expression and mRNA transcript levels
There was a significant variation in FR protein expression after 24 h of exposure in FDM without FB1. The inhibition of FR protein expression was –12 ± 2% after 48 h and –43 ± 1% after 72 h of exposure (Figure 4A). There was no variation at 72 h of exposure in the absence and in the presence of different concentrations of FB1. For hRFC1 protein, expression in cells exposed to FB1 in FDM was decreased in a dose- and time-dependent manner (Figure 4B). Significant decrease of expression was observed at 72 h. Decreases in fr and hrfc transcripts were observed in FDM without FB1, as well as in presence of FB1 at 48 and 72 h with a plateau for all FB1 concentrations (Figure 4C and D).


Figure 4
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  		Fig. 4. Effect of FDM and different doses of FB1 on the folate transporters expression in HepG2 cells after 24, 48, and 72 h. Bioanalyzer analysis of FR protein expression (A); hRFC1 protein expression (B) and quantitative reverse transcriptase–PCR for fr (C) and hrfc1 (D) transcript copy number. Error bars represent standard error to the mean. Results are means of a triplicate assay. Statistical analysis is performed between treated cells and controls. *P< 0.05 versus control.

 
Effect of the 5-MTHF supplementation on apoptosis and transporters expression
The first significant level of apoptosis in ECM was observed with 1 µM of FB1 at 72 h of exposure (Figure 1). Based on that result, we evaluated the effect of the reduced 5-MTHF supplementation in the medium with different FB1 concentrations following 72 h of exposure. As for the folic acid level in ECM, final concentrations of the 5-MTHFR in the MSM was 2.27 µM. In Table I, no significant differences were observed for apoptosis of cells grown in MSM without FB1 compared with control. When the concentrations of FB1 were increased, the degree of apoptosis increased significantly in a dose-dependent manner, reaching 35 ± 1% at 100 µM. As in ECM containing folic acid, apoptosis was significantly increased at 72 h for 1 µM. However, for the same concentration, the difference between both mediua was 18% (12 ± 2% in MSM versus 30 ± 1% in ECM). In the same time, difference of apoptosis with 100 µM was 14% between both media (35 ± 1% in MSM versus 49 ± 1% in ECM). Concerning FRs, a dramatic decrease of FR and hRFC1 protein expression was observed after incubation with FB1 from 1 µM. FR protein and transcript levels in cells exposed to 100 µM FB1 were –30 ± 1%, and –11.55 x 103 ± 0.002 x 103 copies in MSM compared with –10 ± 1% and –7.25 x 103 ± 0.108 x 103 in ECM. For hRFC1, protein and transcript levels in cells exposed to 100 µM FB1 were –18 ± 1% and –16.19 x 105 ± 0.05 x 105 copies in MSM compared with 4 ± 2% and –9.19 x 105 ± 1.05 x 105 copies in ECM.


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  		Table I. Effect of 5-MTHF supplementation on FB1-treated cells after 72 h of incubationa,b

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
FB1-induced apoptosis has been previously reported in studies using human cell lines (21), including HepG2 (22). In the present study, we examined FB1-induced apoptosis to determine the toxic effect of different concentrations of this toxin so as to study its impact on the expression of folate transporters. We first investigated its effects on the expression of these critically important membrane transporters in the presence of optimal level of folic acid (2.27 µM). We subsequently investigated the effect of a folate-depleted growth condition, and finally we evaluated a potential protective effect of media supplementation with 5-MTHF. The choice of the HepG2 cells for this study is of great importance, as it has been stated that the liver is the target organ and the main reservoir of FB1 (23,24). Furthermore, it is well documented that folate and folate derivatives are accumulated in the liver and that the later plays a major role in controlling folate supply in mammalians (25). Results from the current study indicate that FB1 induced HepG2 apoptosis in concentration- and time-dependent manner in ECM, as well as in FDM. It has been reported that the dose level causing apoptosis can vary in rodent from 0.9 to 12 mg FB1/kg body weight in short term (7 days) and long term (2 years), respectively (26). Report from the European Commission reported that no data are available for enabling a risk assessment on humans; however, it has been suggested that 2 µg/kg body weight should serve as the "tolerable daily intake" (26). The mechanisms underlying the FB1-induced apoptosis are unclear. It has been suggested that FB1 kills cells by disrupting sphingolipid biosynthesis, leading to an accumulation of sphingoid bases (27). Several studies reported additional systems that are affected by FB1, including the accumulation of excess sphinganine, which could also induce apoptosis. As previously reported by Seefelder et al. (28), the induction of apoptosis by FB1 cannot be initiated by an increased sphinganine concentration alone, but must occur in combination with the depletion of ceramide or complex sphingolipids derived from ceramide. The mechanism of apoptosis probably involves the disruption of many cell regulatory pathways, including inhibition of protein kinase C (29), and disruption of the endothelial barrier (30) and this may also influence the expression level of fr and hrfc. However, it should be mentioned that ß-actin expression level was not modulated by FB1 increasing concentrations, suggesting that FB1 toxicity did not affect global mRNA expression in cells.

Folate deficiencies can arise for many reasons, including reduction in folate intake, increased metabolism, and/or increased requirements in the case of pregnant women. There are numerous genetic polymorphisms in folate pathway genes, such as MTHFR 677C > T or the RFC80G > A single-nucleotide polymorphisms (11), which can impact cellular folate concentrations. Folate deficiency alone was capable to induce apoptosis in this study (Figure 3). Folate metabolites are essential for de novo purine and thymidine biosynthesis. Deficiency in folic acid can induce an imbalance in the deoxynucleotide precursors for DNA replication/repair and negatively affect the fidelity of DNA synthesis, leading to apoptosis (31). Moreover, Tolleson et al. 1996 (22) demonstrated that FB1 inhibited thymidine incorporation that might lead to DNA breakage followed by apoptosis induction.

The addition of FB1 in FDM increased the toxicity, and the cells became more sensitive, developing apoptosis within 24 h at 1 µM instead of 100 µM concentration in ECM. These results may suggest that under folate-deficient conditions, cells are more sensitive and vulnerable to FB1 toxicity, leading to apoptosis at a lower FB1 concentration. As FB1 and folate deficiency separately induced apoptosis, it will be interesting to determine if the mechanism of apoptosis under the two conditions, i.e. ECM + FB1 and FDM, are similar. Huang et al. (32) demonstrated that when HepG2 cells are cultured under folate-deficient conditions for at least 2 weeks, there was cycle-specific apoptosis and DNA damage. These findings are in agreement with our study; however, apoptosis in this study was detected in the cells much sooner with a limited exposure time. The difference can be explained by the differences in the methods used, since the authors studied the DNA fragmentation by agarose electrophoresis. Many studies reported the association between cytological abnormalities of tissues exposed to FB1 and vitamin deficiencies (33,34). Lipotrope deficiencies have been associated with the risk for esophageal cancer (35). Concentrations of red cell and plasma folate were significantly lower in patients (adults at risk for esophageal carcinoma in Transkei and Ciskei, Southern Africa) presenting with cytological signs of folic acid deficiency or cellular atypical.

Recently, Lamers et al. (36) demonstrated that the administration of [6S]-5-MTHF is more effective than is folic acid supplementation in improving the folate status of red blood cells. These investigators suggested that 5-MTHFmight be an efficient and safe alternative to folic acid. As expected in the present study, the addition of 5-MTHF to a folate-deficient medium completely rescued cells from apoptosis. However, this was not the case for FB1-treated cells, although the percentage of apoptosis was decreased by 44% in comparison to the FDM + 100 µM of FB1 and by 10% in comparison to ECM (containing folic acid) + 100 µM of FB1. Results for apoptosis showed a positive effect of 5-MTHF compared with folic acid. The possible explanation for the positive effect of 5-MTHF may be the chemical form of this folate derivative. In cells, the reduced folate 5-MTHFR does not require reduction before being incorporated into the active cellular folate pool to be used for genomic stability (30), in regards to folic acid that has to be converted into methyltetrahydrofolate to become biologically active.

In FB1-treated cells, FR protein expression in cells grown in ECM was inhibited at the FB1 concentration as high as 100 µM (Figure 2A). Although inhibition with lower concentrations was not significant, Figure 2A shows an inhibition in a concentration- and time-dependent manner. It is interesting to note that the decrease in FR protein expression occurred after the apoptosis event, confirming that the inhibition was not caused by a direct effect of the FB1, but as a consequence of the disruption of sphingolipid metabolism. FB1 inhibiting FR protein was in agreement with an earlier study on Caco-2 cells treated with FB1 (19) and with the study in cultured embryonic cells incubated with FB1 (37). Findings in both studies suggested adverse effect of FB1 and folate uptake that potentially compromise cellular processes of this vitamin (38). The FR is a GPI-anchored protein characterized by its attachment to the plasma membrane through a covalent link to a GPI anchor (39) and by its location in a membrane domain that is rich in sphingolipids and cholesterol (40). Parton and Simons (41) suggested that GPI-anchored proteins might form a complex with sphingolipids and cholesterol in specific plasma membrane domains. Thus, the inhibition of sphingolipids by the action of FB1 would alter and disturb the attachment of GPI-anchored proteins such as FR to the membrane leading to a decrease in its protein expression. To better understand this inhibition, we studied the transcriptional modulation of the fr in FB1-treated cells. Whereas FR protein expression was inhibited by FB1, results in Figure 2B showed that the hRFC1 protein expression was rather positively regulated. There was significant increase in hRFC1 protein expression at 48 h for 100 µM of FB1. Although we did not observe a direct relationship in the balance between FR and hRFC1, in contrast to the observed FB1 effects, increases of hRFC1 protein were detected in cells exposed to high FB1 concentrations (10 and 100 µM). This might be explained by the cell's attempt to compensate for the decreased expression of FR protein or by the increased folate requirements of the stressed cells for survival and division at 48 h of exposure. It may well be a cellular rescue mechanism by increasing the production of hRFC1. More transcriptional studies should be conducted to determine whether this modulation is controlled by one or several transcriptional factors directly or indirectly related to FB1 treatment. It is noteworthy to mention that ß-actin expression levels were not modulated by any FB1 concentration, suggesting that FB1 toxicity do not affect global mRNA expression in cells. With regard to the hRFC1 transcription decreasing the mRNA copies in the treated cells, this can possibly be explained by the increased degree of apoptosis. It is noteworthy to mention that the expression of hrfc1 was higher than that of FR. Although the liver is a major storage site of folate, it expresses a relatively low level of FR (42), which is in agreement with our findings.

The analysis of the combined effects of exposure to FB1 and folates deficiency would suggest that depletion in folate magnified with FB1 treatment inhibit the normal activities of the two folate transporters and lead to an aggravation of apoptosis secondary to a cellular folate insufficiency. Under folate-deficient conditions, dramatic changes were observed on both transcriptional and post-transcriptional expression of the two transporters. Folate-deficient medium alone reduced FR protein expression by 12 ± 2% and 43 ± 1% at 48 and 72 h, respectively. FR protein expression decreased independently of the FB1 concentrations. However, hRFC1 protein expression was altered in an FB1-dependent manner. Although 5-MTHF had a more positive effect on apoptosis relative to supplemental folic acid, its effects on both folate transporters has not been shown in this study. The 5-MTHF supplementation did not prevent the decrease in the transcript copy numbers for both fr and hrfc1 at any of the FB1 concentrations, when compared with data from ECM with FB1. Nevertheless, compared with data from FDM, 5-MTHFR effects were more noteworthy.

In conclusion, this study provides novel information concerning the link between the consumption of food contaminated by the FB1 toxin and folate metabolism. The study shows the adverse effects of a pre-existing folate deficiency, leading to greater sensitivity and vulnerability of cells to FB1 toxicity. We observed a protective effect of the 5-MTHF supplementation with respects to the prevention of apoptosis observed under both normal and deficient folate conditions. However, its effect in preventing a decrease of both folate transporters has not been shown. Further studies will expand our knowledge of the importance of folate, in reducing diseases associated to FB1 such as hepatocarcinoma. Larger studies taking into account folate intake, blood folate status and hRFC1 or FR common polymorphisms are needed.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary table 1 can be found at http://carcin.oxfordjournals.org/.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Comité de l'Oise de la Ligue Contre le Cancer, France.


   Acknowledgments
 
The authors acknowledge Professor Richard H. Finnell, Texas Institute for Genomic Medicine and Institute of Biosciences and Technology, Houston, TX, for revising the manuscript.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received March 9, 2007; revised May 25, 2007; accepted June 22, 2007.


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