Effects of β-glucan extracted from Agaricus blazei on the expression of ERCC5,CASP9,and CYP1A1 genes and metabolic profile in HepG2 cells

Abstract

The polysaccharide β-glucan has biological properties that stimulate the immune system and can prevent chronic pathologies, including cancer. It has been shown to prevent damage to DNA caused by the chemical and physical agents to which humans are exposed. However, the mechanism of β-glucan remains poorly understood. The objective of the present study was to verify the protective effect of β-glucan on the expression of the genes ERCC5 (involved in excision repair of DNA damage), CASP9 (involved in apoptosis), and CYP1A1 (involved in the metabolism of xenobiotics) using real-time polymerase chain reaction and perform metabolic profile measurements on the HepG2 cells. Cells were exposed to only benzo[a]pyrene (B[a]P), β-glucan, or a combination of B[a]P with β-glucan. The results demonstrated that 50 µg/mL β-glucan significantly repressed the expression of the ERCC5 gene when compared with the untreated control cells in these conditions. No change was found in the CASP9 transcript level. However, the CYP1A1 gene expression was also induced by HepG2 cells exposed to B[a]P only or in association with β-glucan, showing its effective protector against damage caused by B[a]P, while HepG2 cells exposed to only β-glucan did not show CYP1A1 modulation. The metabolic profiles showed moderate bioenergetic metabolism with an increase in the metabolites involved in bioenergetic metabolism (alanine, glutamate, creatine and phosphocholine) in cells treated with β-glucan and to a lesser extent treated with B[a]P. Thus, these results demonstrate that the chemopreventive activity of β-glucan may modulate bioenergetic metabolism and gene expression.

Keywords

β-glucan gene expression

Introduction

Several diet products have been outstanding for having chemopreventive properties among which is the Basidiomycete fungus Agaricus blazei (Murill), native to Brazil, that is commonly consumed as tea. Because of its biological activity, it has been evaluated for its nutraceutical potential and as a medicinal supplement used for the prevention and/or as an adjuvant with cancer chemotherapeutic drugs after the removal of a malignant tumor.¹

The importance of A. blazei has been attributed to the β-glucan component of the intermediate layer of the cell wall. This polysaccharide consists primarily of a linear central backbone of d-glucose linked in the β(1–3) position with glucose side branches (linkage β(1-6)) of various sizes.² In vitro and in vivo studies in animals and humans show that β-glucans derived from fungi and yeast have immune-modulating properties.^3
–5

β-Glucans are naturally occurring agents with stimulating effects on the defense mechanisms of living organisms. These polysaccharides are grouped within the class of drugs known as biological response modifiers. These drugs influence the host’s biological response by stimulating the immune system.^6,7 Studies on β-glucan receptors, such as dectin-1, have begun to elucidate the mechanisms underlying the properties of these carbohydrates.⁸

Several studies have indicated that the consumption of foods that produce high levels of these polysaccharides results in antimutagenic activity and might contribute to a reduction in the risk of cancer. Animal studies indicate that β-glucans, when used as a nutritional supplement, stimulate growth and improve nutrient retention.⁹ Thus, the scientific community has focused on the study of β-glucan as a protective compound. Most β-glucans enter the proximal small intestine and some are captured by the macrophages. Consequently, β-glucans can modulate both innate and adaptive responses, and they can enhance phagocytosis.¹⁰ Studies show that β-glucans enhance the tumor response to photodynamic therapy (PDT), resulting in pronounced necrosis of PDT-treated tumors and suppression of the DNA damage repair system in mice.¹¹

To verify the probable protective effect of β-glucan, the study used the standard damage-inducing agent benzo[a]pyrene (B[a]P) whose toxicity has been previously purchased by Uno et al.¹² using knockout mice (Cyp1a1-/-). B[a]P is a polyaromatic hydrocarbon present in cooked meat and cigarette smoke and is formed by the pyrolysis of fat¹³ and the compound represents a standard agent to induce DNA damage. The oxidation of this xenobiotic compound has been done for several P450 enzymes as CYP1A1.¹⁴ The major intermediate metabolites of B[a]P oxidation as the 7,8-diol-9,10-epoxides are toxic and in the nucleus may covalently bind to DNA mainly forming deoxyguanosine DNA adducts.^15,16 In addition, the effects of B[a]P have been shown to induce apoptosis in vitro in Hepa1c1c7 cells,¹⁷ because of this, the B[a]P is an appropriate agent to damage and induce in HepG2 cells. The human hepatocellular carcinoma (HepG2) cell line represents an alternative system for the metabolism and modulation studies of human P450. The cell line has the essential phase I and phase II enzymes required for the metabolism of drugs and carcinogens,¹⁸ and represents the model to genetic toxicology studies.¹⁹ Moreover, the HepG2 P450 system is responsive to the modulatory effects of xenobiotics and this cell line has been chosen for the development of several experiments in genetic toxicology studies such as Brandon et al.²⁰

Hence, the objective if this study was to verify the protective effect of β-glucan on the expression of the cytochrome P450 type CYP1A1, excision repair cross-complementing rodent repair deficiency, complementation group 5 (ERCC5), and caspase 9 (CASP9) genes involved in xenobiotic metabolism, excision repair nucleotide, and apoptosis process, respectively. We also checked the metabolites produced in the HepG2 cells exposed to only β-glucan, only B[a]P, and β-glucan and B[a]P.

Material and methods

Human hepatocellular carcinoma cells

Human hepatocellular carcinoma (HepG2) cells derived from a human hepatocellular carcinoma were obtained from the Cell Bank of Rio de Janeiro, Brazil. The cells were cultured in 25 cm² tissue culture flasks in 10 mL of Dulbecco’s modified Eagle medium (Gibco, BRL, USA) that was supplemented with 20% fetal bovine serum (Gibco, BRL, USA) and 1% sodium pyruvate (Gibco, BRL, USA). The cells were grown at 37°C in an atmosphere of 5%CO₂.

Chemicals

The DNA-damaging agent was B[a]P that had been prepared in dimethylsulfoxide and added to the culture at a final concentration of 20 μg/mL. The chemopreventive agent, β-glucan, was extracted from A. blazei obtained from the State University Paulista (UNESP, Botucatu Campus, Sao Paulo) in Brazil. The β-glucan was extracted following Gonzaga et al.²¹ and prepared in Ca⁺²- and Mg⁺²-free phosphate-buffered saline with a pH of 7.4.

Cytotoxicity assays

For the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays, 2.5 × 10⁴ HepG2 cells were incubated for 24 h in 96-well plates at 37°C in an atmosphere of 5% CO₂. Then, the cells were treated for 24 h with β-glucan at 20, 40, 80 and 120 μg/mL concentrations. The criterion of selection of the concentration of β-glucan was based on the study of Angeli et al.²² Using the same conditions, we used 20 μg/mL B[a]P as a DNA damaging agent and 20 μg/mL of doxorubicin as an internal control of cytotoxicity. Controls were incubated without treatment. The protocol used for the MTT assay was described by Mosmann.²³

Real-time Adding the Reverse Transcriptase - Polymerase Chain Reaction polymerase chain reaction

The HepG2 cells (4.0 × 10⁶ per flask) were incubated for 24 h and subsequently treated for 6 h. The treatments were carried out using B[a]P (20 µg/mL), β-glucan (50 µg/mL) or simultaneous B[a]P and β-glucan. Use of 50 µg/mL β-glucan was based on the analysis of cytotoxicity and the results obtained by Angeli et al.²⁴ Total RNA was extracted using TRIzol LS reagent (Invitrogen, Life Technologies, USA).

First-strand complementary DNA (cDNA) synthesis was performed with reverse transcriptase (RT M-MLV, Invitrogen) according to the manufacturer’s protocol and using 1 μg of total RNA as the template.

Real-time polymerase chain reactions (RT-PCRs) were performed in a PTC 200 DNA Engine Cycler using a Chromo 4 Detection System (MJ Research, Waltham, MA, USA). The oligonucleotides utilized in these experiments are listed in Table 1. The Platinum^® SYBR^® Green qPCR Supermix-UDG (Invitrogen, Life Technologies, USA) was used as the reaction mixture to which 0.4 µM of each primer and 2 μL of template cDNA were added. The final reaction volume was 25 μL. The PCR thermal-cycling conditions included an initial step at 95°C for 5 min; 40 cycles at 95°C for 20s, 60°C for 30s, and 72°C for 20s; another step at 95°C for 10s; and finally, 40°C for 1 min. A melting curve analysis was consistently performed at the end of the reaction with temperatures between 50 and 90°C. The data were normalized using the β-actin housekeeping gene²⁶ cDNA that was amplified in each set of PCR experiments. The relative expression data were obtained using the Pfaffl method.²⁷

Table 1.

List of primers used in this study.

Name	Sequence 5′–3′	Reference
ERCC5	F- 5′GCTCCCATTAGTGCCGTC	Present study
ERCC5	R- 5′CCCTGCTCCTACACAACAA	Present study
CASP9	F- 5′GCTCTTCCTTTGTTCATCTCC	Present study
CASP9	R-5′GTTTTCTAGGGTTGGCTTCG	Present study
CYP1A1	F- 5′TCATCCCTATTCTTCGCTACC	Jiao et al.²⁵
CYP1A1	R- 5′CAGGAGATAGCAGTTGTGAC	Present study
β-Actin	F- 5′CTGGCACCCAGCACAATG	Westerink and Schoonen²⁶
β-Actin	R- 5′GCCGATCCACACGGAGTACT	Westerink and Schoonen²⁶

Each experiment was performed in triplicate in two independent experiments. Data are expressed as the mean values ± SD. The Pairwise Fixed Reallocation Randomisation Test²⁸ was used, and the values of p < 0.05 were considered significant. Instat software was used for all statistical analyses. For messenger RNA induction studies, only the induction levels greater than twofold with statistically significant differences were considered relevant.

Cell extraction

Reagent-grade methanol and chloroform (4°C) in a ratio of 2:1 (v/v, 250 μL/cell pellet) were added to the frozen cell pellets. The cell pellet–solvent mixture was sonicated. After approximately 15 min of contact with the first solvents, chloroform and distilled water were added to the cold samples in a ratio of 1:1 (250 μL/cell pellet) to form an emulsion. The samples were then centrifuged at 13,000 r/min for 20 min at 4°C. The upper phase (methanol and water) was separated from the lower organic phase using a glass syringe and both fractions were dried at room temperature by centrifugation under high vacuum. The solvent free extracts were resuspended in 1.0 mMol/L sodium trimethylsylilpropionate D₂O.

Metabolic profile measurements

The metabolic profile spectra were acquired using a Varian INOVA-500 spectrometer (B0 = 11.7 T) operating at 499.886 MHz for ¹H using a 5-mm triple resonance (H, C, and N) inverse probe with a z-gradient. Each experiment was acquired with a 256 transient at 25°C, 3.277s acquisition time, 10,000.0 Hz spectral width, 65,536 points, and 30°C pulse angle. The intense residual water signal was suppressed using presaturation (presat). Chemical shifts were compared with 3-(trimethylsilyl)-tetradeutero-sodium propionate as a reference. Chemical shifts of ¹H metabolic profile and coupling constants of the most characteristic metabolites were assigned using previously published data. The peaks were normalized, and the difference between the treatments was calculated from the intensity of the peak area by the program Origin 8.0

Results

Comparisons of the expression of the ERCC5, CASP9, and CYP1A1 genes in HepG2 cells exposed to B[a]P and β-glucan

In this study, neither 50 µg/mL of β-glucan nor 20 µg/mL of B[a]P were cytotoxic for HepG2 cells based on the results of MTT cytotoxicity assays. Under these conditions, and knowing the doubling time (24 h) of HepG2 cells, we analyzed the expression of the CYP1A1, ERCC5, and CASP9 genes.

Expression of the genes was analyzed by RT-PCR. The transcript levels of CYP1A1 were 96-fold and 36-fold higher in cells treated with B[a]P and with B[a]P and β-glucan simultaneously, respectively (Figure 1(a)). In the treatments using only β-glucan, this transcript did not show any significant difference in the level compared with the controls. In the simultaneous treatment, the expression level was only 36-fold higher than the controls compared with 96-fold higher in the cells treated with B[a]P alone. This result demonstrates that the β-glucan treatment had a protective effect.

Figure 1.

Real time RT-PCR analysis of the CYP1A1, ERCC5 and CASP9 transcripts of the HepG2 cells treated for 6 h. Control cells, and cells treated with B[a]P,b-glucan, or simultaneous B[a]P and b-glucan were analyzed. A) The relative expression of the CYP1A1 gene was significantly enhanced in the cells treated with B[a]P and in cells treated with both B[a]P and b-glucan compared to the control cells. B) The relative expression of the ERCC5 gene was significantly less in cells treated with β-glucan than the control cells. C) The relative expression of the CASP9 gene was not significantly affected by the treatment with b-glucan. The amount of mRNA was normalized using the Ct obtained for the β-actin gene.

The levels of the ERCC5 transcript did not show significant differences from the controls after B[a]P or the simultaneous treatment; however, after treatment with only β-glucan, the transcript level of ERCC5 was significantly lower (twofold) than in the controls (Figure 1(b)).

The transcript level of CASP9 gene was also analyzed, but no significant differences were found (Figure 1(c)).

Metabolic profile of HepG2 cells exposed to B[a]P and β-glucan

Analyses of differential areas of intensity of the normalized spectra showed increases in the metabolites involved in bioenergetic metabolism in the cells treated with β-glucan (Figure 2(a)). The same metabolites were found in smaller amounts in the cells treated with B[a]P. Intermediate quantities were found in the cells treated simultaneously with B[a]P and β-glucan (Figure 2(a)). Alanine, glutamate, creatine, and phosphocholine were the metabolites that showed the most variations after the treatments.

Figure 2.

A) A representative metabolic profile of the HepG2 control cells. The main metabolites identified were: (Ala) alanine, (Glu) glutamate, (Cr) creatine and (PC) phosphocholine. The reference for the chemical shift was TSP (3-(trimethylsilyl)-tetradeutero-sodium propionate). B1-3) Differential glutamate metabolic intensity between treatments C) Differential alanine metabolic intensity between treatments. D1-2) Differential creatine metabolic intensity between treatments, E) Differential phosphocholine metabolic intensity between treatments. Differential metabolic profiles between treatments (Control, β-glucan, B[a]P and B[a]P + β-glucan (simultaneous)) of the HepG2 cells were calculated from the intensity of the peak areas.

Alanine and glutamate showed the greatest variation in peak intensities between the treatments (34% and 32%, respectively), with creatine and phosphocholine (14% and 24%, respectively) showing the next highest peak intensity variation (Figure 2(b) to (e).

Discussion

The polysaccharide β-glucan has antigenotoxic, antimutagenic, and antioxidative properties^6,29

–33 that may protect against DNA damage caused by various chemical agents.

Polycyclic aromatic hydrocarbons, such as B[a]P, are potent inducers of the CYP1A1 gene in animals and good substrates for this enzyme.³⁴ The mechanism of action of B[a]P begins with their binding to the aryl-hydrocarbon receptor and subsequently activation of a transcription factor that translocates to the cell nucleus and associates with a nuclear protein forming a complex that can interact with promoter elements which induces the transcription of genes involved in the metabolism of B[a]P, including cytochromes P450 such as CYP1A1.^35,36 Our results confirm that the expression of the CYP1A1 gene is induced in HepG2 cells exposed to B[a]P. However, when the cells were treated with B[a]P and β-glucan, the β-glucan had a protective effect against damage caused by B[a]P. Angeli et al.²² previously reported the protective effect of β-glucan using simultaneous measurements pre- and post-treatment in HepG2 cells. These authors showed that the polysaccharide could modulate cell metabolism and/or bind to the B[a]P. The gene expression results showed significantly induced expression of CYP1A1 transcript in HepG2 cells treated with β-glucan and B[a]P together, while in HepG2 cells treated with only β-glucan, this transcript did not show significant difference. In contrast to Okamoto et al.,³⁷ β-glucan of fungal origin represses the expression of the cytochrome P450 (CYP) enzymes in rats.

It has been shown that B[a]P is metabolized to reactive intermediate compounds produced by P450 enzymes that participate the detoxification of the cells and has specificities substrate. The reactive intermediates cause DNA damage, and the capacity of HepG2 cells to repair the damage affects the sensitivity of the cells to the pro-carcinogen B[a]P, as does the amount of the endogenous intermediate compounds. Using published information about the DNA damage induced by B[a]P reactive intermediates, we investigated the possible alterations in the expression of the ERCC5 repair gene. We observed only minor differences due to β-glucan treatment, probably because the time of treatment, 6 h, corresponded to G₁/G₀ phase, that is, when the reactive intermediate metabolites of B[a]P are just beginning to be produced. The exposure of cells to noncytotoxic B[a]P concentrations results in the cells accumulating in the S-phase. This has led to the hypothesis that escape from G₁-phase arrest contributes to the transforming activity of carcinogens that form DNA adducts.³⁸ Jiao et al.²⁵ reported that increased DNA adduct levels in B[a]P-treated cultures of MCF-7 enriched for S-phase cells resulted from the CYP1A1-mediated activation of B[a]P to a reactive intermediate metabolite compared with the cultures enriched for the other cell cycle phases. In accordance with the modulating activity of β-glucan, Angeli et al.²² showed that β-glucan reduced DNA damage in the presence of a DNA polymerase inhibitor. These results confirm that the protective action of β-glucan does not involve a repair pathway.

To verify the protective effect of β-glucan, we also analyzed the transcript level of the CASP9 gene, and no changes were found. These results are consistent with the protective effects of β-glucan, and they reinforce the hypothesis that the polysaccharide probably acts as a metabolism modulator.

Under the same conditions, the ¹H NMR spectra were analyzed to verify the possible modulator effect of β-glucan. Alanine and glutamate metabolites showed the highest variation between treatments. These metabolites are essential for cells. For example, hepatoma cells actively consume these metabolites. An increase in glutamate utilization is related to an increase in the consumption of the other metabolites.³⁹ Alanine can participate in the diverse reactions of primary metabolism, and glutamate contributes to essentially every core metabolite. It participates in bioenergetics, supports cell defenses against oxidative stress, and complements glucose metabolism in the production of macromolecules.⁴⁰ The importance of these metabolites in the β-glucan treated cells is the indicative of high-energy metabolism. The β-glucan is a source of glucose for the cell and provides more energy for recovery from cell damage. However, in cells treated with B[a]P or B[a]P together with β-glucan, the quantities of metabolites were smaller because the B[a]P caused cell damage. When cells treated with B[a]P were compared with those treated with B[a]P and β-glucan, we observed more quantities of this metabolites in the cells treated with B[a]P and β-glucan.

The other metabolites, creatine and phosphocholine, were found in low amounts indicating that β-glucan can maintain the bioenergetic status in spite of cell damage. Several studies have shown that a progressive decline in bioenergetic status, including decreases in phosphocreatine and ATP levels and consequent increase in creatine levels, are associated with cellular hypoxia.⁴¹ Additionally, cells show high lactate levels in hypoxic conditions, but we did not find any high levels of this metabolite in HepG2 cells. This result is in agreement with Loiseau et al.,⁴² who showed that HepG2 cells use oxidative phosphorylation to produce increased energy, which confers a survival advantage. This energy production can be regulated by the availability of β-glucan, which is a source of glucose in cells.

The metabolic adaptation required to produce cellular energy was also observed with the quantities of phosphocholine metabolite that are involved in the synthesis of cellular membranes containing choline. Choline kinase is an important enzyme that catalyzes the phosphorylation of choline to form phosphocholine in the presence of ATP and magnesium. With more cellular energy supplied by β-glucan, this metabolite can help the cell recover from the damage caused by B[a]P. The β-glucan treatments do not cause cell damage, but they improve bioenergetic metabolism and protective properties.

In summary, this study showed cell damage caused by B[a]P, as an active enzyme modulator of phase I (CYP1A1), and also demonstrated that β-glucan polysaccharide has a protective effect in the HepG2 cells. This protection can be produced by binding B[a]P or by modulating bioenergetic metabolism.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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