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Mar 20, 2016 - FIP-gat, an immunomodulatory protein isolated from Ganoderma atrum, is a new member of the FIP family. Little is known, however, about ...
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Recombinant FIP-gat, a Fungal Immunomodulatory Protein from Ganoderma atrum, Induces Growth Inhibition and Cell Death in Breast Cancer Cells Hui Xu,†,∥ Ying-Yu Kong,†,∥ Xin Chen,§ Meng-Yuan Guo,† Xiao-Hui Bai,# Yu-Jia Lu,‡ Wei Li,*,‡ and Xuan-Wei Zhou*,† †

Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, and Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, People’s Republic of China ‡ Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China § Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut 06032, United States # State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China S Supporting Information *

ABSTRACT: FIP-gat, an immunomodulatory protein isolated from Ganoderma atrum, is a new member of the FIP family. Little is known, however, about its expressional properties and antitumor activities. It was availably expressed in Escherichia coli with a total yield of 29.75 mg/L. The migration of recombinant FIP-gat (rFIP-gat) on SDS-PAGE corresponded to the predicted molecular mass, and the band was correctly detected by a specific antibody. To characterize the direct effects of rFIP-gat on MDA-MB-231 breast cancer cells, MDA-MB-231 cells were treated with different concentrations of rFIP-gat in vitro; the results showed that this protein could reduce cell viability dose-dependently with a median inhibitory concentration (IC50) of 9.96 μg/ mL and agglutinate the MDA-MB-231 cells at a concentration as low as 5 μg/mL. Furthermore, FIP-gat at a concentration of 10 μg/mL can induce significant growth inhibition and cell death in MDA-MB-231 cells. Notably, FIP-gat treatment triggers significant cell cycle arrest at the G1/S transition and pronounced increase in apoptotic cell population. Molecular assays based on microarray and real-time PCR further revealed the potential mechanisms encompassing growth arrest, apoptosis, and autophagy underlying the phenotypic effects. KEYWORDS: Ganoderma atrum, fungal immunomodulatory protein, agglutination, growth inhibition, cell death

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INTRODUCTION Ganoderma atrum, also known as black Ganoderma (other names include Xuanzhi, black Yunzhi, false Lingzhi), is a fungus belonging to the phylum Basidiomycota, class Agaricomycetes, order Agaricomycetes, family Ganodermataceae.1 It is widely distributed in Taiwan and the southern provinces of China, for example, Sichuan, Guangdong, Guizhou, Hubei, Hainan, and Yunnan.2 G. atrum is an important medicinal mushroom and contains various bioactive components with potential antitumor,3 antioxidant,4 and bacteriostatic activities.5 To date, studies on the bioactive components and pharmacological actions of G. atrum have mainly concentrated on its polysaccharides,5−7 ganoderic acid,4 and other components; the functions of its bioactive proteins have not yet been elucidated.8 Fungal immunomodulatory proteins (FIPs) are a group of small molecular weight proteins with structure and immunoregulatory activity similar to those of phytohemagglutinin and immunoglobulins. Since Kino and colleagues first isolated FIP from Ganoderma lucidum mycelia, another 10 FIPs, ling zhi-8 (LZ-8/FIP-glu),9 and FIP-LZ9, FIP-gts, FIP-fve, FIP-vvo, FIPgja, FIP-gmi,10 FIP-gsi,11 FIP-tvc,12 and FIP-nha,13 have been © 2016 American Chemical Society

isolated and identified from G. lucidum, Ganoderma tsugae, Flammulina velutipes, Volvariella volvacea, Ganoderma japonicum, Ganoderma microsporum, Ganoderma sinensis, Trametes versicolor, and Nectria hematococca, respectively. Previous studies have demonstrated that FIPs possess biological properties similar to those of plant lectins in their ability to agglutinate red blood cells.2 Both types of proteins are able to bind to cell surface sugar moieties.14,15 FIPs promote blood cell agglutination and lymphocyte proliferation14,16 and stimulate lymphocytes to produce a variety of cytokines.15,17 They are also involved in immunoregulation, cholesterol lowering, antitumor18,19 and antiobesity processes, etc.20 However, less is known about the direct effects of FIPs on tumor cells. Previously, we cloned a FIP gene from G. atrum mycelia and designated it FIP-gat (Genbank accession no. KM077027). Bioinformatic analysis revealed that the FIP-gat gene encodes a protein consisting of 111 amino acids with a theoretical Received: Revised: Accepted: Published: 2690

February 1, 2016 March 18, 2016 March 20, 2016 March 20, 2016 DOI: 10.1021/acs.jafc.6b00539 J. Agric. Food Chem. 2016, 64, 2690−2698

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Journal of Agricultural and Food Chemistry

blotting was confirmed using the standard protocol.23 The membranes were incubated in blocking buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Tween 20, 3% BSA] for 1 h. The rabbit anti-FIP-gsi antibody12 was then added at 1:5000 dilution into the blocking buffer to react with rFIP-gat. After washing, the membranes were treated with alkaline phosphatase (ALP) conjugated donkey anti-rabbit antibody (Promega, Madison, WI, USA) at 1:5000 dilution. Finally, membranes were soaked in Western Blue stabilized color substrate for alkaline phosphatase (Promega) until the bands recognized by antibody appeared. Purification of rFIP-gat. The rFIP-gat was subjected to purification using a nickel−nitrilotriacetic acid (Ni-NTA) agarose resin column (Qiagen, Shanghai, Chian). This system had previously been successfully used for purification of the rFIP-gsi and rFIP-SN fusion proteins.22,24 Fifty milliliter cultures was grown at 37 °C until OD600 was approximately 0.6. IPTG was then added to the culture to a final concentration of 1 mM to induce recombinant protein expression at 28 °C overnight. The induced cells were harvested by centrifugation at 5000 rpm for 20 min at 4 °C and resuspended in 5 mL of lysis buffer (pH 8.0) containing 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole. After freezing in liquid nitrogen and thawing in cold water, samples were lysed by sonication using six 10-s bursts at 200− 300 W with a 10-s cooling period between each burst. Cell debris was removed by centrifugation at 12000 rpm for 20−30 min at 4 °C; the supernatant containing the 6× His-tagged rFIP-gat was purified using a Ni-NTA agarose resin column. The column was washed using 10 bed volumes of wash buffer (pH 8.0) containing 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole. Finally, the rFIP-gat was eluted with elution buffer (pH 8.0) containing 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole and collected in five 0.5-mL aliquots. The proteins in collected fractions were then analyzed by SDS-PAGE. Determination of rFIP-gat and Lipopolysaccharide (LPS) Concentration. The rFIP-gat concentration was determined according to the Bradford method using a Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China). The total yield of rFIP-gat for 1 L of culture was calculated on the basis of rFIP-gat recovered from 50 mL of culture. The relative expression of the rFIP-gat in total cellular protein and the purity of purified product was quantified by scanning the SDS-PAGE gels using an imaging densitometer (model GS-700, Bio-Rad, Hercules, CA, USA) followed by analysis using the BandScan V 5.0 software (Glyko, Novato, CA, USA). LPS levels in the purified rFIP-gat were measured using an ELISA kit (Cloude-Clone Corp., Houston, TX, USA) by following the manufacturer’s instruction. The content of LPS was converted to picograms of LPS per microgram of protein to facilitate the calculation of LPS amount present in culture media when different dosages of rFIP-gat were applied in the subsequent cell experiments. Identification of rFIP-gat. The target protein band was excised from the polyacrylamide gel, and the gel slice was cut into pieces. After destaining, the gel pieces were dried under vacuum and digested with sequencing grade modified trypsin at 37 °C overnight. The peptides were extracted and subjected to LC-MS/MS analysis for identification. Liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC/Q-TOF MS, Applied Biosystems, Foster City, CA, USA) was used to analyze the tryptin-digested rFIP-gat, which was performed at the Instrumental Analysis Center of SJTU.22 The observed peptide masses were matched to the rFIP-gat peptide sequences. Cell Culture. MDA-MB-231 human breast cancer cells were cultured in monolayers in DMEM containing 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified CO2 incubator. Cells were trypsinized with 0.25% trypsin containing 0.04% EDTA, and cell density was adjusted to 5 × 103 cells/100 μL and seeded into 96-well plates 24 h before experiment. Cell morphology was observed using an inverted microscope (Axiovert 40 CFL, Zeiss, Germany), and images were taken using a charge-coupled discharge (CCD) camera coupled to the microscope (Axiocam MRm, Zeiss). In all subsequent functional studies, FIP-glu, a precharacterized FIP protein that has been shown to

molecular weight of 12.45 kDa.21 In this study, we achieved high-yield production of the recombinant FIP-gat (rFIP-gat) protein by using the Escherichia coli BL21 strain and obtained enough protein with satisfactory purity by employing Ni-NTA affinity purification. In addition, we conducted a preliminary study to test the effects of rFIP-gat protein on the morphology and growth of MDA-MB-231 breast cancer cells and found that rFIP-gat protein agglutinated the cells as well as inhibited cell growth in vitro. We also analyzed the effects of rFIP-gat protein on cell cycle and apoptosis in MDA-MB-231 cells and clarified the functions of this protein in inducing growth arrest and apoptotic cell death. Molecular assays based on microarray and real-time PCR further revealed the potential underlying mechanisms of the phenotypic effects. This basic work has laid a foundation for the development of rFIP-gat protein products and further assessment of its antitumor activities.

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MATERIALS AND METHODS

Strain, Plasmids, and Media. G. atrum (ACCC 510001) was purchased from the Agricultural Culture Collection of the People’s Republic of China (ACCC). The pMD18-T vector was purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China); the pET30a expression vector and E. coli strain BL21 are maintained in our laboratory. Human breast cancer cells MDA-MB-231 were obtained from the Cell Bank of the Chinese Academy of Sciences. Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Expression Vector Construction. The FIP-gat gene had previously been cloned into a pMD18-T vector (TaKaRa), and the resulting plasmid was designated p18T::FIP-gat.21 The FIP-gat gene sequence was reamplified from the p18T::FIP-gat vector by PCR and then subcloned into the pET30a expression vector (Novagen). To facilitate cloning of the FIP-gat gene into pET30a, we added a BamHI restriction site (underlined) to the forward primer FIP-gatB, 5′CGGGATCCATGTCCGACACTGCCTTGATCTTCAGG-3′, and a HindIII site (underlined) to the reverse primer FIP-gatH, 5′CCCAAGCTTCTAGTTCCACTGGGCGATGATGAAGTC-3′. PCR was performed using Taq plus DNA polymerase (TaKaRa). The PCR condition was the same as previously described.22 The resulting recombinant plasmid, designated pET30a::FIP-gat, was transformed into E. coli strain BL21 for the expression of rFIP-gat. Analysis of rFIP-gat Expression. A single colony of pET30a::FIP-gat transformants was inoculated into Luria−Bertani (LB) medium supplemented with 100 mg/L ampicillin (Amp) and 25 mg/L kanamycin (Kan) and grown at 37 °C overnight; 0.1 mL of the overnight cultures was diluted into 10 mL of fresh LB medium containing antibiotics and grown with shaking (200 rpm) at 37 °C until the optical density (OD600) reached 0.5−0.7. Protein expression was induced by adding isopropyl-β-D- thiogalactoside (IPTG) to a final concentration of 1 mM. After another 5 h of culturing, 1 mL of cultured cells was harvested by centrifugation at 5000g for 20 min at 4 °C. Pellets were resuspended in 100 μL of 2× sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer [100 mM Tris-Cl (pH 6.8), 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (w/v) glycerin, and 2% (w/v) β-mercaptoethanol] and boiled for 5 min at 95 °C. After centrifugation, the supernatants were loaded onto a 15% SDS-PAGE gel and separated by electrophoresis. The gel was then stained with Coomassie Brilliant Blue R-250 and then destained to reveal the separated protein bands. Optimization of Induction Duration. To find the optimal time length for inducing recombinant FIP-gat expression, 1 mL cultures were collected at 0.5, 1, 2, 3, 4, 5, and 6 h after the addition of IPTG. The cell lysates from each time point were subjected to SDS-PAGE followed by CBB R-250 staining. To detect specific rFIP-gat bands, the gels were electroblotted onto 0.2 μm polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) using a Mini Trans-Blot Electrophoretic Transfer Cell System (Bio-Rad, Shanghai, China). The success of electro2691

DOI: 10.1021/acs.jafc.6b00539 J. Agric. Food Chem. 2016, 64, 2690−2698

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ncifcrf.gov). Only functional groups with p values ≤0.05 were reported. Confirmation of Differentially Expressed Genes by RealTime PCR. Total RNA was isolated from approximately 2 × 105 cells from each group and reverse transcribed using oligo-dT primer in a two-step quantitative real-time PCR (qRT-PCR). Briefly, first-strand cDNA syntheses were conducted on 1 μg of total RNA using SuperScript-II RNase H-Reverse Transcriptase (Invitrogen). qRTPCR was performed using the Cyber Green-based method or inventoried TaqMan gene expression assay kits. Custom-designed primer sequences are summarized in Table S1. The mRNA levels were quantified relative to endogenous GAPDH controls. The reactions were monitored using the real-time PCR instrument (ABI 7300, Applied Biosystems, Foster City, CA, USA). PCR conditions for Cyber Green-based assays were as follows: 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The PCR conditions for TaqMan assays were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s. Delta CT values (CT value for genes of interest minus CT value for controls) were determined. 2∧-ddCt was used for the relative quantification of gene expression. Statistical Analysis. Calculations of mean, SD, and p values were performed on triplicate experiments using XLSTAT 2006 (New York, NY, USA). The Student t test was used to calculate p values for comparison. Significant statistics were set at a p value 90%. The total yield of purified rFIP-gat was 29.75 mg/L bacteria cultures. The results of optimizing the induction duration for rFIP-gat expression showed that optimal protein expression was obtained in 5 h induction periods (Figure 2A). Subsequent Western blot detection using a well-characterized polyclonal antibody against Ganoderma FIPs confirmed the identity of the target band (Figure 2B). In addition, the purified rFIP-gat protein was identified by LC/Q-TOF-MS, and the results showed that eight peptides (Table 1) matched the amino acid sequences derived from rFIP-gat. The total sequence coverage was 62.2%. Due to the fact that lipopolysacharide (LPS) is a major component in the outer membrane of E. coli cells, it is one of the most common contaminants in proteins purified from E. coli. Therefore, we measured the LPS concentration in the purified rFIP-gat, the LPS concentration was determined to be 2.74 pg/μg proteins. We then performed a prior experiment to test if a comparable amount of LPS per se is able to influence the morphology and growth of MDA-MB-231 breast cancer cells. It was found that including 50 or 100 pg/mL LPS in the culture media did not have obvious effects on either the growth 2692

DOI: 10.1021/acs.jafc.6b00539 J. Agric. Food Chem. 2016, 64, 2690−2698

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Figure 2. Analysis of rFIP-gat protein expression after IPTG induction by SDS-PAGE and Western blot. (A) Accumulation of rFIP-gat protein in E. coli BL21 cells. Band patterns of total cellular proteins from BL21 cells collected at different time points after induction were visualized by Coomassie Brilliant Blue (CBB) R-250 staining following SDS-PAGE separation. Lanes: 1, sample collected before IPTG induction; 2−7, samples collected 0.5, 1, 2, 3, 4, 5, and 6 h after IPTG induction, respectively; M, low molecular mass protein markers (arrow indicates rFIP-gat). (B) Western blot detection of rFIP-gat bands. Electroblotted membranes were incubated with primary antibody against Ganoderma FIPs followed by specific secondary antibody conjugated with alkaline phosphatase (ALP). The recognized bands were developed with the Western Blue stabilized substrate for ALP (Promega). Lanes: 1, sample collected before IPTG induction; 2−7, samples collected 0.5, 1, 2, 3, 4, 5, and 6 h after IPTG induction, respectively.

Figure 1. Analysis of the expression (A) and solubility of rFIP-gat and purity of purified rFIP-gat (B) by SDS-PAGE. (A) Lanes: M, protein molecular mass markers; 1, E. coli BL21 cells without IPTG treatment; 2, E. coli BL21 cells induced with IPTG; 3, E. coli BL21 cells containing expression vector pET30a without IPTG treatment; 4, E. coli BL21 cells containing expression vector pET30a induced with IPTG; 5, E. coli BL21 cells containing expression vector pET30a::FIP-gat without IPTG treatment; 6, E. coli BL21 cells containing expression vector pET30a::FIP-gat induced with IPTG. (B) Lanes: M, protein molecular mass marker; 1, sample without IPTG treatment; 2, sample induced with IPTG; 3, soluble protein fraction; 4, insoluble protein fraction; 5, purified protein.

Table 1. Peptide Fragments Identified from the Tryptic Digests of rFIP-gat

or morphology of MDA-MB-231 cells (data not shown). These results suggest that the effects of LPS can be excluded only if the final concentration of rFIP-gat added to the media does not exceed 36.5 μg/mL in the subsequent experiments. Assessment of the Effects of rFIP-gat on the Growth of Breast Cancer Cells. To assess the effects of rFIP-gat on the growth of MDA-MB-231 breast cancer cells, purified rFIPgat proteins were added to the culture media at a final concentration of 2, 4, 8, 16, 32, 64, or 128 μg/mL, respectively. After incubation for 48 h, the cell viability was determined by CCK-8 assay. It was found that rFIP-gat reduced tumor cell viability dose-dependently (Figure 3), and the deduced 50% inhibitory concentration (IC50) was 9.96 μg/mL. Observation of Morphological Changes in Breast Cancer Cells after rFIP-gat Treatment. To investigate similar effects of the rFIP-gat with fungal lectins, following rFIP-gat treatment (5, 10, 15, and 20 μg/mL), morphological changes of MDA-MB-231 cells were monitored periodically under an inverted microscope. It was observed that cells cultured with 5, 10, 15, or 20 μg/mL rFIP-gat underwent apparent detachment and aggregation as early as 6 h after treatment (Figure 4). After treatment for 12−24 h, prominent cell shrinkage appeared in all dosage groups (Figure 4II,III). In contrast, untreated cells kept their normal spindle shape and grew normally (data not shown). These results demonstrate that rFIP-gat has agglutinating activity against breast cancer cells.

parent ion (m/z)

peptide sequence

366.2097 527.2669 579.2885 584.0603 671.8269 735.8682 749.7155 796.9164

RLAWDVKK MSDTALIFRL RDLGVRPSYAVGSDGSQKV RVVVSGRDLGVRPSYAVGSDGSQKV KLSFDYTPTWGRG KKLSFDYTPTWGRG RFVDNVTFPQVLADKAYTYRV RFVDNVTFPQVLADKA

Figure 3. Reduction of the viability of MDA-MB-231 cells by FIP-gat. MDA-MB-231 cells were treated with different concentrations of FIPgat. Cell viability was measured using CCK-8 colorimetric assay, and the half-maximal inhibitory concentration (IC50) was calculated using Xlfit 4 software. 2693

DOI: 10.1021/acs.jafc.6b00539 J. Agric. Food Chem. 2016, 64, 2690−2698

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than the control ones (31.62%, Figure 5A,a) in S phase. The results demonstrate that both FIP-gat and FIP-glu could effectively inhibit the transition of MDA-MB-231 cells from G1 phase to S phase. Statistical analysis of three independent experiments showed that the changes in percentages of cells in G1 phase and S phase caused by FIP-gat or FIP-glu treatment were statistically significant (p < 0.05) (Figure 5B). Induction of Apoptotic Cell Death. To investigate the effects of rFIPs on apoptotic cell death of human MDA-MB231 cells, we performed flow fluorescence activated cell sorting analysis on MDA-MB-231 cells treated with FIP-gat (10 μg/ mL) or FIP-glu (10 μg/mL; Figure 6). We found that the cells treated with either FIP-gat or FIP-glu had a significantly higher percentage of cells (33.62 in Figure 6A,b and 36.94% in Figure 6A,c) than the control cells (10.39% Figure 6A,a) in late apoptotic stage. These results demonstrated that FIP-gat and FIP-glu have similar pro-apoptotic activities in MDA-MB-231 cells. Statistical analysis of three independent experiments showed that the percentage of cells in late apoptotic stage (upper right quadrant) was significantly higher (p < 0.05) upon FIP-gat or FIP-glu treatment, and a concomitant decrease in the living cell populations was also observed (p < 0.05) (Figure 6B). Identification and Verification of Differentially Expressed Genes Caused by FIP-gat Treatment. Microarray

Figure 4. Induction of MDA-MB-231 cell agglutination by rFIP-gat treatment: I−III, cells treated with rFIP-gat for 6, 12, and 24 h, respectively; (A) control cells after PBS treatment; (B−E) cells treated with 5, 10, 15, and 20 μg/mL rFIP-gat, respectively.

Inhibition of Cell Cycle Progression. The distribution of cells in different phases of cell cycle was determined on the basis of DNA content analysis; MDA-MB-231 cells treated with rFIP-gat or rFIP-glu had a higher percentage (83.85 or 84.42%, Figure 5A,b;Ac) than control cells (21.41%, Figure 5A,a) in G1 phase but lower percentages (4.33 or 4.13%, Figure 5A,b;A,c)

Figure 5. Effect of FIP-gat and FIP-glu on cell cycle distribution of MDA-MB-231 cells. MDA-MB-231 cells were seeded into a six-well plates at a density of 5 × 104 cells/m: in 2 mL of medium and cultured at 37 °C in a CO2 incubator for 24 h. Cells were then subjected to respective treatments including PBS (mock), FIP-gat (10 μg/mL), or FIP-glu (10 μg/mL) at 37 °C in a CO2 incubator for 48 h. Finally, the cells were trypsinized, counted, and stained with hypotonic propidium iodide staining solution and analyzed by FACS. The calculated proportion of the G1, S, and G2/M cells is also indicated. All experiments were performed in duplicate. The data from one representative experiment of three independent experiments are shown. (A, a) FACS analysis of MDA-MB-231 cells treated with (a) PBS, (b) FIP-gat, or (c) FIP-glu. (B) Statistical analysis of three independent experiments (∗, p < 0.05). 2694

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Figure 6. FACS analysis of apoptotic cells induced by FIP-gat and FIPglu. MDA-MB231 cells were seeded into a six-well plate at a density of 5 × 104 cells/mL in 2 mL of medium and cultured at 37 °C in a CO2 incubator for 24 h. Cells were then subjected to respective treatments including PBS (mock), FIP-gat (10 μg/mL), or FIP-glu (10 μg/mL) at 37 °C in a CO2 incubator for 48 h. Finally, the cells were trypsinized, counted, and stained with annexin V-PE and 7-AAD and analyzed by FACS. All experiments were performed in duplicates. The data from one representative experiment of three independent experiments are shown. (A) (a) Control cells treated with PBS; (b) cells treated with FIP-gat; (c) cells treated with FIP-glu. (B) Statistical analysis of three independent experiments (∗, p < 0.05).

analysis identified 669 differentially expressed genes with a minimum fold change of 2 (p < 0.05) upon rFIP-gat treatment. Among these genes, 343 genes were up-regulated and 326 genes were down-regulated (data not shown). Functional clustering of these genes using the online PATHER classification system revealed that these genes are involved in 96 biological processes (data not shown). Among these functional processes, cell death, cell growth, and cell adhesion are closely correlated with the phenotypic effects incurred by FIP-gat treatment. We then selected 10 genes (TNFSF8, SQSTM1, DUSP1, SMPD1, BCL-2, JAK2, ITPR1, CCR10, CCKBR, and DRD1) known to play important roles in regulating cell death and cell growth for subsequent confirmation using real-time PCR. The results showed that nine of these genes were up-regulated by >2-fold, the exception being the CCR10 gene (Figure 7).

Figure 7. Verificationof differentially expressed genes resulting from FIP-gat treatment by quantitative real-time PCR experiments. Ten upregulated genes relevant to the phenotypic effects of FIP-gat were analyzed by using the Cyber Green-based fluorescence assay or the TaqMan-based assay (marked with ∗). n = 3; data are presented as the mean (error bars indicate SD) and represented as fold changes from controls.

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DISCUSSION The FIP-gat gene isolated from G. atrum mycelia is a new member of the fungal immunomodulatory protein family. To functionally characterize the protein, the current study efficiently expressed the recombinant FIP-gat protein as Histagged fusion in E. coli BL21 strain. Pure rFIP-gat proteins were successfully recovered by routine NTA affinity purification. By using MDA-MB-231 breast cancer cells as a model, direct

effects of the protein on cell morphorlogy, cell growth, cell cycle, and apoptosis were explored. In the current studies, immunomodulatory activities of fungal proteins should refer to their indirect/direct inhibitory effect on cancer/tumor cells.2,20 For studying the indirect inhibitory 2695

DOI: 10.1021/acs.jafc.6b00539 J. Agric. Food Chem. 2016, 64, 2690−2698

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Figure 8. Proposed model for the potential molecular mechanisms of FIP-gat action on MDA-MB231 cancer cells. In this model, pro-death/death pathways are indicated in red, and the pro-survival/growth pathways are indicated in green.

direct clue for rFIP-gat triggered apoptosis.30,31 Activation of the type-1 receptor of the neurotransmitter dopamine (DRD1) has been reported to induce apoptosis in breast cancer cells.32 Furthermore, SMPD1, which encodes a lysosomal acid sphingomyelinase that converts sphingomyelin to ceramide, should also contribute to apoptotic cell death.33−35 Notably, increased expression of SQSTM1,which is a stress-inducible protein and a selective autophagy receptor,35 may lead to autophagic cell death.34,36−39 DUSP1 up-regulation contributes to the inhibition of breast cancer cell growth.40,41 Conversely, the up-regulated expression of several other genes including CCKBR, JAK2, ITPR1, and BCL-2 indicates the adaptive cell survival responses to stress. CCKBR is a growth factor receptor for cholecystokinin type-2; it has been shown to be positively correlated with cancer cell growth in gastric cancer, colon cancer, and pancreatic cancer.41−44 The Janus kinase 2(JAK2), a nonreceptor tyrosine kinase, is able to promote breast cancer cell growth.44−46 The inositol 1,4,5-trisphosphate receptor type 1(ITPR1), which is an endoplasmic reticulum (ER) Ca2+ release channel, can enhance calcium release from the ER into cytosol to benefit cell survival.47,48 The increased expression of the well-known anti-apoptosis protein BCL-2 is undoubtedly a manifestation of cell survival.49 On the basis of all these findings, we propose a model illustrating the potential biological processes through which the FIP-gat proteins exert their direct effects on breast cancer cells (Figure 8). At present, many questions pertaining to the molecular mechanisms of FIP-gat function remain to be answered. First, the binding specificity of FIP-gat toward cell surface glycoconjugates/receptors needs to be characterized. Second, the significance of agglutinating activity in triggering anticancer effects needs to be addressed. Third, the signaling pathways activated upon FIP-gat binding to its receptor need to be deciphered. Fourth, the roles of key intracellular molecules in executing FIP-gat function need to be elucidated. In short, indepth mechanistic studies need to be pursued further to

effect on the cancer cells, Liao et al. found that FIP-gts inhibited the growth of human adenocarcinoma A549 cells significantly and predicted that its effect was indirect, in which FIP-gts could regulate immunologic function via promoting NK cell and macrophage activation for inhibiting the growth of cancer cells,25 or controlling the transformation of the A549 cells for exhibiting antitumor activity.26 For studying the direct inhibitory effect on the cancer cells, Chang et al. reported that FIP-five, a fungal immunomodulatory protein isolated from F. velutipes, suppresses the proliferation of A549 lung cancer cells by causing cell cycle arrest in vitro.18 Cong et al. found that rFIPs (rFIP-SN and rFIP-glu) could directly inhibit cell cycle progression by retardation of G1/S transition as well as promote apoptosis in cultured human glioblastoma U-251 MG cells.24 In another study, Li et al. demonstrated that recombinant protein-rFIP-ppl (a fungal immunomodulatory protein isolated from Postia placenta) expressed in E. coli has significant growth-inhibiting and apoptosis-inducing effects on MGC823 gastric tumor cells.27 As demonstrated by morphological observation in the present study, the purified rFIP-gat protein is capable of mediating the agglutination of MDA-MB231 cells. The agglutinating activity could be mediated through interaction with either sugar moieties or proteins that are present on cell surface; the authentic mechanisms remain to be elaborated.20 The protein also exhibited a growth inhibition effect on breast cancer cells as determined by the CCK-8-based cell viability assay. Flow cytometry analyses confirmed that this protein significantly induces cell cycle arrest and apoptotic cell death. All of these results corroborate each other very well. These results are also consistent with several publications addressing the direct effects of FIPs on tumor cells.19,28,29 Gene expression microarray hybridization and quantitative real-time PCR assay were also conducted to probe the molecular mechanisms underlying the phenotypic effects incurred by rFIP-gat. Marked up-regulation of TNFSF8, a member of the tumor necrosis factor superfamily, provides a 2696

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Journal of Agricultural and Food Chemistry

(6) Li, W. J.; Nie, S. P.; Chen, Y.; Wang, X. Y.; Li, C.; Xie, M. Y. Enhancement of cyclophosphamide-induced antitumor effect by a novel polysaccharide from Ganoderma atrum in sarcoma 180-bearing mice. J. Agric. Food Chem. 2011, 59, 3707−3716. (7) Chen, Y.; Xie, M.; Zhang, H.; Wang, Y.; Nie, S.; Li, C. Quantification of total polysaccharides and triterpenoids in Ganoderma lucidum and Ganoderma atrum by near infrared spectroscopy and chemometrics. Food Chem. 2012, 135, 268−275. (8) Zhou, X. W.; Lin, J.; Li, Q. Z.; Yin, Y. Z.; Sun, X. F.; Tang, K. X. Study progress on bioactive proteins from Ganoderma spp. Nat. Prod. Res. Dev. 2007, 19, 916−924 (in Chinese). (9) Murasugi, A.; Tanaka, S.; Komiyama, N.; Iwata, N.; Kino, k.; Tsunoo, H.; Sakuma, S. Molecular cloning of a cDNA and a gene encoding an immunomodulatory protein, LingZhi-8, from a fungus, Ganoderma lucidum. J. Biol. Chem. 1991, 266, 2486−2493. (10) Li, Q. Z.; Zheng, S. B.; Wang, X. F.; Bao, T. W.; Zhou, X. W. Preparation of rabbit anti-Ganoderma sinensis immunomodulatory protein polyclonal antibody. Afr. J. Microbiol. Res. 2011, 5, 1562−1564. (11) Zhou, X. W.; Xie, M. Q.; Hong, F.; Li, Q. Z.; Lin, J. Genomic cloning and characterization of a FIP-gsi gene encoding a fungal immunomodulatory protein from Ganoderma sinense. Int. J. Med. Mushrooms 2009, 11, 77−86. (12) Li, F.; Wen, H. A.; Zhang, Y. J.; An, M.; Liu, X. Z. Purification and characterization of a novel immunomodulatory protein (FIP-tvc) from Trametes versicolor. Sci. China: Life Sci. 2011, 54 (4), 379−385. (13) Bastiaan-Net, S.; Chanput, W.; Hertz, A.; Zwittink, R. D.; Mes, J. J.; Wichers, H. J. Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs). Int. Immunopharmacol. 2013, 15, 167−175. (14) Debray, H.; Decout, D.; Strecker, G.; Spik, G.; Montreuil, J. Specificity of twelve lectins towards oligosaccharides and glycopeptides related to N-glycosylproteins. Eur. J. Biochem. 1981, 117, 41−55. (15) Liu, Y. F.; Chang, S. H.; Sun, H. L.; Chang, Y. C.; Hsin, I. L.; Lue, K. H.; Ko, J. L. IFN-γ induction on carbohydrate binding module of fungal immunomodulatory protein in human peripheral mononuclear cells. J. Agric. Food Chem. 2012, 60, 4914−4922. (16) Li, F.; Wen, H.; Liu, X.; Zhou, F.; Chen, G. Gene cloning and recombinant expression of a novel fungal immunomodulatory protein from Trametes versicolor. Protein Expression Purif. 2012, 82, 339−344. (17) Lee, Y. T.; Lee, S. S.; Sun, H. L.; Lu, K. H.; Ku, M. S.; Sheu, J. N.; Ko, J. L.; Lue, K. H. Effect of the fungal immunomodulatory protein FIP-fve on airway inflammation and cytokine production in mouse asthma model. Cytokine+ 2013, 61, 237−244. (18) Chang, Y. C.; Hsiao, Y. M.; Wu, M. F.; Ou, C. C.; Lin, Y. W.; Lue, K. H.; Ko, J. L. Interruption of lung cancer cell migration and proliferation by fungal immunomodulatory protein FIP-fve from Flammulina velutipes. J. Agric. Food Chem. 2013, 61, 12044−12052. (19) Li, J.-W.; Jia, J.; Shen, Y. H.; Zhong, M.; Chen, L. J.; Li, H. G.; Ma, H.; Guo, Z. F.; Qi, M. F.; Liu, L. X.; Li, T. L. Functional expression of FIP-fve, a fungal immunomodulatory protein from the edible mushroom Flammulina velutipes in Pichia pastoris GS115. J. Biotechnol. 2013, 168 (4), 527−533. (20) Wang, X. F.; Su, K. Q.; Bao, T. W.; Cong, W. R.; Chen, Y. F.; Li, Q. Z.; Zhou, X. W. Immunomodulatory effects of fungal proteins. Curr. Top. Nutraceut. 2012, 10 (1), 1−11. (21) Su, K. Q.; Wang, X. F.; Zhou, X. W. Cloning and bioinformatics analysis of fugal immunomodulatory protein gene from Ganoderma atrum. J. Shanghai Jiaotong Univ. (Agric. Sci.) 2012, 30, 65−71 (in Chinese). (22) Li, Q. Z.; Wang, X. F.; Chen, Y. Y.; Lin, J.; Zhou, X. W. Cytokines expression induced by Ganoderma sinensis fungal immunomodulatory proteins (FIP-gsi) in mouse spleen cells. Appl. Biochem. Biotechnol. 2010, 162 (5), 1403−1413. (23) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning; Cold Spring Harbor Laboratory Press: NewYork, 1989; Vol. 2, pp 14−19. (24) Cong, W. R.; Xu, H.; Liu, Y.; Li, Q. Z.; Li, W.; Zhou, X. W. Production and functional characterization of a novel fungal immunomodulatory protein FIP-SN shuffled from two genes of

understand the bioprocesses implicated in the anticancer function of FIP-gat. In conclusion, this study has clarified the direct cytotoxic effect of recombinant FIP-gat protein on breast cancer cells and discovered some potential molecular mechanisms on a gene expression level. This has laid a foundation for further assessment of pharmacological functions and deeper elucidation of biological pathways of this G. atrum-derived protein. All relevant works are necessary for evaluating the potential of rFIP-gat as an anticancer drug candidate.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00539. Primers and amplicons for Cyber Green-based real-time PCR (Table S1) (PDF) Characterization of pET30a::FIP-gat plasmid by PCR and restriction enzyme digestion (Figure S1) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(X.-W.Z.) E-mail: [email protected]. Phone: +86-2134205778. Fax: +86-21-34205778. *(W.L.) E-mail: [email protected]. Phone: +86-21-34205885. Fax: +86-21-34206059. Author Contributions ∥

H.X. and Y.-Y.K. contributed equally to this work.

Funding

This work was supported by Grant 30771500 from the National Natural Science Foundation of China. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED FIPs, fungal immunomodulatory proteins; rFIP, recombinat FIP; ALP, alkaline phosphatase; Ni-NTA, nickel−nitrilotriacetic acid; LPS, lipopolysaccharide; SDS-PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide; LC/Q-TOF MS, liquid chromatography coupled with quadrupole time-of-flight mass spectrometry; ESIQ-TOF, electrospray ionization−quadrupole time-of-flight; qRT-PCR, quantitative real-time PCR

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REFERENCES

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DOI: 10.1021/acs.jafc.6b00539 J. Agric. Food Chem. 2016, 64, 2690−2698

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