Article pubs.acs.org/JAFC
Antiproliferative Protein from the Culture Supernatant of Lentinula Edodes C91‑3 Mycelia Xingyun Li, Mintao Zhong, Ben Liu, Xiaoli Wang, Lei Liu, Wei Zhang, and Min Huang* Department of Microbiology, Colleges of Basic Medical Sciences, Dalian Medical University, 9 Western Section, Lvshun South Street, Lvshunkou District, Dalian 116044, Liaoning Province, P.R. China ABSTRACT: We purified and isolated a novel protein (LFP91‑3A2) with antitumor effect from Lentinula edodes C91‑3 liquid mycelial culture supernatant. LFP91‑3A2 was purified by (NH4)2SO4 precipitation, ion-exchange chromatography (DEAEcellulose) and gel filtration chromatography (Sephacryl S-200HR). SDS-PAGE and MALDI-TOF/MS analysis Mascot search showed LFP91‑3A2 is a new protein with apparent molecular weight of 26 kDa. The effect on tumor cell proliferation was assessed by using MTT assay in vitro, and the LFP91‑3A2 reduced tumor cell growth obviously in a dose dependent manner (5−15 μg/ mL) (p < 0.05), while it exhibited no toxic effect on normal chick embryo fibroblasts. The antiproliferative mechanism of LFP91‑3A2 was found to be associated with inducing cell apoptosis by flow cytometry analysis and transmission electron microscopy. The LFP91‑3A2 is a novel protein from Lentinula edodes with tumor-suppressive activity via inducing apoptosis of tumor cells without toxicity on normal cells and may be beneficial to natural products in clinical treatment. KEYWORDS: Lentinula edodes, protein, apoptosis
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Pharmacia Biotech) and a Sephacryl S-200 HR column (Sigma). The eluted fractions were collected and detected at 280 nm using a UV detector (BIO-RAD Econo). All isolation processes were performed at 4 °C. The molecular weight of protein was determined according to the method of Laemmli (1970).25 SDS−PAGE was prepared with 12.5% running gel and 5% stacking gels. A series of molecular weight marker proteins (6−205 kDa) (Takara) was run as marker to determine the molecular weight. MALDI-TOF-MS Identification of Protein. The protein band was cut from the gel and sequenced from the amino terminus using the trypsase digestion procedure. The proteolytic sample was identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Peptide mass fingerprints analyzed by the MALDITOF MS were sequenced in the Mascot search engine based on three databases (MSDB, SwissProt, NCBInr). Assay of Antiproliferative Activity on Tumor Cell Lines. The cell lines A549, H22 and S180 were prepared for MTT assays to test cytotoxicity on tumor cells, while the chick embryo fibroblasts were tested as normal cells. Cells (1 × 105) were dispensed into 96-well plates in the exponential growth phase and incubated with LFP91‑3A2 at given concentrations (5, 10 and 15 μg/mL) for 24, 48 and 72 h, respectively. Under the same conditions, to the negative control group was added 10% FBS RPMI-1640, and to the positive control group was added 5 μg/mL of chemotherapeutics DDP (Cisplatin). The effects of LFP91‑3A2 on cell proliferation were assessed using the MTT assay with a plate reader (model 550; Bio-Rad, CA, USA) with absorbance at 570 nm. The experiments were performed in triplicate, and the results were presented as mean ± SD (n = 3 measurements). Cell inhibitory rate was calculated in comparison with a control group treated with 10% FBS RPMI-1640 flow, with the following formula: inhibitory rate (%) = (1 − Atreated/Acontrol) × 100%. Flow Cytometry Analysis (Annexin V/PI) Antiproliferation. Cells (1 × 106) were seeded in 6-well plates and treated with different
INTRODUCTION Lentinula edodes (Berk.) Pegler, known in the English-speaking world by its Japanese name shiitake, is one of the most commonly cultivated edible mushrooms native to East Asia.1 In the last few decades, Lentinus edodes have been invested deeply for their medicinal benefits, especially their antitumor properties. Several well-studied components, such as lentinan, lectins and eritadenine, have been isolated and characterized from Lentinula edodes mycelium or fruiting body and proven to have significant pharmacological properties, and some of them have been used clinically.2,3 Among these bioactive constituents from Lentinula edodes, polysaccharides are the most extensively invested and are used clinically for cancer treatment. Thus, little is known about the protein with antitumor activity from Lentinula edodes. We isolated a strain of Lentinula edodes from Basidiomycetes Umbelliferae fungi in1991, named Lentinula edodes C91‑3, and extracted crude protein (LFP91‑3) from the mycelium fermentation broth which exhibited significantly effects on inducing tumor cell apoptosis both in vivo and in vitro.4,5 In this study, we described a novel protein, designated LFP91‑3A2, isolated and purified from the Lentinula edodes C91‑3 mycelium fermentative liquid. The objective of the present project is concerned with the isolation of its molecular characterization and investigation of its bioactivities based on the cell growth inhibitory effect on tumor cells in vitro.
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EXPERIMENTAL PROCEDURE
Purification of LFP91‑3-A2. The Lentinula edodes C91‑3 strain was obtained from the Department of Microbiology at Dalian Medical University, and the mycelium of Lentinula edodes C91‑3 was cultured with integrated potato culture medium.6 The incubation broth was treated with (NH4)2SO4 to 95% of saturation and centrifuged (12000g, 20 min, 4 °C) to precipitate the soluble proteins in the supernatant. The precipitates were dialyzed against 10 mM Tris-HCl buffer, pH 8.5 for 72 h and then subjected to a DEAE−cellulose column (Amersham © 2014 American Chemical Society
Received: Revised: Accepted: Published: 5316
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Journal of Agricultural and Food Chemistry
Article
concentrations of LFP91‑3A2 for 24 h. The cell pellets were washed with PBS after centrifugation and having supernatant removed, and they were stained with annexin V-FITC and PI in binding buffer according to the manufacturer’s protocol. Cells were incubated at room temperature for 15 min in the dark, and flow cytometry was conducted using emission filters of 525 and 575 nm within 10 min, respectively. Transmission Electron Microscope. For conventional transmission electron microscopy, the sample was postfixed with 1% buffered osmium at 4 °C for 2 h, dehydrated by an ethanol series and embedded in EPON 812. The semithin sections were stained by 1% Toluidine blue dye (TB) . Statistical Analysis. All data are expressed as mean ± SD. Statistical analysis was evaluated by one-way analysis of variance (Anova), and calculations were done by the SPSS/Win 13.0 software. P < 0.05 was considered as the statistically significant difference.
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RESULTS Purification of LFP91‑3A2. The Lentinula edodes C91‑3 mycelium fermentative liquid extract from (NH 4 ) 2 SO 4 precipitation (95% saturation) was applied on the DEAE− cellulose column. Four peaks (A, B, C and D) were eluted (Figure 1), and peak A (LFP91‑3A2) showed distinctive biological activity and then was subjected to a Sephacryl S200 HR column in the next step; two peaks were obtained from the Sephacryl S-200 HR column, and the biological activity was largely confined to the fraction peak A2 (LPF91‑3A2) (Figure 1). The homogeneity of purified LFP91‑3A2 checked by SDS− PAGES appeared as a single band with a molecular mass of 26 kDa (Figure 1). At the same time, a single peak with the same molecular mass was obtained upon rechromatography on Superdex 75. The protein gel did not stain with the periodic acid-Schiff (PAS) reagent after being subjected to glycoprotein staining. Polysaccharides isolated from mushrooms are best known for their antitumor macromolecules both in vitro and in vivo; some are already used in clinical cancer treatment.7,8 Recently, various protein compounds, such as polysaccharide−peptides and polysaccharide−protein complexes, have been proven to possess effective antitumor activities;9 however, only a few reports about proteins from mushroom have been identified and even fewer characterized, although mushrooms are an abounding source of diverse proteins. SDS-PAGE results and PAS negatively demonstrated that the LPF91‑3A2 is a pure protein without any polysaccharide conjugates. Mushrooms contain a large number of biologically active proteins and peptides. The general protocol for isolating and purifying the target bioactive protein in a pure form consists of several steps.10,11 In our study, two-step serial gel chromatography and ion exchange chromatography to isolate and purify protein LPF91‑3A2 proved to be simpler and more rapid than the frequently used protocol for separating protein from mushroom. Assay of Antiproliferative Activity of LPF91‑3A2 on Tumor Cell Lines in Vitro. The cytotoxicity of LFP91‑3A2 on tumor cells and normal cells was examined in vitro by using the MTT microculture assay. H22, S180, A549 and the chick embryo fibroblasts growth proliferation were investigated after different incubation periods with different concentrations of LPF91‑3A2 (Figure 2). H22 and A549 cell growth were pressed in a dose-dependent manner which responded to increasing concentrations of LPF91‑3A2 (5−15 μg/mL). S180 growth also proliferated by LFP91‑3A2; although it had low significance in contrast to the other tumor cell lines, the difference was
Figure 1. (a) Ion exchange chromatography on DEAE-cellulose of the mycelium fermentative liquid extract of Lentinula edodes C91-3. Adsorbed fraction A showed prominent biological activity. (b) Gel filtration on a Sephacryl S-200 HR column of peak A. Two peaks were obtained, and biological activity was largely confined to fraction peak A2. (c) SDS-PAGE of LFP91-3A2. Left lane: Amersham Biosciences molecular mass markers. Right lane: LFP91-3A2. Samples were electrophoresced on a 15% gel and stained with silver.
significant compared with the negative control groups (p < 0.05). H22 cells had a more insignificant inhibition rate after being treated with LPF91‑3A2, so this lineage was used to test exact the proliferation mechanism further. The growth of the chick embryo fibroblasts was not suppressed at given concentrations of LFP91‑3A2, and the difference was not significant compared with the negative control groups. The tumor cells were also inhibited by 5 μg/mL of DDP but with no more pronounced effects compared with the groups treated 5317
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were significantly higher compared with those of the control group at 5, 10 and 15 μg/mL of LPF91‑3A2, and it induced H22 cell apoptosis in a dose-dependent manner. All the experiments were repeated three times and obtained similar results. Although the MTT and the flow cytometry assay results present strong evidence that LFP91‑3A2 could activate the apoptotic pathway in tumor cells, the morphological characteristics in apoptosis need to be observed by transmission electron microscopy to consolidate the results of cytotoxicity tests. Morphological changes of the H22 cells were observed after treatment by 5 μg/mL of LPF91‑3A2 in 24 h. Transmission electron microscopy observation showed that H22 cells had the early changes of apoptosis, such as cell shrinkage, floating of the cell bodies and microvilli disappearance, nuclear chromatin condensation and formation of a crescent clump, which are the typical morphological appearance of apoptosis (Figure 4a). After treatment with 15 μg/mL of LPF91‑3A2, chromatin was condensed strongly, forming a high-density area, and the apoptotic body was observed in the H22 cell (Figure 4b). The morphological changes in LPF91‑3A2 treated tumor cells observed by transmission electron microscopy gave further evidence of the antitumor activity. The antitumor activities of polysaccharides and polysaccharide−protein complexes from Lentinula edodes are considered dependent on activation of the host immune system rather than direct cytotoxic effects on the cancer cells.13−15 Recently, a few studies demonstrated that polysaccharide from Lentinula edodes inhibited tumor cell proliferation not only by enhancing the host immunity but also by directly inducing tumor cell apoptosis.11,16,17 LPF91‑3A2 showed direct cytotoxic and cell growth inhibitory effects without being accompanied by
Figure 2. Effect of LFP91‑3A2 on viability of cell lines S180, H22, A549 and the normal chick embryo fibroblasts (CEF). The cytotoxicity of LFP91‑3A2 on the tumor lines S180, H22, A549 and against the normal chick embryo fibroblasts was performed by MTT reduction assay. The test cells were treated with different concentrations of LFP91‑3A2 (5− 15 μg/mL) for 24, 48, and 72 h of culture in microplates. 5 μg/mL of DDP was used as positive control. Results represent the mean ± SD (standard deviation) of three experiments run in three replicates. *p < 0.001 compared to control.
with different concentrations of LPF91‑3A2 under the same conditions. Our findings revealed that LFP91‑3A2 suppressed tumor cells remarkably without side effects on the normal cells. This is particularly important, since many traditional chemotherapeutic agents and ratios exhibit severe toxicity against the normal cells, causing undesirable side effects and thus limiting their application in the clinical field.10,12 For further characterization, LPF91‑3A2 induced tumor cell death was analyzed by flow cytomery after staining H22 cells with annexin V-FITC/PI. As shown in Figure 3, the early and late apoptosis rates of the cells which were incubated for 24 h
Figure 3. Assessment of apoptosis using flow cytometry with Annexin V-FITC/PI staining, showing the dot-plot graph of H22cells: (a) control group; (b) H22 treated with 5 μg/mL of LFP91-3A2; (c) H22 treated with 10 μg/mL of LFP91‑3A2; (d) H22 treated with 15 μg/mL of LFP91-3A2. 5318
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Figure 4. Tumor cell apoptosis morphology examined by transmission electron microscopy. (A, left) Native control. (B, right) The short microvilli disappeared and chromatin was condensed strongly, forming a high-density area.
Figure 5. Peptide fragments of LFP91‑3A2 generated by trypsin cleavage analysis by MS/MS.
stimulating the immune system, which is distinct from extracts previously mentioned. Mass Spectrometric Identification of Novel Proteins. The purified sample of LPF91‑3A2 was excised, digested by trypure in-gel and characterized by matrix assisted laser desorption/ionization/time of flight (MALDI-TOF) MS. Using the Mascot search engine (http://www.matrixscience. com), the MALDI-TOF data obtained from LPF91‑3A2 was matched significantly with adenylate kinase isoenzyme2 mitochondrial [Harpengnathos saltalor](gi|307198091). The score of mascot search matching was 85, and sequence coverage was 27%. Adenylate kinase isoenzyme2 is localized in the space of mitochondrial intermembrane and plays a role in mediating a novel intrinsic apoptotic pathway that may be involved in tumorigenesis processes. Adenylate kinase isoenzyme2 is released from mitochondria during the early phase of the apoptotic process.18,19 The antitumor activity of LFP91‑3A2 was supposed to be related to the similar molecular structure with adenylate kinase isoenzyme2. Three peptide fragments generated from LFP91‑3A2 by trypsin cleavage were subjected to MS/MS analysis for sequencing (Figure 5). The amino acid sequences were (a) NVAVPLYNR, (b)
SDGANGLLTK and (c) NKYEDELNKR. However, the BLAST searches in several databases of de novo amino acid sequences derived from analysis of the 26 kDa band failed to identify any homologous proteins.20 The paucity of related fungi protein amino acid sequences in accessible databases may be the reason why we found no homologous proteins from mushroom. In order to well know the properties of LPF91‑3A2, we leveraged Solexa sequencing technology in de novo assembly of Lentinula edodes C91‑3 transcriptome and obtained 28,923 unigene sequences including 18,120 unigenes with a coding sequence.6 Based on this gene information, we can obtain deep insight into the medical function, especially antitumor activity at the protein level in Lentinula edodes. The present study was carried out to purify and characterize active antitumor protein LPF91‑3A2 from the mycelium fermentative liquid extract of Lentinula edodes C91‑3. LPF91‑3A2 was isolated and purified to homogeneity after (NH4)2SO4 precipitation and two steps of chromatography analysis. SDSPAGE results are in accordance with MALDI/MS analysis, which show an approximate molecular weight of 26 kDa. The role of protein molecules from natural resources in drug and toxin-induced apoptosis is not clearly defined. In fact, very little 5319
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(14) Lull, C.; Wichers, H.; Savelkoul, H. Antiinflammatory and immunomodulating properties of fungal metabolites. Mediators Inflamm. 2005, 63−80. (15) Lo, T. C.; Hsu, F. M.; Chang, C. A.; Cheng, J. C. Branched α(1,4) glucans from Lentinula edodes (L10) in combination with radiation enhance cytotoxic effect on human lung adenocarcinoma through the Toll-like receptor 4 mediated induction of THP-1. J. Agric. Food Chem. 2011, 59, 11997−12005. (16) Israilides, C.; Kletsas, D.; Arapoglou, D.; Philippoussis, A.; Pratsinis, H.; Ebringerova, A.; Hribalova, V.; Harding, S. E. In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes. Phytomed.: Int. J. Phytother. Phytopharmacol. 2008, 15, 512−9. (17) Yukawa, H.; Ishikawa, S.; Kawanishi, T.; Tamesada, M.; Tomi, H. Direct cytotoxicity of Lentinula edodes mycelia extract on human hepatocellular carcinoma cell line. Biol. Pharm. Bull. 2012, 35, 1014− 21. (18) Kohler, C.; Gahm, A.; Noma, T.; Nakazawa, A.; Orrenius, S.; Zhivotovsky, B. Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis. FEBS Lett. 1999, 447, 10−2. (19) Lee, H.; Pyo, J.; Oh, Y.; Kim, H.; Hong, S.; Jeon, Y.; Kim, H.; Cho, D.; Woo, H.; Song, S.; Nam, J.; Kim, H.; Kim, K.; Jung, Y. AK2 activates a novel apoptotic pathway through formation of a complex with FADD and caspase-10. Nat. Cell Biol. 2007, 1303−1310. (20) Au, C. H.; Wong, M. C.; Bao, D.; Zhang, M.; Song, C.; Song, W.; Law, P. T.; Kues, U.; Kwan, H. S. The genetic structure of the A mating-type locus of Lentinula edodes. Gene 2014, 535, 184−90. (21) Tsoi, A. Y.; Ng, T. B.; Fong, W. P. Antioxidative effect of a chymotrypsin inhibitor from Momordica cochinchinensis (Cucurbitaceae) seeds in a primary rat hepatocyte culture. J. Peptide Sci. 2005, 11, 665−8. (22) Lee, S.; Oh, P.; Ko, J.; Lim, K.; Lim, K. Protective effect of glycoprotein isolated from Ulmus davidiana Nakai on carbon tetrachloride-induced mouse liver injury. J. Pharm. Pharmacol. 2006, 58, 143−152. (23) Oh, P.; Lim, K. Plant originated glycoprotein has anti-oxidative and anti-inflammatory effects on dextran sulfate sodium-induced colitis in mouse. J. Biomed. Sci. 2006, 13, 549−560. (24) Liu, B.; Zhong, M.; Lun, Y.; Wang, X.; Sun, W.; Li, X.; Ning, A.; Cao, J.; Zhang, W.; Liu, L.; Huang, M. A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C(91-3). Int. J. Mol. Sci. 2012, 13, 6246−65. (25) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680− 685.
information is available in the literature about this regard. A few articles, published recently, revealed that some protein molecules from various plant sources possess activities like those of our protein, although these reports do not describe any mechanism of protective action or structural features of the active principles.21−23 In this study, we isolated and partially characterized antitumor protein LFP91‑3A2 from Lentinula edodes C91‑3 However, the identities and detailed characteristic properties of LFP91‑3A2 remain unknown up to now. Further studies are needed such as molecular properties, gene clone, expression and characterization of LFP91‑3A2. All these experiments are now under investigation in our laboratory based on the transcriptome sequence.24 These antitumor studies would be helpful for its potential medical application in the future.
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AUTHOR INFORMATION
Corresponding Author
*Tel.:0411 86110007. Fax: 0411 86110007. E-mail address: [email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Watanabe, A.; Kobayashi, M.; Hayashi, S.; Kodama, D.; Isoda, K.; Kondoh, M.; Kawase, M.; Tamesada, M.; Yagi, K. Protection against D-galactosamine-induced acute liver injury by oral administration of extracts from Lentinus edodes mycelia. Biol. Pharm. Bull. 2006, 29, 1651−4. (2) Lee, H. H.; Lee, J. S.; Cho, J. Y.; Kim, Y. E.; Hong, E. K. Study on immunostimulating activity of macrophage treated with purified polysaccharides from liquid culture and fruiting body of Lentinus edodes. J. Microbiol. Biotechnol. 2009, 19, 566−72. (3) Bisen, P. S.; Baghel, R. K.; Sanodiya, B. S.; Thakur, G. S.; Prasad, G. B. Lentinus edodes: a macrofungus with pharmacological activities. Curr. Med. Chem. 2010, 17, 2419−2430. (4) Takehara, M.; Mori, K.; Kuida, K.; Hanawa, M. A. Antitumor effect of virus-like particles from Lentinus edodes (Shiitake) on Ehrlich ascites carcinoma in mice. Arch. Virol. 1981, 68, 297−301. (5) Enman, J.; Hodge, D.; Berglund, K. A.; Rova, U. Production of the bioactive compound eritadenine by submerged cultivation of shiitake (Lentinus edodes) mycelia. J. Agric. Food Chem. 2008, 56, 2609−12. (6) Zhong, M.; Liu, B.; Wang, X.; Liu, L.; Lun, Y.; Li, X.; Ning, A.; Cao, J.; Huang, M. De novo characterization of Lentinula edodes C913 transcriptome by deep Solexa sequencing. Biochem. Biophys. Res. Commun. 2012, 431, 111−115. (7) Lindequist, U.; Niedermeyer, T. H.; Julich, W. D. The pharmacological potential of mushrooms. Evidence-based complementary and alternative medicine: eCAM 2005, 2, 285−99. (8) Wasser, S. P. Current findings, future trends, and unsolved problems in studies of medicinal mushrooms. Appl. Microbiol. Biotechnol. 2011, 89, 1323−32. (9) Erjavec, J.; Kos, J.; Ravnikar, M.; Dreo, T.; Sabotic, J. Proteins of higher fungifrom forest to application. Trends Biotechnol. 2012, 30, 259−73. (10) Newman, D.; Cragg, G. Natural products from marine invertebrates and microbes as modulators of antitumor targets. Curr. Drug Targets 2006, 279−304. (11) Xu, X.; Yan, H.; Chen, J.; Zhang, X. Bioactive proteins from mushrooms. Biotechnol. Adv. 2011, 29, 667−674. (12) Ren, R. Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat. Rev. Cancer 2005, 5, 172−83. (13) Wasser, S. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 2002, 60, 258−274. 5320
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Antiproliferative Protein from the Culture Supernatant of Lentinula Edodes C91‑3 Mycelia Xingyun Li, Mintao Zhong, Ben Liu, Xiaoli Wang, Lei Liu, Wei Zhang, and Min Huang* Department of Microbiology, Colleges of Basic Medical Sciences, Dalian Medical University, 9 Western Section, Lvshun South Street, Lvshunkou District, Dalian 116044, Liaoning Province, P.R. China ABSTRACT: We purified and isolated a novel protein (LFP91‑3A2) with antitumor effect from Lentinula edodes C91‑3 liquid mycelial culture supernatant. LFP91‑3A2 was purified by (NH4)2SO4 precipitation, ion-exchange chromatography (DEAEcellulose) and gel filtration chromatography (Sephacryl S-200HR). SDS-PAGE and MALDI-TOF/MS analysis Mascot search showed LFP91‑3A2 is a new protein with apparent molecular weight of 26 kDa. The effect on tumor cell proliferation was assessed by using MTT assay in vitro, and the LFP91‑3A2 reduced tumor cell growth obviously in a dose dependent manner (5−15 μg/ mL) (p < 0.05), while it exhibited no toxic effect on normal chick embryo fibroblasts. The antiproliferative mechanism of LFP91‑3A2 was found to be associated with inducing cell apoptosis by flow cytometry analysis and transmission electron microscopy. The LFP91‑3A2 is a novel protein from Lentinula edodes with tumor-suppressive activity via inducing apoptosis of tumor cells without toxicity on normal cells and may be beneficial to natural products in clinical treatment. KEYWORDS: Lentinula edodes, protein, apoptosis
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Pharmacia Biotech) and a Sephacryl S-200 HR column (Sigma). The eluted fractions were collected and detected at 280 nm using a UV detector (BIO-RAD Econo). All isolation processes were performed at 4 °C. The molecular weight of protein was determined according to the method of Laemmli (1970).25 SDS−PAGE was prepared with 12.5% running gel and 5% stacking gels. A series of molecular weight marker proteins (6−205 kDa) (Takara) was run as marker to determine the molecular weight. MALDI-TOF-MS Identification of Protein. The protein band was cut from the gel and sequenced from the amino terminus using the trypsase digestion procedure. The proteolytic sample was identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Peptide mass fingerprints analyzed by the MALDITOF MS were sequenced in the Mascot search engine based on three databases (MSDB, SwissProt, NCBInr). Assay of Antiproliferative Activity on Tumor Cell Lines. The cell lines A549, H22 and S180 were prepared for MTT assays to test cytotoxicity on tumor cells, while the chick embryo fibroblasts were tested as normal cells. Cells (1 × 105) were dispensed into 96-well plates in the exponential growth phase and incubated with LFP91‑3A2 at given concentrations (5, 10 and 15 μg/mL) for 24, 48 and 72 h, respectively. Under the same conditions, to the negative control group was added 10% FBS RPMI-1640, and to the positive control group was added 5 μg/mL of chemotherapeutics DDP (Cisplatin). The effects of LFP91‑3A2 on cell proliferation were assessed using the MTT assay with a plate reader (model 550; Bio-Rad, CA, USA) with absorbance at 570 nm. The experiments were performed in triplicate, and the results were presented as mean ± SD (n = 3 measurements). Cell inhibitory rate was calculated in comparison with a control group treated with 10% FBS RPMI-1640 flow, with the following formula: inhibitory rate (%) = (1 − Atreated/Acontrol) × 100%. Flow Cytometry Analysis (Annexin V/PI) Antiproliferation. Cells (1 × 106) were seeded in 6-well plates and treated with different
INTRODUCTION Lentinula edodes (Berk.) Pegler, known in the English-speaking world by its Japanese name shiitake, is one of the most commonly cultivated edible mushrooms native to East Asia.1 In the last few decades, Lentinus edodes have been invested deeply for their medicinal benefits, especially their antitumor properties. Several well-studied components, such as lentinan, lectins and eritadenine, have been isolated and characterized from Lentinula edodes mycelium or fruiting body and proven to have significant pharmacological properties, and some of them have been used clinically.2,3 Among these bioactive constituents from Lentinula edodes, polysaccharides are the most extensively invested and are used clinically for cancer treatment. Thus, little is known about the protein with antitumor activity from Lentinula edodes. We isolated a strain of Lentinula edodes from Basidiomycetes Umbelliferae fungi in1991, named Lentinula edodes C91‑3, and extracted crude protein (LFP91‑3) from the mycelium fermentation broth which exhibited significantly effects on inducing tumor cell apoptosis both in vivo and in vitro.4,5 In this study, we described a novel protein, designated LFP91‑3A2, isolated and purified from the Lentinula edodes C91‑3 mycelium fermentative liquid. The objective of the present project is concerned with the isolation of its molecular characterization and investigation of its bioactivities based on the cell growth inhibitory effect on tumor cells in vitro.
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EXPERIMENTAL PROCEDURE
Purification of LFP91‑3-A2. The Lentinula edodes C91‑3 strain was obtained from the Department of Microbiology at Dalian Medical University, and the mycelium of Lentinula edodes C91‑3 was cultured with integrated potato culture medium.6 The incubation broth was treated with (NH4)2SO4 to 95% of saturation and centrifuged (12000g, 20 min, 4 °C) to precipitate the soluble proteins in the supernatant. The precipitates were dialyzed against 10 mM Tris-HCl buffer, pH 8.5 for 72 h and then subjected to a DEAE−cellulose column (Amersham © 2014 American Chemical Society
Received: Revised: Accepted: Published: 5316
March 5, 2014 May 18, 2014 May 18, 2014 May 19, 2014 dx.doi.org/10.1021/jf500316f | J. Agric. Food Chem. 2014, 62, 5316−5320
Journal of Agricultural and Food Chemistry
Article
concentrations of LFP91‑3A2 for 24 h. The cell pellets were washed with PBS after centrifugation and having supernatant removed, and they were stained with annexin V-FITC and PI in binding buffer according to the manufacturer’s protocol. Cells were incubated at room temperature for 15 min in the dark, and flow cytometry was conducted using emission filters of 525 and 575 nm within 10 min, respectively. Transmission Electron Microscope. For conventional transmission electron microscopy, the sample was postfixed with 1% buffered osmium at 4 °C for 2 h, dehydrated by an ethanol series and embedded in EPON 812. The semithin sections were stained by 1% Toluidine blue dye (TB) . Statistical Analysis. All data are expressed as mean ± SD. Statistical analysis was evaluated by one-way analysis of variance (Anova), and calculations were done by the SPSS/Win 13.0 software. P < 0.05 was considered as the statistically significant difference.
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RESULTS Purification of LFP91‑3A2. The Lentinula edodes C91‑3 mycelium fermentative liquid extract from (NH 4 ) 2 SO 4 precipitation (95% saturation) was applied on the DEAE− cellulose column. Four peaks (A, B, C and D) were eluted (Figure 1), and peak A (LFP91‑3A2) showed distinctive biological activity and then was subjected to a Sephacryl S200 HR column in the next step; two peaks were obtained from the Sephacryl S-200 HR column, and the biological activity was largely confined to the fraction peak A2 (LPF91‑3A2) (Figure 1). The homogeneity of purified LFP91‑3A2 checked by SDS− PAGES appeared as a single band with a molecular mass of 26 kDa (Figure 1). At the same time, a single peak with the same molecular mass was obtained upon rechromatography on Superdex 75. The protein gel did not stain with the periodic acid-Schiff (PAS) reagent after being subjected to glycoprotein staining. Polysaccharides isolated from mushrooms are best known for their antitumor macromolecules both in vitro and in vivo; some are already used in clinical cancer treatment.7,8 Recently, various protein compounds, such as polysaccharide−peptides and polysaccharide−protein complexes, have been proven to possess effective antitumor activities;9 however, only a few reports about proteins from mushroom have been identified and even fewer characterized, although mushrooms are an abounding source of diverse proteins. SDS-PAGE results and PAS negatively demonstrated that the LPF91‑3A2 is a pure protein without any polysaccharide conjugates. Mushrooms contain a large number of biologically active proteins and peptides. The general protocol for isolating and purifying the target bioactive protein in a pure form consists of several steps.10,11 In our study, two-step serial gel chromatography and ion exchange chromatography to isolate and purify protein LPF91‑3A2 proved to be simpler and more rapid than the frequently used protocol for separating protein from mushroom. Assay of Antiproliferative Activity of LPF91‑3A2 on Tumor Cell Lines in Vitro. The cytotoxicity of LFP91‑3A2 on tumor cells and normal cells was examined in vitro by using the MTT microculture assay. H22, S180, A549 and the chick embryo fibroblasts growth proliferation were investigated after different incubation periods with different concentrations of LPF91‑3A2 (Figure 2). H22 and A549 cell growth were pressed in a dose-dependent manner which responded to increasing concentrations of LPF91‑3A2 (5−15 μg/mL). S180 growth also proliferated by LFP91‑3A2; although it had low significance in contrast to the other tumor cell lines, the difference was
Figure 1. (a) Ion exchange chromatography on DEAE-cellulose of the mycelium fermentative liquid extract of Lentinula edodes C91-3. Adsorbed fraction A showed prominent biological activity. (b) Gel filtration on a Sephacryl S-200 HR column of peak A. Two peaks were obtained, and biological activity was largely confined to fraction peak A2. (c) SDS-PAGE of LFP91-3A2. Left lane: Amersham Biosciences molecular mass markers. Right lane: LFP91-3A2. Samples were electrophoresced on a 15% gel and stained with silver.
significant compared with the negative control groups (p < 0.05). H22 cells had a more insignificant inhibition rate after being treated with LPF91‑3A2, so this lineage was used to test exact the proliferation mechanism further. The growth of the chick embryo fibroblasts was not suppressed at given concentrations of LFP91‑3A2, and the difference was not significant compared with the negative control groups. The tumor cells were also inhibited by 5 μg/mL of DDP but with no more pronounced effects compared with the groups treated 5317
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were significantly higher compared with those of the control group at 5, 10 and 15 μg/mL of LPF91‑3A2, and it induced H22 cell apoptosis in a dose-dependent manner. All the experiments were repeated three times and obtained similar results. Although the MTT and the flow cytometry assay results present strong evidence that LFP91‑3A2 could activate the apoptotic pathway in tumor cells, the morphological characteristics in apoptosis need to be observed by transmission electron microscopy to consolidate the results of cytotoxicity tests. Morphological changes of the H22 cells were observed after treatment by 5 μg/mL of LPF91‑3A2 in 24 h. Transmission electron microscopy observation showed that H22 cells had the early changes of apoptosis, such as cell shrinkage, floating of the cell bodies and microvilli disappearance, nuclear chromatin condensation and formation of a crescent clump, which are the typical morphological appearance of apoptosis (Figure 4a). After treatment with 15 μg/mL of LPF91‑3A2, chromatin was condensed strongly, forming a high-density area, and the apoptotic body was observed in the H22 cell (Figure 4b). The morphological changes in LPF91‑3A2 treated tumor cells observed by transmission electron microscopy gave further evidence of the antitumor activity. The antitumor activities of polysaccharides and polysaccharide−protein complexes from Lentinula edodes are considered dependent on activation of the host immune system rather than direct cytotoxic effects on the cancer cells.13−15 Recently, a few studies demonstrated that polysaccharide from Lentinula edodes inhibited tumor cell proliferation not only by enhancing the host immunity but also by directly inducing tumor cell apoptosis.11,16,17 LPF91‑3A2 showed direct cytotoxic and cell growth inhibitory effects without being accompanied by
Figure 2. Effect of LFP91‑3A2 on viability of cell lines S180, H22, A549 and the normal chick embryo fibroblasts (CEF). The cytotoxicity of LFP91‑3A2 on the tumor lines S180, H22, A549 and against the normal chick embryo fibroblasts was performed by MTT reduction assay. The test cells were treated with different concentrations of LFP91‑3A2 (5− 15 μg/mL) for 24, 48, and 72 h of culture in microplates. 5 μg/mL of DDP was used as positive control. Results represent the mean ± SD (standard deviation) of three experiments run in three replicates. *p < 0.001 compared to control.
with different concentrations of LPF91‑3A2 under the same conditions. Our findings revealed that LFP91‑3A2 suppressed tumor cells remarkably without side effects on the normal cells. This is particularly important, since many traditional chemotherapeutic agents and ratios exhibit severe toxicity against the normal cells, causing undesirable side effects and thus limiting their application in the clinical field.10,12 For further characterization, LPF91‑3A2 induced tumor cell death was analyzed by flow cytomery after staining H22 cells with annexin V-FITC/PI. As shown in Figure 3, the early and late apoptosis rates of the cells which were incubated for 24 h
Figure 3. Assessment of apoptosis using flow cytometry with Annexin V-FITC/PI staining, showing the dot-plot graph of H22cells: (a) control group; (b) H22 treated with 5 μg/mL of LFP91-3A2; (c) H22 treated with 10 μg/mL of LFP91‑3A2; (d) H22 treated with 15 μg/mL of LFP91-3A2. 5318
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Figure 4. Tumor cell apoptosis morphology examined by transmission electron microscopy. (A, left) Native control. (B, right) The short microvilli disappeared and chromatin was condensed strongly, forming a high-density area.
Figure 5. Peptide fragments of LFP91‑3A2 generated by trypsin cleavage analysis by MS/MS.
stimulating the immune system, which is distinct from extracts previously mentioned. Mass Spectrometric Identification of Novel Proteins. The purified sample of LPF91‑3A2 was excised, digested by trypure in-gel and characterized by matrix assisted laser desorption/ionization/time of flight (MALDI-TOF) MS. Using the Mascot search engine (http://www.matrixscience. com), the MALDI-TOF data obtained from LPF91‑3A2 was matched significantly with adenylate kinase isoenzyme2 mitochondrial [Harpengnathos saltalor](gi|307198091). The score of mascot search matching was 85, and sequence coverage was 27%. Adenylate kinase isoenzyme2 is localized in the space of mitochondrial intermembrane and plays a role in mediating a novel intrinsic apoptotic pathway that may be involved in tumorigenesis processes. Adenylate kinase isoenzyme2 is released from mitochondria during the early phase of the apoptotic process.18,19 The antitumor activity of LFP91‑3A2 was supposed to be related to the similar molecular structure with adenylate kinase isoenzyme2. Three peptide fragments generated from LFP91‑3A2 by trypsin cleavage were subjected to MS/MS analysis for sequencing (Figure 5). The amino acid sequences were (a) NVAVPLYNR, (b)
SDGANGLLTK and (c) NKYEDELNKR. However, the BLAST searches in several databases of de novo amino acid sequences derived from analysis of the 26 kDa band failed to identify any homologous proteins.20 The paucity of related fungi protein amino acid sequences in accessible databases may be the reason why we found no homologous proteins from mushroom. In order to well know the properties of LPF91‑3A2, we leveraged Solexa sequencing technology in de novo assembly of Lentinula edodes C91‑3 transcriptome and obtained 28,923 unigene sequences including 18,120 unigenes with a coding sequence.6 Based on this gene information, we can obtain deep insight into the medical function, especially antitumor activity at the protein level in Lentinula edodes. The present study was carried out to purify and characterize active antitumor protein LPF91‑3A2 from the mycelium fermentative liquid extract of Lentinula edodes C91‑3. LPF91‑3A2 was isolated and purified to homogeneity after (NH4)2SO4 precipitation and two steps of chromatography analysis. SDSPAGE results are in accordance with MALDI/MS analysis, which show an approximate molecular weight of 26 kDa. The role of protein molecules from natural resources in drug and toxin-induced apoptosis is not clearly defined. In fact, very little 5319
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(14) Lull, C.; Wichers, H.; Savelkoul, H. Antiinflammatory and immunomodulating properties of fungal metabolites. Mediators Inflamm. 2005, 63−80. (15) Lo, T. C.; Hsu, F. M.; Chang, C. A.; Cheng, J. C. Branched α(1,4) glucans from Lentinula edodes (L10) in combination with radiation enhance cytotoxic effect on human lung adenocarcinoma through the Toll-like receptor 4 mediated induction of THP-1. J. Agric. Food Chem. 2011, 59, 11997−12005. (16) Israilides, C.; Kletsas, D.; Arapoglou, D.; Philippoussis, A.; Pratsinis, H.; Ebringerova, A.; Hribalova, V.; Harding, S. E. In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes. Phytomed.: Int. J. Phytother. Phytopharmacol. 2008, 15, 512−9. (17) Yukawa, H.; Ishikawa, S.; Kawanishi, T.; Tamesada, M.; Tomi, H. Direct cytotoxicity of Lentinula edodes mycelia extract on human hepatocellular carcinoma cell line. Biol. Pharm. Bull. 2012, 35, 1014− 21. (18) Kohler, C.; Gahm, A.; Noma, T.; Nakazawa, A.; Orrenius, S.; Zhivotovsky, B. Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis. FEBS Lett. 1999, 447, 10−2. (19) Lee, H.; Pyo, J.; Oh, Y.; Kim, H.; Hong, S.; Jeon, Y.; Kim, H.; Cho, D.; Woo, H.; Song, S.; Nam, J.; Kim, H.; Kim, K.; Jung, Y. AK2 activates a novel apoptotic pathway through formation of a complex with FADD and caspase-10. Nat. Cell Biol. 2007, 1303−1310. (20) Au, C. H.; Wong, M. C.; Bao, D.; Zhang, M.; Song, C.; Song, W.; Law, P. T.; Kues, U.; Kwan, H. S. The genetic structure of the A mating-type locus of Lentinula edodes. Gene 2014, 535, 184−90. (21) Tsoi, A. Y.; Ng, T. B.; Fong, W. P. Antioxidative effect of a chymotrypsin inhibitor from Momordica cochinchinensis (Cucurbitaceae) seeds in a primary rat hepatocyte culture. J. Peptide Sci. 2005, 11, 665−8. (22) Lee, S.; Oh, P.; Ko, J.; Lim, K.; Lim, K. Protective effect of glycoprotein isolated from Ulmus davidiana Nakai on carbon tetrachloride-induced mouse liver injury. J. Pharm. Pharmacol. 2006, 58, 143−152. (23) Oh, P.; Lim, K. Plant originated glycoprotein has anti-oxidative and anti-inflammatory effects on dextran sulfate sodium-induced colitis in mouse. J. Biomed. Sci. 2006, 13, 549−560. (24) Liu, B.; Zhong, M.; Lun, Y.; Wang, X.; Sun, W.; Li, X.; Ning, A.; Cao, J.; Zhang, W.; Liu, L.; Huang, M. A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C(91-3). Int. J. Mol. Sci. 2012, 13, 6246−65. (25) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680− 685.
information is available in the literature about this regard. A few articles, published recently, revealed that some protein molecules from various plant sources possess activities like those of our protein, although these reports do not describe any mechanism of protective action or structural features of the active principles.21−23 In this study, we isolated and partially characterized antitumor protein LFP91‑3A2 from Lentinula edodes C91‑3 However, the identities and detailed characteristic properties of LFP91‑3A2 remain unknown up to now. Further studies are needed such as molecular properties, gene clone, expression and characterization of LFP91‑3A2. All these experiments are now under investigation in our laboratory based on the transcriptome sequence.24 These antitumor studies would be helpful for its potential medical application in the future.
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AUTHOR INFORMATION
Corresponding Author
*Tel.:0411 86110007. Fax: 0411 86110007. E-mail address: [email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
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