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Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells.

ABSTRACT

Polygonatum odoratum lectin (POL), a mannose-binding GNA-related lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which POL induces cancer cell death are still elusive. In the current study, we found that POL could induce both apoptosis and autophagy in human MCF-7 breast cancer cells. Subsequently, we found that POL induced MCF-7 cell apoptosis via the mitochondrial pathway. Additionally, we also found that POL induces MCF-7 cell apoptosis via EGFR-mediated Ras-Raf-MEK-ERK pathway, suggesting that POL may be a potential EGFR inhibitor. Finally, we used proteomics analyses for exploring more possible POL-induced pathways with EGFR, Ras, Raf, MEK and ERK, some of which were consistent with our in silico network prediction. Taken together, these results demonstrate that POL induces MCF-7 cell apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK signaling pathway, which would provide a new clue for exploiting POL as a potential anti-neoplastic drug for future cancer therapy.

Keywords:

Polygonatum odoratum lectin (POL)

Apoptosis

Autophagy

Epidermal growth factor receptor (ECFR)

MCF-7 cell

Introduction

Plant lectins are a group of highly diverse non-immune origin proteins ubiquitously distributed in a variety of plant species and they contain at least one non-catalytic domain that enables them to selectively recognize and reversibly bind to specific free sugars or glycans present on glycoproteins and glycolipids without altering the structure of the carbohydrate (Van Damme et al., 1998). Of note, plant lectins can be divided into 12 different families, including ABA (Agaricus bisporus agglutinin), Amaranthin, CRA (chitinase-related agglutinin), Cyanovirin, EEA (Euonymus europaeus agglutinin), GNA (Galanthus nivalis agglutinin), Hevein, Jacalins, Legume lectin, LysM (lysin motif), Nictaba and Ricin_B families (Van Damme et al., 2008). Amongst the above-mentioned families, Galanthus nivalis agglutinin (GNA)-related lectins have been reported to possess a broad range of biological activities, such as anti-tumor activities (Tian et al., 2008; Van Damme et al., 2007; Li et al., 2009).

In the previous studies, several GNA-related lectins, such as Polygonatum cyrtonema lectin (PCL), Ophiopogon japonicus lectin (OJL) and Liparis noversa lectin (LNL), were reported to possess remarkable anti-proliferative and apoptosis-inducing activities toward various cancer cells (Liu et al., 2009a, 2009b, 2009c, 2009d; Wang et al., 2011). Moreover, Polygonatum odoratum lectin (POL), isolated from crude extracts from rhizomes of polygonatum odoratum (Mill.), is a homo-tetramer with molecular weight of 11,953.623 Da per subunits as purified by gel filtration, SDS-PAGE, and determined by mass spectrometry. And, POL bears three conserved motif of 'QXDXNXVXY', which is essential in the mannose recognition, and it can agglutinate rabbit erythrocytes at a minimal concentration ([greater than or equal to]3.75 p-g/ml) (Yang et al., 2011). Interestingly, Polygonatum odoratum lectin (POL) has been recently reported to induce apoptosis via death-receptor and mitochondrial apoptotic pathways in murine fibrosarcoma L929 cells (Liu et al., 2009a,2009b,2009c,2009d; Fu et al., 2011; Yu et al., 2011). However, the precise mechanisms by which POL induces cancer cell death are still rudimentarily understood.

In this study, we report for the first time that POL induces mitochondrial apoptosis and autophagic cell death via targeting EGFR-mediated Ras-Raf-MEK-ERK signaling pathways in MCF-7 cells. These results may provide new evidence for further understanding key apoptotic and autophagic pathways induced by such GNA-related lectin for future cancer drug discovery.

Materials and methods

Reagents

Polygonatum cyrtonema lectin (POL) was purified as previously described. The purification was first applied to a CM-Sepharose column and eluted with 0 - 0.5 M NaCl gradient in 40 mM, NaAc--HAc buffer (pH 4.6) and peak fractions with hemagglutinating activity were collected. Then the pooled active fractions were concentrated and loaded on the column of Sephacyl S100. Finally, the peak fraction showing hemagglutinating activity was purified POL (Yang et al., 2011). Fetal bovine serum (FBS) was purchased from TBD Biotechnology Development (Tianjin, China); 3-(4,5-dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3,3-diaminobenzidine tetrahydrochloride (DAB), and propidium iodide (PI) were purchased from Sigma Chemical (St. Louis, MO, USA). Rabbit polyclonal antibodies against EGFR, Ras, Raf, MEK, ERK, LC3, Beclin-1, [beta]-actin, and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell culture

The MCF-7 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 [micro]g/ml streptomycin, 100U/ml penicillin, and 0.03% L-glutamine and maintained at 37 [degrees]C with 5% C[O.sub.2] at a humidified atmosphere. All the experiments were performed on logarithmically growing cells.

Growth inhibition assay

The cytotoxic effect of POL in MCF-7 cells was measured by the MTT assay as described elsewhere (Cheng et al., 2010). The MCF-7 cells were incubated in 96-well tissue culture plates (NUNU, Roskilde, Denmark) at a density of 5 x [10.sup.4] cells/well. The cytotoxic effect was measured with a plate reader by the MTT assay. The percentage of cell growth inhibition was calculated as follows: Cell growth inhibition (%) = [[A.sub.492] (control) - [A.sub.492] (POL)]/[[A.sub.492] (control)-[A.sub.492] (blank)] x 100.

Apoptosis assay

Human breast adenocarcinoma MCF-7 cells were seeded into 96-well culture plates with or without POL and cultured for 24 h. The ultrastructure of cell apoptosis was observed under the electron microscope (Hitachi7000, Japan) (Cheng et al., 2008). The collected cells were fixed with 500 [micro]l PBS and 10 ml 70% ethanol at 4[degrees]C overnight; then after washing twice with PBS, the cells were incubated with 1 ml Hoechst staining solution for 30 min at 4[degrees]C. The percentage of cells at different phases of the Sub-G1 DNA content was measured by flow cytometry (Becton Dickinson, Franklin Lakes, NJ).

Autophagy assay

After incubation with POL for the fixed times, the MCF-7 cells were cultured with 0.05 mM MDC at 37[degrees]C for 60 min. The fluorescence intensity of cells was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ) (Cheng et al., 2009a,2009b). Cells were transfected with GFP-LC3 plasmid (kindly provided by Prof. Canhua Huang, Sichuan University) using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. The fluorescence of GFP-LC3 was observed under a fluorescence microscope.

Small interfering RNA (siRNA) transfection

siRNA against human EGFR and control siRNA was purchased from Invitrogen (Carlsbad, CA). Cells were transfected with siRNAs at 33 nM final concentration using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The transfected cells were used for subsequent experiments 24 h later.

Western blot analysis

The MCF-7 cells were treated with 10 [micro]g/ml POL for 6, 12, 18 and 24 h, respectively. Both adherent and floating cells were collected, and then Western blot analysis was carried out as previously described (Cheng et al., 2009a,2009b). Briefly, the cell pellets were resuspended with lysis buffer consisting of Hepes 50 mmol/l pH 7.4, Triton-X-100 1%, sodium orthovanada 2 mmol/l, sodium fluoride 100 mmol/l, edetic acid 1 mmol/1, PMSF 1 mmol/1, aprotinin (Sigma, MO, USA) 10 mg/l and leupeptin (Sigma) 10 mg/l and lysed at 4[degrees]C for 1 h. After 12,000g centrifugation for 15 min, the protein content of supernatant was determined by the BioRad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of the total protein were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes, the membranes were soaked in blocking buffer (5% skimmed milk). Proteins were detected using polyclonal antibodies and visualized using anti-rabbit or anti-mouse IgG conjugated with peroxidase (HRP) and 3,3-diaminobenzidine tetrahydrochloride (DAB) as the HRP substrate.

2-DE and MS/MS analysis

2-DE and MS/MS analysis were performed as described previously (Zhang et al., 2013). Briefly, cells were dissolved in lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 100 mM DTT, 0.2% pH 3-10 ampholyte, Bio-Rad, USA) in presence of protease inhibitor (Sigma). Samples were loaded into IPG strips (17 cm, pH 3-10 NL, Bio-Rad) using a passive rehydration method, and then subjected to isoelectric focusing (Bio-Rad). The second dimension separation was performed using 12% SDS-PAGE after equilibration. The gels were stained with CBB R-250 (BioRad). Identification and quantitation of protein spots in a gel was performed by using PDQuest software (Bio-Rad). In-gel protein digestion was performed using mass spectrometry grade trypsin according to the manufacturer's instructions. The gel spots were destained with 100 mM NH4HC03/50% acetonitrile (ACN) and dehydrated with 100% ACN. The gels were then incubated with trypsin (Promega, V5280), followed by double extraction with 50% ACN/5% trifluoroacetic acid (TFA). The peptide extracts were dried in a speed-VAC concentrator (Thermo), and subjected to mass spectrometric analysis using a Q-TOF mass spectrometer (Micromass, Manchester, UK) fitted with an ESI source.

Prediction of ECFR-modulated Ras-Raf-MEK-ERK pathway in MCE-7 cells

To build the global human PPI network, we collected diverse sets of biological evidence from five online databases. To predict pair-wise PPIs, all the data were preprocessed into pair-wise scores, reflecting the similarity between protein pairs. And, we used several online databases, including Protein interaction data were from Human Protein Reference Database (HPRD) (Mishra et al., 2006), Biomolecular Object Network Databank (BOND) (Alfarano et al., 2005), IntAct (Kerrien et al., 2007), HomoMINT (Persico et al., 2005) and BioGRID (Winter et al., 2011) to construct the global PPI network and EGFR subnetwork. To identify core EGFR-regulated Ras-Raf-MEK-ERK pathway, we used microarray data (No. GSE22368) from human breast adenocarcinoma MCF-7 cells, treated with 2.5 mM DTT to measure the pair-wise co-expression treated with tamoxifen, respectively (Taylor et al., 2010). The coexpression level is calculated as Pearson Correlation Coefficient [rho]:

[[rho].sub.X,Y] = [n.summation over (i=1)] ([X.sub.i] - [bar.X])([Y.sub.i] - [bar.Y])/(n - 1)[[sigma].sub.X][[sigma].sub.Y]

where X and Y are expression level data vectors of length n for two genes, [bar.X] and [bar.Y] are means, and [[sigma].sub.*] and [[sigma].sub.y] are the standard deviations. Then, based on microarray analysis, we identified dynamic EGFR-1-modulated Ras-Raf-MEK-ERK pathway in MCF-7 cells by the methods as previously described (Fu et al., 2013; Wang et al., 2012).

Statistical analysis

All the presented data and results were confirmed in at least three independent experiments. The data are expressed as means [+ or -] SD. Statistical comparisons were made by Student's t-test. P < 0.05 was considered statistically significant.

Results

POL induces apoptosis in MCF-7 cells

POL caused a remarkable anti-proliferative effect on MCF-7 cell growth in a time- and dose-dependent manner, and the treatment with 10 [micro]g/ml POL for 24 h resulted in almost 50% inhibition (Fig. 1A). Then, we examined the ultrastructure of POL-treated MCF-7 cells by transmission electron microscopy. As shown in Fig. 1 B, control cells displayed normal cell phenotype. In contrast, POL-treated MCF-7 cells showed typical apoptotic features, including chromatin condensation and margination at the nuclear periphery. And, the marked apoptotic morphologic alterations were observed by inverted microscopy, as well as by Hoechst staining under florescence microscopy (Fig. 1 C). In addition, apoptosis was further evaluated by the measurement of cell number in SubG1 region. POL markedly induced the increase of SubG1 cells proportion in MCF-7 cells (Fig. ID). Altogether, these results suggest that POL induces apoptosis in MCF-7 cells.

POL induces autophagy in MCF-7 cells

Meanwhile, typical characteristics of autophagy were also observed in POL-treated cells. These characteristic changes included extensive cytoplasm vacuolization, and some autophagic vacuoles contained degraded organelles by transmission electron microscopy (Fig. 2A). The morphologic changes were observed under a fluorescence microscope by MDC staining. MCF-7 Cells were treated with POL for indicated time periods, and the MDC fluorescent intensity was analyzed by flow cytometry (Fig. 2B). The formation of autophagic vacuoles was further assessed by GFP-LC3 distribution. As shown in Fig. 2C, control cells presented diffused staining, and POL treatment resulted in extensive GFP-LC3 localization, suggesting that POL induces autophagy in MCF-7 cells. To further investigate the role of autophagy in POL-induced death in MCF-7 cells, we examined the expressions of Beclin-1 and LC3. As shown in Fig. 2D, these treatments reduced POL-induced Beclin1 or LC3 level, respectively. In addition, treated 3-MA (a specific autophagic inhibitor) with POL showed that the cell viability was increased, indicating that autophagy plays a type II programmed cell death role (Fig. 2E). Thus, these results show that autophagy is involved in POL-induced MCF-7 cell death.

POL-induced MCF-7 cell apoptosis is via mitochondrial pathway and EGFR-mediated Ras-Raf-MEK-ERK pathway

Next, the amount of cytochrome c In the cytosol of the cells was increased; Bax expression was increased whereas Bcl-2 expression was decreased in MCF-7 cells (Fig. 3). These results clearly indicate that POL-induced apoptosis in MCF-7 cells is mediated by a mitochondrial pathway. Since the release of cytochrome c from mitochondria can activate caspase cascade, we investigated the involvements of caspase-9 and caspase-3 in POL-induced apoptosis. Caspase-9 activation was determined by measurement of the active forms of caspase-9. The active form of caspase-3 was observed during POL treatment (Fig. 3). These results suggest that mitochondrial pathway was involved in POL-induced apoptosis. Moreover, western blot data shown that the treatment of MCF-7 cells with POL resulted in down-regulation in the level of EGFR and p-EGFR, the levels of Ras, Raf, p-MEK and p-ERK in a time-dependent manner (Fig. 4). These results indicate that POL plays the promoting-death role in MCF-7 cells by inhibiting EGFR-mediated Ras-Raf-MEK-ERK pathway.

POL-induced apoptosis and autophagy is mainly dependent on EGFR in MCF-7 cells

In this study, we found that p-EGFR expression was significantly decreased in POL-treated MCF-7 cell apoptosis and autophagy (Fig. 5). To examine EGFR siRNA effect, we found that the expression of capase-9 was almost disappeared while the expressions of caspase-3 and p62 were both abruptly decreased in this condition, suggesting caspase-9 activation is dependent on EGFR and caspase3 and p62 activation are mainly dependent on EGFR in POL-induced apoptosis and autophagy (Fig. 5). Moreover, we found that POL with EGFR siRNA can upregulate the expressions of caspase-9, caspase-3 and p62 at some level, suggesting that POL is not totally dependent on EGFR inhibition but mainly dependent on EGFR (Fig. 5).

Proteomics and bioinformatics analyses of POL-induced apoptosis and autophagy in MCF-7 cells

To explore the molecular mechanisms underlying POL-induced apoptosis and autophagy, 2-DE based proteomics was employed to profile differentially expressed proteins in MCF-7 cells treated with POL. Representative 2-DE maps were shown in Fig. 6A. By comparing 2-DE patterns, differentially expressed proteins were defined as statistically meaningful (p < 0.05) based on both of the following two criteria: (1) intensity alterations of >2-fold and (2) observed in at least three individual experiments. Using these criteria, a total of 11 spots were identified as being differentially expressed. Of these, five proteins including M24, M23, N15, M22 and N13 were down-regulated whilst six proteins including M18, N14, M17, M19, M21 and M20 were up-regulated after treated with POL in MCF-7 cells (Fig. 6B). Then, these 11 spots were subjected to MS/MS analysis and were identified based on a number of criteria including pi, MW, the number of matched-peptides and MOWSE score (Table 1). The identified 11 proteins were further analyzed based on their interactions with EGFR, Ras, Raf, MEK and ERK.

In this study, we computationally constructed the global human PPI network covering almost all PPIs (about 2,00,000 protein pairs), based on IntAct, HPRD, HomoMINT, BOND and BioGRID. To construct the set of true-positive gene pairs, we achieved physical protein-protein interactions derived from manually curated PPI databases that include 58,976 protein pairs (Table SI). Thus, we got a total number of unique 85,806 protein pairs (13,128 proteins) prepared as the global human PPI network. Subsequently, we further modified the PPI network into the EGFR subnetwork. Since significance analysis of microarray (SAM) analysis was performed on data of different expression microarrays from tamoxifen-treated MCF-7 cells to identify divergent expression genes between normal and cancer cells (Table S2). We identified core EGFR-modulated pathways in MCF-7 cells (Fig. 6C). As a result, we demonstrate that seven proteins could interact with them in apoptosis and autophagy, suggesting some of proteomics analyses may be in good agreement with in silico prediction of EGFR-Ras-Raf-MEK-ERK pathway (Fig. 6C).

Discussion

Recently, GNA-related lectins have been drawn a rising attention for cancer biologists due to their remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells (Liu et a!., 2010). In our study, we report herein that POL, one of GNA-related lectins, induces apoptosis and autophagy in MCF-7 cells. Previous studies reported that garlic lectin, one member of GNA-related lectin family, could induce apoptosis in U937 cells [26]; moreover, other reports demonstrated that Ophiopogon japonicus lectin (OJL) and Liparis noversa lectin (LNL) could induce apoptosis in human MCF-7 breast cancer cells (Liu et al., 2009a, 2009b, 2009c, 2009d). POL bears three conserved motif of 'QXDXNXVXY', which is essential in the mannose recognition, whereas OJL has two conserved motifs and one mutated site. It might be the reason why POL and OJL have different anti-tumor activities or mechanisms in cancer therapy. In addition, our previous study reported that POL induced MCF-7 cell death via both intrinsic and extrinsic apoptotic pathways (Yang et al., 2011). Thus, these results further confirm that POL is the first reported plant lectin that can induce apoptosis and autophagy in MCF-7 cells.

Previous studies showed that other plant lectins induced cancer cell apoptosis through classical apoptotic pathways or pro-apoptotic pathways (De Mejia and Prisecaru, 2005; Heike et al., 1999; Hostanska et al., 2003). For instance, mistletoe lectins, the typical type II ribosome-inactivating proteins, induced apoptotic death via the caspase-8/FLICE independent of death receptor or p53-independent pathways (Kim et al., 2004; Kima et al., 2003), as well as via the caspase-activated ROS-mediated JNK pathway (Liu et al., 2009a,2009b,2009c,2009d; Ruhul Amin et al., 2007). Concanavalin A (ConA), the first typical legume lectin, could induce tumor cell apoptosis via the caspase-dependent mitochondrial pathways or p73-regulated Akt-Foxola-Bim Signaling pathway (Liu et al., 2009a, 2009b, 2009c, 2009d; Li et al., 2010), In contrast to these previous studies, our results show that POL can induce apoptosis and autophagy in MCF-7 cells via blocking several antiapoptotic pathways.

However, in this study, we found that POL induced apoptosis and autophagy in MCF-7 cells by inhibiting such as EGFR-mediated Ras-Raf-MEK-ERK pathway, which was different from our previous study that focused on exploring important apoptotic or autophagic signaling pathways (Liu et al., 2010). Thus, these results may provide a new molecular basis on further exploring the precise signaling pathways by which POL induces apoptosis and autophagy in cancer cells. Previous studies have reported that several sugar-containing receptors such as epidermal growth factor receptor (EGFR) can be involved in anti-apoptotic/survival pathways in cancer cells (Sreenath et al., 2007; Mukesh et al., 2006). POL, a typical GNA-related lectin binding mannose, can be speculated to bind directly the sugar-chains of EGFR on the surface of cancer cells, and thus POL could block EGFR-mediated survival/anti-apoptotic pathways such as Ras-Raf and other signaling pathways.

In conclusion, we found that POL could induce mitochondrial apoptosis and autophagic cell death via EGFR-mediated Ras-Raf-MEK-ERK pathway in MCF-7 cells (Fig. 7). These findings would provide us a new clue to exploit POL as a potential anti-tumor drug for future cancer therapy.

http://dx.doi.org/10.1016/j.phymed.2014.08.002

Conflict of interest

None.

Acknowledgments

This work was supported by grants from the National 973 Basic Research Program of China (Nos. 2010CB529900 and 2013CB911300), the Key Projects of the National Science and Technology Pillar Program (No. 2012BAI30B02), and National Natural Science Foundation of China (Nos. 81260628,81303270,81402496 and 81473091).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.phymed.2014.08.002.

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ARTICLE INFO

Article history:

Received 4 May 2014

Received in revised form 18 July 2014

Accepted 16 August 2014

Liang Ouyang (a,1), Yi Chen (a,1), Xiao-yan Wang (b,1), Rui-feng Lu (c), Shou-yue Zhang (a), Mao Tian (a), Tao Xie (a), Bo Liu (a),*, Gu He (a),*

(a) State Key Laboratory of Biotherapy and Collaborative Innovation Center of Biotherapy, Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu 610041, China

(b) Analytical and Testing Center, Sichuan University, Chengdu 610064, China

(c) Department of Pediatrics, Key Laboratory of Obstetric and Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu 610064, China

* Corresponding authors. Tel.: +86 28 85411914; fax: +86 28 85411914.

E-mail addresses: liubo2400@163.com (B. Liu), heguscu@163.com (G. He).

(1) These authors contributed equally to this work.

Table 1
MALDI Q-TOF MS identification of differential protein expressions
in POL-induced apoptosis Supplementary materials.

Spot     Accession
number   number      Protein name

M17      K22E        Keratin, type II cytoskeletal 2 epidermal
M18      Q6NTA2      HNRNPL protein
M19      Q32Q12      Nucleoside diphosphate kinase
M20      ALDOA       Fructose-bisphosphate aldolase A
M21      PEBP1       Phosphatidylethanolamine-binding protein 1
M22      PGAM1       Phosphoglycerate mutase 1
N13      GMFB        Glia maturation factor beta
N14      HSPB1       Heat shock protein beta-1
N15      TP1S        Triosephosphate isomerase
M23      PGAM1       Phosphoglycerate mutase 1
M24      ENOA        Alpha-enolase

Spot                 Theoretical   Theoretical   Number of
number   Gene name   Mr            pl            peptide     Score

M17      KRT2        65,678.3      8.07          24           420
M18      HNRNPL      62,515.3      7.07          38           543
M19      NME1-NME2   32,906        8.7           26           738
M20      ALDOA       39,851.5      8.3           30          1000
M21      PEBP1       21,157.7      7.01          22           708
M22      PGAM1       28,899.9      6.67          17           408
N13      GMFB        16.873.6      5.19          12           181
N14      HSPB1       22,825.5      5.98          23           730
N15      TPI1        31,056.8      5.65          33           941
M23      PGAM1       28,899.9      6.67          19           482
M24      ENOI        47,481.4      7.01          36           651

Protein spots were identified by ES1-Q-TOF in
POL-induced apoptosis and autophagy.
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Author:Ouyang, Liang; Chen, Yi; Wang, Xiao-yan; Lu, Rui-feng; Zhang, Shou-yue; Tian, Mao; Xie, Tao; Liu, Bo
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Oct 15, 2014
Words:5124
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