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Grifolin, a potential antitumor natural productfrom the mushroom Albatrellus confluens, induces cell-cycle

arrest in G1 phase via the ERK1/2 pathway

Mao Ye a,c,1, Xiangjian Luo a,1, Lili Li a, Ying Shi a, Ming Tan a, Xinxian Weng a,Wei Li a, Jikai Liu b,*, Ya Cao a,*

a Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, PR China

b Department of Phytochemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204, PR Chinac School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang 325027, PR China

Received 27 May 2007; received in revised form 31 August 2007; accepted 3 September 2007

Abstract

Grifolin, a natural product isolated from the mushroom Albatrellus confluens, has been shown to inhibit the growth ofsome cancer cell lines and induce significant apoptosis. However, the molecular targets and the signaling mechanism under-lying the anticancer effect of this compound are not completely understood. Here, we undertook a gene expression profiling

study to identify novel targets of grifolin. We found that the effect of grifolin on the human nasopharyngeal carcinoma cellline CNE1 occurs primarily via the ERK1/2 pathway. At high doses, both the ERK1/2 and the ERK5 pathways may beinvolved in the inhibition. Because inhibition of the ERK1/2 or the ERK5 pathway has been associated with cell-cyclearrest and growth inhibition, we evaluated the cell cycle distribution after grifolin treatment. We found that grifolin sig-nificantly caused cell-cycle arrest in G1 phase. To investigate the underlying mechanisms, G1-related proteins were assayedby Western blotting. Following grifolin treatment, a concomitant inhibition of cyclin D1, cyclin E, CDK4 expression, andsubsequent reduction in pRB phosphorylation occurred. Meanwhile, grifolin treatment also resulted in a significant upreg-ulation of CKI (p19INK4D). These results suggest that the inhibition of the ERK1/2 or the ERK5 pathway is responsiblefor at least part of the induction of cell-cycle arrest in G1 phase by grifolin. These results are significant in that they providea mechanistic framework for further exploring the use of grifolin as a novel antitumor agent. 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Grifolin; Cell cycle; ERK1/2; ERK5

1. Introduction

Natural products are the sources of innovativelead compounds. Higher fungi belong to very pro-ductive biological sources, which produce a largeand diverse variety of secondary metabolites, andplay crucial roles in drug discovery and develop-

0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.canlet.2007.09.001

* Corresponding authors. Tel.: +86 731 4805448; fax: +86 7314470589 (Y. Cao).

E-mail address: [email protected] (Y. Cao).1 These authors contributed equally to this work.

Available online at www.sciencedirect.com

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2. Materials and methods

2.1. Cell culture

The human nasopharyngeal carcinoma cell line CNE1was grown in RPMI 1640 medium supplemented with

10% heat-inactivated fetal bovine serum, 1% glutamine,and 1% antibiotics, and incubated at 37 C in a humidifiedincubator containing 5% CO2.

2.2. Antibodies and chemicals

Anti-MEK1 and anti-ERK5 were presented fromAbgent (San Diego, CA). Anti-cyclin E was purchasedfrom Oncogene Sciences Products (Boston, MA). Anti-MEKK3, anti-MEK5, anti-ERK1/2, anti-phospho-ERK1/2 (anti-pERK1/2), anti-phospho-ERK5 (anti-pERK5),anti-pRB, anti-cyclin D1, anti-CDK4, anti-CDK2, anti-

a-tubulin, and horseradish peroxidase-conjugated goatanti-mouse or anti-rabbit secondary antibodies werepurchased from Santa Cruz Biotechnology (Santa Cruz,CA). The antibody against pRB (sc-50, Santa Cruz) is arabbit polyclonal affinity purified antibody raised againsta peptide mapping at the C-terminus of RB of humanorigin. Based on published data from other group [20,21],it revealed that the antibody was able to recognize thehypophosphorylated and hyperphosphorylated isoformsof pRB. The p44/42 MAP kinase assay kit was obtainedfrom Cell Signaling Technology (Beverly, MA). Grifolin(2-trans, trans-farnesyl-5-methylresorcinol) was provided

by Kunming Institute of Botany, the Chinese Academy ofSciences (purity >99%, HPLC analysis) [11]. Dimethylsulfoxide (DMSO; Sigma Chemical, St. Louis, MO) wasused to dissolve grifolin.

2.3. RNA preparation, cDNA synthesis, hybridization, and

scanning of microarray

CNE1 cells were treated with or without 40 lM grifolinfor 48 h respectively. Cells were washed with phosphate-buffered saline (PBS), and total RNA samples wereextracted using Trizol reagent (Invitrogen, Carlsbad, CA,

USA) according to the manufacturers instructions, fol-lowed by treatment with RNase-free DNase at 37 C for20 min to avoid contamination of genomic DNA. TheRNA quality and concentration were assessed using aga-rose gel electrophoresis and spectrophotometer readings.Probe mixtures were synthesized by reverse transcribing5 lg of total RNA using CDS primer mix (Clontech, SanDiego, CA, USA), [a-32P]dATP (Amersham PharmaciaBiotech, Piscataway, NJ, USA), and MMLV reverse trans-criptase. The 32P-labeled cDNA probes were purified usinga NucleoSpin extraction spin column. The hybridizationexperiment was performed with the human apoptosis array(Clontech) at 68 C overnight in a hybridization oven. The

membranes were washed thrice with 250 ml of 2 sodium

chloride-sodium citrate (SSC) buffer containing 1% sodiumdodecyl sulfate (SDS) and once with 250 ml of 1 SSCcontaining 0.1% SDS solution at 68 C for 30 min. Themembranes were then exposed to X-ray film for autoradi-ography at 70 C. Data were analyzed using AtlasImage1.0 software (Clontech). Normalization of the signal inten-

sity between two arrays was based on the overall value of allgenes on the arrays (global normalization). Weak signalswere filtered out by applying a background-based signalthreshold of 200%. To define different gene induction, weused a twofold threshold value.

2.4. Protein extraction and Western blotting

After treatment with grifolin for 48 h, cells were washedwith cold PBS and subjected to lysis in lysis buffer (50 mM/L TrisCl,1 mM/L EDTA, 20 g/LSDS, 5 mM/L dithiothre-itol, 10 mM/L phenylmethyl sulfonylfluoride). Equal

amounts of whole cell lysates (containing 50 lg protein)and rainbow molecular weight markers (Amersham Phar-macia Biotech, Piscataway, NJ, USA) were separated by12% sodium dodecyl sulfatepolyacrylamide gel electropho-resis (SDSPAGE) and then electrotransferred to a nitrocel-lulose membrane. Membranes were blocked with buffercontaining 5% fat-free milk in PBS with 0.05% Tween 20for 2 h, and incubated with antibody overnight at 4 C. Aftera second wash with PBS containing 0.05% Tween 20, themembranes were incubated with peroxidase-conjugated sec-ondary antibodies (Santa Cruz Biotech) and developed witha chemiluminescence detection kit (ECL; Pierce Chemical,

Rockford, IL). a-Tubulin was used as a loading control.

2.5. Assay of ERK activities

After treatment with different concentrations (0, 30, 40,and 50 lM) of grifolin for 48 h, cells were washed with coldPBS and subjected to lysis. Equal amounts of whole celllysate (containing 200 lg protein each) were dilutedby add-ing 15 ll of immobilized antibody bead slurry to bring thetotal volume to 200 ll and incubated with gentle rockingovernight at 4 C, then microcentrifuged at 14,000g for30 s at 4 C. The pellet was washed two times with 500 llof 1 cell lysis buffer, and twice with 500 ll o f 1 kinase buf-fer, and then suspended in 50 ll of 1 kinase buffer supple-mented with 200 lM ATP and 1 lg of kinase substrate(Elk-1). After incubation at 30 C for 30 min, the reactionwas terminated with 25 ll 3 SDS sample buffer, followedby vortexing and microcentrifugation for 30 s. Then Elk-1phosphorylation was detected using phospho-Elk-1 anti-body by Western blotting and chemiluminescence.

2.6. Flow cytometry analysis

Flow cytometry was used to quantitatively detect thecell-cycle distribution. Cells (1 106) were plated into

10-cm tissue culture dishes 1 day before treatment with

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grifolin at various concentrations. After treatment for48 h, cells were harvested, washed with PBS, fixed in70% ethanol overnight at 4 C for at least 2 h, and stainedwith 50 lg/ml propidium iodide (PI) by incubation at 4 Cfor at least 15 min. The stained cells were analyzed by flowcytometry (BectonDickinson).

3. Results

3.1. Expression profiles of genes reveal the potential

antitumor mechanism of grifolin

We showed previously that grifolin inhibited the growthand induced significant apoptosis of some cancer cell linesin a dose-dependent manner (3050 lM) [19]. To furtherdetermine the mechanism of grifolin-induced tumor celldeath, RNA isolated from untreated or grifolin-treatedCNE1 cells at 40 lM was reversely transcripted into 32P-

labeled cDNA, followed by hybridization to the Atlashuman apoptosis array. This expression array containsDNA fragments, in duplicate, for 205 apoptosis- and cell-cycle-related genes and nine housekeeping genes. We iden-tified seven mRNAs that were upregulated at least twofold(Table 2) and four transcripts that were downregulated atleast twofold in response to the grifolin treatment (Table1). Of the four downregulated genes, three (MEKK3,MEK1, and MEK5) are involved in MAPK pathways. Thisfinding strongly implies that inhibition of MAPK pathwaysmight very well contribute to the antitumor effect of grifo-lin. Theupregulation of cyclin-dependent kinase 4 inhibitor

2D (CDKN2D), a cell-cycle regulator, was also found,which suggests that the expression changes of CDKN2Dmight be involved in causing the cell-cycle arrestby grifolin.

3.2. Grifolin inhibited the activation of ERK1/2 and ERK5

pathways

Because of the important role of the ERK1/2 and theERK5 pathways in cancer biology, the microarray resultsprompted us to further explore their expression at the pro-tein level. Western blotting showed that grifolin was able toinhibit the expression of MEK1 in a dose-dependent man-

ner (Fig. 1). It has been reported that ERK1/2 can be acti-

vated by MEK1. To determine if the inhibition of MEK1resulting from grifolin treatment was associated with theactivation of ERK1/2, a downstream target of MEK1, weanalyzed the expression and phosphorylation status ofERK1/2. Immunoblot analysis using an antibody that rec-ognizes the total level of ERK1/2 revealed no significantchange in total protein levels between control and grifo-

lin-treated CNE1 cells. In contrast, grifolin induced a

Table 1Genes downregulated by grifolin in CNE1 cells

Accession No. Definition Total foldchange

U78876 Mitogen-activated protein kinasekinase kinase 3 (MEKK3)

2.1

X66362 PCTAIRE protein kinase 3(PCTK3)

2.2

L05624 Mitogen-activated protein kinasekinase 1 (MEK1)

2.5

U25265 Mitogen-activated protein kinase

kinase 5 (MEK5)

2.6

Table 2Genes upregulated by grifolin in CNE1 cells

Accession No. Definition Total foldchange

D89667 C-myc-binding protein MM-1 4.9M36981 Nucleoside diphosphate kinase B

(NDPKB)

4.2

M62402 Insulin-like growth factor-binding protein 6 (IGFBP6)

2.6

M35410 Insulin-like growth factor-binding protein 2 (IGFBP2)

2.3

X76104 Death-associated protein kinase1 (DAPK1)

2.3

U21092 CD40-receptor-associated factor(CRAF1)

2.2

U40343 Cyclin-dependent kinase 4inhibitor 2D (CDKN2D)

2.2

Fig. 1. Effects of grifolin on the expression of MEKK3, MEK1,and MEK5 in CNE1 cells. CNE1 cells were cultured in thepresence of various concentrations of grifolin as indicated. After

treatment, cells were removed from culture, washed, and lysedwith cell lysis buffer. Equal amounts of whole cell lysates(containing 50 lg protein) and rainbow molecular weight mark-ers were subjected to SDSPAGE and Western blotting analysiswith antibodies against MEKK3, MEK1, and MEK5.

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dose-dependent decrease in phospho-ERK1/2 (Fig. 2a.Theprevention of ERK1/2 activation by grifolin indicated thatthis compound inhibited the expression of kinase upstreamof ERK1/2. However, these data raised one importantquestion: Can the activity of ERK1/2 be directly inhibitedby grifolin? To further substantiate this point, we per-

formed an in vitro kinase assay of ERK1/2 in the absenceor presence of grifolin. ERK1/2 was immunoprecipitatedwith a specific antibody and incubated with Elk-1, a specificsubstrate for ERK1/2. As shown in Fig 2a, the in vitro

kinase assay with Elk-1 as substrate revealed that grifolininhibited ERK1/2 kinase in a similar pattern to the regula-tion of the pERK1/2 level (Fig. 2b).

ERK5, which is also a member of the MAPK family,is phosphorylated and activated by MEK5, a specificMAPKK for ERK5. However, MEKK3, a member of

the MAPKKK family, is an upstream kinase ofMEK5. Unlike the ERK1/2 pathways, grifolin treatmentat low dose levels (30 or 40 lM) did not alter the levelsof MEKK3 and MEK1 proteins, whereas significantinhibition of these proteins was observed only at thehighest dose (50 lM) (Fig. 1). Similarly, a significantdecrease in ERK5 activity was found only at the highestdose of grifolin (Fig. 2a).

3.3. Cell-cycle arrest induced by grifolin

To investigate the effects of grifolin on cell-cycle

status, CNE1 cells were treated with different concentra-tions of grifolin for 48 h and then analyzed for cell-cyclealteration by flow cytometry. We observed that grifolincaused a dose-dependent accumulation of cells in G1.The percentage of G1 cells increased from 60.75% inthe control to 91.87% in populations treated with50 lM grifolin; consequently, fewer cells progressed toS phase, from 26.74% in the untreated group to 2.94%in the treated group (Fig. 3). This finding indicated thatcell cycle progression was dramatically blocked in G1when CNE1 cells were treated with grifolin. In addition,we also found that PD98059, a small molecule inhibitor

of ERK1/2 or ERK5 pathways, induced a remarkableG1 cell-cycle arrest in CNE1 cells (data not shown).The result is consistent with the effect of grifolin onCNE1 cells.

3.4. Effects of grifolin on the expression of G1-related

protein in cells treated with grifolin

To reveal the mechanism of cell-cycle arrest, weinvestigated whether the effects induced by grifolin wereassociated with the level of G1-S transition-related pro-tein. After treating CNE1 cells with grifolin, we

observed a dose-dependent decrease in cyclin D1 andcyclin E (Fig. 4). Meanwhile, a decrease in cyclin D1expression observed in grifolin-treated cells was accom-panied by a reduction in the amount of CDK4 that isassociated with cyclin D1. CDK4 was rarely detectablein these cells, especially at 50 lM grifolin. However,the expression pattern of CDK2 that associated withcyclin E was not significantly altered after treatmentwith grifolin (Fig. 4).

Cyclin-dependent kinase inhibitor 2D (CDKN2D)encodes cyclin-dependent kinase inhibitor (CKI)p19INK4D, which specifically inhibits CDK4 and CDK6activities during G1 phase. The expression array showed

that CDKN2D was upregulated >twofold after 40 lM

Fig. 2. (a) Grifolin inhibits the phosphorylation of ERK1/2 andERK5. CNE1 cells were treated with various concentrations of

grifolin as indicated. Cell lysates were prepared and examined byWestern blotting analysis with antibodies against ERK1/2,pERK1/2, ERK5, and pERK5. The level of a-tubulin in eachlane was comparable, indicating equivalent amounts of celllysates were loaded on the gel. The experiments were repeatedthree times with similar results. (b) Effects of grifolin on theactivity of ERK1/2. CNE1 cells were cultured in the presence ofvarious concentrations of grifolin as indicated. After treatment,cells were removed from culture, washed, and lysed with 1 celllysis buffer. A monoclonal phospho-antibody to p44/42 MAPKinase (Thr202 and Tyr204) is used to selectively immunopre-cipitate active MAP Kinase from whole cell lysates. In vitrokinase assays were done using a Elk-1 fusion protein as thesubstrate. Western blots were incubated with an antibody

directed against phosphorylated Ser383 of Elk-1.


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grifolin treatment in CNE1 cells. We further validated theexpression of p19INK4D at the protein level and found thatgrifolin can induce a dose-dependent increase of p19INK4D

in CNE1 cells (Fig. 5).During G1-phase progression, the retinoblastoma

protein (pRB) is sequentially phosphorylated by the cyclinD-CDK4 and cyclin E-CDK2 complexes. Hyperphosph-orylation of pRB leads to the dissociation of the E2Ftranscription factor from pRB and entry into the S phase.To identify the phosphorylation state of pRB, CNE1 cellswere treated with different concentrations of grifolin, andproteins were assayed using the antibody that recognizesthe total pRB (both phosphorylated pRB [ppRB] and

non-phosphorylated pRB). We observed that treating

CNE1 cells with grifolin significantly reduced the totalpRB (both pRB and ppRB) in a dose-dependent manner(Fig. 6).

4. Discussion

Grifolin is a natural product isolated from thefresh fruiting bodies of the mushroom A. confluens.Previous studies by our group have showed that grif-olin is able to inhibit the growth of some cancer celllines in vitro by induction of apoptosis [19].Tofurtheridentify molecular targets in signal transduction

pathways, cDNA microarray analysis was employed

Fig. 3. Grifolin induces G1 cell-cycle arrest in CNE1 cells by flow cytometry analysis. After CNE1 cells were grown in medium alone ortreated with grifolin at the indicated concentrations, cells were harvested and washed with PBS, fixed with ice-cold 70% ethanol, stainedwith PI, and treated with RNase A. Cell-cycle distribution was analyzed by flow cytometry. Data shown are a representative result fromthree independent experiments.


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to investigate the mechanism of grifolin-induced celldeath at the gene expression level. Our studies identi-fied three genes related to MAPK pathways that weresignificantly downregulated. These results stronglyindicated that MAPK pathways may be involved inthe antitumor effect of grifolin.

The MAPK pathways serve to coordinate key

cellular processes. The ERK1/2 pathway and the

BMK1/ERK5 pathway play key roles in the regu-lation of multiple biological activities, includingcell proliferation, differentiation, cell-cycle transi-tion, and survival. The bulk of the evidence sug-gests that constitutive activation of the ERK1/2pathway contributes to tumorigenesis, or cancergrowth, and increases the cell death threshold[22]. In nasopharyngeal carcinoma, high ERK1/2activation was correlated with poor prognosis[23]. Recent reports have demonstrated that theERK5 pathway is constitutively activated in breast

cancer [24] and metastatic prostate cancer [25]. Thecomponents of MAPK signaling pathways areknown to be attractive therapeutic targets for can-cer. Our results showed that the activity of ERK1/2 in CNE1 cells can be inhibited by grifolin in adose-dependent manner following 48 h of treat-ment. No significant differences in the expressionof total ERK1/2 protein were detected. Meanwhile,the observation that grifolin inhibits the expressionof MEK1, the upstream activator of ERK, sug-gests that grifolin inhibits the activity of ERK1/2involving the action of MEK1. Furthermore, grifo-lin inhibits the phosphorylation and kinase activityof ERK1/2 at doses that are consistent with apop-totic induction in CNE1 cells [19]. In contrast,ERK5 activity was inhibited only at the highestdose (50 lM) of grifolin. At low doses (30 or40 lM) of grifolin, no significant changes in theexpression of total MEKK3 and MEK5 proteins,two upstream kinases of ERK5, were detected.This finding showed that the ERK5 pathway wasless sensitive to grifolin than the ERK1/2 pathway.In addition, the decrease in the expression of total

MEKK3, MEK1, and MEK5 proteins indicates

Fig. 4. Effects of grifolin on G1-related protein in CNE1 cells.CNE1 cells were cultured and treated with various concentrationsof grifolin. At the end of the treatments, total cell lysates wereprepared and subjected to SDSPAGE followed by Westernblotting. Membranes were probed with anti-cyclin D1, anti-CDK4, anti-cyclin E, and anti-CDK2. The level of a-tubulin ineach lane was comparable, indicating equivalent amounts of celllysates were loaded on the gel. The experiments were repeatedthree times with similar results.

Fig. 5. Effects of grifolin on the level of p19. CNE1 cells weretreated with grifolin as described in Fig. 4. The expression of p19was detected by Western blotting. The level of a-tubulin in eachlane was comparable, indicating equivalent amounts of whole celllysates were loaded on the gel. The experiments were repeatedthree times with similar results.

Fig. 6. Effects of grifolin on the phosphorylation level of pRB.CNE1 cells were treated with grifolin as described in Fig. 4. Theexpression of pRB and ppRB was detected with antibody againstpRB by Western blotting. The level ofa-tubulin in each lane wascomparable, indicating equivalent amounts of cell lysates loadedon the gel. The experiments were repeated three times with similarresults.


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that grifolin may induce proteolytic cleavage orinhibit the transcription or translation of theseproteins.

Studies have shown a link between the MAPKpathways and cell-cycle mechanism. The ERK1/2

and the ERK5 pathways can exert their cell-cycleprogression effects through the induction of cell-cycle regulatory proteins, such as CDKs, cyclins,and CK inhibitors (CKIs) [26]. Cell cycle progres-sion is stimulated by protein kinase complexes,including a cyclin and a CDK [27]. During G1-phase progression, cyclin D1/CDK4/6 complexesare activated in mid-G1, whereas cyclin E/CDK2complexes are required for the G1/S transition.CDK-mediated hyperphosphorylation of pRBresults in the dissociation of pRB and E2F andentry into the S phase. CDKs are regulated by a

group of functionally related proteins calledCDK inhibitors (CKIs), which include the INK4family and the Cip/Kip family. The INK4 familyinhibits CDK4/6 activity specifically during G1phase. There is increasing evidence that perturba-tion of cell cycle regulation is an important con-tributing factor to cancer. Our studies indicatedthat grifolin can induce cell-cycle arrest in G1 ina dose-dependent manner. The results obtainedfrom evaluating the effects of grifolin on theexpression of G1-related protein suggested that cell

cycle arrest is associated with the downregulationof cyclin D1, CDK4, and cyclin E. However, grif-olin had little effect on CDK2 expression. In con-trast, INK4 family member p19, which mediatesthe inhibition of CDK4, was obviously upregulatedby grifolin in a dose-dependent manner. Progres-sion through G1 phase of the cell cycle requirespRB phosphorylation, a major cellular target ofthe cyclin D1/CDK4 and the cyclin E/CDK2 com-plexes. The current study showed that the phos-phorylation of pRB in CNE1 cells wassignificantly inhibited by grifolin. These resultsrevealed that grifolin-induced cell-cycle arrest inG1 phase, which involved the alternation of theG1-related protein.

In summary, our data show that grifolin inhibitsthe proliferation of CNE1 cells through G1 phasearrest, which is mediated by the regulation of G1-related protein. The effect of grifolin on CNE1 cellsprimarily involves the ERK1/2 pathway. At highdoses, both the ERK1/2 and the ERK5 pathwaysmay be involved in the inhibition. Further indepthin vivo studies are presently under investigation in

our laboratory.

Acknowledgements

This work was supported by Grant No.2007FJ4012 and No. 02SSY2001-2 from theResearch Programs of Science and Technology

Commission Foundation of Hunan Province andby Grant No. 30500621 from the National NaturalScience Foundation of China.

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