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Differential inhibitory potencies and mechanisms of the type I ribosome inactivating protein marmorin on estrogen receptor (ER)-positive and ER-negative breast cancer cells Wen Liang Pan a , Jack Ho Wong a , Evandro Fei Fang b , Yau Sang Chan a , Xiu Juan Ye c , Tzi Bun Ng a, a School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China b National Institute on Ageing, National Institutes of Health, Baltimore, MD, USA c Key Laboratory of Plant Virology of Fujian Province, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China abstract article info Article history: Received 3 August 2012 Received in revised form 24 November 2012 Accepted 18 December 2012 Available online 26 December 2012 Keywords: Marmorin Ribosome inactivating protein Breast cancer Apoptosis Estrogen receptor α Endoplasmic reticulum stress Breast cancer is the second most common cancer with a high incidence rate worldwide. One of the promising therapeutic approaches on breast cancer is to use the drugs that target the estrogen receptor (ER). In the present investigation, marmorin, a type I ribosome inactivating protein from the mushroom Hypsizigus marmoreus, inhibited the survival of breast cancer in vitro and in vivo. It evinced more potent cytotoxicity toward estrogen receptor (ER)-positive MCF7 breast cancer cells than ER-negative MDA-MB-231 cells. Further study disclosed that marmorin undermined the expression level of estrogen receptor α (ERα) and signicantly inhibited the proliferation of MCF7 cells induced by 17β-estradiol. Knockdown of ERα in MCF7 cells signicantly attenuated the inhibitory effect of marmorin on proliferation, suggesting that the ERα-mediated pathway was implicated in the suppressive action of marmorin on ER-positive breast cancer cells. Moreover, marmorin induced time-dependent apoptosis in both MCF7 and MDA-MB-231 cells. It brought about G2/M-phase arrest, mitochon- drial membrane potential depolarization and caspase-9 activation in MCF7 cells, and to a lesser extent in MDA-MB-231 cells. Marmorin triggered the death receptor apoptotic pathway (e.g. caspase-8 activation) and endoplasmic reticulum stress (ERS, as evidenced by phosphorylation of PERK and IRE1α, cleavage of caspase-12, and up-regulation of CHOP expression) in both MCF7 and MDA-MB-231 cells. In summary, marmorin exhibited inhibitory effect on breast cancer partially via diminution of ERα and apoptotic pathways mediated by mitochondrial, death receptor and ERS. The results advocate that marmorin is a potential candidate for breast cancer therapy. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Breast cancer is the second most common cancer with a high incidence rate worldwide [1]. More than 70% of breast cancer tissue exhibited an elevated level of expression of estrogen receptor α (ERα) compared with normal breast tissue, suggesting that ERα plays a pivotal role in breast carcinogenesis [2,3]. ERα is a steroid hormone receptor localized in the nucleus where it exerts its transcrip- tional effect by interacting with 17β-estradiol (E 2 ) and subsequently binding to specic estrogen response elements in the promoter of target gene [4]. In addition, ERα can trigger transduction signaling pathways through directly or indirectly interacting with G-coupled proteins [46]. Accumulating evidence suggest that ERα mediates E2-dependent G1S phase transition by transcriptional up-regulation of helix-turn protein Id1 [7] or Bcl-2-homology domain (BH3)-only protein Noxa [8] 222which both counteract cell cycle checkpoints. Furthermore, ERα-positive tumors manifest an overexpression of the anti-apoptotic protein Bcl-2 [9] that prevents cells from undergoing mitochondrial- dependent apoptosis. Therefore, the progression of ERα-positive cells in benign breast lesions and tumors may escape from tumorigenesis barrier that involves DNA replication stress leading to an activation of the DNA damage checkpoint, cell cycle arrest and apoptosis [10,11]. The presence of ERα in breast cancer at the time of diagnosis is an indi- cation for endocrine therapy [6] and many therapeutic strategies thereby focus on the drugs that target the estrogen receptor (ER). This kind of drugs, which are categorized into two groups including selective estrogen receptor modulators (SERM, such as tamoxifen) and selective estrogen receptor down-regulator (SERD, such as fulvestrant), are highly effective in breast cancer treatment, but their utility is restricted by endocrine resistance [12]. Consequently, development of drugs that Biochimica et Biophysica Acta 1833 (2013) 987996 Abbreviations: CHOP, C/EBP homologous protein; E 2 , 17β-estradiol; ER, Estrogen receptor; ERα, Estrogen receptor α; ERβ, Estrogen receptor β; ERS, Endoplasmic reticulum stress; GRP75, A 75 kDa glucose regulated protein; IRE1α, Inositol-requiring protein 1 α; PERK, Protein kinase RNA-activated-like endoplasmic reticulum kinase; RIP, Ribosome inactivating protein; SERM, Selective estrogen receptor modulator; SERD, Selective estrogen receptor down-regulator; ΔΨm, Mitochondrial membrane potential Corresponding author. Tel.: +852 26096872 (ofce), +852 26098031 (lab); fax: +852 26035123. E-mail address: [email protected] (T.B. Ng). 0167-4889/$ see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2012.12.013 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector

Biochimica et Biophysica Acta · 2017. 1. 3. · (School of Biomedical Sciences, The Chinese University of Hong Kong). Transformed human nasopharyngeal epithelial NP 69 cells were

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Page 1: Biochimica et Biophysica Acta · 2017. 1. 3. · (School of Biomedical Sciences, The Chinese University of Hong Kong). Transformed human nasopharyngeal epithelial NP 69 cells were

Biochimica et Biophysica Acta 1833 (2013) 987–996

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbamcr

CORE Metadata, citation and similar papers at core.ac.uk

Provided by Elsevier - Publisher Connector

Differential inhibitory potencies and mechanisms of the type I ribosomeinactivating protein marmorin on estrogen receptor (ER)-positive andER-negative breast cancer cells

Wen Liang Pan a, Jack Ho Wong a, Evandro Fei Fang b, Yau Sang Chan a, Xiu Juan Ye c, Tzi Bun Ng a,⁎a School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, Chinab National Institute on Ageing, National Institutes of Health, Baltimore, MD, USAc Key Laboratory of Plant Virology of Fujian Province, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China

Abbreviations: CHOP, C/EBP homologous protein; Ereceptor; ERα, Estrogen receptor α; ERβ, Estrogen rreticulum stress; GRP75, A 75 kDa glucose regulated proprotein 1 α; PERK, Protein kinase RNA-activated-like eRIP, Ribosome inactivating protein; SERM, SelectiveSERD, Selective estrogen receptor down-regulator; ΔΨpotential⁎ Corresponding author. Tel.: +852 26096872 (office), +

26035123.E-mail address: [email protected] (T.B

0167-4889/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbamcr.2012.12.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 August 2012Received in revised form 24 November 2012Accepted 18 December 2012Available online 26 December 2012

Keywords:MarmorinRibosome inactivating proteinBreast cancerApoptosisEstrogen receptor αEndoplasmic reticulum stress

Breast cancer is the second most common cancer with a high incidence rate worldwide. One of the promisingtherapeutic approaches on breast cancer is to use the drugs that target the estrogen receptor (ER). In the presentinvestigation, marmorin, a type I ribosome inactivating protein from the mushroom Hypsizigus marmoreus,inhibited the survival of breast cancer in vitro and in vivo. It evinced more potent cytotoxicity toward estrogenreceptor (ER)-positive MCF7 breast cancer cells than ER-negative MDA-MB-231 cells. Further study disclosedthat marmorin undermined the expression level of estrogen receptor α (ERα) and significantly inhibited theproliferation of MCF7 cells induced by 17β-estradiol. Knockdown of ERα in MCF7 cells significantly attenuatedthe inhibitory effect of marmorin on proliferation, suggesting that the ERα-mediated pathway was implicatedin the suppressive action of marmorin on ER-positive breast cancer cells. Moreover, marmorin inducedtime-dependent apoptosis in bothMCF7 andMDA-MB-231 cells. It brought about G2/M-phase arrest, mitochon-drial membrane potential depolarization and caspase-9 activation in MCF7 cells, and to a lesser extent inMDA-MB-231 cells. Marmorin triggered the death receptor apoptotic pathway (e.g. caspase-8 activation) andendoplasmic reticulum stress (ERS, as evidenced by phosphorylation of PERK and IRE1α, cleavage ofcaspase-12, and up-regulation of CHOP expression) in both MCF7 and MDA-MB-231 cells. In summary,marmorin exhibited inhibitory effect on breast cancer partially via diminution of ERα and apoptotic pathwaysmediated bymitochondrial, death receptor and ERS. The results advocate that marmorin is a potential candidatefor breast cancer therapy.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Breast cancer is the second most common cancer with a highincidence rate worldwide [1]. More than 70% of breast cancer tissueexhibited an elevated level of expression of estrogen receptor α(ERα) compared with normal breast tissue, suggesting that ERαplays a pivotal role in breast carcinogenesis [2,3]. ERα is a steroidhormone receptor localized in the nucleus where it exerts its transcrip-tional effect by interacting with 17β-estradiol (E2) and subsequentlybinding to specific estrogen response elements in the promoter of target

2, 17β-estradiol; ER, Estrogeneceptor β; ERS, Endoplasmictein; IRE1α, Inositol-requiringndoplasmic reticulum kinase;estrogen receptor modulator;m, Mitochondrial membrane

852 26098031 (lab); fax: +852

. Ng).

rights reserved.

gene [4]. In addition, ERα can trigger transduction signaling pathwaysthrough directly or indirectly interactingwith G-coupled proteins [4–6].

Accumulating evidence suggest that ERα mediates E2-dependentG1–S phase transition by transcriptional up-regulation of helix-turnprotein Id1 [7] or Bcl-2-homology domain (BH3)-only proteinNoxa [8] 222which both counteract cell cycle checkpoints. Furthermore,ERα-positive tumors manifest an overexpression of the anti-apoptoticprotein Bcl-2 [9] that prevents cells from undergoing mitochondrial-dependent apoptosis. Therefore, the progression of ERα-positive cellsin benign breast lesions and tumors may escape from tumorigenesisbarrier that involves DNA replication stress leading to an activation ofthe DNA damage checkpoint, cell cycle arrest and apoptosis [10,11].The presence of ERα in breast cancer at the time of diagnosis is an indi-cation for endocrine therapy [6] andmany therapeutic strategies therebyfocus on the drugs that target the estrogen receptor (ER). This kindof drugs, which are categorized into two groups including selectiveestrogen receptor modulators (SERM, such as tamoxifen) and selectiveestrogen receptor down-regulator (SERD, such as fulvestrant), are highlyeffective in breast cancer treatment, but their utility is restricted byendocrine resistance [12]. Consequently, development of drugs that

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aims at both ERα and the pathways which could conquer the resistanceturns out to be a promising approach for breast cancer therapy.

Ribosome inactivating proteins (RIPs), which exhibit enzymaticactivity that brings about the cleavage of a specific adenine basefrom ribosomal RNA, were first isolated and characterized from theseeds of the castor oil plant Ricinus communis [13]. RIPs were subse-quently purified from more plants [14], bacteria [15], and fungi [16].RIPs are classified into 3 types: (i) type I with a single polypeptidechain displaying a molecular mass close to 30 kDa, (ii) type II with aRIP chain and a lectin chain that facilitates internalization of the RIPchain, and (iii) maize RIP, first synthesized as an inactive precursor,and considered as an atypical type I RIP/a type III RIP [16]. Recently,small RIPs with a molecular mass of 20 kDa [17,18] or 10 kDa [19]have also been identified from mushrooms.

It has been reported that type II RIPs, as well as free or conjugatedtype I RIPs, induced cell apoptosis in vitro and in vivo [20,21]. Someinvestigations have delineated the mechanism and pathways ofRIP-induced apoptosis. Ricin caused cell cycle arrest in Hela cells atG2/M phase [22] and induced unfolded protein response in HCT116and MDA-MB-231 cells [23]. Abrin induced apoptosis in Jurkat cellsvia the mitochondrial pathway, independent of caspase-8 and hencedeath receptors [21]. Shih et al. [24] observed that abrin-inducedapoptosis was independent of N-glycosidase activity, but attributedto reduction of anti-oxidant activity of mitochondrial protein anti-oxidant protein-1, with a consequent rise in reactive oxygen speciesand cytochrome c release into the cytosol. Saporin induced apoptosisthrough a mitochondrial cascade independent of translation inhibi-tion [25]. Xiong et al. [26] demonstrated that bitter melon RIPinduced apoptosis in prostate cancer cells by inhibiting Wnt signal-ing activity and histone deacetylase-1 activity. Viscumin evokedapoptosis via a caspase-dependent pathway, with the expressionlevel of the anti-apoptotic protein Mcl-1 was down-regulated [27].Trichosanthin has been demonstrated to induce a nitric oxide(NO)-mediated apoptosis and also by generating reactive oxygenspecies (ROS) [28]. Immunotoxins containing saporin and bouganincan inhibit cell protein synthesis, induce apoptosis, and block clonogenicgrowth of target cells, although with different kinetics. Thus there is adiversity of mechanisms of cell killing [29]. It seems that the pathwaydepends on the type of tumor cell and the type of RIP.

We have isolated a 9,567-Da RIP marmorin with a novel N-terminal sequence from fresh fruiting bodies of the common ediblemushroom Hypsizigus marmoreus in a previous study [19]. Biologicalcharacterization showed that marmorin displayed higher inhibitorypotency toward breast cancer MCF7 cells, liver cancer HepG2 cellsand HIV-1 reverse transcriptase compared with plant RIPs fromfamily Cucurbitaceae [19]. Since marmorin manifests a small molecu-lar mass and a distinctive N-terminal sequence compared with otherRIPs, we were interested in ascertaining the mechanism of its inhibi-tion of breast cancer cells. In this study, purified marmorin was foundto exert its anti-tumor activity on breast cancer cells in vitroand in vivo. Interestingly, marmorin evinced more potent cytotox-icity toward ER-positive MCF7 breast cancer cells than ER-negative MDA-MB-231 cells. Both death receptor apoptotic path-way (e.g. caspase-8 activation) and endoplasmic reticulum stress(e.g. phosphorylation of PERK and IRE1α, cleavage of caspase-12,and up-regulation of CHOP expression) were involved in marmorin-induced apoptosis in MCF7 and MDA-MB-231 cells. The underlyingmechanism of the differential sensitivity of the two types of breast cancercells to marmorin was contributed by (a) diminution of the expressionlevel of ERα in ER-positive MCF7 cells, but deficiency of ERα inER-negative MDA-MB-231 cells, (b) induction of G2/M-phase arrest inMCF7 cells, and also in MDA-MB-231 cells but to a much smaller extent,(c) activation of a mitochondrial-mediated apoptotic pathway in MCF7cells (as witnessed in reduction of GRP75 expression, cleavage ofcaspase-9 and mitochondrial membrane potential depolarization), andnot in MDA-MB-231 cells (as evidenced by negligible mitochondrial

membrane potential depolarization and lack of an effect on GRP75expression and cleavage of caspase-9).

2. Materials and methods

2.1. Materials

Human breast cancer MCF7 and MDA-MB-231 cells were obtainedfrom American Type Culture Collection. Human umbilical veinendothelial cells (HUVEC) were generously provided by Prof. Y. Huang(School of Biomedical Sciences, The Chinese University of Hong Kong).Transformed human nasopharyngeal epithelial NP 69 cells weregenerously provided by Prof. S.W. Tsao (Department of Anatomy, TheUniversity of Hong Kong). Primary antibodies against PERK (#3192),IRE1α (#3294), CHOP (#2895), β-actin (#4970), caspase-12 (#2202),caspase-9 (#9502) and caspase-3 (#9662), and secondary antibodiesfor HRP-linked anti-mouse IgG (#7076) and anti-rabbit IgG (#7074)were purchased from Cell Signaling Technology (Danvers, MA, USA).Primary antibodies for ERα (sc-542), GRP75 (sc-31652) and p53(sc-6243) were acquired from Santa Cruz Biotechnology (CA, USA).Primary antibody for ERβ (Q92731) was obtained from Millipore(Darmstadt, Germany). Primary antibody for capase-8 (551243) waspurchased from BD Pharmingen (CA, USA).

2.2. Preparation of marmorin

Marmorin was isolated as described in a previous study [19]. Briefly,fresh fruiting bodies of Hypsizigus marmoreus (2 kg) were extracted byhomogenizing in 2 l of 0.15 M NaCl at 4 °C. The supernatant obtainedafter centrifugation was subsequently subjected to ion exchangechromatography on a (DEAE)–cellulose column (5 cm diameter×10 cmlength, Sigma, USA) in 10mM Tris–HCl buffer (pH 7), an Affi-gel blue gelcolumn (2.5 cm×15 cm, Bio-Rad, UK) in 20 mM NH4HCO3 buffer(pH 9.4) and a gel-filtration Superdex 75 HR 10/30 FPLC column(GE Healthcare) in 20 mM NH4HCO3 buffer (pH 9.4).

2.3. Cell culture and cell viability assessment

Cells were cultured in Dulbecco's modified Eagle's medium (DMEM,Gibco, Grand Island, USA) containing 10% fetal bovine serum (Hyclone,Gramlington, UK), 100 U/ml penicillin and 100 μg/ml streptomycin(Millipore, Darmstadt, Germany). After growing to 70–80% confluence,cells (5×103–6×103)were trypsinized and seeded into a 96-well plate.After incubation for 24 h, the cells were treatedwith increasing concen-trations (0–20 μM) ofmarmorin for 48 h, and cell viability was assessedby the MTT assay as previously described [30].

2.4. Annexin V and propidium iodide (PI) dual staining

The percentage of cells undergoing apoptosis was determinedby flow cytometry (FACSCanto I, BD, New Jersey, USA) using dualstaining with Annexin V–FITC (BD Phamingen, CA, USA) andpropidium iodide (PI, Sigma) as previously described [31]. Briefly,after seeding on a 12-well culture plate for 24 h, MCF7 and MDA-MB-231 cells (105) were treated with 5 and 15 μM marmorin fordifferent durations (0, 12, 24, 36, and 48 h), respectively. Then thecells were trypsinized, centrifuged and washed thrice with phosphatebuffered saline (PBS). The cell pellets were resuspended in bindingbuffer (0.01 M HEPES, pH 7.4, containing 140 mM NaCl and 25 mMCaCl2) for subsequent Annexin V/PI staining and flow cytometryanalysis.

2.5. siRNA assay

Double-stranded siRNA against ERα (ESR1-homo-1014) and non-specific siRNA (negative control) were purchased from Shanghai

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GenePharma Co., Ltd. ESR1-homo-1014 forward: 5′-GAG GGA GAAUGU UGA AAC ATT-3′; ESR1-homo-1014 reverse: 5′-UGU UUC AACAUU CUC CCU CTT-3′. Negative control forward: 5′-UUC UCC GAACGU GUC ACG UTT-3′; negative control reverse: 5′-ACG UGA CACGUU CGG AGA-3′. ESR1-homo-1014 or negative control siRNA (finalconcentration: 50 nmol/l) were transfected into MCF7 cells by usingLipofectamine reagent (GIBCOBRL, MD, USA) for 36 h accordingto the manufacturer's instructions. Then the transfected cells wereused for cell viability and apoptosis assays. The efficiency of knockdownof ERα was monitored by western blotting analysis of proteinexpression.

2.6. Cell cycle analysis and measurement of mitochondrial membranepotential (ΔΨm) depolarization

MCF7 and MDA-MB-231 cells (105) were treated for differentdurations with 5 and 15 μM marmorin, respectively. For cell cycleanalysis, cells were trypsinized and stained with PI (50 μg/ml PI inPBS, containing 0.05% Triton X-100 and 1 mg/ml RNase A) at 4 °Covernight with avoidance of exposure to light. Then the cell cyclewas analyzed using flow cytometry. For ΔΨm detection, cells werestainedwith 2.5 mg/ml JC-1,which can accumulate in themitochondriaas aggregates that emit red fluorescence. When ΔΨm depolarizationoccurs, JC-1 is localized in monomeric form in the cytosol and emitsgreen fluorescence.

2.7. Western blot analysis

After cells had been treated with marmorin for different durations,cell lysates were extracted by using RIPA lysis buffer (50 mM Tris-HClbuffer, pH 8.0, containing 150 mM NaCl, 1% NP40, 0.5% sodiumdeoxycholate, 0.1% SDS) containing 1 mM phenylmethylsulfonylfluoride (PMSF) and protease cocktail inhibitor. Then cell lysateswere subjected to Western blot analysis using antibody mentionedin Materials, as previously described [32]. The results were quantifiedby plotting the density of the band using Quantity One Version 4.6.2.

2.8. Tumor xenografted assay

The animal assayswere approved by Animal Experimentation EthicsCommittee of The Chinese University of Hong Kong. Five-week-oldnude mice (16–20 g) were kept in isolated pathogen-free ventilationchambers. Cell suspension was obtained by trypsinization of confluentbreast cancer cells. The carcinoma was established by subcutaneouslyinjecting 107 MCF7 or MDA-MB-2311 cells into nude mice. MCF7 cellsrequire estrogen for xenografted tumor formation; thus 100 μl agonistagent (2 mg 17-β estradiol dissolved in 1 ml olive oil) was subcutane-ously injected into the nude mice every 5 days since the day beforetumor cells injection. Tumor volume (V) was measured according tothe following formula: V=L×W2/2, where L is the mid-axis lengthand W is the mid-axis width. When tumors reached a mean sizeof 100–200 3, xenograft-bearing mice were divided into groups with 6animals per group. Marmorin (2 mg/kg) dissolved in PBS was adminis-tered daily by intraperitoneal injection for 19 days. Tumor volume wasmeasured every 3 days using a digital caliper. When the experimentended, all animals were sacrificed by cervical dislocation. Their tumorswere excised and weighed.

2.9. TUNEL staining and immunoblotting analysis of tumor specimens

The tumor specimens collected from xenograft-bearing mice weredivided into two portions: one for histological analysis by TUNELstaining assay, and the other for immunoblotting. For TUNEL staining,tumor specimens were fixed in 4% formalin and embedded in paraffinwax. Sections (4-μm-thick) were cut and subjected to immunostainingwith In Situ Cell Death Detection Kit (Roche, Germany) as per

manufacturer's instructions, and then viewed under a fluorescencemicroscope. For immunoblotting, tumor specimens were homogenizedin RIPA lysis buffer at 4 °C. Then cell lysates were extracted by centrifu-gation (14,000 g) for 30 min at 4 °C and subsequent immunoblottinganalysis was performed as previously described [32].

2.10. Statistics analysis

Results were collected from 3 independent experiments conductedin triplicate, and data are expressed as mean±SD. The histogramswere drawn using Origin Version 6.0. Student’s t test and two-wayANOVA were used for between-group comparisons. A P valueb0.05was considered as statistically significant.

3. Results

3.1. Cytotoxicity of marmorin on breast cancer cells partially mediated byreduction of ERα expression

MTT assay was used to investigate the in vitro antitumor activity ofmarmorin. Breast cancer MCF7 and MDA-MB-231 cells, transformedhuman nasopharyngeal epithelial NP 69 cells and human umbilicalvein endothelial cells (HUVEC) were exposed to increasing concen-trations (0–20 μM) of marmorin for 48 h, respectively. As shownin Fig. 1A, marmorin exerted more potent cytotoxicity on estrogenreceptor (ER)-positive MCF7 breast cancer cells (IC50=5 μM) thanER-negative MDA-MB-231 cells (IC50=15 μM). In contrast, therewas no inhibitory effect toward NP 69 cells (Fig. 1A). Interestingly,marmorin inhibited, in a dose-dependent manner, proliferation ofHUVEC which is critical to angiogenesis (Fig. 1A). Further investigationdisclosed that marmorin reduced the expression level of estrogenreceptor α (ERα) but not that of estrogen receptor β (ERβ) (Fig. 1B;Fig. S1A). The proliferation of MCF7 cells induced by 10 nM 17β-estradiol (E2), which can up-regulate the expression level of ERα(Fig. 1C; Fig. S1B), was significantly suppressed by 0.5 or 5 μMmarmorin (Fig. 1C). Moreover, knockdown of ERα in MCF7 cells signif-icantly attenuated the inhibitory effect of marmorin on proliferation(Fig. 1D). These results suggested that the ERα-mediated signalingpathway was involved in the inhibitory action of marmorin on ER-positive breast cancer cells.

3.2. Marmorin induced apoptosis in MCF7 and MDA-MB-231 cells

To characterize the type of cell death induced by marmorin,Annexin V/PI dual staining was used to evaluate the apoptotic cells.MCF7 and MDA-MB-231 cells were treated with 5 and 15 μMmarmorin respectively for different durations (0, 12, 24, 36, and48 h). As shown in Fig. 1E, marmorin induced time-dependentapoptosis in both MCF7 and MDA-MB-231 cells, with the effect onMCF7 cells being more pronounced. Furthermore, the percentage ofapoptotic cells induced by marmorin decreased when ERα wasknocked down by siRNA in MCF7 cells (Fig. 1F). These resultsunveiled that marmorin-induced apoptosis was partially dependenton the reduction of ERα in ER-positive MCF7 cells.

3.3. Exposure to marmorin induced G2/M phase arrest and DNA breakagein MCF7 cells, and to a lesser extent in MDA-MB-231 cells

ERα has been demonstrated to indirectly promote G1/S progressionthrough counteracting cell cycle checkpoint in breast cancer cells [7,8].To further investigate the role of ERα-mediated signaling in thecytotoxicity of marmorin, cell cycle analysis of MCF7 and MDA-MB-231 cells was conducted after treatment with 5 and 15 μM marmorin,respectively, for different durations (0, 6, 12, 24, 36, and 48 h).As shown in Fig. 2A, marmorin induced a drastic time-dependent G2/Mphase arrest with a decline in the proportion of G1/S-phase cells and a

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Fig. 1. Cytotoxicity of marmorin to breast cancer cells was partially mediated by reduction of ERα expression. (A) Breast cancer MCF7 and MDA-MB-231 cells, transformed humannasopharyngeal epithelial NP 69 cells and human umbilical vein endothelial cells (HUVEC) were respectively exposed to increasing concentrations (0–20 μM) of marmorin for 48 h.MTT assay was used to detect cell viability. *Pb0.05, **Pb0.01 and ***Pb0.001, when compared with control. (B) Western blot analysis of estrogen receptor α (ERα) and estrogenreceptor β (ERβ) expression after MCF7 cells had been treated with 5 μM marmorin for different durations as indicated, respectively. β-Actin served as an intrinsic control.*Pb0.05 and **Pb0.01, when compared with control. #Pb0.05, when compared with E2 treatment alone. (C) MCF7 cells were treated with 10 nM 17β-estradiol (E2), 0.5 or 5 μMmarmorin alone or co-treated with 10 nM E2 and 0.5 or 5 μM marmorin for 48 h, respectively. Then the cells were subjected to MTT assay (upper panel) or western blot analysis(lower panel). (D) The double-stranded siRNA against ERα (siRNA ERα) and non-specific siRNA (siRNA NC) were transfected into MCF7 cells for 36 h. Then the cells were usedfor western analysis (upper panel) or cell viability assessment after treatment with 5 μM marmorin for 48 h (lower panel) as indicated. **Pb0.01 and ***Pb0.001, when comparedwith control. #Pb0.05, when compared with siRNA NC cells treated with 5 μM marmorin for 48 h. (E) Marmorin induced time-dependent apoptosis in breast cancer cells in vitro.MCF7 and MDA-MB-231 cells were treated with 5 and 15 μM marmorin for different durations as indicated, respectively. Flow cytometry analysis of marmorin-induced apoptosisusing Annexin V/PI dual staining. (F) The double-stranded siRNA against ERα (siRNA ERα) and non-specific siRNA (siRNA NC) were transfected into MCF7 cells for 36 h. Then thecells were treated with 5 μM marmorin for 36 h and subsequent apoptosis analysis using Annexin V/PI dual staining.

990 W.L. Pan et al. / Biochimica et Biophysica Acta 1833 (2013) 987–996

rise in the Sub G0 cells which represents an indicator of DNA damage inMCF7 cells. An analogous change, albeit to a lesser extent, was detectedin MDA-MB-231 cells (Fig. 2B). Western blotting analysis revealedthat the expression level of p53, which plays a pivotal role incell cycle checkpoint, was up-regulated when MCF7 cells wereexposed to marmorin (Fig. 2C; Fig. S1D). However, marmorin didnot enhance p53 expression level in MDA-MB-231 cells (Fig. 2C;Fig. S1D). These results confirmed that marmorin exhibited cytotoxic-ity via the induction of p53-dependent G2/M arrest in ER-positive

breast cancer MCF7 cells, and also in ER-negative MDA-MB-231 cells,albeit to a lesser extent.

3.4. Marmorin induced differentiated mitochondrial collapse and deathreceptor apoptotic pathway in MCF7 and MDA-MB-231 cells

In view of RIP-induced apoptosis referred to two classical pathwaysmediated respectively by mitochondria and death receptor [21], thefactors contributing to these two pathways were investigated after

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Fig. 2. Exposure of MCF7 cells to marmorin induced G2/M phase arrest and DNA breakage, and to a lesser extent of MDA-MB-231 cells. (A) MCF7 cells and (B) MDA-MB-231 cellswere treated with 5 and 15 μM marmorin for different durations as indicated. Flow cytometry analysis of cell cycle using PI staining. The percentage of cells in G0/G1, S, and G2/Mphases of the cell cycle is presented in the upper panel and sub-G0 phase cells which represents an indicator of DNA damage is shown in lower panel. (C) Western blot analysis ofp53 expression. MCF7 and MDA-MB-231 cells were respectively treated with 5 and 15 μM marmorin for different durations as indicated. β-Actin served as an intrinsic control.

991W.L. Pan et al. / Biochimica et Biophysica Acta 1833 (2013) 987–996

treatment of MCF7 and MDA-MB-231 cells for different durations (0, 6,12, 24, 36, and 48 h) with 5 and 15 μM marmorin, respectively.Western blot analysis unveiled that cleavage of caspase-8 accountingfor the death receptor pathway was found in both MCF7 and MDA-MB-231 cells (Fig. 3A; Fig. S1E and F). Caspase-3, which is deficient inMCF7 cells, downstream of caspase-8, was also activated in MDA-MB-231 cells (Fig. 3A; Fig. S1F). Regarding the mitochondrial pathway,marmorin suppressed the expression level of mitochondrial chaperoneGRP75 (mtHsp70) and cleavage of caspase-9 (Fig. 3A; Fig. S1E) inMCF7cells, but not in MDA-MB-231 cells. Furthermore, flow cytometryanalysis of mitochondrial membrane potential (ΔΨm) disclosed thatmarmorin treatment produced a marked time-dependent ΔΨmdepolarization in MCF7 cells, and also in MDA-MB-231 cells, though toa lesser extent (Fig. 3B). Overall, marmorin triggered bothmitochondrialand death receptor apoptotic pathways in MCF7 cells, while it preferen-tially activated the death receptor-mediated pathway in MDA-MB-231cells.

3.5. Marmorin induced endoplasmic reticulum stress (ERS) in both MCF7and MDA-MB-231 cells

In addition to the pathways of death receptor- and mitochondria-mediated apoptosis, prolonged activation of the unfolded proteinresponse (UPR) in the endoplasmic reticulum stress (ERS) would leadto programmed cell death [33]. Three known pro-apoptotic pathwaysin response to ERS are mediated by PERK, IRE1 and caspase-12, respec-tively [34,35], which converge to a b-ZIP transcription factor CHOP thatinduces expression of genes favoring apoptosis [35] or subsequentlyactivates caspase-3 [36].

The factors responsible for ERS hereby were considered after treat-ment of MCF7 and MDA-MB-231 cells for different durations with 5and 15 μMmarmorin, respectively (0, 6, 12, 24, 36, and 48 h). Westernblot analysis revealed that marmorin induced phosphorylation ofIRE1α, cleavage of caspase-12, and up-regulation of the expressionlevel of CHOP in both MCF7 and MDA-MB-231 cells (Fig. 4; Fig. S1G

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Fig. 3. Marmorin induced differentiated mitochondrial collapse and death receptor apoptotic pathway in MCF7 and MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were treatedwith 5 and 15 μM marmorin for different durations as indicated, respectively. (A) Western blot analysis of the factors contributed to mitochondrial and death receptor pathways.Primary antibodies against GRP75, capsase-8, caspase-9 and caspase-3 were used. β-Actin served as an intrinsic control. (B) Flow cytometry analysis of mitochondrial membranepotential depolarization using JC-1 staining. Percentage of cells undergoing mitochondrial membrane potential depolarization was recorded.

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and H). In addition, phosphorylation of PERK was detected in MCF7cells, but not in MDA-MB-231 cells (Fig. 4; Fig. S1G and H). The resultsindicated that the apoptotic effect of marmorin on MCF7 and MDA-MB-231 cells was partially attributed to ERS.

3.6. Marmorin suppressed the growth of xenografted tumor bearingMCF7 or MDA-MB-231 cells

In order to determine the in vivo anti-tumor activity of marmorin,MCF7 and MDA-MB-231 cells (107) were injected subcutaneouslyinto nude mice to facilitate development of tumor xenografted,respectively. When tumors attained a mean size of 100–200 mm3,

Fig. 4. Marmorin induced endoplasmic reticulum stress (ERS) in both MCF7 and MDA-MB-different durations as indicated, respectively. The factors responsible for ERS, such as PERKserved as an intrinsic control.

the mice received intraperitoneal injections of marmorin (2 mg/kg/day).In contrast to the vehicle control, marmorin treatment for 13 days ledto a decline of tumor volume in the xenografted tumor (Fig. 5A and D).At the termination of the experiment, all nude mice were sacrificed bycervical dislocation, and their tumors were excised (Fig. 5C and F). Asrevealed in Fig. 5B and E, marmorin significantly suppressed the tumorweight compared with the vehicle control. For further investigation,MDA-MB-231 xenografted tumor specimens were used for histologicaland immunoblot analysis. As shown in Fig. 6A, marmorin greatlyincreased the amount of TUNEL-positive cells compared with the vehiclecontrol, signifying that marmorin stimulated DNA breakage in the MDA-MB-231 xenografted tumor. Furthermore, immunoblotting analysis

231 cells. MCF7 and MDA-MB-231 cells were treated with 5 and 15 μM marmorin for, IRE1α, caspase-12 and CHOP, were investigated using western blot analysis. β-Actin

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Fig. 5. Marmorin halted the growth of xenografted tumor involving MCF7 or MDA-MB-231 cells. (A, D) MCF7 cells or MDA-MB-231 cells (107) were injected subcutaneously intothe nude mice. Marmorin (2 mg/kg/day) and vehicle control were administered intraperitoneally into xenografted mice, respectively. After treatment for 19 days, mice weresacrificed by cervical dislocation, and their tumor specimens were excised (C, F) and weighed (B, E). *Pb0.05, when compared with control.

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disclosed that marmorin elevated the level of ERS marker such asIRE1α and cleavage of caspase-12, but not PERK (Fig. 6B; Fig. S1I).Intriguingly, marmorin induced cleavage of caspase-9 in xenograftedtumor involving MDA-MB-231 cells (Fig. 6B), which was contra-dictory to the in vitro outcome (Fig. 3A). These results furnished

evidence that marmorin halted the growth of MDA-MB-231xenografted tumor through cleavage of DNA and ERS. Moreover,marmorin caused the diminution of ERα expression in MCF7xenografted tumor (Fig. S2), which was in line with the findingin vitro (Fig. 1B).

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Fig. 6. Marmorin induced apoptosis in MDA-MB-231 xenografted tumor by eliciting DNA damage and ERS. (A) MDA-MB-231 xenografted tumor specimens were subjected toimmunostaining with In Situ Cell Death Detection Kit, and then viewed under fluorescent microscope. DNase I recombinant served as a positive control of a reagent causingDNA breakage. (B) Western blot analysis of cell lysates extracted from MDA-MB-231 xenografted tumor specimens using primary antibodies of PERK, IRE1α, caspase-12 andcaspase-9. β-Actin served as an intrinsic control.

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4. Discussion

It is well documented that RIPs are capable of inducing apoptosisin cancer cells by provoking several cellular stresses [21]. It is demon-strated herein that the type I RIP marmorin suppresses the in vitroand in vivo growth of breast tumors with a mechanism that entailsreduction of ERα expression, endoplasmic reticulum stress (ERS), andmitochondrial- and death receptor-mediated apoptotic pathways. It isreported for the first time here that an RIP could down-regulate theexpression level of ERα in ER-positive breast cancer cells. The resultsimply that marmorin is a candidate for treatment of breast cancersand provide a new insight into cancer therapy using RIPs.

The major finding of this study is that marmorin inhibits theexpression level of ERα and the proliferation induced by E2 in ER-positive MCF-7 cells. This implies that an ER-mediated signaling path-way is required for the manifestation of the effects of marmorin. ERαis a well-known promoter of both the onset and malignant progressionof breast cancer through ligand-dependent transcription or ligand-independent activation [6]. Drugs that target ER are classified intotwo groups: selective estrogen receptor modulators (SERM, such astamoxifen) [37] and selective estrogen receptor down-regulator(SERD, such as fulvestrant) [38]. Here, we present evidence that atype I RIP marmorin can specifically down-regulate the expressionlevel of ERα but not that of ERβ, although it is not known whethermarmorin modulates ERα by a direct or indirect mode. Furthermore,the observation that marmorin brings about a prominent G2/M phasecell cycle arrest in MCF7 cells, but not inMDA-MB-231 cells, is probablyassociated with the down-regulation of the expression of ERα whichinduces carcinogenesis predominantly bymeans of cell cyclemanipula-tion [7,8]. As expected,marmorinproduced inMCF7 cells, after treatmentfor 6 h, a pronounced increase of cells remaining in G2/M-phase (Fig. 2A)concurrent with the marked decrement of ERα expression (Fig. 1B). p53response and DNA damage, in turn, participate in marmorin-inducedapoptosis of MCF7 cells (Fig. 1E).

Another outcome that contributed to the attenuation of ERαexpression is that marmorin dramatically activates mitochondrial-mediated apoptotic pathway in MCF7 cells, but to a less remarkableextent inMDA-MB-231 cells. Generally, themitochondrial-mediated ap-optotic pathway is associatedwith factors anchored on themitochondria.

The insertion of these pro-apoptotic proteins on mitochondria altersthe mitochondrial membrane permeability, ensuing in the release ofcytochrome c that can activate caspase-9 and downstream caspase-3,eventually leading to cell apoptosis [39]. Consistent with this paradigm,a considerable number of MCF7 cells undergoing ΔΨm depolarizationand caspase-9 activation (Fig. 3) were observed after treatment withmarmorin for 12 h, and the continuing stressfinally brought about exten-sive apoptosis at 36 h (Fig. 1E). In contrast, even though a large numberof MDA-MB-231 cells underwent apoptosis after treatment withmarmorin for 24 h (Fig. 1E), the cells demonstrated only slight ΔΨmdepolarization and there was no caspase-9 activation (Fig. 3). It hasbeen reported that ERα influenced the survival of breast cancer cellsthrough regulation of Bcl-2/BclXL [7–9]. Therefore, abrogation of thestimulatory effect of ERα on overexpression of Bcl-2 that prevents theinsertion of pro-apoptotic proteins on mitochondria facilitates the initia-tion of mitochondrial apoptotic pathway in MCF7 cells. Collectively,the above phenomena may account for the discrepancy between thesensitivity of MCF7 and MDA-MB-231 cells to marmorin.

Apart from modulation of ERα expression, results of this studyindicate that marmorin induces death receptor and ERS apoptoticpathway in both MCF7 and MDA-MB-231 cells. Caspase-8 activationis a conspicuous feature of the death receptor-mediated apoptoticpathway [39]. Although the death receptor-mediated apoptotic path-way was engaged in both MCF7 and MDA-MB-231 cells after admin-istration of marmorin (e.g. cleavage of capsase-8, Fig. 4A), caspase-8stimulated different downstream signaling events in these twocell lines due to deficiency of caspase-3 in MCF7 cells. Caspase-8amplified the mitochondrial apoptotic loop through cleavage of theBH3 protein Bid that creates holes in themitochondria leading tomito-chondrial collapse [39], whichwas expected inmarmorin-treatedMCF7cells (e.g.ΔΨmdepolarization and caspase-9 activation, Fig. 3). Alterna-tively, caspase-8 directly cleaved caspase-3 culminating in cell death[39], which was discernible at 12 h in MDA-MB-231 cells followingexposure to marmorin (Fig. 3A). Interestingly, the effect of marmorinon cleavage of caspase-3 was sustained until 12 h in MDA-MB-231cells, hinting that this action contributed to the early cytotoxicity ofmarmorin. To further prolong the impact, marmorin needs to launchanother apoptotic pathway, such as ERS. CHOP, which is regulated bythree known pathways [34,35], is a key mediator of ERS-induced

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apoptosis [40] and could block AKT activation through binding to thepromoter of TRB3 gene [41]. As anticipated, we found that marmorinmarkedly heightened the level of expression of CHOP later, at 24, 36and 48 h, in both MDA-MB-231 and MCF7 cells. In MCF7 cells, long-term anti-ER treatment engenders drug resistance regarding prolifera-tive AKT/PI3K signaling [42]. Hence, marmorin, as a SERD, seems to becompetent for circumventing this resistance to endocrine therapy viaup-regulation of CHOP expression.

In a nude mouse model involving MDA-MB-231 cells, we discoverthat marmorin restrains the growth of breast cancer in vivo. Devoid ofinduction of inflammation, apoptosis is the favorable mode of killingcancer cells in vivo [39]. In conformity with in vitro results, marmorininduced DNA damage and ERS that elicited apoptosis in mice bearingMDA-MB-231 xenografted tumor (Fig. 6). Also, marmorin down-regulated the expression level of ERα (Fig. S2) in MCF7 xenograftedtumor. In addition, marmorin hindered angiogenesis through lower-ing the viability of HUVEC (Fig. 1A) in vitro. These observationssuggest that marmorin also starves tumors to death throughdiminishing the amount of blood vessels in vivo. Our data alsounveiled that marmorin exerted no cytotoxicity on normal NP69cells (Fig. 1A), as well as WRL-68 embryonic liver cells [19] in vitro.At the conclusion of the in vivo experiment, the tumor-bearing micewere dissected. No abnormalities were noted in the viscera andorgans (data not shown), signifying that the dosage of marmorin(2 mg/kg/day) is safe for usage in mice. Surprisingly, inconsistentwith the upshot in MDA-MB-231 cells, marmorin provoked cleavageof caspase-9 in tumor bearing nude mice. Zhang et al. [43] showedthat the apoptotic rate in maspin-expressing tumor cells was increasedupon treatment with serum starvation, and that maspin-mediatedapoptosis was partially offset by an inhibitor of caspase-9. This discrep-ancy was probably attributed to the inhibitory effect of marmorin onangiogenesis, which caused the xenografted tumor to go into starvationwhich increased the likelihood of the MDA-MB-231 xenografted tumorundergoing caspase-9 mediated apoptotic pathway.

In summary, marmorin exerts cytotoxicity on breast cancers bothin vitro and in vivo. Marmorin initiates the mitochondrial apoptoticpathway by modulation of ERα expression in ER-positive MCF7cells. Furthermore, marmorin targets the death receptor and ERSapoptotic pathways in both ER-positive and -negative breast cancers.It seems that triggering a variety of apoptotic pathways enhanced theanti-tumor effect of marmorin. Moreover, the anti-proliferative activityof marmorin on HUVEC cells implies an anti-angiogenic and henceanti-metastatic potential. These observations indicate the potentialutility of marmorin for breast cancer therapy.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbamcr.2012.12.013.

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