Induction of ROS-Dependent Mitochondria-Mediated IntrinsicApoptosis in MDA-MB-231 Cells by Glycoprotein from CodiumdecorticatumRamar Thangam,*,†,#,∥ Dharmaraj Senthilkumar,§,∥ Veeraperumal Suresh,*,□ Malairaj Sathuvan,⊥

Srinivasan Sivasubramanian,# Kalailingam Pazhanichamy,□ Praveen Kumar Gorlagunta,▽

Soundarapandian Kannan,*,†,△ Palani Gunasekaran,# Ramasamy Rengasamy,⊥ and Jayanthi Sivaraman*,§

†Proteomics and Molecular Cell Physiology Laboratory, Department of Zoology, Bharathiar University, Coimbatore, Tamil Nadu,India§School of Bio-sciences and Technology, VIT University, Vellore, Tamil Nadu, India#Department of Virology, King Institute of Preventive Medicine and Research, Chennai, Tamil Nadu, India⊥Centre for Advanced Studies in Botany, University of Madras, Chennai, Tamil Nadu, India□Department of Biotechnology, IIT Madras, Chennai, Tamil Nadu, India△Department of Zoology, Periyar University, Salem, Tamil Nadu, India▽Department of Zoology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India

*S Supporting Information

ABSTRACT: Marine macroalgae consist of a range of bioactive molecules exhibiting different biological activities, and many ofthese properties are attributed to sulfated polysaccharides, fucoxanthin, phycobiliproteins, and halogenated compounds. In thisstudy, a glycoprotein (GLP) with a molecular mass of ∼48 kDa was extracted and purified from Codium decorticatum andinvestigated for its cytotoxic properties against human MDA-MB-231 breast cancer cells. The IC50 values of GLP against MDA-MB-231 and normal breast HBL-100 cells (control) were 75 ± 0.23 μg/mL (IC25), 55 ± 0.32 μg/mL (IC50), and 30 ± 0.43 μg/mL (IC75) and 90 ± 0.57 μg/mL (IC25), 80 ± 0.48 μg/mL (IC50), and 60 ± 0.26 μg/mL (IC75), respectively. Chromatincondensation and poly(ADP-ribose) polymerase (PARP) cleavage studies showed that the GLP inhibited cell viability byinducing apoptosis in MDA-MB-231 cells. Induction of mitochondria-mediated intrinsic apoptotic pathway by GLP wasevidenced by the events of loss of mitochondrial membrane potential (ΔΨm), bax/bcl-2 dysregulation, cytochrome c release, andactivation of caspases 3 and 9. Apoptosis-associated factors such as reactive oxygen species (ROS) formation and loss of ΔΨmwere evaluated by DCFH-DA staining and flow cytometry, respectively. Cell cycle arrest of G2/M phase and expression ofapoptosis associated proteins were determined using flow cytometry and Western blotting, respectively.

KEYWORDS: Codium decorticatum, glycoprotein, ROS, cell cycle arrest, mitochondrial membrane potential (ΔΨm) loss, apoptosis

■ INTRODUCTION

Recently, there has been an interest in the investigation ofnaturally occurring products such as carbohydrates, proteins,and secondary metabolites for their potential in treating severalcancers. Multiple epidemiological studies have reported thatdiets rich in flavonoids, proteins, etc., can reduce the incidenceof various cancers due to their antioxidant properties.1

Edible seaweeds are rich in bioactive antioxidants, solubledietary fibers, proteins, minerals, vitamins, phytochemicals, andpolyunsaturated fatty acids. Although seaweeds find applica-tions as gelling and thickening agents in the food andpharmaceutical industries, recent research has revealed theirpotential as complementary medicine. Red, brown, and greenseaweeds have been shown to possess therapeutic properties forhealth and disease management such as anticancer, antiobesity,antidiabetic, antihypertensive, antihyperlipidemic, antioxidant,anticoagulant, anti-inflammatory, immunomodulatory, anties-trogenic, thyroid stimulating, neuroprotective, antiviral, anti-fungal, and antibacterial properties in vitro and in vivo.2 Active

compounds from seaweeds include sulfated polysaccharides,phlorotannins, carotenoids (e.g., fucoxanthin), minerals,peptides, and sulfolipids that have proven benefits againstdegenerative metabolic diseases.3

Several of the currently available drugs for cancer therapyaffect both cancer and normal cells by exhibiting toxicity, andthis necessitates searching for novel, effective, and nontoxicanticancer compounds from natural sources. Recently,glycoproteins from natural sources have been gaining attentionfor their anticancer properties. Glycoprotein from Lonicerajaponica stimulates the growth of the ICE-6 normal murineintestinal epithelial cells but exhibits antiproliferative effects onHT-29 colon cancer cells.4 However, reports are limited foralgal glycoproteins having anticancer properties.5

Received: November 15, 2013Revised: March 14, 2014Accepted: March 23, 2014Published: April 2, 2014

Article

pubs.acs.org/JAFC

© 2014 American Chemical Society 3410 dx.doi.org/10.1021/jf405329e | J. Agric. Food Chem. 2014, 62, 3410−3421

Apoptosis is a highly regulated programmed cell deathprocess that eliminates dysfunctional cells from the body byself-degradation.6 Apoptosis can be triggered in a cell througheither the extrinsic pathway or the intrinsic pathway. Theextrinsic pathway is initiated through the stimulation oftransmembrane death receptors such as Fas receptors, locatedon the cell membrane.7 In contrast, the intrinsic pathway ofapoptosis is driven by a mitochondria- mediated death signalingcascade through the release of death signal factors, and thesetwo apoptotic pathways are executed mainly by a class ofcysteine proteases known as caspases.8

During apoptosis, engagement of the mitochondrial pathwayinvolves mitochondrial membrane permeabilization (MMP),which depends on activation, translocation, and oligomerizationof multidomain Bcl-2 family proteins such as Bax or Bak. MMPtriggers the release of cytochrome c and other apoptogenicproteins into the cytosol followed by the activation of caspasecascades, which eventually results in poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis.9 Besides, MMPinduces apoptosis through altering the levels of expression ofBcl-2 family proteins.10 PARP are critical to DNA repair andapoptosis signaling, and play an important role in variousmechanisms of cellular stress.7,8

Apoptosis can also occur via the caspase-independentapoptotic pathway in which apoptosis-inducing factor (APIF)plays a key role.11,12 APIF protein is normally confined tomitochondria and is released by cleavage of calpains uponapoptotic stimulation.13,14 The truncated form of APIF proteinacts as a pro-apoptotic mediator, which translocates first frommitochondria into the cytosol followed by entry into thenucleus, where it promotes chromatin condensation and DNAdegradation.15,16

Codium decorticatum is a dark green alga belonging to thefamily Codiales, and it is commonly found in the Gulf ofMannar region, India. Thus far, there has been no report on thebiological properties of glycoprotein (GLP) from seaweeds.Hence, in the present study, GLP from C. decorticatum wasextracted, purified, and investigated for its cytotoxicity potentialon human breast cancer (MDA-MB-231) cells against normal(HBL-100) breast epithelial cells that served as control. Also,the apoptotic effect of GLP through activation of intrinsicsignaling cascade pathway in MDA-MB-231 breast cancer cellswas evaluated with respect to the expression of mitochondrialmembrane proteins, ROS generation and cleavage of PARP,and up- and downstream events of bcl-2 family genes.

■ MATERIALS AND METHODSMaterials and Chemicals. Toluidine Blue O and ammonium

sulfate were purchased from SRL Chemicals, Mumbai, India. Q-Sepharose anionic exchanger, Dulbecco’s minimal essential medium(DMEM), penicillin, streptomycin, 2′,7′-dichlorofluorescein-diacetate(DCFH-DA), DAPI stain, Acridine orange (AO), ethidium bromide(EtBr), and propidium iodide (PI) were purchased from SigmaAldrich, USA. Dialysis membrane (MWCO 12−14 kDa) and methylthiazol tetrazolium salt were purchased from HiMedia, India.Coomassie Brilliant Blue (CBB) stain, periodic acid−Schiff (PAS)stainm and medium-range protein marker (14.3−97.4 kDa) for SDS-PAGE were purchased from Genei, India. Fetal bovine serum waspurchased from Gibco, Inc., USA. LDH assay kit was procured fromCayman Chemicals, USA. cDNA synthesis kit (SuPrimeScript RTMaster Mix, catalog no. SR2000) was purchased from GeNetBio.Primary antibodies against cytochrome c and caspase 3 were purchasedfrom Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-PARP antibody was procured from Pharmingen, San Diego, CA, USA.

Antibody against β-actin was purchased from Bio-World (St. LouisPark, MN, USA). Horseradish peroxidase-conjugated secondaryantibodies were purchased from Jackson Immuno Research (WestGrove, PA, USA). All other reagents and chemicals were of the highestpurity grade.

Extraction and Purification of GLP from C. decorticatum. Thecollection of fresh and healthy C. decorticatum specimens was madebetween December 2011 and January 2012 during low tide at thedepth of 1−3 m along the coast of Kilakarai, Gulf of Mannar, TamilNadu, India. The collected algae were washed with seawater anddistilled water. The fresh thallus of the alga was powdered with liquidnitrogen and stored at −80 °C until used. One hundred and fiftygrams of powder was added to 1 L of 0.05 mM phosphate buffer (pH7.2) and dispersed for 6 h with constant stirring at 4 °C followed byfiltration using cheesecloth to remove insoluble debris. Furthermore,the sample was centrifuged at 12500 rpm for 20 min to removeinsoluble matter. The supernatant was saturated with 20% followed by70% ammonium sulfate and centrifuged at 12000 rpm for 20 min toobtain pellets, which were dissolved and dialyzed against 0.05 Mphosphate buffer at pH 7.2. The dialyzed sample was loaded onto Q-Sepharose anionic exchange column (30 cm × 2.5 cm) and washedwith 0.05 M phosphate buffer at pH 7.2. The sample was elutedserially with 50 mL of 50, 100, 150, 200, 300, 400, 500, and 700 mMNaCl in the same buffer with a flow rate of 2.0 mL/min. Fractions of 5mL volume were collected and read at 280 and 490 nm using a UV−vis spectrophotometer (DU-40 spectrophotometer, Beckman, USA)for identification of protein- and carbohydrate-rich fractions,respectively. Furthermore, GLP-rich fractions showing the presenceof GLP band by CBB and PAS staining methods on sodium dodecylsulfate−polyacrylamide gel electrophoresis (SDS-PAGE) were col-lected, pooled, dialyzed against the same buffer, lyophilized (Vir TisFTS systems, Warminster, PA, USA), and stored in vials at 4 °C forfurther use (Scheme 1). The purity of the GLP fraction from anion

Scheme 1. Extraction and Purification Process ofGlycoprotein from C. decorticatum

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exchange chromatography was analyzed by the presence of a singleband detected with CBB staining (for protein) and PAS staining (forpolysaccharides) on SDS-PAGE. Besides, Toluidine Blue O stainingwas employed for the detection of polysaccharides resolved in agarosegel by electrophoresis.Cell Culture and Maintenance. The selected cell lines of human

MDA-MB-231 and HBL-100 cells were obtained from NationalCentre for Cells Science (NCCS), Pune, India. Detailed cultureconditions are provided in the Supporting Information.Cell Proliferation Assay. Cell proliferation was analyzed by 3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)assay, as described by Mosmann.17 Briefly, exponentially growingMDA-MB-231 and HBL-100 cells (1 × 104 cells/mL) were seeded in96-well plates in a final volume of 100 μL/well and were treated with10 μL of test sample (GLP) in FCS-free complete medium at variousconcentrations (5−100 μg/mL) for 48 h. One hundred microliters ofMTT (5 mg/mL) was added to treated cells, and the plates wereincubated at 37 °C for 4 h. The supernatant was aspirated, and 100 μLof dimethyl sulfoxide (DMSO) was added to each well to dissolve theFormosan crystals. Absorbance was measured at 620 nm using a 96-well microplate reader (THERMO Multiskan, USA), and theinhibitory concentrations (IC25, IC50, and IC75) were determined forGLP causing reduction in cell viability. The percentage of cell survivalwas calculated using the following formula:

=

×

cytotoxicity (%) (mean exptl absorbance

/mean control absorbance) 100

The control wells were not treated with GLP.Lactate Dehydrogenase (LDH) Assay. The LDH assay was

performed with an LDH colorimetric assay kit using an ELISA readeraccording to the manufacturer’s protocol. Detailed experimentalconditions are provided in the Supporting Information.Determination of Apoptosis Induction and Cell Death. The

induction of apoptosis by GLP-treated cells was analyzed by AO/EtBrstaining. Detailed conditions for this method are provided in theSupporting Information.Fluorescent Microscopic Studies on Chromatin Condensa-

tion. The condensation of chromatin and nuclear fragmentation inGLP-treated cells were analyzed by DAPI staining. Detailed conditionsare provided in the Supporting Information.Measurement of ROS Generation. 2′,7′-Dichlorofluorescein-

diacetate (DCFH-DA) can be deacetylated by intracellular esterase tononfluorescent DCFH, which can be oxidized by ROS, resulting in theformation of fluorescent compound 2′,7′-dichloroflorescein (DCF).The fluorescence intensity of DCF is proportional to the amount ofROS produced by the cells. After seeding of 5 × 105 cells/well to a 6-well plate, the cells were treated with GLP for 24 h in dose-dependentconcentrations. Subsequently, cells were washed once with ice-coldPBS and incubated with DCFH-DA (50 μM final concentration) at 37°C for 30 min in the dark. Then, the cells were washed twice andmaintained in 1 mL of PBS. ROS generation was assessed using afluorescence microscope (Nikon Eclipse, Inc., Japan) at excitation andemission wavelengths of 488 and 530 nm, respectively, and the meanfluorescence intensity of DCF was evaluated.Determination of ΔΨm by Flow Cytometry. MDA-MB-231

cells (1 × 105/mL) were seeded in a 6-well plate containing completemedium and allowed to attach for 24 h. The cells were treated withGLP for 24 h in dose-dependent concentrations and harvested. Themedium was removed, and the adherent cells were trypsinized. Thecells were pelleted by centrifugation at 2000 rpm for 10 min followedby resuspension with 1 mL of Rhodamine 123 (Rh 123; 5 μg/mL inmethanol) for 30 min at 37 °C in dark, washed with PBS twice, andresuspended in PBS. Then, Rh 123-stained cells were fixed with 4%paraformaldehyde for 10 min. Finally, the cells were washed withmethanol and analyzed for change in ΔΨm using BD FACS caliber(FL-1 detector with 530 nm band-pass filter). Data were analyzedusing Cell Quest Pro 6.0 software (BD Biosciences, San Jose, CA,USA).

Cell Cycle Analysis. Measurement of cellular DNA content anddistribution of cells for cell cycle analysis were carried out by flowcytometry. Detailed experimental conditions are provided in theSupporting Information.

Semiquantitative Reverse Transcription PCR (RT-PCR) forMitochondrial Gene Expression. RT-PCR was performed todetermine changes in the expression of bax, bcl-2, and internal controlβ-actin genes. TriZol reagent was used to isolate total RNA andreverse transcribed. Briefly, the cDNA was synthesized using a cDNAsynthesis kit and amplified according to the manufacturer’s protocol ina 25 μL reaction mixture containing random primer pairs (1.0 μL):10× buffer (5.0 μL), cDNA (2.0 μg), 25 mM/L MgCl (3.0 μL), 10mM/L dNTPs (1.0 μL), and Taq polymerase (2.5 units).Amplification cycles consisted of denaturation at 94 °C for 1 min,primer annealing at 57 °C for 45 s, and extension at 72 °C for 45 s, fora total of 30 cycles followed by a final extension at 72 °C for 10 min.The following primer sequences were used for amplification: (a) baxgene, forward 5′-TTTGCTTCAGGGTTTCATCC-3′ and reverse 5′-CAGTTGAAGTTGCCGTCAGA-3′; (b) bcl-2 gene, forward 5′-CATCCATTATAAGCTGTCGCA-3′ and reverse 5′-TGCCGG-TTCAGGTACTCAGT-3′; and (c) β-actin, forward 5′-GTTGCT-ATCCAGGCTGTGC-3′ and reverse 5′-GCATCCTGTCGGCAA-TGC-3′. The PCR products were electrophoresed and visualized bytransillumination system (Alpha Innotech Image viewer 6.0.0, Japan).

Analysis of Expression of Mitochondrial Membrane Proteinsby Western Blot. Cells (1 ×106 cells/well) were transferred to aculture dish (100 mm × 20 mm) for 24 h; the cells were treated for 24h with GLP in dose-dependent concentration, harvested, and washedtwice with ice-cold PBS. Subsequently, a 1.5 mL microfuge tubecontaining cell extract was centrifuged at 2500 rpm for 5 min todiscard the supernatant. Treated cells were washed in PBS and lysed in100 μL of buffer containing 50 mM Tris-HCl (pH 8.0), 150 mMNaCl, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 10μg/mL pepstatin, and 10 μg/mL leupeptin. After 20 min, extracts werecentrifuged at 12000 rpm for 10 min at 4 °C, and the supernatantswere stored at −80 °C until further use. Proteins (30 μg/lane) wereseparated using 10% SDS-PAGE and transferred to PVDF membranes.The membranes were blocked in TBST solution containing 5% (w/v)nonfat milk for 2 h, followed by overnight incubation at 4 °C withprimary antibodies such as cytochrome c, caspase 9, caspase 3, and β-actin. After being washed with TBST buffer, the membranes wereincubated for 1 h with the secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG. Antibody-bound proteins weredetected using enhanced chemiluminescence reagents. Blots werewashed with washing buffer and incubated with secondary antibodiesconjugated with horseradish peroxidase for 1 h at room temperature.

Poly(ADP-ribose) Polymerase (PARP) Cleavage Analysis.Protein samples (50 μg) in nuclear fractions were resolved on 7.5%SDS-PAGE gels and transferred to nitrocellulose membranes. Themembranes were blocked overnight with BSA solution (5% bovineserum albumin in PBS) and 0.1% Tween 20 at 4 °C and thereafterincubated with anti-PARP antibody (1:2000). The immune complexeswere visualized as described for immune-blot analysis.

Statistical Analysis. All of the experiments were done intriplicates, and the experiments were repeated at least three times.The statistical software SPSS version 17.0 (SPSS Inc., Chicago, IL,USA) was used for analysis. p values were determined using Student’s ttest; (*) p ≤ 0.05 and (**) p ≤ 0.01 were considered statisticallysignificant.

■ RESULTSExtraction and Purification of GLP from C. decortica-

tum. The crude extract from C. decorticatum was precipitatedwith 20 and 70% ammonium sulfate fractionation, and theprecipitate of the 70% ammonium sulfate saturated fraction wassubjected to dialysis. The dialyzed sample was loaded onto theQ-Sepharose anion exchange column chromatograph andfractions were collected. A total of 80 fractions were collectedand the content of protein and carbohydrate was determined

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for each fraction. Eight GLP-rich fractions (Figure 1A), elutedwith 500 mM NaCl solution, were identified, and each fraction

was again checked for purity by SDS-PAGE stained with CBBand PAS for protein and GLP, respectively. Among the 8eightfractions, six were homogeneous and were pooled, dialyzed, andlyophilized. We confirmed the presence of a GLP band havingthe molecular mass of ∼48 kDa (Figure 1B). The yield of thepurified GLP was found to be 195 mg (0.13%) from 150 g ofdried extract of C. decorticatum, and the contents ofcarbohydrate and protein in GLP were found to be 36.24and 63.76%, respectively. The detailed process of extraction andpurification of GLP is depicted in Scheme 1.GLP Reduces Cell Viability and Causes Membrane

Damage. To investigate the effects of apoptotic cell deathinduced by GLP, MDA-MB-231 and HBL-100 cells weretreated with GLP at different dose levels for 24 h and subjectedto MTT assay. Results showed that MDA-MB-231 cellsexhibited strong growth inhibitory effects when compared toHBL-100 cells (Supporting Information Figure S2). Weevaluated the effect of GLP upon cell viability and plasmamembrane damage of normal cell lines against untreatedcontrols (Supporting Information Figure S2; Figure 2A,B). Thecytotoxicities of GLP in dose-dependent concentration forMDA-MB-231 and HBL-100 cells were found to be 75 ± 0.23μg/mL (IC25), 55 ± 0.32 μg/mL (IC50), and 30 ± 0.43 μg/mL(IC75) and 90 ± 0.57 μg/mL (IC25), 80 ± 0.48 μg/mL (IC50),and 60 ± 0.26 μg/mL (IC75), respectively (Figure 2A).The extent of cell membrane damage of GLP-treated MDA-

MB-231 cells at dose-dependent concentrations in 24 h wasmeasured by LDH release assay (Figure 2B). The percentage of

added LDH protein either absorbed or inactivated by dose-dependent concentrations in 24 h is shown in Figure 2B. Thefigure shows that the release of LDH was greater when the cellswere treated with increasing concentrations of GLP.In addition, direct observation using an inverted microscope

revealed numerous morphological changes in MDA-MB-231cells treated with GLP (data not shown). There are nosignificant cytotoxic effects of GLP in normal HBL-100 cells;hence, additional apoptotic parameters were performed only forcancer cells. In MDA-MB-231 cells, chromatin condensation,loss of nuclear construction, and formation of apoptosis bodiesappeared in a dose-dependent manner after GLP treatment. Toexamine whether GLP inhibits the proliferation of MDA-MB-231 cells by inducing apoptosis, cells were investigated bynuclear DAPI staining (Figure 2C). The GLP-treated cellsexhibited typical morphological features of apoptosis withnuclear fragmentation, whereas control (untreated) cells didnot show these features.

Detection of Apoptosis Induction and Cell Death byAO/EtBr and DAPI Staining. Using fluorescence microscopy,necrotic and apoptotic cells were distinguished on the basis ofoverall cell morphology and cell membrane integrity. Uponstaining with AO/EtBr, apoptotic cells containing apoptoticbodies and necrotic cells were observed as orange and red,respectively, whereas untreated MDA-MB-231 cells were foundto exhibit green fluorescence with the absence of morphologicalchanges (Figure 3). However, morphological changes as well asthe induction of apoptosis in these cells were detected aftertreatment with GLP at dose-dependent concentrations for aperiod of 24 h, and the percentage of apoptotic induction isdepicted in Figure 3.DAPI is also known to form fluorescent complexes with

double-stranded DNA and is useful in finding the apoptoticcondensed nuclei. The 80−90% confluent MDA-MB-231 cellswere seeded in 24-well plates and exposed to GLP for 24 h.The morphology and the condensed nuclei chromatins wereobserved in the cells using a fluorescence microscope after cellfixation. Apoptotic nuclei were identified by the reducednuclear size and gathering of condensed chromatin at theperiphery of the nuclear membrane besides the fragmentedmorphology of nuclear bodies (Figure 2C).

GLP Increases Intracellular ROS Level in MDA-MB-231Cells. ROS play a central role in the regulation of cellularapoptosis. We examined the effect of GLP in intracellular ROSgeneration due to increase in ΔΨm loss that could mediateintrinsic apoptotic signaling pathways (Figure 4). ROSscavenger DCFH-DA was used to confirm the role of ROS inGLP-induced apoptosis. The results showed that there was areduction of DCFH2 into DCF through esterase activity due tothe release of peroxidase cytochrome c in the cytosol leading toincreased production of ROS in GLP-treated MDA-MB-231cells (Figure 4A,B) when compared to untreated cells (data notshown). After exposure of MDA-MB-231 cells to 75 ± 0.23 μg/mL (IC25), 55 ± 0.32 μg/mL (IC50), and 30 ± 0.43 μg/mL(IC75) GLP for 24 h in a dose-dependent manner withuntreated control cells, the ROS levels in these cells weremeasured as 21 ± 4.76, 43 ± 6.60, 62 ± 4.30, and 76.04 ±5.34%, respectively (Figure 4B). The production of ROS isincreased in a dose-dependent manner in GLP-treated MDA-MB-231 cells (Figure 4A,C). These data suggested that ROSgeneration is required for ΔΨm loss and subsequent inductionof intrinsic apoptotic signaling pathways in GLP-treated MDA-MB-231 cells.

Figure 1. (A) Elution profile of crude GLP-rich extract on Q-Sepharose Fast Flow chromatography. (B) Electrophoretic analysis ofGLP from C. decorticatum. CBB staining reveals (i) GLP-rich crudefraction and (ii) purified GLP by SDS-PAGE; PAS staining shows (iii)purified GLP by SDS-PAGE; Toluidine Blue staining shows (iv)purified GLP by agarose gel electrophoresis.

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GLP Triggers Mitochondria-Dependent Apoptosis. Toinvestigate the molecular mechanism of GLP-induced apopto-sis, we examined the effect of GLP on mitochondrial functionin MDA-MB-231 cells using Rh 123 fluorescence staining.Treatment with increasing concentrations of GLP induced adose-dependent loss of ΔΨm (Figure 5A). In addition, wefound the dose-dependent release of cytochrome c frommitochondria into the cytosol, which was associated withchanges in the ratio of expression of bcl-2/bax. These datareveal that GLP could cause mitochondrial dysfunction inMDA-MB-231 cells. The role of GLP in intracellular ΔΨm

dysfunction and the subsequent induction of mitochondria-mediated intrinsic apoptotic signaling pathways in MDA-MB-231 cells were evaluated by FACS analysis. The results showedthat the loss of ΔΨm increased in a dose-dependent manner incells treated with GLP for 24 h, and this resulted in induction ofintrinsic apoptotic signaling pathway in the cells (Figure 5B,C).GLP Promotes G2/M Arrest in MBA-MB-231 Cells. To

understand the cell death mechanism, GLP was evaluated for its

effect on cell cycle and induction of apoptosis in cancer cells byflow cytometry. The results on cell cycle analysis for untreatedcontrol cells and GLP-treated cells with dose-dependentconcentrations are depicted in Figure 6A. From the figure, itwas observed that there was a significant increase in cells at sub-G0 as well as G2/M phases (∗∗, p ≤ 0.01). The cells at G0/G1

phase decreased significantly (∗, p ≤ 0.05), whereas those at Sphase did not change significantly (Figure 6B). The studyfindings revealed the effect of GLP on the accumulation of cellsin G2/M phase accompanied by cell cycle arrest (Figure 6). Itwas shown that GLP arrested the growth of cancer MDA-MB-231 cells in G2/M phase. This suggested that GLP might act asa G0/G1 checkpoint that controlled the progression of cellsfrom G1 to S phase and thereby prevented the replication ofDNA. These results suggested that the GLP delayed cell cycleprogression by arresting cells in the G2/M phase of cell cycle,resulting in apoptosis.

Expression of bcl-2 Family Genes, Attenuation ofMMP, Loss of ΔΨm, and Induction of Apoptosis. As

Figure 2. (A) GLP-induced cytotoxicity in MDA-MB-231 and HBL-100 cells treated with dose-dependent concentrations of GLP for 24 h intriplicates. (B) Plasma membrane damage of MDA-MB-231 breast cancer cells was assessed by LDH assay. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01,compared with untreated cells (control groups). (C) Effects of GLP on nuclear fragmentations in MDA-MB-231 cells by DAPI staining. Intenselystained condensed apoptotic nuclei bodies besides nuclear change and relocations were observed in GLP-treated cells by fluorescence microscope(400× magnification).

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shown in Figure 7A, an additional experiment was carried outto elucidate whether GLP-induced apoptosis is associated withthe expression of bcl-2 family member genes such as bax andbcl-2. Cells were exposed to dose-dependent concentrations ofGLP for 24 h with untreated control cells, and the expressionprofile of pro-apoptotic bax and anti-apoptotic bcl-2 familygenes in response to dose-dependent manner was elucidated(Figure 7A). The gene expression profiles in GLP-inducedapoptosis of MDA-MB-231 cells were further confirmed byinvestigating with densitometry calculations (Figure 7B). Asshown in Figure 7B, exposure of cells to dose-dependentconcentrations led to change in the expression of mitochondrialpro-apoptotic and anti-apoptotic genes. Furthermore, weinvestigated the role of mitochondria in GLP-induced apoptosisof MDA-MB-231 cells by measuring Rh 123 dye retention. Asshown in Figure 7B, MDA-MB-231 cells treated with GLPexhibited a significant reduction in mitochondria dysfunction ina dose-dependent manner.GLP Induces Apoptosis through a Caspase 3-Depend-

ent Pathway. The mitochondrial pathway is one of the majorapoptotic pathways, which is often related to the loss of ΔΨm.The release of cytochrome c from mitochondria to cytosol isthe limiting factor in the mitochondrial pathway, and the loss ofΔΨm has been suggested to cause the release of cytochrome c.Here, we investigated the activation of caspases and thesubsequent proteolytic cleavage of PARP proteins in MDA-

MB-231 cells by Western blotting. Western blot analysissuggested that GLP treatment with dose-dependent concen-trations led to a significant increase in caspase 3, caspase 9, andPARP proteins (Figure 8A and B). The release of cytochrome c(14 kDa) could initiate apoptosis via the mitochondrialpathway by activation of caspase 9 and caspase 3, and cleavageof PARP. 37/35 kDa caspase 9, upon cleavage, was convertedto active caspase 9 having the molecular weight of 17 kDa. Themolecular weight of PARP before and after cleavage was 116and 89 kDa, respectively (Figure. 8). After MDA-MB-231 cellswere treated with GLP in dose dependent concentration for 24h, the release of cytochrome c increased in a concentration-corresponding manner (Figure 8). The ratios for densitometricvalues of expression of cytochrome c/β-actin, caspase 9/β-actin,caspase 3/β-actin, and PARP/β-actin for control and cancercells were also calculated (Figure 8B). These results suggestedthat GLP induced caspase 3-dependent apoptosis in MDA-MB-231 cells.

■ DISCUSSIONCancer is one of the most common diseases that threatenhuman life. Unfortunately, the drugs used for cancer therapyare toxic and affect not only cancer cells but also normal cells.Thus, finding novel, effective, and nontoxic compounds fromnatural sources is important now more than ever before.Induction of apoptosis has been the target mechanism forcancer treatments, and DNA fragmentation is a marker of celldeath. There have been limited studies on cytotoxic effects andapoptotic induction activities of GLP specifically against cancercells;18 however, there are no reports of GLP from greenseaweeds having antiproliferative activities toward cancercells.19 Studies report that oral consumption of seaweedssignificantly decreases the incidence of carcinogenesis in vivo.5

Hence, we attempted to isolate and purify GLP from greenseaweed C. decorticatum and evaluated its cytotoxic activitiesand antiproliferative mechanisms by induction of apoptosisusing an in vitro cancer cell line model.In Figure 1, purified GLP from C. decorticatum was shown as

a single band on SDS-PAGE with a molecular mass of about 48kDa consisting carbohydrate (36.24%) and protein (63.76%)moieties. The gels stained with Schiff’s reagent and CBBreagent confirmed it as glycoprotein (GLP).To elucidate clearly the specificity of GLP toward cancer

cells, the cytotoxic effect of GLP on MDA-MB-231 cells wascompared with normal breast HBL-100 cells. GLP of the study,with its inhibitory concentrations on cancer cells, produced notoxicity or variations in proliferative effects in HBL-100 cells asevidenced by MTT assay. Our data regarding MTT assay, LDHassay, and nuclear fragmentation were consistent, and theseresults suggested that GLP could penetrate test cancer cells(MDA-MB-231) and destroy plasma membrane integrity,which consequently caused apoptosis in the cells and LDHleakage (Figure 2B). The LDH assay was found to besusceptible to interference from particle adsorption or proteininactivation. The exact mechanism could not be determined byour experiments because both could cause a decrease in themeasurable LDH protein, resulting in a false indication of anontoxic response. There was a reduction in the measurableLDH content with increasing dose-dependent concentrationsof GLP until a seemingly steady state condition was observed.The amount of LDH reduction was also affected by theconcentration of GLP in 24 h. Han et al.14 found indications ofLDH binding to titanium dioxide nanoparticles during cell

Figure 3. (A) Images of fluorescent microscopic analysis of GLP-induced apoptosis in MDA-MB-231 cells stained with AO/EtBr.Viable cells are shown in green, necrotic cells in red, and apoptoticcells in orange. (B) Graph shows apoptotic cells (%) after treatingMDA-MB-231 cells with GLP. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01,compared with untreated cells (control groups).

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Figure 4. (A) Fluorescence microscopy at 530 nm (400× magnification) shows the effect of GLP on ROS generation due to change in ΔΨm ofMDA-MB-231 cells. (B) Schematic representation of mechanism involved in ROS production from cancer cells treated with GLP. The schemeprovided was prepared on the basis of our study according to the report of Curtin et al.48 (C) Release of ROS (%) from GLP-treated MDA-MB-231cells with the dose-dependent concentrations of GLP for 24 h. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01, compared with untreated cells (control groups).

Figure 5. (A) Fluorescence microscopy studies using Rh 123 on the effect of dose-dependent concentrations of GLP in induction of mitochondria-mediated apoptosis in MDA-MB-231 cells via changes in ΔΨm at 530 nm (400× magnification). (B) Flow cytometry analysis using Rh 123 on theeffect of dose-dependent concentrations of GLP in induction of mitochondria-mediated apoptosis in MDA-MB-231 cells via changes in ΔΨm. (C)ΔΨm loss (%) in GLP-treated MDA-MB-231 cells by flow cytometry. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01, compared with untreated cells (controlgroups).

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exposure but did not confirm with cell-free measurements ofLDH absorption. The differences between our results and thoseof other groups may be due to the presence of other proteins orbiological compounds that affect LDH absorption to particles.14

Our measurements were performed with pure GLP in PBS withadherent cells, whereas other studies used different media orhad the additional presence of intracellular proteins and celldebris generated during cell exposure.The present study showed that after exposure to the GLP,

MDA-MB-231 cells displayed typical apoptotic appearancessuch as nuclei condensation, cell shrinkage, and apoptoticbodies along with accumulation of a sub-G1 cell cyclepopulation, substantiating the activation of an intrinsicapoptotic pathway in cancer cells.18−22 Although the caspaseactivation occurred in MDA-MB-231 cells, an increase in theexpression of pro-apoptotic bcl-2 member genes resulted innuclear translocation of APIF, which could be due to loss ofΔΨm.

23

GLP induced apoptosis and cell death via cell cycle arrest inhuman breast cancer cell lines and not in normal cells. Resultsfrom Figure 2A clearly show that GLP induced cytotoxicity andreduced cell viability in breast cancer cell lines throughinduction of G2/M phase arrest. It was reported that cellcycle control represents a major regulatory mechanism for cellgrowth,24 wherein anticancer agents can play a role in blockingthe progression of cell cycle, and development of new cancertherapeutics can be promising with this approach.25,26 Cellcycle progression is partly controlled by a family of protein

Figure 6. (A) GLP treated MDA-MB-231 cells stained with PI for cell cycle analysis by flow cytometry. Panels indicate the occurrence of cells ineach phase of the cell cycle. (B) Data on cell cycle distribution represent the percentage of cell death in the total population. Experiments wereperformed in duplicates. Data show percentage of live cells in each phase of the cell cycle (G0/G1, S, and G2/M). A dose-dependent increase in thepercentage of cell death was observed in G2/M cycle. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01, compared with untreated cells (control groups).

Figure 7. (A) RT-PCR studies on down-regulation of bax and up-regulation of bcl-2 genes induced by GLP in MDA-MB-231 cells. Theevents are associated with changes in the levels of ΔΨm loss. β-Actingene was used as an internal loading control for RT-PCR analysis. (B)Densitometry analysis of RT-PCR of intrinsic apoptotic related geneexpression of GLP treated cells. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01,compared with untreated cells (control groups).

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kinase complexes in eukaryotic cells including cyclin-dependentkinases (CDKs) and their activating partners, the cyclins.27

Together, these results suggested that GLP delayed cell cycleprogression by arresting cells in the G2/M phase of the cellcycle, resulting in significant induction of apoptosis.Mitochondrial characteristics, such as mitochondrial mass,

mitochondrial DNA, MMP, oxygen/glucose consumption,ROS, and ATP,28,29 can aid in understanding the differencesbetween sensitive and resistant cancer cells. An increasingnumber of studies have suggested that the function andintegrity of mitochondria may affect the viability, proliferation/division, cytotoxic resistance, and hypoxic tolerance of cells.Hence, properties of mitochondria of cancer cells mightbecome the basis of designing chemotherapeutic agents foreffective cancer treatment. Mitochondrial targets such as (i)mitochondrial permeability transition, (ii) outer membranepermeabilization, and (iii) mitochondrial metabolism andmetabolic reprogramming30,31 have been recognized fordeveloping potential strategies to overcome chemotherapeuticresistance. It is well-known that an increase in intracellular ROScan lead to apoptosis. On the other hand, a decrease of ROScan also ruin the stability of mitochondria, which is followed by

a loss of ΔΨm, release of cytochrome c into cytosol, andactivation of the caspase cascade.32,33 To support thisstandpoint, GLP could be employed to circumvent cancerresistance due to its ability to target the hyperpolarizedmitochondria.Mitochondrial membrane dysfunction and the release of pro-

apoptotic factors from the intermembrane space are controlledby Bcl-2 members.34 The intrinsic mitochondrial-dependentpathway involves the disruption of outer mitochondrialmembrane integrity that was followed by the reduction inMMP and the release of cytochrome c and other pro-apoptoticmolecules from the mitochondria to the cytosol.35 Additionally,the bcl-2 family genes and proteins are the essential regulatorsof apoptosis through controlling mitochondrial permeability.36

A low concentration of ROS is important in redox balance andcell proliferation.37 However, excessive ROS accumulationinduces protein oxidation, lipid peroxidation, and DNA damagein cells, followed by cell death or apoptosis. It was reported thatphenolic acids provoked ROS generation in HepG2 cells andIMR-32 cells.38,39 Our results also showed that GLP couldinduce ROS generation in MDA-MB-231 cells. Furthermore,pretreatment of ROS significantly blocked GLP-induced celldeath, confirming the involvement of ROS in this process. Ithas been demonstrated that ROS-mediated apoptosis ismodulated by Akt and MAPK signaling pathways in HepG2cells and U-937 cells, respectively.38,39 Therefore we hypothe-sized that ROS might play a key role in regulating intrinsicapoptotic signaling pathways in GLP-induced apoptosis, whichwas substantiated in the present study by Western blotting andsemiquantitative RT-PCR analysis of expression of apoptoticproteins and bcl-2 family genes, respectively (Figures 4−8).The caspase cascade is a key pathway in the apoptotic signal

transduction. Caspases include two types of subfamilies:upstream initiator caspases such as caspase 9, which areinvolved in regulatory events, and downstream effector caspasesuch as caspases 3 and 6, which correspond to the change incell morphological events and to the cleavage on nuclearprotein, PARP.28,40 Thus, the increased activity of caspases incells may enhance the risk of apoptosis. The present studyshowed that the treatment of cancer cells by GLP markedlyelevated the activity of cytochrome c, caspase 9, and caspase 3,suggesting the activation of both upstream and downstreamcaspase cascades in these cells. Following caspase activation, anincreasing number of proteins including PARP are degraded orcleaved,36,37,41−46 which consequently promotes nuclearcondensation and cell shrinkage, resulting in cell death. Theresults of our present study revealed that GLP activated caspasecascades and promoted the release of apoptotic factors tofacilitate apoptosis in the cancer cells (Figure 8). Theinvolvement of PARP in DNA repair is based on an earlierobservation that the activity of this enzyme is increased severalhundred-fold following DNA damage.41,47 Many insights intopossible functions of PARP in different biological processeswere obtained from in vitro experiments employing NAD+

analogues as inhibitors of poly(ADP-ribosylation) (e.g.,nicotinamide, benzamide, and their derivatives).47 Thesefindings were further supported by PARP inhibition in intactMDA-MB-231 cells (Figure 8). Inhibition of PARP activity hasbeen shown to protect cells from undergoing apoptosis.42−46

In summary, this study focused on the potential of a purifiedglycoprotein (GLP) from C. decorticatum for inducingapoptosis in human breast cancer MDA-MB-231 cells. GLPinhibited the growth of cancer cells when they were treated

Figure 8. (A) Effect of GLP on activation of caspases, cleavage ofPARP, release of cytochrome c, and ΔΨm loss was evaluated byWestern blotting and densitometry. Activities of caspases are indicatedin percentage against unstimulated cells. Percentage of apoptoticpopulation is represented on the basis of densitometry profilecompared with its internal control β-actin. (B) Densitometry analysisof intrinsic apoptotic related protein expression of MDA-MB-231 cellstreated with GLP. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01, compared withuntreated cells (control groups).

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with dose-dependent concentrations of GLP for 24 h.Observation of morphological changes in GLP-treated cancercells suggested that apoptosis was induced in these cells, whichwas further confirmed by PARP cleavage (Figure 8). We alsoshowed that mitochondria played a central role in the apoptoticinduction process, and the mitochondria destabilizationinvolves formation of ROS, release of cytochrome c, andactivation of multiple caspases in MDA-MB-231 cells treatedwith GLP. GLP regulated mitochondrial membrane proteinexpression for the induction of intrinsic apoptotic pathway bycausing G2/M phase cell cycle arrest and halting cell cycleprogression. Besides, it induced apoptosis through ROSgeneration from mitochondrial membranes of cancer cellsand caused ΔΨm stress, resulting in mitochondrial dysfunction.A schematic model depicting the actions of GLP is presented inScheme 2. Our results supported the emerging picture of GLPfrom biological sources, especially from C. decorticatum, withrespect to their therapeutic potential to treat breast cancers.

■ ASSOCIATED CONTENT

*S Supporting InformationDetails of experimental conditions for cell culture andmaintenance, lactate dehydrogenase (LDH) assay, determi-nation of apoptosis induction and cell death by AO/EtBrstaining, fluorescent microscopic studies on chromatin

condensation by DAPI staining, cell cycle analysis by FACS,and results of cell viability of normal and cancer cells (FigureS2). This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors*(R.T.) Phone: +91-8807293688. E-mail: thangam1985@yahoo.co.in.*(V.S.) +91-9976619607. E-mail: veeraperumalsuresh@gmail.com.*(S.K.) Phone +91-9486252052. Fax: +91-4222425706. E-mail:skperiyaruniv@gmail.com, sk_protein@buc.edu.in.*(J.S.) E-mail: jays66@gmail.com.Author Contributions∥R.T. and D.S. equally contributed.NotesThe authors declare no competing financial interest.

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Scheme 2. Proposed Schematic Diagram of GLP-Mediated Apoptosis in MDA-MB-231 Cellsa

aGLP induces ROS generation, which promotes ΔΨm loss and intrinsic apoptotic signaling pathways resulting in changes in the expression levels ofbax/bcl-2. This leads to mitochondrial dysfunction and caspase 3 activation. Finally, GLP induces PARP cleavage and apoptosis in MDA-MB-231cells via G2/M cell cycle arrest, which mediates the nuclear damage of the cells.

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