Induction of ROS-Dependent Mitochondria-Mediated Intrinsic

Article pubs.acs.org/JAFC

Induction of ROS-Dependent Mitochondria-Mediated Intrinsic Apoptosis in MDA-MB-231 Cells by Glycoprotein from Codium decorticatum Ramar 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 of these properties are attributed to sulfated polysaccharides, fucoxanthin, phycobiliproteins, and halogenated compounds. In this study, a glycoprotein (GLP) with a molecular mass of ∼48 kDa was extracted and purified from Codium decorticatum and investigated for its cytotoxic properties against human MDA-MB-231 breast cancer cells. The IC50 values of GLP against MDAMB-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. Chromatin condensation and poly(ADP-ribose) polymerase (PARP) cleavage studies showed that the GLP inhibited cell viability by inducing apoptosis in MDA-MB-231 cells. Induction of mitochondria-mediated intrinsic apoptotic pathway by GLP was evidenced by the events of loss of mitochondrial membrane potential (ΔΨm), bax/bcl-2 dysregulation, cytochrome c release, and activation of caspases 3 and 9. Apoptosis-associated factors such as reactive oxygen species (ROS) formation and loss of ΔΨm were evaluated by DCFH-DA staining and flow cytometry, respectively. Cell cycle arrest of G2/M phase and expression of apoptosis 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

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INTRODUCTION Recently, there has been an interest in the investigation of naturally occurring products such as carbohydrates, proteins, and secondary metabolites for their potential in treating several cancers. Multiple epidemiological studies have reported that diets rich in flavonoids, proteins, etc., can reduce the incidence of various cancers due to their antioxidant properties.1 Edible seaweeds are rich in bioactive antioxidants, soluble dietary fibers, proteins, minerals, vitamins, phytochemicals, and polyunsaturated fatty acids. Although seaweeds find applications as gelling and thickening agents in the food and pharmaceutical industries, recent research has revealed their potential as complementary medicine. Red, brown, and green seaweeds have been shown to possess therapeutic properties for health and disease management such as anticancer, antiobesity, antidiabetic, antihypertensive, antihyperlipidemic, antioxidant, anticoagulant, anti-inflammatory, immunomodulatory, antiestrogenic, thyroid stimulating, neuroprotective, antiviral, antifungal, and antibacterial properties in vitro and in vivo.2 Active © 2014 American Chemical Society

compounds from seaweeds include sulfated polysaccharides, phlorotannins, carotenoids (e.g., fucoxanthin), minerals, peptides, and sulfolipids that have proven benefits against degenerative metabolic diseases.3 Several of the currently available drugs for cancer therapy affect both cancer and normal cells by exhibiting toxicity, and this necessitates searching for novel, effective, and nontoxic anticancer compounds from natural sources. Recently, glycoproteins from natural sources have been gaining attention for their anticancer properties. Glycoprotein from Lonicera japonica stimulates the growth of the ICE-6 normal murine intestinal epithelial cells but exhibits antiproliferative effects on HT-29 colon cancer cells.4 However, reports are limited for algal glycoproteins having anticancer properties.5 Received: Revised: Accepted: Published: 3410

November 15, 2013 March 14, 2014 March 23, 2014 April 2, 2014 dx.doi.org/10.1021/jf405329e | J. Agric. Food Chem. 2014, 62, 3410−3421

Journal of Agricultural and Food Chemistry

Article

Antibody against β-actin was purchased from Bio-World (St. Louis Park, MN, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson Immuno Research (West Grove, PA, USA). All other reagents and chemicals were of the highest purity grade. Extraction and Purification of GLP from C. decorticatum. The collection of fresh and healthy C. decorticatum specimens was made between December 2011 and January 2012 during low tide at the depth of 1−3 m along the coast of Kilakarai, Gulf of Mannar, Tamil Nadu, India. The collected algae were washed with seawater and distilled water. The fresh thallus of the alga was powdered with liquid nitrogen and stored at −80 °C until used. One hundred and fifty grams of powder was added to 1 L of 0.05 mM phosphate buffer (pH 7.2) and dispersed for 6 h with constant stirring at 4 °C followed by filtration using cheesecloth to remove insoluble debris. Furthermore, the sample was centrifuged at 12500 rpm for 20 min to remove insoluble matter. The supernatant was saturated with 20% followed by 70% ammonium sulfate and centrifuged at 12000 rpm for 20 min to obtain pellets, which were dissolved and dialyzed against 0.05 M phosphate buffer at pH 7.2. The dialyzed sample was loaded onto QSepharose anionic exchange column (30 cm × 2.5 cm) and washed with 0.05 M phosphate buffer at pH 7.2. The sample was eluted serially with 50 mL of 50, 100, 150, 200, 300, 400, 500, and 700 mM NaCl in the same buffer with a flow rate of 2.0 mL/min. Fractions of 5 mL 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 presence of GLP band by CBB and PAS staining methods on sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) were collected, pooled, dialyzed against the same buffer, lyophilized (Vir Tis FTS systems, Warminster, PA, USA), and stored in vials at 4 °C for further use (Scheme 1). The purity of the GLP fraction from anion

Apoptosis is a highly regulated programmed cell death process that eliminates dysfunctional cells from the body by self-degradation.6 Apoptosis can be triggered in a cell through either the extrinsic pathway or the intrinsic pathway. The extrinsic pathway is initiated through the stimulation of transmembrane death receptors such as Fas receptors, located on the cell membrane.7 In contrast, the intrinsic pathway of apoptosis is driven by a mitochondriamediated death signaling cascade through the release of death signal factors, and these two apoptotic pathways are executed mainly by a class of cysteine proteases known as caspases.8 During apoptosis, engagement of the mitochondrial pathway involves mitochondrial membrane permeabilization (MMP), which depends on activation, translocation, and oligomerization of multidomain Bcl-2 family proteins such as Bax or Bak. MMP triggers the release of cytochrome c and other apoptogenic proteins into the cytosol followed by the activation of caspase cascades, which eventually results in poly(ADP-ribose) polymerase (PARP) cleavage and apoptosis.9 Besides, MMP induces apoptosis through altering the levels of expression of Bcl-2 family proteins.10 PARP are critical to DNA repair and apoptosis signaling, and play an important role in various mechanisms of cellular stress.7,8 Apoptosis can also occur via the caspase-independent apoptotic pathway in which apoptosis-inducing factor (APIF) plays a key role.11,12 APIF protein is normally confined to mitochondria and is released by cleavage of calpains upon apoptotic stimulation.13,14 The truncated form of APIF protein acts as a pro-apoptotic mediator, which translocates first from mitochondria into the cytosol followed by entry into the nucleus, where it promotes chromatin condensation and DNA degradation.15,16 Codium decorticatum is a dark green alga belonging to the family Codiales, and it is commonly found in the Gulf of Mannar region, India. Thus far, there has been no report on the biological properties of glycoprotein (GLP) from seaweeds. Hence, in the present study, GLP from C. decorticatum was extracted, purified, and investigated for its cytotoxicity potential on 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 intrinsic signaling cascade pathway in MDA-MB-231 breast cancer cells was evaluated with respect to the expression of mitochondrial membrane proteins, ROS generation and cleavage of PARP, and up- and downstream events of bcl-2 family genes.

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Scheme 1. Extraction and Purification Process of Glycoprotein from C. decorticatum

MATERIALS AND METHODS

Materials and Chemicals. Toluidine Blue O and ammonium sulfate were purchased from SRL Chemicals, Mumbai, India. QSepharose 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 Sigma Aldrich, USA. Dialysis membrane (MWCO 12−14 kDa) and methyl thiazol 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 SDSPAGE were purchased from Genei, India. Fetal bovine serum was purchased from Gibco, Inc., USA. LDH assay kit was procured from Cayman Chemicals, USA. cDNA synthesis kit (SuPrimeScript RT Master Mix, catalog no. SR2000) was purchased from GeNetBio. Primary antibodies against cytochrome c and caspase 3 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). AntiPARP antibody was procured from Pharmingen, San Diego, CA, USA. 3411

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


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Cell Cycle Analysis. Measurement of cellular DNA content and distribution of cells for cell cycle analysis were carried out by flow cytometry. Detailed experimental conditions are provided in the Supporting Information. Semiquantitative Reverse Transcription PCR (RT-PCR) for Mitochondrial Gene Expression. RT-PCR was performed to determine changes in the expression of bax, bcl-2, and internal control β-actin genes. TriZol reagent was used to isolate total RNA and reverse transcribed. Briefly, the cDNA was synthesized using a cDNA synthesis kit and amplified according to the manufacturer’s protocol in a 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), 10 mM/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, for a total of 30 cycles followed by a final extension at 72 °C for 10 min. The following primer sequences were used for amplification: (a) bax gene, forward 5′-TTTGCTTCAGGGTTTCATCC-3′ and reverse 5′CAGTTGAAGTTGCCGTCAGA-3′; (b) bcl-2 gene, forward 5′CATCCATTATAAGCTGTCGCA-3′ and reverse 5′-TGCCGGTTCAGGTACTCAGT-3′; and (c) β-actin, forward 5′-GTTGCTATCCAGGCTGTGC-3′ and reverse 5′-GCATCCTGTCGGCAATGC-3′. The PCR products were electrophoresed and visualized by transillumination system (Alpha Innotech Image viewer 6.0.0, Japan). Analysis of Expression of Mitochondrial Membrane Proteins by Western Blot. Cells (1 ×106 cells/well) were transferred to a culture dish (100 mm × 20 mm) for 24 h; the cells were treated for 24 h with GLP in dose-dependent concentration, harvested, and washed twice with ice-cold PBS. Subsequently, a 1.5 mL microfuge tube containing cell extract was centrifuged at 2500 rpm for 5 min to discard the supernatant. Treated cells were washed in PBS and lysed in 100 μL of buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 10 μg/mL pepstatin, and 10 μg/mL leupeptin. After 20 min, extracts were centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatants were stored at −80 °C until further use. Proteins (30 μg/lane) were separated 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 with primary antibodies such as cytochrome c, caspase 9, caspase 3, and βactin. After being washed with TBST buffer, the membranes were incubated for 1 h with the secondary antibody, horseradish peroxidaseconjugated goat anti-rabbit IgG. Antibody-bound proteins were detected using enhanced chemiluminescence reagents. Blots were washed with washing buffer and incubated with secondary antibodies conjugated 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. The membranes were blocked overnight with BSA solution (5% bovine serum albumin in PBS) and 0.1% Tween 20 at 4 °C and thereafter incubated with anti-PARP antibody (1:2000). The immune complexes were visualized as described for immune-blot analysis. Statistical Analysis. All of the experiments were done in triplicates, 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 t test; (*) p ≤ 0.05 and (**) p ≤ 0.01 were considered statistically significant.

exchange chromatography was analyzed by the presence of a single band detected with CBB staining (for protein) and PAS staining (for polysaccharides) on SDS-PAGE. Besides, Toluidine Blue O staining was employed for the detection of polysaccharides resolved in agarose gel by electrophoresis. Cell Culture and Maintenance. The selected cell lines of human MDA-MB-231 and HBL-100 cells were obtained from National Centre for Cells Science (NCCS), Pune, India. Detailed culture conditions 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 growing MDA-MB-231 and HBL-100 cells (1 × 104 cells/mL) were seeded in 96-well plates in a final volume of 100 μL/well and were treated with 10 μL of test sample (GLP) in FCS-free complete medium at various concentrations (5−100 μg/mL) for 48 h. One hundred microliters of MTT (5 mg/mL) was added to treated cells, and the plates were incubated at 37 °C for 4 h. The supernatant was aspirated, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the Formosan crystals. Absorbance was measured at 620 nm using a 96well microplate reader (THERMO Multiskan, USA), and the inhibitory concentrations (IC25, IC50, and IC75) were determined for GLP causing reduction in cell viability. The percentage of cell survival was 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 reader according to the manufacturer’s protocol. Detailed experimental conditions 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/EtBr staining. Detailed conditions for this method are provided in the Supporting Information. Fluorescent Microscopic Studies on Chromatin Condensation. The condensation of chromatin and nuclear fragmentation in GLP-treated cells were analyzed by DAPI staining. Detailed conditions are provided in the Supporting Information. Measurement of ROS Generation. 2′,7′-Dichlorofluoresceindiacetate (DCFH-DA) can be deacetylated by intracellular esterase to nonfluorescent DCFH, which can be oxidized by ROS, resulting in the formation of fluorescent compound 2′,7′-dichloroflorescein (DCF). The fluorescence intensity of DCF is proportional to the amount of ROS produced by the cells. After seeding of 5 × 105 cells/well to a 6well plate, the cells were treated with GLP for 24 h in dose-dependent concentrations. Subsequently, cells were washed once with ice-cold PBS and incubated with DCFH-DA (50 μM final concentration) at 37 °C for 30 min in the dark. Then, the cells were washed twice and maintained in 1 mL of PBS. ROS generation was assessed using a fluorescence microscope (Nikon Eclipse, Inc., Japan) at excitation and emission wavelengths of 488 and 530 nm, respectively, and the mean fluorescence 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 complete medium and allowed to attach for 24 h. The cells were treated with GLP for 24 h in dose-dependent concentrations and harvested. The medium was removed, and the adherent cells were trypsinized. The cells were pelleted by centrifugation at 2000 rpm for 10 min followed by resuspension with 1 mL of Rhodamine 123 (Rh 123; 5 μg/mL in methanol) for 30 min at 37 °C in dark, washed with PBS twice, and resuspended in PBS. Then, Rh 123-stained cells were fixed with 4% paraformaldehyde for 10 min. Finally, the cells were washed with methanol and analyzed for change in ΔΨm using BD FACS caliber (FL-1 detector with 530 nm band-pass filter). Data were analyzed using Cell Quest Pro 6.0 software (BD Biosciences, San Jose, CA, USA).

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RESULTS Extraction and Purification of GLP from C. decorticatum. The crude extract from C. decorticatum was precipitated with 20 and 70% ammonium sulfate fractionation, and the precipitate of the 70% ammonium sulfate saturated fraction was subjected to dialysis. The dialyzed sample was loaded onto the Q-Sepharose anion exchange column chromatograph and fractions were collected. A total of 80 fractions were collected and the content of protein and carbohydrate was determined 3412



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added LDH protein either absorbed or inactivated by dosedependent concentrations in 24 h is shown in Figure 2B. The figure shows that the release of LDH was greater when the cells were treated with increasing concentrations of GLP. In addition, direct observation using an inverted microscope revealed numerous morphological changes in MDA-MB-231 cells treated with GLP (data not shown). There are no significant cytotoxic effects of GLP in normal HBL-100 cells; hence, additional apoptotic parameters were performed only for cancer cells. In MDA-MB-231 cells, chromatin condensation, loss of nuclear construction, and formation of apoptosis bodies appeared in a dose-dependent manner after GLP treatment. To examine whether GLP inhibits the proliferation of MDA-MB231 cells by inducing apoptosis, cells were investigated by nuclear DAPI staining (Figure 2C). The GLP-treated cells exhibited typical morphological features of apoptosis with nuclear fragmentation, whereas control (untreated) cells did not show these features. Detection of Apoptosis Induction and Cell Death by AO/EtBr and DAPI Staining. Using fluorescence microscopy, necrotic and apoptotic cells were distinguished on the basis of overall cell morphology and cell membrane integrity. Upon staining with AO/EtBr, apoptotic cells containing apoptotic bodies and necrotic cells were observed as orange and red, respectively, whereas untreated MDA-MB-231 cells were found to exhibit green fluorescence with the absence of morphological changes (Figure 3). However, morphological changes as well as the induction of apoptosis in these cells were detected after treatment with GLP at dose-dependent concentrations for a period of 24 h, and the percentage of apoptotic induction is depicted in Figure 3. DAPI is also known to form fluorescent complexes with double-stranded DNA and is useful in finding the apoptotic condensed nuclei. The 80−90% confluent MDA-MB-231 cells were seeded in 24-well plates and exposed to GLP for 24 h. The morphology and the condensed nuclei chromatins were observed in the cells using a fluorescence microscope after cell fixation. Apoptotic nuclei were identified by the reduced nuclear size and gathering of condensed chromatin at the periphery of the nuclear membrane besides the fragmented morphology of nuclear bodies (Figure 2C). GLP Increases Intracellular ROS Level in MDA-MB-231 Cells. ROS play a central role in the regulation of cellular apoptosis. We examined the effect of GLP in intracellular ROS generation due to increase in ΔΨm loss that could mediate intrinsic apoptotic signaling pathways (Figure 4). ROS scavenger DCFH-DA was used to confirm the role of ROS in GLP-induced apoptosis. The results showed that there was a reduction of DCFH2 into DCF through esterase activity due to the release of peroxidase cytochrome c in the cytosol leading to increased production of ROS in GLP-treated MDA-MB-231 cells (Figure 4A,B) when compared to untreated cells (data not shown). 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 with untreated control cells, the ROS levels in these cells were measured as 21 ± 4.76, 43 ± 6.60, 62 ± 4.30, and 76.04 ± 5.34%, respectively (Figure 4B). The production of ROS is increased in a dose-dependent manner in GLP-treated MDAMB-231 cells (Figure 4A,C). These data suggested that ROS generation is required for ΔΨm loss and subsequent induction of intrinsic apoptotic signaling pathways in GLP-treated MDAMB-231 cells.

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

Figure 1. (A) Elution profile of crude GLP-rich extract on QSepharose Fast Flow chromatography. (B) Electrophoretic analysis of GLP from C. decorticatum. CBB staining reveals (i) GLP-rich crude fraction 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.

was again checked for purity by SDS-PAGE stained with CBB and PAS for protein and GLP, respectively. Among the 8eight fractions, six were homogeneous and were pooled, dialyzed, and lyophilized. We confirmed the presence of a GLP band having the molecular mass of ∼48 kDa (Figure 1B). The yield of the purified GLP was found to be 195 mg (0.13%) from 150 g of dried extract of C. decorticatum, and the contents of carbohydrate and protein in GLP were found to be 36.24 and 63.76%, respectively. The detailed process of extraction and purification of GLP is depicted in Scheme 1. GLP Reduces Cell Viability and Causes Membrane Damage. To investigate the effects of apoptotic cell death induced by GLP, MDA-MB-231 and HBL-100 cells were treated with GLP at different dose levels for 24 h and subjected to MTT assay. Results showed that MDA-MB-231 cells exhibited strong growth inhibitory effects when compared to HBL-100 cells (Supporting Information Figure S2). We evaluated the effect of GLP upon cell viability and plasma membrane damage of normal cell lines against untreated controls (Supporting Information Figure S2; Figure 2A,B). The cytotoxicities of GLP in dose-dependent concentration for MDA-MB-231 and HBL-100 cells were found to be 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 (Figure 2A). The extent of cell membrane damage of GLP-treated MDAMB-231 cells at dose-dependent concentrations in 24 h was measured by LDH release assay (Figure 2B). The percentage of 3413



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Figure 2. (A) GLP-induced cytotoxicity in MDA-MB-231 and HBL-100 cells treated with dose-dependent concentrations of GLP for 24 h in triplicates. (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. Intensely stained condensed apoptotic nuclei bodies besides nuclear change and relocations were observed in GLP-treated cells by fluorescence microscope (400× magnification).

GLP Triggers Mitochondria-Dependent Apoptosis. To investigate the molecular mechanism of GLP-induced apoptosis, we examined the effect of GLP on mitochondrial function in MDA-MB-231 cells using Rh 123 fluorescence staining. Treatment with increasing concentrations of GLP induced a dose-dependent loss of ΔΨm (Figure 5A). In addition, we found the dose-dependent release of cytochrome c from mitochondria into the cytosol, which was associated with changes in the ratio of expression of bcl-2/bax. These data reveal that GLP could cause mitochondrial dysfunction in MDA-MB-231 cells. The role of GLP in intracellular ΔΨm dysfunction and the subsequent induction of mitochondriamediated intrinsic apoptotic signaling pathways in MDA-MB231 cells were evaluated by FACS analysis. The results showed that the loss of ΔΨm increased in a dose-dependent manner in cells treated with GLP for 24 h, and this resulted in induction of intrinsic 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 by flow cytometry. The results on cell cycle analysis for untreated control cells and GLP-treated cells with dose-dependent concentrations are depicted in Figure 6A. From the figure, it was observed that there was a significant increase in cells at subG0 as well as G2/M phases (∗∗, p ≤ 0.01). The cells at G0/G1 phase decreased significantly (∗, p ≤ 0.05), whereas those at S phase did not change significantly (Figure 6B). The study findings revealed the effect of GLP on the accumulation of cells in G2/M phase accompanied by cell cycle arrest (Figure 6). It was shown that GLP arrested the growth of cancer MDA-MB231 cells in G2/M phase. This suggested that GLP might act as a G0/G1 checkpoint that controlled the progression of cells from G1 to S phase and thereby prevented the replication of DNA. These results suggested that the GLP delayed cell cycle progression by arresting cells in the G2/M phase of cell cycle, resulting in apoptosis. Expression of bcl-2 Family Genes, Attenuation of MMP, Loss of ΔΨm, and Induction of Apoptosis. As 3414



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MB-231 cells by Western blotting. Western blot analysis suggested that GLP treatment with dose-dependent concentrations led to a significant increase in caspase 3, caspase 9, and PARP proteins (Figure 8A and B). The release of cytochrome c (14 kDa) could initiate apoptosis via the mitochondrial pathway by activation of caspase 9 and caspase 3, and cleavage of PARP. 37/35 kDa caspase 9, upon cleavage, was converted to active caspase 9 having the molecular weight of 17 kDa. The molecular weight of PARP before and after cleavage was 116 and 89 kDa, respectively (Figure. 8). After MDA-MB-231 cells were treated with GLP in dose dependent concentration for 24 h, the release of cytochrome c increased in a concentrationcorresponding manner (Figure 8). The ratios for densitometric values of expression of cytochrome c/β-actin, caspase 9/β-actin, caspase 3/β-actin, and PARP/β-actin for control and cancer cells were also calculated (Figure 8B). These results suggested that GLP induced caspase 3-dependent apoptosis in MDA-MB231 cells.

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DISCUSSION Cancer is one of the most common diseases that threaten human life. Unfortunately, the drugs used for cancer therapy are toxic and affect not only cancer cells but also normal cells. Thus, finding novel, effective, and nontoxic compounds from natural sources is important now more than ever before. Induction of apoptosis has been the target mechanism for cancer treatments, and DNA fragmentation is a marker of cell death. There have been limited studies on cytotoxic effects and apoptotic induction activities of GLP specifically against cancer cells;18 however, there are no reports of GLP from green seaweeds having antiproliferative activities toward cancer cells.19 Studies report that oral consumption of seaweeds significantly decreases the incidence of carcinogenesis in vivo.5 Hence, we attempted to isolate and purify GLP from green seaweed C. decorticatum and evaluated its cytotoxic activities and antiproliferative mechanisms by induction of apoptosis using 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 48 kDa consisting carbohydrate (36.24%) and protein (63.76%) moieties. The gels stained with Schiff’s reagent and CBB reagent 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 was compared with normal breast HBL-100 cells. GLP of the study, with its inhibitory concentrations on cancer cells, produced no toxicity or variations in proliferative effects in HBL-100 cells as evidenced by MTT assay. Our data regarding MTT assay, LDH assay, and nuclear fragmentation were consistent, and these results suggested that GLP could penetrate test cancer cells (MDA-MB-231) and destroy plasma membrane integrity, which consequently caused apoptosis in the cells and LDH leakage (Figure 2B). The LDH assay was found to be susceptible to interference from particle adsorption or protein inactivation. The exact mechanism could not be determined by our experiments because both could cause a decrease in the measurable LDH protein, resulting in a false indication of a nontoxic response. There was a reduction in the measurable LDH content with increasing dose-dependent concentrations of GLP until a seemingly steady state condition was observed. The amount of LDH reduction was also affected by the concentration of GLP in 24 h. Han et al.14 found indications of LDH binding to titanium dioxide nanoparticles during cell

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

shown in Figure 7A, an additional experiment was carried out to elucidate whether GLP-induced apoptosis is associated with the expression of bcl-2 family member genes such as bax and bcl-2. Cells were exposed to dose-dependent concentrations of GLP for 24 h with untreated control cells, and the expression profile of pro-apoptotic bax and anti-apoptotic bcl-2 family genes in response to dose-dependent manner was elucidated (Figure 7A). The gene expression profiles in GLP-induced apoptosis of MDA-MB-231 cells were further confirmed by investigating with densitometry calculations (Figure 7B). As shown in Figure 7B, exposure of cells to dose-dependent concentrations led to change in the expression of mitochondrial pro-apoptotic and anti-apoptotic genes. Furthermore, we investigated the role of mitochondria in GLP-induced apoptosis of MDA-MB-231 cells by measuring Rh 123 dye retention. As shown in Figure 7B, MDA-MB-231 cells treated with GLP exhibited a significant reduction in mitochondria dysfunction in a dose-dependent manner. GLP Induces Apoptosis through a Caspase 3-Dependent Pathway. The mitochondrial pathway is one of the major apoptotic pathways, which is often related to the loss of ΔΨm. The release of cytochrome c from mitochondria to cytosol is the limiting factor in the mitochondrial pathway, and the loss of ΔΨm has been suggested to cause the release of cytochrome c. Here, we investigated the activation of caspases and the subsequent proteolytic cleavage of PARP proteins in MDA3415



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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 of MDA-MB-231 cells. (B) Schematic representation of mechanism involved in ROS production from cancer cells treated with GLP. The scheme provided 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-231 cells 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 mitochondriamediated apoptosis in MDA-MB-231 cells via changes in ΔΨm at 530 nm (400× magnification). (B) Flow cytometry analysis 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. (C) ΔΨm loss (%) in GLP-treated MDA-MB-231 cells by flow cytometry. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01, compared with untreated cells (control groups).

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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 in each phase of the cell cycle. (B) Data on cell cycle distribution represent the percentage of cell death in the total population. Experiments were performed 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 the percentage of cell death was observed in G2/M cycle. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01, compared with untreated cells (control groups).

exposure but did not confirm with cell-free measurements of LDH absorption. The differences between our results and those of other groups may be due to the presence of other proteins or biological compounds that affect LDH absorption to particles.14 Our measurements were performed with pure GLP in PBS with adherent cells, whereas other studies used different media or had the additional presence of intracellular proteins and cell debris generated during cell exposure. The present study showed that after exposure to the GLP, MDA-MB-231 cells displayed typical apoptotic appearances such as nuclei condensation, cell shrinkage, and apoptotic bodies along with accumulation of a sub-G1 cell cycle population, substantiating the activation of an intrinsic apoptotic pathway in cancer cells.18−22 Although the caspase activation occurred in MDA-MB-231 cells, an increase in the expression of pro-apoptotic bcl-2 member genes resulted in nuclear translocation of APIF, which could be due to loss of ΔΨm.23 GLP induced apoptosis and cell death via cell cycle arrest in human breast cancer cell lines and not in normal cells. Results from Figure 2A clearly show that GLP induced cytotoxicity and reduced cell viability in breast cancer cell lines through induction of G2/M phase arrest. It was reported that cell cycle control represents a major regulatory mechanism for cell growth,24 wherein anticancer agents can play a role in blocking the progression of cell cycle, and development of new cancer therapeutics can be promising with this approach.25,26 Cell cycle progression is partly controlled by a family of protein

Figure 7. (A) RT-PCR studies on down-regulation of bax and upregulation of bcl-2 genes induced by GLP in MDA-MB-231 cells. The events are associated with changes in the levels of ΔΨm loss. β-Actin gene was used as an internal loading control for RT-PCR analysis. (B) Densitometry analysis of RT-PCR of intrinsic apoptotic related gene expression of GLP treated cells. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01, compared with untreated cells (control groups). 3417



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a loss of ΔΨm, release of cytochrome c into cytosol, and activation of the caspase cascade.32,33 To support this standpoint, GLP could be employed to circumvent cancer resistance due to its ability to target the hyperpolarized mitochondria. Mitochondrial membrane dysfunction and the release of proapoptotic factors from the intermembrane space are controlled by Bcl-2 members.34 The intrinsic mitochondrial-dependent pathway involves the disruption of outer mitochondrial membrane integrity that was followed by the reduction in MMP and the release of cytochrome c and other pro-apoptotic molecules from the mitochondria to the cytosol.35 Additionally, the bcl-2 family genes and proteins are the essential regulators of apoptosis through controlling mitochondrial permeability.36 A low concentration of ROS is important in redox balance and cell proliferation.37 However, excessive ROS accumulation induces protein oxidation, lipid peroxidation, and DNA damage in cells, followed by cell death or apoptosis. It was reported that phenolic acids provoked ROS generation in HepG2 cells and IMR-32 cells.38,39 Our results also showed that GLP could induce ROS generation in MDA-MB-231 cells. Furthermore, pretreatment of ROS significantly blocked GLP-induced cell death, confirming the involvement of ROS in this process. It has been demonstrated that ROS-mediated apoptosis is modulated by Akt and MAPK signaling pathways in HepG2 cells and U-937 cells, respectively.38,39 Therefore we hypothesized that ROS might play a key role in regulating intrinsic apoptotic signaling pathways in GLP-induced apoptosis, which was substantiated in the present study by Western blotting and semiquantitative RT-PCR analysis of expression of apoptotic proteins 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 are involved in regulatory events, and downstream effector caspase such as caspases 3 and 6, which correspond to the change in cell morphological events and to the cleavage on nuclear protein, PARP.28,40 Thus, the increased activity of caspases in cells may enhance the risk of apoptosis. The present study showed that the treatment of cancer cells by GLP markedly elevated the activity of cytochrome c, caspase 9, and caspase 3, suggesting the activation of both upstream and downstream caspase cascades in these cells. Following caspase activation, an increasing number of proteins including PARP are degraded or cleaved,36,37,41−46 which consequently promotes nuclear condensation and cell shrinkage, resulting in cell death. The results of our present study revealed that GLP activated caspase cascades and promoted the release of apoptotic factors to facilitate apoptosis in the cancer cells (Figure 8). The involvement of PARP in DNA repair is based on an earlier observation that the activity of this enzyme is increased several hundred-fold following DNA damage.41,47 Many insights into possible functions of PARP in different biological processes were obtained from in vitro experiments employing NAD+ analogues as inhibitors of poly(ADP-ribosylation) (e.g., nicotinamide, benzamide, and their derivatives).47 These findings were further supported by PARP inhibition in intact MDA-MB-231 cells (Figure 8). Inhibition of PARP activity has been shown to protect cells from undergoing apoptosis.42−46 In summary, this study focused on the potential of a purified glycoprotein (GLP) from C. decorticatum for inducing apoptosis in human breast cancer MDA-MB-231 cells. GLP inhibited the growth of cancer cells when they were treated

Figure 8. (A) Effect of GLP on activation of caspases, cleavage of PARP, release of cytochrome c, and ΔΨm loss was evaluated by Western blotting and densitometry. Activities of caspases are indicated in percentage against unstimulated cells. Percentage of apoptotic population is represented on the basis of densitometry profile compared with its internal control β-actin. (B) Densitometry analysis of intrinsic apoptotic related protein expression of MDA-MB-231 cells treated with GLP. (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01, compared with untreated cells (control groups).

kinase complexes in eukaryotic cells including cyclin-dependent kinases (CDKs) and their activating partners, the cyclins.27 Together, these results suggested that GLP delayed cell cycle progression by arresting cells in the G2/M phase of the cell cycle, 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 differences between sensitive and resistant cancer cells. An increasing number of studies have suggested that the function and integrity of mitochondria may affect the viability, proliferation/ division, cytotoxic resistance, and hypoxic tolerance of cells. Hence, properties of mitochondria of cancer cells might become the basis of designing chemotherapeutic agents for effective cancer treatment. Mitochondrial targets such as (i) mitochondrial permeability transition, (ii) outer membrane permeabilization, and (iii) mitochondrial metabolism and metabolic reprogramming30,31 have been recognized for developing potential strategies to overcome chemotherapeutic resistance. It is well-known that an increase in intracellular ROS can lead to apoptosis. On the other hand, a decrease of ROS can also ruin the stability of mitochondria, which is followed by 3418



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

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

a

condensation by DAPI staining, cell cycle analysis by FACS, and results of cell viability of normal and cancer cells (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

with dose-dependent concentrations of GLP for 24 h. Observation of morphological changes in GLP-treated cancer cells suggested that apoptosis was induced in these cells, which was further confirmed by PARP cleavage (Figure 8). We also showed that mitochondria played a central role in the apoptotic induction process, and the mitochondria destabilization involves formation of ROS, release of cytochrome c, and activation of multiple caspases in MDA-MB-231 cells treated with GLP. GLP regulated mitochondrial membrane protein expression for the induction of intrinsic apoptotic pathway by causing G2/M phase cell cycle arrest and halting cell cycle progression. Besides, it induced apoptosis through ROS generation from mitochondrial membranes of cancer cells and caused ΔΨm stress, resulting in mitochondrial dysfunction. A schematic model depicting the actions of GLP is presented in Scheme 2. Our results supported the emerging picture of GLP from biological sources, especially from C. decorticatum, with respect to their therapeutic potential to treat breast cancers.

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AUTHOR INFORMATION

Corresponding 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: [email protected], [email protected]. *(J.S.) E-mail: [email protected]. Author Contributions ∥

R.T. and D.S. equally contributed.

Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

S Supporting Information *

REFERENCES

(1) Okada, H.; Mak, T. W. Pathways of apoptotic and non-apoptotic death in tumour cells. Nat. Rev. Cancer 2004, 4, 592−603. (2) Suresh, V.; Anbazhagan, C.; Thangam, R.; Senthilkumar, D.; Senthilkumar, N.; Kannan, S.; Rengasamy, R.; Palani, P. Stabilization of mitochondrial and microsomal function of fucoidan from Sargassum

Details of experimental conditions for cell culture and maintenance, lactate dehydrogenase (LDH) assay, determination of apoptosis induction and cell death by AO/EtBr staining, fluorescent microscopic studies on chromatin 3419



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morbifera against chloroquine-sensitive strains of Plasmodium falciparum. Phytother. Res. 2009, 23, 1634−1637. (23) Jeong, H. W.; Han, D. C.; Son, K. H.; Han, M. Y.; Lim, J. S.; Ha, J. H.; Lee, C. W.; Kim, H. M.; Kim, H. C.; Kwon, B. M. Antitumor effect of the cinnamaldehyde derivative CB403 through the arrest of cell cycle progression in the G2/M phase. Biochem. Pharmacol. 2003, 65, 1343−1350. (24) Buolamwini, J. K. Cell cycle molecular targets in novel anticancer drug discovery. Curr. Pharm. Des. 2000, 6, 379−392. (25) McDonald, E. R.; El-Deiry, W. S. Cell cycle control as a basis for cancer drug development (review). Int. J. Oncol. 2000, 16, 871−886. (26) Malumbres, M.; Barbacid, M. Mammalian cyclin-dependent kinases. Trends Biochem. Sci. 2005, 30, 630−641. (27) Bonnet, S.; Archer, S. L.; Allalunis-Turner, J.; Haromy, A.; Beaulieu, C.; Thompson, R.; Lee, C. T.; Lopaschuk, G. D.; Puttagunta, L.; Harry, G.; Hashimoto, K.; Porter, C. J.; Andrade, M. A.; Thebaud, B.; Michelakis, E. D. A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell 2007, 11, 37−51. (28) Ye, X. Q.; Li, Q.; Wang, G. H.; Sun, F. F.; Huang, G. J.; Bian, X. W.; Yu, S. C.; Qian, G. S. Mitochondrial and energy metabolismrelated properties as novel indicators of lung cancer stem cells. Int. J. Cancer 2011, 129, 820−831. (29) Fulda, S.; Galluzzi, L.; Kroemer, G. Targeting mitochondria for cancer therapy. Nat. Rev. Drug Discovery 2011, 9, 447−464. (30) Don, A. S.; Hogg, P. J. Mitochondria as cancer drug targets. Trends Mol. Med. 2004, 10, 372−378. (31) Zhang, J. Y.; Wu, H. Y.; Xia, X. K.; Liang, Y. J.; Yan, Y. Y.; She, Z. G.; Lin, Y. C.; Fu, L. W. Anthracenedione derivative 1403P-3 induces apoptosis in KB and KBv200 cells via reactive oxygen speciesindependent mitochondrial pathway and death receptor pathway. Cancer Biol. Ther. 2007, 6, 1413−1421. (32) Wang, X. H.; Jia, D. Z.; Liang, Y. J.; Yan, S. L.; Ding, Y.; Chen, L. M.; Shi, Z.; Zeng, M. S.; Liu, G. F.; Fu, L. W. Lgf-YL-9 induces apoptosis in human epidermoid carcinoma KB cells and multidrug resistant KBv200 cells via reactive oxygen species-independent mitochondrial pathway. Cancer Lett. 2007, 249, 256−270. (33) Green, D. R.; Evan, G. I. A matter of life and death. Cancer Cell 2002, 1, 19−30. (34) Gross, A.; McDonnell, J. M.; Korsmeyer, S. J. BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 1999, 13, 1899−1911. (35) Martinou, J. C.; Green, D. R. Breaking the mitochondrial barrier. Nat. Rev. Mol. Cell Biol. 2001, 2, 63−67. (36) Sauer, H.; Wartenberg, M.; Hescheler, J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell. Physiol. Biochem. 2001, 11, 173−186. (37) Mallis, R. J.; Buss, J.; Thomas, J. A. Oxidative modification of Hras: S-thiolation and S-nitrosylation of reactive cysteines. Biochem. J. 2001, 355, 145−153. (38) Lee, Y. S. Role of NADPH oxidase-mediated generation of reactive oxygen species in the mechanism of apoptosis induced by phenolic acids in HepG2 human hepatoma cells. Arch. Pharm. Res. (Seoul) 2005, 28, 1183−1189. (39) Ranger, A. M.; Malynn, B. A.; Korsmeyer, S. J. Mouse models of cell death. Nat. Genet. 2001, 28, 113−118. (40) Wu, J.; Suzuki, H.; Zhou, Y. W.; Liu, W.; Yoshihara, M.; Kato, M.; Akhand, A. A.; Hayakawa, A.; Takeuchi, K.; Hossain, K.; Kurosawa, M.; Nakashima, I. Cepharanthine activates caspases and induces apoptosis in Jurkat and K562 human leukemia cell lines. J. Cell. Biochem. 2001, 82, 200−214. (41) Benjamin, R. C.; Gill, D. M. ADP-ribosylation in mammalian cell ghosts. Dependence of poly(ADP-ribose) synthesis on strand breakage in DNA. J. Biol. Chem. 1980, 255, 10493−10501. (42) Green, D. R.; Reed, J. C. Mitochondria and apoptosis. Science 1998, 281, 1309−1312. (43) Tong, W. M.; Cortes, U.; Wang, Z. Poly(ADP-ribose) polymerasea guardian angel protecting the genome and suppressing tumorigenesis. Biochim. Biophys. Acta 2001, 1552, 27−37.

plagiophyllum in diethylnitrosamine induced hepatocarcinogenesis. Carbohydr. Polym. 2013, 92, 1377−1385. (3) Mohamed, S.; Hashim, S. N.; Rahman, A. H. Seaweeds: a sustainable functional food for complementary and alternative therapy. Trends Food Sci. Technol. 2012, 23, 83−96. (4) Go, H.; Hwang, H. J.; Nam, T. J. Glycoprotein extraction from Laminaria japonica promotes IEC-6 cell proliferation. Int. J. Mol. Med. 2009, 24, 819−824. (5) Sheu, J. H.; Huang, S. Y.; Duh, C. Y. Cytotoxic oxygenated desmosterols of the red alga Galaxaura marginata. J. Nat. Prod. 1996, 59, 23−26. (6) Ashkenazi, A.; Dixit, V. M. Death receptors: signaling and modulation. Science 1998, 281, 1305−1308. (7) Ashkenazi, A. Targeting death and decoy receptors of the tumour-necrosis factor super family. Nat. Rev. Cancer 2002, 2, 420− 430. (8) Susin, S. A.; Lorenzo, H. K.; Zamzami, N.; Marzo, I.; Snow, B. E.; Brothers, G. M.; Mangion, J.; Jacotot, E.; Costantini, P.; Loeffler, M.; Larochette, N.; Goodlett, D. R.; Aebersold, R.; Siderovski, D. P.; Penninger, J. M.; Kroemer, G. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999, 397, 441−446. (9) Gatenby, R. A.; Gillies, R. J. Why do cancers have high aerobic glycolysis? Nat. Rev. Cancer 2004, 4, 891−899. (10) Moubarak, R. S.; Yuste, V. J.; Artus, C.; Bouharrour, A.; Greer, P. A.; Menissier-de Murcia, J.; Susin, S. A. Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis. Mol. Cell. Biol. 2007, 27, 4844−4862. (11) Fulda, S.; Galluzzi, L.; Kroemer, G. Targeting mitochondria for cancer therapy. Nat. Rev. Drug Discovery 2011, 9, 447−464. (12) Er, E.; Oliver, L.; Cartron, P. F.; Juin, P.; Manon, S.; Vallette, F. M. Mitochondria as the target of the pro-apoptotic protein Bax. Biochim. Biophys. Acta, Bioenerg. 2006, 1757, 1301−1311. (13) Wu, M.; Xu, L. G.; Li, X.; Zhai, Z.; Shu, H. B. AMID, an apoptosis-inducing factor-homologous mitochondrion-associated protein, induces caspase-independent apoptosis. J. Biol. Chem. 2002, 277, 25617−25623. (14) Han, X.; Gelein, R.; Corson, N.; Wade-Mercer, P.; Jiang, J.; Biswas, P.; Finkelstein, J. N.; Elder, A.; Oberdörster, G. Validation of an LDH assay for assessing nanoparticle toxicity. Toxicology 2011, 287, 99−104. (15) U.K. Department for Environment, Food and Rural Affairs. Characterising the potential risks posed by engineered nanoparticles, London, 2006. (16) Ohiro, Y.; Garkavtsev, I.; Kobayashi, S.; Sreekumar, K. R.; Nantz, R.; Higashikubo, B. T.; Duffy, S. L.; Higashikubo, R.; Usheva, A.; Gius, D.; Kley, N.; Horikoshi, N. A. Novel p53-inducible apoptogenic gene, PRG3, encodes a homologue of the apoptosisinducing factor (AIF). FEBS Lett. 2002, 524, 163−171. (17) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (18) Sun, H. K.; Lee, S. J.; Lim, K. T. Cytotoxic effect of glycoprotein isolated from Solanum nigrum L. through the inhibition of hydroxyl radical-induced DNA-binding activities of NF-κB in HT-29 cells. Environ. Toxicol. Pharmacol. 2004, 17, 45−54. (19) Jung, S. J.; Lim, K. T. Apoptosis induced by glycoprotein (150kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT-116 cells. Cancer Chemother. Pharmacol. 2006, 57, 507−516. (20) Barlow, P.; Clouter-Baker, A.; Donaldson, K.; Maccallum, J.; Stone, V. Carbon black nanoparticles induce type II epithelial cells to release chemotaxins for alveolar macrophages. Part. Fibre Toxicol. 2005, 2, 11−23. (21) Hurt, R. H.; Monthioux, M.; Kane, A. Toxicology of carbon nanomaterials: status, trends, and perspectives on the special issue. Carbon 2006, 44, 1028−1033. (22) Chung, I. M.; Kim, M. Y.; Park, S. D.; Park, W. H.; Moon, H. I. In vitro evaluation of the antiplasmodial activity of Dendropanax 3420



Article

(44) Shall, S.; de Murcia, G. Poly(ADP-ribose) polymerase-1 what have we learned from the deficient mouse model? Mutat. Res. 2000, 460, 1−15. (45) Uchida, K.; Hanai, S.; Ishikawa, K.; Ozawa, Y.; Uchida, M.; Sugimura, T.; Miwa, M. Cloning of cDNA encoding Drosophila poly(ADP-ribose) polymerase leucine zipper in the auto-modification domain. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3481−3485. (46) Masutani, M.; Nozaki, T.; Hitomi, Y.; Ikejima, M.; Nagasaki, K.; de Prati, A. C.; Kurata, S.; Natori, S.; Sugimura, T.; Esumi, H. Cloning and functional expression of poly(ADP-ribose) polymerase cDNA from Sarcophaga peregrine. Eur. J. Biochem. 1994, 200, 607−614. (47) Susin, S. A.; Lorenzo, H. K.; Zamzami, N.; Marzo, I.; Snow, B. E.; Brothers, G. M.; Mangion, J.; Jacotot, E.; Costantini, P.; Loeffler, M.; Larochette, N.; Goodlett, D. R.; Aebersold, R.; Siderovski, D. P.; Penninger, J. M.; Kroemer, G. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999, 397, 441−446. (48) Curtin, J. F.; Donovan, M.; Cotter, T. G. Regulation and measurement of oxidative stress in apoptosis. J. Immunol. Methods 2002, 265, 49−72.

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Induction of ROS-Dependent Mitochondria-Mediated Intrinsic

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