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View Article OnlineView Journal | View Issue

Nanostructured

aDepartment of Cell and Molecular Biology, F

Sciences, Universiti Putra Malaysia, 43400,bCollege of Veterinary Medicine, University

Sulaimany City, Kurdistan Region, NortherncDepartment of Microbiology and Pathology,

Putra Malaysia, 43400, Serdang, Selangor,dDepartment of Agriculture Genetics and Bre

Biology, Cantho University, 3/2 Street, CanTeSchool of Biomedical Sciences, The Universi

Broga, 43500 Semenyih, Selangor, MalaysiafFaculty of Medicine and Health Sciences,

21144, Jalan Sungai Long, Bandar Sungai LgInstitute of Bioscience, Universiti Putra

Malaysia. E-mail: skyeap2005@gmail.com

Cite this: RSC Adv., 2015, 5, 22066

Received 5th January 2015Accepted 10th February 2015

DOI: 10.1039/c5ra00144g

www.rsc.org/advances

22066 | RSC Adv., 2015, 5, 22066–220

lipid carrier improved in vivo anti-tumor and immunomodulatory effect ofZerumbone in 4T1 challenged mice

Nurul Elyani Mohamad,a Nadiah Abu,a Heshu Sulaiman Rahman,bc Huynh Ky,d

Wan Yong Ho,e Kian Lam Lim,f Chee Wun How,g Abdullah Rasedee,c

Noorjahan Banu Alitheena and Swee Keong Yeap*g

Zerumbone is a potential anti-inflammatory, immunomodulatory and anti-tumor agent. However,

the major problems that prevent the in vivo and even clinical applications of Zerumbone are its

water insolubility that compromises its bioavailability. To enhance the bioavailability and efficacy of

Zerumbone, nanostructured lipid carriers (NLC) were used to encapsulate Zerumbone. NLC–Zerumbone

has been reported to have a slow release, comparable in vitro cytotoxicity against leukemia and breast

cancer, and no observed in vivo oral acute toxicity. However, the in vivo antitumor effect of NLC–

Zerumbone is still unknown. The anti-tumor, immunostimulatory and anti-inflammatory effects of

Zerumbone and NLC–Zerumbone in 4T1 challenged mice were compared in this study. Zerumbone

effectively controlled the tumor growth and metastasis via delaying the cancer cell cycle progression

and apoptosis. Besides, Zerumbone also enhanced the anti-tumor immunity and anti-inflammatory

effects. NLC encapsulation was found to further improve the anti-tumor, immunomodulation, anti-

inflammation and anti-metastatic efficacy of Zerumbone in 4T1 challenged mice. This study provides the

possibility of utilizing NLC encapsulated Zerumbone for breast cancer therapy.

Introduction

Cancer has become a prime disease that is globally feared. Theincreasing number of newly diagnosed cancer cases throughoutthe world is amassing rapidly. Breast cancer, in particular, hasbeen the center of attention among other cancers because therate of women being diagnosed with breast cancer is approxi-mately 1 in 8.1 Though there are a number of options that can beselected to treat breast cancer, the likelihood of these treat-ments to work is still low. One of the most approachablemethods of treating breast cancer is through chemotherapy anddrug therapy.2 There are various potent anti-cancer agents that

aculty of Biotechnology and Biomolecular

Serdang, Selangor, Malaysia

of Sulaimany, Sulaimany Nwe, Street 27,

Iraq

Faculty of Veterinary Medicine, Universiti

Malaysia

eding, College of Agriculture and Applied

ho City, Vietnam

ty of Nottingham Malaysia Campus, Jalan

Universiti Tunku Abdul Rahman, Lot PT

ong, Cheras, 43000 Selangor, Malaysia

Malaysia, 43400, Serdang, Selangor,

74

have been discovered in recent years, particularly in naturallyderived products. Natural products from plants and secondarymetabolites from fungus and bacteria have attracted great atten-tion as major sources of drug discovery because of their diverseunique chemical structures.3 Among the plant based naturalcompounds, Zerumbone, a sesquiterpene compound isolatedfrom the ginger family, especially Zingiber zerumbet Smith, hasbeen reported to have pronounced antitumor and anti-inammatory effects.4 Zerumbone has been reported to be cyto-toxic to a wide range of cancer cell types including blood, skin,breast, liver, cervical, colon, bile duct, ovarian, pancreatic, lung,renal, brain, prostate, gastric, oral, head and neck and pharyngealcancers. This cytotoxicity was mainly attributed to its G2/M cellcycle arrest and apoptotic induction against the tested cancercell.5 Besides, its wide spectrum of cytotoxicity to cancer cell lines,Zerumbone has also been reported to have anti-inammatoryeffects on oncogenesis6 and diabetis7 induced inammation;thus, it can prevent the progression of these diseases.

Although natural compounds including Zerumbone havegained overwhelming success in the in vitro cytotoxic effect onvarious cancer cell lines, the poor solubility and oral bioavail-ability of these compounds have hindered the in vivo antitumorapplication.8 Glucosylation, oil emulsion and micro or nano-encapsulation have been utilized to enhance the in vivobioavailability and efficacy of low soluble compounds suchas curcumin.9 Besides the bioavailability, carriers of these

This journal is © The Royal Society of Chemistry 2015

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insoluble or slightly soluble compounds with a rate-controlledrelease, especially with the ability to release the compoundover an extended period of time at a controlled rate, possessextra advantages and are generally more effective and safe.This is because the slow release of this compound aer oraladministration overcomes the possible toxicity associated withthe drastic accumulation of the drug in the body in a shortperiod. In addition, it allows the consumption of fewercompounds due to the achievement of maximum pharmo-logical effectiveness. Thus, delivery systems, especially nano-carrier with features of slow rate controlled release and higherbioavailability, are the preferable solution for low solublenatural compounds.10 For Zerumbone, nano-Zerumbone-zeoliteY-gelatin has been prepared and recorded to have effectivesustained release of Zerumbone in vitro.10 More recently, theencapsulation of Zerumbone using nanostructured lipidcarriers (NLC) was reported, and the in vitro controlled releasehas shown that this nanoparticle complex possesses a slowerand more sustainable release of Zerumbone. Furthermore, theslow release feature of NLC encapsulation did not affect the invitro efficacy of Zerumbone; similar in vitro cytotoxicity has beenrecorded on human leukemia11 and breast cancer cell lines12 byboth Zerumbone and NLC–Zerumbone. Moreover, the in vivoacute toxicity study also revealed that NLC–Zerumbone has a50% lethal dose (LD50), which is higher than 200 mg kg�1 and issafe for oral administration.13 However, the in vivo efficacy ofthe orally administrated NLC–Zerumbone has not been testedon the mice breast cancer animal model. Thus, this study isaimed to evaluate the in vivo antitumor and anti-inammatoryeffect of NLC–Zerumbone in 4T1 challenged mice.

Experimental sectionPreparation of Zerumbone and NLC–Zerumbone

Zerumbone was isolated from Z. Zerumbet rhizomes by steamhydro-distillation, and the purity was determined by highperformance liquid chromatography (Waters, USA) according toa previous report.14 The preparation and characterization ofNLC–Zerumbone was performed according to our previousreport.11 In brief, the lipid phase consisted of hydrogenatedpalm oil, olive oil and lipoid S100 (in a ratio of 7 : 3 : 3). Inaddition, 4.75% of sorbitol, 1% of Tween-80 and 0.005% ofthimerosal were added into the aqueous phase. Zerumbone(0.4% w/v) was added in the lipid phase and dispersed in theaqueous solution using a high-pressure homogenizer to obtainthe NLC–Zerumbone.11 The loading of Zerumbone to NLC wasapproximately 99% with �50% of in vitro release, which can becompared to >90% of release of the Zerumbone aer 48 hoursaccording to the drug release study using dialysis.11

Culture of 4T1 cell line

4T1 cells were obtained from the ATCC collection (ATCC, USA)and were maintained in RPMI-1640 (Sigma, USA). The mediawas supplemented with 10% FBS and 1% penicillin–strepto-mycin. The cells were kept in a humidied equipped with 5%CO2 at 37 �C.

This journal is © The Royal Society of Chemistry 2015

Animal

Female balb/c mice (n ¼ 21), 5–6 weeks old, were purchasedfrom Universiti Putra Malaysia's Animal House. The mice werekept under standard housing conditions with a 12 hour regularlight–dark cycle at a 25 � 2 �C. Moreover, the mice were givenstandard diet pellets and tap water. The mice were divided into7 mice per group, and all the animal experiments were per-formed under the Universiti Putra Malaysia's ethical guidelinesfor the care of lab animals (UPM/FPV/PS/3.2.1.551/AUP-R152).

Tumor inoculation and treatment

4T1 cells (1 � 106 cell per mice) were prepared and inoculatedsubcutaneously in the lemammary fat pad of NLC control (n¼7), Zerumbone (n ¼ 7) and NLC–Zerumbone (n ¼ 7) treatedmice. The mice were fed with 50 mg kg�1 body weight of Zer-umbone (solubilized in olive oil), NLC–Zerumbone or NLC (asthe untreated group) daily aer a week of 4T1 inoculation whenthe tumor was visible (�5mm3). The dose was selected based ona study by Rahman et al., 2013.8,11 Tumors were measured withvernier calipers twice a week, and tumor volume was calculatedusing the following formula: tumor volume (mm3) ¼ [(width)2

� length]/2. Aer 28 days of treatment, the mice were intra-peritoneal injected with ketamine-HCl and xylazine and sacri-ced via cervical dislocation. The tumor, spleen and lung wereharvested and blood was collected. Harvested lung was xed in10% formaldehyde (Sigma-Aldrich, USA) overnight, dried andviewed under a dissection microscope to calculate the meta-static tumor nodules on the lung.15

Tumor histopathological evaluation

The harvested tumors were xed in 10% formalin, embedded inparaffin, sliced into thin sections, stained with haematoxylinand eosin and viewed under bright-eld microscope (Nikon,Japan). The mitotic and apoptotic cells were counted andcompared between the groups. Mitotic and apoptotic cells wereclassied based on the criteria set by Diest et al.16 and Carneiroet al.17

Tumor immunohistology by TUNEL assay

The tumor sections obtained from the samples treated withNLC, Zerumbone and Zerumbone–NLC were paraffin-embedded on glass slides. To detect the presence of cellsundergoing DNA fragmentation, the DeadEnd™ ColorimetricApoptosis Detection System (Promega, USA) was employedaccording to the manufacturer's protocol. First, the slides werede-paraffinized in xylene twice before being rehydrated indifferent concentrations of ethanol (100%, 95%, 85%, 70% and50% of ethanol). The slides were then washed in 0.85% NaCland PBS. To detect apoptosis, the slides were xed in 4%paraformaldehyde, and the tumor sections were permeabilizedusing Proteinase K. Subsequently, the slides were equilibratedusing the provided equilibration buffer and were labeledusing the TdT reaction mix; the reaction was then stoppedby immersing the slides in the provided SSC buffer. Then,the slides were blocked using 0.3% hydrogen peroxide, and

RSC Adv., 2015, 5, 22066–22074 | 22067

Table 1 The sequence and accession number of the primers used inthe real-time PCR analysis

Gene name Accession number Sequence

iNOS NM_010927.3 F: 5-GCACCGAGATTGGAGTTC-3R: 5-GAGCACAGCCACATTGAT-3

NF-kB NM_008689.2 F: 5-CATTCTGACCTTGCCTATCT-3R: 5-CTGCTGTTCTGTCCATTCT-3

ACTB NM_007393.3 F: 5-TTCCAGCCTTCCTTCTTG-3R: 5-GGAGCCAGAGCAGTAATC-3

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streptavidin-HRP was added to bind to those biotinylatednucleotides. The slides were then stained with DAB for 30min at room temperature before being mounted in glycerol.The slides were nally observed using a light microscope at amagnication of 40� (Nikon, Japan). Apoptotic cells werestained dark brown.

Serum cytokine ELISA

Cytokine analysis was performed using the serum harvestedfrom the NLC control and Zerumbone or NLC–Zerumbonetreated mice using the ELISA MAX IL-1b, IL-2, IL-6 and IFN-gkits (Biolegend, USA). Basically, a 96-well plate was pre-coatedwith the designated capture antibody at 4 �C overnight. Subse-quently, the plate was washed three times for 5 min eachwith PBS, before being blocked with bovine serum albumin for1 hour. Then, the standard dilution as well as the samples wereadded to the plate and were le to incubate for 2 hours on arocking platform. The plate was then again washed with PBS toremove any non-binding samples, and it was further incubatedwith the diluted detection antibody for 30 min. Then, the TMBsubstrate was added to the plate until the resulting color(yellow) was developed. The provided stop solution was thenadded to terminate the reaction of the substrate. The plate wasthen read at 450 nm and 570 nm using a mquant microplatereader (Biotek Instruments, USA).

Lipid peroxidation and nitric oxide detection

Nitric oxide in the tumors was detected using the Griess reagent(Sigma, USA), whereas malondialdehyde (MDA) formation intumor, which indicated lipid peroxidation, was detected bymeasuring thiobarbituric acid-reactive substance (TBARS).18

Harvested tumors were meshed with an 80 mm wire mesh in icecold PBS (0.1 g of tumor per mL of ice cold PBS) and pelleted at500�g for 5 min. For the Griess assay, 10 mL of supernatant wasmixed with the Griess reagent according to the user's manual,and it was le to incubate at room temperature for 30 min.Then, the absorbance of the samples was measured at 548 nmusing a mquant microplate reader (Biotek Instruments, USA).For the TBARS assay, 100 mL of supernatant was mixed with400 mL of PBS, 12.5 mL of butylated hydroxytoluene (BHT, 88 mg/10 mg absolute ethanol) and 250 mL of 30% trichloroacetic acid,and it was then incubated on ice for 2 hours. Subsequently, themixture was centrifuged at 500�g for 15 min and mixed with37.5 mL of 0.1 M EDTA and 125 mL of 1% thiobarbituric acid in0.1 M NaOH. The mixture was boiled for 15min, chilled to roomtemperature, and the absorbance was read at 532 nm using amquant microplate reader (Biotek Instruments, USA).

Spleen ow cytometry immunophenotyping analysis

Splenocytes harvested from the NLC control, Zerumbone andNLC–Zerumbone treatedmice were washed several times in PBSbefore being mechanically disrupted using a syringe plungerand a 70 mm cell strainer. Aer obtaining a single cell suspen-sion, the red blood cells were removed from the splenocytesusing a lysis buffer. Then, the splenocyte cells were stainedwith the immune markers: uorochrome (CD3-FITC, CD4-PE,

22068 | RSC Adv., 2015, 5, 22066–22074

CD8-APC, NK1.1-APC) (Abcam, USA) for 2 hours. Aer theincubation period, the stained splenocytes were xed in 1%paraformaldehyde and were kept at 4 �C until the ow cytom-eter analysis (BD, USA).

Splenocyte cytotoxicity against YAC-1 and 4T1 cells

Single cell suspensions of splenocytes were obtained from thespleens harvested from the NLC, Zerumbone and Zerumbone–NLC treated mice aer their sacrice. The splenocytes wereco-cultured with either YAC-1 or 4T1 cells at 2 : 1 and 5 : 1 ratioin a 96 well-plate and were le to incubate overnight in ahumidied incubator maintained at 37 �C. 50 mL of mediafrom each of the wells was collected next day, and the release ofLDH to the supernatant was assayed by a CytoTox 96 none-radioactive cytotoxic assay kit (Promega, USA) according to theprotocol provided by the manufacturer.19

Reverse transcription quantitative PCR on the expression ofiNOS and NF-kB in tumors

The tumors were kept in RNAlater® and were snapped frozen inliquid nitrogen. Total RNA was isolated from the tumors bymechanical disruption and using a Qiagen RNAeasy Mini Kit(Qiagen, USA). Subsequently, 1 mg of the RNA was converted tocDNA using a cDNA MAXIME Reverse Transcription Kit(Thermo Scientic, USA). Then, the real time PCR reaction wasperformed using a Maxima Sybr Green (Thermo Scientic, USA)on the iQ5 (Bio-Rad, USA) to quantify the differential expressionof inducible nitric oxide synthase (iNOS) and nuclear factorkappa-light-chain-enhancer of activated B cells (NF-kB) betweentumors harvested from NLC control, Zerumbone and NLC–Zerumbone. The forward and reverse sequences of the targetgenes (iNOS and NF-kB) and housekeeping genes, namely, hypo-xanthine phosphoribosyltransferase (HPRT), beta actin (ACTB)and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), arelisted in Table 1. The PCR condition was 1 cycle of 50 �C/2min forUDG activation; 1 cycle of 95 �C/2 min for DNA polymerase acti-vation; 40 cycles of 95 �C/2 seconds for denaturation, 52 �C for30 seconds for annealing and extending. All the samples wereassayed in triplicate, and the template-less controls wereprepared. The quantity of the target and housekeeping genes,namely, ACTB, HPRT and GAPDH, were calculated according to astandard curve, and the expressions of NF-kB and iNOS weremeasured by Bio-Rad CFX Manager (Bio-Rad, USA). The expres-sion levels of Zerumbone and NLC–Zerumbone were compared tothe NLC control group.

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Western blot

Total protein lysate was isolated from the tumors via mechan-ical disruption using liquid nitrogen. The meshed tumor lysateswere then lysed using RIPA buffer supplemented with aprotease cocktail tablet (Roche, USA). Subsequently, the lysateswere centrifuged at 12 000�g for 15 min, and the supernatantswere collected. Before proceeding to the western blot, the har-vested protein lysates were quantied using the Bradfordreagent (Sigma, USA). 50 mg of protein from each sample wassubjected to 10% SDS-Page and was run at 120 V for an hour.Then, the proteins from the gel were electro-blotted onto anitrocellulose membrane using an electro-blotter (Bio-Rad,USA). Subsequently, the membrane was blocked using 5%skimmed milk overnight to prevent non-specic binding. Thefollowing day, the membrane was washed several times with 1�TBST before being incubated with the designated primaryantibody for 1 hour. Then, the membrane was washed againwith TBST and was probed with the respective secondary anti-body for 1 hour. Subsequently, the membrane was exposed to achemi-luminescence light using the ChemiDocx aer theaddition of the substrate (Thermo, USA).

Statistical analysis

All assays were carried out with three biological replicates andperformed in duplicates. The results were expressed as mean �standard deviation. Statistical signicance (p < 0.05) was assayedby one-way ANOVA with a post-hoc Duncan test compared to theNLC control group.

Fig. 1 The values of the (a) tumor volume (cm3), (b) tumor weight (g) andZerumbone treated and NLC–Zerumbone treated mice after orally feedrepresents mean � S.E.M (n ¼ 7), with *p value < 0.05.

This journal is © The Royal Society of Chemistry 2015

ResultsNLC–Zerumbone reduced the size of the 4T1 tumors

This study was carried out to compare the in vivo efficacy ofZerumbone and NLC–Zerumbone as an anti-tumor agent. Asshown in Fig. 1, the size and weight of the tumor excised fromthe NLC–Zerumbone mice had a lower value as compared tothat of the tumor obtained from the NLC control andZerumbone-treated mice. NLC–Zerumbone treated mouse alsoexhibited lower numbers of metastatic lung nodules ascompared to the NLC control and Zerumbone treated mice. Inaddition, the number of the metastatic lung nodules in theZerumbone mice was also signicantly lower than that of theNLC control mice (Fig. 1c). The same pattern can be observed inthe weight of Zerumbone treated tumors in comparison withthe NLC treated tumor.

NLC–Zerumbone reduced mitosis and induced apoptosis intumor

The histology analysis of tumor from NLC control displayed anundifferentiated feature that high number of cancer cellsunderwent mitotic division. Moreover, the number of mitotic/dividing cells in the tumor harvested from NLC–Zerumbonetreated mice decreased as compared to those in NLC andZerumbone treated tumors. Besides, the number of apoptoticcells (indicated by the cells with condensed nuclear thatstained by hematoxylin as dark blue indicated by the redarrow in Fig. 2) were higher in the tumor harvested from

(c) number of metastatic lung nodules harvested from the NLC control,ing 50 mg kg�1 per day of the respective drug for 28 days. The value

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Fig. 2 Representative histopathology (H&E staining, magnification 100�) and TUNEL (DeadEnd™ Colorimetric Apoptosis Detection Kit,magnification 100�) analyses of the tumor sections excised from the NLC control, Zerumbone treated and NLC–Zerumbone treated mice after28 days of treatment via oral feeding. In the H&E stained tumor, mitotic cells are circular with a square box, while apoptotic cells were labeledwith red arrow. For TUNEL histology, apoptotic cells can be seen as dark brown (red arrow) against a light brown background, as indicated by thearrows. Scale: 50 mm.

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NLC–Zerumbone treated mice (Fig. 2). This result was sup-ported by the TUNEL assay. In the TUNEL assay, apoptoticcells are categorized as dark brown cells indicating thebinding of the substrate. The number of cells that underwentDNA fragmentation in the NLC–Zerumbone and Zerumboneincreased substantially (Fig. 2).

Fig. 3 (a) The level of cytokines (IL-1b, IL-2, IL-6 IFN-g) in the harvestedcells ex vivo at a ratio of 5 : 1 or 2 : 1 of splenocyte to Yac-1. The level (ctumor. Values represent mean � S.E.M with significance set at p < 0.05.

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Zerumbone–NLC regulated cytokines expression

The levels of IL-1b, IL-2, IL-6 and IFN-g were measured in theserum of the NLC, Zerumbone and NLC–Zerumbone treatedmice. As shown in Fig. 3a, the level of antitumor related cyto-kines IL-2 and IFN-g increased signicantly (p < 0.05) in the

serum. (b) Cytotoxicity of splenocytes against lymphoma Yac-1 or 4T1) nitric oxide (NO) and (d) malondialdehyde (MDA) lipid peroxidation in

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NLC–Zerumbone treated mice as compared to both the NLC-treated and Zerumbone-treated mice. However, the levels ofinammatory related cytokines IL-1b and IL-6 in the NLC–Zer-umbone treated mice were signicantly lower than those of theNLC control and Zerumbone treated mice.

Zerumbone–NLC reduced lipid peroxidation and nitric oxide(NO) levels in tumors

As illustrated in Fig. 3c and d, the level of inammation-relatedNO and lipid peroxidation malondialdehyde (MDA) in the Zer-umbone serum was reduced 1.5 and 1.12 fold, respectively, ascompared to the NLC control mice. Furthermore, NLC–Zer-umbone further reduced 1.6 and 1.16 fold of the NO and MDAlevels, respectively, as compared to the NLC control mice.

Zerumbone–NLC enhanced splenocyte cytotoxicity againstmouse lymphoma Yac-1 and breast cancer 4T1 cells

Splenocyte harvests from all the mice possessed cytotoxicityagainst mouse lymphoma Yac-1 cells, which are sensitive tocytolytic T and natural killer cells. However, it was found thatsplenocytes isolated from Zerumbone and NLC–Zerumbonetreated mice show 11% and 20% higher cytotoxicity, respec-tively, than that of the NLC control splenocyte when co-culturedin a ratio of 2 : 1 splenocyte to Yac-1 cells. In a 5 : 1 ratio of

Fig. 4 Flow cytometry immunophenotyping analysis of CD3, CD4, CD8Zerumbone treated and NLC Zerumbone treated mice after 28 days of tre0.05.

This journal is © The Royal Society of Chemistry 2015

splenocyte to Yac-1 cells, the percentage of cytotoxicity of all thegroups was higher than that in ratio of 2 to 1. However, sple-nocytes isolated from Zerumbone and NLC–Zerumbone stillshow 9% and 21% higher cytotoxicity, respectively, than thesplenocyte isolated from NLC control. Unlike the cytolytic T andNK cell sensitive Yac-1 lymphoma cells, splenocytes isolatedfrom all the mice exhibited a lower cytotoxicity level againstbreast cancer 4T1 cells. However, splenocytes harvested fromNLC–Zerumbone treated mice still recorded the best cytotox-icity against 4T1 cells in both a 5 to1 and a 2 to 1 ratio of co-culture (Fig. 3b).

Zerumbone–NLC enhanced helper T cells, cytotoxic T cellsand natural killer T cells in spleens

The same pattern can also be observed in the immuno-phenotyping results. As shown in Fig. 4, the population ofHelper T cells (CD3+/CD4+), cytotoxic T cells (CD3+/CD8+) andnatural killer T (NKT) cells (CD3+NK1.1+) in the spleen ofZerumbone-treated mice was signicantly (p < 0.05) elevated1.2, 1.9 and 1.3 folds higher as compared to NLC control treatedmice (Fig. 4). In addition, NLC–Zerumbone treatment furtherenhanced the population of T helper, cytotoxic T, and NKT cellsby 1.6, 2.5 and 1.4 folds, respectively, as compared to the NLCcontrol.

and NK1.1 markers in the splenocytes harvested from the NLC control,atment. The values represent mean� S.E.M with significance set at p <

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Zerumbone–NLC reduced the expression of inammation-related mediators

As shown in Fig. 5a, based on the qPCR results, the mRNA levelsof the iNOS gene and NF-kB gene in both Zerumbone and NLC–Zerumbone tumor lysates decreased as compared to the NLCcontrol group (Fig. 5a). More signicantly, a 35 fold of down-regulation was observed in tumors harvested from NLC–Zer-umbone treated mice. A similar pattern can also be seen in thewestern blot results (Fig. 5b). The levels of COX-2 and VEGFprotein were reduced signicantly in the NLC–Zerumbone tumorlysates as compared to the NLC and Zerumbone-treated tumors.

Discussion

Breast cancer has the highest incidence in women.1 Althoughbetter therapeutic strategies have improved the survival rateof women with estrogen positive breast cancer, women withtriple-negative breast cancer, which contributed to 10–20% ofbreast cancer cases, have low survival rates due to the aggressivecharacteristics of the cancer, including drug resistance andmetastasis.20 Researches have recorded multiple bioactivitiesincluding anti-tumor and anti-inammatory effects. However,low water solubility has limited the in vivo and clinical poten-tial of Zerembone.11 Nano-encapsulation delivery has beenproposed as solution to improve the bioavailability and hasachieved a slow release of Zerumbone.9,11 In vitro studies haveshown that nanostructured lipid carriers (NLC) encapsulatedZerumbone possess characteristics of slow release, comparablecytotoxicity on leukemia11 and breast cancer cells,12 and it issuitable for oral administration without causing acute toxicityin mice.13 However, the in vivo oral efficacy of NLC–Zerumboneis yet to be compared with Zerumbone in the breast canceranimal model. Because both Zerumbone and NLC–Zerumbonehave shown similar in vitro cytotoxicities on triple negativeMDA-MB-231 cell lines,12 it is valuable to compare the advan-tages of slow in vivo release of NLC encapsulated and freeZerumbone against triple negative breast cancer cells. NLC

Fig. 5 (a) The level of mRNA expression of iNOS and NF-kB in the tumZerumbone treated mice after 28 days of treatment. The values represenanalysis of two proteins, namely, COX-2 and VEGF, in the tumors harvetreated mice after 28 days of treatment.

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encapsulation has various advantages as an oral deliverysystem, including increase in the bioavailability of waterinsoluble compounds, enabling a slow release mechanism,and reaching a higher peak concentration (Cmax) than non-encapsulated compounds such as vinpocetine. In terms oforal administration, it was proposed that an NLC encapsulatedcompound was transported via the lymphatic system, and thusit avoids rst-pass metabolism, which helps to achieve a higherpeak concentration aer oral administration. Besides, it wasalso reported that an NLC encapsulated compound has a higherpermeability in duodenum, jejunum, ileum and colon, whichimproves the absorption in the gastrointestinal tract post oraladministration.21 With all these advantages reported for theNLC encapsulation, this study was carried out to compare theanti-inammatory and anti-tumor effects of Zerumbone andNLC–Zerumbone on triple negative 4T1 breast cancer cellchallenged mice. Based on the measurement of tumors aer28 days of treatment, both Zerumbone and NLC–Zerumbonesignicantly (p < 0.05) limited the size and weight of tumorscompared to NLC control mice; moreover, the NLC–Zerumboneoutperformed Zerumbone treatment. The substantial reductionof the tumor size and weight of NLC–Zerumbone signies thatthis treatment is more effective compared to Zerumbone alone.DNA fragmentation is one of the steps executed in the apoptosiscascade. Therefore, the higher the number of DNA fragmentedcells, the more likely the cancer cells are approaching cell death.Cancer cells are classically known to have uncontrollable celldivision;17 thus by reducing the number of actively dividingcells, the growth of the tumor could eventually be impeded,as shown in Fig. 2. Based on the tumor histology and the TUNELassay, NLC–Zerumbone and Zerumbone limited the growthof 4T1 tumors through control of mitosis and induction ofapoptosis in the tumor (Fig. 2). Previous studies have reportedthat the in vitro cytotoxicity of Zerumbone against variouscancerous cell lines was mainly attributed to the G2/M cellcycle arrest and apoptosis induction.5 This study has furtherconrmed that both NLC–Zerumbone and Zerumbone control

ors harvested from the NLC control, Zerumbone treated and NLC–t mean � S.E.M with significance set at >2 fold change. (b) Western blotsted from the NLC control, Zerumbone treated and NLC–Zerumbone

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the progression of 4T1 tumor in mice via arresting cell cycleprogression and inducing apoptosis.

Besides the direct induction of cell cycle arrest and apoptosison cancer cells, the activation of anti-tumor immune responsesmediated by cytotoxic CD8+ T cells and natural killer cells mayalso synergistically enhance the antitumor response. A healthyand alerted immune system could be benecial to cancerpatients because it can wade off cancer cells in a more robustapproach. Various anti-cancer treatments have side effectsresulting in a compromised immune system, whereas thisphenomenon could actually delay the cancer progression andprolong survival.22 The depletion of cytotoxic T and NK cell wereassociated with increased cancer risk.23 However, the mainte-nance of the lymphocyte population may not necessarily reectthe role in tumor rejection. Thus, an evaluation of the expres-sion of antitumor cytokines and cytotoxicity of these lympho-cytes should be carried out to conrm the interaction betweenthis treatment and the cytotoxic lymphocytes towards the tumorrejection. IL-2 is a T helper 1 cytokine that induces the prolif-eration of cytotoxic-T and NK cells. In addition, IFN-g is acytokine classied under type II interferons. A high expressionof IFN-g activates cytotoxic T and NK cells. Previous studieshave reported both IL-2 and IFN-g as antitumor cytokines,where they induced primary tumor rejection.24 Zerumbone hasbeen reported with immunostimulatory effects, which promotesplenocyte and peripheral blood mononuclear cell (PBMC)proliferation and anti-tumor cytokine (IL-2 and IFN-g) secre-tion.25 This study has further conrmed the immunomodula-tory effect of Zerumbone to enhance the splenocyte T cell andNK cell populations (Fig. 4) and their cytotoxicity (Fig. 3b)against cytotoxic lymphocyte sensitive lymphoma Yac-1 cells. Interms of a co-culture with 4T1 cells ex vivo, splenocytes har-vested from the NLC control mice possessed the lowest cyto-toxicity. More signicantly (p < 0.05), splenocytes harvestedfrom NLC–Zerumbone treated mice showed the highest cyto-toxic effect on 4T1 cells ex vivo; this cytotoxic effect wasconsiderably greater as compared to that achieved by Zer-umbone treatment (Fig. 3b). Previous studies have shown thatnano-encapsulation delivered via lymph nodes exhibited betterimmune regulation.26 Thus, the further enhanced immuno-modulatory effect in NLC–Zerumbone treated mice may beattributed to the delivery of the NLC via lymph nodes.

In terms of the stimulation of antitumor immunity, Zer-umbone has been well studied with its anti-inammatory effectthrough the inhibition of lipid peroxidation (MDA), pro-inammatory cytokines (IL-1b and IL-6) and inammatorymediators (NF-kB, iNOS, COX-2 and NO).5 Extrinsic (chronicinammation) or intrinsic (oncogene activation) pathwaysinduced inammatory microenvironment resulted in the acti-vation of nuclear factor-kB (NF-kB) in cancer cells and coordi-nated the production of inammatory mediators cyclooxygenase2 (COX-2), inducible nitric oxide synthase (iNOS) and pro-inammatory cytokines IL-1b and IL-6. This cancer-relatedinammatory microenvironment can subsequently enhancetumor progression.27 Besides, the massive accumulation of lipidperoxidation may also induce overexpression of COX-2.28 In thisstudy, NLC control mice were found with higher levels of lipid

This journal is © The Royal Society of Chemistry 2015

peroxidation (Fig. 3d), inammatory mediators (NF-kB, iNOS,COX-2 and NO) (Fig. 5) and pro-inammatory cytokines (IL-1band IL-6) (Fig. 3a). Similar to the in vitro report,5 Zerumbonewas able to reduce lipid peroxidation, inammatory mediatorsand pro-inammatory cytokines. More signicantly, NLC–Zer-umbone was able to further reduce inammation in tumorsharvested from 4T1 challenged mice. Chronic inammationtumor microenvironments may further contribute to migration,invasion and metastasis. Tumors harvested from NLC controlmice were found with a high expression of pro-angiogenicprotein vascular endothelial growth factor (VEGF), whichcontributed to the metastatic niche.27 This phenomenon wasassociated with higher metastatic lung nodules in NLC controlmice. Zerumbone lowers inammation induces lower expres-sions of VEGF and reduces lung metastasis. The NLC–Zer-umbone was found to outperform Zerumbone in control lungmetastasis in 4T1 challenged mice. This higher anti-metastaticeffect of NLC–Zerumbone may be attributed to the possiblelymphatic targeting effects of NLC, which has been previouslyreported more effectively to control tumor metastasis.29

Conclusion

This study has shown that Zerumbone was able to control invivo 4T1 breast cancer progression via controlling cancer cellmitosis, inducing apoptosis, activating anti-tumor immunity,controlling tumor microenvironment inammation and delay-ing tumor metastasis. This effect was even signicant in NLC–Zerumbone treated mice, indicating that the NLC encapsula-tion enhanced the anti-tumor efficacy of Zerumbone. Thus, NLCis a potential nano-encapsulator of Zerumbone, which maybring benets to the clinical treatment of breast cancer.

Conflict of interest

All authors disclose no potential conict of interest.

Acknowledgements

This project was funded by Research University Grants (RUGS)91194, Universiti Putra Malaysia and eScience Fund 5495308,Ministry of Science, Technology and Innovation, Malaysia.

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