Research Article Antimetastatic and Anti-Inflammatory ...downloads.hindawi.com/journals/tswj/2014/239508.pdf · Research Article Antimetastatic and Anti-Inflammatory Potentials of

Research ArticleAntimetastatic and Anti-Inflammatory Potentials ofEssential Oil from Edible Ocimum sanctum Leaves

Thamilvaani Manaharan1 Ramaraj Thirugnanasampandan2 Rajarajeswaran Jayakumar1

Gunasekar Ramya2 Gogul Ramnath2 and M S Kanthimathi1

1 Department of Molecular Medicine Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia2 Department of Biotechnology Kongunadu Arts and Science College GN Mills Coimbatore Tamil Nadu 641029 India

Correspondence should be addressed to Ramaraj Thirugnanasampandan rtsampandanyahoocom

Received 22 July 2014 Revised 24 September 2014 Accepted 2 October 2014 Published 5 November 2014

Academic Editor Urs Feller

Copyright copy 2014 Thamilvaani Manaharan et alThis is an open access article distributed under theCreative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Antimetastatic and anti-inflammatory activities of Ocimum sanctum essential oil (OSEO) have been assessed in this study OSEOat the concentration of 250 120583gmL and above showed a significant (lowast119875 lt 005) decrease in the number of migrated cancer cellsIn addition OSEO at concentration of 250 120583gmL and above suppressed MMP-9 activity in lipopolysaccharide (LPS) inducedinflammatory cells A dose-dependent downregulation of MMP-9 expression was observed with the treatment of OSEO comparedto the control Our findings indicate that OSEO has both antimetastatic and anti-inflammatory potentials advocating furtherinvestigation for clinical applications in the treatment of inflammation associated cancer

1 Introduction

Inflammation is one of the hallmarks of cancer initiation andprogression It contributes to tumor initiation by inducingDNAdamage and chromosomal instability as well as enhanc-ing tumor cell proliferation Inflammation also stimulatesangiogenesis and tissue remodeling which contribute totumor cell invasion and metastasis [1] Many studies haveshown that chronic inflammation stimulates developmentand progression of cancer due to the release of matrix metal-loproteinases (MMPs) from the inflammatory cells [2 3]TheMMPs function as essential regulators for the degradationof extracellular matrix (ECM) and basement membranethereby it contributes to the development and progression ofhuman malignancies [4] The MMPs can be classifiedinto four major subgroups collagenases (MMP-1 MMP-3and MMP-8) gelatinases (MMP-2 MMP-9) stromelysins(MMP-3 MMP-7 MMP-10 and MMP-12) and membrane-type metalloproteinases (MTMMP-1 through MTMMP-5)[5] The MMP-9 (92KDa) plays a crucial role in the mech-anism of tumor invasion of many types of cancer [2] A pre-vious report suggested a role for MMP-9 in tumor invasionrelates to the fact that the release ofMMP-9 is associated with

themetastatic phenotype of transformed rat embryo cells [6]Recent studies revealed that overexpression of MMP-9 ininflammation associated breast cancer [7] colon cancer [5]and ovarian cancer [8] led to tumor metastasis Thus inhi-bition of MMP-9 activity could reduce inflammation andprevent cancer progression and metastasis as well [9]

It is widely recognized that the prevention of cancer andinflammatory diseases could be associated with the intakeof fresh fruits and vegetables Ocimum sanctum Linn com-monly known as Tulsi or holy basil is widely known acrossSouth Asia as an aromatic medicinal herb and is distributedand cultivated worldwide [10 11] O sanctum leaves arecategorized as functional foods and have a variety of pharma-cological effects like antimicrobial immunomodulatory anti-stress anti-inflammatory antiulcer antidiabetic hepatopro-tective chemoprotective hypolipidemic cardioprotectiveantioxidant antitussive radioprotective memory enhancingantiarthritic antifertility antihypertensive anticoagulantanticataract anthelmintic and antinociceptive effects [12ndash16] The essential oil from the leaves of O sanctum (OSEO)on the other hand has been evaluated pharmacologicallyfor antimicrobial [17] anticandidal [18] and antifungal [1920] activities However to the best of our knowledge there

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 239508 5 pageshttpdxdoiorg1011552014239508

2 The Scientific World Journal

is limited information about the antimetastatic and anti-inflammatory effects of OSEO The aim of the present studywas to investigate the antimetastatic and anti-inflammatorypotential of OSEO using in vitro assays

2 Materials and Methods

21 Essential Oil Extraction and Preparation Freshly col-lected leaves of O sanctum (1 kg) were hydrodistilled for4 h using Clevenger apparatus for essential oil extractionExtracted oil was treated with sodium sulphate anhydrous toremove excess water The purified oil was then filled in smallvials tightly sealed and stored in a refrigerator (4∘C) forfurther studies For the cell culture assays stock of OSEOwasprepared in dimethyl sulphoxide (1 gmL) and was furtherdissolved in the culture medium (RPMI-1640)

22 Cell Migration Assay The cell migration assay wascarried out according to themethod described by [21] Photo-graphs of three random fields were taken through an invertedmicroscope (Nikon ECLIPSE TI-S) and the numbers ofmigrating cells were counted to calculate the average numberof migrated cells from three independent experiments

23 Anti-Inflammatory Effects of OSEO Lipopolysaccharides(LPS) also known as lipoglycans are an endotoxin localizedin the outer membrane of Gram negative bacteria (E coli) Inthis study LPS was used to induce inflammation [22] Lym-phocytes (1 times 105 cellswell) cultured in 96 well plates usingRPMI-1640 medium were induced with inflammation usingLPS (1 120583gmL in culture medium) for 24 h After induction ofinflammation different concentrations (50 100 150 200 and250120583gmL) of OSEO were added to each well and incubatedovernight At the end of incubation the cell free mediumwas collected and assayed for MMP-9 inhibition by gelatinzymography

24 Gelatin Zymography SDS-PAGEwas carried out accord-ing to the method of [23] Zymogram gel consisting of 75polyacrylamide gel copolymerized with gelatin (1mgmL)was prepared for electrophoresis Following electrophoresisthe gel was washed successively with 50mL of 25 (vv)TritonX-100 in distilledwater for an hour to remove SDSThegel was then incubated with developing solution (CaCl

2

10mM Triton X-100 1 and Tris buffer 50mM pH 74) at32∘C for 18 h Further the gel was stained with Coomassiebrilliant blue R250 for 2 h and destained overnight to revealthe bands The bands on gel reflect the MMP-9 inhibitoryeffects of OSEO

25 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR Analysis) The LPS-induced inflammatory cells weretreated with different concentrations (50 100 150 200 and250120583gmL) of OSEO for 24 h Total RNA was obtained andsuspended in 9 120583L of deionised autoclaved DEPC treatedwater Following that 2 120583L of dNTP 5 120583L of cDNA synthesisbuffer 1 120583L of oligo d (T) 1120583L of reverse transcriptase enzymemix (Thermo Fischer Scientific India) and 9 120583L of nuclease

free water were added and reverse transcription was carriedout in a thermocycler (Eppendorf USA) to synthesize cDNAPrimers for human MMP-9 and 120573-actin (Helini India) wereas follows forward primer 51015840-AAG ATG CTG CTG TTCAGC GGG-31015840 and reverse primer 51015840-GTC CTC AGG GCACTG CAG GAT-31015840 for MMP-9 [24] and forward primer 51015840-AGG GAA ATC GTG CGT GAC-31015840 and reverse primer 51015840-CGC TCA TTG CCG ATA GTG-31015840 for 120573-actin The PCRconditions for MMP-9 and 120573-actin are as follows initialdenaturation (94∘C for 5min) denaturation (94∘C for 30 sec)annealing (57∘C for 30 sec for MMP-9) and (60∘C for 30 secfor 120573-actin) extension (72∘C for 1min) and final exten-sion (72∘C for 10min) The amplified fragments of MMP-9were then loaded into the wells of 05 agarose gel and elec-trophoresis was run for 30min at 50 voltsThe gel was viewedand photographed using Gel Doc 2000 (Bio-Rad Philadel-phia USA)

26 Statistical Analysis Analysis at every time point fromeach experiment was carried out in triplicate Means stan-dard errors standard deviations studentrsquos paired 119905-test andone way ANOVA were calculated from replicates within theexperiments and analyses were done using SPSS version 16Statistical significance was accepted at a level of 119875 lt 005

3 Results and Discussion

31 Suppression of MMP-9 and Inhibition of Cancer CellMigration by OSEO Matrix metalloproteinases (MMPs) inparticular the MMP-9 are essential regulators of extracellu-lar matrix (ECM) and recruit the inflammatory cells duringchronic inflammation which involves a series of complexmorphological changes in cell barrier cell-cell interactionand cell matrix interaction [25] In this study inhibition ofcancer cell migration was also observed following treatmentwith OSEO (50ndash500120583gmL) (Figure 1) The OSEO at theconcentration of 250120583gmL and above showed a significant(lowast119875 lt 005) decrease in the number of migrated cancer cells(Figure 2) In this study we assessed the ability of OSEO as ananti-inflammatory agent to inhibit MMP-9 activity Gelatinzymography clearly showed that OSEO inhibits MMP-9activity in LPS-induced inflammatory cells dose-dependentlycompared to the control (Figure 3) No specific laddermarkerwas used in this study since the inhibition of MMP-9by OSEOwas compared to the control (cells treated with LPSonly) Out of five different concentrations tested 250120583gmLof oil showed the strongest MMP-9 inhibitory activity com-pared to the control (Figure 3) In addition our RT-PCRanalysis revealed that treatment with OSEO significantlydownregulated MMP-9 expression dose-dependently com-pared to the LPS-induced control cells (Figure 4(a)) and thereference marker 120573-actin (Figure 4(b)) The ladder marker100ndash1000 kb was used as a reference for the amplifiedfragments of MMP-9 and 120573-actin genes LPS has been rec-ognized as the major inducer of proinflammatory cytokinesincluding tumor necrosis factor- (TNF-) 120572 and interleukin-(IL-) 6 which in turn stimulates nitric oxide synthase (iNOS)production during the inflammatory process [26] Thus

The Scientific World Journal 3

50120583gmL 100120583gmL 250120583gmL 500120583gmL

OSE

OO

SEO

Con

trol (01

D

MSO

)

Resv

erat

rol300120583

gm

L

0hr 24hr 0hr 24hr0

hr24

hr

Figure 1 Effect of OSEO on migration of MCF-7 cells at various concentrations (50ndash500 120583gmL) Cells treated with vehicle (01 DMSO)served as negative control and cells treated with resveratrol (300120583gmL) served as positive control Photographs were captured at 0 h and 24 hof treatment on an inverted microscope (Nikon ECLIPSE TI-S) at 20x magnification The gap or ldquoscarringrdquo area is marked with yellow lines

0

200

400

600

800

1000

1200

1400

Control 50 100 250 500

Concentration (120583gmL)

lowast

lowast

Num

ber o

f mig

rate

d ce

lls

Figure 2 The number of migrated cells per three random fieldswas counted using an inverted microscope (Nikon ECLIPSE TI-S)at 400x magnification Data are expressed as means plusmn SD 119899 = 3Studentrsquos paired 119905-test showed significant values lowast119875 lt 005 versuscontrol

C 1 2 3 4 5

Figure 3 Gelatin zymography of MMP-9 activity MMP-9 inhi-bition of different concentrations of OSEO after being inducedwith LPS Denotation C (Control) 1 (50 120583gmL) 2 (100 120583gmL) 3(150 120583gmL) 4 (200120583gmL) and 5 (250 120583gmL) of OSEO Effect ofOSEO on downregulation of MMP-9

OSEO could also suppress the expression of proinflammatorycytokines However furthermolecular studies are required tounderstand its anti-inflammatory mechanism Plant derivedmolecules have inhibitory effects on tumour cell migrationprimarily by suppression of the activity of MMPs [27] Ourresults indicate that OSEO has anti-inflammatory poten-tial by suppressing MMP-9 expression and prevents cancermetastasis by inhibiting cancer cell migration


M 1 2 3 4 5

(a)

M 1 2 3 4

(b)

Figure 4 (a) Downregulation of MMP-9 was assessed after 24 h treatment with various concentrations of OSEO in LPS-induced cellsDenotation for lanes M (100ndash1000 kb ladder marker) 1 (50 120583gmL) 2 (100120583gmL) 3 (150 120583gmL) 4 (200 120583gmL) and 5 (250 120583gmL) ofOSEO (b) 120573-Actin (452 bp) served as an internal marker

4 Conclusion

In our studyOSEO showed strong anti-inflammatory activityby suppressing the expression of MMP-9 in LPS-inducedinflammatory cells which in turn triggered the prevention ofcancer cell migration These findings provide evidence thatthe antimetastatic and anti-inflammatory potential of OSEOcould be useful in the development of new therapeutic strate-gies for inflammation associated cancer Thus appropriateaddition of OSEO in the diet may prevent cancer andimmune-mediated inflammatory diseases

Abbreviations

LPS LipopolysaccharideMMP Matrix metalloproteinasesOSEO Ocimum sanctum essential oil

Conflict of Interests

The authors declare that there is no conflict of interestsregarding publication of this paper

Acknowledgments

This research was supported by the High Impact ResearchGrant (HIR-MOHEUMC6251HIRE000043-20001) fromthe University of Malaya Malaysia and KASC ResearchGrant India (52014) The authors would like to thank MrsNur Faezah for the technical contributions

References

[1] Y Wu S Antony J L Meitzler and J H Doroshow ldquoMolec-ular mechanisms underlying chronic inflammation-associatedcancersrdquo Cancer Letters vol 345 no 2 pp 164ndash173 2014

[2] E I Deryugina and J P Quigley ldquoMatrix metalloproteinasesand tumor metastasisrdquo Cancer and Metastasis Reviews vol 25no 1 pp 9ndash34 2006

[3] P Garg D Sarma S Jeppsson et al ldquoMatrix metalloproteinase-9 functions as a tumor suppressor in colitis-associated cancerrdquoCancer Research vol 70 no 2 pp 792ndash801 2010

[4] J Westermarck and V-M Kahari ldquoRegulation of matrix metal-loproteinase expression in tumor invasionrdquoThe FASEB Journalvol 13 no 8 pp 781ndash792 1999

[5] L Walter C Harper and P Garg ldquoRole of matrix metallo-proteinases in inflammationcolitis-associated colon cancerrdquoImmuno-Gastroenterology vol 2 pp 22ndash28 2013

[6] E J Bernhard S B Gruber and R J Muschel ldquoDirect evi-dence linking expression ofmatrixmetalloproteinase 9 (92-kDagelatinasecollagenase) to the metastatic phenotype in trans-formed rat embryo cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 91 no 10 pp4293ndash4297 1994

[7] K S Leifler S Svensson A Abrahamsson et al ldquoInflammationinduced byMMP-9 enhances tumor regression of experimentalbreast cancerrdquo The Journal of Immunology vol 190 no 8 pp4420ndash4430 2013

[8] B Schmalfeldt D Prechtel K Harting et al ldquoIncreased expres-sion of matrix metalloproteinases (MMP)-2 MMP-9 andthe urokinase-type plasminogen activator is associated withprogression from benign to advanced ovarian cancerrdquo ClinicalCancer Research vol 7 no 8 pp 2396ndash2404 2001

[9] M Hidalgo and S G Eckhardt ldquoDevelopment of matrix metal-loproteinase inhibitors in cancer therapyrdquo Journal of theNational Cancer Institute vol 93 no 3 pp 178ndash193 2001

[10] J Sethi S Sood S Seth and A Talwar ldquoProtective effect ofTulsi (Ocimum sanctum) on lipid peroxidation in stress inducedby anemic hypoxia in rabbitsrdquo Indian Journal of Physiology andPharmacology vol 47 no 1 pp 115ndash119 2003

[11] E I Nweze and E E Eze ldquoJustification for the use of Ocimumgratissimum L in herbal medicine and its interaction with discantibioticsrdquo BMC Complementary and Alternative Medicinevol 9 article 37 2009

[12] D Runyoro O Ngassapa K Vagionas N Aligiannis KGraikou and I Chinou ldquoChemical composition and antimicro-bial activity of the essential oils of fourOcimum species growingin Tanzaniardquo Food Chemistry vol 119 no 1 pp 311ndash316 2010

[13] H Harnafi H S Caid N H Bouanani M Aziz and S AmranildquoHypolipemic activity of polyphenol-rich extracts from Oci-mum basilicum in Triton WR-1339-induced hyperlipidemicmicerdquo Food Chemistry vol 108 no 1 pp 205ndash212 2008

[14] E M Kwee and E D Niemeyer ldquoVariations in phenolic com-position and antioxidant properties among 15 basil (Ocimumbasilicum L) cultivarsrdquo Food Chemistry vol 128 no 4 pp1044ndash1050 2011


[15] K Carovic-Stanko S Orlic O Politeo et al ldquoComposition andantibacterial activities of essential oils of seven Ocimum taxardquoFood Chemistry vol 119 no 1 pp 196ndash201 2010

[16] P Prakash andN Gupta ldquoTherapeutic uses ofOcimum sanctumLinn (tulsi) with a note on eugenol and its pharmacologicalactions a short reviewrdquo Indian Journal of Physiology andPharmacology vol 49 no 2 pp 125ndash131 2005

[17] AKumar R Shukla P Singh andNKDubey ldquoChemical com-position antifungal and antiaflatoxigenic activities of Ocimumsanctum L essential oil and its safety assessment as plant basedantimicrobialrdquo Food and Chemical Toxicology vol 48 no 2 pp539ndash543 2010

[18] K Amber A Aijaz X Immaculata K A Luqman and MNikhat ldquoAnticandidal effect of Ocimum sanctum essential oiland its synergy with fluconazole and ketoconazolerdquo Phytome-dicine vol 17 no 12 pp 921ndash925 2010

[19] A Khan AAhmad F Akhtar et al ldquoOcimum sanctum essentialoil and its active principles exert their antifungal activity bydisrupting ergosterol biosynthesis and membrane integrityrdquoResearch in Microbiology vol 161 no 10 pp 816ndash823 2010

[20] A Kumar N K Dubey and S Srivastava ldquoAntifungal evalu-ation of Ocimum sanctum essential oil against fungal deterio-ration of raw materials of Rauvolfia serpentina during storagerdquoIndustrial Crops and Products vol 45 pp 30ndash35 2013

[21] R Jayakumar and M S Kanthimathi ldquoDietary spices protectagainst hydrogen peroxide-induced DNA damage and inhibitnicotine-induced cancer cell migrationrdquo Food Chemistry vol134 no 3 pp 1580ndash1584 2012

[22] D Vijayalakshmi R Dhandapani S Jayaveni P S JithendraC Rose and A B Mandal ldquoIn vitro anti inflammatory activityof Aloe vera by down regulation of MMP-9 in peripheral bloodmononuclear cellsrdquo Journal of Ethnopharmacology vol 141 no1 pp 542ndash546 2012

[23] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[24] S Rayalam J-Y Yang S Ambati M A Della-Fera and C ABaile ldquoResveratrol induces apoptosis and inhibits adipogenesisin 3T3-L1 adipocytesrdquo Phytotherapy Research vol 22 no 10 pp1367ndash1371 2008

[25] M J W Meijer M A C Mieremet-Ooms A M van der Zonet al ldquoIncreasedmucosal matrix metalloproteinase-1 -2 -3 and-9 activity in patients with inflammatory bowel disease and therelation with Crohnrsquos disease phenotyperdquo Digestive and LiverDisease vol 39 no 8 pp 733ndash739 2007

[26] L-P Chang Y-S Lai C-J Wu and T-C Chou ldquoLiquid perflu-orochemical inhibits inducible nitric oxide synthase expressionand nitric oxide formation in lipopolysaccharide-treated RAW2647 macrophagesrdquo Journal of Pharmacological Sciences vol111 no 2 pp 147ndash154 2009

[27] S Shishodia and B B Aggarwal ldquoNuclear factor-120581B a friend ora foe in cancerrdquo Biochemical Pharmacology vol 68 no 6 pp1071ndash1080 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


is limited information about the antimetastatic and anti-inflammatory effects of OSEO The aim of the present studywas to investigate the antimetastatic and anti-inflammatorypotential of OSEO using in vitro assays

2 Materials and Methods

21 Essential Oil Extraction and Preparation Freshly col-lected leaves of O sanctum (1 kg) were hydrodistilled for4 h using Clevenger apparatus for essential oil extractionExtracted oil was treated with sodium sulphate anhydrous toremove excess water The purified oil was then filled in smallvials tightly sealed and stored in a refrigerator (4∘C) forfurther studies For the cell culture assays stock of OSEOwasprepared in dimethyl sulphoxide (1 gmL) and was furtherdissolved in the culture medium (RPMI-1640)

22 Cell Migration Assay The cell migration assay wascarried out according to themethod described by [21] Photo-graphs of three random fields were taken through an invertedmicroscope (Nikon ECLIPSE TI-S) and the numbers ofmigrating cells were counted to calculate the average numberof migrated cells from three independent experiments

23 Anti-Inflammatory Effects of OSEO Lipopolysaccharides(LPS) also known as lipoglycans are an endotoxin localizedin the outer membrane of Gram negative bacteria (E coli) Inthis study LPS was used to induce inflammation [22] Lym-phocytes (1 times 105 cellswell) cultured in 96 well plates usingRPMI-1640 medium were induced with inflammation usingLPS (1 120583gmL in culture medium) for 24 h After induction ofinflammation different concentrations (50 100 150 200 and250120583gmL) of OSEO were added to each well and incubatedovernight At the end of incubation the cell free mediumwas collected and assayed for MMP-9 inhibition by gelatinzymography

24 Gelatin Zymography SDS-PAGEwas carried out accord-ing to the method of [23] Zymogram gel consisting of 75polyacrylamide gel copolymerized with gelatin (1mgmL)was prepared for electrophoresis Following electrophoresisthe gel was washed successively with 50mL of 25 (vv)TritonX-100 in distilledwater for an hour to remove SDSThegel was then incubated with developing solution (CaCl

2

10mM Triton X-100 1 and Tris buffer 50mM pH 74) at32∘C for 18 h Further the gel was stained with Coomassiebrilliant blue R250 for 2 h and destained overnight to revealthe bands The bands on gel reflect the MMP-9 inhibitoryeffects of OSEO

25 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR Analysis) The LPS-induced inflammatory cells weretreated with different concentrations (50 100 150 200 and250120583gmL) of OSEO for 24 h Total RNA was obtained andsuspended in 9 120583L of deionised autoclaved DEPC treatedwater Following that 2 120583L of dNTP 5 120583L of cDNA synthesisbuffer 1 120583L of oligo d (T) 1120583L of reverse transcriptase enzymemix (Thermo Fischer Scientific India) and 9 120583L of nuclease

free water were added and reverse transcription was carriedout in a thermocycler (Eppendorf USA) to synthesize cDNAPrimers for human MMP-9 and 120573-actin (Helini India) wereas follows forward primer 51015840-AAG ATG CTG CTG TTCAGC GGG-31015840 and reverse primer 51015840-GTC CTC AGG GCACTG CAG GAT-31015840 for MMP-9 [24] and forward primer 51015840-AGG GAA ATC GTG CGT GAC-31015840 and reverse primer 51015840-CGC TCA TTG CCG ATA GTG-31015840 for 120573-actin The PCRconditions for MMP-9 and 120573-actin are as follows initialdenaturation (94∘C for 5min) denaturation (94∘C for 30 sec)annealing (57∘C for 30 sec for MMP-9) and (60∘C for 30 secfor 120573-actin) extension (72∘C for 1min) and final exten-sion (72∘C for 10min) The amplified fragments of MMP-9were then loaded into the wells of 05 agarose gel and elec-trophoresis was run for 30min at 50 voltsThe gel was viewedand photographed using Gel Doc 2000 (Bio-Rad Philadel-phia USA)

26 Statistical Analysis Analysis at every time point fromeach experiment was carried out in triplicate Means stan-dard errors standard deviations studentrsquos paired 119905-test andone way ANOVA were calculated from replicates within theexperiments and analyses were done using SPSS version 16Statistical significance was accepted at a level of 119875 lt 005

3 Results and Discussion

31 Suppression of MMP-9 and Inhibition of Cancer CellMigration by OSEO Matrix metalloproteinases (MMPs) inparticular the MMP-9 are essential regulators of extracellu-lar matrix (ECM) and recruit the inflammatory cells duringchronic inflammation which involves a series of complexmorphological changes in cell barrier cell-cell interactionand cell matrix interaction [25] In this study inhibition ofcancer cell migration was also observed following treatmentwith OSEO (50ndash500120583gmL) (Figure 1) The OSEO at theconcentration of 250120583gmL and above showed a significant(lowast119875 lt 005) decrease in the number of migrated cancer cells(Figure 2) In this study we assessed the ability of OSEO as ananti-inflammatory agent to inhibit MMP-9 activity Gelatinzymography clearly showed that OSEO inhibits MMP-9activity in LPS-induced inflammatory cells dose-dependentlycompared to the control (Figure 3) No specific laddermarkerwas used in this study since the inhibition of MMP-9by OSEOwas compared to the control (cells treated with LPSonly) Out of five different concentrations tested 250120583gmLof oil showed the strongest MMP-9 inhibitory activity com-pared to the control (Figure 3) In addition our RT-PCRanalysis revealed that treatment with OSEO significantlydownregulated MMP-9 expression dose-dependently com-pared to the LPS-induced control cells (Figure 4(a)) and thereference marker 120573-actin (Figure 4(b)) The ladder marker100ndash1000 kb was used as a reference for the amplifiedfragments of MMP-9 and 120573-actin genes LPS has been rec-ognized as the major inducer of proinflammatory cytokinesincluding tumor necrosis factor- (TNF-) 120572 and interleukin-(IL-) 6 which in turn stimulates nitric oxide synthase (iNOS)production during the inflammatory process [26] Thus


50120583gmL 100120583gmL 250120583gmL 500120583gmL

OSE

OO

SEO

Con

trol (01

D

MSO

)

Resv

erat

rol300120583

gm

L

0hr 24hr 0hr 24hr0

hr24

hr


0

200

400

600

800

1000

1200

1400

Control 50 100 250 500


lowast

lowast

Num

ber o

f mig

rate

d ce

lls


C 1 2 3 4 5




M 1 2 3 4 5

(a)

M 1 2 3 4

(b)


4 Conclusion


Abbreviations




Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


50120583gmL 100120583gmL 250120583gmL 500120583gmL

OSE

OO

SEO

Con

trol (01

D

MSO

)

Resv

erat

rol300120583

gm

L

0hr 24hr 0hr 24hr0

hr24

hr


0

200

400

600

800

1000

1200

1400

Control 50 100 250 500


lowast

lowast

Num

ber o

f mig

rate

d ce

lls


C 1 2 3 4 5




M 1 2 3 4 5

(a)

M 1 2 3 4

(b)


4 Conclusion


Abbreviations




Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2 3 4 5

(a)

M 1 2 3 4

(b)


4 Conclusion


Abbreviations




Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Antimetastatic and Anti-Inflammatory ...downloads.hindawi.com/journals/tswj/2014/239508.pdf · Research Article Antimetastatic and Anti-Inflammatory Potentials of