ResearchArticle Curcuma longa Is Able to Induce Apoptotic...

Research ArticleCurcuma longa Is Able to Induce Apoptotic Cell Death ofPterygium-Derived Human Keratinocytes

Silvia Sancilio1 Silvio Di Staso2 Stefano Sebastiani3 Lucia Centurione4

Nick Di Girolamo5 Marco Ciancaglini2 and Roberta Di Pietro4

1Department of Pharmacy G drsquoAnnunzio University of Chieti-Pescara Via dei Vestini 31 66100 Chieti Italy2Ophthalmic Clinic Department of Life Health and Environmental Sciences LrsquoAquila University Piazzale S Tommasi 167100 LrsquoAquila Italy3Sebastiani Pharmacy Via Italia 70 65010 Spoltore Italy4Department of Medicine and Ageing Science School of Medicine and Health Sciences G drsquoAnnunzio University of Chieti-PescaraVia dei Vestini 31 66100 Chieti Italy5Inflammatory Diseases Research Unit School of Medical Sciences The University of New South Wales Sydney NSW 2052 Australia

Correspondence should be addressed to Roberta Di Pietro rdipietrounichit

Received 26 July 2017 Accepted 11 October 2017 Published 17 December 2017

Academic Editor Mirella Falconi

Copyright copy 2017 Silvia Sancilio et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour To evaluate the in vitro effectsof Curcuma longa on human pterygium-derived keratinocytes specimens of pterygium from 20 patients undergoing pterygiumsurgical excision were collected Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholicextract of 13Curcuma longa in 0001 BenzalkoniumChloride for 3 6 and 24 h Cultured cells were examined for CAM52 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expression between 3th and 16th passageto assess cell homogeneity TUNEL technique and Annexin-VPI staining in flow cytometry were used to detect keratinocyteapoptosis We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 htreatmentMoreover after 24 h treatmentCurcuma longa induces a significant increase in TUNEL as well as Annexin-VPI positivecells in comparison to untreated samples Our study confirms previous observations highlighting the expression in pterygiumkeratinocytes of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1 All inall these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of humanpterygium

1 Introduction

Ocular surface epithelium is composed of corneal conjunc-tival and limbal epithelial layers [1] Epithelia covering thecorneal and the conjunctival surfaces are both stratified andnonkeratinized but only the conjunctival epithelium containsgoblet cells involved in a gel-forming mucina secretion [2 3]Functionally corneal and conjunctival epithelium play simi-lar roles since both are able to be wettable so as to maintaincomfort while providing smooth optical surface [4] and toprotect the underlying tissues from infection [2] Limbalepithelium has a key role in the maintenance of cornealtransparency by means of a population of limbal epithelial

stem cells Depletion of stratospheric ozone has increased theflux of ultraviolet-B (UV-B) radiation at the surface of theearth increasing the rate of UV-induced eye damage [5] Anocular surface disease attributed to chronic UV-B exposureand to lesions of the limbal epithelium is pterygium [6]Pterygium has been defined by Duke-Elder as a triangularshaped degenerative and hyperplastic process occurringmedially and laterally in the rhyme eyelid inwhich the bulbarconjunctiva encroaches on the cornea [7] Pterygium is avery common fibrovascular lesionwidespread in the so calledldquopterygium zonerdquo [8] This is an area defined by a geograph-ical latitude of 40∘ north and south of the equator Herea prevalence rate of up to 22 in the population has been

HindawiBioMed Research InternationalVolume 2017 Article ID 2956597 9 pageshttpsdoiorg10115520172956597

2 BioMed Research International

reported Outside this area prevalence rates usually do notovercome 2of the general population and the disease affectsmainly patients with an increased exposure to sunlight orinvolved in outdoor activities [9] Although previous studieshave highlighted the involvement of genetic factors in thepathogenesis of pterygium [10] the etiology of pterygiumstill remains unclear [8] Current management strategies forpterygium imply surgical excision [11] that is a complex andinvasive procedure that often results in the recurrence of alesion more clinically aggressive than the original one [9] Inaddition surgery can lead to further clinical manifestationssuch as symblepharon corneal clouding corneal or scleraldellen [9 12] keloid formation [13] and scleral necrosis [14]

Curcuma longa is a plant belonging to the family ofZingiberaceae The rhizomes of this plant are the sourceof turmeric which has been used for centuries all overthe world in cooking cosmetics and medical treatments[15] The curcuminoids present in the rhizome which areresponsible for the yellow colour of turmeric consist ofa mixture of curcumin (also known as diferuloylmethaneNatural Yellow and E100) demethoxycurcumin and bis-demethoxycurcumin [16] Curcumin makes up sim90 of thecurcuminoid content Due to its chemical structure cur-cumin ismuch less soluble inwater at acid and neutral pHbutsoluble in methanol ethanol dimethyl sulfoxide (DMSO)and acetone [17] Its traditional uses as a strong therapeutic orpreventive agent in several human diseases such as diabetesinflammation atherosclerosis and cancer [18] is due to itsbeneficial properties including anti-inflammatory antioxi-dant antineoplastic pro- and antiapoptotic antiangiogeniccytotoxic immune-modulatory and antimicrobial effects viathemodulation of various targets (grow factors enzymes andgenes) [19]

Based on available literature and with the aim to identifyan alternative strategy to currently available surgical proce-dures in this study we investigated the effects of in vitrotreatment with Curcuma longa of keratinocytes derived fromexplants of human pterygium

2 Materials and Methods

21 Experimental Design Human pterygium specimens wereobtained from 20 patients with primary pterygium (averageage 682 plusmn 97 years) undergoing routine pterygiectomy Allthe patients affected with pterygia displayed inflammatorysigns of the ocular surface such as chemosis and rednessof the conjunctiva Normal conjunctival tissue specimenswere obtained from 3 patients (average age 712 plusmn 83 years)undergoing cataract surgery All patients were treated at theSS Annunziata Hospital in Chieti Italy Signed informedconsent was obtained from the donors according to Italianlegislation and to the code of Ethical Principles for MedicalResearch involving Human Subjects of the World MedicalAssociation (Declaration ofHelsinki) After surgical excisionthe clinical specimens were properly treated or preserved forimmunohistochemistry or primary cell culture

22 Primary Culture and Subculture of Pterygium-DerivedKeratinocytes Pterygium specimens were placed in a Petri

dish and washed with 1x Dulbeccorsquos Phosphate-BufferedSaline (PBS) Samples were cut into several 1-2mm2 piecesunder sterile conditions and placed into six-well plates at37∘C in an atmosphere of 5 carbon dioxide in air Explantswere allowed to attach to the plastic dish for at least 8 h in adrop of complete medium selective for human keratinocytessurvival and consisting of Eaglersquos MEM (GIBCO by Invit-rogen Carlsbad CA USA) supplemented with 10 foetalbovine serum (FBS Hyclone Logan UT USA) 100Umlpenicillin 100 120583gml streptomycin and 25 120583gml fungizone(Sigma-Aldrich St Louis MO USA) Cell migration fromexplants was observed within 3ndash5 days Cells were used forexperiments between the 4th and 7th passage and only forcharacterization up to the 16th passage Pterygium-derivedkeratinocytes were grown on 6-well culture plates and whencells reached confluence culture medium was replaced andwhere required treated with an alcoholic extract of 13Curcuma longa in 0001 Benzalkonium Chloride (BCA)(Sigma-Aldrich) awell knownpreservative commonly addedto several eye drops Control cultures were left untreatedor treated with the vehicle alone (BCA) for the same timeintervals After 3 6 and 24 h observations were carried outunder a phase contrast light microscope (LEICA WetzlarGermany) equipped with a CoolSNAP video camera foracquiring computerized images (Photometrics Tucson AZ)

23 Histochemical Analysis Explants of human pterygiumwere fixed with 10 (volvol) phosphate-buffered formalindehydrated in a series of graded increases in alcohol con-centrations embedded in paraffin and cut at the microtome(Leica RM 2265 Germany) Sections were routinely stainedwith Haematoxylin-Eosin staining solution and mounted inBioMount (Bio-Optica Italy) For periodic acidndashSchiff (PAS)staining samples were fixed with 4 paraformaldehydewashed with PBS oxidized in 1 periodic acid for 5min andrinsed in several changes of deionized water The cells werethen incubatedwith Schiff reagent (containing basic fuchsinepotassium metabisulfite and hydrochloric acid) for 15minand washed with tap water for 5min and their nuclei werecounterstained with Mayerrsquos Haematoxylin for 1min All thesamples were observed under a ZEISS Axioskop 40 (CarlZeiss Gottingen Germany) light microscope equipped witha CoolSNAP video camera (Photometrics Tucson AZ USA)for acquiring digital images

24 Characterization Studies Cultured cells were examinedfor CAM 52 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expressionbetween 3th and 16th passages Cells were fixed for 10minwith a 3 paraformaldehyde solution at room temperaturein 1x Dulbeccorsquos PBS pH 76 supplemented with 2 sucroseThen cell membranes were permeabilized for 5min atroom temperature with a pH 76 solution containing 05Triton X-10020mM HEPES 300 120583M sucrose 50mM NaCland 3mM MgCl

2 After membrane permeabilization cells

were incubated with 10 BSA in 1x Dulbeccorsquos PBS for30min at room temperature followed by a 45min incubationat room temperature with FITC-conjugated CAM 52 atthe final concentration of 5 120583gml (in 1 BSAPBS) and

BioMed Research International 3

PE-conjugated CD140 antibody at the final concentrationof 5 120583gml (in 1 BSAPBS) (all purchased from BD SanJose CA USA) All the slides were mounted with Slowfade(Molecular Probes Eugene OR) and observed with a ZEISSAxioskop 40 light microscope equipped with a CoolSNAPvideo camera (Photometrics) for acquiring digital images

25 Immunofluorescent Staining of DNA Strand Breaks(TUNEL) The TUNEL assay detects single or double DNAstrand breaks with the use of labelled nucleotides poly-merized to free 31015840-hydroxyl termini in a reaction catalysedby Terminal deoxynucleotidyl Transferase (TdT) Cells weregrown in a complete medium in 24-well plates and treatedwith 13 Curcuma longa in 0001 BCA up to 24 h Sampleswere fixed with 4 paraformaldehyde firstly washed andthen incubated in a permeabilizing solution (01 Triton X-100 01 sodium citrate) for 2min on ice Deoxyribonucleicacid strand breaks were identified with the use of an ldquoin situcell death detection kitrdquo (BoehringerMannheimMannheimGermany) according to the manufacturerrsquos instructions aselsewhere reported [20]

26 Annexin-VPI Detection of Apoptotic and Necrotic Cells inFlow Cytometry In order to assess apoptosis a commercialAnnexin-V-FITCPI kit (Bender Med System Vienna Aus-tria) was used according to the manufacturerrsquos instructionsas elsewhere reported [21] Analyses were performed usinga Coulter FC500 flow cytometer with the FL1 and the FL3in a log mode with the use of the CXP Software (Beck-man Coulter FL USA) For each sample 10000ndash20000events were collected Viable cells were Annexin-VnegPIneg(unlabelled) early apoptotic cells were Annexin-VposPInegand late apoptotic cells were Annexin-VposPIpos whereasnecrotic cells were Annexin-VnegPIpos

27 Immunofluorescent Staining of VEGF and VEGF-R1 Toevaluate the expression of vascular endothelial growth factor(VEGF) and its receptor (VEGF-R1) adherent cells were fixedpermeabilized and blocked as mentioned above Sampleswere then incubated with anti-human VEGF and VEGF-R1(Becton Dickinson Biosciences San Jose California USA)(working dilution 1 500) followed by FITC-conjugated IgGand TRITC-conjugated IgG (working dilution 1 100) (Jack-son ImmunoResearch West Grove PA USA) respectivelyas secondary antibodies Cell nuclei were counterstainedwithDAPI (Vector Laboratories Inc Burlingame CA) All obser-vations were performed under a ZEISS Axioskop 40 lightmicroscope equipped with a CoolSNAP video camera (Pho-tometrics) to acquire images to analyse withMetaMorph 61software (Universal Imaging Corp Downingtown PA USA)

28 Statistics Statistical analysis was performed using theanalysis of variance (ANOVA) test Results were expressed asmeans plusmn SD Values of 119901 lt 005 were considered statisticallysignificant

3 Results

31 Histochemistry Surgical explants were subjected to histo-chemical analysis to assess morphological features As shown

in Figures 1(a) and 1(b) the pterygium epithelium that cen-tripetally invades the cornea displays squamous metaplasiaand goblet cell hyperplasia As expected the connective tissueunderlying themultilayered epithelium is highly vascularizedand full of inflammatory infiltrates (Figures 1(a) and 1(b))

32 Primary Culture and Subculture of Pterygium-DerivedEpithelial Cells Cell outgrowth from explants was observedwithin 5ndash8 days in serum-supplemented media except fornormal conjunctiva where only few cells were detectable nearor far away from the explant (Figures 2(a) and 2(b)) Phasecontrast microscopy revealed that primary outgrowths ofcells exhibited a polygonal morphology typical of epithelialcells (Figures 2(b) and 2(c)) After 2 weeks in primaryculture cells were trypsinised and seeded onto uncoatedtissue culture dishes in the presence of 10 FBS Subculturedpterygium-derived epithelial cells appeared more irregular(Figure 2(d)) and grew rapidly in less well-organized mono-layers up to 16 passages in culture

33 Characterization Studies To characterize pterygium-derived epithelial cells immune-histochemical detection ofboth keratinocyte (CAM 52) and fibroblast (CD140)markerswas performed The anti-cytokeratin (CAM 52) antibody iscommonly used for qualitative identification of both normaland malignant cells of epithelial origin To ascertain whetherthe primary cell population growing in culture did not con-tain contaminating fibroblasts we performed the immune-histochemical staining of cultured keratinocytes with anti-CD140 antibody As shown in Figure 3 primary pterygium-derived cells express CAM 52 staining at cytoplasm level(98 plusmn 6 positive cells) As expected no CD140 labellingwas found in any of the samples due to the absence ofcontaminating fibroblasts

To investigate the presence of angiogenic pathways inpterygium-derived epithelial cells that could result in neo-vascularization and pterygium development and growthimmunofluorescence staining for VEGF and VEGF-R1 wasperformed Figure 4 shows the presence of VEGF-R1 (greenlabel) in both the nucleus and the cytoplasm (96plusmn7positivecells) whereas VEGF (red label) is present only in the nuclearcompartment (78 plusmn 3 positive cells)

34 Effects of Curcuma longa on Pterygium-Derived Ker-atinocytes To assess the effects induced by Curcuma longaadministration samples were treated with 13 Curcumalonga dissolved in 0001 BCA up to 24 h and compared withsamples treated with BCA alone or left untreated as controls

As shown by phase contrast microscopy (Figure 5) clearchanges in cell morphology occurred as early as after 3 htreatment with Curcuma longa leading a number of cells tolose the cobblestone-like appearance and to detach from theculture dish resembling the typical hallmarks of apoptoticcell death Interestingly detachment started at the peripheryof the culture dish moving towards the explant in a time-dependent manner On the other hand samples treated withBCA alone displayed the presence of granular material andcell debris due to the occurrence of necrotic cell death


lowastlowast

lowast

lowast

lowast

lowast

50 m

(a)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)

Figure 1 Typical histological features of pterygium Two serial tissue sections of pterygiumwere stained (a) withHaematoxylin-Eosin and (b)with periodic acidndashSchiff (PAS) staining solutions Red asterisks show goblet cells grouped as intraepithelial glands while black asterisks showgoblet cells randomly placed within pterygium epithelium Black arrows show a rich vascular network orange asterisks show inflammatoryinfiltrates Original magnification 10x

Normal conjunctiva

50 m

(a)

Normal conjunctiva

(b)Pterygium

50 m

(c)

Pterygium

(d)

Figure 2 Phase contrast images of human keratinocytes derived from normal conjunctiva (a b) and pterygium explants (c d) On the leftimages of the explants of normal conjunctiva (a) and pterygium (c) are shownThe cell overgrowth in the normal conjunctiva (a b) was highlyreduced compared with pterygium where the overgrowth was really extensive In both cases cells displayed a typical polygonal morphologyOriginal magnification 20x

To better evaluate the possible apoptotic effects inducedby the different treatments (Curcuma longa plus BCA or BCAalone) samples were subjected to TUNEL assay Analysisof DNA strand breaks revealed the absence of apoptosisinduction in untreated control cells (1 plusmn 02) converselyBCA treatment showed a slight increase in TUNELpos cells(27 plusmn 2 119901 lt 005 versus Control) whereas Curcuma longa-treated samples showed a significantly increased number ofTUNELpos cells (93 plusmn 7 119901 lt 003 versus Control 119901 lt 005

vs BCA) Of note in Curcuma longa-treated samples thefluorescent labelling was both located at nuclear level andfinely dispersed in the cytoplasm of apoptotic cells (Figure 6)

Finally flow cytometric analysis of Annexin-VPI stain-ing was performed to further confirm the occurrence ofapoptotic and necrotic cell death after Curcuma longa andBCA treatment of pterygium-derived keratinocytes In apop-totic cells the membrane phospholipid phosphatidylserine(PS) is translocated from the inner to the outer leaflet


50 m

CAM 52 CD140 Merge

Figure 3 Immunofluorescence analysis of cultured human keratinocytes derived from pterygium explants labelled with anti-cytokeratinCAM 52 antibody to determine the purity of cultured cells (98 plusmn 6 positive cells) and with CD140 (no positive cells were detected) to verifyfibroblastic contaminationOriginalmagnification 40xTheobservationswere carried out using aZEISSAxioskop lightmicroscope equippedwith a Coolsnap video camera to acquire images analysed with the MetaMorph 61 Software (Universal Imaging Corp Downingtown PA)Images displayed are representative of 3 independent experiments

VEGF-R1 VEGF Merge

125 m

Figure 4 Immunofluorescence analysis of cultured human keratinocytes derived from pterygium explants labelled with anti-VEGF (redlabel at nuclear level 78 plusmn 3) and anti-VEGF-R1 (green label located both in the nucleus and cytoplasm 96 plusmn 7) Original magnification100x The observations were carried out using a ZEISS Axioskop light microscope equipped with a Coolsnap video camera to acquireimages analysed with the MetaMorph 61 Software (Universal Imaging Corp Downingtown PA) Images displayed are representative of3 independent experiments

3 h 6 h 24 h

C

BCA

CL

50 m 50 m 50 m

Figure 5 Effect of BCA andCurcuma longa (CL) on human keratinocytes derived frompterygium explants after 3 6 and 24 h treatment Lossof adhesion and morphological changes occur in a time-dependent manner in treated samples Original magnification 20x Observationswere carried out with a phase contrast light microscope (LEICA) equipped with a CoolSNAP video camera to acquire computerized images(Photometrics)The browndark regions are pterygium explants in culture Images displayed are representative of 5 independent experiments


C

BCA

CL

50 m

Figure 6 Immune fluorescence labelling of human keratinocytes derived from pterygium explants after treatment with BCA and Curcumalonga (CL) for 24 h A significantly increased positivity for the terminal deoxynucleotidyl transferase- (TdT-) mediated nick end labelling(TUNEL) was found in samples treated with BCA (27 plusmn 2) In Curcuma longa-treated samples a different pattern of apoptosis was revealeddue to the presence of DNA also in the cytoplasm (93 plusmn 7) Red arrow heads indicate some apoptotic cells Nuclei were counterstained with6-diamino-2-phenylindole (DAPI) (blue fluorescence) Green and blue fluorescence single emissions are overlapped in the merge panelsOriginal magnification 40x Images displayed are representative of 3 independent experiments

of the plasma membrane Annexin-V is a Ca2+-dependentphospholipid binding protein with high affinity for PS that isexposed on the cell surface of apoptotic cells PS membranetranslocation precedes the loss of membrane integrity whichaccompanies the later stages of cell death resulting fromeitherapoptotic or necrotic processes Annexin-V is typically usedin combination with PI which can enter the nucleus due tocell membrane permeability changes occurring in the laterstage of apoptosis or necrosis This analysis revealed that thepercentage of viable cells decreased in the presence of BCA aswell as of Curcuma longa As shown in Figures 7(a) and 7(b)the 24 h treatment with Curcuma longa lead to an increasein early (Annexin-VposPIneg) and late (Annexin-VposPIpos)apoptotic cells compared to untreated controls (for both119901 lt 005) and BCA-treated samples (for both 119901 lt 005)whereas the number of necrotic cells (Annexin-VnegPIposcells) was significantly higher in samples treated with BCAalone compared to controls (119901 lt 003) and to Curcumalonga (119901 lt 005) In fact after Curcuma longa treatmentthe percentage of Annexin-VposIpos cells was 143 fold higherthan control (119901 lt 005) whereas after BCA treatment thepercentage of Annexin-VposPIpos cells was 45-fold higherthan untreated controls (119901 lt 003) and more importantlythe percentage of Annexin-VnegPIpos cells was even 184-foldhigher compared to untreated controls (119901 lt 003)

4 Discussion

Pterygium is a common ocular surface disease which isbelieved to be closely associated with ultraviolet exposure[22] Until recently surgery was considered as the onlyfeasible option of pterygium treatment However apart frombeing invasive and associated with a variety of potential seri-ous complications surgery often results in a more aggressiveclinical behaviour of pterygium [9]Herewe propose an alter-native strategy for the removal of human pterygium based onthe dramatic apoptogenic effect of Curcuma longa on humanpterygium-derived keratinocytes In fact as early as after3 h of in vitro treatment pterygium-derived keratinocytesincubated even with a low concentration (13) of the totalextract of Curcuma longa are able to undergo apoptotic celldeath as shown with light microscopy TUNEL assay andAnnexin-VPI staining in flow cytometry Instead BCA usedas a single treatment induces keratinocytes to necrotic celldeath that unlike apoptosis is a type of inflammatory celldeath Cells undergoing apoptosis were recognizable in lightmicroscopy for their typical shrinkage leading them to detachvery early from the plastic surface of the culture dish Inaddition typical hallmarks of apoptotic cell death includingDNA strand breaks and phosphatidylserine exposition at thecell membrane were detected with the TUNEL techniqueand with Annexin-VPI staining respectively Interestingly


100

101

102

103

PI L

og

100 101 102 103

Annexin-V

C

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

BCA

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

CL

(a)

CBCACL

25

20

15

10

5

0

Fold

incr

ease

lowastsect

lowastsect

∘A

nnex

in-V

pos P

ineg

Ann

exin

-Vpo

s PIpo

s

Ann

exin

-Vne

g PIpo

s

(b)

Figure 7 Annexin-VPI evaluation in human keratinocytes derived from pterygium explants (a) Flow cytometric dot plots of onerepresentative experiment Apoptotic cells can be discriminated from viable late apoptotic or necrotic cells according to their fluorescenceemission B3 viable cells B4 early apoptotic cells B2 late apoptotic cells B1 necrotic cells (b) The histogram shows the fold increase ofAnnexin-VposPIneg Annexin-VposPIpos and Annexin-VnegPIpos cells plusmn SD of three independent experiments (119901 lt 00214 BCA versus Clowast119901 lt 00497 CL versus C sect119901 lt 00459 CL versus BCA ∘119901 lt 00288 BCA versus CL)

the use of Curcuma longa in combination with BCA wasable to attenuate necrotic effects of BCA used as a singletreatment We cannot assign this apoptogenic effect to spe-cific components of Curcuma longa since it is known thatthis compound contains a number of curcuminoids includ-ing mainly curcumin Curcumin is considered the activeprinciple of Curcuma longa due to its well-recognized anti-inflammatory effects [17] In fact studies carried out by otherlaboratories have identified a number of different moleculesinvolved in inflammation that are inhibited by curcuminincluding phospholipase lipoxygenase cyclooxygenase 2leukotrienes thromboxane prostaglandins nitric oxide col-lagenase elastase hyaluronidase monocyte chemoattrac-tant protein-1 (MCP-1) interferon-inducible protein tumournecrosis factor (TNF) and interleukin-12 (IL-12) [23] Morerecently it has become evident that curcumin also modulatessurvival-associated signalling pathways causing apoptosis [1719] and exerting potent anticancer effects [24] Moreover

curcumin has been demonstrated in six human trials to behighly safe when orally administered [22] and to significantlyinhibit the proliferation of human pterygium fibroblastsarresting them in G

0G1phase and inducing apoptosis in a

dose- and time-dependent manner [25] Our results togetherwith the findings of Zhang et al [25] pave the way to apossible medical treatment of pterygium and are particularlyinteresting in light of previous studies showing that ratesof proliferation in pterygium samples are similar to thosefound in the normal conjunctival surface suggesting thatpterygium may represent a failure of appropriate apoptosis[26] Furthermore our study confirms previous observationshighlighting the expression in pterygium keratinocytes ofnuclear VEGF [27ndash29] and gives evidence for the first time tothe expression of nuclear and cytoplasmic VEGF-R1 Theseobservations although still to be further characterized areparticularly intriguing since they suggest a possible role ofepithelial cells in new blood vessels formation that is one


of pathogenetic mechanisms involved in pterygium growthand recurrence [26] In fact as a benign type of uncontrolledcell proliferation pterygium requires cell migration andlocal angiogenesis that is triggered and controlled by severalangiogenic and inhibitory factors [8] Among these factorsVEGF is a potent angiogenic cytokine that has a pivotal rolein normal and pathological angiogenesis [30] by interactingwith three different receptors VEGF-R1 VEGF-R2 andVEGF-R3 [1] VEGF-R2 appears to mediate almost all ofthe known cellular responses to VEGF whereas the functionof VEGF-R1 is less well defined although it is thought tomodulate VEGF-R2 signalling acting as a dummydecoyreceptor and to promote angiogenesis mainly by increasingbone marrow-derived macrophage recruitment [31] In thisrespect it is worth noting that curcumin appears to be ableto inhibit tumour growth and vasculogenesis in vivo throughinterrupting VEGFVEGF-R2 signalling pathway [32]

All in all previous and present findings suggest thatCurcuma longa or its components could have a promisingtherapeutic potential in the pterygium treatment by interact-ing both with apoptotic disorder and potentially with bloodvessels formation Further studies are needed to better clarifythe pharmacokinetics and tolerability in the human eye of thisnutraceutical compound

Conflicts of Interest

None of the authors have any conflicts of interest in themanuscript

Authorsrsquo Contributions

Silvia Sancilio and Silvio Di Staso contributed equally to thiswork and should be considered co-first authors

Acknowledgments

This work was performed with funds of the Italian Ministryof University and Research granted to Professor Roberta DiPietro and Professor Marco Ciancaglini in 2009 2010 and2017

References

[1] N Di Girolamo J Chui M T Coroneo and D WakefieldldquoPathogenesis of pterygia role of cytokines growth factorsand matrix metalloproteinasesrdquo Progress in Retinal and EyeResearch vol 23 no 2 pp 195ndash228 2004

[2] R AThoft and J Friend ldquoBiochemical transformation of regen-erating ocular surface epitheliumrdquo Investigative Ophthalmologyand Visual Science vol 16 no 1 pp 14ndash20 1977

[3] N Di Girolamo N Tedla R K Kumar et al ldquoCulture andcharacterisation of epithelial cells from human pterygiardquoBritishJournal of Ophthalmology vol 83 no 9 pp 1077ndash1082 1999

[4] W Li Y Hayashida Y-T Chen and S C G Tseng ldquoNicheregulation of corneal epithelial stem cells at the limbusrdquo CellResearch vol 17 no 1 pp 26ndash36 2007

[5] P Das A Gokani K Bagchi G Bhaduri S Chaudhuri andS Law ldquoLimbal epithelial stem-microenvironmental alteration

leads to pterygium developmentrdquo Molecular and Cellular Bio-chemistry vol 402 no 1-2 pp 123ndash139 2015

[6] J C Bradley W Yang R H Bradley T W Reid and I RSchwab ldquoThe science of pterygiardquo British Journal of Ophthal-mology vol 94 no 7 pp 815ndash820 2010

[7] S Duke-Elder ldquoTextbook of ophthalmologyrdquoMosby vol 7 pp570ndash586 1954

[8] T Liu Y Liu L Xie X He and J Bai ldquoProgress in the patho-genesis of pterygiumrdquo Current Eye Research vol 38 no 12 pp1191ndash1197 2013

[9] E T Detorakis and D A Spandidos ldquoPathogenetic mecha-nisms and treatment options for ophthalmic pterygium trendsand perspectives (review)rdquo International Journal of MolecularMedicine vol 23 no 4 pp 439ndash447 2009

[10] J H C Hilgers ldquoPterygium Its incidence heredity and etiol-ogyrdquoAmerican Journal of Ophthalmology vol 50 no 4 pp 635ndash644 1960

[11] J ChuiM T Coroneo L T Tat R CrouchDWakefield andNDi Girolamo ldquoOphthalmic pterygium a stem cell disorder withpremalignant featuresrdquoThe American Journal of Pathology vol178 no 2 pp 817ndash827 2011

[12] J J Garcia-Medina M Del-Rio-Vellosillo V Zanon-MorenoA Ortiz-Gomariz M Morcillo-Guardiola and M D Pinazo-Duran ldquoSevere scleral dellen as an early complication of ptery-gium excision with simple conjunctival closure and review ofthe literaturerdquo Arquivos Brasileiros de Oftalmologia vol 77 no3 pp 182ndash184 2014

[13] H B Kal R E Veen and I M Jurgenliemk-Schulz ldquoDose-effect relationships for recurrence of keloid and pterygium aftersurgery and radiotherapyrdquo International Journal of RadiationOncology Biology Physics vol 74 no 1 pp 245ndash251 2009

[14] R R Doshi G J Harocopos I R Schwab and E T Cunning-ham ldquoThe spectrum of postoperative scleral necrosisrdquo Survey ofOphthalmology vol 58 no 6 pp 620ndash633 2013

[15] A R Vaughn A Branum and R K Sivamani ldquoEffects ofturmeric (curcuma longa) on skin health a systematic reviewof the clinical evidencerdquo Phytotherapy Research pp 1243ndash12642016

[16] G Calapai M Miroddi P L Minciullo A P Caputi SGangemi and R J Schmidt ldquoContact dermatitis as an adversereaction to some topically used European herbal medicinalproducts - Part 1 Achillea millefolium-Curcuma longardquo Con-tact Dermatitis vol 71 no 1 pp 1ndash12 2014

[17] S Ghosh S Banerjee and P C Sil ldquoThe beneficial role ofcurcumin on inflammation diabetes and neurodegenerativedisease a recent updaterdquo Food and Chemical Toxicology vol 83pp 111ndash124 2015

[18] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[19] M Mazidi E Karimi M Meydani M Ghayour-Mobarhanand G A Ferns ldquo Potential effects of curcumin on peroxisomeproliferator-activated receptor-120574 rdquo World Journal of Methodol-ogy vol 6 no 1 p 112 2016

[20] G Mincione M C Di Marcantonio C Tarantelli et al ldquoIden-tification of the zinc finger 216 (ZNF216) in human carcinomacells A potential regulator of EGFR activityrdquoOncotarget vol 7no 46 pp 74947ndash74965 2016

[21] C Di Nisio S Sancilio V Di Giacomo et al ldquoInvolvement ofcyclic-nucleotide response element-binding family members inthe radiation response of Ramos B lymphoma cellsrdquo Interna-tional Journal of Oncology vol 48 no 1 pp 28ndash36 2016


[22] Y H Cui H Y Li Z X Gao et al ldquoRegulation of apoptosis bymiR-122 in pterygium via Targeting Bcl-wrdquo Investigative Oph-thalmology and Visual Science vol 57 no 8 pp 3723ndash37302016

[23] N Chainani-Wu ldquoSafety and anti-inflammatory activity ofcurcumin a component of tumeric (Curcuma longa)rdquo TheJournal of Alternative and Complementary Medicine vol 9 no1 pp 161ndash168 2003

[24] A Bernd ldquoVisible light andor UVA offer a strong amplificationof the anti-tumor effect of curcuminrdquo Phytochemistry Reviewsvol 13 no 1 pp 183ndash189 2014

[25] M Zhang F Bian C Wen and N Hao ldquoInhibitory effect ofcurcumin on proliferation of human pterygium fibroblastsrdquoJournal of Huazhong University of Science and Technology(Medical Sciences) vol 27 no 3 pp 339ndash342 2007

[26] D T H Tan W Y Tang Y P Liu H-S Goh and D R SmithldquoApoptosis and apoptosis related gene expression in normalconjunctiva and pterygiumrdquo British Journal of Ophthalmologyvol 84 no 2 pp 212ndash216 2000

[27] D-H Lee H J Cho J-T Kim J S Choi and C-K JooldquoExpression of vascular endothelial growth factor and induciblenitric oxide synthase in pterygiardquoCornea vol 20 no 7 pp 738ndash742 2001

[28] A L Marcovich Y Morad J Sandbank et al ldquoAngiogenesisin pterygiummorphometric and immunohistochemical studyrdquoCurrent Eye Research vol 25 no 1 pp 17ndash22 2002

[29] G van SettenM Aspiotis T D Blalock G Grotendorst andGSchultz ldquoConnective tissue growth factor in pterygium simul-taneous presence with vascular endothelial growth factor -Possible contributing factor to conjunctival scarringrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 241no 2 pp 135ndash139 2003

[30] Y Rosenbaum-Dekel A Fuchs E Yakirevich et al ldquoNuclearlocalization of long-VEGF is associated with hypoxia and tumorangiogenesisrdquo Biochemical and Biophysical Research Communi-cations vol 332 no 1 pp 271ndash278 2005

[31] Z Fu X Chen S Guan Y Yan H Lin and Z-CHua ldquoCurcumin inhibits angiogenesis and improves defectivehematopoiesis induced by tumor-derived VEGF in tumormodel throughmodulatingVEGF-VEGFR2 signaling pathwayrdquoOncotarget vol 6 no 23 pp 19469ndash19482 2015

[32] MMurakami Y ZhengMHirashima et al ldquoVEGFR1 tyrosinekinase signaling promotes lymphangiogenesis as well as angio-genesis indirectly viamacrophage recruitmentrdquoArteriosclerosisThrombosis and Vascular Biology vol 28 no 4 pp 658ndash6642008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


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Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


reported Outside this area prevalence rates usually do notovercome 2of the general population and the disease affectsmainly patients with an increased exposure to sunlight orinvolved in outdoor activities [9] Although previous studieshave highlighted the involvement of genetic factors in thepathogenesis of pterygium [10] the etiology of pterygiumstill remains unclear [8] Current management strategies forpterygium imply surgical excision [11] that is a complex andinvasive procedure that often results in the recurrence of alesion more clinically aggressive than the original one [9] Inaddition surgery can lead to further clinical manifestationssuch as symblepharon corneal clouding corneal or scleraldellen [9 12] keloid formation [13] and scleral necrosis [14]

Curcuma longa is a plant belonging to the family ofZingiberaceae The rhizomes of this plant are the sourceof turmeric which has been used for centuries all overthe world in cooking cosmetics and medical treatments[15] The curcuminoids present in the rhizome which areresponsible for the yellow colour of turmeric consist ofa mixture of curcumin (also known as diferuloylmethaneNatural Yellow and E100) demethoxycurcumin and bis-demethoxycurcumin [16] Curcumin makes up sim90 of thecurcuminoid content Due to its chemical structure cur-cumin ismuch less soluble inwater at acid and neutral pHbutsoluble in methanol ethanol dimethyl sulfoxide (DMSO)and acetone [17] Its traditional uses as a strong therapeutic orpreventive agent in several human diseases such as diabetesinflammation atherosclerosis and cancer [18] is due to itsbeneficial properties including anti-inflammatory antioxi-dant antineoplastic pro- and antiapoptotic antiangiogeniccytotoxic immune-modulatory and antimicrobial effects viathemodulation of various targets (grow factors enzymes andgenes) [19]

Based on available literature and with the aim to identifyan alternative strategy to currently available surgical proce-dures in this study we investigated the effects of in vitrotreatment with Curcuma longa of keratinocytes derived fromexplants of human pterygium

2 Materials and Methods

21 Experimental Design Human pterygium specimens wereobtained from 20 patients with primary pterygium (averageage 682 plusmn 97 years) undergoing routine pterygiectomy Allthe patients affected with pterygia displayed inflammatorysigns of the ocular surface such as chemosis and rednessof the conjunctiva Normal conjunctival tissue specimenswere obtained from 3 patients (average age 712 plusmn 83 years)undergoing cataract surgery All patients were treated at theSS Annunziata Hospital in Chieti Italy Signed informedconsent was obtained from the donors according to Italianlegislation and to the code of Ethical Principles for MedicalResearch involving Human Subjects of the World MedicalAssociation (Declaration ofHelsinki) After surgical excisionthe clinical specimens were properly treated or preserved forimmunohistochemistry or primary cell culture

22 Primary Culture and Subculture of Pterygium-DerivedKeratinocytes Pterygium specimens were placed in a Petri

dish and washed with 1x Dulbeccorsquos Phosphate-BufferedSaline (PBS) Samples were cut into several 1-2mm2 piecesunder sterile conditions and placed into six-well plates at37∘C in an atmosphere of 5 carbon dioxide in air Explantswere allowed to attach to the plastic dish for at least 8 h in adrop of complete medium selective for human keratinocytessurvival and consisting of Eaglersquos MEM (GIBCO by Invit-rogen Carlsbad CA USA) supplemented with 10 foetalbovine serum (FBS Hyclone Logan UT USA) 100Umlpenicillin 100 120583gml streptomycin and 25 120583gml fungizone(Sigma-Aldrich St Louis MO USA) Cell migration fromexplants was observed within 3ndash5 days Cells were used forexperiments between the 4th and 7th passage and only forcharacterization up to the 16th passage Pterygium-derivedkeratinocytes were grown on 6-well culture plates and whencells reached confluence culture medium was replaced andwhere required treated with an alcoholic extract of 13Curcuma longa in 0001 Benzalkonium Chloride (BCA)(Sigma-Aldrich) awell knownpreservative commonly addedto several eye drops Control cultures were left untreatedor treated with the vehicle alone (BCA) for the same timeintervals After 3 6 and 24 h observations were carried outunder a phase contrast light microscope (LEICA WetzlarGermany) equipped with a CoolSNAP video camera foracquiring computerized images (Photometrics Tucson AZ)

23 Histochemical Analysis Explants of human pterygiumwere fixed with 10 (volvol) phosphate-buffered formalindehydrated in a series of graded increases in alcohol con-centrations embedded in paraffin and cut at the microtome(Leica RM 2265 Germany) Sections were routinely stainedwith Haematoxylin-Eosin staining solution and mounted inBioMount (Bio-Optica Italy) For periodic acidndashSchiff (PAS)staining samples were fixed with 4 paraformaldehydewashed with PBS oxidized in 1 periodic acid for 5min andrinsed in several changes of deionized water The cells werethen incubatedwith Schiff reagent (containing basic fuchsinepotassium metabisulfite and hydrochloric acid) for 15minand washed with tap water for 5min and their nuclei werecounterstained with Mayerrsquos Haematoxylin for 1min All thesamples were observed under a ZEISS Axioskop 40 (CarlZeiss Gottingen Germany) light microscope equipped witha CoolSNAP video camera (Photometrics Tucson AZ USA)for acquiring digital images

24 Characterization Studies Cultured cells were examinedfor CAM 52 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expressionbetween 3th and 16th passages Cells were fixed for 10minwith a 3 paraformaldehyde solution at room temperaturein 1x Dulbeccorsquos PBS pH 76 supplemented with 2 sucroseThen cell membranes were permeabilized for 5min atroom temperature with a pH 76 solution containing 05Triton X-10020mM HEPES 300 120583M sucrose 50mM NaCland 3mM MgCl

2 After membrane permeabilization cells

were incubated with 10 BSA in 1x Dulbeccorsquos PBS for30min at room temperature followed by a 45min incubationat room temperature with FITC-conjugated CAM 52 atthe final concentration of 5 120583gml (in 1 BSAPBS) and







3 Results









lowastlowast

lowast

lowast

lowast

lowast

50 m

(a)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)


Normal conjunctiva

50 m

(a)

Normal conjunctiva

(b)Pterygium

50 m

(c)

Pterygium

(d)






50 m

CAM 52 CD140 Merge


VEGF-R1 VEGF Merge

125 m


3 h 6 h 24 h

C

BCA

CL

50 m 50 m 50 m



C

BCA

CL

50 m



4 Discussion



100

101

102

103

PI L

og

100 101 102 103

Annexin-V

C

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

BCA

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

CL

(a)

CBCACL

25

20

15

10

5

0

Fold

incr

ease

lowastsect

lowastsect

∘A

nnex

in-V

pos P

ineg

Ann

exin

-Vpo

s PIpo

s

Ann

exin

-Vne

g PIpo

s

(b)













Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results









lowastlowast

lowast

lowast

lowast

lowast

50 m

(a)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)


Normal conjunctiva

50 m

(a)

Normal conjunctiva

(b)Pterygium

50 m

(c)

Pterygium

(d)






50 m

CAM 52 CD140 Merge


VEGF-R1 VEGF Merge

125 m


3 h 6 h 24 h

C

BCA

CL

50 m 50 m 50 m



C

BCA

CL

50 m



4 Discussion



100

101

102

103

PI L

og

100 101 102 103

Annexin-V

C

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

BCA

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

CL

(a)

CBCACL

25

20

15

10

5

0

Fold

incr

ease

lowastsect

lowastsect

∘A

nnex

in-V

pos P

ineg

Ann

exin

-Vpo

s PIpo

s

Ann

exin

-Vne

g PIpo

s

(b)













Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast

lowast

lowast

lowast

lowast

50 m

(a)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)


Normal conjunctiva

50 m

(a)

Normal conjunctiva

(b)Pterygium

50 m

(c)

Pterygium

(d)






50 m

CAM 52 CD140 Merge


VEGF-R1 VEGF Merge

125 m


3 h 6 h 24 h

C

BCA

CL

50 m 50 m 50 m



C

BCA

CL

50 m



4 Discussion



100

101

102

103

PI L

og

100 101 102 103

Annexin-V

C

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

BCA

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

CL

(a)

CBCACL

25

20

15

10

5

0

Fold

incr

ease

lowastsect

lowastsect

∘A

nnex

in-V

pos P

ineg

Ann

exin

-Vpo

s PIpo

s

Ann

exin

-Vne

g PIpo

s

(b)













Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















50 m

CAM 52 CD140 Merge


VEGF-R1 VEGF Merge

125 m


3 h 6 h 24 h

C

BCA

CL

50 m 50 m 50 m



C

BCA

CL

50 m



4 Discussion



100

101

102

103

PI L

og

100 101 102 103

Annexin-V

C

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

BCA

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

CL

(a)

CBCACL

25

20

15

10

5

0

Fold

incr

ease

lowastsect

lowastsect

∘A

nnex

in-V

pos P

ineg

Ann

exin

-Vpo

s PIpo

s

Ann

exin

-Vne

g PIpo

s

(b)













Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C

BCA

CL

50 m



4 Discussion



100

101

102

103

PI L

og

100 101 102 103

Annexin-V

C

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

BCA

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

CL

(a)

CBCACL

25

20

15

10

5

0

Fold

incr

ease

lowastsect

lowastsect

∘A

nnex

in-V

pos P

ineg

Ann

exin

-Vpo

s PIpo

s

Ann

exin

-Vne

g PIpo

s

(b)













Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

101

102

103

PI L

og

100 101 102 103

Annexin-V

C

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

BCA

100

101

102

103

PI L

og

100 101 102 103

Annexin-V

CL

(a)

CBCACL

25

20

15

10

5

0

Fold

incr

ease

lowastsect

lowastsect

∘A

nnex

in-V

pos P

ineg

Ann

exin

-Vpo

s PIpo

s

Ann

exin

-Vne

g PIpo

s

(b)













Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















ResearchArticle Curcuma longa Is Able to Induce Apoptotic...

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Transcript of ResearchArticle Curcuma longa Is Able to Induce Apoptotic...