Research ArticleImmunohistochemical Expression ofSurvivin and Its Relationship with Cell Apoptosisand Proliferation in Ameloblastomas

Rogelio Gonzaacutelez-Gonzaacutelez12 Nelly Molina-Frechero3 Pablo Damian-Matsumura4

Sirced Salazar-Rodriguez56 and Ronell Bologna-Molina17

1Research Department School of Dentistry Universidad Juarez del Estado de Durango (UJED) 34000 Durango Mexico2Doctorado en Ciencias Biologicas y de la Salud Universidad Autonoma Metropolitana 04960 Mexico City Mexico3Health Care Department Universidad Autonoma Metropolitana Xochimilco 04960 Mexico City Mexico4Department of Biology of Reproduction Universidad Autonoma Metropolitana Iztapalapa 09340 Mexico City Mexico5Society for Fight Against Cancer Portoviejo 130105 Manabi Ecuador6National Institute of Oncology and Radiobiology 10400 La Habana Cuba7School of Dentistry Universidad de la Republica (UDELAR) 19200 Montevideo Uruguay

Correspondence should be addressed to Ronell Bologna-Molina ronellbolognahotmailcom

Received 25 November 2014 Revised 25 February 2015 Accepted 9 March 2015

Academic Editor Lance A Liotta

Copyright copy 2015 Rogelio Gonzalez-Gonzalez et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Ameloblastoma behavior is related to the potential of tumor cells to inhibit apoptosis and to initiate a proliferative phase Thisstudy was performed to compare the immunoexpression of Survivin with Bcl-2 Bax and Ki-67 and to associate them with thehistopathological type of each variant of ameloblastoma Material and Methods Using the World Health Organization (WHO)criteria for ameloblastoma 110 cases were selected The cases were classified as solidmulticystic and unicystic ameloblastomasCellular counts of cytoplasmic immunoexpression were assessed for cytoplasmic Survivin Bcl-2 and Bax while the nuclearimmunoexpression of Survivin and Ki-67 was assessed using label index Results Cytoplasmic Survivin and Bcl-2 showed higherpercentages of immunoexpression in solid multicystic ameloblastomas compared to unicystic ameloblastomas (119875 lt 005) Bax Ki-67 and nuclear Survivin were expressed in higher percentages in unicystic ameloblastomasConclusions Cytoplasmic Survivin andBcl-2 immunoexpression levels were elevated in relation to Bax immunoexpression suggesting aggressive ameloblastoma behaviorwhile Ki-67 and nuclear Survivin immunoexpressionmay be associated with the type of tumormorphology that influences cellularcounts or with the greater capacity for cellular proliferation and tumor growth

1 Introduction

Ameloblastoma (AM) is considered one of the most frequentodontogenic tumors of epithelial origin and is a benignneoplasm with an aggressive behavior [1 2]

In 2005 the World Health Organization (WHO) clas-sified AMs as solidmulticystic (SMA) unicystic (UA)peripheral or desmoplastic according to their clinical andhistopathological characteristics [3] SMA is an aggressiveslow growing and locally invasive neoplasm characterized bytwo main histopathological growth patterns the follicular

and the plexiform and by cellular variations that do notimpact the clinical behavior [3] UA is classified accordingto cellular growth as intraluminal UA (IUA) and luminalUA (LUA) where the cystic wall remains uninvaded andthe clinical behavior unaffected or as mural UA (MUA)where the cells invade the cystic wall and depending on thedepth of invasion affect the clinical behavior [3] Apoptosisis a physiological process that maintains tissues duringhomeostasis [4] During tumorigenesis tumor cells mayinvade the proapoptotic processes by overregulating theantiapoptotic proteins [4] Apoptosis inhibition is regulated

Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 301781 7 pageshttpdxdoiorg1011552015301781

2 Disease Markers

by a group of molecules called the inhibitor of apoptosisproteins (IAPs) a group that includes Survivin [5ndash7] Sur-vivin is expressed during embryo-fetal development [8] israrely expressed in adult tissue and may be overregulatedin proliferative processes [6ndash9] Survivin is present in twosubcellular compartments (the cytoplasm and the nucleus)plays antiapoptotic and promitotic roles and takes part inBax inhibition Furthermore this protein not only inhibitsapoptosis but also promotes cell proliferation in malignanttumors [6 10 11]

The Bcl-2 protein family includes Bcl-2 and Bax whichplay important roles in the regulation of the apoptosisprocess Bcl-2 is a protooncogene that codes for an internalmitochondrial protein Bcl-2 an inhibitor of apoptosis [7 8]Bax is a proapoptotic protein that induces mitochondrialouter membrane permeabilization through translocation orinsertion causing membrane oligomerization pore forma-tion and the release of mitochondrial proteins from theintermembrane space (ie cytochrome C SmacDiablo andOmiHtra2) [5 12 13]

The Ki-67 nuclear antigen is a widely used cell prolif-eration marker [14 15] and a nuclear protein detected inall cell division phases except for the G0 phase [16 17] Ki-67 is frequently used to determine the growth fraction indifferent tumors [16ndash18] which is why a low growth fractionis associated with a favorable prognosis in several neoplasms

The purpose of this study was to identify and to correlatecytoplasmic and nuclear Survivin immunoexpression withBcl-2 Bax and Ki-67 immunoexpression in the SMA and UAvariants

2 Materials and Methods

21 Sample Tissues The study group consisted of 110 casesof ameloblastoma from the Oral and Maxillofacial PathologyService of public and private hospitals in Mexico the OralPathology Service of the School of Dentistry of JuarezUniversity of the State of Durango the University of theRepublic of Uruguay the Pathology Services of the NationalInstitute of Oncology and Radiobiology and Calixto GarciaHospital in Havana Cuba All cases were reevaluated bytwo pathologists with experience in odontogenic tumors andfollowing the 2005 WHO classification [3] When two ormore histological subtypes of AM were present the mostaggressive or predominant subtype was considered Sectionspresenting severe inflammation in the connective tissue wereexcluded

The study was evaluated and accepted by the EthicsCommittee of the Faculty of Dentistry of Juarez Universityof the State Durango (Folio CE-FO-UJED-01-13)

22 Immunohistochemistry Sections (3 120583m thick) were cutand placed in poly-L-lysine-coated slides The sections weredeparaffinized in a 60∘C oven for 30 minutes and placedin xylol for 5 minutes The sections were rehydrated indecreasing alcohol concentrations (absolute 90 70 and 50)and washed in distilled water To unmask the epitopes theantigen recovery was performed with 10mM sodium citrate

solution with either high or low pH depending on thecharacteristics of each antibodyThis recoverywas performedin a microwave pressure cooker with a maximum powerof 750W in two cycles of 5 minutes The samples werethen cooled to room temperature and washed with distilledwater The endogenous peroxidases were blocked with 09hydrogen peroxide and the samples were again washedwith distilled water and phosphate-buffered saline solution(PBS pH 74) The primary antibodies were incubated for30 minutes for Survivin (Clone 12C4 1 100 Dako CorpCarpinteria CA USA) Bcl-2 (Clone 124 1 50 Dako CorpCarpinteria CA USA) Bax (Polyclonal 1 150 Dako CorpCarpinteria CA USA) and Ki-67 (Clone MIB-1 1 50 DakoCorp Carpinteria CAUSA) Subsequently the sectionswereincubated with the biotinylated anti-mouseanti-rabbit sec-ondary antibody and streptavidinperoxidase complex (LSA-B + labeled streptavidin-biotin Dako Corp CarpinteriaCA USA) for 30 minutes each The reaction products weredisplayed with 331015840-diaminobenzidine-H

2O2substrate (Dako

Corp Carpinteria CA USA) Fragments of tonsillar tissuewere used as positive controls and incubation with theprimary antibodies was omitted for the negative controls

Survivin Bcl-2 and Bax proteins express immunohisto-chemically on the cytoplasm and the cytoplasmicmembranetherefore the percentages of positive tumor cells were evalu-ated on all slides The percentage of positive Survivin Bcl-2andBax immunoexpressionwas calculated using a 10x opticalmicroscope objective corresponding to an area of 53mm2The photomicrographs were taken with a digital camera(Olympus C-7070) from five random areas with abundantneoplastic ameloblastoma cells The results were gatheredaccording to the percentage of positive cells in each case

The Ki-67 assessment was performed in selected neoplas-tic cell-rich areas on the SMA cases and throughout the cysticlines and mural follicles of the UA variants The nuclear Sur-vivin immunoexpression was evaluated in neoplastic SMAand UA cells where nuclear and nuclearcytoplasmic immu-noexpression was observed In both cases (Ki-67 and nuclearand nuclearcytoplasmic Survivin immunoexpression) thecell counts were performed as follows microphotographywith a 40x objective (five fields per case)was taken the imageswere transferred to an image processor to aid in the counting(ImageJ 147d Rasband W ImageJ National Institutes ofHealth USA httpimagejnihgovij) and rack countingwas performed using ImageJ and then manually using thecell counter method to obtain the cellular proliferation index(number of positive tumor cellstotal number of tumor cellsexpressed as a percentage) This technique was used in allcases

23 Statistical Analysis Statistical analysis was performedwith Pearsonrsquos 1205942 method using the expected values TheKruskal-Wallis test was applied to detect the differencesbetween ameloblastomas and Survivin Bcl-2 Bax and Ki-67immunoexpression Results with 119875 le 005 were consideredsignificant The results were tabulated and the data analyzedwith SPSS 200 statistical software (SPSS Professional Statis-tics SPSS Inc Chicago IL USA)

Disease Markers 3

(a) (b)

(c) (d)

(e) (f)

Figure 1 Differences in the cytoplasmic immunoexpression of Survivin Bcl-2 and Bax in ameloblastomas Differences in the cytoplasmicimmunoexpression of Survivin in (a) solidmulticystic follicular ameloblastoma (100x) and (b) luminal unicystic ameloblastoma (400x)Differences in Bcl-2 immunoexpression in (c) solidmulticystic follicular ameloblastoma (400x) and (d) luminal unicystic ameloblastoma(400x) Differences in Bax immunoexpression in (e) solidmulticystic plexiform ameloblastoma (100x) and (f) luminal unicysticameloblastoma (100x)

3 Results

Akappa value of 096 between the two calibrated pathologistswas first obtained by histologically classifying the AMs asSMA and UA following the WHO criteria subsequentlythe cases without agreement were reevaluated until totalconformity was obtained Of a total of 113 cases three wereexcluded as the evaluated tissue did not correspond withany variant of ameloblastoma The remaining total studysample consisted of 110 ameloblastomas of which 38 wereSMA six were diagnosed as follicular cases (FSA) ten asacanthomatous ameloblastomas (ASA) and 22 as plexiformameloblastomas (PSA) Of the UA (119899 = 72) eight cases wereluminal unicystic (LUA) 24 were mural unicystic (MUA)and 40 were intraluminal unicystic ameloblastoma (IUA)

The positivity index for cytoplasmic Survivin was higherin the SMA than the UA samples (119875 lt 005) Similar tocytoplasmic Survivin the Bcl-2 positivity index was higher inthe SMA samples (119875 lt 005 versus UA) while the UA casesshowed a higher Bax positivity index (119875 lt 005 versusMUA)(Figure 1)

Table 1 shows the positivity index of the cellular apoptosisregulatory proteins among the AM histological subtypesThe UA cases had a higher cellular proliferation index

for Ki-67 and greater nuclear Survivin immunoexpressionthan the SMA cases (119875 lt 005) (Figure 2) Table 2 showsthe cellular proliferation index of Ki-67 and the nuclearimmunoexpression of Survivin among the histological AMsubtypes

4 Discussion

Interestingly UA was the predominant type of AM in ourseries which is consistent with previous studies in LatinAmerica [19 20] while SMA are the most frequent AM typesin North America and Asia [21] Therefore it is possible thatthis variant is predominant in someLatinAmerican countriesdue to geographical ethnic and social differences howeverthese results might be associated with a lack of uniformdiagnostic criteria or with the inclusion of incision biopsiesand surgical resections that could explain the variation in thefrequency of the AM subtypes

Several studies have identifiedmolecular alterations asso-ciated with AM development and progression [22] includingthose associated with proteins involved in the apoptotic andcellular proliferation pathways [23 24]

Survivin is a protein from the IAP family that regulatescellular death suppresses cellular apoptosis and regulates

4 Disease Markers

Table 1 Average expression percentages of apoptosis regulatory proteins according to the ameloblastic histological types and subtypes

Histological type Number of cases Mean plusmn SDCytoplasmic Survivin Bcl-2 Bax

Solidmulticystic 38 45 plusmn 316 46 plusmn 30a 402 plusmn 258FSA 6 433 plusmn 344 583 plusmn 292 466 plusmn 301ASA 10 43 plusmn 275 58 plusmn 304 46 plusmn 227PSA 22 463 plusmn 338 372 plusmn 283 359 plusmn 263Unicystic 72 395 plusmn 297 402 plusmn 273b 431 plusmn 254LUA 8 275 plusmn 301 337 plusmn 192 375 plusmn 311MUA 24 446 plusmn 346 433 plusmn 266 483 plusmn 241IUA 40 39 plusmn 296 367 plusmn 283 412 plusmn 253c

FSA follicular solidmulticystic ameloblastoma ASA acanthomatous solidmulticystic ameloblastoma PSA plexiform solidmulticystic ameloblastoma LUAluminal unicystic ameloblastoma MUA Mural unicystic ameloblastoma IUA intraluminal unicystic ameloblastoma Kruskal-Wallis test the 119875 values in boldindicate statistical significance (119875 lt 005) Solidmulticystic ameloblastoma acytoplasmic Survivin vs Bcl-2 119875 = 0007 Unicystic ameloblastoma bBaxvs Bcl-2 119875 lt 005 Intraluminal ameloblastoma ccytoplasmic Survivin vs Bax 119875 lt 005

(a) (b)

(c) (d)

Figure 2 Ki-67 and nuclear Survivin immunoexpression (a) Ki-67 immunoexpression in solidmulticystic follicular ameloblastoma (400x)(b) Nuclear Survivin immunoexpression in solidmulticystic plexiform ameloblastoma (400x) (c) Ki-67 immunoexpression in intraluminalunicystic ameloblastoma (400x) (d) Nuclear Survivin immunoexpression in intraluminal unicystic ameloblastoma (400x)

cellular division [25] High Survivin immunoexpression ispresent in several malignant neoplasms [26ndash29]

In our study SMA exhibited higher cytoplasmic Survivinimmunoexpression in the ameloblastic tumor cells than inUA Furthermore Survivin immunoexpression was highercompared with Bax immunoexpression in SMA while in UABax immunoexpression was higher than Survivin immuno-expression

The Bcl-2 and Survivin proteins are involved in theinhibition of apoptosis processes and possibly play roles in

cellular proliferation tumor progression aggressive clinicalbehavior and oncogenesis of the odontogenic epithelium[1 10] Similar to cytoplasmic Survivin Bcl-2 inhibits theproapoptotic functions of Bax and promotes cellular prolif-eration which is ultimately reflected in the biological tumorbehavior of each type of AM [23 24] The Bcl-2 protein hasbeen identified in B-cell non-Hodgkin follicular lymphomasand is an important regulator of programmed cell death Thepositive immunoreaction of the Bcl-2 product is observed inlong-lived cellular populations or in those with a proliferative

Disease Markers 5

Table 2 Average percentages of Ki-67 and nuclear Survivin expres-sion according to ameloblastic histological types and subtypes

Histological type Number ofcases

Mean plusmn SDKi-67 Nuclear Survivin

Solidmulticystic 38 128 plusmn 104 045 plusmn 13FSA 6 111 plusmn 6 mdashASA 10 99 plusmn 74 mdashPSA 22 146 plusmn 143 077 plusmn 17Unicystic 72 164 plusmn 143 072 plusmn 15LUA 8 241 plusmn 178 05 plusmn 14MUA 24 171 plusmn 178 067 plusmn 17IUA 40 145 plusmn 109 08 plusmn 16FSA follicular solidmulticystic ameloblastomaASA acanthomatous solidmulti-cystic ameloblastoma PSA plexiform solidmulticystic ameloblastomaLUA luminal unicystic ameloblastoma MUA mural unicystic ameloblas-toma IUA intraluminal unicystic ameloblastoma Kruskal-Wallis test Nostatistical significance was observed between Ki-67 and Nuclear Survivin insubtypes of ameloblastomas

high capacity [23 24] In this study a higher percentageof Bcl-2 immunoexpression was observed in ameloblast-like tumor cells confirming the high Bcl-2 protein immu-noexpression of either most or a considerable number ofameloblastic tumor cells our results are in agreement withpublished data from other authors and could be consideredas a type of inhibition of themechanisms of cellular apoptosisinduction [24 30 31]

According to Mitsuyasu et al [31] the Bcl-2 proteinsignificantly inhibits AM tumor cell apoptosis and our resultscould support this suggestion The high percentage of Bcl-2 immunoexpression in AM cells especially in basal cellswith inverse polarization possibly affects the behavior of theAMs In this study SMA had a higher percentage of Bcl-2immunoexpression than did UA

However Bax is a promoter of apoptosis is similar toBcl-2 at the amino acid level and is capable of forminghomodimers and heterodimers with Bcl-2 The relationshipbetween Bcl-2 and Bax determines the range of survivalor cellular death When Bax predominates Bcl-2 activityit becomes repressed and the apoptotic pathway of Bax isactivated [30 32]

Bax immunoexpression was lower in SMA than in UAThis result suggests that SMA tumor cells inhibit apoptosisdue to the difference in Bcl-2 and Bax immunoexpressionlevels The relationship between these two proteins wouldjustify the more aggressive biological behavior of SMASandra et al [24] and Soluk Tekkesin et al [30] reportedthat over-immunoexpression of anti-apoptotic proteins in theodontogenic epithelium is linked to cellular proliferationAM cell differentiation and apoptosis inhibition influencingon the clinical behavior of the AM

Survivin can be detected in the nuclei of tumor cellswhich may generally indicate a poor prognosis in severalmalignant tumors [26 28] Furthermore it is associated withmetastasis to lymph nodes in oral and oropharyngeal cancers[28] suggesting an involvement of nuclear and cytoplasmic

Survivin immunoexpression in cellular proliferation poorcellular differentiation and metastasis [26]

To the best of our knowledge this is the first evidence oflocalization of Survivin protein in the nucleus of ameloblasticcells In this study nuclear Survivin immunoexpression wasslightly exclusive in UA and only expressed in the plexiformvariant of SMA Stasikowska-Kanicka et al [6] suggested thatnuclear Survivin immunoexpression promotes cell prolifer-ation while cytoplasmic immunoexpression is involved inthemechanisms of apoptosis regulationThenuclear Survivinimmunoexpression was the most novel result that was foundin this study However the percentage of nuclear positivitywas very low so more studies focusing on nuclear Survivinlevels are needed to clarify whether nuclear Survivin couldreally be involved in promoting cell proliferation

The Ki-67 protein is present in proliferative cells andas with nuclear Survivin an increased proliferation indexindicated poor prognosis The results involving Ki-67 in thisstudy are in agreement with Meer et al [33] Rosenstein etal [34] and Bologna-Molina et al [20] suggesting that theimmunoexpression index of Ki-67 in UA could be associatedwith bone destruction and aggressive behavior however thedifferences in tumor morphology may be the reason for thisfinding as SMAs form large follicles or plexiform nests whileUAs present only unicystic areas with thin epithelia thusincluding great numbers of basal and suprabasal tumor cellswith a subsequent increase in the cellular proliferation indexIn other words such a difference in the cellular count couldbe associated with a smaller number of stellate reticulum-likecells thus most cells correspond to the basal and suprabasallayers that have a greater tendency to proliferate and havehigher levels of Ki-67 nuclear immunoexpression [20]

The correlations between Survivin and BaxBcl2Ki-67have already been done in many types of tumors Althoughthe immunoexpression of the proteins included in this studyhas already been studied in several malignancies there existslittle information in ameloblastic and odontogenic tumorsOur work has the advantage of including one of the biggestameloblastoma series reported

However our study has the limitations of using only a sin-gle experimental approach and the lack of comparison witha malignant odontogenic tumor It is necessary to performa second confirmatory technique for nuclear Survivin andfunctional studies to analyze the presence and biologic role ofnuclear Survivin protein in tumorigenesis and in the biologicbehavior of ameloblastomas and other odontogenic lesions

5 Conclusion

In conclusion our data show that cytoplasmic expression ofSurvivin and the expression of Bcl-2 and Bax are relate withbehavior of ameloblastomas and possibly the higher expres-sion for Ki-67 and nuclear Survivin in UA are associate withthe epithelial morphology Future studies in the mechanismsassociated with apoptosis and with cell proliferation processin ameloblastic tumors including malignant tumors such asameloblastic carcinomas are required to help to clarify theassociation of these mechanisms in the tumoral biologicalbehavior

6 Disease Markers

Conflict of Interests

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

References

[1] H Kumamoto and K Ooya ldquoExpression of survivin and Xchromosome-linked inhibitor of apoptosis protein in amelo-blastomasrdquo Virchows Archiv vol 444 no 2 pp 164ndash170 2004

[2] L R Eversole ldquoMalignant epithelial odontogenic tumorsrdquo Sem-inars in Diagnostic Pathology vol 16 no 4 pp 317ndash324 1999

[3] D G Gardner K Heikinheimo M Shear H P Philipsen andH Coleman ldquoAmeloblastomasrdquo in Pathology and Genetics ofHead and Neck Tumours J W Eveson P Reichart and DSidransky Eds pp 296ndash300 World Health Organization Clas-sification of Tumours IARC Press Lyon France 2005

[4] J C Reed ldquoDysregulation of apoptosis in cancerrdquo Journal ofClinical Oncology vol 17 no 9 pp 2941ndash2953 1999

[5] T T Renault O Teijido B Antonsson L M Dejean and SManon ldquoRegulation of Bax mitochondrial localization by Bcl-2and Bcl-x L keep your friends close but your enemies closerrdquoInternational Journal of Biochemistry and Cell Biology vol 45no 1 pp 64ndash67 2013

[6] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[7] Q L Deveraux and J C Reed ldquoIAP family proteins suppressorsof apoptosisrdquoGenes andDevelopment vol 13 no 3 pp 239ndash2521999

[8] C Adida P L Crotty J McGrath D Berrebi J Diebold and DC Altieri ldquoDevelopmentally regulated expression of the novelcancer anti-apoptosis gene survivin in human and mouse dif-ferentiationrdquoThe American Journal of Pathology vol 152 no 1pp 43ndash49 1998

[9] N K Sah Z Khan G J Khan and P S Bisen ldquoStructuralfunctional and therapeutic biology of survivinrdquo Cancer Lettersvol 244 no 2 pp 164ndash171 2006

[10] J A Rodrıguez SW Span C GM Ferreira F A E Kruyt andG Giaccone ldquoCRM1-mediated nuclear export determines thecytoplasmic localization of the antiapoptotic protein survivinrdquoExperimental Cell Research vol 275 no 1 pp 44ndash53 2002

[11] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[12] L L Loro O K Vintermyr and A C Johannessen ldquoApoptosisin normal and diseased oral tissuesrdquoOral Diseases vol 11 no 5pp 274ndash287 2005

[13] H Kumamoto and K Ooya ldquoDetection of mitochondria-mediated apoptosis signaling molecules in ameloblastomasrdquoJournal of Oral Pathology and Medicine vol 34 no 9 pp 565ndash572 2005

[14] A Abdel-Aziz andMMAmin ldquoEGFR CD10 and proliferationmarker Ki67 expression in ameloblastoma possible role in localrecurrencerdquo Diagnostic Pathology vol 7 no 1 article 14 2012

[15] P Sah A Menon A Kamath C Chandrashekar S Carnelioand R Radhakrishnan ldquoRole of immunomarkers in the clin-icopathological analysis of unicystic ameloblastomardquo DiseaseMarkers vol 35 no 5 pp 481ndash488 2013

[16] J Gerdes U Schwab H Lemke and H Stein ldquoProduction ofa mouse monoclonal antibody reactive with a human nuclearantigen associated with cell proliferationrdquo International Journalof Cancer vol 31 no 1 pp 13ndash20 1983

[17] J Gerdes H Lemke and H Baisch ldquoCell cycle analysis of a cellproliferation associated human nuclear antigen defined by themonoclonal antibody Ki-67rdquo The Journal of Immunology vol133 no 4 pp 1710ndash1715 1984

[18] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[19] C Ledesma-Montes A Mosqueda-Taylor R Carlos-Bregni etal ldquoAmeloblastomas a regional Latin-American multicentricstudyrdquo Oral Diseases vol 13 no 3 pp 303ndash307 2007

[20] R Bologna-Molina A Mosqueda-Taylor E Lopez-Corella etal ldquoSyndecan-1 (CD138) and Ki-67 expression in differentsubtypes of ameloblastomasrdquo Oral Oncology vol 44 no 8 pp805ndash811 2008

[21] K Dhanuthai S Chantarangsu S Rojanawatsirivej et al ldquoAmelo-blastoma a multicentric studyrdquo Oral Surgery Oral MedicineOral Pathology and Oral Radiology vol 113 no 6 pp 782ndash7882012

[22] C C Gomes A P Duarte M G Diniz and R S Gomez ldquoCur-rent concepts of ameloblastoma pathogenesis review articlerdquoJournal of Oral Pathology and Medicine vol 39 no 8 pp 585ndash591 2010

[23] D M Hockenbery M Zutter W Hickey M Nahm and SJ Korsmeyer ldquoBcl2 protein is topographically restricted intissues characterized by apoptotic cell deathrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 16 pp 6961ndash6965 1991

[24] F Sandra N Nakamura T Mitsuyasu Y Shiratsuchi and MOhishi ldquoTwo relatively distinct patterns of ameloblastoma ananti-apoptotic proliferating site in the outer layer (periphery)and a pro-apoptotic differentiating site in the inner layer(centre)rdquo Histopathology vol 39 no 1 pp 93ndash98 2001

[25] G Ambrosini C Adida and D C Altieri ldquoA novel anti-apoptosis gene survivin expressed in cancer and lymphomardquoNature Medicine vol 3 no 8 pp 917ndash921 1997

[26] G Qi Y Kudo T Ando et al ldquoNuclear Survivin expression iscorrelated with malignant behaviors of head and neck cancertogether with Aurora-BrdquoOral Oncology vol 46 no 4 pp 263ndash270 2010

[27] L Sui Y Dong M Ohno Y Watanabe K Sugimoto and MTokuda ldquoSurvivin expression and its correlation with cellproliferation and prognosis in epithelial ovarian tumorsrdquo Inter-national journal of oncology vol 21 no 2 pp 315ndash320 2002

[28] C Jane A V Nerurkar N V Shirsat R B Deshpande A DAmrapurkar and F R Karjodkar ldquoIncreased survivin expres-sion in high-grade oral squamous cell carcinoma a studyin Indian tobacco chewersrdquo Journal of Oral Pathology andMedicine vol 35 no 10 pp 595ndash601 2006

[29] G Marioni A Bedogni L Giacomelli et al ldquoSurvivin expres-sion is significantly higher in pN+ oral and oropharyngealprimary squamous cell carcinomas than in pN0 carcinomasrdquoActa Oto-Laryngologica vol 125 no 11 pp 1218ndash1223 2005

[30] M Soluk Tekkesin S Mutlu and V Olgac ldquoExpressions ofbax bcl-2 and Ki-67 in odontogenic keratocysts (Keratocystic

Disease Markers 7

odontogenic tumor) in comparison with ameloblastomas andradicular cystsrdquo Turkish Journal of Pathology vol 28 no 1 pp49ndash55 2012

[31] TMitsuyasu H Harada Y Higuchi et al ldquoImmunohistochem-ical demonstration of bcl-2 protein in ameloblastomardquo Journalof Oral Pathology andMedicine vol 26 no 8 pp 345ndash348 1997

[32] L Lalier P-F Cartron P Juin et al ldquoBax activation andmitochondrial insertion during apoptosisrdquo Apoptosis vol 12no 5 pp 887ndash896 2007

[33] S Meer J S Galpin M Altini H Coleman and H Ali ldquoPro-liferating cell nuclear antigen and Ki67 immunoreactivity inameloblastomasrdquo Oral Surgery Oral Medicine Oral PathologyOral Radiology and Endodontics vol 95 no 2 pp 213ndash2212003

[34] T RosensteinMA Pogrel RA Smith and J A Regezi ldquoCysticameloblastomamdashbehavior and treatment of 21 casesrdquo Journal ofOral andMaxillofacial Surgery vol 59 no 11 pp 1311ndash1316 2001

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2 Disease Markers

by a group of molecules called the inhibitor of apoptosisproteins (IAPs) a group that includes Survivin [5ndash7] Sur-vivin is expressed during embryo-fetal development [8] israrely expressed in adult tissue and may be overregulatedin proliferative processes [6ndash9] Survivin is present in twosubcellular compartments (the cytoplasm and the nucleus)plays antiapoptotic and promitotic roles and takes part inBax inhibition Furthermore this protein not only inhibitsapoptosis but also promotes cell proliferation in malignanttumors [6 10 11]

The Bcl-2 protein family includes Bcl-2 and Bax whichplay important roles in the regulation of the apoptosisprocess Bcl-2 is a protooncogene that codes for an internalmitochondrial protein Bcl-2 an inhibitor of apoptosis [7 8]Bax is a proapoptotic protein that induces mitochondrialouter membrane permeabilization through translocation orinsertion causing membrane oligomerization pore forma-tion and the release of mitochondrial proteins from theintermembrane space (ie cytochrome C SmacDiablo andOmiHtra2) [5 12 13]

The Ki-67 nuclear antigen is a widely used cell prolif-eration marker [14 15] and a nuclear protein detected inall cell division phases except for the G0 phase [16 17] Ki-67 is frequently used to determine the growth fraction indifferent tumors [16ndash18] which is why a low growth fractionis associated with a favorable prognosis in several neoplasms

The purpose of this study was to identify and to correlatecytoplasmic and nuclear Survivin immunoexpression withBcl-2 Bax and Ki-67 immunoexpression in the SMA and UAvariants

2 Materials and Methods

21 Sample Tissues The study group consisted of 110 casesof ameloblastoma from the Oral and Maxillofacial PathologyService of public and private hospitals in Mexico the OralPathology Service of the School of Dentistry of JuarezUniversity of the State of Durango the University of theRepublic of Uruguay the Pathology Services of the NationalInstitute of Oncology and Radiobiology and Calixto GarciaHospital in Havana Cuba All cases were reevaluated bytwo pathologists with experience in odontogenic tumors andfollowing the 2005 WHO classification [3] When two ormore histological subtypes of AM were present the mostaggressive or predominant subtype was considered Sectionspresenting severe inflammation in the connective tissue wereexcluded

The study was evaluated and accepted by the EthicsCommittee of the Faculty of Dentistry of Juarez Universityof the State Durango (Folio CE-FO-UJED-01-13)

22 Immunohistochemistry Sections (3 120583m thick) were cutand placed in poly-L-lysine-coated slides The sections weredeparaffinized in a 60∘C oven for 30 minutes and placedin xylol for 5 minutes The sections were rehydrated indecreasing alcohol concentrations (absolute 90 70 and 50)and washed in distilled water To unmask the epitopes theantigen recovery was performed with 10mM sodium citrate

solution with either high or low pH depending on thecharacteristics of each antibodyThis recoverywas performedin a microwave pressure cooker with a maximum powerof 750W in two cycles of 5 minutes The samples werethen cooled to room temperature and washed with distilledwater The endogenous peroxidases were blocked with 09hydrogen peroxide and the samples were again washedwith distilled water and phosphate-buffered saline solution(PBS pH 74) The primary antibodies were incubated for30 minutes for Survivin (Clone 12C4 1 100 Dako CorpCarpinteria CA USA) Bcl-2 (Clone 124 1 50 Dako CorpCarpinteria CA USA) Bax (Polyclonal 1 150 Dako CorpCarpinteria CA USA) and Ki-67 (Clone MIB-1 1 50 DakoCorp Carpinteria CAUSA) Subsequently the sectionswereincubated with the biotinylated anti-mouseanti-rabbit sec-ondary antibody and streptavidinperoxidase complex (LSA-B + labeled streptavidin-biotin Dako Corp CarpinteriaCA USA) for 30 minutes each The reaction products weredisplayed with 331015840-diaminobenzidine-H

2O2substrate (Dako

Corp Carpinteria CA USA) Fragments of tonsillar tissuewere used as positive controls and incubation with theprimary antibodies was omitted for the negative controls

Survivin Bcl-2 and Bax proteins express immunohisto-chemically on the cytoplasm and the cytoplasmicmembranetherefore the percentages of positive tumor cells were evalu-ated on all slides The percentage of positive Survivin Bcl-2andBax immunoexpressionwas calculated using a 10x opticalmicroscope objective corresponding to an area of 53mm2The photomicrographs were taken with a digital camera(Olympus C-7070) from five random areas with abundantneoplastic ameloblastoma cells The results were gatheredaccording to the percentage of positive cells in each case

The Ki-67 assessment was performed in selected neoplas-tic cell-rich areas on the SMA cases and throughout the cysticlines and mural follicles of the UA variants The nuclear Sur-vivin immunoexpression was evaluated in neoplastic SMAand UA cells where nuclear and nuclearcytoplasmic immu-noexpression was observed In both cases (Ki-67 and nuclearand nuclearcytoplasmic Survivin immunoexpression) thecell counts were performed as follows microphotographywith a 40x objective (five fields per case)was taken the imageswere transferred to an image processor to aid in the counting(ImageJ 147d Rasband W ImageJ National Institutes ofHealth USA httpimagejnihgovij) and rack countingwas performed using ImageJ and then manually using thecell counter method to obtain the cellular proliferation index(number of positive tumor cellstotal number of tumor cellsexpressed as a percentage) This technique was used in allcases

23 Statistical Analysis Statistical analysis was performedwith Pearsonrsquos 1205942 method using the expected values TheKruskal-Wallis test was applied to detect the differencesbetween ameloblastomas and Survivin Bcl-2 Bax and Ki-67immunoexpression Results with 119875 le 005 were consideredsignificant The results were tabulated and the data analyzedwith SPSS 200 statistical software (SPSS Professional Statis-tics SPSS Inc Chicago IL USA)

Disease Markers 3

(a) (b)

(c) (d)

(e) (f)

Figure 1 Differences in the cytoplasmic immunoexpression of Survivin Bcl-2 and Bax in ameloblastomas Differences in the cytoplasmicimmunoexpression of Survivin in (a) solidmulticystic follicular ameloblastoma (100x) and (b) luminal unicystic ameloblastoma (400x)Differences in Bcl-2 immunoexpression in (c) solidmulticystic follicular ameloblastoma (400x) and (d) luminal unicystic ameloblastoma(400x) Differences in Bax immunoexpression in (e) solidmulticystic plexiform ameloblastoma (100x) and (f) luminal unicysticameloblastoma (100x)

3 Results

Akappa value of 096 between the two calibrated pathologistswas first obtained by histologically classifying the AMs asSMA and UA following the WHO criteria subsequentlythe cases without agreement were reevaluated until totalconformity was obtained Of a total of 113 cases three wereexcluded as the evaluated tissue did not correspond withany variant of ameloblastoma The remaining total studysample consisted of 110 ameloblastomas of which 38 wereSMA six were diagnosed as follicular cases (FSA) ten asacanthomatous ameloblastomas (ASA) and 22 as plexiformameloblastomas (PSA) Of the UA (119899 = 72) eight cases wereluminal unicystic (LUA) 24 were mural unicystic (MUA)and 40 were intraluminal unicystic ameloblastoma (IUA)

The positivity index for cytoplasmic Survivin was higherin the SMA than the UA samples (119875 lt 005) Similar tocytoplasmic Survivin the Bcl-2 positivity index was higher inthe SMA samples (119875 lt 005 versus UA) while the UA casesshowed a higher Bax positivity index (119875 lt 005 versusMUA)(Figure 1)

Table 1 shows the positivity index of the cellular apoptosisregulatory proteins among the AM histological subtypesThe UA cases had a higher cellular proliferation index

for Ki-67 and greater nuclear Survivin immunoexpressionthan the SMA cases (119875 lt 005) (Figure 2) Table 2 showsthe cellular proliferation index of Ki-67 and the nuclearimmunoexpression of Survivin among the histological AMsubtypes

4 Discussion

Interestingly UA was the predominant type of AM in ourseries which is consistent with previous studies in LatinAmerica [19 20] while SMA are the most frequent AM typesin North America and Asia [21] Therefore it is possible thatthis variant is predominant in someLatinAmerican countriesdue to geographical ethnic and social differences howeverthese results might be associated with a lack of uniformdiagnostic criteria or with the inclusion of incision biopsiesand surgical resections that could explain the variation in thefrequency of the AM subtypes

Several studies have identifiedmolecular alterations asso-ciated with AM development and progression [22] includingthose associated with proteins involved in the apoptotic andcellular proliferation pathways [23 24]

Survivin is a protein from the IAP family that regulatescellular death suppresses cellular apoptosis and regulates

4 Disease Markers

Table 1 Average expression percentages of apoptosis regulatory proteins according to the ameloblastic histological types and subtypes

Histological type Number of cases Mean plusmn SDCytoplasmic Survivin Bcl-2 Bax

Solidmulticystic 38 45 plusmn 316 46 plusmn 30a 402 plusmn 258FSA 6 433 plusmn 344 583 plusmn 292 466 plusmn 301ASA 10 43 plusmn 275 58 plusmn 304 46 plusmn 227PSA 22 463 plusmn 338 372 plusmn 283 359 plusmn 263Unicystic 72 395 plusmn 297 402 plusmn 273b 431 plusmn 254LUA 8 275 plusmn 301 337 plusmn 192 375 plusmn 311MUA 24 446 plusmn 346 433 plusmn 266 483 plusmn 241IUA 40 39 plusmn 296 367 plusmn 283 412 plusmn 253c

FSA follicular solidmulticystic ameloblastoma ASA acanthomatous solidmulticystic ameloblastoma PSA plexiform solidmulticystic ameloblastoma LUAluminal unicystic ameloblastoma MUA Mural unicystic ameloblastoma IUA intraluminal unicystic ameloblastoma Kruskal-Wallis test the 119875 values in boldindicate statistical significance (119875 lt 005) Solidmulticystic ameloblastoma acytoplasmic Survivin vs Bcl-2 119875 = 0007 Unicystic ameloblastoma bBaxvs Bcl-2 119875 lt 005 Intraluminal ameloblastoma ccytoplasmic Survivin vs Bax 119875 lt 005

(a) (b)

(c) (d)

Figure 2 Ki-67 and nuclear Survivin immunoexpression (a) Ki-67 immunoexpression in solidmulticystic follicular ameloblastoma (400x)(b) Nuclear Survivin immunoexpression in solidmulticystic plexiform ameloblastoma (400x) (c) Ki-67 immunoexpression in intraluminalunicystic ameloblastoma (400x) (d) Nuclear Survivin immunoexpression in intraluminal unicystic ameloblastoma (400x)

cellular division [25] High Survivin immunoexpression ispresent in several malignant neoplasms [26ndash29]

In our study SMA exhibited higher cytoplasmic Survivinimmunoexpression in the ameloblastic tumor cells than inUA Furthermore Survivin immunoexpression was highercompared with Bax immunoexpression in SMA while in UABax immunoexpression was higher than Survivin immuno-expression

The Bcl-2 and Survivin proteins are involved in theinhibition of apoptosis processes and possibly play roles in

cellular proliferation tumor progression aggressive clinicalbehavior and oncogenesis of the odontogenic epithelium[1 10] Similar to cytoplasmic Survivin Bcl-2 inhibits theproapoptotic functions of Bax and promotes cellular prolif-eration which is ultimately reflected in the biological tumorbehavior of each type of AM [23 24] The Bcl-2 protein hasbeen identified in B-cell non-Hodgkin follicular lymphomasand is an important regulator of programmed cell death Thepositive immunoreaction of the Bcl-2 product is observed inlong-lived cellular populations or in those with a proliferative

Disease Markers 5

Table 2 Average percentages of Ki-67 and nuclear Survivin expres-sion according to ameloblastic histological types and subtypes

Histological type Number ofcases

Mean plusmn SDKi-67 Nuclear Survivin

Solidmulticystic 38 128 plusmn 104 045 plusmn 13FSA 6 111 plusmn 6 mdashASA 10 99 plusmn 74 mdashPSA 22 146 plusmn 143 077 plusmn 17Unicystic 72 164 plusmn 143 072 plusmn 15LUA 8 241 plusmn 178 05 plusmn 14MUA 24 171 plusmn 178 067 plusmn 17IUA 40 145 plusmn 109 08 plusmn 16FSA follicular solidmulticystic ameloblastomaASA acanthomatous solidmulti-cystic ameloblastoma PSA plexiform solidmulticystic ameloblastomaLUA luminal unicystic ameloblastoma MUA mural unicystic ameloblas-toma IUA intraluminal unicystic ameloblastoma Kruskal-Wallis test Nostatistical significance was observed between Ki-67 and Nuclear Survivin insubtypes of ameloblastomas

high capacity [23 24] In this study a higher percentageof Bcl-2 immunoexpression was observed in ameloblast-like tumor cells confirming the high Bcl-2 protein immu-noexpression of either most or a considerable number ofameloblastic tumor cells our results are in agreement withpublished data from other authors and could be consideredas a type of inhibition of themechanisms of cellular apoptosisinduction [24 30 31]

According to Mitsuyasu et al [31] the Bcl-2 proteinsignificantly inhibits AM tumor cell apoptosis and our resultscould support this suggestion The high percentage of Bcl-2 immunoexpression in AM cells especially in basal cellswith inverse polarization possibly affects the behavior of theAMs In this study SMA had a higher percentage of Bcl-2immunoexpression than did UA

However Bax is a promoter of apoptosis is similar toBcl-2 at the amino acid level and is capable of forminghomodimers and heterodimers with Bcl-2 The relationshipbetween Bcl-2 and Bax determines the range of survivalor cellular death When Bax predominates Bcl-2 activityit becomes repressed and the apoptotic pathway of Bax isactivated [30 32]

Bax immunoexpression was lower in SMA than in UAThis result suggests that SMA tumor cells inhibit apoptosisdue to the difference in Bcl-2 and Bax immunoexpressionlevels The relationship between these two proteins wouldjustify the more aggressive biological behavior of SMASandra et al [24] and Soluk Tekkesin et al [30] reportedthat over-immunoexpression of anti-apoptotic proteins in theodontogenic epithelium is linked to cellular proliferationAM cell differentiation and apoptosis inhibition influencingon the clinical behavior of the AM

Survivin can be detected in the nuclei of tumor cellswhich may generally indicate a poor prognosis in severalmalignant tumors [26 28] Furthermore it is associated withmetastasis to lymph nodes in oral and oropharyngeal cancers[28] suggesting an involvement of nuclear and cytoplasmic

Survivin immunoexpression in cellular proliferation poorcellular differentiation and metastasis [26]

To the best of our knowledge this is the first evidence oflocalization of Survivin protein in the nucleus of ameloblasticcells In this study nuclear Survivin immunoexpression wasslightly exclusive in UA and only expressed in the plexiformvariant of SMA Stasikowska-Kanicka et al [6] suggested thatnuclear Survivin immunoexpression promotes cell prolifer-ation while cytoplasmic immunoexpression is involved inthemechanisms of apoptosis regulationThenuclear Survivinimmunoexpression was the most novel result that was foundin this study However the percentage of nuclear positivitywas very low so more studies focusing on nuclear Survivinlevels are needed to clarify whether nuclear Survivin couldreally be involved in promoting cell proliferation

The Ki-67 protein is present in proliferative cells andas with nuclear Survivin an increased proliferation indexindicated poor prognosis The results involving Ki-67 in thisstudy are in agreement with Meer et al [33] Rosenstein etal [34] and Bologna-Molina et al [20] suggesting that theimmunoexpression index of Ki-67 in UA could be associatedwith bone destruction and aggressive behavior however thedifferences in tumor morphology may be the reason for thisfinding as SMAs form large follicles or plexiform nests whileUAs present only unicystic areas with thin epithelia thusincluding great numbers of basal and suprabasal tumor cellswith a subsequent increase in the cellular proliferation indexIn other words such a difference in the cellular count couldbe associated with a smaller number of stellate reticulum-likecells thus most cells correspond to the basal and suprabasallayers that have a greater tendency to proliferate and havehigher levels of Ki-67 nuclear immunoexpression [20]

The correlations between Survivin and BaxBcl2Ki-67have already been done in many types of tumors Althoughthe immunoexpression of the proteins included in this studyhas already been studied in several malignancies there existslittle information in ameloblastic and odontogenic tumorsOur work has the advantage of including one of the biggestameloblastoma series reported

However our study has the limitations of using only a sin-gle experimental approach and the lack of comparison witha malignant odontogenic tumor It is necessary to performa second confirmatory technique for nuclear Survivin andfunctional studies to analyze the presence and biologic role ofnuclear Survivin protein in tumorigenesis and in the biologicbehavior of ameloblastomas and other odontogenic lesions

5 Conclusion

In conclusion our data show that cytoplasmic expression ofSurvivin and the expression of Bcl-2 and Bax are relate withbehavior of ameloblastomas and possibly the higher expres-sion for Ki-67 and nuclear Survivin in UA are associate withthe epithelial morphology Future studies in the mechanismsassociated with apoptosis and with cell proliferation processin ameloblastic tumors including malignant tumors such asameloblastic carcinomas are required to help to clarify theassociation of these mechanisms in the tumoral biologicalbehavior

6 Disease Markers

Conflict of Interests

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

References

[1] H Kumamoto and K Ooya ldquoExpression of survivin and Xchromosome-linked inhibitor of apoptosis protein in amelo-blastomasrdquo Virchows Archiv vol 444 no 2 pp 164ndash170 2004

[2] L R Eversole ldquoMalignant epithelial odontogenic tumorsrdquo Sem-inars in Diagnostic Pathology vol 16 no 4 pp 317ndash324 1999

[3] D G Gardner K Heikinheimo M Shear H P Philipsen andH Coleman ldquoAmeloblastomasrdquo in Pathology and Genetics ofHead and Neck Tumours J W Eveson P Reichart and DSidransky Eds pp 296ndash300 World Health Organization Clas-sification of Tumours IARC Press Lyon France 2005

[4] J C Reed ldquoDysregulation of apoptosis in cancerrdquo Journal ofClinical Oncology vol 17 no 9 pp 2941ndash2953 1999

[5] T T Renault O Teijido B Antonsson L M Dejean and SManon ldquoRegulation of Bax mitochondrial localization by Bcl-2and Bcl-x L keep your friends close but your enemies closerrdquoInternational Journal of Biochemistry and Cell Biology vol 45no 1 pp 64ndash67 2013

[6] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[7] Q L Deveraux and J C Reed ldquoIAP family proteins suppressorsof apoptosisrdquoGenes andDevelopment vol 13 no 3 pp 239ndash2521999

[8] C Adida P L Crotty J McGrath D Berrebi J Diebold and DC Altieri ldquoDevelopmentally regulated expression of the novelcancer anti-apoptosis gene survivin in human and mouse dif-ferentiationrdquoThe American Journal of Pathology vol 152 no 1pp 43ndash49 1998

[9] N K Sah Z Khan G J Khan and P S Bisen ldquoStructuralfunctional and therapeutic biology of survivinrdquo Cancer Lettersvol 244 no 2 pp 164ndash171 2006

[10] J A Rodrıguez SW Span C GM Ferreira F A E Kruyt andG Giaccone ldquoCRM1-mediated nuclear export determines thecytoplasmic localization of the antiapoptotic protein survivinrdquoExperimental Cell Research vol 275 no 1 pp 44ndash53 2002

[11] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[12] L L Loro O K Vintermyr and A C Johannessen ldquoApoptosisin normal and diseased oral tissuesrdquoOral Diseases vol 11 no 5pp 274ndash287 2005

[13] H Kumamoto and K Ooya ldquoDetection of mitochondria-mediated apoptosis signaling molecules in ameloblastomasrdquoJournal of Oral Pathology and Medicine vol 34 no 9 pp 565ndash572 2005

[14] A Abdel-Aziz andMMAmin ldquoEGFR CD10 and proliferationmarker Ki67 expression in ameloblastoma possible role in localrecurrencerdquo Diagnostic Pathology vol 7 no 1 article 14 2012

[15] P Sah A Menon A Kamath C Chandrashekar S Carnelioand R Radhakrishnan ldquoRole of immunomarkers in the clin-icopathological analysis of unicystic ameloblastomardquo DiseaseMarkers vol 35 no 5 pp 481ndash488 2013

[16] J Gerdes U Schwab H Lemke and H Stein ldquoProduction ofa mouse monoclonal antibody reactive with a human nuclearantigen associated with cell proliferationrdquo International Journalof Cancer vol 31 no 1 pp 13ndash20 1983

[17] J Gerdes H Lemke and H Baisch ldquoCell cycle analysis of a cellproliferation associated human nuclear antigen defined by themonoclonal antibody Ki-67rdquo The Journal of Immunology vol133 no 4 pp 1710ndash1715 1984

[18] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[19] C Ledesma-Montes A Mosqueda-Taylor R Carlos-Bregni etal ldquoAmeloblastomas a regional Latin-American multicentricstudyrdquo Oral Diseases vol 13 no 3 pp 303ndash307 2007

[20] R Bologna-Molina A Mosqueda-Taylor E Lopez-Corella etal ldquoSyndecan-1 (CD138) and Ki-67 expression in differentsubtypes of ameloblastomasrdquo Oral Oncology vol 44 no 8 pp805ndash811 2008

[21] K Dhanuthai S Chantarangsu S Rojanawatsirivej et al ldquoAmelo-blastoma a multicentric studyrdquo Oral Surgery Oral MedicineOral Pathology and Oral Radiology vol 113 no 6 pp 782ndash7882012

[22] C C Gomes A P Duarte M G Diniz and R S Gomez ldquoCur-rent concepts of ameloblastoma pathogenesis review articlerdquoJournal of Oral Pathology and Medicine vol 39 no 8 pp 585ndash591 2010

[23] D M Hockenbery M Zutter W Hickey M Nahm and SJ Korsmeyer ldquoBcl2 protein is topographically restricted intissues characterized by apoptotic cell deathrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 16 pp 6961ndash6965 1991

[24] F Sandra N Nakamura T Mitsuyasu Y Shiratsuchi and MOhishi ldquoTwo relatively distinct patterns of ameloblastoma ananti-apoptotic proliferating site in the outer layer (periphery)and a pro-apoptotic differentiating site in the inner layer(centre)rdquo Histopathology vol 39 no 1 pp 93ndash98 2001

[25] G Ambrosini C Adida and D C Altieri ldquoA novel anti-apoptosis gene survivin expressed in cancer and lymphomardquoNature Medicine vol 3 no 8 pp 917ndash921 1997

[26] G Qi Y Kudo T Ando et al ldquoNuclear Survivin expression iscorrelated with malignant behaviors of head and neck cancertogether with Aurora-BrdquoOral Oncology vol 46 no 4 pp 263ndash270 2010

[27] L Sui Y Dong M Ohno Y Watanabe K Sugimoto and MTokuda ldquoSurvivin expression and its correlation with cellproliferation and prognosis in epithelial ovarian tumorsrdquo Inter-national journal of oncology vol 21 no 2 pp 315ndash320 2002

[28] C Jane A V Nerurkar N V Shirsat R B Deshpande A DAmrapurkar and F R Karjodkar ldquoIncreased survivin expres-sion in high-grade oral squamous cell carcinoma a studyin Indian tobacco chewersrdquo Journal of Oral Pathology andMedicine vol 35 no 10 pp 595ndash601 2006

[29] G Marioni A Bedogni L Giacomelli et al ldquoSurvivin expres-sion is significantly higher in pN+ oral and oropharyngealprimary squamous cell carcinomas than in pN0 carcinomasrdquoActa Oto-Laryngologica vol 125 no 11 pp 1218ndash1223 2005

[30] M Soluk Tekkesin S Mutlu and V Olgac ldquoExpressions ofbax bcl-2 and Ki-67 in odontogenic keratocysts (Keratocystic

Disease Markers 7

odontogenic tumor) in comparison with ameloblastomas andradicular cystsrdquo Turkish Journal of Pathology vol 28 no 1 pp49ndash55 2012

[31] TMitsuyasu H Harada Y Higuchi et al ldquoImmunohistochem-ical demonstration of bcl-2 protein in ameloblastomardquo Journalof Oral Pathology andMedicine vol 26 no 8 pp 345ndash348 1997

[32] L Lalier P-F Cartron P Juin et al ldquoBax activation andmitochondrial insertion during apoptosisrdquo Apoptosis vol 12no 5 pp 887ndash896 2007

[33] S Meer J S Galpin M Altini H Coleman and H Ali ldquoPro-liferating cell nuclear antigen and Ki67 immunoreactivity inameloblastomasrdquo Oral Surgery Oral Medicine Oral PathologyOral Radiology and Endodontics vol 95 no 2 pp 213ndash2212003

[34] T RosensteinMA Pogrel RA Smith and J A Regezi ldquoCysticameloblastomamdashbehavior and treatment of 21 casesrdquo Journal ofOral andMaxillofacial Surgery vol 59 no 11 pp 1311ndash1316 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 3

(a) (b)

(c) (d)

(e) (f)

Figure 1 Differences in the cytoplasmic immunoexpression of Survivin Bcl-2 and Bax in ameloblastomas Differences in the cytoplasmicimmunoexpression of Survivin in (a) solidmulticystic follicular ameloblastoma (100x) and (b) luminal unicystic ameloblastoma (400x)Differences in Bcl-2 immunoexpression in (c) solidmulticystic follicular ameloblastoma (400x) and (d) luminal unicystic ameloblastoma(400x) Differences in Bax immunoexpression in (e) solidmulticystic plexiform ameloblastoma (100x) and (f) luminal unicysticameloblastoma (100x)

3 Results

Akappa value of 096 between the two calibrated pathologistswas first obtained by histologically classifying the AMs asSMA and UA following the WHO criteria subsequentlythe cases without agreement were reevaluated until totalconformity was obtained Of a total of 113 cases three wereexcluded as the evaluated tissue did not correspond withany variant of ameloblastoma The remaining total studysample consisted of 110 ameloblastomas of which 38 wereSMA six were diagnosed as follicular cases (FSA) ten asacanthomatous ameloblastomas (ASA) and 22 as plexiformameloblastomas (PSA) Of the UA (119899 = 72) eight cases wereluminal unicystic (LUA) 24 were mural unicystic (MUA)and 40 were intraluminal unicystic ameloblastoma (IUA)

The positivity index for cytoplasmic Survivin was higherin the SMA than the UA samples (119875 lt 005) Similar tocytoplasmic Survivin the Bcl-2 positivity index was higher inthe SMA samples (119875 lt 005 versus UA) while the UA casesshowed a higher Bax positivity index (119875 lt 005 versusMUA)(Figure 1)

Table 1 shows the positivity index of the cellular apoptosisregulatory proteins among the AM histological subtypesThe UA cases had a higher cellular proliferation index

for Ki-67 and greater nuclear Survivin immunoexpressionthan the SMA cases (119875 lt 005) (Figure 2) Table 2 showsthe cellular proliferation index of Ki-67 and the nuclearimmunoexpression of Survivin among the histological AMsubtypes

4 Discussion

Interestingly UA was the predominant type of AM in ourseries which is consistent with previous studies in LatinAmerica [19 20] while SMA are the most frequent AM typesin North America and Asia [21] Therefore it is possible thatthis variant is predominant in someLatinAmerican countriesdue to geographical ethnic and social differences howeverthese results might be associated with a lack of uniformdiagnostic criteria or with the inclusion of incision biopsiesand surgical resections that could explain the variation in thefrequency of the AM subtypes

Several studies have identifiedmolecular alterations asso-ciated with AM development and progression [22] includingthose associated with proteins involved in the apoptotic andcellular proliferation pathways [23 24]

Survivin is a protein from the IAP family that regulatescellular death suppresses cellular apoptosis and regulates

4 Disease Markers

Table 1 Average expression percentages of apoptosis regulatory proteins according to the ameloblastic histological types and subtypes

Histological type Number of cases Mean plusmn SDCytoplasmic Survivin Bcl-2 Bax

Solidmulticystic 38 45 plusmn 316 46 plusmn 30a 402 plusmn 258FSA 6 433 plusmn 344 583 plusmn 292 466 plusmn 301ASA 10 43 plusmn 275 58 plusmn 304 46 plusmn 227PSA 22 463 plusmn 338 372 plusmn 283 359 plusmn 263Unicystic 72 395 plusmn 297 402 plusmn 273b 431 plusmn 254LUA 8 275 plusmn 301 337 plusmn 192 375 plusmn 311MUA 24 446 plusmn 346 433 plusmn 266 483 plusmn 241IUA 40 39 plusmn 296 367 plusmn 283 412 plusmn 253c

FSA follicular solidmulticystic ameloblastoma ASA acanthomatous solidmulticystic ameloblastoma PSA plexiform solidmulticystic ameloblastoma LUAluminal unicystic ameloblastoma MUA Mural unicystic ameloblastoma IUA intraluminal unicystic ameloblastoma Kruskal-Wallis test the 119875 values in boldindicate statistical significance (119875 lt 005) Solidmulticystic ameloblastoma acytoplasmic Survivin vs Bcl-2 119875 = 0007 Unicystic ameloblastoma bBaxvs Bcl-2 119875 lt 005 Intraluminal ameloblastoma ccytoplasmic Survivin vs Bax 119875 lt 005

(a) (b)

(c) (d)

Figure 2 Ki-67 and nuclear Survivin immunoexpression (a) Ki-67 immunoexpression in solidmulticystic follicular ameloblastoma (400x)(b) Nuclear Survivin immunoexpression in solidmulticystic plexiform ameloblastoma (400x) (c) Ki-67 immunoexpression in intraluminalunicystic ameloblastoma (400x) (d) Nuclear Survivin immunoexpression in intraluminal unicystic ameloblastoma (400x)

cellular division [25] High Survivin immunoexpression ispresent in several malignant neoplasms [26ndash29]

In our study SMA exhibited higher cytoplasmic Survivinimmunoexpression in the ameloblastic tumor cells than inUA Furthermore Survivin immunoexpression was highercompared with Bax immunoexpression in SMA while in UABax immunoexpression was higher than Survivin immuno-expression

The Bcl-2 and Survivin proteins are involved in theinhibition of apoptosis processes and possibly play roles in

cellular proliferation tumor progression aggressive clinicalbehavior and oncogenesis of the odontogenic epithelium[1 10] Similar to cytoplasmic Survivin Bcl-2 inhibits theproapoptotic functions of Bax and promotes cellular prolif-eration which is ultimately reflected in the biological tumorbehavior of each type of AM [23 24] The Bcl-2 protein hasbeen identified in B-cell non-Hodgkin follicular lymphomasand is an important regulator of programmed cell death Thepositive immunoreaction of the Bcl-2 product is observed inlong-lived cellular populations or in those with a proliferative

Disease Markers 5

Table 2 Average percentages of Ki-67 and nuclear Survivin expres-sion according to ameloblastic histological types and subtypes

Histological type Number ofcases

Mean plusmn SDKi-67 Nuclear Survivin

Solidmulticystic 38 128 plusmn 104 045 plusmn 13FSA 6 111 plusmn 6 mdashASA 10 99 plusmn 74 mdashPSA 22 146 plusmn 143 077 plusmn 17Unicystic 72 164 plusmn 143 072 plusmn 15LUA 8 241 plusmn 178 05 plusmn 14MUA 24 171 plusmn 178 067 plusmn 17IUA 40 145 plusmn 109 08 plusmn 16FSA follicular solidmulticystic ameloblastomaASA acanthomatous solidmulti-cystic ameloblastoma PSA plexiform solidmulticystic ameloblastomaLUA luminal unicystic ameloblastoma MUA mural unicystic ameloblas-toma IUA intraluminal unicystic ameloblastoma Kruskal-Wallis test Nostatistical significance was observed between Ki-67 and Nuclear Survivin insubtypes of ameloblastomas

high capacity [23 24] In this study a higher percentageof Bcl-2 immunoexpression was observed in ameloblast-like tumor cells confirming the high Bcl-2 protein immu-noexpression of either most or a considerable number ofameloblastic tumor cells our results are in agreement withpublished data from other authors and could be consideredas a type of inhibition of themechanisms of cellular apoptosisinduction [24 30 31]

According to Mitsuyasu et al [31] the Bcl-2 proteinsignificantly inhibits AM tumor cell apoptosis and our resultscould support this suggestion The high percentage of Bcl-2 immunoexpression in AM cells especially in basal cellswith inverse polarization possibly affects the behavior of theAMs In this study SMA had a higher percentage of Bcl-2immunoexpression than did UA

However Bax is a promoter of apoptosis is similar toBcl-2 at the amino acid level and is capable of forminghomodimers and heterodimers with Bcl-2 The relationshipbetween Bcl-2 and Bax determines the range of survivalor cellular death When Bax predominates Bcl-2 activityit becomes repressed and the apoptotic pathway of Bax isactivated [30 32]

Bax immunoexpression was lower in SMA than in UAThis result suggests that SMA tumor cells inhibit apoptosisdue to the difference in Bcl-2 and Bax immunoexpressionlevels The relationship between these two proteins wouldjustify the more aggressive biological behavior of SMASandra et al [24] and Soluk Tekkesin et al [30] reportedthat over-immunoexpression of anti-apoptotic proteins in theodontogenic epithelium is linked to cellular proliferationAM cell differentiation and apoptosis inhibition influencingon the clinical behavior of the AM

Survivin can be detected in the nuclei of tumor cellswhich may generally indicate a poor prognosis in severalmalignant tumors [26 28] Furthermore it is associated withmetastasis to lymph nodes in oral and oropharyngeal cancers[28] suggesting an involvement of nuclear and cytoplasmic

Survivin immunoexpression in cellular proliferation poorcellular differentiation and metastasis [26]

To the best of our knowledge this is the first evidence oflocalization of Survivin protein in the nucleus of ameloblasticcells In this study nuclear Survivin immunoexpression wasslightly exclusive in UA and only expressed in the plexiformvariant of SMA Stasikowska-Kanicka et al [6] suggested thatnuclear Survivin immunoexpression promotes cell prolifer-ation while cytoplasmic immunoexpression is involved inthemechanisms of apoptosis regulationThenuclear Survivinimmunoexpression was the most novel result that was foundin this study However the percentage of nuclear positivitywas very low so more studies focusing on nuclear Survivinlevels are needed to clarify whether nuclear Survivin couldreally be involved in promoting cell proliferation

The Ki-67 protein is present in proliferative cells andas with nuclear Survivin an increased proliferation indexindicated poor prognosis The results involving Ki-67 in thisstudy are in agreement with Meer et al [33] Rosenstein etal [34] and Bologna-Molina et al [20] suggesting that theimmunoexpression index of Ki-67 in UA could be associatedwith bone destruction and aggressive behavior however thedifferences in tumor morphology may be the reason for thisfinding as SMAs form large follicles or plexiform nests whileUAs present only unicystic areas with thin epithelia thusincluding great numbers of basal and suprabasal tumor cellswith a subsequent increase in the cellular proliferation indexIn other words such a difference in the cellular count couldbe associated with a smaller number of stellate reticulum-likecells thus most cells correspond to the basal and suprabasallayers that have a greater tendency to proliferate and havehigher levels of Ki-67 nuclear immunoexpression [20]

The correlations between Survivin and BaxBcl2Ki-67have already been done in many types of tumors Althoughthe immunoexpression of the proteins included in this studyhas already been studied in several malignancies there existslittle information in ameloblastic and odontogenic tumorsOur work has the advantage of including one of the biggestameloblastoma series reported

However our study has the limitations of using only a sin-gle experimental approach and the lack of comparison witha malignant odontogenic tumor It is necessary to performa second confirmatory technique for nuclear Survivin andfunctional studies to analyze the presence and biologic role ofnuclear Survivin protein in tumorigenesis and in the biologicbehavior of ameloblastomas and other odontogenic lesions

5 Conclusion

In conclusion our data show that cytoplasmic expression ofSurvivin and the expression of Bcl-2 and Bax are relate withbehavior of ameloblastomas and possibly the higher expres-sion for Ki-67 and nuclear Survivin in UA are associate withthe epithelial morphology Future studies in the mechanismsassociated with apoptosis and with cell proliferation processin ameloblastic tumors including malignant tumors such asameloblastic carcinomas are required to help to clarify theassociation of these mechanisms in the tumoral biologicalbehavior

6 Disease Markers

Conflict of Interests

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

References

[1] H Kumamoto and K Ooya ldquoExpression of survivin and Xchromosome-linked inhibitor of apoptosis protein in amelo-blastomasrdquo Virchows Archiv vol 444 no 2 pp 164ndash170 2004

[2] L R Eversole ldquoMalignant epithelial odontogenic tumorsrdquo Sem-inars in Diagnostic Pathology vol 16 no 4 pp 317ndash324 1999

[3] D G Gardner K Heikinheimo M Shear H P Philipsen andH Coleman ldquoAmeloblastomasrdquo in Pathology and Genetics ofHead and Neck Tumours J W Eveson P Reichart and DSidransky Eds pp 296ndash300 World Health Organization Clas-sification of Tumours IARC Press Lyon France 2005

[4] J C Reed ldquoDysregulation of apoptosis in cancerrdquo Journal ofClinical Oncology vol 17 no 9 pp 2941ndash2953 1999

[5] T T Renault O Teijido B Antonsson L M Dejean and SManon ldquoRegulation of Bax mitochondrial localization by Bcl-2and Bcl-x L keep your friends close but your enemies closerrdquoInternational Journal of Biochemistry and Cell Biology vol 45no 1 pp 64ndash67 2013

[6] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[7] Q L Deveraux and J C Reed ldquoIAP family proteins suppressorsof apoptosisrdquoGenes andDevelopment vol 13 no 3 pp 239ndash2521999

[8] C Adida P L Crotty J McGrath D Berrebi J Diebold and DC Altieri ldquoDevelopmentally regulated expression of the novelcancer anti-apoptosis gene survivin in human and mouse dif-ferentiationrdquoThe American Journal of Pathology vol 152 no 1pp 43ndash49 1998

[9] N K Sah Z Khan G J Khan and P S Bisen ldquoStructuralfunctional and therapeutic biology of survivinrdquo Cancer Lettersvol 244 no 2 pp 164ndash171 2006

[10] J A Rodrıguez SW Span C GM Ferreira F A E Kruyt andG Giaccone ldquoCRM1-mediated nuclear export determines thecytoplasmic localization of the antiapoptotic protein survivinrdquoExperimental Cell Research vol 275 no 1 pp 44ndash53 2002

[11] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[12] L L Loro O K Vintermyr and A C Johannessen ldquoApoptosisin normal and diseased oral tissuesrdquoOral Diseases vol 11 no 5pp 274ndash287 2005

[13] H Kumamoto and K Ooya ldquoDetection of mitochondria-mediated apoptosis signaling molecules in ameloblastomasrdquoJournal of Oral Pathology and Medicine vol 34 no 9 pp 565ndash572 2005

[14] A Abdel-Aziz andMMAmin ldquoEGFR CD10 and proliferationmarker Ki67 expression in ameloblastoma possible role in localrecurrencerdquo Diagnostic Pathology vol 7 no 1 article 14 2012

[15] P Sah A Menon A Kamath C Chandrashekar S Carnelioand R Radhakrishnan ldquoRole of immunomarkers in the clin-icopathological analysis of unicystic ameloblastomardquo DiseaseMarkers vol 35 no 5 pp 481ndash488 2013

[16] J Gerdes U Schwab H Lemke and H Stein ldquoProduction ofa mouse monoclonal antibody reactive with a human nuclearantigen associated with cell proliferationrdquo International Journalof Cancer vol 31 no 1 pp 13ndash20 1983

[17] J Gerdes H Lemke and H Baisch ldquoCell cycle analysis of a cellproliferation associated human nuclear antigen defined by themonoclonal antibody Ki-67rdquo The Journal of Immunology vol133 no 4 pp 1710ndash1715 1984

[18] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[19] C Ledesma-Montes A Mosqueda-Taylor R Carlos-Bregni etal ldquoAmeloblastomas a regional Latin-American multicentricstudyrdquo Oral Diseases vol 13 no 3 pp 303ndash307 2007

[20] R Bologna-Molina A Mosqueda-Taylor E Lopez-Corella etal ldquoSyndecan-1 (CD138) and Ki-67 expression in differentsubtypes of ameloblastomasrdquo Oral Oncology vol 44 no 8 pp805ndash811 2008

[21] K Dhanuthai S Chantarangsu S Rojanawatsirivej et al ldquoAmelo-blastoma a multicentric studyrdquo Oral Surgery Oral MedicineOral Pathology and Oral Radiology vol 113 no 6 pp 782ndash7882012

[22] C C Gomes A P Duarte M G Diniz and R S Gomez ldquoCur-rent concepts of ameloblastoma pathogenesis review articlerdquoJournal of Oral Pathology and Medicine vol 39 no 8 pp 585ndash591 2010

[23] D M Hockenbery M Zutter W Hickey M Nahm and SJ Korsmeyer ldquoBcl2 protein is topographically restricted intissues characterized by apoptotic cell deathrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 16 pp 6961ndash6965 1991

[24] F Sandra N Nakamura T Mitsuyasu Y Shiratsuchi and MOhishi ldquoTwo relatively distinct patterns of ameloblastoma ananti-apoptotic proliferating site in the outer layer (periphery)and a pro-apoptotic differentiating site in the inner layer(centre)rdquo Histopathology vol 39 no 1 pp 93ndash98 2001

[25] G Ambrosini C Adida and D C Altieri ldquoA novel anti-apoptosis gene survivin expressed in cancer and lymphomardquoNature Medicine vol 3 no 8 pp 917ndash921 1997

[26] G Qi Y Kudo T Ando et al ldquoNuclear Survivin expression iscorrelated with malignant behaviors of head and neck cancertogether with Aurora-BrdquoOral Oncology vol 46 no 4 pp 263ndash270 2010

[27] L Sui Y Dong M Ohno Y Watanabe K Sugimoto and MTokuda ldquoSurvivin expression and its correlation with cellproliferation and prognosis in epithelial ovarian tumorsrdquo Inter-national journal of oncology vol 21 no 2 pp 315ndash320 2002

[28] C Jane A V Nerurkar N V Shirsat R B Deshpande A DAmrapurkar and F R Karjodkar ldquoIncreased survivin expres-sion in high-grade oral squamous cell carcinoma a studyin Indian tobacco chewersrdquo Journal of Oral Pathology andMedicine vol 35 no 10 pp 595ndash601 2006

[29] G Marioni A Bedogni L Giacomelli et al ldquoSurvivin expres-sion is significantly higher in pN+ oral and oropharyngealprimary squamous cell carcinomas than in pN0 carcinomasrdquoActa Oto-Laryngologica vol 125 no 11 pp 1218ndash1223 2005

[30] M Soluk Tekkesin S Mutlu and V Olgac ldquoExpressions ofbax bcl-2 and Ki-67 in odontogenic keratocysts (Keratocystic

Disease Markers 7

odontogenic tumor) in comparison with ameloblastomas andradicular cystsrdquo Turkish Journal of Pathology vol 28 no 1 pp49ndash55 2012

[31] TMitsuyasu H Harada Y Higuchi et al ldquoImmunohistochem-ical demonstration of bcl-2 protein in ameloblastomardquo Journalof Oral Pathology andMedicine vol 26 no 8 pp 345ndash348 1997

[32] L Lalier P-F Cartron P Juin et al ldquoBax activation andmitochondrial insertion during apoptosisrdquo Apoptosis vol 12no 5 pp 887ndash896 2007

[33] S Meer J S Galpin M Altini H Coleman and H Ali ldquoPro-liferating cell nuclear antigen and Ki67 immunoreactivity inameloblastomasrdquo Oral Surgery Oral Medicine Oral PathologyOral Radiology and Endodontics vol 95 no 2 pp 213ndash2212003

[34] T RosensteinMA Pogrel RA Smith and J A Regezi ldquoCysticameloblastomamdashbehavior and treatment of 21 casesrdquo Journal ofOral andMaxillofacial Surgery vol 59 no 11 pp 1311ndash1316 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Disease Markers

Table 1 Average expression percentages of apoptosis regulatory proteins according to the ameloblastic histological types and subtypes

Histological type Number of cases Mean plusmn SDCytoplasmic Survivin Bcl-2 Bax

Solidmulticystic 38 45 plusmn 316 46 plusmn 30a 402 plusmn 258FSA 6 433 plusmn 344 583 plusmn 292 466 plusmn 301ASA 10 43 plusmn 275 58 plusmn 304 46 plusmn 227PSA 22 463 plusmn 338 372 plusmn 283 359 plusmn 263Unicystic 72 395 plusmn 297 402 plusmn 273b 431 plusmn 254LUA 8 275 plusmn 301 337 plusmn 192 375 plusmn 311MUA 24 446 plusmn 346 433 plusmn 266 483 plusmn 241IUA 40 39 plusmn 296 367 plusmn 283 412 plusmn 253c

FSA follicular solidmulticystic ameloblastoma ASA acanthomatous solidmulticystic ameloblastoma PSA plexiform solidmulticystic ameloblastoma LUAluminal unicystic ameloblastoma MUA Mural unicystic ameloblastoma IUA intraluminal unicystic ameloblastoma Kruskal-Wallis test the 119875 values in boldindicate statistical significance (119875 lt 005) Solidmulticystic ameloblastoma acytoplasmic Survivin vs Bcl-2 119875 = 0007 Unicystic ameloblastoma bBaxvs Bcl-2 119875 lt 005 Intraluminal ameloblastoma ccytoplasmic Survivin vs Bax 119875 lt 005

(a) (b)

(c) (d)

Figure 2 Ki-67 and nuclear Survivin immunoexpression (a) Ki-67 immunoexpression in solidmulticystic follicular ameloblastoma (400x)(b) Nuclear Survivin immunoexpression in solidmulticystic plexiform ameloblastoma (400x) (c) Ki-67 immunoexpression in intraluminalunicystic ameloblastoma (400x) (d) Nuclear Survivin immunoexpression in intraluminal unicystic ameloblastoma (400x)

cellular division [25] High Survivin immunoexpression ispresent in several malignant neoplasms [26ndash29]

In our study SMA exhibited higher cytoplasmic Survivinimmunoexpression in the ameloblastic tumor cells than inUA Furthermore Survivin immunoexpression was highercompared with Bax immunoexpression in SMA while in UABax immunoexpression was higher than Survivin immuno-expression

The Bcl-2 and Survivin proteins are involved in theinhibition of apoptosis processes and possibly play roles in

cellular proliferation tumor progression aggressive clinicalbehavior and oncogenesis of the odontogenic epithelium[1 10] Similar to cytoplasmic Survivin Bcl-2 inhibits theproapoptotic functions of Bax and promotes cellular prolif-eration which is ultimately reflected in the biological tumorbehavior of each type of AM [23 24] The Bcl-2 protein hasbeen identified in B-cell non-Hodgkin follicular lymphomasand is an important regulator of programmed cell death Thepositive immunoreaction of the Bcl-2 product is observed inlong-lived cellular populations or in those with a proliferative

Disease Markers 5

Table 2 Average percentages of Ki-67 and nuclear Survivin expres-sion according to ameloblastic histological types and subtypes

Histological type Number ofcases

Mean plusmn SDKi-67 Nuclear Survivin

Solidmulticystic 38 128 plusmn 104 045 plusmn 13FSA 6 111 plusmn 6 mdashASA 10 99 plusmn 74 mdashPSA 22 146 plusmn 143 077 plusmn 17Unicystic 72 164 plusmn 143 072 plusmn 15LUA 8 241 plusmn 178 05 plusmn 14MUA 24 171 plusmn 178 067 plusmn 17IUA 40 145 plusmn 109 08 plusmn 16FSA follicular solidmulticystic ameloblastomaASA acanthomatous solidmulti-cystic ameloblastoma PSA plexiform solidmulticystic ameloblastomaLUA luminal unicystic ameloblastoma MUA mural unicystic ameloblas-toma IUA intraluminal unicystic ameloblastoma Kruskal-Wallis test Nostatistical significance was observed between Ki-67 and Nuclear Survivin insubtypes of ameloblastomas

high capacity [23 24] In this study a higher percentageof Bcl-2 immunoexpression was observed in ameloblast-like tumor cells confirming the high Bcl-2 protein immu-noexpression of either most or a considerable number ofameloblastic tumor cells our results are in agreement withpublished data from other authors and could be consideredas a type of inhibition of themechanisms of cellular apoptosisinduction [24 30 31]

According to Mitsuyasu et al [31] the Bcl-2 proteinsignificantly inhibits AM tumor cell apoptosis and our resultscould support this suggestion The high percentage of Bcl-2 immunoexpression in AM cells especially in basal cellswith inverse polarization possibly affects the behavior of theAMs In this study SMA had a higher percentage of Bcl-2immunoexpression than did UA

However Bax is a promoter of apoptosis is similar toBcl-2 at the amino acid level and is capable of forminghomodimers and heterodimers with Bcl-2 The relationshipbetween Bcl-2 and Bax determines the range of survivalor cellular death When Bax predominates Bcl-2 activityit becomes repressed and the apoptotic pathway of Bax isactivated [30 32]

Bax immunoexpression was lower in SMA than in UAThis result suggests that SMA tumor cells inhibit apoptosisdue to the difference in Bcl-2 and Bax immunoexpressionlevels The relationship between these two proteins wouldjustify the more aggressive biological behavior of SMASandra et al [24] and Soluk Tekkesin et al [30] reportedthat over-immunoexpression of anti-apoptotic proteins in theodontogenic epithelium is linked to cellular proliferationAM cell differentiation and apoptosis inhibition influencingon the clinical behavior of the AM

Survivin can be detected in the nuclei of tumor cellswhich may generally indicate a poor prognosis in severalmalignant tumors [26 28] Furthermore it is associated withmetastasis to lymph nodes in oral and oropharyngeal cancers[28] suggesting an involvement of nuclear and cytoplasmic

Survivin immunoexpression in cellular proliferation poorcellular differentiation and metastasis [26]

To the best of our knowledge this is the first evidence oflocalization of Survivin protein in the nucleus of ameloblasticcells In this study nuclear Survivin immunoexpression wasslightly exclusive in UA and only expressed in the plexiformvariant of SMA Stasikowska-Kanicka et al [6] suggested thatnuclear Survivin immunoexpression promotes cell prolifer-ation while cytoplasmic immunoexpression is involved inthemechanisms of apoptosis regulationThenuclear Survivinimmunoexpression was the most novel result that was foundin this study However the percentage of nuclear positivitywas very low so more studies focusing on nuclear Survivinlevels are needed to clarify whether nuclear Survivin couldreally be involved in promoting cell proliferation

The Ki-67 protein is present in proliferative cells andas with nuclear Survivin an increased proliferation indexindicated poor prognosis The results involving Ki-67 in thisstudy are in agreement with Meer et al [33] Rosenstein etal [34] and Bologna-Molina et al [20] suggesting that theimmunoexpression index of Ki-67 in UA could be associatedwith bone destruction and aggressive behavior however thedifferences in tumor morphology may be the reason for thisfinding as SMAs form large follicles or plexiform nests whileUAs present only unicystic areas with thin epithelia thusincluding great numbers of basal and suprabasal tumor cellswith a subsequent increase in the cellular proliferation indexIn other words such a difference in the cellular count couldbe associated with a smaller number of stellate reticulum-likecells thus most cells correspond to the basal and suprabasallayers that have a greater tendency to proliferate and havehigher levels of Ki-67 nuclear immunoexpression [20]

The correlations between Survivin and BaxBcl2Ki-67have already been done in many types of tumors Althoughthe immunoexpression of the proteins included in this studyhas already been studied in several malignancies there existslittle information in ameloblastic and odontogenic tumorsOur work has the advantage of including one of the biggestameloblastoma series reported

However our study has the limitations of using only a sin-gle experimental approach and the lack of comparison witha malignant odontogenic tumor It is necessary to performa second confirmatory technique for nuclear Survivin andfunctional studies to analyze the presence and biologic role ofnuclear Survivin protein in tumorigenesis and in the biologicbehavior of ameloblastomas and other odontogenic lesions

5 Conclusion

In conclusion our data show that cytoplasmic expression ofSurvivin and the expression of Bcl-2 and Bax are relate withbehavior of ameloblastomas and possibly the higher expres-sion for Ki-67 and nuclear Survivin in UA are associate withthe epithelial morphology Future studies in the mechanismsassociated with apoptosis and with cell proliferation processin ameloblastic tumors including malignant tumors such asameloblastic carcinomas are required to help to clarify theassociation of these mechanisms in the tumoral biologicalbehavior

6 Disease Markers

Conflict of Interests

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

References

[1] H Kumamoto and K Ooya ldquoExpression of survivin and Xchromosome-linked inhibitor of apoptosis protein in amelo-blastomasrdquo Virchows Archiv vol 444 no 2 pp 164ndash170 2004

[2] L R Eversole ldquoMalignant epithelial odontogenic tumorsrdquo Sem-inars in Diagnostic Pathology vol 16 no 4 pp 317ndash324 1999

[3] D G Gardner K Heikinheimo M Shear H P Philipsen andH Coleman ldquoAmeloblastomasrdquo in Pathology and Genetics ofHead and Neck Tumours J W Eveson P Reichart and DSidransky Eds pp 296ndash300 World Health Organization Clas-sification of Tumours IARC Press Lyon France 2005

[4] J C Reed ldquoDysregulation of apoptosis in cancerrdquo Journal ofClinical Oncology vol 17 no 9 pp 2941ndash2953 1999

[5] T T Renault O Teijido B Antonsson L M Dejean and SManon ldquoRegulation of Bax mitochondrial localization by Bcl-2and Bcl-x L keep your friends close but your enemies closerrdquoInternational Journal of Biochemistry and Cell Biology vol 45no 1 pp 64ndash67 2013

[6] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[7] Q L Deveraux and J C Reed ldquoIAP family proteins suppressorsof apoptosisrdquoGenes andDevelopment vol 13 no 3 pp 239ndash2521999

[8] C Adida P L Crotty J McGrath D Berrebi J Diebold and DC Altieri ldquoDevelopmentally regulated expression of the novelcancer anti-apoptosis gene survivin in human and mouse dif-ferentiationrdquoThe American Journal of Pathology vol 152 no 1pp 43ndash49 1998

[9] N K Sah Z Khan G J Khan and P S Bisen ldquoStructuralfunctional and therapeutic biology of survivinrdquo Cancer Lettersvol 244 no 2 pp 164ndash171 2006

[10] J A Rodrıguez SW Span C GM Ferreira F A E Kruyt andG Giaccone ldquoCRM1-mediated nuclear export determines thecytoplasmic localization of the antiapoptotic protein survivinrdquoExperimental Cell Research vol 275 no 1 pp 44ndash53 2002

[11] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[12] L L Loro O K Vintermyr and A C Johannessen ldquoApoptosisin normal and diseased oral tissuesrdquoOral Diseases vol 11 no 5pp 274ndash287 2005

[13] H Kumamoto and K Ooya ldquoDetection of mitochondria-mediated apoptosis signaling molecules in ameloblastomasrdquoJournal of Oral Pathology and Medicine vol 34 no 9 pp 565ndash572 2005

[14] A Abdel-Aziz andMMAmin ldquoEGFR CD10 and proliferationmarker Ki67 expression in ameloblastoma possible role in localrecurrencerdquo Diagnostic Pathology vol 7 no 1 article 14 2012

[15] P Sah A Menon A Kamath C Chandrashekar S Carnelioand R Radhakrishnan ldquoRole of immunomarkers in the clin-icopathological analysis of unicystic ameloblastomardquo DiseaseMarkers vol 35 no 5 pp 481ndash488 2013

[16] J Gerdes U Schwab H Lemke and H Stein ldquoProduction ofa mouse monoclonal antibody reactive with a human nuclearantigen associated with cell proliferationrdquo International Journalof Cancer vol 31 no 1 pp 13ndash20 1983

[17] J Gerdes H Lemke and H Baisch ldquoCell cycle analysis of a cellproliferation associated human nuclear antigen defined by themonoclonal antibody Ki-67rdquo The Journal of Immunology vol133 no 4 pp 1710ndash1715 1984

[18] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[19] C Ledesma-Montes A Mosqueda-Taylor R Carlos-Bregni etal ldquoAmeloblastomas a regional Latin-American multicentricstudyrdquo Oral Diseases vol 13 no 3 pp 303ndash307 2007

[20] R Bologna-Molina A Mosqueda-Taylor E Lopez-Corella etal ldquoSyndecan-1 (CD138) and Ki-67 expression in differentsubtypes of ameloblastomasrdquo Oral Oncology vol 44 no 8 pp805ndash811 2008

[21] K Dhanuthai S Chantarangsu S Rojanawatsirivej et al ldquoAmelo-blastoma a multicentric studyrdquo Oral Surgery Oral MedicineOral Pathology and Oral Radiology vol 113 no 6 pp 782ndash7882012

[22] C C Gomes A P Duarte M G Diniz and R S Gomez ldquoCur-rent concepts of ameloblastoma pathogenesis review articlerdquoJournal of Oral Pathology and Medicine vol 39 no 8 pp 585ndash591 2010

[23] D M Hockenbery M Zutter W Hickey M Nahm and SJ Korsmeyer ldquoBcl2 protein is topographically restricted intissues characterized by apoptotic cell deathrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 16 pp 6961ndash6965 1991

[24] F Sandra N Nakamura T Mitsuyasu Y Shiratsuchi and MOhishi ldquoTwo relatively distinct patterns of ameloblastoma ananti-apoptotic proliferating site in the outer layer (periphery)and a pro-apoptotic differentiating site in the inner layer(centre)rdquo Histopathology vol 39 no 1 pp 93ndash98 2001

[25] G Ambrosini C Adida and D C Altieri ldquoA novel anti-apoptosis gene survivin expressed in cancer and lymphomardquoNature Medicine vol 3 no 8 pp 917ndash921 1997

[26] G Qi Y Kudo T Ando et al ldquoNuclear Survivin expression iscorrelated with malignant behaviors of head and neck cancertogether with Aurora-BrdquoOral Oncology vol 46 no 4 pp 263ndash270 2010

[27] L Sui Y Dong M Ohno Y Watanabe K Sugimoto and MTokuda ldquoSurvivin expression and its correlation with cellproliferation and prognosis in epithelial ovarian tumorsrdquo Inter-national journal of oncology vol 21 no 2 pp 315ndash320 2002

[28] C Jane A V Nerurkar N V Shirsat R B Deshpande A DAmrapurkar and F R Karjodkar ldquoIncreased survivin expres-sion in high-grade oral squamous cell carcinoma a studyin Indian tobacco chewersrdquo Journal of Oral Pathology andMedicine vol 35 no 10 pp 595ndash601 2006

[29] G Marioni A Bedogni L Giacomelli et al ldquoSurvivin expres-sion is significantly higher in pN+ oral and oropharyngealprimary squamous cell carcinomas than in pN0 carcinomasrdquoActa Oto-Laryngologica vol 125 no 11 pp 1218ndash1223 2005

[30] M Soluk Tekkesin S Mutlu and V Olgac ldquoExpressions ofbax bcl-2 and Ki-67 in odontogenic keratocysts (Keratocystic

Disease Markers 7

odontogenic tumor) in comparison with ameloblastomas andradicular cystsrdquo Turkish Journal of Pathology vol 28 no 1 pp49ndash55 2012

[31] TMitsuyasu H Harada Y Higuchi et al ldquoImmunohistochem-ical demonstration of bcl-2 protein in ameloblastomardquo Journalof Oral Pathology andMedicine vol 26 no 8 pp 345ndash348 1997

[32] L Lalier P-F Cartron P Juin et al ldquoBax activation andmitochondrial insertion during apoptosisrdquo Apoptosis vol 12no 5 pp 887ndash896 2007

[33] S Meer J S Galpin M Altini H Coleman and H Ali ldquoPro-liferating cell nuclear antigen and Ki67 immunoreactivity inameloblastomasrdquo Oral Surgery Oral Medicine Oral PathologyOral Radiology and Endodontics vol 95 no 2 pp 213ndash2212003

[34] T RosensteinMA Pogrel RA Smith and J A Regezi ldquoCysticameloblastomamdashbehavior and treatment of 21 casesrdquo Journal ofOral andMaxillofacial Surgery vol 59 no 11 pp 1311ndash1316 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 5

Table 2 Average percentages of Ki-67 and nuclear Survivin expres-sion according to ameloblastic histological types and subtypes

Histological type Number ofcases

Mean plusmn SDKi-67 Nuclear Survivin

Solidmulticystic 38 128 plusmn 104 045 plusmn 13FSA 6 111 plusmn 6 mdashASA 10 99 plusmn 74 mdashPSA 22 146 plusmn 143 077 plusmn 17Unicystic 72 164 plusmn 143 072 plusmn 15LUA 8 241 plusmn 178 05 plusmn 14MUA 24 171 plusmn 178 067 plusmn 17IUA 40 145 plusmn 109 08 plusmn 16FSA follicular solidmulticystic ameloblastomaASA acanthomatous solidmulti-cystic ameloblastoma PSA plexiform solidmulticystic ameloblastomaLUA luminal unicystic ameloblastoma MUA mural unicystic ameloblas-toma IUA intraluminal unicystic ameloblastoma Kruskal-Wallis test Nostatistical significance was observed between Ki-67 and Nuclear Survivin insubtypes of ameloblastomas

high capacity [23 24] In this study a higher percentageof Bcl-2 immunoexpression was observed in ameloblast-like tumor cells confirming the high Bcl-2 protein immu-noexpression of either most or a considerable number ofameloblastic tumor cells our results are in agreement withpublished data from other authors and could be consideredas a type of inhibition of themechanisms of cellular apoptosisinduction [24 30 31]

According to Mitsuyasu et al [31] the Bcl-2 proteinsignificantly inhibits AM tumor cell apoptosis and our resultscould support this suggestion The high percentage of Bcl-2 immunoexpression in AM cells especially in basal cellswith inverse polarization possibly affects the behavior of theAMs In this study SMA had a higher percentage of Bcl-2immunoexpression than did UA

However Bax is a promoter of apoptosis is similar toBcl-2 at the amino acid level and is capable of forminghomodimers and heterodimers with Bcl-2 The relationshipbetween Bcl-2 and Bax determines the range of survivalor cellular death When Bax predominates Bcl-2 activityit becomes repressed and the apoptotic pathway of Bax isactivated [30 32]

Bax immunoexpression was lower in SMA than in UAThis result suggests that SMA tumor cells inhibit apoptosisdue to the difference in Bcl-2 and Bax immunoexpressionlevels The relationship between these two proteins wouldjustify the more aggressive biological behavior of SMASandra et al [24] and Soluk Tekkesin et al [30] reportedthat over-immunoexpression of anti-apoptotic proteins in theodontogenic epithelium is linked to cellular proliferationAM cell differentiation and apoptosis inhibition influencingon the clinical behavior of the AM

Survivin can be detected in the nuclei of tumor cellswhich may generally indicate a poor prognosis in severalmalignant tumors [26 28] Furthermore it is associated withmetastasis to lymph nodes in oral and oropharyngeal cancers[28] suggesting an involvement of nuclear and cytoplasmic

Survivin immunoexpression in cellular proliferation poorcellular differentiation and metastasis [26]

To the best of our knowledge this is the first evidence oflocalization of Survivin protein in the nucleus of ameloblasticcells In this study nuclear Survivin immunoexpression wasslightly exclusive in UA and only expressed in the plexiformvariant of SMA Stasikowska-Kanicka et al [6] suggested thatnuclear Survivin immunoexpression promotes cell prolifer-ation while cytoplasmic immunoexpression is involved inthemechanisms of apoptosis regulationThenuclear Survivinimmunoexpression was the most novel result that was foundin this study However the percentage of nuclear positivitywas very low so more studies focusing on nuclear Survivinlevels are needed to clarify whether nuclear Survivin couldreally be involved in promoting cell proliferation

The Ki-67 protein is present in proliferative cells andas with nuclear Survivin an increased proliferation indexindicated poor prognosis The results involving Ki-67 in thisstudy are in agreement with Meer et al [33] Rosenstein etal [34] and Bologna-Molina et al [20] suggesting that theimmunoexpression index of Ki-67 in UA could be associatedwith bone destruction and aggressive behavior however thedifferences in tumor morphology may be the reason for thisfinding as SMAs form large follicles or plexiform nests whileUAs present only unicystic areas with thin epithelia thusincluding great numbers of basal and suprabasal tumor cellswith a subsequent increase in the cellular proliferation indexIn other words such a difference in the cellular count couldbe associated with a smaller number of stellate reticulum-likecells thus most cells correspond to the basal and suprabasallayers that have a greater tendency to proliferate and havehigher levels of Ki-67 nuclear immunoexpression [20]

The correlations between Survivin and BaxBcl2Ki-67have already been done in many types of tumors Althoughthe immunoexpression of the proteins included in this studyhas already been studied in several malignancies there existslittle information in ameloblastic and odontogenic tumorsOur work has the advantage of including one of the biggestameloblastoma series reported

However our study has the limitations of using only a sin-gle experimental approach and the lack of comparison witha malignant odontogenic tumor It is necessary to performa second confirmatory technique for nuclear Survivin andfunctional studies to analyze the presence and biologic role ofnuclear Survivin protein in tumorigenesis and in the biologicbehavior of ameloblastomas and other odontogenic lesions

5 Conclusion

In conclusion our data show that cytoplasmic expression ofSurvivin and the expression of Bcl-2 and Bax are relate withbehavior of ameloblastomas and possibly the higher expres-sion for Ki-67 and nuclear Survivin in UA are associate withthe epithelial morphology Future studies in the mechanismsassociated with apoptosis and with cell proliferation processin ameloblastic tumors including malignant tumors such asameloblastic carcinomas are required to help to clarify theassociation of these mechanisms in the tumoral biologicalbehavior

6 Disease Markers

Conflict of Interests

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

References

[1] H Kumamoto and K Ooya ldquoExpression of survivin and Xchromosome-linked inhibitor of apoptosis protein in amelo-blastomasrdquo Virchows Archiv vol 444 no 2 pp 164ndash170 2004

[2] L R Eversole ldquoMalignant epithelial odontogenic tumorsrdquo Sem-inars in Diagnostic Pathology vol 16 no 4 pp 317ndash324 1999

[3] D G Gardner K Heikinheimo M Shear H P Philipsen andH Coleman ldquoAmeloblastomasrdquo in Pathology and Genetics ofHead and Neck Tumours J W Eveson P Reichart and DSidransky Eds pp 296ndash300 World Health Organization Clas-sification of Tumours IARC Press Lyon France 2005

[4] J C Reed ldquoDysregulation of apoptosis in cancerrdquo Journal ofClinical Oncology vol 17 no 9 pp 2941ndash2953 1999

[5] T T Renault O Teijido B Antonsson L M Dejean and SManon ldquoRegulation of Bax mitochondrial localization by Bcl-2and Bcl-x L keep your friends close but your enemies closerrdquoInternational Journal of Biochemistry and Cell Biology vol 45no 1 pp 64ndash67 2013

[6] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[7] Q L Deveraux and J C Reed ldquoIAP family proteins suppressorsof apoptosisrdquoGenes andDevelopment vol 13 no 3 pp 239ndash2521999

[8] C Adida P L Crotty J McGrath D Berrebi J Diebold and DC Altieri ldquoDevelopmentally regulated expression of the novelcancer anti-apoptosis gene survivin in human and mouse dif-ferentiationrdquoThe American Journal of Pathology vol 152 no 1pp 43ndash49 1998

[9] N K Sah Z Khan G J Khan and P S Bisen ldquoStructuralfunctional and therapeutic biology of survivinrdquo Cancer Lettersvol 244 no 2 pp 164ndash171 2006

[10] J A Rodrıguez SW Span C GM Ferreira F A E Kruyt andG Giaccone ldquoCRM1-mediated nuclear export determines thecytoplasmic localization of the antiapoptotic protein survivinrdquoExperimental Cell Research vol 275 no 1 pp 44ndash53 2002

[11] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[12] L L Loro O K Vintermyr and A C Johannessen ldquoApoptosisin normal and diseased oral tissuesrdquoOral Diseases vol 11 no 5pp 274ndash287 2005

[13] H Kumamoto and K Ooya ldquoDetection of mitochondria-mediated apoptosis signaling molecules in ameloblastomasrdquoJournal of Oral Pathology and Medicine vol 34 no 9 pp 565ndash572 2005

[14] A Abdel-Aziz andMMAmin ldquoEGFR CD10 and proliferationmarker Ki67 expression in ameloblastoma possible role in localrecurrencerdquo Diagnostic Pathology vol 7 no 1 article 14 2012

[15] P Sah A Menon A Kamath C Chandrashekar S Carnelioand R Radhakrishnan ldquoRole of immunomarkers in the clin-icopathological analysis of unicystic ameloblastomardquo DiseaseMarkers vol 35 no 5 pp 481ndash488 2013

[16] J Gerdes U Schwab H Lemke and H Stein ldquoProduction ofa mouse monoclonal antibody reactive with a human nuclearantigen associated with cell proliferationrdquo International Journalof Cancer vol 31 no 1 pp 13ndash20 1983

[17] J Gerdes H Lemke and H Baisch ldquoCell cycle analysis of a cellproliferation associated human nuclear antigen defined by themonoclonal antibody Ki-67rdquo The Journal of Immunology vol133 no 4 pp 1710ndash1715 1984

[18] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[19] C Ledesma-Montes A Mosqueda-Taylor R Carlos-Bregni etal ldquoAmeloblastomas a regional Latin-American multicentricstudyrdquo Oral Diseases vol 13 no 3 pp 303ndash307 2007

[20] R Bologna-Molina A Mosqueda-Taylor E Lopez-Corella etal ldquoSyndecan-1 (CD138) and Ki-67 expression in differentsubtypes of ameloblastomasrdquo Oral Oncology vol 44 no 8 pp805ndash811 2008

[21] K Dhanuthai S Chantarangsu S Rojanawatsirivej et al ldquoAmelo-blastoma a multicentric studyrdquo Oral Surgery Oral MedicineOral Pathology and Oral Radiology vol 113 no 6 pp 782ndash7882012

[22] C C Gomes A P Duarte M G Diniz and R S Gomez ldquoCur-rent concepts of ameloblastoma pathogenesis review articlerdquoJournal of Oral Pathology and Medicine vol 39 no 8 pp 585ndash591 2010

[23] D M Hockenbery M Zutter W Hickey M Nahm and SJ Korsmeyer ldquoBcl2 protein is topographically restricted intissues characterized by apoptotic cell deathrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 16 pp 6961ndash6965 1991

[24] F Sandra N Nakamura T Mitsuyasu Y Shiratsuchi and MOhishi ldquoTwo relatively distinct patterns of ameloblastoma ananti-apoptotic proliferating site in the outer layer (periphery)and a pro-apoptotic differentiating site in the inner layer(centre)rdquo Histopathology vol 39 no 1 pp 93ndash98 2001

[25] G Ambrosini C Adida and D C Altieri ldquoA novel anti-apoptosis gene survivin expressed in cancer and lymphomardquoNature Medicine vol 3 no 8 pp 917ndash921 1997

[26] G Qi Y Kudo T Ando et al ldquoNuclear Survivin expression iscorrelated with malignant behaviors of head and neck cancertogether with Aurora-BrdquoOral Oncology vol 46 no 4 pp 263ndash270 2010

[27] L Sui Y Dong M Ohno Y Watanabe K Sugimoto and MTokuda ldquoSurvivin expression and its correlation with cellproliferation and prognosis in epithelial ovarian tumorsrdquo Inter-national journal of oncology vol 21 no 2 pp 315ndash320 2002

[28] C Jane A V Nerurkar N V Shirsat R B Deshpande A DAmrapurkar and F R Karjodkar ldquoIncreased survivin expres-sion in high-grade oral squamous cell carcinoma a studyin Indian tobacco chewersrdquo Journal of Oral Pathology andMedicine vol 35 no 10 pp 595ndash601 2006

[29] G Marioni A Bedogni L Giacomelli et al ldquoSurvivin expres-sion is significantly higher in pN+ oral and oropharyngealprimary squamous cell carcinomas than in pN0 carcinomasrdquoActa Oto-Laryngologica vol 125 no 11 pp 1218ndash1223 2005

[30] M Soluk Tekkesin S Mutlu and V Olgac ldquoExpressions ofbax bcl-2 and Ki-67 in odontogenic keratocysts (Keratocystic

Disease Markers 7

odontogenic tumor) in comparison with ameloblastomas andradicular cystsrdquo Turkish Journal of Pathology vol 28 no 1 pp49ndash55 2012

[31] TMitsuyasu H Harada Y Higuchi et al ldquoImmunohistochem-ical demonstration of bcl-2 protein in ameloblastomardquo Journalof Oral Pathology andMedicine vol 26 no 8 pp 345ndash348 1997

[32] L Lalier P-F Cartron P Juin et al ldquoBax activation andmitochondrial insertion during apoptosisrdquo Apoptosis vol 12no 5 pp 887ndash896 2007

[33] S Meer J S Galpin M Altini H Coleman and H Ali ldquoPro-liferating cell nuclear antigen and Ki67 immunoreactivity inameloblastomasrdquo Oral Surgery Oral Medicine Oral PathologyOral Radiology and Endodontics vol 95 no 2 pp 213ndash2212003

[34] T RosensteinMA Pogrel RA Smith and J A Regezi ldquoCysticameloblastomamdashbehavior and treatment of 21 casesrdquo Journal ofOral andMaxillofacial Surgery vol 59 no 11 pp 1311ndash1316 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Disease Markers

Conflict of Interests

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

References

[1] H Kumamoto and K Ooya ldquoExpression of survivin and Xchromosome-linked inhibitor of apoptosis protein in amelo-blastomasrdquo Virchows Archiv vol 444 no 2 pp 164ndash170 2004

[2] L R Eversole ldquoMalignant epithelial odontogenic tumorsrdquo Sem-inars in Diagnostic Pathology vol 16 no 4 pp 317ndash324 1999

[3] D G Gardner K Heikinheimo M Shear H P Philipsen andH Coleman ldquoAmeloblastomasrdquo in Pathology and Genetics ofHead and Neck Tumours J W Eveson P Reichart and DSidransky Eds pp 296ndash300 World Health Organization Clas-sification of Tumours IARC Press Lyon France 2005

[4] J C Reed ldquoDysregulation of apoptosis in cancerrdquo Journal ofClinical Oncology vol 17 no 9 pp 2941ndash2953 1999

[5] T T Renault O Teijido B Antonsson L M Dejean and SManon ldquoRegulation of Bax mitochondrial localization by Bcl-2and Bcl-x L keep your friends close but your enemies closerrdquoInternational Journal of Biochemistry and Cell Biology vol 45no 1 pp 64ndash67 2013

[6] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[7] Q L Deveraux and J C Reed ldquoIAP family proteins suppressorsof apoptosisrdquoGenes andDevelopment vol 13 no 3 pp 239ndash2521999

[8] C Adida P L Crotty J McGrath D Berrebi J Diebold and DC Altieri ldquoDevelopmentally regulated expression of the novelcancer anti-apoptosis gene survivin in human and mouse dif-ferentiationrdquoThe American Journal of Pathology vol 152 no 1pp 43ndash49 1998

[9] N K Sah Z Khan G J Khan and P S Bisen ldquoStructuralfunctional and therapeutic biology of survivinrdquo Cancer Lettersvol 244 no 2 pp 164ndash171 2006

[10] J A Rodrıguez SW Span C GM Ferreira F A E Kruyt andG Giaccone ldquoCRM1-mediated nuclear export determines thecytoplasmic localization of the antiapoptotic protein survivinrdquoExperimental Cell Research vol 275 no 1 pp 44ndash53 2002

[11] O Stasikowska-Kanicka M Wagrowska-Danilewicz and MDanilewicz ldquoImmunohistochemical study on survivin insinonasal tumors and its relationship with the immunoexpres-sion of Ki67 and Bcl-2rdquo Folia Histochemica et Cytobiologica vol51 no 3 pp 225ndash231 2013

[12] L L Loro O K Vintermyr and A C Johannessen ldquoApoptosisin normal and diseased oral tissuesrdquoOral Diseases vol 11 no 5pp 274ndash287 2005

[13] H Kumamoto and K Ooya ldquoDetection of mitochondria-mediated apoptosis signaling molecules in ameloblastomasrdquoJournal of Oral Pathology and Medicine vol 34 no 9 pp 565ndash572 2005

[14] A Abdel-Aziz andMMAmin ldquoEGFR CD10 and proliferationmarker Ki67 expression in ameloblastoma possible role in localrecurrencerdquo Diagnostic Pathology vol 7 no 1 article 14 2012

[15] P Sah A Menon A Kamath C Chandrashekar S Carnelioand R Radhakrishnan ldquoRole of immunomarkers in the clin-icopathological analysis of unicystic ameloblastomardquo DiseaseMarkers vol 35 no 5 pp 481ndash488 2013

[16] J Gerdes U Schwab H Lemke and H Stein ldquoProduction ofa mouse monoclonal antibody reactive with a human nuclearantigen associated with cell proliferationrdquo International Journalof Cancer vol 31 no 1 pp 13ndash20 1983

[17] J Gerdes H Lemke and H Baisch ldquoCell cycle analysis of a cellproliferation associated human nuclear antigen defined by themonoclonal antibody Ki-67rdquo The Journal of Immunology vol133 no 4 pp 1710ndash1715 1984

[18] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[19] C Ledesma-Montes A Mosqueda-Taylor R Carlos-Bregni etal ldquoAmeloblastomas a regional Latin-American multicentricstudyrdquo Oral Diseases vol 13 no 3 pp 303ndash307 2007

[20] R Bologna-Molina A Mosqueda-Taylor E Lopez-Corella etal ldquoSyndecan-1 (CD138) and Ki-67 expression in differentsubtypes of ameloblastomasrdquo Oral Oncology vol 44 no 8 pp805ndash811 2008

[21] K Dhanuthai S Chantarangsu S Rojanawatsirivej et al ldquoAmelo-blastoma a multicentric studyrdquo Oral Surgery Oral MedicineOral Pathology and Oral Radiology vol 113 no 6 pp 782ndash7882012

[22] C C Gomes A P Duarte M G Diniz and R S Gomez ldquoCur-rent concepts of ameloblastoma pathogenesis review articlerdquoJournal of Oral Pathology and Medicine vol 39 no 8 pp 585ndash591 2010

[23] D M Hockenbery M Zutter W Hickey M Nahm and SJ Korsmeyer ldquoBcl2 protein is topographically restricted intissues characterized by apoptotic cell deathrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 16 pp 6961ndash6965 1991

[24] F Sandra N Nakamura T Mitsuyasu Y Shiratsuchi and MOhishi ldquoTwo relatively distinct patterns of ameloblastoma ananti-apoptotic proliferating site in the outer layer (periphery)and a pro-apoptotic differentiating site in the inner layer(centre)rdquo Histopathology vol 39 no 1 pp 93ndash98 2001

[25] G Ambrosini C Adida and D C Altieri ldquoA novel anti-apoptosis gene survivin expressed in cancer and lymphomardquoNature Medicine vol 3 no 8 pp 917ndash921 1997

[26] G Qi Y Kudo T Ando et al ldquoNuclear Survivin expression iscorrelated with malignant behaviors of head and neck cancertogether with Aurora-BrdquoOral Oncology vol 46 no 4 pp 263ndash270 2010

[27] L Sui Y Dong M Ohno Y Watanabe K Sugimoto and MTokuda ldquoSurvivin expression and its correlation with cellproliferation and prognosis in epithelial ovarian tumorsrdquo Inter-national journal of oncology vol 21 no 2 pp 315ndash320 2002

[28] C Jane A V Nerurkar N V Shirsat R B Deshpande A DAmrapurkar and F R Karjodkar ldquoIncreased survivin expres-sion in high-grade oral squamous cell carcinoma a studyin Indian tobacco chewersrdquo Journal of Oral Pathology andMedicine vol 35 no 10 pp 595ndash601 2006

[29] G Marioni A Bedogni L Giacomelli et al ldquoSurvivin expres-sion is significantly higher in pN+ oral and oropharyngealprimary squamous cell carcinomas than in pN0 carcinomasrdquoActa Oto-Laryngologica vol 125 no 11 pp 1218ndash1223 2005

[30] M Soluk Tekkesin S Mutlu and V Olgac ldquoExpressions ofbax bcl-2 and Ki-67 in odontogenic keratocysts (Keratocystic

Disease Markers 7

odontogenic tumor) in comparison with ameloblastomas andradicular cystsrdquo Turkish Journal of Pathology vol 28 no 1 pp49ndash55 2012

[31] TMitsuyasu H Harada Y Higuchi et al ldquoImmunohistochem-ical demonstration of bcl-2 protein in ameloblastomardquo Journalof Oral Pathology andMedicine vol 26 no 8 pp 345ndash348 1997

[32] L Lalier P-F Cartron P Juin et al ldquoBax activation andmitochondrial insertion during apoptosisrdquo Apoptosis vol 12no 5 pp 887ndash896 2007

[33] S Meer J S Galpin M Altini H Coleman and H Ali ldquoPro-liferating cell nuclear antigen and Ki67 immunoreactivity inameloblastomasrdquo Oral Surgery Oral Medicine Oral PathologyOral Radiology and Endodontics vol 95 no 2 pp 213ndash2212003

[34] T RosensteinMA Pogrel RA Smith and J A Regezi ldquoCysticameloblastomamdashbehavior and treatment of 21 casesrdquo Journal ofOral andMaxillofacial Surgery vol 59 no 11 pp 1311ndash1316 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 7

odontogenic tumor) in comparison with ameloblastomas andradicular cystsrdquo Turkish Journal of Pathology vol 28 no 1 pp49ndash55 2012

[31] TMitsuyasu H Harada Y Higuchi et al ldquoImmunohistochem-ical demonstration of bcl-2 protein in ameloblastomardquo Journalof Oral Pathology andMedicine vol 26 no 8 pp 345ndash348 1997

[32] L Lalier P-F Cartron P Juin et al ldquoBax activation andmitochondrial insertion during apoptosisrdquo Apoptosis vol 12no 5 pp 887ndash896 2007

[33] S Meer J S Galpin M Altini H Coleman and H Ali ldquoPro-liferating cell nuclear antigen and Ki67 immunoreactivity inameloblastomasrdquo Oral Surgery Oral Medicine Oral PathologyOral Radiology and Endodontics vol 95 no 2 pp 213ndash2212003

[34] T RosensteinMA Pogrel RA Smith and J A Regezi ldquoCysticameloblastomamdashbehavior and treatment of 21 casesrdquo Journal ofOral andMaxillofacial Surgery vol 59 no 11 pp 1311ndash1316 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom