Annals of African Medicine
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ORIGINAL ARTICLE
Year : 2015  |  Volume : 14  |  Issue : 4  |  Page : 188-192  

Relative expression of α-smooth muscle actin and matrix metalloproteinases-2 in ameloblastoma of a black African sub-population


1 Department of Oral Pathology, College of Medicine, University of Ibadan, Ibadan, Nigeria
2 Department of Oral and Maxillofacial Surgery, College of Health Sciences, University of Port Harcourt, Rivers, Nigeria; Department for Oral Cranio Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt am Main; REPAIR Laboratory Institute of Pathology, University Medical Center, Johannes Gutenberg University, Mainz, Germany
3 Department for Oral Cranio Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt am Main, Germany; REPAIR Laboratory Institute of Pathology, University Medical Center, Johannes Gutenberg University, Mainz, Germany
4 Department for Oral Cranio Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt am Main, Germany

Date of Web Publication16-Oct-2015

Correspondence Address:
Shahram Ghanaati
Department for Oral Cranio.Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt am Main, Germany

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1596-3519.152075

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   Abstract 

Background: Ameloblastoma although a benign odontogenic tumor, is locally invasive. The abundant presence of myofibroblasts (marked by α-smooth muscle actin [α-SMA]) in the stroma and expression of matrix metalloproteinase-2 (MMP-2) in the neoplastic or stromal cells have been linked with the tumor's ability for both local and distant spread. We aim to estimate the relative expression of α-SMA and MMP-2 in ameloblastoma from a black African subgroup to gauge their relative potential for enhancing local invasiveness and hence, their prospects as possible chemotherapeutic targets. Materials and Methods: Twenty-five formalin-fixed paraffin-embedded blocks of ameloblastoma cases from Nigeria were prepared for antibody processing to α-SMA (Dako Monoclonal Mouse Anti-Human α-SMA antibody clone 1A4) and MMP-2 (Abcam Mouse Monoclonal Anti-MMP-2 antibody [CA-4001/CA719E3C] ab3158). The score for percentage positivity of the tumor cells and the score for staining intensities were then multiplied in order to generate an immunoreactive score. Results: α-smooth muscle actin was only expressed in the fibrous connective tissues adjacent to the tumor islands while MMP-2 was expressed in the ameloblasts, stellate reticulum, and the connective tissues in varying proportions. All the variants analyzed expressed α-SMA mildly or moderately, except for the follicular variant that either did not express α-SMA or expressed it mildly. The highest number of strong immunoreactivity to MMP-2 in the ameloblast region was found in the plexiform variant. Conclusion: Chemotherapeutic targeting of both molecules may, therefore, be a vital step in the control of local ameloblastoma invasiveness.

   Abstract in French 

Résumé
Contexte: Ameloblastoma même si une tumeur bénigne odontogènes, est localement invasive. La présence abondante de myofibroblastes (marquée par l'actine muscle lisse-α [α-SMA]) dans le stroma et l'expression de matrix metalloproteinase-2 (MMP-2) dans les cellules néoplasiques ou stromales ont été associés à la capacité de la tumeur pour la propagation locale et lointaine. Notre objectif est d'estimer l'expression relative de α-SMA et MMP-2 dans l'ameloblastoma d'un sous-groupe d'africain noir afin d'évaluer leur potentiel relatif pour renforcer le pouvoir envahissant local et, par conséquent, leurs perspectives en tant que cibles chimiothérapeutiques possibles.
Matériel et Méthodes: Vingt-cinq fixés au formol des blocs paraffine ameloblastoma cas du Nigéria ont été préparés pour anticorps traitement α-SMA (Dako Monoclonal souris anti-humain α-SMA anticorps clone 1 a 4) et de la MMP-2 (Abcam souris anticorps Monoclonal Anti-MMP-2 anticorps [CA-4001/CA719E3C] ab3158). Le score pour le pourcentage de positivité des cellules tumorales et le score pour les intensités de coloration ont été ensuite multiplié afin de générer un score immunoréactif.
Résultats: actine muscle lisse-α a été seulement exprimée dans les tissus conjonctifs fibreux adjacentes aux îles tumeur tandis que la MMP-2 a été exprimé dans les améloblastes, réticulum étoilé et les tissus conjonctifs dans des proportions variables. Toutes les variantes analysées exprimé α-SMA légèrement ou modérément, à l'exception de la variante folliculaire qui n'exprime pas α-SMA ou légèrement l'a exprimé. Le plus grand nombre de forte immunoréactivité de MMP-2 dans la région de l'Odam a été trouvé dans la variante plexiforme.
Conclusion: Chimiothérapeutiques ciblage de ces deux molécules peut-être, par conséquent, une étape très importante dans le contrôle du pouvoir envahissant local ameloblastoma.
Mots-clés: Ameloblastoma, noire africaine sous-population, actine musculaire lisse α mιtalloprotιinase-2 matrice

Keywords: Ameloblastoma, black African sub-population, matrix metalloproteinase-2, α-smooth muscle actin


How to cite this article:
Adisa AO, Udeabor SE, Adeyemi BF, Alica K, Booms P, Ghanaati S, Sader RA. Relative expression of α-smooth muscle actin and matrix metalloproteinases-2 in ameloblastoma of a black African sub-population. Ann Afr Med 2015;14:188-92

How to cite this URL:
Adisa AO, Udeabor SE, Adeyemi BF, Alica K, Booms P, Ghanaati S, Sader RA. Relative expression of α-smooth muscle actin and matrix metalloproteinases-2 in ameloblastoma of a black African sub-population. Ann Afr Med [serial online] 2015 [cited 2020 Oct 19];14:188-92. Available from: https://www.annalsafrmed.org/text.asp?2015/14/4/188/152075


   Introduction Top


Ameloblastoma is a benign, locally infiltrative odontogenic neoplasm with an unpredictable tendency for metastasis.[1] Some proteins have been described as supporting the local and distant spread of this neoplasm and a significant correlation was found to exist between the presence of myofibroblasts (marked by α-smooth muscle actin [α-SMA]) and matrix metalloproteinase-2 (MMP-2) expression in ameloblastoma.[2] The abundant presence of myofibroblasts in the stroma and expression of MMP-2 in the neoplastic or stromal cells were also significantly correlated with rupture of the cortical jawbone, which was considered an important prognostic marker of ameloblastoma aggressiveness. They then suggested that the abundant presence of myofibroblasts and expression of MMP-2 in solid ameloblastomas might be associated with a more aggressive infiltrative behavior.

Myofibroblasts are found in the stroma of neoplasms and by expressing proteinases they can affect tumor infiltration and advancement. The presence of myofibroblasts has been reported in ameloblastoma as being closely apposed to the neoplastic cells.[2] MMPs are enzymes that can cleave extracellular matrix and basement membrane components. Within the MMP family, the MMP-2 has been most associated with ameloblastoma invasiveness.[3] The presence of this enzyme in ameloblastoma has been used to explain its local invasiveness and tumor advancement. MMP-2 also contributes to angiogenesis by degrading the proteins that keep the vessel walls solid. This proteolysis allows the endothelial cells to escape into the interstitial matrix as seen in sprouting angiogenesis. This activity of MMP encourages tumor growth. Thus, the inhibition of MMPs will prevent the formation of new capillaries and inadvertently prevent tumor advancement.[4]

We aim to estimate the relative expression of α-SMA and MMP-2 in ameloblastoma from a black African subgroup to gauge their relative potential for enhancing local invasiveness and hence their prospects as possible chemotherapeutic targets.


   Materials and Methods Top


Twenty-five formalin-fixed paraffin-embedded blocks of ameloblastoma cases from the Oral Pathology Department of the University College Hospital and University of Ibadan, Nigeria were sectioned and stained with hematoxylin and eosin for re-evaluation and inclusion. At the REPAIR laboratory, Institute of Pathology, School of Medicine, University of Mainz Germany, sections were prepared for antibody processing to α-SMA and MMP-2 for each specimen, using the specification of the manufacturers and established standardized protocol.[5] A material transfer agreement was signed between the institutions, and the tissue blocks were labeled with numbers instead of names to conceal patients' identities. The sections were deparaffinized, hydrated, and then rinsed in phosphate-buffered solution (PBS). They were immersed in heat-induced epitope retrieval citrate buffer of concentration 15 mMol and pH 6.0, diluted 1:10 with distilled water and incubated at 94°C for 10 min. They were then placed in fresh citrate, cooled in water for 20 min, and then rinsed in PBS for 6 min. Positive and negative controls were employed for each antibody.

EnVision FLEX peroxidase blocking reagent was added to each section for 5 min, and the sections were rinsed in PBS for 6 min. The specimen was incubated for 30 min with 1:100 dilutions of Dako Monoclonal Mouse Anti-Human α-SMA antibody clone 1A4 and Abcam Mouse Monoclonal anti-MMP-2 antibody (CA-4001/CA719E3C) ab3158, rinsed with PBS, followed by incubation with undiluted EnVision

FLEX/horseradish peroxidase for 20 min. A volume of 1 ml diaminobenzidine solution was added to cover the specimen, followed by incubation in a humidity chamber for 15 min. The sections were then immersed in aqueous Meyer's hematoxylin and rinsed in distilled water for 5 min. The tissue was dehydrated and subsequently rinsed with xylene. Distyrene plasticizer in xylene mounting fluid was then applied, and a cover slip placed.

The positive cells were quantified as a percentage of the total number of cells and assigned to one of five categories (0, <5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, >75%). The percentage of positivity of the tumor cells and the staining intensities were then multiplied in order to generate an immunoreactive score. The product of the proportion and intensity scores were calculated such that a final score of 0 indicated no expression, 0–4 indicated weak expression, 5–8 indicated moderate expression and 9–12 indicated strong expression. Each sample was examined and scored by two independent oral pathologists using a conventional diagnostic microscope (Eclipse 80i, Nikon, Tokyo, Japan) and further image analysis was done with the NIS-Elements AR software (version 4.10.03, Nikon, Tokyo, Japan).

The data were analyzed using version 20 of the SPSS (IBM corp.). Qualitative data were compared using Chi-square statistics. Quantitative data were summarized using mean, standard deviation, and confidence interval and compared using Student's t-test and/or one-way analysis of variance test. The level of significance was set at P < 0.05.


   Results Top


The gender distribution of patients was approximately equal, and 18 (72.0%) were in the third decade of life. α-SMA was only expressed in the fibrous connective tissues adjacent to the tumor islands. About 75% of the samples that did not express α-SMA were from males while all the cases with strong expression were from females. All the variants analyzed expressed α-SMA mildly or moderately, except for the follicular variant that either did not express α-SMA or expressed it mildly [Table 1].
Table 1: Expression of MMP-2 in variants of ameloblastoma

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Matrix metalloproteinase-2 was expressed in the ameloblasts, stellate reticulum, and the connective tissue in varying proportions. Strong expression of MMP-2 was noted in the ameloblast and stellate reticulum in 16.0% and 8.0% of cases, respectively [Table 2]. The ameloblast in the cystic variant had the highest frequency of zero MMP-2 expression, closely followed by the fibrous connective tissue of the plexiform variant [Table 3]. The highest number of strong immunoreactivity in the ameloblast region was found in the plexiform variant, but there was no strong expression of MMP-2 in the adjacent fibrous connective tissue of any histologic variant [Table 3]. [Figure 1] and {Figure 2] show the various levels of expression for α-SMA and MMP-2.
Table 2: MMP-2 expression in AB and ST

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Table 3: Expression of α-SMA in variants of ameloblastoma

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Figure 1: Fibrous connective tissue adjacent to the odontogenic tumor islands with +++ staining for α-smooth muscle actin (×40)

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Figure 2: Fibrous connective tissue +, stellate reticulum ++ and ameloblast +++ for matrix metalloproteinase-2 (×40)

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   Discussion Top


Myofibroblasts have been found to be the most prominent stromal cell type in the tumor microenvironment [6] and their role in the support of tumor growth and progression through the secretion of growth factors, extracellular matrix proteins, and the stimulation of angiogenesis has been documented.[7],[8],[9] This has, therefore, become the basis for a possible potential target for therapeutic intervention. α-SMA is one of the immunohistochemical markers that are used to define the presence of these myofibroblasts in the tumor immediate environment.[10] In this study, α-SMA was expressed only in the fibrous connective tissue adjacent to the islands of odontogenic epithelium, this is in agreement with the report by Fregnani et al.[2] and that by Vered et al.[11] further linked the level of α-SMA expression with aggressiveness of ameloblastoma and found that solid ameloblastoma expressed levels comparable to that seen in squamous cell carcinoma.

It was therefore suggested that anti-myofibroblast agents be used in odontogenic tumors with high α-SMA expression to reduce their size before surgical intervention. In our study, most of the moderate to strong α-SMA expression was seen in the plexiform and unicystic variants. In a review of 3677 cases of ameloblastoma, approximately one-third were found to be plexiform [12] and this gives a representation of cases that may benefit from possible chemotherapy.

Even though a study by Pechkovsky et al.[13] showed no differential α-SMA expression according to gender, our study showed that 75% of the tissue samples that did not express α-SMA were from males while all the cases with strong expression were from females. Currently, we have no explanation for this variation.

Several studies have demonstrated that MMP-2 plays an important role in tumor invasion and aggressive behavior in ameloblastoma.[14],[15] We found that MMP-2 was expressed in the ameloblast, stellate reticulum, and the adjacent fibrous connective tissue stroma, and this is in agreement with a report by Sah et al.[1] Zhong et al.[16] reported strong MMP-2 expression in the central and peripheral cells of ameloblastoma islands in 65% of studied cases. We found strong expression of MMP-2 in the ameloblast and stellate reticulum in 16.0% and 8.0% of cases, respectively. Except for population disparities, we have no other explanation for this wide margin of difference in percentage expression of MMP-2.

The plexiform variant in this study had moderate to strong α-SMA expression and also strong immunoreactivity for MMP-2 in the ameloblast region compared to other variants, this may suggest a more aggressive biology than the other variants. Histological observation shows that plexiform ameloblastoma does have the most invasive interlacing pattern compared to all the other variants. Considering the fibrous connective tissue microenvironment in this study, there was no strong expression of MMP-2 in the adjacent fibrous connective tissue of any histologic variant but all variants expressed α-SMA mildly or moderately.


   Conclusion Top


We, therefore, suggest that to control the tumor microenvironment of ameloblastoma with chemotherapeutics, targeting myofibroblasts is appropriate while for local tumor invasiveness, anti-MMP-2 agents should be considered. Targeting both molecules simultaneously may, therefore, be more effective for tumor control.


   Acknowledgments Top


The authors wish to thank Mrs. Verena Hoffmann for her excellent technical assistance in the course of this study.

Source of Support:

Nil

Conflict of Interest:

None declared.

 
   References Top

1.
Sah P, Menon A, Kamath A, Chandrashekar C, Carnelio S, Radhakrishnan R. Role of immunomarkers in the clinicopathological analysis of unicystic ameloblastoma. Dis Markers 2013;35:481-8.  Back to cited text no. 1
    
2.
Fregnani ER, Sobral LM, Alves FA, Soares FA, Kowalski LP, Coletta RD. Presence of myofibroblasts and expression of matrix metalloproteinase-2 (MMP-2) in ameloblastomas correlate with rupture of the osseous cortical. Pathol Oncol Res 2009;15:231-40.  Back to cited text no. 2
    
3.
Zhang B, Zhang J, Xu ZY, Xie HL. Expression of RECK and matrix metalloproteinase-2 in ameloblastoma. BMC Cancer 2009;9:427.  Back to cited text no. 3
    
4.
Haas TL, Milkiewicz M, Davis SJ, Zhou AL, Egginton S, Brown MD, et al. Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle. Am J Physiol Heart Circ Physiol 2000;279:H1540-7.  Back to cited text no. 4
    
5.
Ghanaati S, Barbeck M, Lorenz J, Stuebinger S, Seitz O, Landes C, et al. Synthetic bone substitute material comparable with xenogeneic material for bone tissue regeneration in oral cancer patients:First and preliminary histological, histomorphometrical and clinical results. Ann Maxillofac Surg 2013;3:126-38.  Back to cited text no. 5
[PUBMED]  Medknow Journal  
6.
Sappino AP, Skalli O, Jackson B, Schürch W, Gabbiani G. Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues. Int J Cancer 1988;41:707-12.  Back to cited text no. 6
    
7.
Elenbaas B, Weinberg RA. Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp Cell Res 2001;264:169-84.  Back to cited text no. 7
    
8.
Laird AD, Vajkoczy P, Shawver LK, Thurnher A, Liang C, Mohammadi M, et al. SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. Cancer Res 2000;60:4152-60.  Back to cited text no. 8
    
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Teicher BA. Malignant cells, directors of the malignant process: Role of transforming growth factor-beta. Cancer Metastasis Rev 2001;20:133-43.  Back to cited text no. 9
    
10.
Walter-Yohrling J, Pratt BM, Ledbetter S, Teicher BA. Myofibroblasts enable invasion of endothelial cells into three-dimensional tumor cell clusters: A novel in vitro tumor model. Cancer Chemother Pharmacol 2003;52:263-9.  Back to cited text no. 10
    
11.
Vered M, Shohat I, Buchner A, Dayan D. Myofibroblasts in stroma of odontogenic cysts and tumors can contribute to variations in the biological behavior of lesions. Oral Oncol 2005;41:1028-33.  Back to cited text no. 11
    
12.
Reichart PA, Philipsen HP, Sonner S. Ameloblastoma: Biological profile of 3677 cases. Eur J Cancer B Oral Oncol 1995;31B: 86-99.  Back to cited text no. 12
    
13.
Pechkovsky DV, Hackett TL, An SS, Shaheen F, Murray LA, Knight DA. Human lung parenchyma but not proximal bronchi produces fibroblasts with enhanced TGF-beta signaling and alpha-SMA expression. Am J Respir Cell Mol Biol 2010;43:641-51.  Back to cited text no. 13
    
14.
Baum O, Hlushchuk R, Forster A, Greiner R, Clézardin P, Zhao Y, et al. Increased invasive potential and up-regulation of MMP-2 in MDA-MB-231 breast cancer cells expressing the beta3 integrin subunit. Int J Oncol 2007;30:325-32.  Back to cited text no. 14
    
15.
Pinheiro JJ, Freitas VM, Moretti AI, Jorge AG, Jaeger RG. Local invasiveness of ameloblastoma. Role played by matrix metalloproteinases and proliferative activity. Histopathology 2004;45:65-72.  Back to cited text no. 15
    
16.
Zhong M, Wand J, Yue Y. Expression of matrix metalloproteinases in human ameloblastoma. Int Chin J Dent 2004;4:19-26.  Back to cited text no. 16
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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