1

Introduction

Bone morphogenetic proteins (BMPs) are pleiotropic cytokines of the transforming growth factor β family . These proteins are components of an evolutionarily conserved signaling system and are involved in the regulation of cell growth, differentiation, apoptosis, chemotaxis, angiogenesis, and matrix production both during embryo and postnatal animal development .

Studies in the areas of embryology, genetics, and carcinogenesis have demonstrated that disturbances in the BMP signaling pathway contribute to the development of neoplasms. The first evidence of this involvement arose from genetic studies on familial cancer syndromes such as familial juvenile polyposis, in which mutations in Smad-4 and BMPR-IA have been implicated in the origin of the disease. In addition, the BMP signaling pathway is found to be altered in sporadic human cancers. The action of these proteins during carcinogenesis is complex and involves both protumor and antitumor activities, depending on the stage of the disease .

During the initial phase of neoplastic development, BMPs inhibit the cell cycle by stimulating the overexpression of protein p21, a universal inhibitor of cyclin-dependent kinases. Interaction of p21 with the cyclin–cyclin-dependent kinase complex results in the inhibition of the kinase activity of the latter and consequently paralyzes cell cycle progression . The protumor activity of BMPs occurs in more advanced stages of neoplastic development and favors the dissemination of metastasis by inducing the expression of vascular endothelial growth factor (VEGF), thus exerting a proangiogenic effect. Therefore, elevated expression of these cytokines in tumors might be significantly associated wit a poor prognosis .

The role of BMPs in the development of epithelial tumors is still uncertain, and various studies have investigated their mechanisms of action in different tissues of the human organism. In view of the scarcity of reports investigating these proteins in oral epithelial tumors, the present study analyzed the immunohistochemical expression of BMP-2/4 and its receptor BMPR-IA in metastatic and nonmetastatic oral squamous cell carcinoma (OSCC) and its implications for disease prognosis.

2

Materials and methods

The sample consisted of 23 squamous cell carcinoma specimens involving different sites in the oral cavity. The specimens were obtained from incisional biopsies stored in the archives of Hospital Dr Luís Antônio, Natal, Rio Grande do Norte, Brazil. These specimens were divided into 2 groups: a nonmetastatic group consisting of 16 OSCC cases without nodal or distant metastasis and a metastatic group consisting of 7 cases of OSCC with nodal and distant metastasis. The parameter presence or absence of nodal and distant metastasis was collected from the patient records based on the TNM staging system and was used for the selection of the OSCC cases and the evaluation of disease prognosis. For the control group, 10 specimens of fibroepithelial hyperplasia were selected among excisional biopsies involving different sites in the oral cavity. The specimens were obtained from the archives of the Pathological Anatomy Service, Discipline of Oral Pathology, Dentistry School, Federal University of Rio Grande do Norte (UFRN).

Histologic sections (3 μ m thick) were obtained from the selected material and submitted to immunohistochemistry by the streptavidin-biotin method. Bone morphogenetic protein-2/4 and its receptor BMPR-IA were first reconstituted in 1 mL sterile phosphate-buffered saline each, pH 7.6, and immunohistochemistry was performed after 24 hours. For this, the paraffin-embedded sections were deparaffinized in xylene and rehydrated in a decreasing ethanol series. Endogenous tissue peroxidase was blocked by 2 baths of 5 minutes each in 10 volumes of 6% hydrogen peroxide in methanol (1:1, vol/vol). Antigens were retrieved with 0.1% trypsin and 0.1% CaCl ₂ in Tris for 30 minutes at 37°C. After incubation in normal serum for 30 minutes, the sections were incubated with the following primary antibodies: BMP-2/4 (AF355) and BMPR-IA (AF346) (both from R&D Systems, Inc, Minneapolis, MN, USA, overnight at 4°C and 1:50). The LSAB peroxidase kit (Dako Corporation, Glostrup, Denmark) was used as secondary antibody and tertiary complex for the 2 proteins studied. The reaction was developed with 0.3% diaminobenzidine (Sigma Chemical Co, St Louis, MO, USA) diluted in Tris, pH 7.4, and activated with 600 mL 6% hydrogen peroxide in a dark chamber for 3 minutes. The sections were then washed under running water and in distilled water and counterstained with Mayer’s hematoxylin for 10 minutes. Human cartilage was used as positive control, and samples in which the primary antibody was omitted and replaced with buffered 1% bovine serum albumin were used as negative control. Positive and negative controls were processed as described above.

Immunoreactivity to BMP-2/4 and BMPR-IA was analyzed qualitatively in a double-blind fashion by 2 observers. Only the epithelial component of the selected specimens was examined. Scores of 1 (weak expression) and 2 (strong expression), adapted from Wakulich et al , were used for analysis.

Expression of the proteins studied, as well as its correlation with the prognosis of OSCC, was evaluated using the χ ² test, with the level of significance set at 5% ( α = .05). The study was approved by the ethics committee of UFRN (no. 65/06- 2006).

2

Materials and methods

The sample consisted of 23 squamous cell carcinoma specimens involving different sites in the oral cavity. The specimens were obtained from incisional biopsies stored in the archives of Hospital Dr Luís Antônio, Natal, Rio Grande do Norte, Brazil. These specimens were divided into 2 groups: a nonmetastatic group consisting of 16 OSCC cases without nodal or distant metastasis and a metastatic group consisting of 7 cases of OSCC with nodal and distant metastasis. The parameter presence or absence of nodal and distant metastasis was collected from the patient records based on the TNM staging system and was used for the selection of the OSCC cases and the evaluation of disease prognosis. For the control group, 10 specimens of fibroepithelial hyperplasia were selected among excisional biopsies involving different sites in the oral cavity. The specimens were obtained from the archives of the Pathological Anatomy Service, Discipline of Oral Pathology, Dentistry School, Federal University of Rio Grande do Norte (UFRN).

Histologic sections (3 μ m thick) were obtained from the selected material and submitted to immunohistochemistry by the streptavidin-biotin method. Bone morphogenetic protein-2/4 and its receptor BMPR-IA were first reconstituted in 1 mL sterile phosphate-buffered saline each, pH 7.6, and immunohistochemistry was performed after 24 hours. For this, the paraffin-embedded sections were deparaffinized in xylene and rehydrated in a decreasing ethanol series. Endogenous tissue peroxidase was blocked by 2 baths of 5 minutes each in 10 volumes of 6% hydrogen peroxide in methanol (1:1, vol/vol). Antigens were retrieved with 0.1% trypsin and 0.1% CaCl ₂ in Tris for 30 minutes at 37°C. After incubation in normal serum for 30 minutes, the sections were incubated with the following primary antibodies: BMP-2/4 (AF355) and BMPR-IA (AF346) (both from R&D Systems, Inc, Minneapolis, MN, USA, overnight at 4°C and 1:50). The LSAB peroxidase kit (Dako Corporation, Glostrup, Denmark) was used as secondary antibody and tertiary complex for the 2 proteins studied. The reaction was developed with 0.3% diaminobenzidine (Sigma Chemical Co, St Louis, MO, USA) diluted in Tris, pH 7.4, and activated with 600 mL 6% hydrogen peroxide in a dark chamber for 3 minutes. The sections were then washed under running water and in distilled water and counterstained with Mayer’s hematoxylin for 10 minutes. Human cartilage was used as positive control, and samples in which the primary antibody was omitted and replaced with buffered 1% bovine serum albumin were used as negative control. Positive and negative controls were processed as described above.

Immunoreactivity to BMP-2/4 and BMPR-IA was analyzed qualitatively in a double-blind fashion by 2 observers. Only the epithelial component of the selected specimens was examined. Scores of 1 (weak expression) and 2 (strong expression), adapted from Wakulich et al , were used for analysis.

Expression of the proteins studied, as well as its correlation with the prognosis of OSCC, was evaluated using the χ ² test, with the level of significance set at 5% ( α = .05). The study was approved by the ethics committee of UFRN (no. 65/06- 2006).

3

Results

The immunoexpression of BMP-2/4 and of its receptor BMPR-IA showed a typical cytoplasmic location, with variations in the intensity of expression in the epithelial layer depending on the type of tumor studied.

In the experimental group without metastasis (n = 16), expression of BMP-2/4 was predominantly strong (n = 10, 62.5%) ( Fig. 1 , Table 1 ). Strong immunoreactivity to BMPR-IA was observed in all specimens (n = 16, 100%) ( Fig. 2 ). In the experimental group with metastasis (n = 7), strong expression of BMP-2/4 was observed in most cases (n = 5, 71.4%) ( Fig. 3 , Table 1 ), whereas BMPR-IA exhibited weak expression in 87.5% of the specimens analyzed ( Fig. 4 ). Weak expression of BMP-2/4 ( Fig. 5 , Table 1 ) and BMPR-IA ( Fig. 6 ) was observed in all 10 control cases of fibroepithelial hyperplasia.

Overexpression of BMP-2/4 in nonmetastatic OSCC (arrows) (original magnification ×400).

Table 1

Distribution of the expression of BMP-2/4 in metastatic and nonmetastatic OSCC specimens

		Group						Total		Qui 2	P
		Controle		With metastasis		Without metastasis
		n	%	n	%	n	%	n	%
BMP-2/4 Staining intensity	Weak	10	100	2	28.6	6	37.5	18	54.5	12.11	.002
	Strong	0	0	5	71.4	10	62.5	15	45.5
	Total	10	100	7	100.0	16	100.0	33	100.0

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