EXPRESSION OF ADAM10 AND SFASL IN ORAL SQUAMOUS CELL CARCINOMA
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1
Universidad de Guadalajara, Mexico
INTRODUCTION
Oral squamous cell carcinoma (OSCC) is a major health problem, represent the most common malignancy in the head and neck region. The annual estimated incidence is around 299,000 cases in 2012 Despite advances in the field of oncology, the prognosis for OSCC is poor. The overall five year survival rates for cancers of the tongue, oral cavity, and oropharynx are around 50–60%. Understanding the biology of OSCC, could facilitate the diagnosis, staging and monitoring of this malignancy.
A disintegrin and metalloproteinases (ADAM) are a family of proteins, that belong to the metzincin, contain a metalloproteinase domain and are involved in various physiological events such as cell adhesion, cell fusion, cell migration, membrane protein shedding and proteolysis. There is evidence that some ADAMs including ADAM10, are overexpressed in the development of various malignancies including OSCC, through the regulation of the activity of growth factors, integrin, cytokines and angiogenesis, which leads to the promotion of tumor growth and invasion. ADAM10 is an important regulator in the proteolytic processing of at least 40 substrates. Sheddase of these substrates contributes for the pathology of multiple diseases, including cancer and inflammation (Blobel 2005). Fas ligand (FasL, CD95L, Apo-1L, CD178) belongs to the tumor necrosis factor (TNF) family. FasL extrinsic triggers apoptosis through its Fas receptor. It has been shown that ADAM10 is primarily responsible for the cleavage of the ectodomain of FasL from the plasma membrane of some cell types, leading to the generation of a soluble portion of FasL (sFasL) ), also the ADAM10 activity regulates FasL surface expression and modulates Fas/FasL dependent apoptosis. Have been observed in high levels of sFasL inflammatory diseases, and autoimmune diseases. Several reports indicate that there is an association between serum levels of sFasL and progression of several types of malignant tumors. The objective of the study was to examine the association between ADAM10 and sFasL and the clinical progression of the disease in OSCC patients.
MATERIAL AND METHODS
Tissue samples included 20 oral squamous cell carcinomas (age range 31–83 years). And 20 normal oral mucosal tissue of control subjects (NOM). The most common primary site for OSCC was tongue (40%, 8 cases). A total of 20% (5 cases) of OSCC were well differentiated. Fifty-four percent (13 cases) and fifty-two percent (14 cases) of OSCC patients presented with lymph node metastasis and stage IV tumor, respectively.
Western blotting
Tissue lysates were harvested. Rabbit polyclonal antibody for ADAM10 (Cat# H300: sc-255778, Santa Cruz Biotechnology, Inc.), were used at 1:500 dilutions, FasL (Cat# C-178: sc-6237, Santa Cruz Biotechnology, Inc.), at 1:200 and Actin (Cat# (I-19): sc-1616, Santa Cruz Biotechnology, Inc), were used at 1:500 dilutions. The secondary antibodies were horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Inc.). The signals were detected by the Western lightning chemiluminescence reagent plus kit (Millipore IN). The densities of the signals were measured by a densitometer (Microchemi 4.2) and an automatizated software (Gelquant 12.2). Quantification of the ADAM10 signal was achieved by normalization with that of Actin.
Enzyme-Linked Immunosorbent Assay (ELISA)
A 3 ml sample of peripheral venous blood was drawn by venipuncture from each patient before surgery. Serum was obtained by centrifugation at 1500 r/min for 14 min and stored at 80°C until assayed. sFasL concentrations were measured via commercial ELISA kit (ELISA Human sFas Ligand Immunoassay DFL00; R&D Systems) as per the manufacturer’s instructions. The mean minimal detectable limits for sFasL were 2.66 pg ⁄ mL.
RESULTS
Increase of ADAM10 protein expression in OSCC tissues
To examine the expression of ADAM10 in tissues, western blot analysis was performed on 20 paired OSCC/NOM samples. Among the 20 tissue pairs analyzed, 10 (50%) NOM has increased in ADAM10 protein expression in comparision to OSCC. No correlation between clinical parameters and ADAM10 protein expression was observed. Healthy tissues showed an uniform active forms (55-65 kDa) expression. In relation to presence of pro-active forms, none of the OSCC tissues showed the presence of the zymogen (100 kDa), however, more than half of the healthy controls (65%) showed the expression.
Soluble FasL concentration
The mean serum concentration of sFasL was 5.39 ± 3.49 pg/mL in patients and 7.90 ± 2.97 pg/mL in controls. Neither difference was significant.
CONCLUSION
The expression of metalloprotease ADAM-10, as well as the regulation and the role of soluble FasL in cancer has been debated and is poorly understood. It required further studies to examine the role of cell differentiation, the activity of proteases and their inhibitors in regulating the activity of ADAM-10 and FasL in the oral mucosa, as well as assess the levels of these proteins in the various treatment modalities for OSCC.
Keywords:
oral cancer,
FasL,
sFasl,
ADAM10,
oral squamous cell carcinoma
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Tumor immunology
Citation:
Zepeda Nuño
JS,
Bueno Topete
M,
Guerrero Velázquez
C,
VEGA
AN and
Del Toro Arreola
S
(2015). EXPRESSION OF ADAM10 AND SFASL IN ORAL SQUAMOUS CELL CARCINOMA.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00050
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Received:
02 Jun 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Dr. Celia Guerrero Velázquez, Universidad de Guadalajara, Guadalajara, Mexico, celiagv2001@yahoo.com.mx