chronic stress dysregulates the hypothalamic - pituitary - adrenal axis , elevating the production of stress related hormones , epinephrine , and norepinephrine . these catecholamines seem to enhance the expression of vascular endothelial growth factor ( vegf ) and the matrix of metalloproteinases ( mmps ) in malignant tumors , contributing to tumor progression . it is known that 2-ar is a member of a large family of g - protein - coupled receptors and is responsible for transduction signals from catecholamine ligands [ 3 , 4 ] . once 2-ar binds to catecholamine ligands , it stimulates a g - protein receptor , resulting in adenylyl cyclase activity and , subsequently , in the generation of intracellular cyclic amp ( camp ) that activates protein kinase a ( pka ) , which modulates several cellular functions . a growing number of scientific evidences have verified that 2-ar is expressed by malignant neoplastic cells and that , under chronic psychological stress , through catecholamine induced activation , it could regulate a wide spectrum of tumor cell mechanisms including proliferation , apoptosis , angiogenesis , migration , and metastases [ 1 , 2 , 625 ] . particularly , investigations in oral cancer cell lines have demonstrated that 2-ar signaling upregulates interleukin-6 ( il-6 ) mrna , a cytokine that is involved in angiogenesis and tumor progression process , increasing proliferation and invasion of the tumor [ 16 , 18 ] . furthermore , shang et al . also reported that positive 2-ar immunoexpression in oral cancer is significantly correlated with age , tumor size , clinical stage , and cervical lymph node metastasis in oscc patients , suggesting a role of 2-ar in the metastasis of oral cancer . in contrast , our group , in a retrospective clinical study , showed that patients with oscc , who exhibited strong 2-ar expression by malignant epithelial cells , presented higher survival rates compared with weak / negative 2-ar expression . although 2-ar is present in normal oral tissues , the exact differences between 2-ar expression levels in normal oral epithelium and oral cancer are not so clear [ 3 , 16 , 18 , 21 ] ; for example , 2-ar was highly expressed in oscc tissue when compared to adjacent normal oral mucosa . in turn , bernab et al . showed no significant differences between 2-ar mrna levels in normal oral mucosa and oscc specimens . in order to contribute with recent studies and investigate the role of 2-ar in oscc , the present study aimed to analyze the expression of 2-ar in the tumor - free surgical margin and at the invasive front of a large sample of oscc to verify if 2-ar expression is correlated with tumor - free surgical margins and the invasive front of osccs and to explore associations between 2-ar expression levels and clinicopathological features . the present retrospective study was based on the analysis of 62 patients previously studied by bravo - caldern et al . . all patients were submitted to surgical treatment for primary oral squamous cell carcinoma at the head and neck surgery and otorhinolaryngology department of the a.c . the inclusion criteria were ( i ) primary oscc located in the oral tongue , floor of the mouth , retromolar area or inferior gingiva confirmed by biopsy ; ( ii ) patients submitted to surgery as the initial treatment followed or not by radiotherapy ; ( iii ) clinical stages ii , iii , and iv ; and ( iv ) tumor and morphologically normal / nondysplastic tumor - free surgical margin tissues available for microscopic analysis . patients with other simultaneous primary tumors , or with distant metastases at the time of admission , undergoing preoperative chemotherapy and/or radiotherapy were not included . clinical data of the patients were collected from the hospital records and included age , gender , ethnic group , tobacco and alcohol consumption , tumor location , and disease stage according to the tnm system of the international union against cancer ( uicc ) , and treatment ( surgery , postoperative adjuvant radiotherapy ) and clinical follow - up ( recurrence , cervical lymph node metastasis , distant metastasis , or occurrence of second primary tumor ) . a formalin - fixed 3  m thick section of tumor tissue was selected and paraffin - embedded for hematoxylin and eosin staining and immunohistochemistry analysis of 2-ar . three observers ( denise tostes oliveira , gustavo amaral lauand , diego mauricio bravo - caldern ) analyzed the tumor sections without knowledge of the clinical data . tumor infiltration to adjacent structures , vascular embolization , and lymph node metastases ( pn+ ) were also reported . sections of the 62 oscc specimens were deparaffinized in xylene and hydrated using graded alcohol / water baths . antigen retrieval was performed using 10  mm citrate buffer ( ph 6.0 ) in a domestic pressure cooker ( nigro , model eterna 4.5  l , araraquara , sp , brazil ) for 4  min , and then the endogenous peroxidase activity was blocked by incubation in 3% h2o2 for 30  min . tumor sections were incubated for 18 hours at 4c in a humid chamber with the anti - beta-2 adrenergic receptor primary antibody ( santa cruz biotechnology , sc-9042 , santa cruz , ca , usa ) and diluted 1  :  50 in phosphate buffered saline ( pbs ) with bovine serum albumin solution ( sigma , a9647 , st . next , the tumor sections were sequentially incubated with post primary block ( novocastra , novolink max polymer , re7260-k , newcastle upon tyne , uk ) for 30  min , followed by incubation with the polymer from the same kit . the antigen - antibody reactions were revealed using 3.3 diaminobenzidine tetrahydrochloride ( dab / sigma , d-5637 , st . tumor sections were counterstained with harris hematoxylin before being dehydrated and prepared with a cover slip . the vascular smooth muscle within the sections served as the positive internal control . ten microscopic fields of the tumor - free margins were captured using 400x magnification to analyze the immunohistochemical expression of 2-ar . images were obtained digitally with a camera ( axiocam mrc , zeiss , jena , germany ) attached to a light microscope ( axioskop 2 plus , zeiss , jena , germany ) and saved in a computer program system ( axiovision 4.6 , zeiss , jena , germany ) . briefly , the imagej software ( java - based image processing and analysis program of public - domain developed by wayne rasband nih , bethesda , md , usa ) was used to segment the representative tumor images . variations in the brown intensity of the malignant cells positive for 2-ar expression were categorized according to the following rgb channel value ranges :  r ( red ) channel was from 90 to 194 ;  g ( green ) channel was from 50 to 140 ;  b ( blue ) channel was from 45 to 147 ;  the r value should be greater than the b value ;  the g value should be greater than the b value . r ( red ) channel was from 90 to 194 ; g ( green ) channel was from 50 to 140 ; b ( blue ) channel was from 45 to 147 ; the r value should be greater than the b value ; the g value should be greater than the b value . ten images captured from the surgical margins of each oscc were automatically segmented using the matlab computing language - based software according to the criteria listed above . this software measures , based on the number of pixels , each segmented area ( determining the 2-ar immunopositive regions ) . after performing this computer - assisted immunohistochemistry analysis , the average of the 2-ar expression levels in ten surgical margin images was calculated . next , the averages of 62 oscc specimens , previously obtained by bravo - caldern et al . , were placed in ascending order and the median was established as the cut - off point to classify the specimens as exhibiting weak / negative ( averages 0.62 to 23.86 ) or strong ( averages 25.99 to 79.63 ) 2-ar expression . this measurement of 2-ar expression by epithelial cells of surgical margins was then subjectively confirmed by three investigators ( denise tostes oliveira , gustavo amaral lauand , and diego mauricio bravo - caldern ) without knowledge of the histopathological features and patient clinical status . in case of discordance between these analyzers , the criterion of the subjective evaluation was retained because this assessment involved the entire specimen . in addition , knowing that most antigens are influenced adversely by formalin fixation , a confirmation of 2-ar expression pattern was conducted through immunohistochemical staining of 19 additional frozen sections of oscc obtained from the anatomical pathology service , clinical hospital university of santiago , santiago de compostela , spain . all statistical analysis was performed using the spss 13.0 for windows software ( spss inc . , correlation between 2-ar expressions in the tumor - free surgical margins and at the invasive front of osccs was verified by pearson 's correlation coefficient test . the association between the 2-ar expression and clinicopathological variables was analyzed by the chi - square or fisher 's exact tests . for all tests , p  0.05 was considered to represent a statistically significant result . immunohistochemical analysis of the 62 oscc specimens revealed a positive expression of 2-ar in parakeratinized stratified squamous epithelium of all tumor - free surgical margins , and 50 ( 80.6% ) demonstrated strong expression of 2-ar levels . 2-ar immunostaining was detected at the cytoplasm and at the plasma membrane of normal epithelial cells in tumor - free surgical margins . similarly , the same pattern of 2-ar expression ( cytoplasmic and membranous ) was identified in malignant epithelial cells at the invasive front of tumors . interestingly , pearson 's correlation coefficient test detected a statistically significant and positive correlation between the 2-ar expression levels in tumor - free surgical margins and at the invasive front of tumors ( r = 0.383 ; p = 0.002 ) ( figure 1 ) . effectively , most ( 64.5% ) of the 62 oscc specimens had an increase of 2-ar expression levels in the tumor - free surgical margins , and it was accompanied by an increase of the expression levels at the invasive front . in other words , those specimens with weak 2-ar expression in the tumor - free surgical margin also exhibited weak / negative immunostaining of this protein in their respective invasive tumor front ; and similarly , when the immunoexpression of 2-ar was strong in normal oral epithelial cells , at the tumor - free surgical margin , the expression levels of this protein in malignant epithelial cells at the invasive front of the tumor were also high ( figure 1 ) . on the other hand , 22 ( 35.5% ) oscc samples presented no correlation between 2-ar levels in the tumor - free margins and at the invasive front of tumors , as shown in figure 1 . in 20 of these noncorrelated cases ( 32.25% of the total sample ) , the invasive front of tumor presented a decrease of 2-ar expression levels when compared to their respective tumor - free surgical margins ( figure 1 ) . on the other hand , the pattern of 2-ar expression found in the tumor - free surgical margin and at the invasive tumor front of oscc is illustrated in figure 2 . an additional analysis was performed to confirm our findings in formalin - fixed , paraffin - embedded tissue material by the evaluation of 19 new oscc frozen specimens . 2-ar expression , in this sample , was verified in most oscc specimens , being positively immunoexpressed in 87.5% of the tumor - free surgical margins and in 55.5% of the front of invasion . in the basal and corneum layers of the surgical margins or in the keratin pearls of oral cancers , furthermore , the cytoplasmic and membranous expressions of 2-ar were confirmed in oral epithelial cells adjacent to the tumor , as well as in the malignant epithelial cells of oscc . to determine the possible clinical significance of 2-ar expression in oral cancer and the associations between this protein expression and the clinicopathological features of oscc patients , chi - square or fischer 's exact tests were performed . no statistically significant associations were found regarding immunohistochemical expression of 2-ar in the tumor - free surgical margins and clinical variables evaluated ( table 1 ) . on the other hand , 2-ar immunoexpression at the invasive tumor front was statistically associated with alcohol consumption ( p = 0.038 ) , simultaneous consumption of alcohol , and tobacco ( p = 0.010 ) and with the clinical t stage ( p = 0.014 ) , as shown in table 1 . most oscc patients with weak / negative expression of 2-ar at the invasive front of tumor exhibited alcohol consumption or alcohol and tobacco consumption . in addition , strong 2-ar expression by malignant epithelial cells was more frequently detected in patients with early clinical stage of t1/t2 . regardless of the area that was considered for analysis , either the tumor - free surgical margin or the invasive tumor front , no statistically significant association between 2-ar expression and the histopathological characteristics was shown , including grade of malignancy , lymph nodes involvement ( pn+ ) , vascular embolization and perineural invasion , and muscular or bone infiltration ( table 2 ) . although several studies have described the functional localization of 2-ar in a wide variety of cells including those of the tumor microenvironment , the expression pattern of this receptor in normal oral epithelium and in malignant epithelial cells is not well defined yet [ 13 , 6 , 824 ] . our immunohistochemical analysis of a large cohort of oscc specimens has shown a cytomembranous expression of 2-ar in normal oral epithelial cells of all tumor - free surgical margins , except for the corneum and basal layers ( figure 2 ) . these findings corroborate previous reports that identified 2-ar as the main adrenergic receptor subtype in cultured human oral keratinocytes and those in which a rt - pcr assay verified the expression of 2-ar mrna in 14 of 15 specimens of normal oral mucosa [ 3 , 18 ] . collectively , data obtained by us and by mentioned reports [ 3 , 18 ] suggest that 2-ar is constitutively expressed in normal oral epithelium . concerning oscc , similarly to normal epithelium , the invasive front of tumor is considered as the most progressed region by piffk et al . , and approximately three to six tumor cell layers or detached tumor cell groups were positively immunostained for 2-ar at the cytoplasm and plasma membranes of malignant epithelial cells , except for the keratin pearls that were negative ( figure 2 ) . in addition , the present study is the first to demonstrate that the expression of 2-ar in surgical margins is positively correlated with the invasive front of tumor expression levels ( r = 0.383 ; p = 0.002 ) ( figure 1 ) . despite methodological differences , our findings corroborate the observations proposed by bernab et al .   which reported no expressive difference in 2-ar mrna expression in not matched specimens of normal mucosa and oscc . contradictorily , shang et al .   reported that 2-ar immunoexpression is higher in oscc tissue than in the adjacent normal oral mucosa . however , comparing with our present results , it is impracticable because the aforementioned analysis was carried out in a small number of surgical margins . besides , evaluations through correlation tests were not performed by shang et al . , and according to the authors , their findings of 2-ar immunostaining in surgical margins might be caused by the diversity of experiments . moreover , the accuracy of our findings determined in formalin - fixed , paraffin - embedded tissues was validated positively by the analysis of the oscc frozen specimens . biological evidence has demonstrated that chronic psychological stress through catecholamine - induced activation of 2-ar can influence the progression of a wide variety of malignant tumors [ 1 , 2 , 625 ] . however , the effects of stress related hormones can be stimulatory or inhibitory , depending on the type of hormone and the tumor type . in this context , the role of 2-ar in the progression of oral cancer is not well established . thus , the scientific literature has demonstrated that 2-ar immunoexpression by malignant cells is significantly correlated with age , tumor size , clinical stage , and cervical lymph node metastasis in oscc patients . nevertheless , the present clinical study revealed that patients clinically classified as t3 or t4 exhibited a higher frequency of weak / negative 2-ar expression at the invasive tumor front ( table 1 ) . in addition , considering that 2-ar was presented in all tumor - free surgical margins , it was expected that no statistically significant associations would be found regarding immunohistochemical expression in the normal oral epithelial cells and clinicopathological variables evaluated ( tables 1 and 2 ) . on the other hand , the evidence that , in certain osccs , 2-ar expression by tumoral cells may decrease ( figure 1 ) , as previously shown in patients with oral cancer and weak / negative 2-ar expression and low survival rates , compared with strong 2-ar expression , reinforces previous findings obtained by yu et al . . yu et al . demonstrated that genetic silencing of 2-ar increases cell migration and invasion in normal prostate cells and that weak expression of this protein was associated with metastases and worst survival rates in prostate cancer patients . considering the present results , other studies with 2-ar expression should be performed to further elucidate the role of this receptor . in vitro analyses thereby , in light of these results we can conclude that although the present study reinforces that 2-ar is constitutive in normal oral epithelial cells and is positively correlated with the expression levels of 2-ar by oscc cells , further clinical , cellular , and animal studies are needed to elucidate the role of 2-ar in oral cancer , specially , in relation to the importance of its decrease on tumor progression .
background . 
 the beta-2 adrenergic receptor is expressed by neoplastic cells and is correlated with a wide spectrum of tumor cell mechanisms including proliferation , apoptosis , angiogenesis , migration , and metastasis . objectives . 
 the present study aimed to analyze the expression of the beta-2 adrenergic receptor ( 2-ar ) in tumor - free surgical margins of oral squamous cell carcinomas ( oscc ) and at the invasive front . 
 sixty - two patients diagnosed with oscc , confirmed by biopsy , were selected for the study . 
 the clinicopathological data and clinical follow - up were obtained from medical records and their association with 2-ar expression was verified by the chi - square test or fischer 's exact test . to verify the correlation of 2-ar expression in tumor - free surgical margins and at the invasive front of osccs , pearson 's correlation coefficient test was applied . 
 results . 
 the expression of 2-ar presented a statistically significant correlation between the tumor - free surgical margins and the invasive front of oscc ( r = 0.383 ; p = 0.002 ) . 
 the immunohistochemical distribution of 2-ar at the invasive front of oscc was also statistically significant associated with alcohol ( p = 0.038 ) , simultaneous alcohol and tobacco consumption ( p = 0.010 ) , and t stage ( p = 0.014 ) . 
 conclusions . 
 the correlation of 2-ar expression in oscc and tumor - free surgical margins suggests a role of this receptor in tumor progression and its expression in normal oral epithelium seems to be constitutive .