The Expression And Significance Of Smad4,tgf-β1 And P21^waf1 In Laryngeal Squamous Carcinoma(lscc)

BackgroundLaryngeal carcinoma is the most common malignancy in the head and neck, comprising approximately 5.7%-7.6%of all neoplasias.In recent years the incidence trends has increased significantly.Inactivation of tumor supressor gene has been known as one of the important mechanisms of cancer formation.Therefore,it is important to demonstrate the pathogenesis of tumor by studing the expression of anti-oncogene which will develop new thought and method for the diagnosis and therapies for tumor.Smad4 protein is the product of tumor suppressor gene DPC4(deleted in pancreatic carcinoma locus 4).It mainly takes part in intracellular signal transduction of the transforming growth factor bete(TGF-β) superfamilies.Almost all of the biological effects of TGF-βare the result of the interaction of Smad4 with other Smads proteins.Lower expression of Smad4 has been found in a variety of tumors and are closely related with cancer.Transforming growth factor beta 1(TGF-β1) is the mainly isform of TGF-β.It is a multifunctional cytokine that mainly inhibits proliferation and induces differentiation of epithelial cells.It can influence carcinogenesis in various ways,such as by stimulating angiogenesis,suppressing cancerdirected immune functions.Overexpression of TGF-β1 have been observed in many kinds of malignant tumors.P21^waf1 as the product of tumor suppressor gene p21^waf1 is a cyclin dependent kinase inhibitor protein.It mainly involves in inhibition of cell proliferation,DNA repairing.It was shown that expression of P21^waf1 was down regulated in many types of cancer and was significantly correlated with the clinicopathological matters of malignant tumors such as ditterentiation and stage. Early study has shown that p21^waf1 was one of the target genes of Smad4.To our knowledge,there is no similar study about the expression of Smad4 in human laryngeal squamous carcinoma(LSCC) has been reported.We undertook the current study using immunohistochemical methods to examin the expression level of the protein of Smad4,TGF-β1 and P21^waf1 in LSCC and normal laryngeal epithelial tissue.We analyzed the correlation between the expression of the three proteins in LSCC and their expression with clinicopathological matters of LSCC,hoping to supply new thought and method for the diagnosis and therapies for LSCC.Materials and Methods1.Materials50 LSCC and 10 surrounding normal laryngeal mucosa specimens came from First Affiliated Hospital of Zheng Zhou University.All diagnosis were made by its pathology.The surrounding normal laryngeal mucosa was used as the control group. All of the patients had not undergone preoperative radiotherapy or chemotherapy.2.Method and Evaluation of ImmunoassayingThe expressions of Smad4,transforming growth factor beta1(TGF-β1) as well as P21^waf1 were assayed in LSCC and normal laryngeal squamous epithelium specimens by immunohistochemical technics.Clearly brown staining restricted to cytoplasm and /or the nucleus was considered as positive expression of Smad4 protein.Clearly brown staining restricted to cytoplasm were considered as positive expression of TGF-β1 protein.Clearly brown staining restricted to nucleus was considered as positive expression of P21^waf1 protein.The evaluation was carded out by checking the number of positive cells. 3.Statistical AnalysisData was analyzed by Chi-Square Test,Fisher's Exact test and Spearman's rank correlation test by using SPSS version 13.0.P-values＜0.05 were considered statistically significant.Results1.Smad4-positive staining was observed in the cytoplasm and simultaneously in the nucleus of cells.The positive expression rate of Smad4 in LSCC was 46.00%, which was much lower than 100.00%in normal epithelial tissues(P＜0.05).There was a significant difference of the positive expression rate of Smad4 between the group of stageⅠ～Ⅱwith 63.64%and the group of stageⅢ～Ⅳwith 32.14% (P＜0.05).There was a significant difference of the positive expression rate of Smad4 between the group of well differentiated with 62.96%and the group of moderately and/or poorly differentiated with 26.09%(P＜0.05).For all other clinicopathological features such as age,sex,locations,cervical lymph node metastasis,no association could be demonstrated(P＞0.05).2.TGF-β1-positive staining was observed in the cytoplasm of cells.The positive expression rate of TGF-β1 in LSCC was 86.00%,which was much higher than 40.00%in normal epithelial tissues(P＜0.05).There was a significant difference of the positive expression rate of TGF-β1 between the group of stageⅠ～Ⅱwith 72.73%and the group of stageⅢ～Ⅳwith 96.43%(P＜0.05).There was a significant difference of the positive expression rate of Smad4 between the group of well differentiated with 74.07%and the group of moderately and/or poorly differentiated with 100.00%(P＜0.05).For all other clinicopathological features such as age,sex,locations,cervical lymph node metastasis,no association could be demonstrated(P＞0.05).3.P21^waf1-positive staining was observed in the nucleus of cells.The positive expression rate of P21^waf1 in LSCC was 34.00%,which was much lower than 80.00%in normal epithelial tissues(P＜0.05).There was a significant difference of the positive expression rate of P21^waf1 between the group of stageⅠ～Ⅱwith 50% and the group of stageⅢ～Ⅳwith 21.43%(P＜0.05).There was a significant difference of the positive expression rate of P21^waf1 between the group of well differentiated with 48.15%and the group of moderately and/or poorly differentiated with 17.39%(P＜0.05).For all other clinicopathological features such as age,sex,locations,cervical lymph node metastasis,no association could be demonstrated(P＞0.05).4.In 23 cases of positive expression of Smad4,there were 16 cases that also expressed TGF-β1(69.57%).Whereas 27 out of 27 cases with negative expression of Smad4 had TGF-β1 expression.There was negative correlation between the expression of Smad4 and TGF-β1(P＜0.05);In 23 cases of positive expression of Smad4,there were 15 cases that also expressed P21^waf1(65.22%).Whereas 2 out of 27 cases with negative expression of Smad4 had P21^waf1 expression.There was positive correlation between the expression of Smad4 and P21^waf1(P＜0.05)Conclusion1.The expression of Smad4 and P21^waf1 protein was decreased in LSCC compared with that in normal squmous epithelial tissue.The expression of TGF-β1 protein was increased in LSCC compared with that in normal squmous epithelial tissue. The expression of both of the three proteins closly correlated with pathological grading and clinical staging of LSCC.2.The expression of Smad4 in LSCC correlated with the expression of TGF-β1 and P21^waf1.3.The abnormal expression of Smad4,TGF-β1 and P21^waf1 may play an important role in the carcinogenesis and progression of LSCC.Combining detecting the expression of Smad4,TGF-β1 and P21^waf1 will contribute to the diagnosis and therapeutic for LSCC.