The Expression Of Smad4,uPA,PAI-1 And Their Significance In Colorectal Carcinoma

Background and objectives: Colorectal Carcinoma is one of the malignant digestive carcinomas. As other tumors, the mechanism of colorectal carcinogenesis has not been clear. Smad4 gene, also named DPC4 ( deleted in pancreatic carcinoma locus 4) gene, has been recently identified as a tumor suppressor. Its product Smad4 protein takes part in intracellular signal transduction of the transforming growth factor beta (TGF- β ) superfamilies. Being a potent negative regulator of cell proliferation. TGF-β play an important role in inhibiting normal epithelial cell growth. Smad4 is a major component in TGF- β signal transduction. Loss and/or mutation of gene may disturb this transduction, which makes cell be out of control. Urokinase type plasminogen activator (uPA) is serine proteinase, and it's function is to convert the plasminogen to the active proteinase, plasmin .The main functions of uPA include tissue remodeling and cell migration . Since it is involved in extracellular matrix degradation, uPA may play a key role in the invasion and metastasis of carcinoma .Several studies have suggested that uPA may be a useful prognostic indicator for carcinoma. Plasminogen activator inhibitor 1 (PAI-1) is a member of protease inhibitors. PAI-1 can inhibit uPA by forming stable complexes, and it participates in tissue repair processes. Oppositely , many studies have showed that a high expression of plasminogen activator inhibitor-1 in specimens from tumours is associated with a poor prognosis. Some literatures suggested that uPA and PAI-1 were the target genes of Smad4. In order to investigate the possible mechanism of colorectal carcinogenesis and the clinicopathologicsignificance of Smad4, uPA and PAI-1 protein expression in colorectal carcinoma as well as their interaction, an immunohistochemical SP method was used to examine the expression of the three kinds of proteins. It is expected that the study can help us to judge the diagnosis, therapy and prognosis of colorectal carcinoma.Materials and methods: 1. Tissue specimens used for this study were obtained from 83 patients which were resected surgically or endoscopically at Henan provincial People's Hospital and Henan Provincial Tumor Hospital from Jul.2003 to Apr.2004. Tissue specimens were stored at -70°C until needed. All the samples including 53 carcinomas, 19 adenomas, 11 normal colorectal mucosas were fixed in 4% Polyoxymethylene 0.1M PBS (0.1%DEPC) and embedded in paraffin. 2. The expression of Smad4, uPA, PAI-1 protein were detected by the standard streptavidin-peroxidase (SP) technique. 3. The statistical software package SPSS 10.0 was used. A two-tailed P-value less than 0.05 was considered to indicate statistical significance.Results: l.The positive expressions of Smad4 in colorectal carcinomas were much lower than those in normal colorectal mucosas and adenomas (P <0.01) , and there were no difference between normal colorectal mucosas and adenomas (P >0.05) .The positive expressions of uPA in colorectal carcinomas were significantly higher than those in normal colorectal mucosas and adenomas (P <0.01) , but there were no difference between normal colorectal mucosas and adenomas (P>0.05) .The positive expressions of PAI-1 in colorectal carcinomas were higher than those in normal colorectal mucosas and adenomas (P <0.01) , but there were no difference between normal colorectal mucosas and adenomas (P>0.05) .2. The positive expression rate of Smad4 had no correlation with the patients' sex, age, position of tumor and the depth of invasion (P >0.05) . In highly, moderately and poorly differentiated colorectal carcinomas, the positive rates of Smad4 protein were 100%, 87.50% and 33.33%. The positive expression rate of Smad4 in poorly differentiated group was significantly lower than that in highly and moderately differentiated group (P <0.01) , but there was no difference between highly andmoderately differentiated group (P >0.05) . There was significant difference of the positive expression rate of Smad4 between the lymph node metastasis negative group (93.33%) and the lymph node metastasis positive group(60.87%), (P <0.05). In the group with liver metastasis and the group without distant metastasis , the positive rates of Smad4 protein were 50.00% and 86.05% .There was significant difference between the two groups (P <0.05). According to Duke's stage, cases of colorectal carcinoma were divided into A-B stage group and C-D stage group. The positive rates of Smad4 protein were 93.33% and 60.87%. There was significant difference between the two groups(P <0.05).3. The positive expression rates of uPA had no correlation with the patients' sex, age, position (P >0.05) . In highly, moderately and poorly differentiated colorectal carcinomas, the positive rates of uPA protein were 47.06%,70.83%,100.00%. The positive expression rate of uPA in poorly differentiated group was higher than that in highly differentiated group (P <0.01) , but no significant difference was found among other groups (P >0.05 , Fisher's Exact Test) . There was significant difference of the positive expression rate of uPA between the non-serosal infiltration group(41.67%) and the serosal infiltration group(78.05%) , (P <0.05). There was significant difference of the positive expression rate of uPA between the lymph node metastasis negative group(53.33%)and the lymph node metastasis positive group(91.30%),(P <0.01). In the group without distant metastasis and the group with liver metastasis , the positive rates of uPA protein were 62.79% and 100.00%. There was significant difference between the two groups (P<0.05). According to Duke's stage, the positive expressions of uPA in the C-D stage group were much higher than those in the A-B stage group. The positive rates were 91.30% and 53.33%. There was significant difference between the two groups(P<0.0l).4. The positive expression rate of PAI-1 had no correlation with the patients' sex, age, position of tumor (P>0.05). In highly, moderately and poorly differentiated colorectal carcinomas,the positive rates of PAI-1 protein were 47.06%, 58.33%, 100.00%. The positive expression rate of PAI-1 in poorly differentiated group washigher than that in highly and moderately differentiated groups(P<0.05), but there was no difference between highly and moderately differentiated group (P>0.05). There was significant difference of the positive expression rate of PAI-1 between the non-serosal infiltration group (33.33%) and the serosal infiltration group(73.17%) , (P <0.05). There was significant difference of the positive expression rate of PAI-1 between the lymph node metastasis negative group (46.67%) and the lymph node metastasis positive group (86.96%) ,(P <0.01). In the group without distant metastasis and the group with liver metastasis , the positive rates of PAI-1 protein were55.81% and 100.00%. There was significant difference between the two groups (P <0.01). According to Duke's stage, the positive expressions of PAI-1 in the C-D stage group were much higher than those in the A-B stage group. The positive rates were 86.96% and 46.67%. There was significant difference between the two groups(P <0.01).5. A negative correlation was observed between the expression of Smad4 and that of uPA (r=-0.337; P<0.05)o A negative correlation was observed between the expression of Smad4 and that of PAI-1 (r=-0.383; P<0.01)?A positive correlation was observed between the expression of uPA and that of PAI-1 (r=0.880; P<0.01)oConclusion:l. The weakened expression of Smad4 might play a fundamental role in colorectal carcinogenesis. The reduced expression of Smad4 was associated with histological grades, lymph node metastasis, distant metastasis and Duke's stage. The lower expression of Smad4 occurred in the later stage of colorectal tumor. Reduced expression of Smad4 may be closely related to the histological grades of colorectal carcinoma. These results also demonstrated Smad4's tumor suppressive function and suggested a potential role for Smad4 as a modulator of cell adhesion and invasion.2. Overexpression of uPA might play an important role in colorectal carcinogenesis. The positive expression of uPA was associated with histological grades, the depth of colorectal carcinoma invasion, lymph node metastasis, distant metastasis and Duke's stage. Overexpression of uPA might be closely related to histological grades , cell migration and tumor metastasis. uPA might be a useful prognostic indicator for carcinoma.3. Overexpression of PAI-1 might play an important role in colorectal carcinogenesis. The positive expression of PAI-1 was associated with histological grades, the depth of colorectal carcinoma invasion, lymph node metastasis, distant metastasis and Duke's stage. Overexpression of PAI-1 might play a dominant role in histological grades , cell migration and tumor metastasis. High expression of PAI-1 in colorectal carcinoma was associated with a poor prognosis.4. A negative correlation between the expression of Smad4 and that of uPA and PAI-1 suggested that uPA and PAI-1 were the target genes of Smad4. Smad4 can regulate the adhesion and invasion of tumor cells by down-regulating uPA and PAI-1.5. Smad4, uPA and PAI-1 regulate colorectal carcinogenesis and development of colorectal carcinoma. It is much more important to investigate the expression of Smad4, uPA and PAI-1 combinedly in colorectal carcinoma than to investigate the expression of one of them . This method has not been reported in the world until now.