The Expression And Significance Of Bax,Caspase-9 And DFF45 In Large Intestine Adenocarcinoma Tissue

Large intestine carcinoma included carcinoma of colon and rectum, it is one of the common gastrointestinal neoplasms. It is the third leading death rate in all cancer in the west world.The incidence of large intestine carcinoma in China is the second in gastrointestinal neoplasms, and the incidence is increasing year by year. The pathology types of large intestine carcinoma were adenocarcinoma, squmous cell carcinoma, undifferentiated carcinoma and so on. For the past few years, The adenocarcinoma was the most common type, and the percentage was 95%. Although the different available therapies,which including surgical operation,radiotherapy, chemical therapy, and molecule targeted therapy have currently been used in the treatment of colorectal carcinoma, and all of these have markedly improved the prognosis of colorectal carcinoma.However results are not satisfied. As for the patients visiting, majority have already been advanced because there are not overt symptoms in the early period of colorectal carcinoma. So the correct early diagnosis is very important to the patients.Now we know that the occurrence and development of tumor are related to the imbalance between cell proliferation and apoptosis , and the process was involved in multiple genes. The disordered of cell apoptosis was the important mechanism in the occurrence and development of carcinoma. And the inactivation of tumor suppressor gene or the activation of proto-oncogene promoted the malignant proliferation of cells, then suppressed the cell apoptosis, and caused the occurrence of carcinoma. With the deep research in the mechanism between large intestine carcinoma and cell apoptosis, the induce therapy using the mechanism of cell apoptosis in large intestine carcinoma is a new research direction. Now many gene related with the occurrence of large intestine carcinoma has been discovered, such as bcl-2 gene family, RAS gene family, ERBB gene family, MYC gene family, p53, and so on.Bax gene was one of the important members in bcl-2 gene family, which could promote the cell apoptosis. The mechanism of Bax inducing cell apoptosis was to induce the Cytochrome C released from mitochondria to the cytoplasm, then combined with Apaf-1, and then activating Caspase-9, starting the apoptosis cascade reaction, inducing cell apoptosis. Caspase-9 was one of the Caspase family members,discovered in recent years, belong to upstream starting Caspase, which mainly activated in endogenous cell apoptosis pathway, then activated the effector Caspase-3 resulting in cell apoptosis. DFF was called Caspase activating DNA enzyme (CAD), and was heterodimer composed with two subunit which molecular weight were 45kD and 40kD, and they were called DFF45(ICAD) and DFF40(CAD). Caspase-3 was substrate of the down- stream Caspase when the cell apoptosis signal started, and then DFF45 was cracked, releasing DFF40, forming deoxyribonuclease, which could cause DNA cracking and chromosome cohesion and then forming apoptosis bodies, caused cell apoptosis.Now the reports related with Bax, Caspase-9 and DFF45 in large intestine carcinoma were less. Our research aimed to detect the expression and significance of Bax, Caspase-9 and DFF45 in large intestine carcinoma, and then try to find the inherent connections with them and find out the mechanism of the occurrence and development of large intestine carcinoma. And provide some clinical significance for colorectal cancer early diagnosis and therapy and prognosis. ObjectivesTo analyze the expression of three proteins and the relationship between clinical and pathology parameters of Bax, Caspase-9 and DFF45 in colorectal cancer by detecting their expression in large intestine carcinoma, lymph node and normal tissue .The correlation analysis of the three proteins was also done. The aim is to detect the primary role and clinical significance of the expression of Bax, Caspase-9 and DFF45 in large intestine carcinoma.Methods:1. Collected filed paraffin specimens of 62 cases with large intestine carcinoma who diagnosed by pathology detection from 2008-6 to 2010-4 in pathology department of affiliated hospital of Hebei Union University. Among them, 20 patients with lymph node metastasis. The qualitative expression of Bax, Caspase-9 and DFF45 in large intestine carcinoma was detected by immunohistochemistry test with SP method in surgical specimens,normal tissues and lymph node. To analysis the relationship between the expression of three proteins in clinical and pathology parameters, and the expressions of the three proteins in primary tumor in situ and metastases tumor of 20 patients with lymph node, and try to analysis the correlations among the three proteins of Bax, Caspase-9 and DFF45.2. Collected fresh surgical specimens and normal tissue samples (>5cm from the cancerous tissue) of 21 patients with large intestine carcinoma who diagnosed by pathology detection from 2010-1 to 2010-7 in affiliated hospital of Hebei Union University. The semi-quantitative expression of Bax, Caspase-9 and DFF45 in large intestine carcinoma and adjacent normal tissues were detected by Western-blotting in surgical specimens. And try to analysis the relationship between the expression of three proteins and Differentiation Grade of the large intestine adenocarcinoma tissue.Results:1. The expression of Bax, Caspase-9 and DFF45 in large intestine carcinoma surgical specimens and normal tissues could be detected by immunohistochemistry. Bax protein mainly located in cytoplasm, with performance of yellowish brown particles, distributed focal or diffuse. The positive expression rate of Bax protein in cancerous tissues and normal tissues were 45.16%(28/62) and 72.58%(45/62). Caspase-9 protein mainly located in cytoplasm and cell nucleus, with performance of yellowish brown particles, distributed diffuse or focal. The positive expression rate of Caspase-9 protein in cancerous tissues and normal tissues were 41.94%(26/62) and 77.42%(48/62). DFF45 protein mainly located in the cell nucleus, with clear brown particles. The positive expression rate of DFF45 protein in cancerous tissues and normal tissues were 43.55%(27/62) and 74.19%(46/62). The expression of Bax, Caspase-9 and DFF45 protein in large intestine carcinoma tissues was less than the expression in normal tissues, and there were statistical significances(P<0.01).2. The expression of Bax, Caspase-9 and DFF45 in large intestine carcinoma tissues and normal tissues could be detected by Western-blotting. And the expression of Bax, Caspase-9 and DFF45 in carcinoma tissues were 0.218±0.045, 0.156±0.030 and 0.110±0.032respectively, while the ones in normal tissues were 0.394±0.062, 0.323±0.060 and 0.256±0.056 respectively. The expression of three proteins in large intestine carcinoma were lower than the normal tissues, and there were statistical significances(P<0.05).3. The results of Western blotting showed that there were 5 poorly differentiated carcinoma , 7 moderately differentiated carcinoma and 9 well differentiated carcinoma in the 21 patients of large intestine carcinoma. The expression of Bax, Caspase-9 and DFF45 in well differentiated carcinoma tissues were 0.278±0.050, 0.183±0.052 and 0.133±0.035 respectively, while the ones in the poorly differentiated and the moderately differentiated carcinoma tissues were 0.110±0.071, 0.131±0.046 and 0.072±0.024. The expression of three proteins is related with the differentiation of the carcinoma. The expression in well differentiated carcinoma were higher than the one in the poorly differentiated and the moderately differentiated carcinoma,and there were statistical significances(P<0.05).4. The relationship between the qualitative expressions and clinical and pathology parameters of Bax, Caspase-9 and DFF45 proteins indicated that the expression of three proteins has no relationship with age, gender, tumor size and the depth of tumor infiltration(P>0.05), and related with the differentiation, Duke's stage and lymph node metastasis. The expression in low differentiation group was lower than the high differentiation ones. The expression in Duke's C and D periods were lower than that in A and B periods. And the ones with lymph node metastasis were low than the ones without lymph node metastasis. There were statistical significances(P<0.05). With the decrease of differentiation , the expression of Bax, Caspase-9 and DFF45 proteins were lower(P<0.05), and the ones with Dukes'C and D periods or lymph node metastasis were lower than the ones with Dukes'A and B periods or without lymph node metastasis(P<0.05).5. The results of immunohistochemistry indicated that in the metastases tumor of lymph node of the 20 patients, The positive rates of Bax,Caspase-9 and DFF45 in large intestine primary tumor lesion were 30.00%(6/20), 25.00%(5/20) and 15.00%(3/20)respectively; while 5.00%(1/20), 0.00%(0/20) and 0.00%(0/20)in the metastases lesion of lymph node。The positive expression rate of Bax, Caspase-9 and DFF45 in large intestine primary carcinoma were all obviously higher than that in metastases of lymph node, there were statistical significances(P<0.05).6. In 62 large intestine carcinoma tissues, Both Bax protein and Caspase-9 protein positive were 16 cases(25.81%), both negative were 24 cases(38.71%), Bax positive and Caspase-9 negative were 12 cases(19.35%), and Bax negative and Caspase-9 positive were 10 cases(16.13%). The expression of Bax protein and Caspase-9 protein in large intestine carcinoma tissues was positive correlation (P=0.028, r=0.280). Both Caspase-9 protein and DFF45 protein positive were 16 cases (25.81%), both negative were 25 cases(40.32%), Caspase-9 protein positive and DFF45 negative were 10 cases(16.13%), and Caspase-9 protein negative and DFF45 positive were 11cases(17.74%). The expression of DFF45 protein and Caspase-9 protein in large intestine carcinoma tissues were positive correlation(P=0.015, r=0.308). Both Bax protein and DFF45 protein positive were 17 cases(25.81%), both negative were 24 case(s38.71%), Bax protein positive and DFF45 negative were 11 cases(17.74%), and Bax protein negative and DFF45 positive were 10 cases(16.13%). The expression of Bax protein and DFF45 protein in large intestine carcinoma tissues was positive correlation(P=0.013, r=0.314).Conclusions:1. The expression of Bax, Caspase-9 and DFF45 could be detected both in large intestine adenocarcinoma surgical specimens and normal tissues. The expression of three proteins in carcinoma tissues was lower than that in normal tissues, the results was that the cell apoptosis were suppressed,and so promoting the occurrence and development of large intestine carcinoma. And they could be one of the biology parameters of imbalances between cell proliferation and apoptosis of the tumor cell2. The expression of Bax, Caspase-9 and DFF45 proteins in large intestine carcinoma tissues had no relationship with some clinical parameters such as age, gender, the size of tumor in situ, and some pathology parameters such as the depth of tumor infiltration, but related with the differentiation, Duke's stage and lymph node metastasis. The low expression of Bax, Caspase- 9 and DFF45 maybe promote the cell growth and lymph node metastasis.3. The expression of Bax, Caspase-9 and DFF45 proteins in the metastatic lesion of lymph node was lower than that in large intestine primary tumor lesion, which indicated that metastasis of tumor lesions in the presence of heterogeneity, and more significant inhibition of apoptosis, and the degree of malignancy increased. 4. The co-expression of Bax, Caspase-9 and DFF45 proteins in large intestine carcinoma was existed, and the expression of Bax was positively correlated with the expression of Caspase-9, and the expression of Caspase-9 was positively correlated with the expression of DFF45. The expression of Bax was positively correlated with the expression of DFF45. It indicated that Bax may promote cell apoptosis by activating Caspase-9; while DFF45 was activated as downstream Caspase. The combined action of three proteins suppressed the tumor occurrence.