Study On Inactive Of Deleted In Colorectal Cancer Gene And Its Correlation To Bcl-2 And Bax In Human Colorectal Carcinoma

PrefaceLarge intestine cancer is one of the commonest malignancies in the world including china. Although early diagnosis and treatment have somewhat improved outcomes of patients, large intestine carcinoma still remains the major killer among Chinese, the DCC (deleted in colorectal cancer) gene was proposed as a tumor suppressor gene, the DCC protein was identified as a transmembrane receptor for netrins, key factors in axon guidance in the developing nervous system, the established role of DCC and netrin-1 during organization of the spinal cord could be viewed as that DCC inactivation might play a significant role in tumorigenesis, In particular, data indicate that, when engaged by netrin ligands, DCC may activate downstream signaling pathways. Moreover, in settings where netrin is absent or at low levels, DCC can promote apoptosis.However, the detailed mechanism of DCC in regulation and control of cell apoptosis of large intestine carcinoma remains unknown. We used immunohistochemical staining S-P method to detect the expression of DCC, Bcl-2, Bax proteins in large intestine cancer tissue. The aim of this study was to explore whether DCC could regulate and control cell apoptosis mainly via influencing the expression of Bcl-2/Bax proteins in large intestine cancer. Materials and Methods1.Large intestine carcinoma specimensSeventy-four large intestine cancer tissue samples were randomly and retrospectively selected from patients with large intestine carcinoma in the First Affiliated Shangyang Hospital of China Medical College from 2005.5 to 2005.10. Among them, The median age was 51.3years, with a range of 28-77 years. The clinical stage was determined according to Dukes' stage. 14 were Dukes' stages A, 33 were Dukes' stages B and 28 were Dukes stages C, 5 were Dukes stages D. 28were well-differentiat, 31 were moderately-differentiated, 15 were poor-differentiated.Main reagentsThe polyclonal rabbit antibodies against human DCC, monoclonal mouse antibodies against human Bcl-2 and Bax, and immunohistochemical staining kits were all purchased from Beijing Zhongshan Biological Technology Co, Ltd.Immunohistochemical stainingSpecimens obtained at surgery were routinely fixed in 10％neutral formalin and embedded in paraffin. Serial 4μm thick sections were cut. Immunohistochemical staining for Bcl-2, Bax, DCC was performed according to the standard streptavidin-biotin-peroxidase (S-P) method. The detailed manipulation was conducted according to the introductions for users. A previously known positive carcinoma tissue was used as positive controls for DCC, Bcl-2, Bax, respectively. PBS 0.01M was used as a negative control to replace the primary antibody.2.Evaluation of scoresThe standard positive DCC expressions were stained brown-yellow mainly in cell plasma, The standard positive Bcl-2 and Bax expressions were also stained brown-yellow mainly in cell plasma. Both the extent and intensity of immunopositivity of DCC and Bcl-2,Bax expressions were scored according to follows, on the high power lence counted five field of vision positive cell population and total cell population. To seek cell percentage, Bellow percentage 10 was negative expression. On the contrary was positive expression. Patho-image analysis system measured optical density and carried out Statistical analysis.3.Statistical analysisStatistical evaluation was performed using chi-square test to differentiate the rates of different groups and using Spearman test to analyze the correlation between the ratio of DCC and the integral of Bcl-2 and Bax. P＜0.05 was considered statistically significant. SPSS 12.0 software for Windows was employed to analyze all data.ResultsThe positive rates ofDCC, Bcl-2, Bax protein expression were 45.9％, 60.8％,48.6％respectively. No correlation was found loss or reduction expression of DCC protein to histological type (P＞0.05), lymphoma metastasis (P＞0.05).DCC protein expression is significantly correlation with tumors degree of the differentiation, Ducks stage (P＜0.05). Bcl-2 protein expression is significantly correlation with Ducks stage(P＜0.05) and lymphoma metastasis (P＜0.01). No correlation was found expression of Bcl-2 protein to histological type (P＞0.05) and tumors degree of the differentiation(P＞0.05). No correlation was found loss or reduction expression of Bax protein to histological type (P＞0.05), lymphoma metastasis and Ducks stage (P＞0.05). DCC protein expression is significantly positive correlation with Bax (r=0.296 P＜0.01). However, there is a negative correlation with Bcl-2 (r=-0.201 P＜0.05).Conclusion1. loss or reduction of DCC proteion expression is a frequent event and DCC gene involves in carcinogenesis of carcinoma and it is a candidate tumors suppressor gene in carcinoma. 2. DCC protein in cell apoptosis of large intestine carcinoma may be directly related to the abnormal expression of Bcl-2, Bax.