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Background Chloride channel accessory 1 (CLCA1) belongs to the calcium-sensitive chloride conductance protein family, which is mainly expressed in the colon, small intestine and appendix. This study was conducted to investigate the functions and mechanisms of CLCA1 in colorectal cancer (CRC). Methods The CLCA1 protein expression level in CRC patients was evaluated by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and western blotting analysis. Using CRISPR/Cas9 technology, CLCA1-upregulated (CLCA1-ACT) and CLCA1-knockout cells (CLCA1-KO), as well as their respective negative controls (CLCA1-ACT-NC and CLCA1-KO-NC), were constructed from the SW620 cell line. Cell growth and metastatic ability were assessed both in vitro and in vivo. The association of CLCA1 with epithelial-mesenchymal transition (EMT) and other signaling pathways was determined by western blotting assays. Results The expression level of CLCA1 in CRC tissues was significantly decreased compared with that in adjacent normal tissue (P

Objective: To characterize the expression of ST13 protein in human tissues for investigation of the function of colorectal cancer related gene ST13.Methods: ST13 ORF was cloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinity chromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinant protein. Immunoblot and immunohistochemical staining were employed to analyze ST13 protein expression in human tissues. Results:The expression and purification of the recombinant ST13 protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterial culture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from 104 to 105 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed that the apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility was approximately 50000, which was about 10000 larger than the 41324 calculated, but the glycosylation of the protein was excluded. Computer modeling revealed the protein to be a hydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributed in cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in the staining intensity of the protein were observed between normal and cancer tissues as well as among different normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with an apparent Mr of 50000. The protein is expressed in colorectal and other epithelial tissues. The expression level of the protein is down-regulated in colorectal cancer and varies among different normal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicates that ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibility that ST13 protein is involved in the development of colorectal cancer through Hsp70 molecular chaperone machinery.

Purpose: This study aims to address the hypothesis that the high-mobility group A2 (HMGA2), an oncofetal protein, relates to survivability and serves as a prognostic biomarker for colorectal cancer (CRC). Experimental Design: This is a retroprospective multiple center study. The HMGA2 expression level was determined by performing immunohistochemistry on surgical tissue samples of 89 CRCs from a training set and 191 CRCs from a validation set. The Kaplan–Meier analysis and COX proportional hazard model were employed to analyze the survivability. Results: Multivariate logistic analysis indicated that the expression of HMGA2 significantly correlates with distant metastasis in training set (odds ratio, OR = 3.53, 95% CI: 1.37–9.70) and validation set (OR = 6.38, 95% CI: 1.47–43.95). Survival analysis revealed that the overexpression of HMGA2 is significantly associated with poor survival of CRC patients (P < 0.05). The adjusted HRs for overall survival were 2.38 (95% CI: 1.30–4.34) and 2.14 (95% CI: 1.21–3.79) in training and validation sets, respectively. Further investigation revealed that HMGA2 delays the clearance of γ-H2AX in HCT-116 and SW480 cells post γ-irradiation, which supports our finding that CRC patients with HMAG2-positive staining in primary tumors had augmented the efficacy of adjuvant radiotherapy (HR = 0.18, 95% CI: 0.04–0.63). Conclusion: Overexpression of HMGA2 is associated with metastasis and unequivocally occurred in parallel with reduced survival rates of patients with CRC. Therefore, HMGA2 may potentially serve as a biomarker for predicting aggressive CRC with poor survivability and as an indicator for better response of radiotherapy. Clin Cancer Res; 17(8); 2570–80. ©2011 AACR.

In this study, we examined the expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) by immunohistochemical staining in 76 tissue sections collected from hepatocellular carcinoma (HCC) patients undergoing hepatectomy. Microvascular density (MVD) was determined by counting endothelial cells immunostained using anti-CD34 antibody. We performed DNA-flow cytometric analyses to elucidate the impact of iNOS and VEGF expression on the cell cycle of HCC. Most of the HCC cells that invaded stroma were markedly immunostained by iNOS antibody. The iNOS stain intensity of the liver tissue close to the tumor edge was stronger than that of HCC tissue, and the strongest was the hepatocytes closer to the tumor tissue. However, iNOS expression in 10 normal hepatic samples was undetectable. VEGF positive expression ratio was 84.8% in iNOS positive expression cases, and the ratio was 35.3% in negative cases. There was significant correlation (P = 0.000) between iNOS and VEGF expression. Moreover, iNOS expression was significantly associated with bcl-2 and MVD, but without p53 expression. DNA-flow cytometric analyses showed that combined expression of iNOS and VEGF had significant impact on the cell cycle in HCC. PI (Proliferating Index) and SPF (S-phase fraction) in the combined positive expression of iNOS and VEGF group was significantly higher than that in the combined negative group. The present findings suggested that iNOS expression was significantly associated with angiogenesis, bcl-2 and cell proliferation of HCC.