Expression Of CEACAM1in Hepatocellular Carcinoma And Its Function

Hepatocellular carcinoma (HCC) is the fifth most common cancer with characteristics of progressive development, high postsurgical recurrence and extremely poor prognosis. The dismal outcome has been attributed to the highly vascular nature of HCC, which increases the propensity to spread and invade into neighboring or metastasize to distant sites. Especially, the particularly high rate of postsurgical recurrence renders this disease a major challenge that is highly refractory to conventional chemotherapy and radiation. Therefore, identification of novel markers to stratify individuals at high risk of relapse after curative resection to benefit from adjuvant therapy is of great concern to improve clinical outcomes.Carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1), is a member of the CEA subfamily and of the immunoglobulin superfamily, which can be detected on some immune cells as well as on epithelial cells. Different views were held about the function of CEACAM1in tumor cell growth and prognosis in different types of cancer. In HCC, some studies showed that CEAGAM1expression was down-regulated in tumor cells, and loss of CEACAM1expression reflected aggressive tumor biology and thus indicates a poor prognosis. Recently, Zhou,et al indicated that membranous and cytoplastic CEACAM1exerted different function in angiogenesis and lymphangiogenesis in oral carcinoma. However, to the best of our knowledge, no data was available regarding the effects of CEACAM1distribution of tumor cells on tumor angiogenesis and relapse of HCC after curative surgical resection.The presence of human soluble CEACAM1protein can be observed in the serum of healthy donors. Furthermore, variations in serum levels of the soluble CEACAM1protein are observed in various pathologies. For example, increased CEACAM1 levels were observed in the serum of patients with various hepatic diseases such as obstructive jaundice, primary biliary cirrhosis, autoimmune hepatitis and cholangiocarcinoma. Furthermore, it has been recently shown that serum CEACAM1level is increased in some pancreatic adenocarcinoma patients, presenting evidence for the potential role of soluble CEACAM1as a tumor marker. Recently study also indicated that the soluble CEACAM1levels upgrades in melanoma patients. However, there is still no gold standard assay for quantification of serum CEACAM1. However, to the best of our knowledge, no data was available regarding the expression of soluble CEACAM1in patients with HCC. And also, the function of soluble CEACAM1in patients with HCC is still uncertain.Part1The expression of CEACAM1in hepatocellular carcinoma tissues Research ObjectivesWe aimed to investigate the expression of carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1) and its effects on tumor angiogenesis and relapse-free survival (RFS) after curative resection of hepatocellular carcinoma (HCC).Materials and methodsThe HCC specimens were obtained from97patients with primary HCC, who underwent curative partial hepatectomy as an initial treatment at Department of Gastroenterology and General Surgery, Qilu Hospital, Shandong University, between January2003and December2005. This study was approved by the investigation and Ethical Committee of Qilu hospital according to the standards of the Declaration of Helsinki.1. The detection of CEACAM1and CD34expression in hepatocellular carcinoma tissues. The HCC specimens were obtained from97patients with primary HCC. Expression of CEACAM1and CD34was immunohistochemically detected in HCC specimens, microvessel density (MVD) was determined by counting CD-34positive endothelial cells.2. Association between CEACAM1expression and clinicopathologic variables. Statistical analyses were performed using SPSS13.0for windows. The Chi-square test was used to analyze the association between CEACAM1expression and clinicopathologic variables and MVD.3. The effects of different CEACAM1expression on clinicopathologic relapse-free survival. Kaplan-Meier analysis was used to calculate the survival curves, and log-rank test was used to compare the survival differences of patient subgroups. Multivariate analysis was used to identify significant independent prognostic factors for RFS.Results1. CEACAM1expression was detected in specimens of91patients,53cases showed membranous expression and38cases showed cytoplastic expression. Six patients with loss of CEACAM1expression were excluded in this study.2. CEACAM1cytoplastic expression was significantly associated with larger tumor size (P=0.02), multiple number (P=0.004), vascular invasion (P=0.032), satellite nodules (P=0.007), Edmondson-Steiner Grades (P=0.019), TNM stage (P=0.023) and higher MVD(P=0.001).3. Patients with CEACAM1cytoplastic expression had a significantly lower3-year RFS (26.3%, median21.5months) than those with CEACAMl membranous expression (52.8%, median36months)(P=0.005). According to the Cox proportional hazard regression analyses, CEACAM1cytoplastic expression was an independent prognostic factor for RFS (P=0.031).Conclusions1. CEACAM1expression was common in HCC tissues. Two different patterns of CEACAMl can be detected in HCC tissues.2. CEACAM1cytoplastic expression was closely associated with tumor progression, angiogenesis and poorer RFS.3. Cytoplastic CEACAM1expression in HCC tissue might be a predictor of relapsing phenotype and a possible novel target of antiangiogenic therapy for patients with HCC. Part2The soluble CEACAM1expression in serum of patients with HCC Research ObjectivesWe aimed to investigate the expression of soluble CEACAM1in patients with HCC and the association between different levels of soluble CEACAM1and clinicopathologic factors. Furthermore we explored the function of this soluble protein in the progression of HCC.Materials and methodsSerum was obtained from50healthy volunteers and124patients with HCC. Informed consent was obtained from each healthy volunteers and patients with HCC, and the study protocol conformed to the ethical guidelines of the1975Declaration of Helsinki.1. The detection of soluble CEACAM1in serum of healthy volunteer and patients with hepatocellular carcinoma. The peripheral blood were obtained from50healthy volunteer and124patients with primary HCC. The serum was separated by centrifugal. The level of CE AC AMI in serum was detected by ELISA.2. Association between soluble CEACAM1levels and clinicopathologic variables. Statistical analyses were performed using SPSS13.0for windows. The Chi-square test was used to analyze the association between soluble CEACAM1levels and clinicopathologic variables and MVD.Results1. Soluble CEACAM1can be detected in serum of healthy volunteer and patients with primary HCC. The level of soluble CEACAM1in serum of patients with primary HCC was significantly higher than that in healthy people (p<0.05).2. High level of soluble CEACAM1expression in patients with primary HCC was significantly associated with positive HBsAg status in serum, larger tumor size, multiple number, vascular invasion, TNM stage and higher MVD in HCC tissues (p<0.05).Conclusions 1. Soluble CEACAM1can be detected in healthy people and patients with HCC. The level of soluble CEACAM1in serum of patients with primary HCC was significantly higher.2. High level of soluble CEACAM1expression in patients with primary HCC was closely associated with tumor progression and aggressive tumor biology. The soluble CEACAM1may play an important role in tumor angiogenesis. Part3The effect and mechanism of soluble CEACAM1in the angiogenesis in vitro experimentResearch ObjectivesWe aimed to investigate the effect and mechanism of the soluble CEACAM1in the angiogenesis in vitro experiment.Materials and methods1. Supernatant preparation of HepG2.2.15and HepG2cells.5×105cells were seeded in100ml flasks. The cells were cultured in new culture medium for48hours after the cells reached60%-80%confluence. Then the supernatant was collected and conserved at the environment of-80℃.2. The detection of CEACAM1expression in HepG2.2.15and HepG2cells. Real-time-PCR was used to detected the gene expression of CEACAM1in HepG2.2.15and HepG2cells, whereas ELISA was used to detected the CEACAM1expression in supernatant of those two cell lines.3. The effect of HepG2.2.15and HepG2cells derived supernatants on tubulogenesis. A96-well plates with80u1matrigel in every well was incubated for30minutes at37℃. HUVECs (10000cells/well) cells were cultured with the supernatants derived from HeG2.2.15or HepG2cells. Then the cells with supernatant was incubated in the96-well plates with matrigel for6-8hours.4. The detection of VEGF expression in HepG2.2.15and HepG2cells. Real-time PCR was used to detected the gene expression of VEGF in HepG2.2.15and HepG2cells, whereas ELISA was used to detected the VEGF expression in supernatant of those two cell lines.5. The effect of human recombinant CEACAM1on tubulogenesis. A96-well plates with80ul matrigel in every well was incubated for30minutes at37℃. HUVECs (10000cells/well) cells were cultured with different concentration of human recombinant CEACAM1. Then the cells with supernatant was incubated in the96-well plates with matrigel for6-8hours.6. The signaling pathways of CEACAM1on tubulogenesis. A96-well plates with80ul matrigel in every well was incubated for30minutes at37℃. HUVECs (10000cells/well) cells were cultured with PI3K inhibitor, JAK3inhibitor, MEK inhibitor and NF-kB inhibitor1hour. Then the cells were stimulated by CEACAM1(50ng/ml)6-8hours.Results1. The level of CEACAM1is higher in HepG2.2.15cells than that in HepG2cells, both in gene and protein expression.2. The supernatant of HepG2.2.15cells has stronger effect on tubulogenesis than that of HepG2cells.3. The VEGF expression was similar between HepG2.2.15cells and HepG2cells, both in gene and protein expression.4. The human recombinant CEACAM1has direct stong effect on tubulogenesis and was dose-dependent.5. PI3K inhibitor had strong inhibitory effects on tubulogenesis of supernatant derived from HepG2.2.15cells. Whereas JAK3inhibitor, MEK inhibitor and NF-kB inhibitor had no inhibitory effects on tubulogenesis.Conclusion1. Hepatitis B virus infection may induce the expression of CEACAM1.2. CEACAM1has direct strong effects on tubulogenesis. However this function has no correlation with VEGF.3. PI3K signaling pathways may play an important role in tubulogenesis of CEACAM1.