Sequential Changes Of Serum Proteasome Or VCP Levelsand Its Significance In Radiotherapy For Esophageal Carcinoma

Background:The ubiquitin-proteasome pathway constitutes the major system for nuclear and extralysosomal cytosolic protein degradation in eukaryotic cells。They were found to catalyze the degradation of various proteins in an ATP-dependent fashion. Thus, it appears to be essential for selective removal of unnecessary proteins, such as those with a rapid turnover and abnormal proteins generated in cells. Valosin-containing protein (VCP), a member of the type II AAA (ATPases associated with a variety of activities), is known to be involved in the ubiquitin-dependent proteasome degradation pathway. Accumulating evidence indicates that the UPP plays an essential role in malignant transformation and correlates with disease activity. In the present study, we analyzed the circulating expression levels of proteasome and VCP in esophageal squamous cell carcinoma by a self-developed sandwich enzymelinked immunosorbent assay (ELISA) and assessed its correlation with tumor recurrence and prognosis. We could provide theoretical basis of forepart diagnosis and genetic treatment of esophageal carcinoma.Objective:To immunize the rabbit by VCP antigen to generate VCP polyclonal antibody, the titer was detected by ELISA. To find out the sequential changes of serum proteasome and VCP levels and their significance in radiotherapy and the association between the expression of VCP and proteasome, the circulating expression level of proteasome and VCP in esophageal carcinoma patients and normal people were examined by ELISA. Method:1. PatientsA total of 37 esophageal carcinoma patients who underwent radiotherapy at the Department of Radiation Oncology, Qilu Hospital, Shandong University during the period from July 2008 to February 2009 were selected. The patients who had undergone surgery or other anti-tumor therapy and the patients with any disease which can influence the level of serum proteasome and VCP were not included. And all the 37 patients finished the radical radiation treatment. Pre- and post-treatment blood samples were available.30 normal people who excluded other disease which can influence the level of serum proteasome and VCP were selected as controls.2. Preparation of antibodyImmunize the rabbit by VCP antigen to generate VCP polyclonal antibody. Thetiter was detected by ELISA.3. Proteasome and VCP ELISAThe circulating level of proteasome and VCP in esophageal squamous cellcarcinoma and normal people was detected by ELISA and Western-bloting.4. Statistical AnalysisStatistical analyses were performed with JMTJFX10.34 software. Comparisons between the independent groups of esophageal carcinoma patients and normal people were calculated by the two-sample t-test. For the longitudinal comparison of pre-versus post-treatment samples, the Wilcoxon test was applied. Comparisons of each stage was performed by one-way ANOVA test and the Newman-Keuls methods. The correlation test between proteasome level and VCP level were calculated by linear correlation test and Spearman rank correlation test. P values of <0.05 were considered to be statistically significant.Result:1. There are 29 males and 8 females (male/female=3.6/1) in the 37 patients. Age range from 34 to 68 years (mean 64 years).Every patients were executed electron esophagogastroscopy and biopsy, and diagnosed as esophageal squamous cell carcinoma. Neck, chest, and abdominal computed tomography (CT) were used to determine the clinical staging. stage I :5 cases, stage II :13 cases, stage III:14 cases, stage IV: 5 cases.2. Immunize the rabbit by VCP angiten, ELISA suggested VCP polyclonal antibody's tilter was> 1: 8000.3. The level of proteasome and VCP in sera derived from esophageal carcinoma patients and normal people were detected by ELISA. Both the proteasome contents and the VCP contents of esophageal carcinoma patients were significantly elevated compared to normal people (P=0.0015 and P=0.0026).And the level of proteasome in esophageal carcinoma was correlative with the level of VCP (P=0.0226). The proteasome contents of stage IV cases were much higher than those of stage I,stage II and stageIII cases (P<0.05). In esophageal carcinoma patients, there was a significant decrease from pre- to post- actinotheraphy proteasome concentrations (P =0.0125). To evaluate the short-term follow-up result of radical radiation treatment, there was a significant decrease of effective group than non-effective groups (P =0.0135).4.The seurm level of proteasome of esophageal carcinoma patients is higher than that of normal people by western bloting.Conclusion:1. It is feasible to generate antibody against VCP with VCP antigen. The VCP antibody can be used in ELISA.2. The abnormal higher level of proteasome could be found in esophageal squamous cell carcinoma. Increased serum proteasome concentrations correlate with advanced disease and are an independent prognostic factor in esophageal squamous cell carcinoma.3. The level of VCP in esophageal carcinoma was correlative with the level of proteasome. It implied that VCP involved in the ubiquitin-dependent proteasome degradation pathway.