The aim of this study was to determine the correlation of S100A4 expression with the progression, prognosis and clinical pathology of gastric cancer (GC) in young pateints. A total of 85 tumor tissues with corresponding adjacent normal tissues and 62 non-metastatic lymph nodes (LNs) with corresponding metastatic LNs were obtained from young GC patients (<40 years old) who underwent surgery between January 2001 and December 2006. The expression of S100A4 was detected by RT-PCR and immunohistochemistry. Differences in the expression of S100A4 mRNA or protein were observed among the GC tissues, matched normal gastric mucosa, non-metastatic LNs and metastatic LNs. The expression of S100A4 mRNA and protein in GC tissues and metastatic LNs was significantly higher compared with that in the matched normal gastric mucosa and non-metastatic LNs, respectively (P<0.05). The overexpression of S100A4 was significantly associated with parameters involved in tumor progression and poor prognosis, including tumor size (P=0.017), Lauren classification (P=0.002), histological classification (P= 0.010), histological differentiation (P= 0.000), Borrmann classification (P=0.020), tumor-node-metastasis (TNM) stage (P=0.000), LN metastasis (P=0.000) and distant metastasis (P=0.024). Multivariate analysis suggested that patient age (P=0.035), tumor size (P=0.002), TNM stage (P=0.001) and S100A4 upregulation (P=0.000) were independent prognostic indicators for the disease. The overexpression of S100A4 in young GC patients is significantly associated with the clinicopathological characteristics. S100A4 may be used as a biomarker to predict the progression and poor prognosis of GC in young patients.
Preparations utilizing monoclonal antibodies against S100A4 provide useful tools for functional studies to investigate the clinical applications of the human S100A4 protein. In the present study, human S100A4 protein was expressed in Escherichia coli (E. coli) BL21 (DE3), successfully purified by diethylaminoethyl cellulose anion-exchange chromatography and identified by western blot analysis. Soluble S100A4 bioactivity was confirmed by Transwell migration and invasion assays in the human HeLa cell line. Monoclonal antibodies (mAbs) were generated utilizing the standard hybridoma method and were validated by enzyme-linked immunosorbent assay and western blot analysis. The antibody was then used to examine human gastric carcinoma specimens by immunohistochemistry. Recombinant S100A4 was functionally expressed in E. coli and promoted the migration and invasion of HeLa cells. Four hybridoma cell lines, which secreted mAbs specifically against human S100A4 protein, were obtained. One of the four mAbs, namely 2A12D10B2, recognized human S100A4 as indicated by immunohistochemical staining of human gastric carcinoma specimens and recombinant S100A4 was functionally expressed in E. coli. The mAbs of recombinant S100A4 were suitable for detecting S100A4 expression in human tissues and for investigating the subsequent clinical applications of the protein.
S100A4 protein is associated with Ca2+-dependent regulation of intracellular activities and is significant in the invasion, growth and metastasis of cancer. In order to express rat S100A4 functionally and identify its biological activity following purification, an S100A4 gene fragment was optimized and fully synthesized via overlapping polymerase chain reaction. The gene was inserted into the prokaryotic expression vector, pBV220, with phage λ PRPL promoters following confirmation by DNA sequencing. The pBV220-S100A4 plasmid was constructed and transformed into Escherichia coli DH5α. Following temperature induction, rat S100A4 was overexpressed and the protein was observed to be located in the supernatant of the lysates, which was ~30–40% of the total protein within the host. The protein was isolated and purified by metal-chelate affinity chromatography. High purity protein (>98% purity) was obtained and in vitro western blot analysis identified that the recombinant S100A4 was able to bind to the antibody against wild-type S100A4. The bioactivity of the recombinant protein was detected via Transwell migration and invasion assays. The polyclonal antibody of rat S100A4 protein was prepared for rabbit immunization and exhibited similar efficacies when compared with commercial S100A4. Therefore, rat S100A4 was functionally expressed in E. coli; thus, the production of active recombinant S100A4 protein in E. coli may further aid with the investigation and application of S100A4.
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