ABSTRACT
Early molecular detection of the colorectal dysplasia-carcinoma transition may augment the accuracy of diagnosis in case of biopsy orientation errors. The combination of high-throughput microarray-based biomarker screening with tissue microarray-based prospective protein biomarker expression analysis could represent an additional test in routine automated diagnostic procedures. Our aim was to test and select protein markers to identify protein expression profile alterations, focusing on the dysplasia-carcinoma transition in sporadic colorectal tumors. Dysplasia-carcinoma transition-specific transcript sets were previously identified using HGU133plus2 microarrays and Taqman RT-PCR cards. Here, 26 potential dysplasia-carcinoma transition-specific markers were tested by immunohistochemistry at the protein level using tissue microarrays in a total of 168 independent colonic biopsy samples. A set of 26 transcripts [including matrix metalloproteinase-3 (MMP3) and chemokine (C-X-C motif) ligand 1 (CXCL1)] has been determined recently, indicating a linear expression correlation with the adenoma-dysplasia-carcinoma sequence, thereby having the potential to discriminate between dysplasia and early malignancy. Currently, we find that high-grade dysplastic sessile adenomatous-stage and early-stage colorectal cancer conditions can be differentiated correctly by the stromal expression of MMP3 and CXCL1, respectively, on tissue microarray-based analysis. Furthermore, in cases of sporadic colorectal tumors, MMP3 protein expression in the lamina propria itself seems to be highly specific for the detection of tumorous transition. Our current and recent results indicate that appropriate antibody marker combinations are highly suitable for tissue microarray-based and digital microscopy-based, automated, high-capacity diagnostic application in tumorous colonic diseases.
Subject(s)
Adenoma/diagnosis , Carcinoma, Transitional Cell/diagnosis , Chemokine CXCL1/metabolism , Colon/pathology , Colorectal Neoplasms/diagnosis , Hyperplasia/diagnosis , Matrix Metalloproteinase 3/metabolism , Stromal Cells/pathology , Adenoma/genetics , Adenoma/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Transitional Cell/genetics , Carcinoma, Transitional Cell/metabolism , Chemokine CXCL1/genetics , Colon/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Female , Follow-Up Studies , Gene Expression Profiling , Humans , Hyperplasia/genetics , Hyperplasia/metabolism , Immunoenzyme Techniques , Male , Matrix Metalloproteinase 3/genetics , Middle Aged , Neoplasm Grading , Neoplasm Staging , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/metabolism , Tissue Array AnalysisABSTRACT
In inflammatory bowel diseases the presence of free-circulating DNA (fcDNA) sequences in the sera is an established phenomenon, albeit its real biological function still remains unclear. In our study the immunobiologic effects of a single-dose, intravenously administered fcDNA of normal and colitic origin were assayed in DSS-colitic and control mice. In parallel with disease and histological activity evaluations changes of the TLR9 and inflammatory cytokine signaling gene expression profiles were assayed in isolated cells of the lamina propria. Intravenously administered colitis-derived fcDNA displayed a more prominent beneficial action regarding the clinical and histological severity of DSS-colitis than that of fcDNA of normal origin. Systemic administration of colitis-derived fcDNA significantly altered the expression of certain TLR9-related and proinflammatory cytokine genes in a clinically favorable manner. Presumably due to induction of severe colitis, the subsequent marked inflammatory environment may result changes in fcDNA with a potential to promote the downregulation of inflammation and improvement of tissue regeneration. Elucidating mechanisms of innate immune alterations by nucleic acids may provide further insight into the etiology of inflammatory bowel diseases, and develop the basis of novel nucleic acid-based immunotherapies.
Subject(s)
Colitis/chemically induced , Colitis/prevention & control , DNA/pharmacology , Dextran Sulfate/toxicity , Disease Models, Animal , Inflammation/prevention & control , Administration, Intravenous , Animals , Colitis/pathology , DNA/administration & dosage , DNA/blood , Gene Expression Regulation , Inflammation/immunology , Male , Mice , Mice, Inbred C57BL , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/metabolismABSTRACT
Toll-like receptors (TLRs) recognize specific motifs which are frequently present in bacteria, fungi, prokaryotes and viruses. Amongst TLRs, TLR9 can be activated by such bacterial or viral DNA fragments, immunoglobulin-DNA complexes or synthetic oligonucleotides, which all contain unmethylated cytosine-guanine nucleotide sequences (CpGs). Emerging data indicate that TLR9 signaling has a role in, and may influence, colorectal carcinogenesis and colonic inflammation. CpGs are classified into three groups according to their influence on both the antigen-specific humoral- and cellular immunity, and the production of type 1 interferons and proinflammatory cytokines. TLR9 activation via CpGs may serve as a new therapeutic target for several cancerous and various inflammatory conditions. Due to its probable anti-cancer effects, the application possibilities of TLR9-signaling modulation may be extremely diverse even in colorectal tumors. In this review we aimed to summarize the current knowledge about TLR-signaling in the pathogenesis and therapy of inflammatory bowel diseases and colorectal cancer. Due to the species-specific differences in TLR9 expression, however, one must be careful in translating the animal model data into the human system, because of the differences between CpG-oligodeoxynucleotide-responsive cells. TLR9 agonist DNA-based immunomodulatory sequences could also represent a promising therapeutic alternative in systemic inflammatory conditions and chronic colonic inflammations as their side effects are not significant.
