The Immunoexpression of YAP1 and LATS1 Proteins in Clear Cell Renal Cell Carcinoma: Impact on Patients' Survival.

The aim of the study was to determine by immunohistochemistry cellular localization and immunoreactivity levels of YAP1 and LATS1 proteins in paired sections of tumor and unchanged renal tissues of 54 clear cell renal cell carcinoma (ccRCC) patients. Associations between clinical-pathological and overall survival (OS; median follow-up was 40.6 months) data of patients and YAP1 and LATS1 immunoreactivity were analyzed by uni- and multivariate Cox regression model and log-rank test. YAP1 immunoreactivity was found in the nuclei of tumor cells in 64.8% of ccRCC patients, whereas only 24.1% of tumors revealed cytoplasmic YAP1 expression. LATS1 immunoexpression was observed only in the cytoplasm of tumor cells in 59.3% of patients. LATS1 immunoreactivity in cancer cells negatively correlated with the size of primary tumor. The overall YAP1 immunoreactivity did not correlate with clinical-pathological data of patients. However, the subgroup of ccRCC patients who presented with cytoplasmic YAP1 immunoexpression had significantly shorter OS (median = 26.8 months) than patients without cytoplasmic YAP1 expression (median undefined). Multivariate Cox analysis revealed that increased cytoplasmic YAP1 (HR = 4.53) and decreased LATS1 immunoreactivity levels (HR = 0.90) were associated with worse prognosis, being independent prognostic factors. These results suggest that YAP1 and LATS1 can be considered as new prognostic factors in ccRCC.

Differential antitumor effects of vitamin D analogues on colorectal carcinoma in culture.

Colorectal cancer (CRC) is an emerging global problem with the rapid increase in its incidence being associated with an unhealthy lifestyle. Epidemiological studies have shown that decreased levels of vitamin D3 significantly increases the risk of CRC. Furthermore, negative effects of vitamin D3 deficiency can be compensated by appropriate supplementation. Vitamin D3 was shown to inhibit growth and induce differentiation of cancer cells, however, excessive vitamin D3 intake leads to hypercalcemia. Thus, development of efficient vitamin D3 analogues with limited impact on calcium homeostasis is an important scientific and clinically relevant task. The aims of the present study were to compare the antiproliferative potential of classic vitamin D3 metabolites (1α,25(OH)2D3 and 25(OH)D3) with selected low calcemic analogues (calcipotriol and 20(OH)D3) on CRC cell lines and to investigate the expression of vitamin D-related genes in CRC cell lines and clinical samples. Vitamin D3 analogues exerted anti-proliferative effects on all CRC cell lines tested. Calcipotriol proved to be as potent as 1α,25(OH)2D3 and had more efficacy than 20-hydroxyvitamin D3. In addition, the analogs tested effectively inhibited the formation of colonies in Matrigel. The expression of genes involved in 1α,25(OH)2D3 signaling and metabolism varied in cell lines analysed, which explains in part their different sensitivities to the various analogues. In CRC biopsies, there was decreased VDR expression in tumor samples in comparison to the surgical margin and healthy colon samples (p<0.01). The present study indicates that vitamin D3 analogues which have low calcemic activity, such as calcipotriol or 20(OH)D3, are very promising candidates for CRC therapy. Moreover, expression profiling of vitamin D-related genes is likely to be a powerful tool in the planning of anticancer therapy. Decreased levels of VDR and increased CYP24A1 expression in clinical samples underline the importance of deregulation of vitamin D pathways in the development of CRC.

TL1A as a Potential Local Inducer of IL17A Expression in Colon Mucosa of Inflammatory Bowel Disease Patients.

Interaction between TL1A and death receptor 3 (DR3) co-stimulates T cells, induces production of several pro-inflammatory cytokines and has been linked to pathogenesis of inflammatory bowel disease (IBD). This study aimed to establish a link between expression of TL1A and selected TL1A-induced pro-inflammatory cytokines involved in IBD pathogenesis (IL-4, IL-13, IL-17A and IFN-γ) and to investigate a connection between serum concentration of TL1A in patients with IBD and activation of peripheral blood T cells. Elevated levels of IL-4 (2.91-fold) and IL-13 (4.05-fold) mRNA were detected in the inflamed colon mucosa of patients with ulcerative colitis (UC), IFN-γ mRNA was upregulated (3.23-fold) in the inflamed colon mucosa of patients with Crohn's disease (CD), whereas upregulation of IL-17A and TL1A mRNA was present in the inflamed colon mucosa of patients with both CD and UC (IL-17A: 4.48-fold and 2.74-fold, TL1A: 3.19-fold and 3.22-fold, respectively) vs. control subjects. We did not detect any changes in DR3 mRNA expression in the investigated groups of patients. TL1A mRNA level in colon mucosa of patients with IBD correlated only with the level of IL-17A mRNA but no other investigated cytokines. In colon mucosa, expression of TL1A and DR3 was localized to enterocytes and lamina propria mononuclear cells. We did not find any correlation between serum concentrations of TL1A and IL-17A or changes of CD4(+) or CD8(+) lymphocytes phenotype in patients with IBD. Therefore, our data indicate that TL1A may contribute to pathogenesis of IBD via local but not systemic induction of IL-17A but not IL-4, IL-13 or IFN-γ.

Influence of receptor activator of nuclear factor kappa B ligand, osteoprotegerin and interleukin-33 on bone metabolism in patients with long-standing ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic disease with periods of remission and recurrences. Dysfunction of the local immune response leads to chronic inflammation within the large intestine which triggers morphological changes in the intestinal wall as well as induces the synthesis of numerous factors that have an adverse impact on the bone metabolism. The aim of the study was to determine the expression of RANKL, OPG and IL-33 in mucosal biopsies of UC patients with long disease duration as well as serum level of these cytokines in the context of bone density and bone metabolism. MATERIALS AND METHODS: The UC group consisted of 56 patients with average disease duration of 16y. The control group comprised 37 healthy individuals. Local expression of cytokines was assessed in the biopsies of colonic mucosa by the real-time PCR and immunohistochemistry (IHC), and their serum concentration was measured by ELISA. RESULTS: The increased bone resorption observed in patients with UC was reflected by low bone density and high serum level of C-terminal telopeptide (CTX). Mucosal RANKL expression and serum concentration were similar in UC group and healthy subjects, however, UC patients had higher local expression of OPG and serum OPG concentration. Increased IL-33 gene expression was observed only in UC at the mRNA level. We propose that bone resorption in UC patients despite OPG up-regulation could be caused by IL-33-induced mucosal synthesis of a potent proinflammatory cytokine, such as TNF-α, known as a possible inducer of osteoclastogenesis in the way independent of RANKL.

Overexpression of the fragile histidine triad (FHIT) gene in inflammatory bowel disease.

OBJECTIVE: FHIT gene encodes human diadenosine triphosphate hydrolase involved in the regulation of cell cycle and nucleotide metabolism and is a candidate tumor suppressor gene. AIM: To investigate expression of FHIT gene at the mRNA and protein levels in sporadic inflammatory bowel disease (IBD). MATERIALS AND METHODS: FHIT mRNA was quantified by the validated real-time PCR (QPCR) and FHIT protein was detected by immunohistochemistry (IHC) in mucosal biopsies of 139 ulcerative colitis (UC), 19 Crohn's disease (CD) and 37 control patients. RESULTS: Significant FHIT gene overexpression was found in 78% of active UC but not in CD. IHC showed comparable results to QPCR. CONCLUSION: The local up-regulation of FHIT gene and protein expression in active UC may represent an adequate response against inflammatory challenge of epithelial cell homeostasis and protect against DNA damage and cell cycle disturbances.

Fragile histidine triad (FHIT) gene is overexpressed in colorectal cancer.

OBJECTIVE: FHIT gene, mapped at FRA3B site, encodes human diadenosine triphosphate hydrolase involved in the regulation of cell cycle and nucleotide metabolism. Decreased FHIT gene expression was previously observed in various types of human cancer, however, quantification of FHIT mRNA was seldom performed. AIM: To investigate loss of heterozygosity (LOH) at FRA3B, expression of FHIT gene at the mRNA and protein levels in sporadic colorectal carcinoma (CRC) and benign colon adenoma. MATERIALS AND METHODS: FHIT mRNA was quantified by the validated realtime PCR (QPCR) in tumor samples of 84 CRC patients and mucosal biopsies of 15 adenomas, in comparison to 37 control patients, whereas subgroup of 57 CRC, 10 adenoma and 10 control cases were selected for immunohistochemical (IHC) detection of the native FHIT protein and LOH determination at FRA3B. RESULTS: Higher level of FHIT mRNA was found in 86% of CRC (P<0.001) and 60% of adenomas (P=0.016). IHC showed comparable results to QPCR (P=0.003), revealing the strongest presence of FHIT protein in Dukes' C/D stages (P<0.001) and N1/N2 lymph nodes metastasis in CRC (P=0.04). FHIT gene expression and Dukes' and G staging were positively correlated in CRC as analyzed by QPCR and IHC. Deletion analysis of the fragile FRA3B site revealed the highest LOH frequency at D3S1234 in 32.5% of CRC informative cases, however, LOH did not correspond to QPCR, IHC or clinical-pathological variables. CONCLUSION: Our data suggest that reduction or absence of the FHIT gene expression is not a prerequisite for colorectal cancer development and progression.

Decreased Toll-like receptor-5 (TLR-5) expression in the mucosa of ulcerative colitis patients.

OBJECTIVE: Although there is a convincing evidence supporting an important role for microorganisms in the pathogenesis of Inflammatory Bowel Disease (IBD) which comprises ulcerative colitis (UC) and Crohn's disease (CD), the specific mechanisms involved remain unclear. Toll-like receptors (TLR) recognize various molecules of microbiota including flagellin, the principal protein of motile comensal and pathogenic bacteria implicated in the pathogenesis of IBD. AIM: To investigate the expression of the TLR-5 receptors at the mRNA and protein levels in the mucosa of UC patients. MATERIALS AND METHODS: TLR-5 mRNA was quantified by the validated real-time PCR (QPCR) in mucosal biopsies of 99 UC patients and 34 control patients and TLR-5 protein was detected by immunohistochemistry (IHC) in 57 UC and 10 control patients. RESULTS: Significantly decreased TLR-5 gene expression at mRNA and protein level was found in the mucosa of patients with moderate and severe disease activity as compared to patients with low UC activity and control. TLR-5 immunoreactivity was found in the mucosa of UC patients and normal controls in the cytoplasm of enterocytes and at their basolateral domain. However, the intensity of the IHC reaction in specimens from UC patients was substantially lower than in control samples. CONCLUSION: The decreased expression of TLR-5 gene and protein in the mucosa of UC patients suggests that down-regulation of TLR-5 is probably caused by the increased number of ligand molecules in the proximity of epithelial cells in the inflamed tissue.