Beclin 1 Expression in Ovarian Tissues and Its Effects on Ovarian Cancer Prognosis

Beclin 1 is an autophagy-associated protein involved in apoptosis and drug resistance, as well as various malignancies. We investigated the expression of Beclin 1 protein in ovarian epithelial tissues and correlated it with the prognosis of ovarian cancer. Beclin 1 protein expression was determined using immunohistochemistry in 148 patients with ovarian epithelial cancer, 26 with ovarian borderline tumor, 25 with benign ovarian tumor, and 30 with normal ovarian tissue. The relationships between Beclin 1 protein expression and ovarian cancer pathological characteristics were analyzed. The risk factors for ovarian cancer prognosis were analyzed using Cox’s regression model. A survival curve was plotted from the follow-up data of 93 patients with ovarian cancer to analyze the effects of Beclin 1 expression on the prognosis of ovarian cancer. The positive rates of Beclin 1 were significantly higher in ovarian epithelial cancer (148) and borderline tumor (26) than in benign ovarian tumor (25) or normal ovarian tissue (30) (all p < 0.001). The surgical stage and Beclin 1 expression were both independent risk factors for ovarian cancer prognosis (both p < 0.05). Patients with high Beclin 1 levels showed better survival than those with low Beclin 1 levels (p = 0.009). Beclin 1 protein is upregulated in ovarian epithelial cancer and is a prognostic factor of ovarian cancer.     

Analysis of the Circulating Tumor Cell Capture Ability of a Slit Filter-Based Method in Comparison to a Selection-Free Method in Multiple Cancer Types

Circulating tumor cells (CTCs) are a promising biomarker for cancer liquid biopsy. To evaluate the CTC capture bias and detection capability of the slit filter-based CTC isolation platform (CTC-FIND), we prospectively compared it head to head to a selection-free platform (AccuCyte^®-CyteFinder^® system). We used the two methods to determine the CTC counts, CTC positive rates, CTC size distributions, and CTC phenotypes in 36 patients with metastatic cancer. Between the two methods, the median CTC counts were not significantly different and the total counts were correlated (r = 0.63, p < 0.0001). The CTC positive rate by CTC-FIND was significantly higher than that by AccuCyte^®-CyteFinder^® system (91.7% vs. 66.7%, p < 0.05). The median diameter of CTCs collected by CTC-FIND was significantly larger (13.0 μm, range 5.2–52.0 vs. 10.4 μm, range 5.2–44.2, p < 0.0001). The distributions of CTC phenotypes (CK+EpCAM+, CK+EpCAM− or CK−EpCAM+) detected by both methods were similar. These results suggested that CTC-FIND can detect more CTC-positive cases but with a bias toward large size of CTCs.     

Study on the Expression and Clinical Significances of Lewis y Antigen and Integrin αv, β3 in Epithelial Ovarian Tumors

Objective

To detect the expression and clinical significances of Lewis y antigen and integrin αv, β3 in epithelial ovarian tumors, and to explore the expression correlation between Lewis y antigen and integrin αv, β3.

Methods

Immunohistochemical staining was performed in 95 cases of epithelial ovarian cancer, 37 cases of borderline tumors, 20 cases of benign tumors, and 20 cases of normal ovarian tissue, for the detection of Lewis y antigen and integrin αv, β3 expressions, and to analyze the relationship between Lewis y antigen and integrin, and the relationship between clinical and pathological parameters of ovarian cancer. In addition, immunofluorescence double labeling was utilized to detect the expression correlation between Lewis y antigen and integrin αv, β3 in ovarian cancer.

Results

In epithelial ovarian tumors, the expression rate of Lewis y antigen was 81.05%, significantly higher than that of borderline (51.53%) (P < 0.05) and benign (25%) (P < 0.01) tumors, and normal ovarian tissues (0) (P < 0.01). The expression rate of integrin αv, β3 in malignant epithelial ovarian tumors was 78.95% and 82.11%, respectively, significantly higher than that of the borderline (45.94%, 40.54%) (both P < 0.05), benign group (10.00%, 15.00%) (both P < 0.01) and normal ovary group (5%, 15%) (both P < 0.01).

Conclusions

Lewis y and integrins αv, β3 are relevant to pelvic and abdominal diffusion and metastasis of ovarian cancer cells, suggesting that these two molecules mediate a boosting function for tumor metastasis.     

Over-Expression of Semaphorin4D, Hypoxia-Inducible Factor-1�� and Vascular Endothelial Growth Factor Is Related to Poor Prognosis in Ovarian Epithelial Cancer

Semaphorin4D (SEMA4D) has been regarded as an important protein in tumor angiogenesis, though originally identified in neurodevelopment. SEMA4D is extensively expressed in several malignant solid tumors. Nevertheless, the function and expression of SEMA4D in epithelial ovarian cancer (EOC) is as yet not well understood. The aim of this study was to investigate SEMA4D expression in EOC and evaluate its clinical–pathological and prognostic significance. Immunohistochemistry was used to analyze SEMA4D expression and tumor angiogenesis-related proteins (HIF-1α and VEGF) in tissues from 40 patients with normal ovarian epithelia and 124 EOC patients. SEMA4D was found to be expressed in 61.3% of the 124 EOC tissues, which was significantly higher than in the normal ovarian epithelia (p < 0.001). SEMA4D expression correlated with HIF-1α and VEGF closely (ρ = 0.349 and 0.263, p < 0.001). Positive SEMA4D staining was significantly higher in tissues from patients with low histological grade, FIGO stage III-IV, lymph node metastasis and residual disease ≥1 cm (p < 0.05). In the Cox proportional hazard mode, SEMA4D expression and histologic grade were independent indicators of overall survival (OS) and progress-free survival (PFS) for EOC patients. These findings suggest that the cooperation of SEMA4D, HIF-1α, and VEGF may indicate poor prognosis for patients with EOC, thereby demonstrating that SEMA4D and its role in angiogenesis in EOC warrants further study.     

Wnt3a Promotes the Vasculogenic Mimicry Formation of Colon Cancer via Wnt/β-Catenin Signaling

Our previous study provided evidence that non-canonical Wnt signaling is involved in regulating vasculogenic mimicry (VM) formation. However, the functions of canonical Wnt signaling in VM formation have not yet been explored. In this study, we found the presence of VM was related to colon cancer histological differentiation (p < 0.001), the clinical stage (p < 0.001), and presence of metastasis and recurrence (p < 0.001). VM-positive colon cancer samples showed increased Wnt3a expression (p < 0.001) and β-catenin nuclear expression (p < 0.001) compared with the VM-negative samples. In vitro, over-regulated Wnt3a expression in HT29 colon cancer cells promoted the capacity to form tube-like structures in the three-dimensional (3-D) culture together with increased expression of endothelial phenotype-associated proteins such as VEGFR2 and VE-cadherin. The mouse xenograft model showed that Wnt3a-overexpressing cells grew into larger tumor masses and formed more VM than the control cells. In addition, the Wnt/β-catenin signaling antagonist Dickkopf-1(Dkk1) can reverse the capacity to form tube-like structures and can decrease the expressions of VEGFR2 and VE-cadherin in Wnt3a-overexpressing cells. Taken together, our results suggest that Wnt/β-catenin signaling is involved in VM formation in colon cancer and might contribute to the development of more accurate treatment modalities aimed at VM.     

Expression of NGF/proNGF and Their Receptors TrkA,p75NTR and Sortilin in Melanoma

There is increasing evidence that nerve growth factor (NGF) and its receptors, the neurotrophic receptor tyrosine kinase 1 (NTRK1/TrkA), the common neurotrophin receptor (NGFR/p75^NTR) and the membrane receptor sortilin, participate in cancer growth. In melanoma, there have been some reports suggesting that NGF, TrkA and p75^NTR are dysregulated, but the expression of the NGF precursor (proNGF) and its membrane receptor sortilin is unknown. In this study, we investigated the expression of NGF, proNGF, TrkA, p75^NTR and sortilin by immunohistochemistry in a series of human tissue samples (n = 100), including non-cancerous nevi (n = 20), primary melanomas (n = 40), lymph node metastases (n = 20) and distant metastases (n = 20). Immunostaining was digitally quantified and revealed NGF and proNGF were expressed in all nevi and primary melanomas, and that the level of expression decreased from primary tumors to melanoma metastases (p = 0.0179 and p < 0.0001, respectively). Interestingly, TrkA protein expression was high in nevi and thin primary tumors but was strongly downregulated in thick primary tumors (p < 0.0001) and metastases (p < 0.0001). While p75^NTR and sortilin were both expressed in most nevi and melanomas, there was no significant difference in expression between them. Together, these results pointed to a downregulation of NGF/ProNGF and TrkA in melanoma, and thus did not provide evidence to support the use of anti-proNGF/NGF or anti-TrkA therapies in advanced and metastatic forms of melanoma.     

PD-L1 Expression in Non-Small Cell Lung Cancer Specimens: Association with Clinicopathological Factors and Molecular Alterations

Immune checkpoint inhibitors (ICI) targeting programmed cell death-1 or its ligand (PD-L1) have improved outcomes in non-small cell lung cancer (NSCLC). High tumor PD-L1 expression, detected by immunohistochemistry (IHC) typically on formalin-fixed paraffin-embedded (FFPE) histological specimens, is linked to better response. Following our previous investigation on PD-L1 in cytological samples, the aim of this study was to further explore the potential impacts of various clinicopathological and molecular factors on PD-L1 expression. Two retrospective NSCLC cohorts of 1131 and 651 specimens, respectively, were investigated for PD-L1 expression (<1%/1–49%/≥50%), sample type, sample site, histological type, and oncogenic driver status. In both cohorts, PD-L1 was positive (≥1%) in 55% of the cases. Adenocarcinomas exhibited lower PD-L1 expression than squamous cell carcinomas (p < 0.0001), while there was no difference between sample types, tumor locations, or between the two cohorts in multivariate analysis (all p ≥ 0.28). Mutational status correlated significantly with PD-L1 expression (p < 0.0001), with the highest expression for KRAS-mutated cases, the lowest for EGFR-mutated, and the KRAS/EGFR wild-type cases in between. There was no difference in PD-L1 levels between different prevalent KRAS mutations (all p ≥ 0.44), while mucinous KRAS-mutated adenocarcinomas exhibited much lower PD-L1 expression than non-mucinous (p < 0.0001). Our data indicate that cytological and histological specimens are comparable for PD-L1 evaluation. Given the impact of KRAS mutations and the mucinous growth pattern on PD-L1 expression, these factors should be further investigated in studies on ICI response.     

Twist-1 Up-Regulation in Carcinoma Correlates to Poor Survival

Epithelial-to-mesenchymal transition (EMT) facilitates tumor metastasis. Twist is a basic helix-loop-helix protein that modulates many target genes through E-box-responsive elements. There are two twist-like proteins, Twist-1 and Twist-2, sharing high structural homology in mammals. Twist-1 was found to be a key factor in the promotion of metastasis of cancer cells, and is known to induce EMT. Twist-1 participation in carcinoma progression and metastasis has been reported in a variety of tumors. However, controversy exists concerning the correlation between Twist-1 and prognostic value with respect to carcinoma. A systematic review and meta-analysis were performed to determine whether the expression of Twist-1 was associated with the prognosis of carcinoma patients. This analysis included 17 studies: four studies evaluated lung cancer, three evaluated head and neck cancer, two evaluated breast cancer, two evaluated esophageal cancer, two evaluated liver cancer and one each evaluated osteosarcoma, bladder, cervical and ovarian cancer. A total of 2006 patients were enrolled in these studies, and the median trial sample size was 118 patients. Twist-1 expression was associated with worse overall survival (OS) at both 3 years (hazard ratio “HR” for death = 2.13, 95% CI = 1.86 to 2.45, p < 0.001) and 5 years (HR for death = 2.01, 95% CI = 1.76 to 2.29, p < 0.001). Expression of Twist-1 is associated with worse survival in carcinoma.     

Effects of Different Sera Conditions on Olfactory Ensheathing Cells in Vitro

Transplantation of olfactory ensheathing cells (OEC) is a promising therapy in spinal cord injury (SCI) treatment. However, the therapeutic efficacy of this method is unstable due to unknown reasons. Considering the alterations in the culture environment that occur during OEC preparation for transplantation, we hypothesize that these changes may cause variations in the curative effects of this method. In this study, we compared OEC cultured in medium containing different types and concentrations of serum. After purification and passage, the OEC were cultured for 7 days in different media containing 5%, 10%, 15% or 20% fetal bovine serum (FBS) or rat serum (RS), or the cells were cultured in FBS-containing medium first, followed by medium containing RS. In another group, the OEC were first cultured in 10% FBS for 3 days and then cultured with rat spinal cord explants with 10% RS for another 4 days. An MTT assay and P75 neurotrophin receptor immunofluorescence staining were used to examine cell viability and OEC numbers, respectively. The concentration of neurotrophin-3 (NT-3), which is secreted by OEC into the culture supernatant, was detected using the enzyme-linked immunosorbent assay (ELISA). RT-PCR was applied to investigate the NT-3 gene expression in OEC according to different groups. Compared with FBS, RS reduced OEC proliferation in relation to OEC counts (χ² = 166.279, df = 1, p < 0.01), the optical density (OD) value in the MTT assay (χ² = 34.730, df = 1, p < 0.01), and NT-3 concentration in the supernatant (χ² = 242.997, df = 1, p < 0.01). OEC cultured with spinal cord explants secreted less NT-3 than OEC cultured alone (F = 9.611, df = 5.139, p < 0.01). Meanwhile, the order of application of different sera was not influential. There was statistically significant difference in NT-3 gene expression among different groups when the serum concentration was 15% (χ² = 64.347, df = 1, p < 0.01). In conclusion, different serum conditions may be responsible for the variations in OEC proliferation and function.     

Association of CDKN2BAS Polymorphism rs4977574 with Coronary Heart Disease: A Case-Control Study and a Meta-Analysis

The goal of our study was to explore the significant association between a non-protein coding single nucleotide polymorphism (SNP) rs4977574 of CDKN2BAS gene and coronary heart disease (CHD). A total of 590 CHD cases and 482 non-CHD controls were involved in the present association study. A strong association of rs4977574 with CHD was observed in females (genotype: p = 0.002; allele: p = 0.002, odd ratio (OR) = 1.57, 95% confidential interval (CI) = 1.18–2.08). Moreover, rs4977574 was more likely to be a risk variant of CHD under the recessive model in females (χ² = 10.29, p = 0.003, OR = 2.14, 95% CI = 1.31–2.77). A breakdown analysis by age had shown that there was an 87% increased risk of CHD for females younger than 65 years (genotype: χ² = 14.64, degrees of freedom (df) = 2, p = 0.0002; allele: χ² = 11.31, df = 1, p = 0.0008, OR = 1.87, 95% CI = 1.30–2.70). Similar observation was also found in males younger than 65 years (genotype: χ² = 8.63, df = 2, p = 0.04; allele: χ² = 7.55, df = 1, p = 0.006, OR = 1.45, 95% CI = 1.11–1.90). p values were adjusted by age, sex, smoking, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). Meta-analysis of 23 studies among 36,452 cases and 39,781 controls showed a strong association between rs4977574 and the risk of CHD (p < 0.0001, OR = 1.27, 95% CI = 1.22–1.31).     

Different Role of Tumor Necrosis Factor-α Polymorphism in Non-Hodgkin Lymphomas among Caucasian and Asian Populations: A Meta-Analysis

Tumor necrosis factor-α (TNF-α) is an immunoregulatory cytokine involved in B- and T-cell function, and also plays an important role in inflammation and cancer. TNF-α-308G>A has been associated with constitutively elevated TNF-α expression. Several studies have reported the association between the TNF-α-308G>A polymorphism and non-Hodgkin lymphomas (NHL) risk, however, results are still inconsistent. To solve these conflicts, we conducted the first meta-analysis to assess the effect of TNF-α-308G>A polymorphism on the risk of NHL and various subtypes (additive model) including 10,619 cases and 12,977 controls in Caucasian and Asian populations. Our meta-analysis indicated that TNF-α-308G>A polymorphism is not associated with NHL risk when pooling all studies together (OR = 1.06, 95% CI: 0.92–1.23, p = 0.413). In stratified analyses, we found TNF-α-308A allele was significantly associated with higher risk of NHL, B-cell lymphomas (BCL), T-cell lymphomas (TCL) and diffuse large B-cell lymphomas (DLBCL) in Caucasians (OR = 1.22, 95% CI: 1.06–1.40, p = 0.007; OR = 1.18, 95% CI: 1.03–1.34, p = 0.014; OR = 1.20, 95% CI: 1.01–1.42, p = 0.040; OR = 1.21, 95% CI: 1.11–1.32, p < 0.001, respectively). Interestingly, it was associated with decreased risk of NHL, BCL and DLBCL in Asians (OR = 0.75, 95% CI: 0.66–0.86, p < 0.001; OR = 0.70, 95% CI: 0.52–0.94, p = 0.018; OR = 0.70, 95% CI: 0.57–0.86, p = 0.001). These findings also suggest TNF-α might play a distinct role in pathogenesis of NHL in different populations.     

Impact of Wisteria floribunda Agglutinin-Positive Mac-2-Binding Protein in Patients with Hepatitis C Virus-Related Compensated Liver Cirrhosis

We aimed to examine the effect of Wisteria floribunda agglutinin-positive Mac-2-binding protein (WFA⁺-M2BP) level on survival comparing with other laboratory liver fibrosis markers in hepatitis C virus (HCV)-related compensated liver cirrhosis (LC) (n = 165). For assessing prognostic performance of continuous fibrosis markers, we adapted time-dependent receiver operating characteristics (ROC) curves for clinical outcome. In time-dependent ROC analysis, annual area under the ROCs (AUROCs) were plotted. We also calculated the total sum of AUROCs in all time-points (TAAT score) in each fibrosis marker. WFA⁺-M2BP value ranged from 0.66 cutoff index (COI) to 19.95 COI (median value, 5.29 COI). Using ROC analysis for survival, the optimal cutoff point for WFA⁺-M2BP was 6.15 COI (AUROC = 0.79348, sensitivity = 80.0%, specificity = 74.78%). The cumulative five-year survival rate in patients with WFA⁺-M2BP ≥ 6.15 COI (n = 69) was 43.99%, while that in patients with WFA⁺-M2BP < 6.15 COI (n = 96) was 88.40% (p < 0.0001). In the multivariate analysis, absence of hepatocellular carcinoma (p = 0.0008), WFA⁺-M2BP < 6.15 COI (p = 0.0132), achievement of sustained virological response (p < 0.0001) and des-γ-carboxy prothrombin < 41 mAU/mL (p = 0.0018) were significant favorable predictors linked to survival. In time-dependent ROC analysis in all cases, WFA⁺-M2BP had the highest TAAT score among liver fibrosis markers. In conclusion, WFA⁺-M2BP can be a useful predictor in HCV-related compensated LC.     

VEGFR-1 Overexpression Identifies a Small Subgroup of Aggressive Prostate Cancers in Patients Treated by Prostatectomy

The VEGFR-1 is suggested to promote tumor progression. In the current study we analyzed prevalence and prognostic impact of the VEGFR-1 by immunohistochemistry on a tissue microarray containing more than 3000 prostate cancer specimens. Results were compared to tumor phenotype, ETS-related gene (ERG) status, and biochemical recurrence. Membranous VEGFR-1 expression was detectable in 32.6% of 2669 interpretable cancers and considered strong in 1.7%, moderate in 6.7% and weak in 24.2% of cases. Strong VEGFR-1 expression was associated with TMPRSS2:ERG fusion status as determined by fluorescence in situ hybridization (FISH) and immunohistochemistry (p < 0.0001 each). Elevated VEGFR-1 expression was linked to high Gleason grade and advanced pT stage in TMPRSS2:ERG negative cancers (p = 0.0008 and p = 0.001), while these associations were absent in TMPRSS2:ERG positive cancers. VEGFR-1 expression was also linked to phosphatase and tensin homolog (PTEN) deletions. A comparison with prostate specific antigen (PSA) recurrence revealed that the 1.7% of prostate cancers with the highest VEGFR-1 levels had a strikingly unfavorable prognosis. This could be seen in all cancers, in the subsets of TMPRSS2:ERG positive or negative, PTEN deleted or undeleted carcinomas (p < 0.0001 each). High level VEGFR-1 expression is infrequent in prostate cancer, but identifies a subgroup of aggressive cancers, which may be candidates for anti-VEGFR-1 targeted therapy.     

Significant Overexpression of DVL1 in Taiwanese Colorectal Cancer Patients with Liver Metastasis

Undetected micrometastasis plays a key role in the metastasis of cancer in colorectal cancer (CRC) patients. The aim of this study is to identify a biomarker of CRC patients with liver metastasis through the detection of circulating tumor cells (CTCs). Microarray and bioinformatics analysis of 10 CRC cancer tissue specimens compared with normal adjacent tissues revealed that 31 genes were up-regulated (gene expression ratio of cancer tissue to paired normal tissue > 2) in the cancer patients. We used a weighted enzymatic chip array (WEnCA) including 31 prognosis-related genes to investigate CTCs in 214 postoperative stage I–III CRC patients and to analyze the correlation between gene expression and clinico-pathological parameters. We employed the immunohistochemistry (IHC) method with polyclonal mouse antibody against DVL1 to detect DVL1 expression in 60 CRC patients. CRC liver metastasis occurred in 19.16% (41/214) of the patients. Using univariate analysis and multivariate proportional hazards regression analysis, we found that DVL1 mRNA overexpression had a significant, independent predictive value for liver metastasis in CRC patients (OR: 5.764; 95% CI: 2.588–12.837; p < 0.0001 on univariate analysis; OR: 3.768; 95% CI: 1.469–9.665; p = 0.006 on multivariate analysis). IHC staining of the immunoreactivity of DVL1 showed that DVL1 was localized in the cytoplasm of CRC cells. High expression of DVL1 was observed in 55% (33/60) of CRC tumor specimens and was associated significantly with tumor depth, perineural invasion and liver metastasis status (all p < 0.05). Our experimental results demonstrated that DVL1 is significantly overexpressed in CRC patients with liver metastasis, leading us to conclude that DVL1 could be a potential prognostic and predictive marker for CRC patients.     

Loss of ZNF677 Expression Is an Independent Predictor for Distant Metastasis in Middle Eastern Papillary Thyroid Carcinoma Patients

Thyroid cancer incidence has increased in recent decades. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Approximately 30% of PTC patients develop recurrence or distant metastasis and tend to have poor prognosis. Therefore, the identification of targetable biomarkers in this subset of patients is of great importance. Accumulating evidence indicates that zinc finger protein 677 (ZNF677), which belongs to the zinc finger protein family, is an important effector during the progression of multiple malignancies. However, its role in Middle Eastern PTC patients has not been fully illustrated. Here, we uncovered the molecular mechanism and the clinical impact of ZNF677 expression in a large cohort of more than 1200 Middle Eastern PTC and 15 metastatic tissues. We demonstrated that ZNF677 is frequently downregulated in primary PTC (13.6%, 168/1235) and showed that complete loss of expression of ZNF677 is significantly associated with aggressive clinico-pathological markers such as extrathyroidal extension (p = 0.0008) and distant metastases (p < 0.0001). We also found a significantly higher incidence of ZNF677 loss in primary tumors with distant metastases (33.3%; p < 0.0001) as well as in distant metastatic tissues (46.7%; p = 0.0002) compared to the overall cohort (13.6%). More importantly, PTC with loss of ZNF677 expression showed significantly lower metastasis-free survival (p = 0.0090). Interestingly, on multivariate logistic regression analysis, ZNF677 loss was an independent predictor of distant metastasis in PTC (Odds ratio = 2.60, 95% Confidence interval = 1.20–5.62, p = 0.0155). In addition, we found a significant association between ZNF677 loss and phospho-AKT expression (p < 0.0001). Our functional molecular results suggest that ZNF677 acts as a tumor suppressor, mediating its effect by inhibiting AKT phosphorylation. Taken together, our results highlight the pivotal role played by ZNF677 during carcinogenesis and metastasis formation in Middle Eastern PTC patients.     

β-Conglycinin Reduces the Tight Junction Occludin and ZO-1 Expression in IPEC-J2

Soybean allergy presents a health threat to humans and animals. The mechanism by which food/feed allergen β-conglycinin injures the intestinal barrier has not been well understood. In this study, the changes of epithelial permeability, integrity, metabolic activity, the tight junction (TJ) distribution and expression induced by β-conglycinin were evaluated using IPEC-J2 model. The results showed a significant decrease of trans-epithelial electrical resistance (TEER) (p < 0.001) and metabolic activity (p < 0.001) and a remarkable increase of alkaline phosphatase (AP) activity (p < 0.001) in a dose-dependent manner. The expression levels of tight junction occludin and ZO-1 were decreased (p < 0.05). The reduced fluorescence of targets and change of cellular morphology were recorded. The tight junction occludin and ZO-1 mRNA expression linearly declined with increasing β-conglycinin (p < 0.001).     

Expression of Vitamin D Receptor (VDR) Positively Correlates with Survival of Urothelial Bladder Cancer Patients

Vitamin D3 shows tumoristatic and anticancer effects by acting through the vitamin D receptor (VDR), while hydroxylation of 25-hydroxyvitamin D3 at position 1α by CYP27B1 is an essential step in its activation. The expression of both the VDR and CYP27B1 has been found in many normal and cancer tissues, but there is a lack of information about its expression in human bladder cancers. The aim of the present research was to examine whether the expression of the VDR and CYP27B1 in bladder cancer was related to the prognostic markers and disease outcome. We analyzed VDR and CYP27B1 in samples of tumor and normal tissues obtained from 71 urinary bladder cancer patients. The highest VDR immunostaining was found in normal epithelium and was significantly lower in bladder cancer cells (p < 0.001 with Mann–Whitney U test). VDR expression was lowest in more advanced (pT2b–pT4) (p = 0.005 with Mann–Whitney U test) and metastasizing cancers (p < 0.05 and p = 0.004 with Mann–Whitney U test for nuclear and cytoplasmic VDR immunostaining, respectively). The lack of cytoplasmic and nuclear VDR was also related to shorter overall survival (for cytoplasmic VDR immunolocalization 13.3 vs. 55.3 months of survival, HR = 1.92, p = 0.04 and for nuclear VDR immunostaining 13.5 vs. 55.3 months of survival, HR = 2.47, p = 0.002 with Mantel-Cox test). In cases with the lack of high cytoplasmic VDR staining the non-classic differentiations (NDs) was observed in higher percentage of tumor area. CYP27B1 expression was lower in cancer cells than in normal epithelial cells (p = 0.03 with Mann–Whitney U test), but its expression did not correlate with tumor stage (pT), metastasizing, grade, mitotic activity or overall survival. In conclusion, expression of the VDR and CYP27B1 are deregulated in urothelial bladder cancers. Although our results showing a relationship between the decreased VDR expression and prognostic markers and survival time indicate potential usefulness of VDR as a new indicator of a poorer prognosis, further studies are needed in different patient cohorts by independent groups to validate this hypothesis. We also suggest that vitamin D-based therapies may represent an adjuvant strategy in treatment for bladder cancers expressing VDR.     

Advanced Glycation End-Products (AGEs) of Lysine and Effects of Anti-TCR/Anti-TNF-α Antibody-Based Therapy in the LEW.1AR1-iddm Rat,an Animal Model of Human Type 1 Diabetes

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). Previously, we have shown that combination with anti-TCR/anti-TNF-α antibody-based therapy re-established normoglycemia and increased proteinic arginine-dimethylation in the spleen, yet not in the pancreas. High blood glucose is often associated with elevated formation of advanced glycation end-products (AGEs) which act via their receptor (RAGE). Both anti-TCR and anti-TNF-α are inhibitors of RAGE. The aim of the present work was to investigate potential biochemical changes of anti-TCR/anti-TNF-α therapy in the LEW.1AR1-iddm rat. We determined by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS) the content of free and proteinic AGEs and the N^ε-monomethylation of lysine (Lys) residues in proteins of pancreas, kidney, liver, spleen and lymph nodes of normoglycemic control (ngCo, n = 6), acute diabetic (acT1D, n = 6), chronic diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Analyzed biomarkers included Lys and its metabolites N^ε-carboxymethyl lysine (CML), furosine and N^ε-monomethyl lysine (MML). Other amino acids were also determined. Statistical methods including ANOVA, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to evaluate the effects. Most statistical differences between the study groups were observed for spleen, pancreas and kidney, with liver and lymph nodes showing no such differences. In the pancreas, the groups differed with respect to proteinic furosine (p = 0.0289) and free CML (p = 0.0023). In the kidneys, the groups differed with respect to proteinic furosine (p = 0.0076) and CML (p = 0.0270). In the spleen, group differences were found for proteinic furosine (p = 0.0114) and free furosine (p = 0.0368), as well as for proteinic CML (p = 0.0502) and proteinic MML (p = 0.0191). The acT1D rats had lower furosine, CML and MML levels in the spleen than the rats in all other groups. This observation corresponds to the lower citrullination levels previously measured in these rats. PCA revealed diametric associations between PC1 and PC2 for spleen (r = −0.8271, p < 0.0001) compared to pancreas (r = 0.5805, p = 0.0073) and kidney (r = 0.8692, p < 0.0001). These findings underscore the importance of the spleen in this animal model of human T1D. OPLS-DA showed that in total sixteen amino acids differed in the experimental groups.     

The Different Temozolomide Effects on Tumorigenesis Mechanisms of Pediatric Glioblastoma PBT24 and SF8628 Cell Tumor in CAM Model and on Cells In Vitro

It is necessary to elucidate the individual effects of temozolomide (TMZ) on carcinogenesis and tumor resistance to chemotherapy mechanisms. The study aimed to investigate the TMZ 50 and 100 μM dose effect difference between PBT24 and SF8628 cell line high-grade pediatric glioblastoma (phGBM) xenografts in a chicken chorioallantoic membrane (CAM) model, on PCNA and EZH2 immunohistochemical expression in the tumor and on the expression of NKCC1, KCC2, E- and N-cadherin genes in TMZ-treated and control cell groups in vitro. TMZ at a 100 μg dose reduced the incidence of PBT24 xenograft invasion into the CAM, CAM thickening and the number of blood vessels in the CAM (p < 0.05), but did not affect the SF8628 tumor in the CAM model. The TMZ impact on PBT24 and SF8628 tumor PCNA expression was similarly significantly effective but did not alter EZH2 expression in the studied tumors. The TMZ at 50 μM caused significantly increased RNA expression of the NKCC1 gene in both studied cell types compared with controls (p < 0.05). The expression of the KCC2 gene was increased in PBT24 TMZ-treated cells (p < 0.05), and no TMZ effect was found in SF8628-treated cells. The study supports the suggestion that individual sensitivity to TMZ should be assessed when starting treatment.