Bioactive potential of ripened white cheeses manufactured in different geographical regions of Turkey.

This study investigated the potential bioactive properties of white cheeses produced in different regions of Turkey, including their potential antioxidant, antihypertensive, antidiabetic, antimicrobial, and anticancer activities. The cheese samples were analyzed both before and after in vitro digestion. The study found that all cheese samples exhibited significant angiotensin-converting enzyme inhibition activity both before (45.5%-70.1% for 0.03 g cheese/mL) and after in vitro digestion (25.5%-63.5% for 0.0167 g cheese/mL), whereas α-amylase inhibition activity was present in all samples (in the range of 5.1%-50.0% for 3.0 × 10-5  g cheese/mL) but disappeared after digestion, and α-glucosidase inhibition activity was only detected after in vitro digestion (from 20.5% to 60.4% for 5.6 × 10-5  g cheese/mL), indicating potential antidiabetic properties. However, antimicrobial and anticancer activities were not observed in any of the samples. The results also suggest that the bioactivity potential of white cheese may vary depending on the region of production, as cheeses from the Marmara region exhibited high α-glucosidase inhibition activity after digestion. In conclusion, while white cheese is a valuable addition to the diet due to its high nutritional value and potential health benefits. This study revealed the bioactive potential of ripened white cheese and in vivo investigations of the cheese components would better show their possible benefits. PRACTICAL APPLICATION: Although the bioactivity potentials varied among the ripened white cheese samples produced in different regions of Turkey, a consistent trend was observed in terms of their presence or absence. Our research revealed that all ripened cheese samples demonstrated significant potential antioxidant and antihypertensive activities, as well as potential antidiabetic properties. Moreover, it can be interpreted that ripened white cheese has the potential to reduce the digestion of carbohydrates when consumed with carbohydrate-rich foods. Although the present results provide limited practical findings directly applicable to the industry, if the data and information obtained from this study are further explored and further studies delving into specific mechanisms of bioactive components could emerge.

Integrative Analysis for Identification of Therapeutic Targets and Prognostic Signatures in Non-Small Cell Lung Cancer.

Differential expressions of certain genes during tumorigenesis may serve to identify novel manageable targets in the clinic. In this work with an integrated bioinformatics approach, we analyzed public microarray datasets from Gene Expression Omnibus (GEO) to explore the key differentially expressed genes (DEGs) in non-small cell lung cancer (NSCLC). We identified a total of 984 common DEGs in 252 healthy and 254 NSCLC gene expression samples. The top 10 DEGs as a result of pathway enrichment and protein-protein interaction analysis were further investigated for their prognostic performances. Among these, we identified high expressions of CDC20, AURKA, CDK1, EZH2, and CDKN2A genes that were associated with significantly poorer overall survival in NSCLC patients. On the contrary, high mRNA expressions of CBL, FYN, LRKK2, and SOCS2 were associated with a significantly better prognosis. Furthermore, our drug target analysis for these hub genes suggests a potential use of Trichostatin A, Pracinostat, TGX-221, PHA-793887, AG-879, and IMD0354 antineoplastic agents to reverse the expression of these DEGs in NSCLC patients.

Slug and Vimentin downregulation at the metastatic site is associated with Skip-N2 metastasis of lung adenocarcinoma.

OBJECTIVE: Lung cancer displays heterogeneity both in the tumor itself and in its metastatic regions. One interesting behavior of the tumor is known as Skip N2 metastasis, which N2 lymph nodes contain tumor cells while N1 are clean. In this study, mRNA levels of epithelial mesenchymal transition (EMT) related genes in skip N2 and normal N2 involvements of non-small cell lung cancer tissues were investigated to evaluate the possible molecular background that may contribute to the pathogenesis of Skip N2 metastasis. MATERIALS AND METHODS: Eighty-three surgically resected and paraffin embedded lymph node samples of lung cancer patients were analyzed in this study, which 40 of them were Skip N2. N2 tissues were sampled from 50% tumor containing areas and total RNA was extracted. mRNA levels for 18S, E-cadherin, Vimentin, ZEB1 and SLUG were analyzed via qPCR and E-cadherin and vimentin protein levels via immunohistochemistry (IHC). Bioinformatic analysis were adopted using online datasets to evaluate significantly co-expressed genes with SLUG in lung cancer tissue samples. RESULTS: Skip-N2 patients who had adenocarcinoma subtype had better survival rates. Comparative analysis of PCR results indicated that Skip N2 tumor tissues had increased E-Cadherin/Vimentin ratio and ZEB1 mRNA expression, and significantly decreased levels of SLUG. E-cadherin IHC staining were higher in Skip N2 and Vimentin were in Non-Skip N2. TP63 had a strong correlation with SLUG expression in the bioinformatics analyses. CONCLUSION: The results indicate that, at molecular level, Skip N2 pathogenesis has different molecular background and regulation of SLUG expression may orchestrate the process.

Nuclear Respiratory Factor-1, a Novel SMAD4 Binding Protein, Represses TGF-ß/SMAD4 Signaling by Functioning as a Transcriptional Cofactor.

SMAD4, a key regulator of transforming growth factor-ß (TGF-ß) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-ß/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-ß signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-ß/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-ß/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-ß/SMAD4-signaling suppression in tumorigenesis.

SMAD4 suppresses AURKA-induced metastatic phenotypes via degradation of AURKA in a TGFß-independent manner.

UNLABELLED: SMAD4 has been suggested to inhibit the activity of the WNT/ß-catenin signaling pathway in cancer. However, the mechanism by which SMAD4 antagonizes WNT/ß-catenin signaling in cancer remains largely unknown. Aurora A kinase (AURKA), which is frequently overexpressed in cancer, increases the transcriptional activity of ß-catenin/T-cell factor (TCF) complex by stabilizing ß-catenin through the inhibition of GSK-3ß. Here, SMAD4 modulated AURKA in a TGFß-independent manner. Overexpression of SMAD4 significantly suppressed AURKA function, including colony formation, migration, and invasion of cell lines. In addition, SMAD4 bound to AURKA induced degradation of AURKA by the proteasome. A luciferase activity assay revealed that the transcriptional activity of the ß-catenin/TCF complex was elevated by AURKA, but decreased by SMAD4 overexpression. Moreover, target gene analysis showed that SMAD4 abrogated the AURKA-mediated increase of ß-catenin target genes. However, this inhibitory effect of SMAD4 was abolished by overexpression of AURKA or silencing of AURKA in SMAD4-overexpressed cells. Meanwhile, the SMAD4-mediated repression of AURKA and ß-catenin was independent of TGFß signaling because blockage of TGFßR1 or restoration of TGFß signaling did not prevent suppression of AURKA and ß-catenin signaling by SMAD4. These results indicate that the tumor-suppressive function of SMAD4 is mediated by downregulation of ß-catenin transcriptional activity via AURKA degradation in a TGFß-independent manner. IMPLICATIONS: SMAD4 interacts with AURKA and antagonizes its tumor-promoting potential, thus demonstrating a novel mechanism of tumor suppression.

Low SP1 expression differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma.

Specificity protein 1 (SP1) is an essential transcription factor that regulates multiple cancer-related genes. Because aberrant expression of SP1 is related to cancer development and progression, we focused on SP1 expression in gastric carcinoma and its correlation with disease outcomes. Although patient survival decreased as SP1 expression increased (P<0.05) in diffuse-type gastric cancer, the lack of SP1 expression in intestinal-type gastric cancer was significantly correlated with poor survival (P<0.05). The knockdown of SP1 in a high SP1-expressing intestinal-type gastric cell line, MKN28, increased migration and invasion but decreased proliferation. Microarray data in SP1 siRNA-transfected MKN28 revealed that the genes inhibiting migration were downregulated, whereas the genes negatively facilitating proliferation were increased. However, both migration and invasion were decreased by forced SP1 expression in a low SP1-expressing intestinal-type gastric cell line, AGS. Unlike the intestinal-type, in a high SP1-expressing diffuse-type gastric cell line, SNU484, migration and invasion were decreased by SP1 siRNA. In contrast to previous studies that did not identify differences between the 2 histological types, our results reveal that low expression of SP1 is involved in cancer progression and metastasis and differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma.

Tob1 induces apoptosis and inhibits proliferation, migration and invasion of gastric cancer cells by activating Smad4 and inhibiting ß­catenin signaling.

Transducer of ErbB-2.1 (Tob1), a tumor suppressor protein, is inactivated in a variety of cancers including stomach cancer. However, the role of Tob1 in gastric carcinogenesis remains elusive. The present study aimed to investigate whether Tob1 could inhibit gastric cancer progression in vitro, and to elucidate its underlying molecular mechanisms. We found differential expression of Tob1 in human gastric cancer (MKN28, AGS and MKN1) cells. The overexpression of Tob1 induced apoptosis in MKN28 and AGS cells, which was associated with sub-G1 arrest, activation of caspase-3, induction of Bax, inhibition of Bcl-2 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, Tob1 inhibited proliferation, migration and invasion, which were reversed in MKN1 and AGS cells transfected with Tob1 siRNA. Overexpression of Tob1 in MKN28 and AGS cells induced the expression of Smad4, leading to the increased expression and the promoter activity of p15, which was diminished by silencing of Tob1 using specific siRNA. Tob1 decreased the phosphorylation of Akt and glycogen synthase kinase-3ß (GSK3ß) in MKN28 and AGS cells, resulting in the reduced protein expression and the transcriptional activity of ß­catenin, which in turn decreased the expression of cyclin D1, cyclin-dependent kinase-4 (CDK4), urokinase plasminogen activator receptor (uPAR) and peroxisome proliferator and activator receptor-δ (PPARδ). Conversely, silencing of Tob1 induced the phosphorylation of Akt and GSK-3ß, and increased the expression of ß­catenin and its target genes. Collectively, our study demonstrates that the overexpression of Tob1 inhibits gastric cancer progression by activating Smad4- and inhibiting ß­catenin-mediated signaling pathways.

The synergistic therapeutic effect of cisplatin with Human papillomavirus E6/E7 short interfering RNA on cervical cancer cell lines in vitro and in vivo.

Human papillomavirus (HPV) types 16 and 18 are the major etiologic factors in the development of cervical epithelial neoplasia. Our study was designed to validate antiviral short interfering RNA (siRNA) targeting the E6 and E7 oncogenes as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) in cervical carcinoma. Specifically, the therapeutic efficacy of combination of CDDP and E6/E7-specific siRNA was assessed in an in vivo cervical cancer xenograft models. The combination of CDDP and E6/E7-specific siRNA had greater efficacy than the combination of CDDP and E6-specific siRNA especially in terms of inducing cellular senescence. Through in vitro and in vivo experiments, the mechanism of synergy between these two treatments was revealed, demonstrating that the combination of E6/E7-specific siRNA and CDDP therapy was significantly superior to either modality alone. In vitro, long-term exposure of HeLa cells to the combination of CDDP and E6/E7-specific siRNA induced apoptosis and cellular senescence. In vivo, E6/E7-specific siRNA potentiated the antitumor efficacy of CDDP via induction of apoptosis, senescence and antiangiogenesis. Our results suggest that E6/E7-specific siRNA may be an effective sensitizer of CDDP chemotherapy in cervical cancer.

High MET copy number and MET overexpression: poor outcome in non-small cell lung cancer patients.

The aim of this study was to evaluate the prevalence and prognostic role of increased gene copy number and protein expression of MET and EGFR in non-small cell lung cancer (NSCLC) patients. Samples were collected from 380 patients with surgically resected NSCLC, and fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) were performed. EGFR amplification and high polysomy (EGFR FISH-positive) were observed in 9.7% and 17.4% of the patients, respectively. EGFR was overexpressed (EGFR IHC-positive) in 19.2% of the patients. Neither EGFR FISH-positive nor EGFR IHC-positive status affected survival after resection. Increased MET copy number (MET FISH-positive by University of Colorado Cancer Center criteria) was observed in 11.1% of the patients (high polysomy, 8.7%; gene amplification, 2.4%). According to the Cappuzzo system, 7.1% of the patients were MET FISH-positive. MET FISH positivity was a negative prognostic factor, especially in patients with adenocarcinoma histology (p=0.040), female gender (p=0.010), old age (p=0.084), and EGFR FISH negativity (p=0.020) at the univariate level but not at the multivariate level. MET was overexpressed (MET IHC-positive) in 13.7% of the patients and associated with shorter overall and disease-free survival (p=0.010 and p=0.056, respectively). Multivariate analysis revealed that MET IHC-positive patients had a significantly increased risk of death (hazard ratio, 1.618; 95% confidence interval, 1.066-2.456; p=0.024). Increased MET copy number and MET overexpression are negative prognostic factors for surgically resected NSCLCs.

Claudin-4 overexpression is associated with epigenetic derepression in gastric carcinoma.

The tight junction (TJ) protein claudin-4 is aberrantly upregulated in gastric cancer, but its clinical significance and the molecular mechanisms underlying claudin-4 overexpression in gastric cancer remain unclear. Here, we investigated its roles and epigenetic mechanisms regulating CLDN4 expression in gastric cancer. We show that increased membranous expression of claudin-4 in gastric carcinoma is associated with better patient prognosis, whereas cytoplasmic claudin-4 expression did not show a significant association with prognosis. Consistent with the correlation of increased membranous claudin-4 with favorable clinicopathological factors, claudin-4 overexpression inhibited the migration and invasion of gastric cancer cells; in contrast, it did not affect cell growth. Claudin-4 expression also increased the barrier function of TJs. Claudin-4 upregulation was strongly correlated with DNA hypomethylation in both gastric tissues and gastric cancer cells. Moreover, CLDN4 expression was repressed in normal gastric tissues in association with bivalent histone modifications, and loss of repressive histone methylations and gain of active histone modifications were associated with CLDN4 overexpression in gastric cancer cells. Interestingly, CLDN4 repression could be markedly derepressed by combined treatments that simultaneously target both histone modifications and DNA demethylation in CLDN4-hypermethylated cells, whereas concomitant changes in histone methylations and acetylations are required for CLDN4 induction in CLDN4-repressed cells with low DNA methylation. Taken together, this study reveals that membranous claudin-4 expression is associated with gastric cancer progression and that it is an independent positive prognosis marker in gastric carcinoma. Furthermore, our findings suggest that epigenetic derepression may be a possible mechanism underlying CLDN4 overexpression in gastric cancer and that claudin-4 may have potential as a promising target for the treatment of gastric cancer.