Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

PURPOSES: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. METHODS: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. RESULTS: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). CONCLUSION: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

DNA methylation-based depiction of the immune microenvironment and immune-associated long non-coding RNAs in oral cavity squamous cell carcinomas.

Oral cavity squamous cell carcinoma (OSCC) is a complex and dynamic disease characterized by clinicopathological and molecular heterogeneity. Spatial and temporal heterogeneity of cell subpopulations has been associated with cancer progression and implicated in the prognosis and therapy response. Emerging evidence indicates that aberrant epigenetic profiles in OSCC may foster an immunosuppressive tumor microenvironment by modulating the expression of immune-related long non-coding RNAs (lncRNAs). DNA methylation analysis was performed in 46 matched OSCC and normal adjacent tissue samples using a genome-wide platform (Infinium HumanMethylation450 BeadChip). Reference-based computational deconvolution (MethylCIBERSORT) was applied to infer the immune cell composition of the bulk samples. The expression levels of genes encoding immune markers and differentially methylated lncRNAs were investigated using The Cancer Genome Atlas dataset. OSCC specimens presented distinct immune cell composition, including the enrichment of monocyte lineage cells, natural killer cells, cytotoxic T-lymphocytes, regulatory T-lymphocytes, and neutrophils. In contrast, B-lymphocytes, effector T-lymphocytes, and fibroblasts were diminished in tumor samples. The hypomethylation of three immune-associated lncRNAs (MEG3, MIR155HG, and WFDC21P) at individual CpG sites was confirmed by bisulfite-pyrosequencing. Also, the upregulation of a set of immune markers (FOXP3, GZMB, IL10, IL2RA, TGFB, IFNG, TDO2, IDO1, and HIF1A) was detected. The immune cell composition, immune markers alteration, and dysregulation of immune-associated lncRNAs reinforce the impact of the immune microenvironment in OSCC. These concurrent factors contribute to tumor heterogeneity, suggesting that epi-immunotherapy could be an efficient alternative to treat OSCC.

Anti-inflammatory effects of α-humulene and ß-caryophyllene on pterygium fibroblasts.

AIM: To investigate the anti-inflammatory effects of the sesquiterpenes α-humulene and ß-caryophyllene on pterygium fibroblasts. METHODS: Primary cultures of pterygium fibroblasts were established. Third passage pterygium fibroblasts were exposed to α-humulene and ß-caryophyllene separately and together. The cell viability was assessed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay at 12, 24, 48, and 72h after exposure. The levels of the inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α and IL-10 in the conditioned culture medium were assessed by enzyme-linked immunosorbent assay (ELISA) at 12, 24 and 48h after exposure. Data were statistically analyzed using Friedman repeated measures analysis of variances on ranks. RESULTS: The 25 µmol/L ß-caryophyllene induced significant decrease in the IL-6 production by pterygium fibroblasts 48h after the exposure (P=0.041). The levels of IL-1ß, IL-8, IL-10, and TNF-α were very low and had no statistically significant variations after exposure to α-humulene, ß-caryophyllene, or both compounds together. CONCLUSION: The exposure to 25 µmol/L of ß-caryophyllene significantly reduce the production of IL-6 by pterygium fibroblasts after 48h. This sesquiterpene may be a potential alternative adjuvant agent for the treatment of pterygium.

Expression of inflammatory cytokines in mesenchymal stem cells derived from proximal humerus fractures.

Background: Mesenchymal stem cells (MSCs) are an excellent treatment option for a wide variety of orthopaedic conditions. This study aimed to establish if bone marrow MSCs obtained from proximal humerus fractures can be an alternative source for obtaining primary cultures of human MSCs. Methods: Human bone marrow was obtained during osteosynthesis surgeries on closed proximal humerus fractures within 48 hours of injury. MSCs were harvested using the Ficoll gradient separation protocol and in vitro cultured until the third passage. Then, the cells were immunophenotyped by flow cytometry using stem cell specific surface markers. The cells were also induced to differentiate into osteoblasts and adipocytes for the characterization and confirmation of MSCs. The production of cytokines interleukin (IL)-1ß, IL-6, IL-8, IL-10, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) was assessed using enzyme-linked immunosorbent assay (ELISA) in the supernatant of the cultures after 3, 5 or 7 days. Results: Immunophenotyping showed high expression of the stem cell surface markers CD73, CD90, and CD105 and negative or very low expression of CD34, CD45, CD11b, CD19, and human leukocyte antigen (HLA)-DR. The bone marrow derived MSCs were able to differentiate into osteoblasts and adipocytes. The quantification of secreted cytokines revealed that IL-8 was the most produced cytokine, followed by IL-6 and IL-10 at similar quantities and lower levels of IL-1ß. TNF-α and IFN-γ were not detected. Conclusions: Proximal humerus fractures can be an alternative source for the collection of bone marrow MSCs. The cytokine production of these cells is very similar to the production profile of fracture haematomas previously reported and may be used for improving bone repair.

The Long Non-Coding RNA SNHG12 as a Mediator of Carboplatin Resistance in Ovarian Cancer via Epigenetic Mechanisms.

Genetic and epigenetic changes contribute to intratumor heterogeneity and chemotherapy resistance in several tumor types. LncRNAs have been implicated, directly or indirectly, in the epigenetic regulation of gene expression. We investigated lncRNAs that potentially mediate carboplatin-resistance of cell subpopulations, influencing the progression of ovarian cancer (OC). Four carboplatin-sensitive OC cell lines (IGROV1, OVCAR3, OVCAR4, and OVCAR5), their derivative resistant cells, and two inherently carboplatin-resistant cell lines (OVCAR8 and Ovc316) were subjected to RNA sequencing and global DNA methylation analysis. Integrative and cross-validation analyses were performed using external (The Cancer Genome Atlas, TCGA dataset, n = 111 OC samples) and internal datasets (n = 39 OC samples) to identify lncRNA candidates. A total of 4255 differentially expressed genes (DEGs) and 14529 differentially methylated CpG positions (DMPs) were identified comparing sensitive and resistant OC cell lines. The comparison of DEGs between OC cell lines and TCGA-OC dataset revealed 570 genes, including 50 lncRNAs, associated with carboplatin resistance. Eleven lncRNAs showed DMPs, including the SNHG12. Knockdown of SNHG12 in Ovc316 and OVCAR8 cells increased their sensitivity to carboplatin. The results suggest that the lncRNA SNHG12 contributes to carboplatin resistance in OC and is a potential therapeutic target. We demonstrated that SNHG12 is functionally related to epigenetic mechanisms.

In vitro effect of triamcinolone and platelet-rich plasma on cytokine levels of elbow lateral epicondylitis-derived cells.

BACKGROUND: The pathogenesis and treatment of lateral elbow epicondylitis (LEE) are still controversial. The purpose of the current study was to evaluate the production of inflammatory cytokines by LEE-derived cells and to compare the anti-inflammatory effect of triamcinolone acetonide with platelet-rich plasma (PRP) on cytokines production in primary culture of these cells. METHODS: Third passage cells from primary cultures of LEE were assessed for the production of the cytokines IL-1ß, IL-6, IL-8, IL-10 and TNF-α by immune-enzymatic assay (ELISA), after the treatment with 1, 10 and 100 µM triamcinolone compared to no treated controls at the time points 6, 12, 18, 24, 48, 72 and 96 h, and to PRP at 48, 72 and 96 h. RESULTS: The cytokines IL-6 and IL-8 were produced in high concentrations by LEE cells. One, 10 and 100 µM triamcinolone induced significant decrease in the production of IL-6 and IL-8 at 48, 72 and 96 h, adding the time point 12 h for IL-8. Compared to controls, PRP caused a significant increase in the production of IL-6 and IL-8 and there was a significant increase in IL-10 production with the use of 100 µM triamcinolone at 48 h. The production of IL1-ß and TNF-α was very low and did not change when the cultures were treated with triamcinolone or PRP. CONCLUSION: LEE-derived cells produce IL-6 and IL-8, confirming the inflammatory nature of this condition. While triamcinolone inhibited the production of IL-6 and IL-8 by LEE cells, PRP induced an increase in these cytokines compared with controls.

Brazilian green propolis: A novel tool to improve the cytotoxic and immunomodulatory action of docetaxel on MCF-7 breast cancer cells and on women monocyte.

Docetaxel (DTX) is used against breast cancer despite its side effects such as toxicity and immunosuppression. Here we investigated the cytotoxic and immunomodulatory effects of the ethanol solution extract of propolis (EEP) in combination with DTX on MCF-7 breast cancer cells and on women's monocyte. The cytotoxic potential of EEP + DTX was assessed by MTT assay and the type of tumor cell death was evaluated by flow cytometry. The effects of EEP + DTX on the migration and invasion of MCF-7 cells were analyzed. Cytokine production by monocytes was assessed by ELISA and the expression of cell surface markers was evaluated by flow cytometry. We also assessed the fungicidal activity of monocytes against Candida albicans and the generation of reactive oxygen species (ROS). Finally, the impact of the supernatants of treated monocytes in the viability, migration, and invasiveness of tumor cells was assessed. EEP enhanced the cytotoxicity of DTX alone against MCF-7 cells by inducing necrosis and inhibiting their migratory ability. EEP + DTX exerted no cytotoxic effects on monocytes and stimulated HLA-DR expression, TNF-α, and IL-6 production, exerted an immunorestorative action in the fungicidal activity, and reduced the oxidative stress. Our findings have practical implications and reveal new insights for complementary medicine.

Systemic treatment of penile squamous cell carcinoma-hurdles and hopes of preclinical models and clinical regimens: a narrative review.

Despite contemporary research efforts, the prognosis of penile squamous cell carcinoma (PeSCC) has not significantly improved over the past decade. Despite frequently encountered patient-related delayed medical consultations impairing outcomes, several other aspects contribute to the lack of advancement in the treatment of this condition. One essential reason is that translational research, a prerequisite for the clinically successful disease management, is still at an early stage in PeSCC as compared to many other malignancies. Preclinical experimental models are indispensable for the evaluation of tumor biology and identification of genomic alterations. However, since neither commercial PeSCC cell lines are available nor xenograft models sustainably established, such analyses are challenging in this field of research. In addition, systemic therapies are less effective and toxic without decisive breakthroughs over recent years. Current systemic management of PeSCC is based on protocols that have been investigated in small series of only up to 30 patients. Thus, there is an unmet medical need for new approaches necessitating research efforts to develop more efficacious systemic strategies. This review aims to highlight the current state of knowledge in the molecular alterations involved in the etiology and ensuing steps for cancer progression, existing preclinical models of translational research, clinically relevant systemic protocols, and ongoing clinical trials.

Master Regulators of Epithelial-Mesenchymal Transition and WNT Signaling Pathways in Juvenile Nasopharyngeal Angiofibromas.

Juvenile nasopharyngeal angiofibroma (JNA) is a rare fibrovascular benign tumor showing an invasive growth pattern and affecting mainly male adolescents. We investigated the role of epithelial-mesenchymal transition (EMT) and WNT signaling pathways in JNA. Gene expression profiles using nine JNA paired with four inferior nasal turbinate samples were interrogated using a customized 2.3K microarray platform containing genes mainly involved in EMT and WNT/PI3K pathways. The expression of selected genes (BCL2, CAV1, CD74, COL4A2, FZD7, ING1, LAMB1, and RAC2) and proteins (BCL2, CAV1, CD74, FZD7, RAF1, WNT5A, and WNT5B) was investigated by RT-qPCR (28 cases) and immunohistochemistry (40 cases), respectively. Among 104 differentially expressed genes, we found a significantly increased expression of COL4A2 and LAMB1 and a decreased expression of BCL2 and RAC2 by RT-qPCR. The immunohistochemistry analysis revealed a low expression of BCL2 and a negative to moderate expression of FZD7 in most samples, while increased CAV1 and RAF1 expression were detected. Moderate to strong CD74 protein expression was observed in endothelial and inflammatory cells. A significant number of JNAs (78%) presented reduced WNT5A and increased WNT5B expression. Overall, the transcript and protein profile indicated the involvement of EMT and WNT pathways in JNA. These candidates are promising druggable targets for treating JNA.

Circulating Plasma miRNA and Clinical/Hemodynamic Characteristics Provide Additional Predictive Information About Acute Pulmonary Thromboembolism, Chronic Thromboembolic Pulmonary Hypertension and Idiopathic Pulmonary Hypertension.

Idiopathic pulmonary artery hypertension (IPAH), chronic thromboembolic pulmonary hypertension (CTEPH), and acute pulmonary embolism (APTE) are life-threatening cardiopulmonary diseases without specific surgical or medical treatment. Although APTE, CTEPH and IPAH are different pulmonary vascular diseases in terms of clinical presentation, prevalence, pathophysiology and prognosis, the identification of their circulating microRNA (miRNAs) might help in recognizing differences in their outcome evolution and clinical forms. The aim of this study was to describe the APTE, CTEPH, and IPAH-associated miRNAs and to predict their target genes. The target genes of the key differentially expressed miRNAs were analyzed, and functional enrichment analyses were carried out. The miRNAs were detected using RT-PCR. Finally, we incorporated plasma circulating miRNAs in baseline and clinical characteristics of the patients to detect differences between APTE and CTEPH in time of evolution, and differences between CTEPH and IPAH in diseases form. We found five top circulating plasma miRNAs in common with APTE, CTEPH and IPAH assembled in one conglomerate. Among them, miR-let-7i-5p expression was upregulated in APTE and IPAH, while miRNA-320a was upregulated in CTEP and IPAH. The network construction for target genes showed 11 genes regulated by let-7i-5p and 20 genes regulated by miR-320a, all of them regulators of pulmonary arterial adventitial fibroblasts, pulmonary artery endothelial cell, and pulmonary artery smooth muscle cells. AR (androgen receptor), a target gene of hsa-let-7i-5p and has-miR-320a, was enriched in pathways in cancer, whereas PRKCA (Protein Kinase C Alpha), also a target gene of hsa-let-7i-5p and has-miR-320a, was enriched in KEGG pathways, such as pathways in cancer, glioma, and PI3K-Akt signaling pathway. We inferred that CTEPH might be the consequence of abnormal remodeling in APTE, while unbalance between the hyperproliferative and apoptosis-resistant phenotype of pulmonary arterial adventitial fibroblasts, pulmonary artery endothelial cell and pulmonary artery smooth muscle cells in pulmonary artery confer differences in IPAH and CTEPH diseases form. We concluded that the incorporation of plasma circulating let-7i-5p and miRNA-320a in baseline and clinical characteristics of the patients reinforces differences between APTE and CTEPH in outcome evolution, as well as differences between CTEPH and IPAH in diseases form.

Relevance of PD-L1 Non-Coding Polymorphisms on the Prognosis of a Genetically Admixed NSCLC Cohort.

PURPOSE: Although non-small cell lung cancer (NSCLC) remains a deadly disease, new predictive biomarkers have emerged to assist in managing the disease, of which one of the most promising is the programmed death-ligand 1 (PD-L1). Each, PD-L1 variant seem to modulate the function of immune checkpoints differently and affect response to adjuvant treatment and outcome in NSCLC patients. We thus investigated the influence of these PD-L1 genetic variations in genetically admixed NSCLC tissue samples, and correlated these values with clinicopathological characteristics, including prognosis. MATERIALS AND METHODS: We evaluated PD-L1 non-coding genetic variants and protein expression in lung adenocarcinomas (ADC), squamous cell carcinomas (SqCC), and large cell carcinomas (LCC) in silico. Microarray paraffin blocks from 70 samples of ADC (N=33), SqCC (N=24), and LCC (N=13) were used to create PD-L1 multiplex immunofluorescence assays with a Cell Signaling E1L3N clone. Fifteen polymorphisms of the PD-L1 gene were investigated by targeted sequencing and evaluated in silico using dedicated tools. RESULTS: Although PD-L1 polymorphisms seemed not to interfere with protein expression, PD-L1 expression varied among different histological subtypes, as did clinical outcomes, with the rs4742098A>G, rs4143815G>C, and rs7041009G>A variants being associated with relapse (P=0.01; P=0.05; P=0.02, respectively). The rs7041009 GG genotype showed a significant correlation with younger and alive patients compared to carriers of the A allele (P=0.02 and P<0.01, respectively). The Cox regression model showed that the rs7041009 GG genotype may influence OS (P<0.01) as a co-dependent factor associated with radiotherapy and recurrence in NSCLC patients. Furthermore, the Kaplan-Meier survival curves showed that rs7041009 and rs4742098 might impact PPS in relapsed patients. In silico approaches identified the variants as benign. CONCLUSION: PD-L1 non-coding variants play an important role in modulating immune checkpoint function and may be explored as immunotherapy biomarkers. We highlight the rs7041009 variant, which impacts OS and PPS in NSCLC patients.

Comprehensive Analysis of DNA Methylation and Prediction of Response to NeoadjuvantTherapy in Locally Advanced Rectal Cancer.

The treatment for locally advanced rectal carcinomas (LARC) is based on neoadjuvant chemoradiotherapy (nCRT) and surgery, which results in pathological complete response (pCR) in up to 30% of patients. Since epigenetic changes may influence response to therapy, we aimed to identify DNA methylation markers predictive of pCR in LARC patients treated with nCRT. We used high-throughput DNA methylation analysis of 32 treatment-naïve LARC biopsies and five normal rectal tissues to explore the predictive value of differentially methylated (DM) CpGs. External validation was carried out with The Cancer Genome Atlas-Rectal Adenocarcinoma (TCGA-READ 99 cases). A classifier based on three-CpGs DM (linked to OBSL1, GPR1, and INSIG1 genes) was able to discriminate pCR from incomplete responders with high sensitivity and specificity. The methylation levels of the selected CpGs confirmed the predictive value of our classifier in 77 LARCs evaluated by bisulfite pyrosequencing. Evaluation of external datasets (TCGA-READ, GSE81006, GSE75546, and GSE39958) reproduced our results. As the three CpGs were mapped near to regulatory elements, we performed an integrative analysis in regions associated with predicted cis-regulatory elements. A positive and inverse correlation between DNA methylation and gene expression was found in two CpGs. We propose a novel predictive tool based on three CpGs potentially useful for pretreatment screening of LARC patients and guide the selection of treatment modality.

The Interplay between Long Noncoding RNAs and Proteins of the Epigenetic Machinery in Ovarian Cancer.

Comprehensive large-scale sequencing and bioinformatics analyses have uncovered a myriad of cancer-associated long noncoding RNAs (lncRNAs). Aberrant expression of lncRNAs is associated with epigenetic reprogramming during tumor development and progression, mainly due to their ability to interact with DNA, RNA, or proteins to regulate gene expression. LncRNAs participate in the control of gene expression patterns during development and cell differentiation and can be cell and cancer type specific. In this review, we described the potential of lncRNAs for clinical applications in ovarian cancer (OC). OC is a complex and heterogeneous disease characterized by relapse, chemoresistance, and high mortality rates. Despite advances in diagnosis and treatment, no significant improvements in long-term survival were observed in OC patients. A set of lncRNAs was associated with survival and response to therapy in this malignancy. We manually curated databases and used bioinformatics tools to identify lncRNAs implicated in the epigenetic regulation, along with examples of direct interactions between the lncRNAs and proteins of the epigenetic machinery in OC. The resources and mechanisms presented herein can improve the understanding of OC biology and provide the basis for further investigations regarding the selection of novel biomarkers and therapeutic targets.

Epigenetics in Inflammatory Breast Cancer: Biological Features and Therapeutic Perspectives.

Evidence has emerged implicating epigenetic alterations in inflammatory breast cancer (IBC) origin and progression. IBC is a rare and rapidly progressing disease, considered the most aggressive type of breast cancer (BC). At clinical presentation, IBC is characterized by diffuse erythema, skin ridging, dermal lymphatic invasion, and peau d'orange aspect. The widespread distribution of the tumor as emboli throughout the breast and intra- and intertumor heterogeneity is associated with its poor prognosis. In this review, we highlighted studies documenting the essential roles of epigenetic mechanisms in remodeling chromatin and modulating gene expression during mammary gland differentiation and the development of IBC. Compiling evidence has emerged implicating epigenetic changes as a common denominator linking the main risk factors (socioeconomic status, environmental exposure to endocrine disruptors, racial disparities, and obesity) with IBC development. DNA methylation changes and their impact on the diagnosis, prognosis, and treatment of IBC are also described. Recent studies are focusing on the use of histone deacetylase inhibitors as promising epigenetic drugs for treating IBC. All efforts must be undertaken to unravel the epigenetic marks that drive this disease and how this knowledge could impact strategies to reduce the risk of IBC development and progression.

Effects of Propolis and Phenolic Acids on Triple-Negative Breast Cancer Cell Lines: Potential Involvement of Epigenetic Mechanisms.

Triple-negative breast cancer is an aggressive disease frequently associated with resistance to chemotherapy. Evidence supports that small molecules showing DNA methyltransferase inhibitory activity (DNMTi) are important to sensitize cancer cells to cytotoxic agents, in part, by reverting the acquired epigenetic changes associated with the resistance to therapy. The present study aimed to evaluate if chemical compounds derived from propolis could act as epigenetic drugs (epi-drugs). We selected three phenolic acids (caffeic, dihydrocinnamic, and p-coumaric) commonly detected in propolis and the (-)-epigallocatechin-3-gallate (EGCG) from green tea, which is a well-known DNA demethylating agent, for further analysis. The treatment with p-coumaric acid and EGCG significantly reduced the cell viability of four triple-negative breast cancer cell lines (BT-20, BT-549, MDA-MB-231, and MDA-MB-436). Computational predictions by molecular docking indicated that both chemicals could interact with the MTAse domain of the human DNMT1 and directly compete with its intrinsic inhibitor S-Adenosyl-l-homocysteine (SAH). Although the ethanolic extract of propolis (EEP) did not change the global DNA methylation content, by using MS-PCR (Methylation-Specific Polymerase Chain Reaction) we demonstrated that EEP and EGCG were able to partly demethylate the promoter region of RASSF1A in BT-549 cells. Also, in vitro treatment with EEP altered the RASSF1 protein expression levels. Our data indicated that some chemical compound present in the EEP has DNMTi activity and can revert the epigenetic silencing of the tumor suppressor RASSF1A. These findings suggest that propolis are a promising source for epi-drugs discovery.

The long non-coding RNA ANRASSF1 in the regulation of alternative protein-coding transcripts RASSF1A and RASSF1C in human breast cancer cells: implications to epigenetic therapy.

Alternative protein-coding transcripts of the RASSF1 gene have been associated with dual functions in human cancer: while RASSF1C isoform has oncogenic properties, RASSF1A is a tumour suppressor frequently silenced by hypermethylation. Recently, the antisense long non-coding RNA RASSF1 (ANRASSF1) was implicated in a locus-specific mechanism for the RASSF1A epigenetic repression mediated by PRC2 (Polycomb Repressive Complex 2). Here, we evaluated the methylation patterns of the promoter regions of RASSF1A and RASSF1C and the expression levels of these RASSF1 transcripts in breast cancer and breast cancer cell lines. As expected, RASSF1C remained unmethylated and RASSF1A was hypermethylated at high frequencies in 75 primary breast cancers, and also in a panel of three mammary epithelial cells (MEC) and 10 breast cancer cell lines (BCC). Although RASSF1C was expressed in all cell lines, only two of them expressed the transcript RASSF1A. ANRASSF1 expression levels were increased in six BCCs. In vitro induced demethylation with 5-Aza-2'-deoxicytydine (5-Aza-dC) resulted in up-regulation of RASSF1A and an inverse correlation with ANRASSF1 relative abundance in BCCs. However, increased levels of both transcripts were observed in two MECs (184A1 and MCF10A) after treatment with 5-Aza-dC. Overall, these findings indicate that ANRASSF1 is differentially expressed in MECs and BCCs. The lncRNA ANRASSF1 provides new perspectives as a therapeutic target for locus-specific regulation of RASSF1A.

Adhesion study of cultured human lens capsule cells on hydrophilic intraocular lenses coated with polyethylene glycol.

PURPOSE: To evaluate the adhesion of human lens capsule cells on hydrophilic acrylic intraocular lenses (IOLs) coated with polyethylene glycol (PEG). SETTING: Department of Ophthalmology, Faculty of Medicine, Universidade Estadual Paulista-Botucatu, São Paulo, Brazil. DESIGN: Experimental study. METHODS: Human anterior lens capsules obtained during cataract surgery were cultured and seeded (200 cells/IOLs) in triplicates on the surface of a copolymer comprising hydroxyethyl methacrylate, ethyl methacrylate, and methyl methacrylate IOLs (Loflex) treated or not treated with PEG. After 26 hours, the number of viable adherent cells was estimated by counting in a hemocytometer. RESULTS: The coating of hydrophilic acrylic IOLs with PEG was effective in inhibiting cell adhesion (P < .05). Cells showing 2 distinct morphologic patterns-epithelial and dendritic-like-were observed during the in vitro establishment of the cultures. A tendency toward greater adhesion of dendritic-like cells was observed in untreated IOLs compared with treated IOLs (P = .095). CONCLUSION: Coating hydrophilic acrylic IOLs with PEG was effective in inhibiting cell adhesion. This treatment might play a role in posterior capsule opacification prevention. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas.

A significant association between DNA losses on 22q13.31 and head and neck squamous cell carcinomas (HNSCC) was previously reported by our group. Our data indicated that PHF21B gene, mapped on 22q13.31 and encoding a protein with function of chromatin-mediated transcriptional regulation, might be a putative tumor suppressor gene. To test this hypothesis, gene copy number was assessed in 75 HNSCC and 49 matched peripheral blood samples. PHF21B losses were detected in 43 tumors and were significantly associated with patients with familial history of cancer (P < 0.0001); i.e., 36/43 cases showed a positive family history of cancer and 22/36 had first-degree relatives with cancer (P = 0.049). In attempt to investigate other mechanisms for PHF21B loss of function, DNA sequencing was performed and no mutations were detected. We next evaluated the gene expression levels after inhibition of DNA methylation in nine HNSCC and breast carcinoma cell lines. Additionally, PHF21B expression levels were evaluated in colon cancer HCT116 cells as well as in its counterpart DKO (double knockout of DNMT1 and DNMT3B). The higher expression levels of PHF21B gene detected in DKO cells were inversely correlated with the DNA methylation. Further, DNA methylation in the specific promoter-associated CpG Island was investigated. Interestingly, gene hypermethylation was detected in 13/37 tumors: 5/13 HNSCC cases had family history of cancer in first-degree relatives and 8/13 showed both, DNA methylation and PHF21B losses in the tumor sample. One patient had PHF21B loss in the peripheral blood cells and PHF21B methylation in the tumor sample. Additionally, overexpression of PHF21B in cell lines drastically reduces clonogenic and migratory abilities. These data suggest that PHF21B is a novel tumor suppressor gene that can be inactivated by genetic and epigenetic mechanisms in the human cancer.

Defining suitable reference genes for RT-qPCR analysis on human sertoli cells after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.

Quantitative real-time RT-PCR (qPCR) has proven to be a valuable molecular technique to quantify gene expression. There are few studies in the literature that describe suitable reference genes to normalize gene expression data. Studies of transcriptionally disruptive toxins, like tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. The present study was designed to validate potential reference genes in human Sertoli cells after exposure to TCDD. 32 candidate reference genes were analyzed to determine their applicability. geNorm and NormFinder softwares were used to obtain an estimation of the expression stability of the 32 genes and to identify the most suitable genes for qPCR data normalization.

DNA methylation patterns of steroid receptor genes ESR1, ESR2 and PGR in deep endometriosis compromising the rectum.

Endometriosis is characterized by the presence of endometrial-like tissue located outside the uterine cavity. Recent evidence suggests that endometriosis may be an epigenetic disease, as well as an estrogen-dependent disease. Based on the unique steroid hormone receptor expression profile observed in endometriotic lesions as compared to eutopic endometrium, the present study aimed to gain further insight into the DNA methylation patterns of alternative promoters of the steroid receptor genes ESR1, ESR2 and PGR in intestinal deep endometriosis, one of the most aggressive forms of endometriosis. The DNA methylation patterns were evaluated by methylation-specific polymerase chain reaction (MS-PCR) after bisulfite modification in 44 endometriotic tissues as well as in 7 matched eutopic endometrium. No differences in the DNA methylation were observed for the ESR1 and ESR2 genes. Methylation of the PGR gene was observed in 39% (17 out of 44) and 19% (7 out of 37) of the cases in the promoter regions B (PGRB) and A (PGRA), respectively. Both PGR promoter regions were methylated in 3 cases. PGRB methylated alleles were detected exclusively in the endometriotic lesions when compared to the eutopic endometrium obtained from the same patient. The effect of DNA methylation in inhibiting the PGR gene expression was corroborated by immuno-staining for PgR protein in a subset of tissue samples. The present study demonstrated that epigenetic changes occur in both promoter regions of the PGR gene in intestinal endometriosis. Since eutopic and ectopic tissues do not respond sufficiently to progesterone in women with endometriosis, further study is necessary to evaluate the effect of epigenetic alterations in progesterone-resistance in this enigmatic disease.