Histone modifications play a key role in the epigenetic regulation of gene transcription in cancer cells. Histone acetylations are regulated by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs are increased in ovarian carcinomas and they are involved in carcinogenesis and resistance to chemotherapeutic agents. In our study we investigated anticancer effect of HDAC inhibitor sodium butyrate (NaBu) on cisplatin-sensitive and cisplatin-resistant ovarian cell lines A2780 and A2780cis. A2780 and A2780cis were treated with NaBu alone or in combination with cisplatin (CP). NaBu inhibited the growth of both cell lines and enhanced cytotoxic effect of CP. Exposure to NaBu for 24 h induced cell cycle arrest. The expressions of EMT-related genes and proteins were further investigated by qPCR and western blot analysis. Loss of E-cadherin has been shown to be crucial in ovarian cancer development. We found that NaBu dramatically induce expression of E-cadherin gene (CDH1) and protein levels in A2780 and A2780cis. We investigated correlation between transcription and methylation of CDH1gene. Methylation level analysis in 32 CpG sites in CDH1 gene (promoter/exon1 regions) was performed using bisulfite NGS (Next Generation Sequencing). We found that cisplatin-resistant cell line A2780cis cells differ from their cisplatin-sensitive counterparts in the CDH1 methylation. Methylation in A2780cis cells is elevated compared to A2780. However, NaBu-induced expression of CDH1 was not accompanied by CDH1 demethylation. NaBu treatment induced changes in expression of EMT-related genes and proteins. Interestingly E-cadherin zinc finger transcriptional repressor SNAIL1 was upregulated in both cell lines. Mesenchymal marker vimentin was downregulated. Matrix metalloproteases (MMPs) are necessary for pericellular proteolysis and facilitate migration and invasion of tumour cells. NaBu induced mRNA expression of MMPs, mild changes in activities of gelatinases MMP2 and MMP9 were detected. Our data demonstrate that NaBu sensitizes cisplatin-resistant ovarian cancer cells, re-established E-cadherin expression, but it was not able to reverse the EMT phenotype completely.

DNA methylation is well-known to be associated with ovarian cancer (OC) and has great potential to serve as a biomarker in monitoring response to therapy and for disease screening. The purpose of this study was to investigate methylation of HNF1B and GATA4 and correlate detected methylation with clinicopathological characteristic of OC patients. The study group consisted of 64 patients with OC and 35 control patients. To determine the most important sites of HNF1B and GATA4, we used next-generation sequencing. For further confirmation of detected methylation of selected regions, we used high-resolution melting analysis and methylation-specific real-time polymerase chain reaction (PCR). Selected regions of HNF1B and GATA4 were completely methylation free in all control samples, whereas methylation-positive pattern was observed in 32.8% (HNF1B) and 45.3% (GATA4) of OC samples. Evaluating both genes together, we were able to detect methylation in 65.6% of OC patients. We observed a statistically significant difference in HNF1B methylation between samples with different stages of OC. We also detected subtype specific methylation in GATA4 and a decrease of methylation in late stages of OC. The combination of unmethylated HNF1B and methylated GATA4 was associated with longer overall survival. In our study, we employed innovative approach of methylation analysis of HNF1B and GATA4 to search for possible epigenetic biomarkers. We confirmed the significance of the HNF1B and GATA4 hypermethylation with emphasis on the need of selecting the most relevant sites for analysis. We suggest selected CpGs to be further examined as a potential positive prognostic factor.

BACKGROUND: Aberrant DNA methylation of protocadherins (PCDHs) has been associated with development and progression of various types of cancer. It could represent possible direction in the search for critically needed tumor biomarkers for ovarian can

Introduction: The amniotic fluid nicotinamide phosphoribosyltransferase (NAMPT) levels have not been compared among women with preterm prelabor rupture of membranes (PPROM) comorbid with intra-amniotic infection, sterile intra-amniotic inflammation (IAI), colonization, or without IAI and microbial invasion of the amniotic cavity (MIAC). Therefore, the main aim was to quantify the amniotic fluid NAMPT in women with PPROM complicated by intra-amniotic infection, sterile IAI, or colonization. The second aim was to characterize the diagnostic indices of NAMPT to reveal IAI. The third aim was to determine whether the cervical fluid and maternal serum NAMPT quantitation might be of value in the identification of intra-amniotic inflammatory complications in PPROM. Methods of study: NAMPT levels in amniotic fluid, cervical fluid, and maternal serum were assessed in three independent cohorts of women with singleton pregnancies complicated by PPROM between 24⁺⁰ and 36⁺⁶ weeks of gestation consisting of 88, 121, and 88 women, respectively. Amniotic fluid samples were obtained by transabdominal amniocentesis, cervical fluid samples were obtained using a Dacron polyester swab, and maternal blood was obtained by venipuncture of the cubital vein. The NAMPT levels were measured by an enzyme linked immunosorbent assay. Testing for MIAC and IAI was performed on all women, who were then categorized into four subgroups: intra-amniotic infection (MIAC and IAI), sterile IAI (IAI alone), colonization (MIAC alone), and without MIAC and IAI. Results: Women with intra-amniotic infection and women with sterile IAI had higher NAMPT levels than did women with colonization and women without MIAC and IAI (intra-amniotic infection: median 73.6 ng/mL, sterile IAI: median 55.5 ng/mL, colonization: median 12.1 ng/mL, without MIAC and IAI: 10.6 ng/mL; p < 0.0001). An amniotic fluid NAMPT level of 37 ng/mL was the best value for the detection of intra-amniotic infection in women with PPROM. Cervical fluid (p = 0.51) and maternal serum (p = 0.50) NAMPT levels did not reflect intra-amniotic inflammatory complications in women with PPROM. Conclusions: Intra-amniotic infection and sterile IAI are associated with higher NAMPT levels in amniotic fluid but not in cervical fluid or maternal serum in women with PPROM. Amniotic fluid NAMPT might be a marker for invasive identification of IAI in PPROM.

Recently, an increasing incidence of HPV-induced oropharyngeal squamous cell carcinoma (OPSCC) has been observed. Moreover, locoregionally advanced stages require a combined modal approach, and the prognosis is poor. Therefore, it is essential to find early diagnostic and prognostic biomarkers. DNA methylation changes play a crucial role in the process of carcinogenesis and are often investigated as promising biomarkers in many types of cancer. For analysis of DNA methylation levels of selected tumour suppressor genes in HPV-positive and HPV-negative samples (including primary tumours and corresponding metastases of metastasizing OPSCCs, primary tumours of non-metastasizing OPSCCs, and control samples), methylation-specific MLPA and methylation-specific high-resolution melting analyses were used. A significant difference in methylation between OPSCCs and the control group was observed in WT1, PAX6 (P < 0.01) and CADM1, RARβ (P < 0.05) genes. CADM1 and WT1 hypermethylation was detected mostly in HPV-positive samples; all but one HPV-negative samples were unmethylated. Moreover, hypermethylation of PAX5 gene was observed in metastases compared with control samples and was also associated with shorter overall survival of all patients (P < 0.05). Associations described herein between promoter methylation of selected genes and clinicopathological data could benefit OPSCC patients in the future by improvement in screening, early detection, and prognosis of the disease.