Ten-eleven translocation proteins are α-ketoglutarate-dependent dioxygenases involved in the conversion of 5-methylcytosines (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine, and 5-carboxylcytosine that play a significant role in DNA demethylation. Deregulation of TET genes expression and changes in the level of 5-hmC are thought to be associated with the onset and progression of several types of cancer, but there are no such data related to endometrial cancer. The aim of the work was to investigate the messenger RNA expression levels of TET1, TET2, and TET3 in relation to clinicopathological characteristics of endometrial cancer as well as the correlation between expression of TET genes and the level of 5-hmC/5-mC. The prognostic significance of TETs expression for overall survival was established. We found that TET1 and TET2 messenger RNA expression was lower and TET3 was higher in cancers compared to normal tissues. Positive correlation between 5-hmC and the relative expression of TET1 and TET2 was found, but no correlation was observed in the case of TET3. Decreased expression of TET1 and TET2 was significantly associated with increased lymph node metastasis and International Federation of Gynecology and Obstetrics stage. Kaplan-Meier analysis indicated that low TET1 expression predicted poor overall survival (p = 0.038). Multivariate analysis identified the TET1 expression in endometrial cancer as an independent prognostic factor. Our results suggest that decreased expression of TET1 correlates with tumor progression and may serve as a potential prognostic biomarker in endometrial cancer.
O-GlcNAcylation is a cell glucose sensor. The addition of O-GlcNAc moieties to target protein is catalyzed by the O-Linked N-acetylglucosamine transferase (OGT). OGT is encoded by a single gene that yields differentially spliced OGT isoforms. One of them is targeted to mitochondria (mOGT). Although the impact of O-GlcNAcylation on cancer cells biology is well documented, mOGT’s role remains poorly investigated. We performed studies using breast cancer cells with up-regulated mOGT or its catalytic inactive mutant to identify proteins specifically modified by mOGT. Proteomic approaches included isolation of mOGT protein partners and O-GlcNAcylated proteins from mitochondria-enriched fraction followed by their analysis by mass spectrometry. Moreover, we analyzed the impact of mOGT dysregulation on mitochondrial activity and cellular metabolism using a variety of biochemical assays. We found that mitochondrial OGT expression is glucose-dependent. Elevated mOGT expression affected the mitochondrial transmembrane potential and increased intramitochondrial ROS generation. Moreover, mOGT up-regulation caused a decrease in cellular ATP level. We identified many mitochondrial proteins as mOGT substrates. Most of these proteins are localized in the mitochondrial matrix and the inner mitochondrial membrane and participate in mitochondrial respiration, fatty acid metabolism, transport, translation, apoptosis, and mtDNA processes. Our findings suggest that mOGT interacts with and modifies many mitochondrial proteins, and its dysregulation affects cellular bioenergetics and mitochondria function.
Enhancer of zest homolog 2 (EZH2) is a histone methyltransferase which plays a crucial role in cancer progression by regulation of genes involved in cellular processes such as proliferation, invasion and self-renewal. Activity and biological function of EZH2 are regulated by posttranslational modifications. It is suggested that EZH2 stability may be regulated by O-GlcNAc transferase (OGT), which is an enzyme catalyzing the addition of GlcNAc moieties to target proteins. In this study, we determined the impact of OGT on expression of EZH2 target genes FOXA1 and FOXC1, that are involved in breast cancer progression. The results of chromatin immunoprecipitation experiments showed that both EZH2 and OGT are targeted to the promoter regions of FOXA1 and FOXC1 and knockdown of EZH2 or OGT affects expression of studied genes in breast non-malignant (MCF10A) and cancer cells (MCF7, T47D and MDA-MB-231). The results showed that OGT silencing affects EZH2 binding to FOXC1 promoter but the effect is cell-context dependent. Despite the slight decrease in EZH2 protein level in cells with OGT depletion, EZH2 binding to FOXC1 was increased. Moreover, OGT binding to promoter regions of FOXA1 and FOXC1 was increased in cells with knockdown of EZH2. Increased expression of FOXA1 and FOXC1 in cells with OGT deregulation was associated with increased acetylation level of histone H3. The results suggest that OGT is involved in regulation of FOXA1 and FOXC1 expression but its role is not associated with regulation of EZH2 protein stability.
BackgroundCells adapt to hypoxia by transcriptional induction of genes that participate in regulation of angiogenesis, glucose metabolism and cell proliferation. The primary factors mediating cell response to low oxygen tension are hypoxia inducible factors (HIFs), oxygen-dependent transcription activators. The stability and activity of the α subunits of HIFs are controlled by hydroxylation reactions that require ascorbate as a cofactor. Therefore, deficiency of intracellular vitamin C could contribute to HIFs overactivation. In this study, we investigated whether vitamin C content of human thyroid lesions is associated with HIF-1α and HIF-2α protein levels.MethodsExpression of HIF-1α and HIF-2α as well as vitamin C content was analyzed in thyroid lesions and cultured thyroid carcinoma cell lines (FTC-133 and 8305c) treated with hypoxia-mimetic agent (cobalt chloride) and ascorbic acid. The expression of HIFs and hypoxia–induced glucose transporters were determined by Western blots while quantitative real-time PCR (qRT-PCR) was performed to detect HIFs mRNA levels. Ascorbate and dehydroascorbate levels were measured by HPLC method.ResultsWe found an inverse correlation between vitamin C level and HIF-1α but not HIF-2α expression in thyroid lesions. These results agree with our in vitro study showing that vitamin C induced a dose - dependent decrease of HIF-1α but not HIF-2α protein level in thyroid cancer cells FTC-133 and 8305C. The decreased HIF-1α expression was correlated with reduced expression of hypoxia-related glucose transporter 1 (GLUT1) in thyroid cancer cells.ConclusionThe results demonstrate that HIF-1α activation is associated with vitamin C content in thyroid lesions. Our study suggests that high tumor tissue ascorbate level could limit the expression of HIF-1α and its targets in thyroid lesions.
The PI3K/AKT pathway is frequently activated in endometrial carcinoma. BMI-1 (Blymphoma Mo-MLV insertion region 1) protein affects expression of PTEN (phosphatase and tensin homolog) in some cancers, but its significance for endometrial tumorigenesis is not known. The objective of this study was to determine the relationship between BMI-1 and expression of factors affecting AKT (protein kinase B) phosphorylation level in endometrial cancer. The expression of proteins and mRNAs was investigated in endometrial cancer specimens and samples of non-neoplastic endometrial tissue by Western blot and RT-PCR, respectively. The impact of BMI-1 down-regulation on AKT phosphorylation and expression of genes coding for several phosphatases were studied in HEC1A cells. The results showed that BMI-1 depletion caused increase in PHLPP1 and PHLPP2 (PH domain and leucine-rich repeat protein phosphatases 1/2) expression and decrease in phospho-AKT (pAKT) level. In more advanced tumours with higher metastatic potential, the expression of BMI-1 was lower compared to tumours less advanced and without lymph node metastasis.There were significant inverse correlations between BMI-1 and PHLPPs, especially PHLPP1 in normal endometrial samples. The inverse correlation between BMI-1 and PHLPP1/PHLPP2 expression was observed in PTEN positive but not PTEN negative cancers. Low PHLPP2 expression in tumours predicted poorer overall survival. BMI-1 impacts on AKT phosphorylation level in endometrial cells by regulation of PHLPP expression. K E Y W O R D SAKT, BMI-1, endometrial cancer, PHLPP, PTEN | 1301 ZACZEK Et Al.
Background. in Poland, the number of parents or caregivers, mainly declaring concern about the occurrence of vaccine injuries, refusing to subject people under the age of 19 to mandatory vaccinations has increased from 3,437 in 2010 to 30,089 in 2017. Material and methods. Legal regulations concerning vaccinations and legal literature in Poland, as well as judicial decisions concerning cases of evasion of mandatory vaccinations were reviewed. Results. the Constitution of the Republic of Poland obliges public authorities to fight epidemic diseases. In judicial decisions, a connection between preventive vaccinations and safety and public health protection is taken for granted-mass vaccinations allow for protection of all people, who would be exposed to infectious diseases without them. Statutory obligation to submit to mandatory vaccinations exists in the law and is directly enforceable; only in the case of refusal, the State Poviat Sanitary Inspectorate (PPIS), as creditor of the obligation, may demand its implementation and indicate an effective enforcement measure; however, it is the voivode who is authorized to conduct enforcement proceedings regarding the non-pecuniary obligation. the imposed fine is a measure leading to fulfilment of the obligation of vaccination, not a penalty for its non-performance. Conclusions. a statutory obligation to submit to protective vaccinations does not constitute a violation of constitutionally guaranteed human freedoms. as a person authorized by a specific law in the form of the Act on Preventing and Combating Infections and Infectious Diseases in Humans, this obligation overcomes the individual's freedom to exercise the right to refuse to undergo health benefits resulting from general regulations, such as the Act on Patients' Rights and Patients' Rights Ombudsman. although the PPIS is the creditor of the statutory obligatory vaccinations, the voivode is the correct enforcement body.
Epigenetic modifications, including DNA methylation and histone modifications, are involved in regulation of gene expression, and alterations in these modifications are implicated in cancer onset and progression. The specific pattern of DNA methylation depends on the balance between methylation and demethylation processes. Recent studies have shown that TET proteins play a key role in DNA demethylation. TET proteins (TET1, TET2, TET3) are iron(II) and α-ketoglutarate dependent dioxygenases, and their enzymatic activity involves hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine and further to 5-formylcytosine and 5-carboxylcytosine. These modified cytosines are removed by enzymes involved in DNA repair. However, the role of TETs in gene expression regulation is not limited to their catalytic activity. TETs can interact with proteins of complexes involved in the modification of histones (i.e. EZH2, OGT, Sin3a or HCF1) and by affecting their activity and, chromatin binding ability, they can cause changes in patterns of histone methylation, acetylation and O-GlcNAcylation. There is growing evidence that decreased expression of TET proteins and mutation in TET genes are associated with cancer onset and progression.
The exchange of metabolites between mitochondria and cytosol occurs through pores formed by voltage-dependent anion channel proteins. Voltage-dependent anion channels appear to be master regulators of mitochondrial bioenergetics and the intracellular flow of energy. Deregulation of voltage-dependent anion channels expression is thought to be related to mitochondrial dysfunction in cancer. The aim of this study was to investigate the mRNA and protein expression levels of VDAC1, VDAC2, and VDAC3 in relation to clinicopathological characteristics of endometrial cancer as well as the prognostic significance of voltage-dependent anion channels expression for overall survival. VDAC1 and VDAC3 expressions were significantly higher in cancer compared to normal tissues. Kaplan–Meier analysis indicated that high expression of all VDAC genes or high VDAC2 protein level predicted poor overall survival. Multivariate analysis identified the VDAC1 and VDAC2 mRNA levels as well as VDAC2 protein level as independent prognostic factors. Our results suggest that increased expression of voltage-dependent anion channels correlates with tumor progression and may serve as a potential prognostic biomarker in endometrial cancer.
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