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.
- MeSH
- buněčný cyklus účinky léků MeSH
- CD antigeny genetika MeSH
- chemorezistence genetika MeSH
- cisplatina farmakologie MeSH
- epigeneze genetická účinky léků MeSH
- epitelo-mezenchymální tranzice účinky léků genetika MeSH
- genetické markery MeSH
- histonový kód účinky léků MeSH
- inhibitory histondeacetylas farmakologie MeSH
- kadheriny genetika MeSH
- kyselina máselná farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory vaječníků farmakoterapie genetika patologie MeSH
- proliferace buněk účinky léků MeSH
- protinádorové látky farmakologie MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rat liver myofibroblasts (MFB) were isolated by repeated passaging of nonparenchymal liver cell fraction. They were cultured on polystyrene Petri dishes, on fibrin or on type I collagen gels for 5 days. Quantitative RT-PCR, Western blotting, zymography and immunocytochemistry were used to study differences in cell morphology and protein expression. MFB were large and spread on plastic substrate, with prominent alpha-smooth muscle (alpha-SMA) fibres. They turned much smaller and elongated on collagen which was accompanied by the rearrangement of the cytoskeleton and a decrease in alpha-SMA and beta-actin content. Collagen gel induced the expression of a group of metalloproteinases (MMP-2, -3, -9, -13), on mRNA and protein level which resulted in the degradation of the gel. This response was accompanied by changes in the mRNA expression of cytokines of TGF-beta family, CTGF and interleukin-6, as well as of osteopontin and thrombospondin-2 that are involved in metalloproteinases (MMPs) regulation. The expression of MMPs substrates, collagen types I, IV and XII did not change or decreased. The effects of fibrin gels on MFB were milder than those of collagen. MFB assumed to deposit collagen and other ECM components in fibrotic liver, besides hepatic stellate cells, also possess a great collagenolytic potential.
- MeSH
- aktiny metabolismus MeSH
- biologické markery metabolismus MeSH
- časové faktory MeSH
- cytokiny metabolismus MeSH
- cytoskelet enzymologie MeSH
- fibrin metabolismus MeSH
- imunohistochemie MeSH
- játra cytologie enzymologie MeSH
- kolagen typu I metabolismus MeSH
- kolagenasy genetika metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- messenger RNA metabolismus MeSH
- myofibroblasty enzymologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- potkani Sprague-Dawley MeSH
- separace buněk metody MeSH
- tvar buňky MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hepatic stellate cells (HSC) and liver myofibroblasts (MFB) are two cell populations most likely responsible for the synthesis of most connective tissue components in fibrotic liver. They differ in their origin and location, and possibly in patterns of gene expression. Normal and carbon tetrachloride-cirrhotic livers from rats were used to isolate HSC. Liver was perfused with pronase and collagenase solutions, followed by centrifugation of the cell suspension on a density gradient. HSC were quiescent 2 days after plating on plastic but they became activated after another 5 days in culture. When the culture was passaged 5 times, its character changed profoundly as HSC were replaced by MFB. Microarray analysis was used to determine gene expression in quiescent HSC, activated HSC and MFB. The expression of 49 genes coding for connective tissue proteins, proteoglycans, metalloproteinases and their inhibitors, growth factors and cellular markers was determined. The pattern of gene expression changed during HSC activation and there were distinct differences between HSC and MFB. Little difference between normal cells and cells isolated from cirrhotic liver was found.
- MeSH
- experimentální cirhóza jater metabolismus MeSH
- exprese genu MeSH
- extracelulární matrix chemie MeSH
- fibroblasty cytologie metabolismus účinky léků MeSH
- financování organizované MeSH
- imunohistochemie MeSH
- játra cytologie metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- messenger RNA biosyntéza účinky léků MeSH
- metaloproteasy genetika metabolismus MeSH
- myocyty hladké svaloviny cytologie metabolismus účinky léků MeSH
- otrava chloridem uhličitým MeSH
- pojivová tkáň metabolismus účinky léků MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteoglykany genetika metabolismus MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- tkáňové inhibitory metaloproteinas MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
