Molecular analysis of mammalian Neu4 sialidase gene promoter

Thesis (Master)--Izmir Institute of Technology, Biotechnology, Izmir, 2011Includes bibliographical referencesText in English; Abstract: Turkish and Englishix, 58 leavesThere are four different mammalian sialidases that have been described; lysosomal (Neu1), cytoplasmic (Neu2), plasma membrane (Neu3), lysosomal/mitochondrial (Neu4). The activity of sialidase Neu4 enzyme against sialic acid containing ganglioside GM2 has been demonstrated. Biological role of sialidase Neu4 enzyme has been shown by the transfection of neuroglia cells from a Tay-Sachs patient with a Neu4-expressing plasmid showed clearance of accumulated ganglioside GM2. It has been also shown that sialidase Neu4 enzyme is responsible for degradation reactions of another ganglioside such as GD1a in brains of Neu4-/- mice. Aim of our study is to identify minimal promoter region of human Neu4 gene and demonstrate binding of transcription factors to this region. In our study, we used bioinformatic approaches to predict the sequence motifs where several specific transcription factors bind using TESS (Transcription Element Seach System) tool. We amplified seven different DNA fragments from human Neu4 promoter region, cloned into luciferase expression vector and performed reporter assay. We also performed electrophoretic mobility shift assay to demonstrate binding of transcription factors to candidate promoter region. We demonstrated that 187 bp upstream of Neu4 gene is minimal promoter region to control transcription from Neu4 gene. Electrophoretic mobility shift assay showed that 187 bp upstream region recruits several transcription factors. Our results demonstrated the minimal promoter region revealing several putative transcription factors such as Sp-1 and c-myc which might be responsible mainly for regulation of Neu4 gene transcription. The data we obtained might be useful to discover small molecules which can control Neu4 gene expression. High expression of Neu4 gene might be controlled using drugs or small molecules and the accumulated GM2 ganglioside in lysosomes of Tay-Sachs patients can be reduced

(R)-4'-metilklavuzon'un karaciğer kanser hücreleri ve karaciğer kanser kök hücreleri üzerindeki antikanser özelliklerinin incelenmesi

Thesis (Doctoral)--İzmir Institute of Technology, Bioengineering, İzmir, 2017Full text release delayed at author's request until 2021.01.17Includes bibliographical references (leaves: 104-121)Text in English; Abstract: Turkish and EnglishHepatocellular carcinoma (HCC) is the fifth most seen cancer type and the third leading cause of death from cancers. HCC is a fatal disease and HCC patients have a 5-year survival rate of 14%. Discovery and identification of mechanisms of action for new therapeutic agents are required for a better treatment of HCC. One of the most important target in cancer treatment is the epigenetic acetylation of histones. Histone deacetylases (HDAC) and sirtuins provide chromatin compaction and transcriptional repression by removing acetyl groups from histone proteins and nonhistone proteins. Re-acetylation of chromatin and re-expression of tumor suppressor genes with the discovery of novel HDAC and/or sirtuin inhibitors are therapeutic targets in cancer research. In this study, (R)-4’-methylklavuzon was found to be cytotoxic in HuH-7 cells with IC50 values of 1.25 μM for HuH-7 parental cells, 2.5 μM for EpCAM+/CD133+ HuH-7 cells and 1.25 μM for EpCAM-/CD133- HuH-7 cells. It was observed that (R)-4’-methylklavuzon causes cell cycle arrest at G1 phase at 1.00 μM concentration in three cell populations, it induces apoptosis at 10 μM concentration at the end of 24 hours incubation. (R)-4’-methylklavuzon does not inhibit Class I/II HDACs in vitro whereas it causes inhibition of endogenous HDACs and/or sirtuins inside the cells sorted by MACS and FACS at 0.10 μM concentration. (R)-4’-methylklavuzon upregulates p21 expression significantly in HuH-7 cell populations to cause G1 arrest. It causes 45% inhibition in p53/MDM2 complex formation when examined with pure p53 and MDM2 proteins. Drug candidate causes 46% SIRT1 inhibition at 100 μM concentration in vitro whereas there was no inhibition of HDAC1 enzyme at the same concentration. SIRT1 protein levels in HuH-7 parental cells were upregulated to 240% within 24 hours of incubation with 3.00 μM of drug candidate. It was found that (R)-4’-methylklavuzon can also inhibit CRM1 protein providing increased retention of tumor suppressor proteins in the nucleus. p53 was overexpressed at 0.10 and 1.00 μM concentrations within 6 and 24 hours in HepG2 cells but slightly overexpressed in HuH-7 parental cells.Hepatoselüler karsinoma (HSK) beşinci en yayın kanser türü olup kanser nedeniyle ölümlerin üçüncü en yüksek nedenidir. HSK ölümcül bir hastalık olup HSK hastalarının 5 yıllık yaşam süreleri %14’tür. Yeni terapötik ajanların keşfi HSK tedavisi için gerekmektedir. Kanser tedavisinde en önemli hedeflerden birisi histonların asetilasyonudur. Histon deasetilazlar (HDAS) ve sirtuinler asetil gruplarını histon proteinlerinden uzaklaştırarak kromatin sıkılaşmasına ve transkripsiyonel represyona sebep olurlar. Yeni HDAS/sirtuin inhibitörleri ile kromatinin tekrar asetillenerek tümör baskılayıcı genlerin tekrar eksprese edilmesi terapötik hedeflerdendir. Bu çalışmada, (R)-4’-metilklavuzon’un HuH-7 parental hücreleri için 1.25 μM, EpCAM+/CD133+ HuH-7 hücreleri için 2,5 μM; EpCAM-/CD133- HuH-7 hücreleri için 1.25 μM olan IC50 değerleri ile sitotoksik olduğu bulunmuştur. (R)-4’- metilklavuzon’un üç hücre populasyonunda 1,00 μM konsantrasyonda G1 fazında hücre döngüsünde birikmeye sebep olduğu ve 24 saatlik inkübasyonda 10 μM konsantrasyonda apoptozu indüklediği gözlemlenmiştir. (R)-4’-metilklavuzon’un in vitro koşullarda sınıf I/II histon deasetilaz enzimlerini inhibe etmediği ancak MACS ve FACS ile ayrımlanmış hücrelerdeki endojen sınıf I/II histon deasetilazları ve/veya sirtuinleri 0,10 μM konsantrasyonda hücre içinde inhibe ettiği bulunmuştur. (R)-4’- metilklavuzon, HuH-7 hücrelerinde p21 ekspresyonunu 3,3 kata kadar arttırabilmiştir. p53/MDM2 kompleks oluşumunu %45 oranında azalttığı görülmüştür. In vitro koşullarda SIRT1 enziminin 100 μM ilaç adayı ile %46 oranında inhibe olduğu bulunmuştur. HuH-7 hücrelerindeki SIRT1 protein seviyelerinin 3,00 μM ilaç adayı ile 24 saatlik inkübasyonunda %240 oranında artmıştır. (R)-4’-metilklavuzon’un CRM1 proteinini inhibe ederek tümör baskılayıcı proteinleri nukleus içinde tutabileceği anlaşılmıştır. 0,10 μM ve 1,00 μM ilaç adayı ile 6 ve 24 saat muamele edilen hücrelerdeki p53 protein seviyelerinin HepG2 hücrelerinde yüksek miktarda artış gösterdiği ancak HuH-7 hücrelerinde çok az bir artış gösterdiği bulunmuştur.The Scientific and Technological Research Council of Turkey (TUBITAK-113S464) and Izmir Institute of Technology Scientific Research Project, (BAP-2015IYTE04

Molecular analysis of mammalian Neu4 sialidase gene promoter

Thesis (Master)--Izmir Institute of Technology, Biotechnology, Izmir, 2011Includes bibliographical referencesText in English; Abstract: Turkish and Englishix, 58 leavesThere are four different mammalian sialidases that have been described; lysosomal (Neu1), cytoplasmic (Neu2), plasma membrane (Neu3), lysosomal/mitochondrial (Neu4). The activity of sialidase Neu4 enzyme against sialic acid containing ganglioside GM2 has been demonstrated. Biological role of sialidase Neu4 enzyme has been shown by the transfection of neuroglia cells from a Tay-Sachs patient with a Neu4-expressing plasmid showed clearance of accumulated ganglioside GM2. It has been also shown that sialidase Neu4 enzyme is responsible for degradation reactions of another ganglioside such as GD1a in brains of Neu4-/- mice. Aim of our study is to identify minimal promoter region of human Neu4 gene and demonstrate binding of transcription factors to this region. In our study, we used bioinformatic approaches to predict the sequence motifs where several specific transcription factors bind using TESS (Transcription Element Seach System) tool. We amplified seven different DNA fragments from human Neu4 promoter region, cloned into luciferase expression vector and performed reporter assay. We also performed electrophoretic mobility shift assay to demonstrate binding of transcription factors to candidate promoter region. We demonstrated that 187 bp upstream of Neu4 gene is minimal promoter region to control transcription from Neu4 gene. Electrophoretic mobility shift assay showed that 187 bp upstream region recruits several transcription factors. Our results demonstrated the minimal promoter region revealing several putative transcription factors such as Sp-1 and c-myc which might be responsible mainly for regulation of Neu4 gene transcription. The data we obtained might be useful to discover small molecules which can control Neu4 gene expression. High expression of Neu4 gene might be controlled using drugs or small molecules and the accumulated GM2 ganglioside in lysosomes of Tay-Sachs patients can be reduced

CRM1 inhibitory and antiproliferative activities of novel 4′-alkyl substituted klavuzon derivatives

Klavuzons are 6-(naphthalen-1-yl) substituted 5,6-dihydro-2H-pyran-2-one derivatives showing promising antiproliferative activities in variety of cancer cell lines. In this work, racemic syntheses of nine novel 4′-alkyl substituted klavuzon derivatives were completed in eight steps and anticancer properties of these compounds were evaluated. It is found that size of the substituent has dramatic effect over the potency and selectivity of the cytotoxic activity in cancerous and healthy pancreatic cell lines. The size of the substituent can also effect the CRM1 inhibitory properties of klavuzon derivatives. Strong cytotoxic activity and CRM1 inhibition can be observed only when a small substituent present at 4′-position of naphthalen-1-yl group. However, these substituents makes the molecule more cytotoxic in healthy pancreatic cells rather than cancerous pancreatic cells. Among the tested compounds 1,2,3,4-tetrahydrophenanthren-9-yl substituted lactone was the most cytotoxic compound and its antiproliferative activity was also tested in 3D spheroids generated from HuH-7 cell lines.Scientific and Technological Research Council of Turkey (TUBITAK 113Z146

Antiproliferative activity of (R)-4 '-methylklavuzon on hepatocellular carcinoma cells and EpCAM(+)/CD133(+) cancer stem cells via SIRT1 and Exportin-1 (CRM1) inhibition

Cytotoxic effects of (R)-4'-methylklavuzon were investigated on hepatocellular carcinoma cells (HuH-7 and HepG2) and HuH-7 EpCAM(+)/CD133(+) cancer stem cells. IC50 of (R)-4'-methylklavuzon was found as 1.25 mu M for HuH-7 parental cells while it was found as 2.50 mu M for HuH-7 EpCAM(+)/CD133(+) cancer stem cells. (R)-4'-methylklavuzon tended to show more efficient in vitro cytotoxicity with its lower IC50 values on hepatocellular carcinoma cell lines compared to its lead molecule, goniothalamin and FDA-approved drugs, sorafenib and regorafenib. Cell-based Sirtuin/HDAC enzyme activity measurements revealed that endogenous Sirtuin/HDAC enzymes were reduced by 40% compared to control. SIRT1 protein levels were upregulated indicating triggered DNA repair mechanism. p53 was overexpressed in HepG2 cells. (R)-4'methylklavuzon inhibited CRM1 protein providing increased retention of p53 and RIOK2 protein in the nucleus. HuH-7 parental and EpCAM(+)/CD133(+) cancer stem cell spheroids lost intact morphology. 3D HepG2 spheroid viabilities were decreased in a correlation with upregulation in p53 protein levels. (C) 2019 Elsevier Masson SAS. All rights reserved