Joanna Fraczek | Universiteit Gent and Vrije Universiteit Brussel (original) (raw)

Papers by Joanna Fraczek

Research paper thumbnail of Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Archives of toxicology, 2013

This review encompasses the most important advances in liver functions and hepatotoxicity and ana... more This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Research paper thumbnail of Histone deacetylase inhibition and the regulation of cell growth with particular reference to liver pathobiology

Journal of Cellular and Molecular Medicine, 2010

• Introduction• Histone Deacetylases- Classification- Histone-dependent mode of action- Non-histo... more • Introduction• Histone Deacetylases- Classification- Histone-dependent mode of action- Non-histone targets• Cell cycle-related histone and non-histone substrates of histone deacetylases- Retinoblastoma- Protein 53 transcription factor- Cyclin-dependent kinase inhibitor p21Cip1- Proliferating cell nuclear antigen• Inhibition of histone deacetylases in liver cells- Physiological condition: effects of histone deacetylase inhibitors on primary hepatocytes- Pathophysiological condition- Liver malignancy – hepatocellular carcinoma- Liver fibrosis- Histone deacetylase inhibitors as modulators of the hepatic stellate cell myofibroblastic phenotype- The role of histone deacetylases in the regulation of pro-fibrogenic and pro-inflammatory cascades• ConclusionsIntroductionHistone Deacetylases- Classification- Histone-dependent mode of action- Non-histone targetsClassificationHistone-dependent mode of actionNon-histone targetsCell cycle-related histone and non-histone substrates of histone deacetylases- Retinoblastoma- Protein 53 transcription factor- Cyclin-dependent kinase inhibitor p21Cip1- Proliferating cell nuclear antigenRetinoblastomaProtein 53 transcription factorCyclin-dependent kinase inhibitor p21Cip1Proliferating cell nuclear antigenInhibition of histone deacetylases in liver cells- Physiological condition: effects of histone deacetylase inhibitors on primary hepatocytes- Pathophysiological condition- Liver malignancy – hepatocellular carcinoma- Liver fibrosis- Histone deacetylase inhibitors as modulators of the hepatic stellate cell myofibroblastic phenotype- The role of histone deacetylases in the regulation of pro-fibrogenic and pro-inflammatory cascadesPhysiological condition: effects of histone deacetylase inhibitors on primary hepatocytesPathophysiological conditionLiver malignancy – hepatocellular carcinomaLiver fibrosisHistone deacetylase inhibitors as modulators of the hepatic stellate cell myofibroblastic phenotypeThe role of histone deacetylases in the regulation of pro-fibrogenic and pro-inflammatory cascadesConclusionsThe transcriptional activity of genes largely depends on the accessibility of specific chromatin regions to transcriptional regulators. This process is controlled by diverse post-transcriptional modifications of the histone amino termini of which reversible acetylation plays a vital role. Histone acetyltransferases (HATs) are responsible for the addition of acetyl groups and histone deacetylases (HDACs) catalyse the reverse reaction. In general, though not exclusively, histone acetylation is associated with a positive regulation of transcription, whereas histone deacetylation is correlated with transcriptional silencing. The elucidation of unequivocal links between aberrant action of HDACs and tumorigenesis lies at the base of key scientific importance of these enzymes. In particular, the potential benefit of HDAC inhibition has been confirmed in various tumour cell lines, demonstrating antiproliferative, differentiating and pro-apoptotic effects. Consequently, the dynamic quest for HDAC inhibitors (HDIs) as a new class of anticancer drugs was set off, resulting in a number of compounds that are currently evaluated in clinical trials. Ironically, the knowledge with respect to the expression pattern and function of individual HDAC isoenzymes remains largely elusive. In the present review, we provide an update of the current knowledge on the involvement of HDACs in the regulation of fundamental cellular processes in the liver, being the main site for drug metabolism within the body. Focus lies on the involvement of HDACs in the regulation of growth of normal and transformed hepatocytes and the transdifferentiation process of stellate cells. Furthermore, extrapolation of our present knowledge on HDAC functionality towards innovative treatment of malignant and non-malignant, hyperproliferative and inflammatory disorders is discussed.

Research paper thumbnail of Preservation of hepatocellular functionality in cultures of primary rat hepatocytes upon exposure to 4Me 2N-BAVAH, a hydroxamate-based HDAC-inhibitor

Toxicology in Vitro, 2011

Great efforts are being put in the development/optimization of reliable and highly predictive mod... more Great efforts are being put in the development/optimization of reliable and highly predictive models for high-throughput screening of efficacy and toxicity of promising drug candidates. The use of primary hepatocyte cultures, however, is still limited by the occurrence of phenotypic alterations, including loss of xenobiotic biotransformation capacity.

Research paper thumbnail of Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation

Journal of Hepatology, 2009

Controlling both growth and differentiation of stem cells and their differentiated somatic progen... more Controlling both growth and differentiation of stem cells and their differentiated somatic progeny is a challenge in numerous fields, from preclinical drug development to clinical therapy. Recently, new insights into the underlying molecular mechanisms have unveiled key regulatory roles of epigenetic marks driving cellular pluripotency, differentiation and self-renewal/proliferation. Indeed, the transcription of genes, governing cell-fate decisions during development and maintenance of a cell's differentiated status in adult life, critically depends on the chromatin accessibility of transcription factors to genomic regulatory and coding regions. In this review, we discuss the epigenetic control of (liver-specific) gene-transcription and the intricate interplay between chromatin modulation, including histone (de)acetylation and DNA (de)methylation, and liver-enriched transcription factors. Special attention is paid to their role in directing hepatic differentiation of primary hepatocytes and stem cells in vitro. Ó

Research paper thumbnail of Histone deacetylase inhibitors: Opening a new field of research in the development of alternative methods

In the present study, the ability of the hydroxamate histone deacetylase (HDAC) inhibitor 4-Me 2 ... more In the present study, the ability of the hydroxamate histone deacetylase (HDAC) inhibitor 4-Me 2 N-BAVAH (5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamate) to prevent dedifferentiation in cultures of primary hepatocytes is investigated. To this end, rat hepatocytes were isolated in the presence of 50µM 4-Me 2 N-BAVAH and exposure was subsequently continued for 7 days in monolayer culture (0.05% v/v ethanol-exposed cultures served as vehicle controls). It was found that in the continuous presence of 4-Me 2 N-BAVAH, albumin secretion into the culture medium was 2.9-and 5.3-fold higher when compared to the control results after 4 and 7 days of cultivation, respectively. CYP1A1 was re-expressed after 4 days culture, but declined again thereafter, whilst CYP2B1 protein levels were maintained throughout the culture time in 4-Me 2 N-BAVAH-treated cells. In addition, pro-caspase-3 cleavage was significantly reduced after 7 days, pointing to decreased apoptosis and thus improved survival in the 4-Me 2 N-BAVAH-exposed hepatocytes. These findings provide additional evidence that HDAC inhibitors could play a major role in the preservation of the differentiated hepatic phenotype in vitro.

Research paper thumbnail of Screening of Trichostatin Analogues Based on Cellular Potency in the Murine Multiple Myeloma 5T33MM Model

Vanderkerken], Toxicology [J. Fraczek, T. Doktorova, T. Vanhaecke, V. Rogiers], Organic Chemistry... more Vanderkerken], Toxicology [J. Fraczek, T. Doktorova, T. Vanhaecke, V. Rogiers], Organic Chemistry [A. Lukaszuk, D. Tourwé], and Cell Biology [A. Geerts], Vrije University Brussel (VUB), Brussels, Belgium AIM: To evaluate the screening of histone deacetylase inhibitors (HDACi) based on cellular potency using the murine multiple myeloma 5T33MM model. METHODS: The cellular potencies of 10 structurally related compounds of the natural HDACi Trichostatin (TSA) were screened by measuring the DNA synthesis in primary murine 5T33MMvv cells. The anti-tumor activity of the three most potent analogues was further confirmed in the 5T33MMvt cell line using proliferation, viability and active caspase-3 assays. The data of this 5T33MM model were compared with the HDAC IC 50 of these compounds which were preliminarily determined in normal hepatocyte extract. , having a pentanoic spacer, significantly reduced the DNA synthesis and the viability. We further observed a significant increase of active caspase-3 in the 5T33MM cells when treated with compound 3 or 4, reflecting their apoptosis-inducing capability. When comparing with the HDAC-inhibitory activities of these compounds in normal hepatocyte extract, a similar result was obtained to suggest that compound 4 had the most potent anti-cancer activity.

Research paper thumbnail of Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors

Investigational New Drugs, 2009

The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug–target (ca... more The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug–target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes.

Research paper thumbnail of Synergetic effects of DNA demethylation and histone deacetylase inhibition in primary rat hepatocytes

Investigational New Drugs

Both, DNA methylation and histone deacetylation play a crucial role in cancer development by sile... more Both, DNA methylation and histone deacetylation play a crucial role in cancer development by silencing the expression of specific tumour suppressor genes. Several studies describe the use of combinations of DNA methyltransferase inhibitors (DNMT-i) and histone deacetylase inhibitors (HDAC-i) as an improved strategy to treat neoplasms. However, no information is available concerning their biological impact on healthy, non-malignant cells, including hepatocytes. Therefore, the effects of the combination of the DNMT-i decitabine (DAC) with the HDAC-i 6-[(4-pyrrolidine-1-ylbenzoyl) amino] hexanoic acid hydroxamate (AN-8) on cell proliferation and differentiation were examined in primary rat hepatocyte cultures. We found that, upon simultaneous exposure of the cells to both compounds, a synergetic anti-proliferative outcome was achieved. This inhibition of DNA synthesis was accompanied by a reduced expression of cyclin-dependent kinase 1 (cdk1), a key cell cycle marker that controls the S/G2/M transition. Compared to exposure of the cells to each agent separately, the combination of lower concentrations of both DAC and AN-8 promoted the maintenance of the differentiated phenotype of the cells as a function of culture time. The functionality of the hepatocytes was evidenced by an increased expression of the phase I biotransformation enzyme cytochrome P 450 (CYP) 1A1 and albumin secretion capacity when both agents were used in combination.

Research paper thumbnail of Effect of Trichostatin A on miRNA expression in cultures of primary rat hepatocytes

Toxicology in Vitro, 2011

In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was in... more In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was investigated on the microRNA (miR, miRNA) expression profile in cultured primary rat hepatocytes by means of microarray analysis. Simultaneously, albumin secretory capacity and morphological features of the hepatocytes were evaluated throughout the culture time. In total, 25 out of 348 miRNAs were found to be differentially expressed between freshly isolated hepatocytes and 7-day cultured cells. Nineteen of these miRNAs were connected with 'general metabolism'. miR-21 and miR-126 were shown to be the most up and down regulated miRs upon cultivation and could be linked to the proliferative response triggered in the hepatocytes upon their isolation from the liver. miR-379 and miR-143, on the other hand, were found to be the most up and down regulated miRs upon TSA treatment. Together with the higher expression of miR-122 observed in TSA-treated versus non-treated cultures, we hypothesize that the changes observed for miR-122, miR-143 and miR-379 could be related to the inhibitory effects of TSA on hepatocellular proliferation.

Research paper thumbnail of Histone deacetylase inhibitors in multiple myeloma

Hematology Reviews, 2009

Novel drugs such as bortezomib and highdose chemotherapy combined with stem cell transplantation ... more Novel drugs such as bortezomib and highdose chemotherapy combined with stem cell transplantation improved the outcome of multiple myeloma patients in the past decade. However, multiple myeloma often remains incurable due to the development of drug resistance governed by the bone marrow microenvironment. Therefore targeting new pathways to overcome this resistance is needed. Histone deacetylase (HDAC) inhibitors represent a new class of anti-myeloma agents. Inhibiting HDACs results in histone hyperacetylation and alterations in chromatine structure, which, in turn, cause growth arrest differentiation and/or apoptosis in several tumor cells.

Research paper thumbnail of DNA methyltransferase 3a expression decreases during apoptosis in primary cultures of hepatocytes

Toxicology in Vitro, 2010

Although it has been acknowledged that DNA methylation is a key factor in the epigenetic control ... more Although it has been acknowledged that DNA methylation is a key factor in the epigenetic control of liver homeostasis, its role in the occurrence of hepatocyte cell death has yet been poorly documented. We therefore have investigated the expression pattern of the effectors of DNA methylation, namely DNA methyltransferase (DNMT) isoenzymes, during Fas-mediated apoptotic cell death in primary hepatocyte cultures. Cell death was assessed by in situ stainings with Annexin V, Hoechst 33342 and Propidium iodide, and measurement of caspase 3-like activity and lactate dehydrogenase release. Similar to the hepatic in vivo situation, DNMT1, DNMT2 and DNMT3b could not be detected, whereas relatively high levels of DNMT3a protein were observed in the in vitro setting, as studied by immunoblotting. Upon induction of cell death, a progressive decrease in DNMT3a protein amount was noticed, reaching a minimum level towards the final stages of the cell death process. This was preceded by parallel changes in DNMT3a mRNA production, measured by qRT-PCR analysis, which became already evident during the early stages of apoptosis. We conclude that downregulated DNMT3a protein production during Fas-mediated hepatocyte apoptosis results from inhibition of DNMT3a gene transcription. This finding further substantiates the existence of an epigenetic signature of apoptosis. Crown

Research paper thumbnail of Biology and pathobiology of gap junctional channels in hepatocytes

Hepatology, 2008

The present review provides the state of the art of the current knowledge concerning gap junction... more The present review provides the state of the art of the current knowledge concerning gap junctional channels and their roles in liver functioning. In the first part, we summarize some relevant biochemical properties of hepatic gap junctional channels, including their structure and regulation. In the second part, we discuss the involvement of gap junctional channels in the occurrence of liver cell growth, liver cell differentiation, and liver cell death. We further exemplify their relevance in hepatic pathophysiology. Finally, a number of directions for future liver gap junctional channel research are proposed, and the up-regulation of gap junctional channel activity as a novel strategy in (liver) cancer therapy is illustrated. (HEPATOLOGY 2007.)

Research paper thumbnail of Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Archives of toxicology, 2013

This review encompasses the most important advances in liver functions and hepatotoxicity and ana... more This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Research paper thumbnail of Histone deacetylase inhibition and the regulation of cell growth with particular reference to liver pathobiology

Journal of Cellular and Molecular Medicine, 2010

• Introduction• Histone Deacetylases- Classification- Histone-dependent mode of action- Non-histo... more • Introduction• Histone Deacetylases- Classification- Histone-dependent mode of action- Non-histone targets• Cell cycle-related histone and non-histone substrates of histone deacetylases- Retinoblastoma- Protein 53 transcription factor- Cyclin-dependent kinase inhibitor p21Cip1- Proliferating cell nuclear antigen• Inhibition of histone deacetylases in liver cells- Physiological condition: effects of histone deacetylase inhibitors on primary hepatocytes- Pathophysiological condition- Liver malignancy – hepatocellular carcinoma- Liver fibrosis- Histone deacetylase inhibitors as modulators of the hepatic stellate cell myofibroblastic phenotype- The role of histone deacetylases in the regulation of pro-fibrogenic and pro-inflammatory cascades• ConclusionsIntroductionHistone Deacetylases- Classification- Histone-dependent mode of action- Non-histone targetsClassificationHistone-dependent mode of actionNon-histone targetsCell cycle-related histone and non-histone substrates of histone deacetylases- Retinoblastoma- Protein 53 transcription factor- Cyclin-dependent kinase inhibitor p21Cip1- Proliferating cell nuclear antigenRetinoblastomaProtein 53 transcription factorCyclin-dependent kinase inhibitor p21Cip1Proliferating cell nuclear antigenInhibition of histone deacetylases in liver cells- Physiological condition: effects of histone deacetylase inhibitors on primary hepatocytes- Pathophysiological condition- Liver malignancy – hepatocellular carcinoma- Liver fibrosis- Histone deacetylase inhibitors as modulators of the hepatic stellate cell myofibroblastic phenotype- The role of histone deacetylases in the regulation of pro-fibrogenic and pro-inflammatory cascadesPhysiological condition: effects of histone deacetylase inhibitors on primary hepatocytesPathophysiological conditionLiver malignancy – hepatocellular carcinomaLiver fibrosisHistone deacetylase inhibitors as modulators of the hepatic stellate cell myofibroblastic phenotypeThe role of histone deacetylases in the regulation of pro-fibrogenic and pro-inflammatory cascadesConclusionsThe transcriptional activity of genes largely depends on the accessibility of specific chromatin regions to transcriptional regulators. This process is controlled by diverse post-transcriptional modifications of the histone amino termini of which reversible acetylation plays a vital role. Histone acetyltransferases (HATs) are responsible for the addition of acetyl groups and histone deacetylases (HDACs) catalyse the reverse reaction. In general, though not exclusively, histone acetylation is associated with a positive regulation of transcription, whereas histone deacetylation is correlated with transcriptional silencing. The elucidation of unequivocal links between aberrant action of HDACs and tumorigenesis lies at the base of key scientific importance of these enzymes. In particular, the potential benefit of HDAC inhibition has been confirmed in various tumour cell lines, demonstrating antiproliferative, differentiating and pro-apoptotic effects. Consequently, the dynamic quest for HDAC inhibitors (HDIs) as a new class of anticancer drugs was set off, resulting in a number of compounds that are currently evaluated in clinical trials. Ironically, the knowledge with respect to the expression pattern and function of individual HDAC isoenzymes remains largely elusive. In the present review, we provide an update of the current knowledge on the involvement of HDACs in the regulation of fundamental cellular processes in the liver, being the main site for drug metabolism within the body. Focus lies on the involvement of HDACs in the regulation of growth of normal and transformed hepatocytes and the transdifferentiation process of stellate cells. Furthermore, extrapolation of our present knowledge on HDAC functionality towards innovative treatment of malignant and non-malignant, hyperproliferative and inflammatory disorders is discussed.

Research paper thumbnail of Preservation of hepatocellular functionality in cultures of primary rat hepatocytes upon exposure to 4Me 2N-BAVAH, a hydroxamate-based HDAC-inhibitor

Toxicology in Vitro, 2011

Great efforts are being put in the development/optimization of reliable and highly predictive mod... more Great efforts are being put in the development/optimization of reliable and highly predictive models for high-throughput screening of efficacy and toxicity of promising drug candidates. The use of primary hepatocyte cultures, however, is still limited by the occurrence of phenotypic alterations, including loss of xenobiotic biotransformation capacity.

Research paper thumbnail of Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation

Journal of Hepatology, 2009

Controlling both growth and differentiation of stem cells and their differentiated somatic progen... more Controlling both growth and differentiation of stem cells and their differentiated somatic progeny is a challenge in numerous fields, from preclinical drug development to clinical therapy. Recently, new insights into the underlying molecular mechanisms have unveiled key regulatory roles of epigenetic marks driving cellular pluripotency, differentiation and self-renewal/proliferation. Indeed, the transcription of genes, governing cell-fate decisions during development and maintenance of a cell's differentiated status in adult life, critically depends on the chromatin accessibility of transcription factors to genomic regulatory and coding regions. In this review, we discuss the epigenetic control of (liver-specific) gene-transcription and the intricate interplay between chromatin modulation, including histone (de)acetylation and DNA (de)methylation, and liver-enriched transcription factors. Special attention is paid to their role in directing hepatic differentiation of primary hepatocytes and stem cells in vitro. Ó

Research paper thumbnail of Histone deacetylase inhibitors: Opening a new field of research in the development of alternative methods

In the present study, the ability of the hydroxamate histone deacetylase (HDAC) inhibitor 4-Me 2 ... more In the present study, the ability of the hydroxamate histone deacetylase (HDAC) inhibitor 4-Me 2 N-BAVAH (5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamate) to prevent dedifferentiation in cultures of primary hepatocytes is investigated. To this end, rat hepatocytes were isolated in the presence of 50µM 4-Me 2 N-BAVAH and exposure was subsequently continued for 7 days in monolayer culture (0.05% v/v ethanol-exposed cultures served as vehicle controls). It was found that in the continuous presence of 4-Me 2 N-BAVAH, albumin secretion into the culture medium was 2.9-and 5.3-fold higher when compared to the control results after 4 and 7 days of cultivation, respectively. CYP1A1 was re-expressed after 4 days culture, but declined again thereafter, whilst CYP2B1 protein levels were maintained throughout the culture time in 4-Me 2 N-BAVAH-treated cells. In addition, pro-caspase-3 cleavage was significantly reduced after 7 days, pointing to decreased apoptosis and thus improved survival in the 4-Me 2 N-BAVAH-exposed hepatocytes. These findings provide additional evidence that HDAC inhibitors could play a major role in the preservation of the differentiated hepatic phenotype in vitro.

Research paper thumbnail of Screening of Trichostatin Analogues Based on Cellular Potency in the Murine Multiple Myeloma 5T33MM Model

Vanderkerken], Toxicology [J. Fraczek, T. Doktorova, T. Vanhaecke, V. Rogiers], Organic Chemistry... more Vanderkerken], Toxicology [J. Fraczek, T. Doktorova, T. Vanhaecke, V. Rogiers], Organic Chemistry [A. Lukaszuk, D. Tourwé], and Cell Biology [A. Geerts], Vrije University Brussel (VUB), Brussels, Belgium AIM: To evaluate the screening of histone deacetylase inhibitors (HDACi) based on cellular potency using the murine multiple myeloma 5T33MM model. METHODS: The cellular potencies of 10 structurally related compounds of the natural HDACi Trichostatin (TSA) were screened by measuring the DNA synthesis in primary murine 5T33MMvv cells. The anti-tumor activity of the three most potent analogues was further confirmed in the 5T33MMvt cell line using proliferation, viability and active caspase-3 assays. The data of this 5T33MM model were compared with the HDAC IC 50 of these compounds which were preliminarily determined in normal hepatocyte extract. , having a pentanoic spacer, significantly reduced the DNA synthesis and the viability. We further observed a significant increase of active caspase-3 in the 5T33MM cells when treated with compound 3 or 4, reflecting their apoptosis-inducing capability. When comparing with the HDAC-inhibitory activities of these compounds in normal hepatocyte extract, a similar result was obtained to suggest that compound 4 had the most potent anti-cancer activity.

Research paper thumbnail of Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors

Investigational New Drugs, 2009

The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug–target (ca... more The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug–target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes.

Research paper thumbnail of Synergetic effects of DNA demethylation and histone deacetylase inhibition in primary rat hepatocytes

Investigational New Drugs

Both, DNA methylation and histone deacetylation play a crucial role in cancer development by sile... more Both, DNA methylation and histone deacetylation play a crucial role in cancer development by silencing the expression of specific tumour suppressor genes. Several studies describe the use of combinations of DNA methyltransferase inhibitors (DNMT-i) and histone deacetylase inhibitors (HDAC-i) as an improved strategy to treat neoplasms. However, no information is available concerning their biological impact on healthy, non-malignant cells, including hepatocytes. Therefore, the effects of the combination of the DNMT-i decitabine (DAC) with the HDAC-i 6-[(4-pyrrolidine-1-ylbenzoyl) amino] hexanoic acid hydroxamate (AN-8) on cell proliferation and differentiation were examined in primary rat hepatocyte cultures. We found that, upon simultaneous exposure of the cells to both compounds, a synergetic anti-proliferative outcome was achieved. This inhibition of DNA synthesis was accompanied by a reduced expression of cyclin-dependent kinase 1 (cdk1), a key cell cycle marker that controls the S/G2/M transition. Compared to exposure of the cells to each agent separately, the combination of lower concentrations of both DAC and AN-8 promoted the maintenance of the differentiated phenotype of the cells as a function of culture time. The functionality of the hepatocytes was evidenced by an increased expression of the phase I biotransformation enzyme cytochrome P 450 (CYP) 1A1 and albumin secretion capacity when both agents were used in combination.

Research paper thumbnail of Effect of Trichostatin A on miRNA expression in cultures of primary rat hepatocytes

Toxicology in Vitro, 2011

In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was in... more In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was investigated on the microRNA (miR, miRNA) expression profile in cultured primary rat hepatocytes by means of microarray analysis. Simultaneously, albumin secretory capacity and morphological features of the hepatocytes were evaluated throughout the culture time. In total, 25 out of 348 miRNAs were found to be differentially expressed between freshly isolated hepatocytes and 7-day cultured cells. Nineteen of these miRNAs were connected with 'general metabolism'. miR-21 and miR-126 were shown to be the most up and down regulated miRs upon cultivation and could be linked to the proliferative response triggered in the hepatocytes upon their isolation from the liver. miR-379 and miR-143, on the other hand, were found to be the most up and down regulated miRs upon TSA treatment. Together with the higher expression of miR-122 observed in TSA-treated versus non-treated cultures, we hypothesize that the changes observed for miR-122, miR-143 and miR-379 could be related to the inhibitory effects of TSA on hepatocellular proliferation.

Research paper thumbnail of Histone deacetylase inhibitors in multiple myeloma

Hematology Reviews, 2009

Novel drugs such as bortezomib and highdose chemotherapy combined with stem cell transplantation ... more Novel drugs such as bortezomib and highdose chemotherapy combined with stem cell transplantation improved the outcome of multiple myeloma patients in the past decade. However, multiple myeloma often remains incurable due to the development of drug resistance governed by the bone marrow microenvironment. Therefore targeting new pathways to overcome this resistance is needed. Histone deacetylase (HDAC) inhibitors represent a new class of anti-myeloma agents. Inhibiting HDACs results in histone hyperacetylation and alterations in chromatine structure, which, in turn, cause growth arrest differentiation and/or apoptosis in several tumor cells.

Research paper thumbnail of DNA methyltransferase 3a expression decreases during apoptosis in primary cultures of hepatocytes

Toxicology in Vitro, 2010

Although it has been acknowledged that DNA methylation is a key factor in the epigenetic control ... more Although it has been acknowledged that DNA methylation is a key factor in the epigenetic control of liver homeostasis, its role in the occurrence of hepatocyte cell death has yet been poorly documented. We therefore have investigated the expression pattern of the effectors of DNA methylation, namely DNA methyltransferase (DNMT) isoenzymes, during Fas-mediated apoptotic cell death in primary hepatocyte cultures. Cell death was assessed by in situ stainings with Annexin V, Hoechst 33342 and Propidium iodide, and measurement of caspase 3-like activity and lactate dehydrogenase release. Similar to the hepatic in vivo situation, DNMT1, DNMT2 and DNMT3b could not be detected, whereas relatively high levels of DNMT3a protein were observed in the in vitro setting, as studied by immunoblotting. Upon induction of cell death, a progressive decrease in DNMT3a protein amount was noticed, reaching a minimum level towards the final stages of the cell death process. This was preceded by parallel changes in DNMT3a mRNA production, measured by qRT-PCR analysis, which became already evident during the early stages of apoptosis. We conclude that downregulated DNMT3a protein production during Fas-mediated hepatocyte apoptosis results from inhibition of DNMT3a gene transcription. This finding further substantiates the existence of an epigenetic signature of apoptosis. Crown

Research paper thumbnail of Biology and pathobiology of gap junctional channels in hepatocytes

Hepatology, 2008

The present review provides the state of the art of the current knowledge concerning gap junction... more The present review provides the state of the art of the current knowledge concerning gap junctional channels and their roles in liver functioning. In the first part, we summarize some relevant biochemical properties of hepatic gap junctional channels, including their structure and regulation. In the second part, we discuss the involvement of gap junctional channels in the occurrence of liver cell growth, liver cell differentiation, and liver cell death. We further exemplify their relevance in hepatic pathophysiology. Finally, a number of directions for future liver gap junctional channel research are proposed, and the up-regulation of gap junctional channel activity as a novel strategy in (liver) cancer therapy is illustrated. (HEPATOLOGY 2007.)