Interest of new alkylsulfonylhydrazide-type compound in the treatment of alcohol use disorders.

RATIONALE: Recent preclinical research suggested that histone deacetylase inhibitors (HDACIs) and specifically class I HDAC selective inhibitors might be useful to treat alcohol use disorders (AUDs). OBJECTIVE: The objective of this study was to find a new inhibitor of the HDAC-1 isoenzyme and to test its efficacy in an animal model of AUDs. METHODS: In the present study, we prepared new derivatives bearing sulfonylhydrazide-type zinc-binding group (ZBG) and evaluated these compounds in vitro on HDAC-1 isoenzyme. The most promising compound was tested on ethanol operant self-administration and relapse in rats. RESULTS: We showed that the alkylsulfonylhydrazide-type compound (ASH) reduced by more than 55% the total amount of ethanol consumed after one intracerebroventricular microinjection, while no effect was observed on motivation of the animals to consume ethanol. In addition, one ASH injection in the central amygdala reduced relapse. CONCLUSIONS: Our study demonstrated that a new compound designed to target HDAC-1 is effective in reducing ethanol intake and relapse in rats and further confirm the interest of pursuing research to study the exact mechanism by which such inhibitor may be useful to treat AUDs.

Variation of the epidermal expression of glucocorticoid receptor-beta as potential predictive marker of bullous pemphigoid outcome.

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease in Western countries. Although topical and/or systemic glucocorticoids treatment efficacy is widely recognized, up to 30% of patients with BP may undergo a relapse during the first year of treatment. We investigated the protein expression of the total glucocorticoid receptor and GRß isoform in the skin biopsy specimens from patients with BP and wondered whether such investigation at baseline provided a tool to predict disease outcome. Total GR and GRß protein expressions were detected by immunohistochemistry at baseline on 12 patients who later relapse and 11 patients who remained on remission in comparison with 14 control patients. The expression of GRß in the epidermis of patients with BP who later relapse was significantly higher than that in the epidermis of patients with BP controlled upon corticosteroid treatment, which was also higher than control patients. Thus, our results suggest that increased protein expression of GRß in skin epithelial cells is predictive of reduced steroid treatment efficacy, and therefore of increased risk of disease relapse in patients with BP.

Histone deacetylases meet microRNA-associated MMP-9 expression regulation in glucocorticoid-sensitive and -resistant cell lines.

Glucocorticoids are largely used in the treatment of inflammatory pathologies and/or hematological malignancies and regulate the expression of a variety of genes involved in inflammation or metastasis such as matrix metalloproteinases (MMP). Long-term exposure to glucocorticoids can result in failure of responsiveness, which is often associated with an unwanted gene expression. Epigenetic mechanisms are involved in gene expression modulated after development of glucocorticoid resistance but how these mechanisms take place must be further studied. The effects of HDAC inhibitors (HDACi) in a context of glucocorticoid resistance are still not well understood and need to be further investigated. We hypothesized that acquired glucocorticoid resistance associated to HDACi could disturbs epigenetic landscape, especially miR expression, leading to a modulation of MMP-9 gene expression and/or protein secretion, described as largely involved in bone remodeling and tumor invasion in multiple myeloma. To this aim, we used sensitive RPMI-8226 cell line and its dexamethasone- and methylprednisolone-resistant derivatives. The resistant cell lines displayed an 'open chromatin' and an MMP-9 overexpression comparatively to the sensitive cell line. HDACi treatment with MS-275 increased even more MMP-9 overexpression not only at an mRNA level but also at the protein level. We showed that MMP-9 expression regulation was not directly linked with HAT/HDAC balance alterations but rather with the deregulation of MMP-9-targeting miRs. Then, we first demonstrated that miR­149 downregulation was directly involved in the MMP-9 overexpression following a chronic glucocorticoid exposure and that MS-275 could amplify this overexpression by inhibition of miR­149 expression and miR­520c overexpression. Taken together, these results indicate that the use of HDACi in a context of acquired glucocorticoid resistance could modify the epigenetic landscape, highlighting the importance of taking the glucocorticoid response status into consideration in treatment with HDACi.

The DNA hypomethylating agent, 5-aza-2'-deoxycytidine, enhances tumor cell invasion through a transcription-dependent modulation of MMP-1 expression in human fibrosarcoma cells.

In diseases such as cancer, cells need to degrade the extracellular matrix (ECM) and therefore require high protease levels. Thus, aberrant tissue degradation is associated to matrix metalloproteinases (MMPs) overexpression resulting from different mechanisms including epigenetic events. One of the most characterized epigenetic mechanisms is DNA methylation causing changes in chromatin conformation, thereby decreasing the accessibility to the transcriptional machinery and resulting in a robust gene silencing. Modulation of DNA methylation by DNA hypomethylating agents such as 5-aza-2'-deoxycytidine (5-azadC) is widely used in epigenetic anticancer treatments. Here, we focus on the effects of this drug on the expression level of MMP-1, -2, and -9 in human HT1080 fibrosarcoma cells. We demonstrate that 5-azadC increases MMP expression at both mRNA and protein levels, and promotes invasion potential of HT1080 cells. Using broad-spectrum and specific MMP inhibitors, we establish that MMP-1, but not MMP-2 and -9, plays a key role in 5-azadC-enhanced cell invasion. We show that 5-azadC induces MMP-1 expression through a transcriptional mechanism without affecting MMP-1 promoter methylation status. Finally, we demonstrate that 5-azadC treatment increases the nuclear levels of Sp1 and Sp3 transcription factors, and modulates their recruitment to the MMP-1 promoter, resulting in chromatin remodeling associated to 5-azadC-induced MMP-1 expression. All together, our data indicate that the hypomethylating agent 5-azadC modulates, mainly via Sp1 recruitment, MMP-1 expression resulting in an increased invasive potential of HT1080 cells.

Raman microspectroscopy detects epigenetic modifications in living Jurkat leukemic cells.

AIMS: Classical biochemical and molecular methods for discerning cells with epigenetic modifications are often biologically perturbing or even destructive. We wondered whether the noninvasive laser tweezer Raman spectroscopy technique allowed the discrimination of single living human cells undergoing epigenetic modifications. MATERIALS & METHODS: Human Jurkat leukemic cells were treated with inhibitors of histone deacetylases (trichostatin A and MS-275). Epigenetic changes were monitored through histone electrophoresis, nuclear image cytometry and laser tweezer Raman spectroscopy. RESULTS: Treatment of Jurkat cells with histone deacetylase inhibitors increased histone acetylation and induced chromatin organization changes. Characteristic vibrations, issued from laser tweezer Raman spectroscopy analyses, mostly assigned to DNA and proteins allowed discerning histone deacetylase inhibitor-treated cells from control with high confidence. Statistical processing of laser tweezer Raman spectroscopy data led to the definition of specific biomolecular fingerprints of each cell group. CONCLUSION: This original study shows that laser tweezer Raman spectroscopy is a label-free rapid tool to identify living cells that underwent epigenetic changes.

Epigenetic regulation of proMMP-1 expression in the HT1080 human fibrosarcoma cell line.

The matrix metalloproteinase (MMP) family members play an important role in various physiological and pathological processes. Although MMP-1 (collagenase-1) has been shown to be involved in tumor invasiveness, the regulation of its expression is still not fully elucidated and could implicate epigenetic mechanisms. The aim of this study was to analyze the effects of the Histone Deacetylase Inhibitor (HDI) trichostatin A (TSA) and the inhibitor of DNA methylation 5-aza-2'-deoxycytidine (5-azadC) on the proMMP-1 expression in the human HT1080 fibrosarcoma cell line. Real-time RT-PCR revealed that 5-azadC or 5-azadC + TSA but not TSA alone, despite global histone H4 hyperacetylation, increased proMMP-1 mRNA levels. This transcription activation was correlated with chromatin decondensation determined by nuclear texture image analysis technique. Western blot analysis of cell culture conditioned media revealed a significant increase in proMMP-1 secretion after 5-azadC or 5-azadC + TSA treatment compared to untreated cells. These results suggested that epigenetic mechanisms could be involved in proMMP-1 gene expression including chromatin supra-organization changes. Indeed, although the proMMP-1 gene promoter does not appear to contain CpG islands, its expression can be induced by the demethylating agent 5-azadC. Further experiments revealed that inhibition of protein neosynthesis by cycloheximide decreased 5-azadC-induced proMMP-1 mRNA, suggesting that epigenetically regulated intermediate molecules could be involved in proMMP-1 expression regulation in these cells.

Thalidomide alters nuclear architecture without ABCB1 gene modulation in drug-resistant myeloma cells.

ABCB1 gene overexpression has been described as an important mechanism for resistance to conventional chemotherapy in multiple myelomas. In the refractory multiple myelomas, other drug regimens have been successfully applied, including thalidomide treatments. Besides its well-documented anti-angiogenic effects, thalidomide therapy could result in a decrease in ABCB1 gene expression. In this study, we analysed the effects of a 24-h short-term treatment by thalidomide or its active metabolite phthaloyl glutamic acid (PGA) on nuclear chromatin higher-order organisation and ABCB1 gene expression in drug-sensitive and drug-resistant 8226 human myeloma cells. As compared to sensitive cells, 8226-Dox40 drug-resistant cells exhibited an increase in chromatin texture condensation and ABCB1 gene overexpression. At this gene promoter level, the -50 and -100 GC boxes displayed an unmethylated profile in drug-sensitive cells whereas drug-resistant cell promoter GC boxes were fully methylated. Thalidomide and PGA induced significant chromatin textural changes in 8226-Dox40 drug-resistant cells only with neither alteration in ABCB1 gene expression nor methylation profile of its promoter. Conversely thalidomide and PGA induced down-regulation of VEGF gene expression in both drug-sensitive and -resistant myeloma cells. These data suggest that short-term treatments by thalidomide or PGA do not induce any significant change on ABCB1 gene expression though they modulate chromatin supra-organisation in drug-resistant 8226 human myeloma cells.

Detection of drug-resistance genes using single bronchoscopy biopsy specimens.

Expression of three major resistance genes MDR1, MRP1 and LRP was investigated in small cell lung cancer, non-small cell lung cancer and metastasis. Single biopsies of bronchoscopy from 73 patients were performed to investigate expression of these three resistance genes by reverse transcriptase-polymerase chain reaction. Relations between gene expression and patient age, smoking status, histology, and chemotherapy were evaluated. A more frequent expression of MDR1 (77 versus 66%), MRP1 (91 versus 72%) and LRP (77 versus 63%) genes was detected in the malignant biopsies than in the non-malignant, respectively. In the metastasis biopsies, expression of these genes was markedly increased. No significant difference was observed between specimens before and after chemotherapy. Biopsies from progressing cancer showed higher MDR1, MRP1 and LRP gene expression. In conclusion, these data reveal a major role of MRP1 in intrinsic resistance and the high gene expression of MDR1 and MRP1 in relapsed diseases.

Differential modulation of nuclear texture, histone acetylation, and MDR1 gene expression in human drug-sensitive and -resistant OV1 cell lines.

Image cytometric study of pathological specimens or cell lines has suggested that epigenetic mechanisms are likely to play a major role in determining chromatin patterns evaluable through nuclear texture analysis. We previously reported that nuclear textural changes observed in the OV1-VCR etoposide-resistant ovarian carcinoma cell line were associated with an increased acetylated histone H4 level. In this study we analyzed the effects of treatments with the HDAC inhibitor trichostatin A (TSA) or with nickel subsulfide on histone H4 acetylation, nuclear texture, and MDR1 gene expression in drug-sensitive IGROV1 and drug-resistant OV1-VCR cell lines. In IGROV1 cells, TSA induced an increase in acetylated H4 level associated with a chromatin textural decondensation and an increase in MDR1 gene expression. In OV1-VCR cells, a similar increase in H4 acetylation was observed, but nuclear texture or MDR1 gene expression remained unchanged. ChIP analysis revealed that MDR1 gene expression remained stable in TSA-treated OV1-VCR cells despite a localized increase in H4 acetylation at the promoter level. Analysis of the methylation status of MDR1 promoter showed an increase in DNA methylation at 3 specific sites in OV1-VCR cells, that could participate to TSA low responsiveness in these cells. Treatment with nickel subsulfide induced a decrease in H4 acetylation without any effect on nuclear texture characteristics in both cell lines. In OV1-VCR cells, nickel subsulfide induced a significant down-regulation of the MDR1 gene expression. These results indicate that modulation of histone H4 acetylation level can be associated with up- or down-regulation of the MDR1 gene in OV1 cells. However, this modulation does not always result in chromatin pattern alterations and these data emphasize the complexity of chromatin texture regulation in tumor cells.

Imaging of P-glycoprotein of H69/VP small-cell lung cancer lines by scanning near-field optical microscopy and confocal laser microspectrofluorometer.

Chemoresistance remains the major obstacle to successful therapy of the lung cancer. The multi-drug resistance (MDR) is generally associated with altered expression of drug transporter proteins, such as P-glycoprotein (P-gp). So the distribution of P-gp on the membrane is of great importance to further study the interaction between drug and P-gp. In the present work, the P-gp of the H69/VP small-lung cancer cells was detected using monoclonal antibody UIC2. A secondary goat-anti mouse antibody coupled with biotin was used. The fluorescence emission was detected from a streptavidin-Texas Red. Results were investigated by a homemade scanning near-field optical microscope (SNOM) coupled to a confocal laser microspectrofluorometer (CLMF). Topographical images and localized spectra were obtained at the level of one cell membrane. It was found that the distribution of P-gp is not homogeneous and this observation is basically in accord with the fluorescent images obtained by classical microscopy. The distribution of P-gp would be localized in a higher region on a cell surface. This methodology would also enhance our understanding of MDR under physiological conditions.

Effects of the histone deacetylase inhibitor trichostatin A on nuclear texture and c-jun gene expression in drug-sensitive and drug-resistant human H69 lung carcinoma cells.

BACKGROUND: Texture analysis of chromatin patterns by image cytometry can be used in the development and refinement of diagnosis and prognosis of cancers and in the follow-up of therapies. However, little is known about the biological mechanisms underlying these patterns. Epigenetic mechanisms as histone posttranslational modifications and particularly histone acetylation could play a major role in the determination of these chromatin patterns and then influence nuclear texture measurements. METHODS: This study examined the consequences of treatment by the histone deacetylase inhibitor trichostatin A (TSA) on the nuclear texture in human cell lines sensitive and resistant to chemotherapy. Small cell lung carcinoma H69 cells and their variant H69-VP, which is resistant to etoposide, were incubated with 100 ng/ml of TSA for 0 to 24 h. Nuclear texture was evaluated by image cytometry and compared with the histone H4 acetylation level measured by western blotting and expression of c-jun gene evaluated by reverse transcription and real-time polymerase chain reaction. RESULTS: TSA treatment induced an increase in histone H4 acetylation level in both cell lines. However, at the level of chromatin texture, sensitive H69 cells displayed a progressive chromatin decondensation up to 24 h, whereas resistant H69-VP showed rapid (8 h) but transient changes. Similarly, expression of c-jun increased regularly in TSA-treated H69 cells. In H69-VP cells, an increase was also observed up to 12 h followed by a decrease after 24 h of treatment. CONCLUSIONS: Analysis of nuclear texture appeared to be a sensitive technique to detect chromatin pattern alterations induced by the histone deacetylase inhibitor TSA in the H69 cell line and enabled the observation of chromatin pattern discrepancies between chemotherapeutic drug-sensitive and drug-resistant cells during this treatment. When c-jun gene expression was analyzed as gene sensitive to epigenetic control, these textural differences seemed to be correlated to gene expression.

Nuclear texture and chromatin structure in OV1/VCR human multidrug-resistant cell line.

Previous studies have demonstrated that multidrug-resistant leukemic cells displayed nuclear texture changes. In this work, the human ovarian carcinoma cell line IGROV1 and its multidrug-resistant variant OV1/VCR were studied. Cell smears of these cell populations were analysed by image cytometry. As compared to sensitive cells, OV1/VCR display a chromatin global decondensation as assessed by textural features analysis. In order to correlate this decondensation with alterations in chromatin structure, DNase I was used. OV1/VCR DNA displayed an increased DNase I sensitivity, suggesting an increased chromatin accessibility. Furthermore, OV1/VCR cells displayed an increased level in acetylated histone H4, a mechanism known to be associated with transcriptionally active chromatin and relaxed chromatin conformation.