Conjugates of Cryptophycin and RGD or isoDGR Peptidomimetics for Targeted Drug Delivery

RGD\u2010cryptophycin and isoDGR\u2010cryptophycin conjugates were synthetized by combining peptidomimetic integrin ligands and cryptophycin, a highly potent tubulin\u2010binding antimitotic agent across lysosomally cleavable Val\u2010Ala or uncleavable linkers. The conjugates were able to effectively inhibit binding of biotinylated vitronectin to integrin \u3b1v\u3b23, showing a binding affinity in the same range as that of the free ligands. The antiproliferative activity of the novel conjugates was evaluated on human melanoma cells M21 and M21\u2010L with different expression levels of integrin \u3b1v\u3b23, showing nanomolar potency of all four compounds against both cell lines. Conjugates containing uncleavable linker show reduced activity compared to the corresponding cleavable conjugates, indicating efficient intracellular drug release in the case of cryptophycin\u2010based SMDCs. However, no significant correlation between the in\u2005vitro biological activity of the conjugates and the integrin \u3b1v\u3b23 expression level was observed, which is presumably due to a non\u2010integrin\u2010mediated uptake. This reveals the complexity of effective and selective \u3b1v\u3b23 integrin\u2010mediated drug delivery

Inhibition of class I histone deacetylase with an apicidin derivative prevents cardiac hypertrophy and failure

Aims: Recent studies have demonstrated the importance of chromatin remodelling via histone acetylation/deacetylation for the control of cardiac gene expression. Specific histone deacetylases (HDACs) can, in fact, play a positive or negative role in determining cardiac myocyte (CM) size. Here, we report on the effect on hypertrophy development of three inhibitors (HDACi) of class I HDACs. Methods and results: The compounds were first analysed in vitro by scoring hypertrophy, expression of foetal genes, and apoptosis of neonatal rat CMs stimulated with phenylephrine, an \u3b11-adrenergic agonist. This initial screening indicated that a truncated derivative of apicidin with class I HDAC specificity, denoted API-D, had the highest efficacy to toxicity ratio, and was thus selected for further analysis in vivo. Administration of this drug significantly decreased myocardial hypertrophy and foetal gene expression after 1 week of pressure overload induced by thoracic aortic constriction (TAC) in mice. After 9 weeks of TAC, when manifest heart failure is encountered, mice treated with API-D presented with significantly improved echocardiographic and haemodynamic parameters of cardiac function when compared with untreated TAC-operated mice. Conclusion: The apicidin derivative, API-D, is capable of reducing hypertrophy and, consequently, the transition to heart failure in mice subjected to TAC. Treatment with this substance, therefore, holds promise as an important therapeutic option for heart failure

Global analysis of DNA methylation in early-stage liver fibrosis

<p>Abstract</p> <p>Background</p> <p>Liver fibrosis is caused by chemicals or viral infection. The progression of liver fibrosis results in hepatocellular carcinogenesis in later stages. Recent studies have revealed the importance of DNA hypermethylation in the progression of liver fibrosis to hepatocellular carcinoma (HCC). However, the importance of DNA methylation in the early-stage liver fibrosis remains unclear.</p> <p>Methods</p> <p>To address this issue, we used a pathological mouse model of early-stage liver fibrosis that was induced by treatment with carbon tetrachloride (CCl₄) for 2 weeks and performed a genome-wide analysis of DNA methylation status. This global analysis of DNA methylation was performed using a combination of methyl-binding protein (MBP)-based high throughput sequencing (MBP-seq) and bioinformatic tools, IPA and Oncomine. To confirm functional aspect of MBP-seq data, we complementary used biochemical methods, such as bisulfite modification and <it>in-vitro</it>-methylation assays.</p> <p>Results</p> <p>The genome-wide analysis revealed that DNA methylation status was reduced throughout the genome because of CCl₄treatment in the early-stage liver fibrosis. Bioinformatic and biochemical analyses revealed that a gene associated with fibrosis, <it>secreted phosphoprotein 1 </it>(<it>Spp1</it>), which induces inflammation, was hypomethylated and its expression was up-regulated. These results suggest that DNA hypomethylation of the genes responsible for fibrosis may precede the onset of liver fibrosis. Moreover, <it>Spp1 </it>is also known to enhance tumor development. Using the web-based database, we revealed that <it>Spp1 </it>expression is increased in HCC.</p> <p>Conclusions</p> <p>Our study suggests that hypomethylation is crucial for the onset of and in the progression of liver fibrosis to HCC. The elucidation of this change in methylation status from the onset of fibrosis and subsequent progression to HCC may lead to a new clinical diagnosis.</p

The human checkpoint sensor Rad9–Rad1–Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase

Human (h) DNA repair enzyme thymine DNA glycosylase (hTDG) is a key DNA glycosylase in the base excision repair (BER) pathway that repairs deaminated cytosines and 5-methyl-cytosines. The cell cycle checkpoint protein Rad9–Rad1–Hus1 (the 9-1-1 complex) is the surveillance machinery involved in the preservation of genome stability. In this study, we show that hTDG interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. The hHus1 interacting domain is mapped to residues 67–110 of hTDG, and Val74 of hTDG plays an important role in the TDG–Hus1 interaction. In contrast to the core domain of hTDG (residues 110–308), hTDG(67–308) removes U and T from U/G and T/G mispairs, respectively, with similar rates as native hTDG. Human TDG activity is significantly stimulated by hHus1, hRad1, hRad9 separately, and by the 9-1-1 complex. Interestingly, the interaction between hRad9 and hTDG, as detected by co-immunoprecipitation (Co-IP), is enhanced following N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment. A significant fraction of the hTDG nuclear foci co-localize with hRad9 foci in cells treated with methylating agents. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of the BER

Genetic Knock-Down of HDAC7 Does Not Ameliorate Disease Pathogenesis in the R6/2 Mouse Model of Huntington's Disease

Huntington's disease (HD) is an inherited, progressive neurological disorder caused by a CAG/polyglutamine repeat expansion, for which there is no effective disease modifying therapy. In recent years, transcriptional dysregulation has emerged as a pathogenic process that appears early in disease progression. Administration of histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) have consistently shown therapeutic potential in models of HD, at least partly through increasing the association of acetylated histones with down-regulated genes and by correcting mRNA abnormalities. The HDAC enzyme through which SAHA mediates its beneficial effects in the R6/2 mouse model of HD is not known. Therefore, we have embarked on a series of genetic studies to uncover the HDAC target that is relevant to therapeutic development for HD. HDAC7 is of interest in this context because SAHA has been shown to decrease HDAC7 expression in cell culture systems in addition to inhibiting enzyme activity. After confirming that expression levels of Hdac7 are decreased in the brains of wild type and R6/2 mice after SAHA administration, we performed a genetic cross to determine whether genetic reduction of Hdac7 would alleviate phenotypes in the R6/2 mice. We found no improvement in a number of physiological or behavioral phenotypes. Similarly, the dysregulated expression levels of a number of genes of interest were not improved suggesting that reduction in Hdac7 does not alleviate the R6/2 HD-related transcriptional dysregulation. Therefore, we conclude that the beneficial effects of HDAC inhibitors are not predominantly mediated through the inhibition of HDAC7

Genetic Modulation of Rpd3 Expression Impairs Long-Term Courtship Memory in Drosophila

There is increasing evidence that regulation of local chromatin structure is a critical mechanism underlying the consolidation of long-term memory (LTM), however considerably less is understood about the specific mechanisms by which these epigenetic effects are mediated. Furthermore, the importance of histone acetylation in Drosophila memory has not been reported. The histone deacetylase (HDAC) Rpd3 is abundant in the adult fly brain, suggesting a post-mitotic function. Here, we investigated the role of Rpd3 in long-term courtship memory in Drosophila. We found that while modulation of Rpd3 levels predominantly in the adult mushroom body had no observed impact on immediate recall or one-hour memory, 24-hour LTM was severely impaired. Surprisingly, both overexpression as well as RNAi-mediated knockdown of Rpd3 resulted in impairment of long-term courtship memory, suggesting that the dose of Rpd3 is critical for normal LTM

Differential expression of 12 histone deacetylase (HDAC) genes in astrocytomas and normal brain tissue: class II and IV are hypoexpressed in glioblastomas

<p>Abstract</p> <p>Background</p> <p>Glioblastoma is the most lethal primary malignant brain tumor. Although considerable progress has been made in the treatment of this aggressive tumor, the clinical outcome for patients remains poor. Histone deacetylases (HDACs) are recognized as promising targets for cancer treatment. In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global <it>HDAC </it>expression in gliomas and its possible correlation to the use of HDACis. The purpose of this study was to evaluate and compare mRNA and protein levels of class I, II and IV of HDACs in low grade and high grade astrocytomas and normal brain tissue and to correlate the findings with the malignancy in astrocytomas.</p> <p>Methods</p> <p>Forty-three microdissected patient tumor samples were evaluated. The histopathologic diagnoses were 20 low-grade gliomas (13 grade I and 7 grade II) and 23 high-grade gliomas (5 grade III and 18 glioblastomas). Eleven normal cerebral tissue samples were also analyzed (54 total samples analyzed). mRNA expression of class I, II, and IV <it>HDACs </it>was studied by quantitative real-time polymerase chain reaction and normalized to the housekeeping gene <it>β-glucuronidase</it>. Protein levels were evaluated by western blotting.</p> <p>Results</p> <p>We found that mRNA levels of class II and IV <it>HDACs </it>were downregulated in glioblastomas compared to low-grade astrocytomas and normal brain tissue (7 in 8 genes, <it>p </it>< 0.05). The protein levels of class II HDAC9 were also lower in high-grade astrocytomas than in low-grade astrocytomas and normal brain tissue. Additionally, we found that histone H3 (but not histone H4) was more acetylated in glioblastomas than normal brain tissue.</p> <p>Conclusion</p> <p>Our study establishes a negative correlation between <it>HDAC </it>gene expression and the glioma grade suggesting that class II and IV <it>HDACs </it>might play an important role in glioma malignancy. Evaluation of histone acetylation levels showed that histone H3 is more acetylated in glioblastomas than normal brain tissue confirming the downregulation of <it>HDAC </it>mRNA in glioblastomas.</p

Introduction to the French GEOTRACES North Atlantic Transect (GA01): GEOVIDE cruise

The GEOVIDE cruise, a collaborative project within the framework of the international GEOTRACES programme, was conducted along the French-led section in the North Atlantic Ocean (Section GA01), between 15 May and 30 June 2014. In this special issue (https://www.biogeosciences.net/special_issue900.html), results from GEOVIDE, including physical oceanography and trace element and isotope cyclings, are presented among 18 articles. Here, the scientific context, project objectives, and scientific strategy of GEOVIDE are provided, along with an overview of the main results from the articles published in the special issue

Concepts of epigenetics in prostate cancer development

Substantial evidence now supports the view that epigenetic changes have a role in the development of human prostate cancer. Analyses of the patterns of epigenetic alteration are providing important insights into the origin of this disease and have identified specific alterations that may serve as useful diagnostic and prognostic biomarkers. Examination of cancer methylation patterns supports a stem cell origin of prostate cancer. It is well established that methylation of GSTpi is a marker of prostate cancer, and global patterns of histone marking appear to be linked to cancer prognosis with levels of acetylated histones H3K9, H3K18, and H4K12, and of dimethylated H4R3 and H3K4, dividing low-grade prostate cancer (Gleason 6 or less) into two prognostically separate groups. Elevated levels of several components of the polycomb group protein complex, EZH2, BMI1, and RING1, can also act as biomarkers of poor clinical outcome. Many components of the epigenetic machinery, including histone deacetylase (whose expression level is linked to the TMPRSS2: ERG translocation) and the histone methylase EZH2, are potential therapeutic targets. The recent discovery of the role of small RNAs in governing the epigenetic status of individual genes offers exciting new possibilities in therapeutics and chemoprevention