Analysis of the genomic response of human prostate cancer cells to histone deacetylase inhibitors

Histone deacetylases (HDACs) have emerged as important targets for cancer treatment. HDAC-inhibitors (HDACis) are well tolerated in patients and have been approved for the treatment of patients with cutaneous T-cell lymphoma (CTCL). To improve the clinical benefit of HDACis in solid tumors, combination strategies with HDACis could be employed. In this study, we applied Analysis of Functional Annotation (AFA) to provide a comprehensive list of genes and pathways affected upon HDACi-treatment in prostate cancer cells. This approach provides an unbiased and objective approach to high throughput data mining. By performing AFA on gene expression data from prostate cancer cell lines DU-145 (an HDACi-sensitive cell line) and PC3 (a relatively HDACi-resistant cell line) treated with HDACis valproic acid or vorinostat, we identified biological processes that are affected by HDACis and are therefore potential treatment targets for combination therapy. Our analysis revealed that HDAC-inhibition resulted among others in upregulation of major histocompatibility complex (MHC) genes and deregulation of the mitotic spindle checkpoint by downregulation of genes involved in mitosis. These findings were confirmed by AFA on publicly available data sets from HDACi-treated prostate cancer cells. In total, we analyzed 375 microarrays with HDACi treated and non-treated (control) prostate cancer cells. All results from this extensive analysis are provided as an online research source (available at the journal’s website and at http://luigimarchionni.org/HDACIs.html). By publishing this data, we aim to enhance our understanding of the cellular changes after HDAC-inhibition, and to identify novel potential combination strategies with HDACis for the treatment of prostate cancer patients.     

Screening assay for blood vessel maturation inhibitors

In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.     

Design,synthesis and evaluation of some new 4-aminopyridine derivatives in learning and memory

Some new anilide and imide derivatives of 4-aminopyridine (4AP) were synthesized and evaluated against antiamnesic, cognition enhancing and anticholinesterase activity through their respective in vitro and in vivo models. These newly synthesized derivatives have illustrated an enhanced cognition effect on elevated plus maze model and also demonstrated a significant reversal in scopolamine-induced amnesia in same model. The IC₅₀ value of synthesized compounds showed maximum activity of 4APMb compared to standard drug donepezil and other derivatives, whereas its enzyme kinetic study revealed a non-competitive inhibition of acetycholinesterase (AChE) and a competetive inhibition of butyrylcholinesterase (BChE). Significant inhibitions in AChE activity by all the synthesized compounds were found in specific brain regions that is prefrontal cortex, hippocampus and hypothalamus. The docking study confirmed their consensual interaction with AChE, showed an affinity and binding with the key peripheral anionic site residues Trp-286, Tyr-124 and Tyr-341 of AChE.     

Synthesis,evaluation and molecular dynamics study of some new 4-aminopyridine semicarbazones as an antiamnesic and cognition enhancing agents

Some new semicarbazones of 4-aminopyridine were synthesized and evaluated for antiamnesic, cognition enhancing and anticholinesterase activities. The results illustrated a significant cognition enhancing effect on elevated plus maze model with a significant reversal of scopolamine-induced amnesia. A significant inhibition in acetycholinesterase (AChE) activity by all the synthesized compounds in specific brain regions that is, prefrontal cortex, hippocampus and hypothalamus was observed. Compound 4APi exhibited significant antiamnesic and cognition enhancing activity which was comparable with standard drug donepezil. Its enzyme kinetic study revealed a non-competitive inhibition of AChE and a competitive inhibition of butyrylcholinesterase (BChE). Docking studies predicted the binding modes of these compounds in AChE active site, which were further processed for molecular dynamics simulation for calculating binding free energies using Molecular Mechanics–Generalized Born Surface Area (MM/GBSA). All the computational study confirmed their consensual interaction with AChE justifying the experimental outcome.     

Arjunarishta alleviates experimental colitis via suppressing proinflammatory cytokine expression,modulating gut microbiota and enhancing antioxidant effect

Molecular Biology Reports - Traditional ayurvedic medicine, Arjunarishta (AA) is used to treat several inflammatory conditions including dysentery associated with blood. The formulation is a...     

25 Years after Vi Typhoid Vaccine Efficacy Study,Typhoid Affects Significant Number of Population in Nepal

Salmonella Typhi, first isolated in 1884, results in infection of the intestines and can end in death and disability. Due to serious adverse events post vaccination, whole cell killed vaccines have been replaced with new generation vaccines. The efficacy of Vi polysaccharide (ViPS) vaccine, a new generation, single-dose intramuscular typhoid vaccine was assessed in Nepal in 1987. However, despite the availability of ViPS vaccine for more than 25 years, Nepal has one of the highest incidence of typhoid fever. Therefore we collected information from hospitals in the Kathmandu Valley from over the past five years. There were 9901 enteric fever cases between January 2008 and July 2012. 1,881 of these were confirmed typhoid cases from five hospitals in the Kathmandu district. Approximately 70% of the cases involved children under 15 years old. 1281 cases were confirmed as S. Paratyphi. Vaccines should be prioritized for control of typhoid in conjunction with improved water and sanitation conditions in Nepal and in endemic countries of Asia and Africa.     

On the characterization and selection of diverse conformational ensembles with applications to flexible docking

To address challenging flexible docking problems, a number of docking algorithms pregenerate large collections of candidate conformers. To remove the redundancy from such ensembles, a central problem in this context is to report a selection of conformers maximizing some geometric diversity criterion. We make three contributions to this problem. First, we resort to geometric optimization so as to report selections maximizing the molecular volume or molecular surface area (MSA) of the selection. Greedy strategies are developed, together with approximation bounds. Second, to assess the efficacy of our algorithms, we investigate two conformer ensembles corresponding to a flexible loop of four protein complexes. By focusing on the MSA of the selection, we show that our strategy matches the MSA of standard selection methods, but resorting to a number of conformers between one and two orders of magnitude smaller. This observation is qualitatively explained using the Betti numbers of the union of balls of the selection. Finally, we replace the conformer selection problem in the context of multiple-copy flexible docking. On the aforementioned systems, we show that using the loops selected by our strategy can improve the result of the docking process.     

Positional cloning of a type 2 diabetes quantitative trait locus; tomosyn-2, a negative regulator of insulin secretion

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lep(ob/ob) and C57BL/6 (B6) Lep(ob/ob) mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16(BT36-38)) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16(BT36-38) mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic β-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion.     

Telomeric injury by KML001 in human T cells induces mitochondrial dysfunction through the p53-PGC-1α pathway

Telomere erosion and mitochondrial dysfunction are prominent features of aging cells with progressive declines of cellular functions. Whether telomere injury induces mitochondrial dysfunction in human T lymphocytes, the major component of adaptive host immunity against infection and malignancy, remains unclear. We have recently shown that disruption of telomere integrity by KML001, a telomere-targeting drug, induces T cell senescence and apoptosis via the telomeric DNA damage response (DDR). In this study, we used KML001 to further investigate the role and mechanism of telomere injury in mitochondrial dysregulation in aging T cells. We demonstrate that targeting telomeres by KML001 induces mitochondrial dysfunction, as evidenced by increased mitochondrial swelling and decreased mitochondrial membrane potential, oxidative phosphorylation, mitochondrial DNA content, mitochondrial respiration, oxygen consumption, glycolysis, and ATP energy production. Mechanistically, we found that the KML001-induced telomeric DDR activated p53 signaling, which in turn repressed the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and nuclear respiratory factor 1 (NRF-1), leading to T cell mitochondrial dysfunction. These results, forging a direct link between telomeric and mitochondrial biology, shed new light on the human T cell aging network, and demonstrate that the p53-PGC-1α-NRF-1 axis contributes to mitochondrial dysfunction in the setting of telomeric DDR. This study suggests that targeting this axis may offer an alternative, novel approach to prevent telomere damage-mediated mitochondrial and T cell dysfunctions to combat a wide range of immune aging-associated human diseases.Subject terms: Immunology, Diseases