Novel naphthyloxy derivatives – Potent histamine H3 receptor ligands. Synthesis and pharmacological evaluation

A series of 1- and 2-naphthyloxy derivatives were synthesized and evaluated for histamine H₃ receptor affinity. Most compounds showed high affinities with K_i values below 100?nM. The most potent ligand, 1-(5-(naphthalen-1-yloxy)pentyl)azepane (11) displayed high affinity for the histamine H₃ receptor with a K_i value of 21.9?nM. The antagonist behaviour of 11 was confirmed both in vitro in the cAMP assay (IC₅₀?=?312?nM) and in vivo in the rat dipsogenia model (ED₅₀?=?3.68?nM). Moreover, compound 11 showed positive effects on scopolamine induced-memory deficits in mice (at doses of 10 and 15?mg/kg) and an analgesic effect in the formalin test in mice with ED₅₀?=?30.6?mg/kg (early phase) and ED₅₀?=?20.8?mg/kg (late phase). Another interesting compound, 1-(5-(Naphthalen-1-yloxy)pentyl)piperidine (13; H₃R K_i?=?53.9?nM), was accepted for Anticonvulsant Screening Program at the National Institute of Neurological Disorders and Stroke/National Institute of Health (Rockville, USA). The screening was performed in the maximal electroshock seizure (MES), the subcutaneous pentylenetetrazole (scPTZ) and the 6-Hz psychomotor animal models of epilepsy. Neurologic deficit was evaluated by the rotarod test. Compound 13 inhibited convulsions induced by the MES with ED₅₀ of 19.2?mg/kg (mice, i.p.), 17.8 (rats, i.p.), and 78.1 (rats, p.o.). Moreover, 13 displayed protection against the 6-Hz psychomotor seizures (32?mA) in mice (i.p.) with ED₅₀ of 33.1?mg/kg and (44?mA) ED₅₀ of 57.2?mg/kg.Furthermore, compounds 11 and 13 showed in vitro weak influence on viability of tested cell lines (normal HEK293, neuroblastoma IMR-32, hepatoma HEPG2), weak inhibition of CYP3A4 activity, and no mutagenicity. Thus, these compounds may be used as leads in a further search for histamine H₃ receptor ligands with promising in vitro and in vivo activity.     

Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells

Epidermal and hepatocyte growth factors can stimulate invasive abilities of melanoma cells, while treatment with combination of their receptors' (EGFR and MET, respectively) inhibitors reduces viability of these cells, as we have previously shown. Proposed therapy has potential; however, used drugs block more than one goal effectively, what raises the question about the real target of analysed inhibitors. For this reason, we analysed direct involvement of these receptors in the invasion of melanoma cells inducing EGFR and MET up‐ and down‐regulations in examined cells. Results were acquired with assays evaluating cell migration and invasion (scratch wound assay, Transwell filter‐based method and single‐cell tracking). We revealed that cells' motile abilities are increased after EGFR overexpression and decreased following EGFR and MET silencing. This outcome correlates with elevated (EGFR up‐regulation) or reduced (EGFR/MET down‐regulation) number of formed invadopodia, visualized with immunofluorescence, and their rate of proteolytic abilities, evaluated by fluorescent gelatin degradation assay, and gelatin zymography, compared to control cells. Above‐mentioned data indicate that both—EGFR and MET signalling is directly connected with melanoma cells invasion, what establishes these receptors as promising targets for anti‐cancer treatment.     

Multiple myeloma-associated hDIS3 mutations cause perturbations in cellular RNA metabolism and suggest hDIS3 PIN domain as a potential drug target

hDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in ∼10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans.     

FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.     

A role of autophagy in PTP4A3-driven cancer progression

Autophagy, a “self-eating” cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promotes multiple oncogenic processes including cell proliferation, invasion, and cancer metastasis. In this study, we demonstrate that PTP4A3 accumulates in autophagosomes upon inhibition of autophagic degradation. Expression of PTP4A3 enhances PIK3C3-BECN1-dependent autophagosome formation and accelerates LC3-I to LC3-II conversion in an ATG5-dependent manner. PTP4A3 overexpression also enhances the degradation of SQSTM1, a key autophagy substrate. These functions of PTP4A3 are dependent on its catalytic activity and prenylation-dependent membrane association. These results suggest that PTP4A3 functions to promote canonical autophagy flux. Unexpectedly, following autophagy activation, PTP4A3 serves as a novel autophagic substrate, thereby establishing a negative feedback-loop that may be required to fine-tune autophagy activity. Functionally, PTP4A3 utilizes the autophagy pathway to promote cell growth, concomitant with the activation of AKT. Clinically, from the largest ovarian cancer data set (GSE 9899, n = 285) available in GEO, high levels of expression of both PTP4A3 and autophagy genes significantly predict poor prognosis of ovarian cancer patients. These studies reveal a critical role of autophagy in PTP4A3-driven cancer progression, suggesting that autophagy could be a potential Achilles heel to block PTP4A3-mediated tumor progression in stratified patients with high expression of both PTP4A3 and autophagy genes.     

General assessment of copy number variation in normal and tumor tissues of the domestic dog (Canis lupus familiaris)

In recent years, characterization of a copy number variation (CNV) of the genomic DNA has provided evidence for the relationship of this type of genetic variation with the occurrence of a broad spectrum of diseases, including cancer lesions. Copy number variants (CNVs) also occur in the genomes of healthy individuals as a result of abnormal recombination processes in germ cells and have a hereditary character contributing to the natural genetic diversity. Recent image analysis methods and advanced computational techniques allow for identification of CNVs using SNPs genotyping microarrays based on the analysis of signal intensity observed for markers located in the specific genomic regions. In this study we used CanineHD BeadChip assay (Illumina) to identify both natural and cancer-induced CNVs in the genomes of different dog breeds and in different cancer types occurring in this species. The obtained results showed that structural aberrations are a common phenomenon arising during a tumor progression and are more complex and widespread in tumors of mesenchymal tissue origin than in epithelial tissue originating tumors. The tumor derived CNVs, in comparison to healthy samples, were characterized by larger sizes of regions, higher number of amplifications, and in some cases encompassed genes with potential effect on tumor progression.     

Influence of 6 MV and 20 MV X-radiation dose rate on in vitro survive of the K-562 cell line

BackgroundAnalysis of the survival rate of cells after irradiation with a specified dose of X-radiation might be one of the basic foundations for assessment of biological implications of ionizing radiation. Investigation of the influence of X-radiation dose rate on cells was carried out in vitro using the SF2 test.AimThe aim of this study was to investigate the influence of X-radiation dose rate on the surviving fraction of the K-562 cell line for two photon energies of 6 MV and 20 MV.Materials/MethodsTo measure the cells' reaction to X-radiation of variable dose rate human leukaemic K-562 cells were used. In order to fulfil the main aim of the study, the cell line was subjected to irradiation at two different dose rates. Total dose applied at once was 2 Gy. A quantitative evaluation of cell survival rate was carried out at every step of the experiment using a clonogenic assay.ResultsHigh dose rate at the energy of 6 MV decreased the percentage of surviving cells to 23%, while lower dose rate decreased it only to 36%. A similar effect is observed at the energy of 20MV-namely at the higher dose rate the percentage of surviving cells is 18%, whereas at the lower one it is only 34%.ConclusionsThe experiment has shown that when using a lower dose rate, the biological effect of ionizing radiation is less pronounced. However, at a higher dose rate higher radiosensitivity of cells is observed.     

The frequency of CD25+CD4+ and FOXP3+ regulatory T cells in ectopic endometrium and ectopic decidua

Background  

The presence of regulatory T (Treg) cells in human endometrium is crucial for maintaining immunological homeostasis within the uterus. For this study we decided to evaluate the subpopulations of Treg cells in conditions where a disturbance in the immunological equilibrium in ectopic endometrium and decidua has been observed, such as in cases of ovarian endometriosis (involving local immune cell suppression) and ectopic pregnancy (involving an increase in local immune system activity). We then compared these findings to what we observed in the normal eutopic endometrium of women during the secretory phase of the menstrual cycle (with immune cells under individual control).