The Sigma-2 Receptor/TMEM97 Agonist PB28 Suppresses Cell Proliferation and Invasion by Regulating the PI3K-AKT-mTOR Signalling Pathway in Renal Cancer

Sigma-2 receptor/TMEM97 is overexpressed in many tumours, and sigma-2 receptor ligands are under investigation for cancer therapy. We intended to evaluate the effect of PB28 on renal cancer in proliferation, migration and invasion in vitro and in vivo. Invasive renal cancer cell lines treated with PB28 (or sigma-2 receptor antagonist 1) were subjected to cell proliferation, migration and invasion assays. The therapeutic effect of PB28 was performed on nude mice. Western blot for proteins in the PI3K-AKT-mTOR signalling pathway was conducted. A CCK-8 assay was used to examine the effect of the combination of PB28 and cisplatin on renal cancer cells. Significant inhibitory effects were observed on proliferation, migration and invasion of 786-O and ACHN cells after culturing with PB28. But, the outcomes of sigma-2 receptor antagonist 1 presented the opposite tendency. PB28 significantly inhibited the proliferative and invasive ability of OS-RC-2 cells in vivo. Treatment resulted in decreased phosphorylation of constituents of the PI3K-AKT-mTOR pathway. The combination of PB28 and cisplatin showed enhanced efficacy in the inhibition of renal cancer cell proliferation. Taken together, PB28 inhibited the tumorigenic behaviours of renal cancer cells by regulating the PI3K-AKT-mTOR signalling pathway and was expected to be a sensitizer of cisplatin.     

The sigma-2 receptor agonist PB28 inhibits calcium release from the endoplasmic reticulum of SK-N-SH neuroblastoma cells

In this paper we demonstrate that PB28 abolishes the Ca2+ release through the inositol 1,4,5-trisphosphate (InsP3) receptors and ryanodine receptors in SK-N-SH cells. Sigma receptors are divided into the subtypes sigma-1 and sigma-2, which are expressed in tumor cell lines and characterized by distinct pharmacological profiles. The sigma-1 receptor has been recently cloned, whereas the sigma-2 receptor is less well characterized. The endogenous ligand(s) of both subtypes remain unclear. In isolated guinea pig ileum, PB28 inhibits the contraction induced by carbachol dose dependently and in a non-competitive manner. In SK-N-SH cells PB28 challenge does not affect the intracellular Ca2+ concentration but incubation with PB28 for 45 min abolishes the cytosolic Ca2+ increases evoked by carbachol or histamine. This effect, not sensitive to cycloheximide, is caused by direct inhibition of the InsP3 receptors, since PB28 abolishes the response elicited by InsP3 administration in permeabilized SK-N-SH cells. Finally, incubation for 45 min with PB28 also abolishes the cytosolic Ca2+ increase evoked by caffeine.     

Involvement of peripheral type of benzodiazepine receptor in social isolation stress-induced decrease in pentobarbital sleep in mice

Our previous studies have shown that central-type benzodiazepine (BZD) receptors (CBR) and neurosteroids capable of modulating GABA(A) receptor function are involved in the decrease of pentobarbital (PB)-induced sleep caused by social isolation stress in mice. In this study, to further clarify the mechanism underlying this decrease, we investigated the possible involvement of peripheral-type BZD receptors (PBR) which play an important role in neurosteroidogenesis in PB sleep in socially isolated mice. Socially isolated mice showed significantly shorter duration of PB-induced sleep than group-housed animals. When injected intracerebroventricularly (i.c.v.), FGIN-1-27 (FGIN, 25-100 nmol), a selective PBR agonist, and PK11195 (PK, 14-28 nmol), a PBR antagonist, and pregnenolone (PREG, 15-30 nmol), a neurosteroid precursor, dose-dependently normalized the PB sleep in isolated mice without having an effect on the group-housed animals. In contrast, pregnenolone sulfate (PS, 24 nmol), an endogenous neurosteroidal negative allosteric modulator of the GABA(A) receptor, reduced PB sleep in group-housed but not isolated mice. PS, at the same dose, significantly attenuated the effects of FGIN (100 nmol), PK (28 nmol) and PREG (30 nmol) in isolated mice, while FGIN (100 nmol), PK (28 nmol) and pregnenolone (30 nmol) significantly blocked the effect of PS (24 nmol) in group-housed mice. These results suggest that the PBR-mediated decrease in the genesis of neurosteroid(s) possessing a GABA(A) receptor agonistic profile is also partly involved in the down regulation of the GABA(A) receptor following long-term social isolation and contributes to the decrease of PB-induced sleep in isolation stressed mice.     

Free energy calculations and binding analysis of two potential anti- influenza drugs with Polymerase basic protein-2 (PB2)

Influenza viruses cause a significant level of morbidity and mortality in the population every year. Their resistance to current anti-influenza drugs increases the difficulty of flu treatment. Thus, development of new anti-influenza drugs is necessary in regards of prevent the tragedy of influenza pandemic. The Polymerase basic protein 2 (PB2) subunit of influenza virus RNA polymerase is one of potential targets for new drugs because the binding of PB2 with the 5' cap of the host pre-mRNAs is the initial step of the virus' protein synthesis. In this study, we compared the binding potency of PB2 cap binding domain with two small molecules, i.e., RO and PPT28, with that of PB2 with cap analog m7GTP. The calculated binding energies showed that RO and PPT28 had higher binding affinity with PB2. Further interaction analysis showed that the important parts for binding were the five- and six-member heterocyclic rings (the 6/5-member rings) of small molecules, as well as the hydrophobic parts of RO and PPT28 which had good interactions with the hydrophobic residues in the binding cavity. Thus, RO and PPT28 are two potential anti-influenza drugs targeted PB2, which may inhibit the growth of influenza virus by competitively binding with the cap structure binding domain of PB2.     

Chemokine receptor expressions and responsiveness of cord blood T cells

Chemokines and their receptors play a critical role in the selective attraction of various subsets of leukocytes. We examined the chemokine receptor expressions and responsiveness of cord blood (CB) T cells. Flow-cytometric analysis revealed that peripheral blood (PB) T cells expressed CCR-1, CCR-2, CCR-5, CCR-6, CXC chemokine receptor-3 (CXCR-3), and CXCR-4, while CB T cells expressed only CXCR-4 on their surface. Chemotactic migratory response of CB T cells to macrophage-inflammatory protein (MIP)-1alpha, monocyte chemoattractant protein-1, RANTES, MIP-3alpha, monokine induced by IFN-gamma, and IFN-gamma-inducible protein-10 was significantly impaired compared with those of PB T cells. In contrast, the ability of CB T cells to migrate to MIP-3beta, 6Ckine, and stromal cell-derived factor-1alpha was greater than that of PB T cells, and these events were correlated with the expression levels of CCR-7 and CXCR-4, respectively. Engagement of CD3 and CD28 specifically up-regulated CXCR-3 expression and chemotaxis to monokine induced by IFN-gamma and IFN-gamma-inducible protein-10, whereas this stimulation down-regulated CCR-7 expression and chemotaxis to MIP-3beta and 6Ckine in PB T cells, but not in CB T cells. These results suggest that PB T cells and CB T cells exhibit distinct chemokine responsiveness via different chemokine receptor repertoire.     

PB183, a sigma receptor ligand, as a potential PET probe for the imaging of prostate adenocarcinoma

PB183, a non-selective sigma receptor ligand displaying high sigma-1 and sigma-2 receptor affinity, was evaluated in prostate tumour cell lines for its suitability as PET radiotracer. The pharmacodynamic and pharmacokinetic properties suggested PB183 as a potential PET radiotracer to visualize prostate adenocarcinoma.     

F281, synthetic agonist of the sigma-2 receptor,induces Ca2+ efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells

We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca²⁺] ([Ca²⁺]_i) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca²⁺]_i. In particular, CB-64D induces a Ca²⁺ response while PB28 supresses Ca²⁺ signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24 h incubation) was more cytotoxic than PB28 (EC₅₀ values 65.4 nM and 8.13 μM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca²⁺ from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca²⁺-entry through the channels activated by store depletion was also observed. After the increase in [Ca²⁺]_i and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death.     

The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene

The endogenous CYP2B6 gene becomes phenobarbital (PB) inducible in androstenol-treated HepG2 cells either transiently or stably transfected with a nuclear receptor CAR expression vector. The PB induction mediated by CAR is regulated by a conserved 51-base pair element called PB-responsive enhancer module (PBREM) that has now been located between -1733 and -1683 bp in the gene's 5'-flanking region. An in vitro translated CAR acting as a retinoid X receptor alpha heterodimer binds directly to the two nuclear receptor sites NR1 and NR2 within PBREM. In a stably transfected HepG2 cell line, both PBREM and NR1 are activated by PB and PB-type compounds such as chlorinated pesticides, polychlorinated biphenyls and chlorpromazine. In addition to PBREM, CAR also transactivates the steroid/rifampicin-response element of the human CYP3A4 gene in HepG2 cells. Thus, activation of the repressed nuclear receptor CAR appears to be a versatile mediator that regulates PB induction of the CYP2B and other genes.     

EGF-Induced receptor autophosphorylation in primary hepatocytes isolated from phenobarbitone-treated mice.

Phenobarbitone (PB) treatment of mice causes a decrease in the growth factor responsiveness of hepatocytes. Here, epidermal growth factor receptor (EGFR) expression and receptor autophosphorylation was determined in hepatocytes isolated from control and PB-treated mice. There was a decrease in the level of EGFR expression in hepatocytes isolated from mice following PB administration when compared to controls. EGF caused an approximate 20-fold increase of the 170 kD phosphotyrosine band in control hepatocytes, which was inhibited by the EGFR specific tyrosine kinase inhibitor 4, 5-dianilinopthalamide. Following PB treatment, the degree of basal receptor phosphorylation (in the absence of EGF) was significantly greater and therefore the fold rise in EGFR phosphorylation in isolated hepatocytes was lower than in controls. However, the overall extent of EGF-induced receptor phosphorylation was not diminished in hepatocytes isolated from PB-treated mice. Therefore the reduction in responsiveness to growth factors seen in hepatocytes ex vivo or the cessation of proliferation observed in vivo following PB administration is unlikely to be attributed to a decrease in ligand binding and subsequent receptor autophosphorylation.     

Association between fibronectin receptor and the substratum: Spare receptors for cell adhesion

We have investigated the association of the recently described 140-kDa cell membrane receptor for fibronectin with the cytoskeleton or with substratum-bound fibronectin. Using a monospecific polyclonal antibody to the 140-kDa receptor, we have demonstrated that most of the receptor molecules are soluble in nonionic detergent either in suspension culture CHO cells or in CHO cells attached to and spread on a fibronectin-coated substratum. This may suggest that putative linkages of the receptor either to fibronectin or to detergent-insoluble cytoskeletal components are labile to nonionic detergent and thus are rather weak. Alternatively, it may mean that only a small fraction of the cell's receptors are needed to mediate adhesion. In order to explore this latter concept, we have coated substrata with various concentrations of PB1, a monoclonal antibody with a high affinity for fibronectin receptor. We demonstrate that coating the substratum with increasing concentrations of PB1 results in increasing amounts of 140-kDa receptor becoming bound to the substratum in detergent-insoluble form. However, the amount of receptor bound does not necessarily correlate with the degree of cell adhesion and spreading. Thus, coating the substratum with 5 μg/ml of PB1 results in essentially complete attachment and spreading of CHO cells, but only a small fraction of the 140-kDa receptor becomes substratum bound. Coating with 50 μg/ml of PB1 produces no further increase in cell adhesion and spreading, but results in the detergent-stable association of a large fraction of the total receptor pool with the substratum. These observations suggest the possibility of there being “spare” receptors for cell adhesion processes.     

AMP-activated protein kinase mediates phenobarbital induction of CYP2B gene expression in hepatocytes and a newly derived human hepatoma cell line

Phenobarbital (PB) administration is known to trigger pleiotropic responses, including liver hypertrophy, tumor promotion, and induction of genes encoding drug-metabolizing enzymes. The induction of human CYP2B6 and the rat (CYP2B1) and mouse (Cyp2b10) homologues by PB is mediated by the nuclear receptor constitutive androstane receptor (CAR). The study of CYP2B gene regulation and CAR activity by PB has been difficult due to the lack of a cellular model. In this study, we describe a novel differentiated human hepatoma cell line (WGA), derived from HepG2, which expresses CYP2B6 and CAR. WGA cells represent a powerful system to study the regulation of CYP2B6 gene expression by PB. There is evidence that CAR activity is regulated by phosphorylation and that regulation of some CYP genes depends on the nutritional status of cells. The AMP-activated protein kinase (AMPK) functions as an energy sensor and is activated when cells experience energy-depleting stresses. In this report, we show that addition of 5-amino-imidazole carboxamide riboside, an AMPK activator, to WGA and human hepatocytes induces CYP2B6 gene expression. Expression of a constitutively active form of AMPK mimics the PB induction of CYP2B6 and CYP2B1 gene expression. Conversely, the expression of a dominant negative form of AMPK inhibits the induction of these genes by PB. Finally, we demonstrate, for the first time, that AMPK activity increases in cells cultured with PB. Our data strongly support a role for AMPK in the PB induction of CYP2B gene expression and provide new insights into the regulation of gene expression by barbiturate drugs.     

Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.     

Molecular systematics of basal subfamilies of ants using 28S rRNA (Hymenoptera: Formicidae)

For many years, the ant subfamily Ponerinae was hypothesized to contain the basal (early branching) lineages of ants. Recently the Ponerinae were reclassified into six poneromorph subfamilies based on morphological analysis. We evaluate this new poneromorph classification using 1240 base pairs of DNA sequence data obtained from 28S rRNA gene sequences of 68 terminal taxa. The molecular tree supported the monophyly of the ant family Formicidae, with 100% parsimony bootstrap (PB) support and posterior probabilities (PP) of 1.00, with the ant subfamily Leptanillinae as a sister group to all other ants (PB=62, PP=93). However, our analyses strongly support the polyphyly of the Poneromorph subfamilies (sensu Bolton). The Ectatomminae and Heteroponerinae are more closely related to the Formicoid subfamilies than to the rest of the poneromophs (PB=96, PP=100). The Amblyoponinae (PB=52, PP=96), Paraponerinae (PB=100, PP=100), Ponerinae (PB<50, PP=71), and Proceratiinae (PB=98, PP=100) appear as distinct lineages at the base of the tree and are identified as a poneroid grade. Monophyletic origins for the poneroid subfamilies Amblyoponinae, Paraponerinae, Ponerinae and Proceratiinae are supported in our analysis. However, the genus Platythyrea forms a distinct sister group to the Ponerini within the Ponerinae. The Heteroponerinae, based on our sample of Heteroponera, are associated with the subfamily Ectatomminae (PB=98, PP=100). Furthermore, our data indicate the genus Probolomyrmex belongs to the Proceratiinae as suggested by recent morphological analysis (PB=98, PP=100).