A compound CP-31398 suppresses excitotoxicity-induced neurodegeneration

Neurodegeneration causes dysfunction and degeneration of neurons and is triggered by various factors including genetic defects, free radicals, injury, and glutamate excitotoxicity. Among those, glutamate excitotoxicity is implicated in chronic disorders including AD and ALS, and in acute insults in the CNS including traumatic brain injury. Neurological disorders show hallmark morphological abnormalities such as axon degeneration and cell body death. The molecular mechanisms underlying excitotoxicity-induced neurodegeneration are complex and deciphering a molecular mechanism from one angle is beneficial to understand the process, however, still difficult to develop strategies to suppress excitotoxicity-induced degeneration due to existence of other mechanisms. Thus, directly identifying compounds that can modulate excitotoxicity-induced neurodegeneration and subsequently clarifiying the molecular mechanism is a valid approach to develop effective strategies to suppress neurodegeneration. We searched for compounds that can suppress excitotoxicity-induced neurodegeneration and found that CP-31398, a known compound that can rescue the structure and function of the tumor suppressor protein p53 mutant form and stabilize the active conformation of the p53 wild-type form, suppresses excitotoxicity-induced axon degeneration and cell body death. Moreover, CP-31398 suppresses mitochondrial dysfunction which has a strong correlation with excitotoxicity. Thus, our findings identify a compound that can serve as a novel modulator of neurodegeneration induced by glutamate excitotoxicity.     

The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: Inhibition by phthalate esters in vitro

This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB₁) receptor, a presynaptic complex involved in the regulation of synaptic activity in mammalian brain. The diesters, n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP), di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP) inhibited the specific binding of the CB₁ receptor agonist [³H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC₅₀s: nBBP 27.4 μM; DnHP 33.9 μM; DnBP 45.9 μM; DEHP 47.4 μM; DiOP 55.4 μM; DnOP 75.2 μM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [³H]CP-55940 binding, whereas DEHP, DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate (M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC₅₀ at 150 μM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [³H]CP-55940 binding assay were positively correlated with inhibition of CB₁ receptor agonist-stimulated binding of [³⁵S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [³H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [³H]SR141716A showed that phthalates reduce the B_max of radioligand without changing its K_d. DnBP and nBBP also rapidly enhanced the dissociation of [³H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB₁ receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB₁ receptor-dependent behavioral and physiological outcomes in the whole animal.     

The nucleoporin Nup358/RanBP2 promotes nuclear import in a cargo- and transport receptor-specific manner

In vertebrates, the nuclear pore complex (NPC), the gate for transport of macromolecules between the nucleus and the cytoplasm, consists of approximately 30 different nucleoporins (Nups). The Nup and SUMO E3-ligase Nup358/RanBP2 are the major components of the cytoplasmic filaments of the NPC. In this study, we perform a structure-function analysis of Nup358 and describe its role in nuclear import of specific proteins. In a screen for nuclear proteins that accumulate in the cytoplasm upon Nup358 depletion, we identified proteins that were able to interact with Nup358 in a receptor-independent manner. These included the importin α/β-cargo DBC-1 (deleted in breast cancer 1) and DMAP-1 (DNA methyltransferase 1 associated protein 1). Strikingly, a short N-terminal fragment of Nup358 was sufficient to promote import of DBC-1, whereas DMAP-1 required a larger portion of Nup358 for stimulated import. Neither the interaction of RanGAP with Nup358 nor its SUMO-E3 ligase activity was required for nuclear import of all tested cargos. Together, Nup358 functions as a cargo- and receptor-specific assembly platform, increasing the efficiency of nuclear import of proteins through various mechanisms.     

Inhibition of glycogen phosphorylase (GP) by CP-91,149 induces growth inhibition correlating with brain GP expression

The role of glycogenolysis in normal and cancer cells was investigated by inhibiting glycogen phosphorylase (GP) with the synthetic inhibitor CP-91,149. A549 non-small cell lung carcinoma (NSCLC) cells express solely the brain isozyme of GP, which was inhibited by CP-91,149 with an IC(50) of 0.5 microM. When treated with CP-91,149, A549 cells accumulated glycogen with associated growth retardation. Treated normal skin fibroblasts also accumulated glycogen with G1-cell cycle arrest that was associated with inhibition of cyclin E-CDK2 activity. Overall, cells expressing high levels of brain GP were growth inhibited by CP-91,149 correlating with glycogen accumulation whereas cells expressing low levels of brain GP were not affected by the drug. Analyses of 59 tumor cell lines represented in the NCI drug screen identified that every cell line expressed brain GP but the profile was dominated by a few highly GP expressing cell lines with lower than mean GP-a enzymatic activities. The correlation program, COMPARE, identified that the brain GP protein measured in the NCI cell lines corresponded with brain GP mRNA expression, ADP-ribosyltransferase 3, and colony stimulating factor 2 receptor alpha in the 10,000 gene microarray database with similar correlation coefficients. These results suggest that brain GP is present in proliferating cells and that high protein levels correspond with the ability of CP-91,149 to inhibit cell growth.     

Cytochrome P450 enzyme activity in five herbivorous,non-passerine bird species

We examined hepatic cytochrome P450 activity in wild and hand-reared grey partridges (Perdix perdix), capercaillies (Tetrao urogallus) and ring-necked pheasants (Phasianus colchicus), as well as the enzyme activity in a variety of tissues of hand-reared Japanese quails (Coturnix coturnix japonica) and pigeons (Columba livia). Post-mortem decrease in hepatic enzyme activity in the grey partridge was measured. Hepatic 7-ethoxyresorufin-O-deethylase activity was similar in wild and hand-reared grey partridges and pheasants, but the activity was significantly lower in wild than in hand-reared capercaillies, probably resulting from their phenolic-rich diet. In the tissues of both quails and pigeons 7-ethoxycoumarin-O-deethylase exhibited the highest and 7-pentoxyresorufin-O-deethylase the lowest activity. Hepatic enzyme activity was significantly higher than that in other tissues. In the small intestine some activity could be found, reflecting some intestinal detoxication capacity. Enzyme activity decreased by 34-69% during the 30-min sampling period, which confirmed the importance of equalising sampling time to obtain comparable data. Because the hand-reared birds in this study were fed the same commercial diets, we assumed that the enzyme activity values detected reflect species differences without any induction by dietary secondary compounds.     

Nup358 integrates nuclear envelope breakdown with kinetochore assembly

Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.     

Phase I trial of intravenous infusion of ex-vivo-activated autologous blood-derived macrophages in patients with non-small-cell lung cancer: Toxicity and immunomodulatory effects

Summary The purpose of this phase I study was to evaluate the toxicity and biological activity of autologous blood-derived macrophages activated ex-vivo with recombinant human interferon (rhuIFN) [monokine-activated killer (MAK) cells] and administered intravenously to 11 lung cancer patients once a week for 6 consecutive weeks. Peripheral blood monocytes were collected by leukapheresis and then purified by counterflow elutriation. The MAK cells were generated by culturing the purified monocytes in Teflon bags for 7 days and adding rhuIFN to the cultured cells for the last 18 h. These MAK cells expressed differentiation-associated surface antigen MAX1, and were cytotoxic in vitro against tumour cell line U937. The MAK cells were infused at dose levels from 1 × 10⁷ to 5 × 10⁸ on an intrapatient dose-escalating schedule. No severe adverse side-effects occurred. Toxicity was mild to moderate [primarly fever (75%) and chills (32%)], non-dose-dependent, and non-cumulative. No consistent change in haemostatic function, or liver or renal function was observed. Dose-limiting toxicity was not reached at 5 × 10⁸ cells (optimal dose reproduced for each patient). The maximum tolerated dose was not determined. The immunomodulatory activity of i.v. infused MAK cells was demonstrated both in vivo by significant increases in granulocyte count and neopterin level in the patients' peripheral blood postinfusion and in vitro by secretory products (IL-1. TNF, neopterin, and thromboplastin-like substance) in the culture supernatants. The in vivo traffic patterns of autologous MAK cells labelled ex-vivo with¹¹¹In oxine were studied in 7 patients. Gamma imaging showed an immediate but transient lung uptake (<24 h), and a progressive uptake of radioactivity in the liver and spleen was seen from 6 h to 72 h post-infusion. Our results indicate that the preparation of high numbers of autologous, blood-derived MAK cells is a feasible procedure, and their transfusion is safe for patients. This immunotherapeutic approach seems to be encouraging from the point of view of establishing an adjuvant therapeutic modality in cancer patients with minimal residual disease.This work was supported in part by a grant 6911 from the Association pour la Recherche contre le Cancer (ARC), grants from the Ligue Nationale contre le cancer and the Ligues Regionales (Bas-Rhin, Haut-Rhin) contre le cancer, and contract 891013 from the Institut National pour la Santé et la Recherche Médicale (INSERM), France     

Cannabinoid-hormone interactions in the regulation of motivational processes

There is a bi-directionality in hormone-cannabinoid interactions: cannabinoids affect prominent endocrine axes (such as the hypothalamic-pituitary-gonadal), and gonadal hormones modulate cannabinoid effects. This review will summarize recent research on these interactions, with a specific focus upon their implications for motivated behavior. Sexual behavior will serve as a “case study.” I will explore the hypothesis that ovarian hormones, in particular estradiol, may serve to release estrous behavior from endocannabinoid inhibition. Hormonal regulation of the endogenous cannabinoid system also affects processes that underlie drug abuse. This review will briefly discuss sex differences in behavioral responses to cannabinoids and explore potential mechanisms by which gonadal hormones alter cannabinoid reward. An examination of this research informs our perspective on how hormones and endocannabinoids may affect drug-seeking behavior as a whole and the development of addiction.     

The expression of macrophage migration inhibitory factor in the non-small cell lung cancer

ObjectiveThe objective was to investigate the expression of macrophage migration inhibitory factor (MIF) in non-small cell lung cancer (NSCLC), as well as the effects of macrophage MIF on tumor cells.MethodsThe human NSCLC cell strains H358 and H524 were selected as research objects. The Real-Time Polymerase Chain Reaction (RT-PCR) and Western Blot were utilized to detect the expression levels of MIF in human NSCLC cell strains. The lentiviral plasmid was utilized for MIF-mRNA interference. The expression levels of MIF before and after transfection were compared. The cell strains were cultured and proliferated for cell count and comparison.ResultsH358 showed MIF high expression while H524 showed MIF low expression. Once the H358 cells were constructed as silent MIF expression, compared with the original H358 cells, the difference was statistically significant. Once the H524 cells were constructed as high MIF expression, compared with original H524 cells, the difference was statistically significant. Being cultured for respective 3, 5, and 7 days, the transfected H358 cells showed a significant decrease in proliferative activity compared with original H358 cells, while the transfected H524 cells showed a significant increase in proliferative activity compared with original H524 cells.ConclusionMIF has high expression in H358 cells while low expression in H524 cells. The expression of MIF could enhance the proliferative activity of NSCLC tumor cells.     

CB1 and CB2 receptors are novel molecular targets for Tamoxifen and 4OH-Tamoxifen

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9–3 μM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.