Characterization of the anticancer effects of S115, a novel heteroaromatic thiosemicarbazone compound,in vitro and in vivo

Aim:

To investigate the anticancer effects of S115, a novel heteroaromatic thiosemicarbazone compound in vitro and in vivo.

Methods:

The anti-proliferative action of S115 was analyzed in 12 human and mouse cancer cell lines using MTT assay. Autograft and xenograft cancer models were made by subcutaneous inoculation of cancer cells into mice or nude mice. The mice were orally treated with S115 (2, 8, 32 mg·kg⁻¹·d⁻¹) for 7 d, and the tumor size was measured every 3 d. Cell apoptosis and cell cycle distribution were examined using flow cytometry, gene expression profile analyses, Western blots and RT-PCR.

Results:

The IC₅₀ values of S115 against 12 human and mouse cancer cell lines ranged from 0.3 to 6.6 μmol/L. The tumor growth inhibition rate caused by oral administration of S115 (32 mg·kg⁻¹·d⁻¹) were 89.7%, 81.7%, 78.4% and 77.8%, respectively, in mouse model of B16 melanoma, mouse model of Colon26 colon cancer, nude mouse model of A549 lung cancer and nude mouse model of SK-OV-3 ovarian cancer. Furthermore, oral administration of S115 (7.5 mg·kg⁻¹·d⁻¹) synergistically enhanced the anticancer effects of cyclophosphamide, cisplatin, or 5-fluorouracil in mouse model of S180 sarcoma. Treatment of A549 human lung cancer cells with S115 (1.5 μmol/L) induced G₀/G₁ cell cycle arrest, and increased apoptosis. Furthermore, S115 downregulated the level of ubiquitin, and upregulated the level of Tob2 in A549 cells.

Conclusion:

S115 exerts anticancer effects against a variety of cancer cells in vitro and in grafted cancer models by inducing apoptosis, downregulating ubiquitin and upregulating Tob2.     

Catecholamine synthesis and metabolism in the central nervous system of mice lacking α2-adrenoceptor subtypes

Background and purpose:

This study investigates the role of α₂-adrenoceptor subtypes, α_2A, α_2B and α_2C, on catecholamine synthesis and catabolism in the central nervous system of mice.

Experimental approach:

Activities of the main catecholamine synthetic and catabolic enzymes were determined in whole brains obtained from α_2A-, α_2B- and α_2C-adrenoceptor knockout (KO) and C56Bl\7 wild-type (WT) mice.

Key results:

Although no significant differences were found in tyrosine hydroxylase activity and expression, brain tissue levels of 3,4-dihydroxyphenylalanine were threefold higher in α_2A- and α_2C-adrenoceptor KO mice. Brain tissue levels of dopamine and noradrenaline were significantly higher in α_2A and α_2CKOs compared with WT [WT: 2.8 ± 0.5, 1.1 ± 0.1; α_2AKO: 6.9 ± 0.7, 1.9 ± 0.1; α_2BKO: 2.3 ± 0.2, 1.0 ± 0.1; α_2CKO: 4.6 ± 0.8, 1.5 ± 0.2 nmol·(g tissue)⁻¹, for dopamine and noradrenaline respectively]. Aromatic L-amino acid decarboxylase activity was significantly higher in α_2A and α_2CKO [WT: 40 ± 1; α_2A: 77 ± 2; α_2B: 40 ± 1; α_2C: 50 ± 1, maximum velocity (V_max) in nmol·(mg protein)⁻¹·h⁻¹], but no significant differences were found in dopamine β-hydroxylase. Of the catabolic enzymes, catechol-O-methyltransferase enzyme activity was significantly higher in all three α₂KO mice [WT: 2.0 ± 0.0; α_2A: 2.4 ± 0.1; α_2B: 2.2 ± 0.0; α_2C: 2.2 ± 0.0 nmol·(mg protein)⁻¹·h⁻¹], but no significant differences were found in monoamine oxidase activity between all α₂KOs and WT mice.

Conclusions and implications:

In mouse brain, deletion of α_2A- or α_2C-adrenoceptors increased cerebral aromatic L-amino acid decarboxylase activity and catecholamine tissue levels. Deletion of any α₂-adrenoceptor subtypes resulted in increased activity of catechol-O-methyltransferase. Higher 3,4-dihydroxyphenylalanine tissue levels in α_2A and α_2CKO mice could be explained by increased 3,4-dihydroxyphenylalanine transport.     

Novel ferrocenyl derivatives exert anti-cancer effect in human lung cancer cells in vitro via inducing G1-phase arrest and senescence

Aim:

To investigate the effects of 7 novel 1-ferrocenyl-2-(5-phenyl-1H-1,2,4-triazol-3-ylthio) ethanone derivatives on human lung cancer cells in vitro and to determine the mechanisms of action.

Methods:

A549 human lung cancer cells were examined. Cell viability was analyzed with MTT assay. Cell apoptosis and senescence were examined using Hoechst 33258 and senescence-associated-β-galactosidase (SA-β-gal) staining, respectively. LDH release was measured using a detection kit. Cell cycle was analyzed using a flow cytometer. Intracellular ROS level was measured with the 2′,7′-dichlorodihydrofluorescein probe. Phosphorylation of p38 was determined using Western blot.

Results:

Compounds 5b, 5d, and 5e (40 and 80 μmol/L) caused significant decrease of A549 cell viability, while other 4 compounds had no effect on the cells. Compounds 5b, 5d, and 5e (80 μmol/L) induced G₁-phase arrest (increased the G₁ population by 22.6%, 24.23%, and 26.53%, respectively), and markedly increased SA-β-gal-positive cells. However, the compounds did not cause nuclear DNA fragmentation and chromatin condensation in A549 cells. Nor did they affect the release of LDH from the cells. The compounds significantly elevated the intracellular ROS level, decreased the mitochondrial membrane potential, and increased p38 phosphorylation in the cells. In the presence of the antioxidant and free radical scavenger N-acetyl-L-cysteine (10 mmol/L), above effects of compounds 5b, 5d, and 5e were abolished.

Conclusion:

The compounds 5b, 5d, and 5e cause neither apoptosis nor necrosis of A549 cells, but exert anti-cancer effect via inducing G₁-phase arrest and senescence through ROS/p38 MAP-kinase pathway.     

Scorpion Peptide Smp24 Exhibits a Potent Antitumor Effect on Human Lung Cancer Cells by Damaging the Membrane and Cytoskeleton In Vivo and In Vitro

Smp24, a cationic antimicrobial peptide identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, shows variable cytotoxicity on various tumor (KG1a, CCRF-CEM and HepG2) and non-tumor (CD34⁺, HRECs, HACAT) cell lines. However, the effects of Smp24 and its mode of action on lung cancer cell lines remain unknown. Herein, the effect of Smp24 on the viability, membrane disruption, cytoskeleton, migration and invasion, and MMP-2/-9 and TIMP-1/-2 expression of human lung cancer cells have been evaluated. In addition, its in vivo antitumor role and acute toxicity were also assessed. In our study, Smp24 was found to suppress the growth of A549, H3122, PC-9, and H460 with IC₅₀ values from about 4.06 to 7.07 µM and show low toxicity to normal cells (MRC-5) with 14.68 µM of IC₅₀. Furthermore, Smp24 could induce necrosis of A549 cells via destroying the integrity of the cell membrane and mitochondrial and nuclear membranes. Additionally, Smp24 suppressed cell motility by damaging the cytoskeleton and altering MMP-2/-9 and TIMP-1/-2 expression. Finally, Smp24 showed effective anticancer protection in a A549 xenograft mice model and low acute toxicity. Overall, these findings indicate that Smp24 significantly exerts an antitumor effect due to its induction of membrane defects and cytoskeleton disruption. Accordingly, our findings will open an avenue for developing scorpion venom peptides into chemotherapeutic agents targeting lung cancer cells.     

Negative regulation of mTOR activity by LKB1-AMPK signaling in non-small cell lung cancer cells

Aim:

To investigate the role of LKB1 in regulation of mTOR signaling in non-small cell lung cancer (NSCLC) cells.

Methods:

LKB1 protein expression and phosphorylation of AMPK, 4E-BP1 and S6K in the cells were assessed using Western blotting in various NSCLC cell lines (A549, H460, H1792, Calu-1 and H1299). Energy stress was mimicked by treating the cells with 2-deoxyglucose (2-DG). Compound C was used to inhibit AMPK activity. Cell growth was measured using the MTS assay.

Results:

LKB1 protein was expressed in LKB1 wild-type Calu-1, H1299 and H1792 cells, but it was undetected in LKB1 mutant A549 and H460 cells. Treatment of the LKB1 wild-type cells with 2-DG (5, 10 and 25 mmol/L) augmented the phosphorylation of AMPK in dose- and time-dependent manners. In the LKB1 wild-type cells, 2-DG dramatically suppressed the phosphorylation of two mTOR targets, 4E-BP1 and S6K, whereas the LKB1 mutant A549 and H460 cells were highly resistant to 2-DG-induced inhibition on mTOR activity. In addition, stable knockdown of LKB1 in H1299 cells impaired 2-DG-induced inhibition on mTOR activity. Pretreatment of H1299 and H1792 cells with the AMPK inhibitor compound C (10 μmol/L) blocked 2-DG-induced inhibition on mTOR activity. 2-DG inhibited the growth of H1299 cells more effectively than that of H460 cells; stable knockdown of LKB1 in H1299 cells attenuated the growth inhibition caused by 2-DG.

Conclusion:

In non-small cell lung cancer cells, LKB1/AMPK signaling negatively regulates mTOR activity and contributes to cell growth inhibition in response to energy stress.     

Myrtucommulone,a natural acylphloroglucinol,inhibits microsomal prostaglandin E2 synthase-1

Background and purpose:

The selective inhibition of prostaglandin (PG)E₂ formation via interference with microsomal PGE₂ synthase (mPGES)-1 could have advantages in the treatment of PGE₂-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1.

Experimental approach:

The effect of MC on PGE₂ formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1β-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF_1α formation.

Key results:

MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH₂ to PGE₂ (IC₅₀ = 1 µmol·L⁻¹). PGE₂ formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 µmol·L⁻¹, and only moderate inhibition of cellular or cell-free COX-1 was evident (IC₅₀ > 15 µmol·L⁻¹).

Conclusions and implications:

MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE₂ formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.     

Pharmacokinetics of PEGylated recombinant human endostatin (M2ES) in rats

Aim:

M₂ES is PEGylated recombinant human endostatin. In this study we investigated the pharmacokinetics, tissue distribution, and excretion of M₂ES in rats.

Methods:

¹²⁵I-radiolabeled M₂ES was administered to rats by intravenous bolus injection at 3 mg/kg. The pharmacokinetics, tissue distribution and excretion of M₂ES were investigated using the trichloroacetic acid (TCA) precipitation method.

Results:

The serum M₂ES concentration-time curve after a single intravenous dose of 3 mg/kg in rats was fitted with a non-compartment model. The pharmacokinetic parameters were evaluated as follows: C_max=28.3 μg·equ/mL, t_1/2=71.5 h, AUC_(0–∞)=174.6 μg·equ·h/mL, Cl=17.2 mL·h⁻¹·kg⁻¹, MRT=57.6 h, and V_ss=989.8 mL/kg for the total radioactivity; C_max=30.3 μg·equ/mL, t_1/2=60.1 h, AUC_(0–∞)=146.2 μg·equ·h/mL, Cl=20.6 mL·h⁻¹·kg⁻¹, MRT=47.4 h, and V_ss=974.6 mL/kg for the TCA precipitate radioactivity. M₂ES was rapidly and widely distributed in various tissues and showed substantial deposition in kidney, adrenal gland, lung, spleen, bladder and liver. The radioactivity recovered in the urine and feces by 432 h post-dose was 71.3% and 8.3%, respectively. Only 0.98% of radioactivity was excreted in the bile by 24 h post-dose.

Conclusion:

PEG modification substantially prolongs the circulation time of recombinant human endostatin and effectively improves its pharmacokinetic behavior. M₂ES is extensively distributed in most tissues of rats, including kidney, adrenal gland, lung, spleen, bladder and liver. Urinary excretion was the major elimination route for M₂ES.     

Antidepressant-like effects of cannabidiol in mice: possible involvement of 5-HT1A receptors

Background and purpose:

Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic- and antipsychotic-like effects in animal models. Effects of CBD may be mediated by the activation of 5-HT_1A receptors. As 5-HT_1A receptor activation may induce antidepressant-like effects, the aim of this work was to test the hypothesis that CBD would have antidepressant-like activity in mice as assessed by the forced swimming test. We also investigated if these responses depended on the activation of 5-HT_1A receptors and on hippocampal expression of brain-derived neurotrophic factor (BDNF).

Experimental approach:

Male Swiss mice were given (i.p.) CBD (3, 10, 30, 100 mg·kg⁻¹), imipramine (30 mg·kg⁻¹) or vehicle and were submitted to the forced swimming test or to an open field arena, 30 min later. An additional group received WAY100635 (0.1 mg·kg⁻¹, i.p.), a 5-HT_1A receptor antagonist, before CBD (30 mg·kg⁻¹) and assessment by the forced swimming test. BDNF protein levels were measured in the hippocampus of another group of mice treated with CBD (30 mg·kg⁻¹) and submitted to the forced swimming test.

Key results:

CBD (30 mg·kg⁻¹) treatment reduced immobility time in the forced swimming test, as did the prototype antidepressant imipramine, without changing exploratory behaviour in the open field arena. WAY100635 pretreatment blocked CBD-induced effect in the forced swimming test. CBD (30 mg·kg⁻¹) treatment did not change hippocampal BDNF levels.

Conclusion and implications:

CBD induces antidepressant-like effects comparable to those of imipramine. These effects of CBD were probably mediated by activation of 5-HT_1A receptors.     

Myoendothelial coupling in the mesenteric arterial bed; segmental differences and interplay between nitric oxide and endothelin-1

Background and purpose:

We tested the hypothesis that activated arterial smooth muscle (ASM) stimulates endothelial vasomotor influences via gap junctions and that the significance of this myoendothelial coupling increases with decreasing arterial diameter.

Experimental approach:

From WKY rats, first-, second-, third-and fourth-order branches of the superior mesenteric artery (MA1, MA2, MA3 and MA4 respectively) were isolated and mounted in wire-myographs to record vasomotor responses to 0.16–20 µmol·L⁻¹ phenylephrine.

Key results:

Removal of endothelium increased the sensitivity (pEC₅₀) to phenylephrine in all arteries. The nitric oxide (NO) synthase inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME) (100 µmol·L⁻¹) did not modify pEC₅₀ to phenylephrine in all denuded arteries, and increased it in intact MA1, MA2 and MA3 to the same extent as denudation. However, in intact MA4, the effect of L-NAME was significantly larger (ΔpEC₅₀ 0.57 ± 0.02) than the effect of endothelium removal (ΔpEC₅₀ 0.20 ± 0.06). This endothelium-dependent effect of L-NAME in MA4 was inhibited by (i) steroidal and peptidergic uncouplers of gap junctions; (ii) a low concentration of the NO donor sodium nitroprusside; and (iii) by the endothelin-receptor antagonist bosentan. It was also observed during contractions induced by (i) calcium channel activation (BayK 8644, 0.001–1 µmol·L⁻¹); (ii) depolarization (10–40 mmol·L⁻¹ K⁺); and (iii) sympathetic nerve stimulation (0.25–32 Hz).

Conclusions and implications:

These pharmacological observations indicated feedback control by endothelium of ASM reactivity involving gap junctions and a balance between endothelium-derived NO and endothelin-1. This myoendothelial coupling was most prominent in distal resistance arteries.     

Central 5-HT1A receptor-mediated modulation of heart rate dynamics and its adjustment by conditioned and unconditioned fear in mice

BACKGROUND AND PURPOSE

The beat-by-beat fluctuation (dynamics) of heart rate (HR) depends on centrally mediated control of the autonomic nervous system (ANS) reflecting the physiological state of an organism. 5-HT_1A receptors are implicated in affective disorders,associated with ANS dysregulation which increases cardiac risk but their role in autonomic HR regulation under physiological conditions is insufficiently characterized.

EXPERIMENTAL APPROACH

The effects of subcutaneously administered 5-HT_1A receptor ligands on HR dynamics were investigated in C57BL/6 mice during stress-free conditions and emotional challenge (recall of fear conditioned to an auditory stimulus and novelty exposure) using time domain and non-linear HR analyses.

KEY RESULTS

Pre-training treatment with of 8-OH-DPAT (0.5 mg·kg⁻¹, s.c.) prevented conditioned tachycardia in the retention test indicating impaired fear memory. Pretest 5-HT_1A receptor activation by 8-OH-DPAT (0.5 but not 0.1 and 0.02 mg·kg⁻¹) caused bradycardia and increased HR variability. 8-OH-DPAT (0.5 mg·kg⁻¹) lowered the unconditioned and conditioned tachycardia from ∼750 to ∼550 bpm, without changing the conditioned HR response to the sound. 8-OH-DPAT induced profound QT prolongation and bradyarrhythmic episodes. Non-linear analysis indicated a pathological state of HR dynamics after 8-OH-DPAT (0.5 mg·kg⁻¹) with ANS hyperactivation impairing HR adaptability. The 5-HT_1A receptor antagonist WAY-100635 (0.03 mg·kg⁻¹) blocked these effects of 8-OH-DPAT.

CONCLUSIONS AND IMPLICATIONS

Pre-training 5-HT_1A receptor activation by 8-OH-DPAT (0.5 mg·kg⁻¹) impaired memory of conditioned auditory fear based on an attenuated HR increase, whereas pretest administration did not prevent the fear-conditioned HR increase but induced pathological HR dynamics through central ANS dysregulation with cardiac effects similar to acute SSRI overdose.     

Maresin 1 mitigates LPS-induced acute lung injury in mice

BACKGROUND AND PURPOSE

Acute lung injury (ALI) is a severe illness with a high rate of mortality. Maresin 1 (MaR1) was recently reported to regulate inflammatory responses. We used a LPS-induced ALI model to determine whether MaR1 can mitigate lung injury.

EXPERIMENTAL APPROACH

Male BALB/c mice were injected, intratracheally, with either LPS (3 mg·kg⁻¹) or normal saline (1.5 mL·kg⁻¹). After this, normal saline, a low dose of MaR1 (0.1 ng per mouse) or a high dose of MaR1 (1 ng per mouse) was given i.v. Lung injury was evaluated by detecting arterial blood gas, pathohistological examination, pulmonary oedema, inflammatory cell infiltration, inflammatory cytokines in the bronchoalveolar lavage fluid and neutrophil–platelet interactions.

KEY RESULTS

The high dose of MaR1 significantly inhibited LPS-induced ALI by restoring oxygenation, attenuating pulmonary oedema and mitigating pathohistological changes. A combination of elisa and immunohistochemistry showed that high-dose MaR1 attenuated LPS-induced increases in pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), chemokines [keratinocyte chemokine, monocyte chemoattractant protein-5, macrophage inflammatory protein (MIP)-1α and MIP-1γ], pulmonary myeloperoxidase activity and neutrophil infiltration in the lung tissues. Consistent with these observations, flow cytometry and Western blotting indicated that MaR1 down-regulated LPS-induced neutrophil adhesions and suppressed the expression of intercellular adhesion molecule (ICAM)-1, P-selection and CD24.

CONCLUSIONS AND IMPLICATIONS

High-dose MaR1 mitigated LPS-induced lung injury in mice by inhibiting neutrophil adhesions and decreasing the levels of pro-inflammatory cytokines.     

Beneficial effect of a hydrogen sulphide donor (sodium sulphide) in an ovine model of burn- and smoke-induced acute lung injury

Background and purpose:

The present study investigated whether the pathophysiological changes induced by burn and smoke inhalation are modulated by parenteral administration of Na₂S, a H₂S donor.

Experimental approach:

The study used a total of 16 chronically instrumented, adult female sheep. Na₂S was administered 1 h post injury, as a bolus injection at a dose of 0.5 mg·kg⁻¹ and subsequently, as a continuous infusion at a rate of 0.2 mg·kg⁻¹·h⁻¹ for 24 h. Cardiopulmonary variables (mean arterial and pulmonary arterial blood pressure, cardiac output, ventricular stroke work index, vascular resistance) and arterial and mixed venous blood gases were measured. Lung wet-to-dry ratio and myeloperoxidase content and protein oxidation and nitration were also measured. In addition, lung inducible nitric oxide synthase expression and cytochrome c were measured in lung homogenates via Western blotting and enzyme-linked immunosorbent assay (elisa) respectively.

Key results:

The H₂S donor decreased mortality during the 96 h experimental period, improved pulmonary gas exchange and lowered further increase in inspiratory pressure and fluid accumulation associated with burn- and smoke-induced acute lung injury. Further, the H₂S donor treatment reduced the presence of protein oxidation and 3-nitrotyrosine formation following burn and smoke inhalation injury.

Conclusions and implications:

Parenteral administration of the H₂S donor ameliorated the pulmonary pathophysiological changes associated with burn- and smoke-induced acute lung injury. Based on the effect of H₂S observed in this clinically relevant model of disease, we propose that treatment with H₂S or its donors may represent a potential therapeutic strategy in managing patients with acute lung injury.     

Adenosine A2A receptors are necessary and sufficient to trigger memory impairment in adult mice

Background and Purpose

Caffeine (a non-selective adenosine receptor antagonist) prevents memory deficits in aging and Alzheimer’s disease, an effect mimicked by adenosine A_2A receptor, but not A₁ receptor, antagonists. Hence, we investigated the effects of adenosine receptor agonists and antagonists on memory performance and scopolamine-induced memory impairment in mice.

Experimental Approach

We determined whether A_2A receptors are necessary for the emergence of memory impairments induced by scopolamine and whether A_2A receptor activation triggers memory deficits in naïve mice, using three tests to assess short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze.

Key Results

Scopolamine (1.0 mg·kg⁻¹, i.p.) impaired short-term memory performance in all three tests and this scopolamine-induced amnesia was prevented by the A_2A receptor antagonist (SCH 58261, 0.1–1.0 mg·kg⁻¹, i.p.) and by the A₁ receptor antagonist (DPCPX, 0.2–5.0 mg·kg⁻¹, i.p.), except in the modified Y-maze where only {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304"}}SCH58261 was effective. Both antagonists were devoid of effects on memory or locomotion in naïve rats. Notably, the activation of A_2A receptors with CGS 21680 (0.1–0.5 mg·kg⁻¹, i.p.) before the training session was sufficient to trigger memory impairment in the three tests in naïve mice, and this effect was prevented by SCH 58261 (1.0 mg·kg⁻¹, i.p.). Furthermore, i.c.v. administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task.

Conclusions and Implications

These results show that A_2A receptors are necessary and sufficient to trigger memory impairment and further suggest that A₁ receptors might also be selectively engaged to control the cholinergic-driven memory impairment.     

Umbelliprenin is cytotoxic against QU-DB large cell lung cancer cell line but anti-proliferative against A549 adenocarcinoma cells

Background

Umbelliprenin is a natural compound, belonging to the class of sesquiterpene coumarins. Recently, umbelliprenin has attracted the researchers'' attention for its antitumor activities against skin tumors. Its effect on lung cancer is largely unknown. The aim of our study was to investigate the effects of this natural compound, which is expected to have low adverse effects, on lung cancer.

Methods

The QU-DB large cell and A549 adenocarcinoma lung cancer cell lines were treated with umbelliprenin. IC₅₀ values were estimated using methyl thiazolely diphenyl-tetrazolium bromide (MTT) assay, in which a decrease in MTT reduction can occur as a result of cell death or cell proliferation inhibition. To quantify the rate of cell death at IC₅₀ values, flow cytometry using Annexin V-FITC (for apoptotic cells), and propidium iodide (for necrotic cells) dyes were employed.

Results

Data from three independent MTT experiments in triplicate revealed that IC₅₀ values for QU-DB and A549 were 47 ± 5.3 μM and 52 ± 1.97 μM, respectively. Annexin V/PI staining demonstrated that umbelliprenin treatment at IC₅₀ induced 50% cell death in QU-DB cells, but produced no significant death in A549 cells until increasing the umbelliprenin concentration to IC₈₀. The pattern of cell death was predominantly apoptosis in both cell lines. When peripheral blood mononuclear cells were treated with 50 μM and less concentrations of umbelliprenin, no suppressive effect was observed.

Conclusions

We found cytotoxic/anti-proliferative effects of umbelliprenin against two different types of lung cancer cell lines.     

Cannabidivarin-rich cannabis extracts are anticonvulsant in mouse and rat via a CB1 receptor-independent mechanism

BACKGROUND AND PURPOSE

Epilepsy is the most prevalent neurological disease and is characterized by recurrent seizures. Here, we investigate (i) the anticonvulsant profiles of cannabis-derived botanical drug substances (BDSs) rich in cannabidivarin (CBDV) and containing cannabidiol (CBD) in acute in vivo seizure models and (ii) the binding of CBDV BDSs and their components at cannabinoid CB₁ receptors.

EXPERIMENTAL APPROACH

The anticonvulsant profiles of two CBDV BDSs (50–422 mg·kg⁻¹) were evaluated in three animal models of acute seizure. Purified CBDV and CBD were also evaluated in an isobolographic study to evaluate potential pharmacological interactions. CBDV BDS effects on motor function were also investigated using static beam and grip strength assays. Binding of CBDV BDSs to cannabinoid CB₁ receptors was evaluated using displacement binding assays.

KEY RESULTS

CBDV BDSs exerted significant anticonvulsant effects in the pentylenetetrazole (≥100 mg·kg⁻¹) and audiogenic seizure models (≥87 mg·kg⁻¹), and suppressed pilocarpine-induced convulsions (≥100 mg·kg⁻¹). The isobolographic study revealed that the anticonvulsant effects of purified CBDV and CBD were linearly additive when co-administered. Some motor effects of CBDV BDSs were observed on static beam performance; no effects on grip strength were found. The Δ⁹-tetrahydrocannabinol and Δ⁹-tetrahydrocannabivarin content of CBDV BDS accounted for its greater affinity for CB₁ cannabinoid receptors than purified CBDV.

CONCLUSIONS AND IMPLICATIONS

CBDV BDSs exerted significant anticonvulsant effects in three models of seizure that were not mediated by the CB₁ cannabinoid receptor and were of comparable efficacy with purified CBDV. These findings strongly support the further clinical development of CBDV BDSs for the treatment of epilepsy.     

Selective endothelin A receptor antagonism with sitaxentan reduces neointimal lesion size in a mouse model of intraluminal injury

Background and Purpose

Endothelin (ET) receptor antagonism reduces neointimal lesion formation in animal models. This investigation addressed the hypothesis that the selective ET_A receptor antagonist sitaxentan would be more effective than mixed ET_A/B receptor antagonism at inhibiting neointimal proliferation in a mouse model of intraluminal injury.

Experimental Approach

Antagonism of ET_A receptors by sitaxentan (1–100 nM) was assessed in femoral arteries isolated from adult, male C57Bl6 mice using isometric wire myography. Neointimal lesion development was induced by intraluminal injury in mice receiving sitaxentan (ET_A antagonist; 15 mg·kg⁻¹·day⁻¹), A192621 (ET_B antagonist; 30 mg·kg⁻¹·day⁻¹), the combination of both antagonists or vehicle. Treatment began 1 week before, and continued for 28 days after, surgery. Femoral arteries were then harvested for analysis of lesion size and composition.

Key Results

Sitaxentan produced a selective, concentration-dependent parallel rightward shift of ET-1-mediated contraction in isolated femoral arteries. Sitaxentan reduced neointimal lesion size, whereas ET_B and combined ET_A/B receptor antagonism did not. Macrophage and α-smooth muscle actin content were unaltered by ET receptor antagonism but sitaxentan reduced the amount of collagen in lesions.

Conclusions and Implications

These results suggest that ET_A receptor antagonism would be more effective than combined ET_A/ET_B receptor antagonism at reducing neointimal lesion formation.     

Cannabidiolic acid prevents vomiting in Suncus murinus and nausea-induced behaviour in rats by enhancing 5-HT1A receptor activation

Background and Purpose

To evaluate the ability of cannabidiolic acid (CBDA) to reduce nausea and vomiting and enhance 5-HT_1A receptor activation in animal models.

Experimental Approach

We investigated the effect of CBDA on (i) lithium chloride (LiCl)-induced conditioned gaping to a flavour (nausea-induced behaviour) or a context (model of anticipatory nausea) in rats; (ii) saccharin palatability in rats; (iii) motion-, LiCl- or cisplatin-induced vomiting in house musk shrews (Suncus murinus); and (iv) rat brainstem 5-HT_1A receptor activation by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and mouse whole brain CB₁ receptor activation by CP55940, using [³⁵S]GTPγS-binding assays.

Key Results

In shrews, CBDA (0.1 and/or 0.5 mg·kg⁻¹ i.p.) reduced toxin- and motion-induced vomiting, and increased the onset latency of the first motion-induced emetic episode. In rats, CBDA (0.01 and 0.1 mg·kg⁻¹ i.p.) suppressed LiCl- and context-induced conditioned gaping, effects that were blocked by the 5-HT_1A receptor antagonist, WAY100635 (0.1 mg·kg⁻¹ i.p.), and, at 0.01 mg·kg⁻¹ i.p., enhanced saccharin palatability. CBDA-induced suppression of LiCl-induced conditioned gaping was unaffected by the CB₁ receptor antagonist, SR141716A (1 mg·kg⁻¹ i.p.). In vitro, CBDA (0.1–100 nM) increased the E_max of 8-OH-DPAT.

Conclusions and Implications

Compared with cannabidiol, CBDA displays significantly greater potency at inhibiting vomiting in shrews and nausea in rats, and at enhancing 5-HT_1A receptor activation, an action that accounts for its ability to attenuate conditioned gaping in rats. Consequently, CBDA shows promise as a treatment for nausea and vomiting, including anticipatory nausea for which no specific therapy is currently available.     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Activation of the lung S1P1 receptor reduces allergen-induced plasma leakage in mice

Background and purpose:

It has been suggested that intratracheal administration of the immunomodulator, FTY720, could have anti-inflammatory effects without causing a decrease in blood lymphocyte counts. However, the receptor responsible for this effect has not been defined.

Experimental approach:

We have described, in a mouse model of allergen-induced inflammation, the use of proton magnetic resonance imaging to non-invasively assess lung fluid accumulation and inflammation. Here, we used this model to investigate the sphingosine-1-phosphate (S1P) receptor responsible for the anti-inflammatory effect of FTY720.

Key results:

When given intranasally, FTY720 (3 and 10 µg·kg⁻¹) inhibited by approximately 50% the allergen-induced accumulation of fluid in the lung detected by magnetic resonance imaging, but had no effect on the cellular inflammation in the airway space or on circulating blood lymphocytes. Inhibition of the infiltration of inflammatory cells into the airways was only observed at a dose of FTY720 that induced lymphopenia (100 µg·kg⁻¹). Similar results were observed in S1P₃-deficient mice. The effect of FTY720 was mimicked by intranasal treatment of wild-type mice with a S1P₁-specific agonist, AUY954.

Conclusions and implications:

Thus, in contrast to previously published work, our results suggest that systemic exposure of FTY720 is necessary to obtain an airway anti-inflammatory effect. On the contrary, inhibition of the allergen-induced accumulation of fluid in the lung, via activation of the S1P₁ receptor, is obtainable without systemic effects.