APS8 Delays Tumor Growth in Mice by Inducing Apoptosis of Lung Adenocarcinoma Cells Expressing High Number of α7 Nicotinic Receptors.

The alkylpyridinium polymer APS8, a potent antagonist of α7 nicotinic acetylcholine receptors (nAChRs), selectively induces apoptosis in non-small cell lung cancer cells but not in normal lung fibroblasts. To explore the potential therapeutic value of APS8 for at least certain types of lung cancer, we determined its systemic and organ-specific toxicity in mice, evaluated its antitumor activity against adenocarcinoma xenograft models, and examined the in-vitro mechanisms of APS8 in terms of apoptosis, cytotoxicity, and viability. We also measured Ca2+ influx into cells, and evaluated the effects of APS8 on Ca2+ uptake while siRNA silencing of the gene for α7 nAChRs, CHRNA7. APS8 was not toxic to mice up to 5 mg/kg i.v., and no significant histological changes were observed in mice that survived APS8 treatment. Repetitive intratumoral injections of APS8 (4 mg/kg) significantly delayed growth of A549 cell tumors, and generally prevented regrowth of tumors, but were less effective in reducing growth of HT29 cell tumors. APS8 impaired the viability of A549 cells in a dose-dependent manner and induced apoptosis at micro molar concentrations. Nano molar APS8 caused minor cytotoxic effects, while cell lysis occurred at APS8 >3 µM. Furthermore, Ca2+ uptake was significantly reduced in APS8-treated A549 cells. Observed differences in response to APS8 can be attributed to the number of α7 nAChRs expressed in these cells, with those with more AChRs (i.e., A549 cells) being more sensitive to nAChR antagonists like APS8. We conclude that α7 nAChR antagonists like APS8 have potential to be used as therapeutics for tumors expressing large numbers of α7 nAChRs.

Links Between Genetic Groups, Host Specificity, and Ergot-Alkaloid Profiles within Claviceps purpurea (Fr.) Tul. on Slovenian Grasses.

In the present study, the genetic relationships and ergot-alkaloid production of the fungus Claviceps purpurea on grasses were investigated, to determine any associations between grass host specificity, ergot-alkaloid production, and geographic origin. C. purpurea sclerotia were obtained from wild and cultivated grasses along a 300-km climatic gradient, from sub-Mediterranean to continental climates. Twenty-one infected grass samples provided 39 sclerotia for analysis of the ergot alkaloids ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, and ergocristine, and their "-inine" epimers, using liquid chromatography-tandem mass spectrometry. C. purpurea ribosomal DNA underwent molecular classification to determine any grass host or geographic specificity of ergot-alkaloid composition for the different operational taxonomic units. Molecular analysis of sclerotia ribosomal DNA showed three genetic groups, with some associations with specific grass host taxonomic groups. The ergot-alkaloid composition data were in agreement with the data obtained by molecular methods. The most frequent ergot-alkaloid epimers were ergocristine, and ergosine. The total ergot-alkaloid concentrations in sclerotia varied from 59 to 4,200 mg kg-1, which corresponds to 0.059 to 4.2 mg kg-1 in animal feed (assuming ergot alkaloids at 1,000 mg kg-1 sclerotia). Therefore, grasses can be associated with significant levels of ergot alkaloids. In addition, the ergot-alkaloid compositions of C. purpurea sclerotia can be different for infections with different C. purpurea genetic groups, because these show different ergot-alkaloid compositions.

Natural polymeric 3-alkylpyridinium salt affects vertebrate skeletal muscle contractility by preferentially blocking neuromuscular transmission.

The effects of natural polymeric alkylpyridinium salt (nPoly-3-APS), a potent acetylcholinesterase inhibitor isolated from the marine sponge Reniera sarai, were studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations using electrophysiological approaches. nPoly-3-APS inhibited nerve-evoked isometric muscle twitch and tetanic contraction in a concentration-dependent manner (IC50=29.4µM and 18.5µM, respectively) and produced a 30-44% decrease of directly muscle-elicited twitch and tetanus amplitudes at 54.4µM. Additionally, nPoly-3-APS (9.1-27.2µM) markedly decreased the amplitude of miniature endplate potentials, while their frequency was only affected at the highest concentration used. Endplate potentials were also inhibited by nPoly-3-APS in a concentration-dependent manner (IC50=20.1µM), without significant change in the resting membrane potential of muscle fibers (up to 54.4µM). In conclusion, our results show, for the first time, that nPoly-3-APS preferentially blocks the neuromuscular transmission, in vitro, by a non-depolarizing mechanism. This strongly suggests that the in vivo toxicity of nPoly-3-APS mainly occurs through an antagonist action of the compound on nicotinic acetylcholine receptors of skeletal muscles.

Beauvericin Inhibits Neuromuscular Transmission and Skeletal Muscle Contractility in Mouse Hemidiaphragm Preparation.

The effects of Beauvericin (BEA) produced by the fungusBeauveria bassianaandFusariumsp. on neuromuscular transmission and contractility were determined in an isolated neuromuscular mouse hemidiaphragm preparation. BEA (5 µM) significantly inhibits indirectly elicited twitch amplitude. At higher concentrations (7.5 and 10 µM), BEA produces a significant reduction of directly elicited, or complete block of indirectly evoked, muscle contraction. BEA also appears to be myotoxic, as indicated by a slowly developing muscle contracture. Development of neuromuscular blockade and contracture is concentration dependent. BEA acted by presynaptically depressing spontaneous acetylcholine release as indicated by the reduction in the frequency of spontaneous miniature endplate potentials (MEPPs), while the membrane potential of muscle fibers remained unchanged. At higher concentrations (7.5 and 10 µM), BEA progressively reduces or completely blocks MEPPs and EPPs amplitudes. Changes in MEPPs and EPPs are associated with substantial depolarization of muscle fibers when exposed to 7.5 and 10 µM of BEA. These results indicate that BEA has neurotoxic and myotoxic effects, which overlap in a narrow range of concentrations.

Pathophysiological effects of synthetic derivatives of polymeric alkylpyridinium salts from the marine sponge, Reniera sarai.

Polymeric 3-alkylpyridinium salts (poly-APS) are among the most studied natural bioactive compounds extracted from the marine sponge, Reniera sarai. They exhibit a wide range of biological activities, and the most prominent among them are the anti-acetylcholinesterase and membrane-damaging activity. Due to their membrane activity, sAPS can induce the lysis of various cells and cell lines and inhibit the growth of bacteria and fungi. Because of their bioactivity, poly-APS are possible candidates for use in the fields of medicine, pharmacy and industry. Due to the small amounts of naturally occurring poly-APS, methods for the synthesis of analogues have been developed. They differ in chemical properties, such as the degree of polymerization, the length of the alkyl chains (from three to 12 carbon atoms) and in the counter ions present in their structures. Such structurally defined analogues with different chemical properties and degrees of polymerization possess different levels of biological activity. We review the current knowledge of the biological activity and toxicity of synthetic poly-APS analogues, with particular emphasis on the mechanisms of their physiological and pharmacological effects and, in particular, the mechanisms of toxicity of two analogues, APS12-2 and APS3, in vivo and in vitro.

Effects of synthetic analogues of poly-APS on contractile response of porcine coronary arteries.

APS12-2 and APS3 are synthetic analogues of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. The aim of the present study was to determine the possible direct contractile effects of these two synthetic molecules on coronary arteries, in order partly to explain hemodynamic and cardiotoxic effects of APS12-2 previously observed in in vivo studies and to reveal possible adverse effects on the organism in the case of their clinical use. In contrast to APS3, APS12-2 caused a concentration-dependent vascular smooth muscle contraction of isolated porcine coronary ring preparations in a concentration-range from 1.36 to 13.60µM. Lanthanum chloride (5mM) and verapamil (10µM) completely abolished the APS12-2 evoked contraction of the coronary rings. Pre-incubation with indomethacin (10µM) had no effect on the contractile responses of coronary ring preparations. These results indicate that APS12-2 contracts vascular smooth muscle in a concentration-dependent manner, due to an increase of Ca(2+) influx through the voltage-gated Ca(2+) channels. Our data show for the first time that APS12-2 induces concentration-dependent contraction of coronary ring preparations, which may contribute to the cardiotoxic effects of APS12-2, in addition to hyperkalemia.

Toxicity of the synthetic polymeric 3-alkylpyridinium salt (APS3) is due to specific block of nicotinic acetylcholine receptors.

The in vivo and in vitro toxic effects of the synthetic polymeric 3-alkylpyridinium salt (APS3), from the Mediterranean marine sponge Reniera sarai, were evaluated on mammals, with emphasis to determine its mode of action. The median lethal doses of APS3 were 7.25 and higher that 20mg/kg in mouse and rat, respectively. Intravenous administration of 7.25 and 20mg/kg APS3 to rat caused a significant fall followed by an increase in mean arterial blood pressure accompanied by tachycardia. In addition, cumulative doses of APS3 (up to 60 mg/kg) inhibited rat nerve-evoked skeletal muscle contraction in vivo, with a median inhibitory dose (ID(50)) of 37.25mg/kg. When administrated locally by intramuscular injection to mouse, APS3 decreased the compound muscle action potential recorded in response to in vivo nerve stimulation, with an ID(50) of 0.5mg/kg. In vitro experiments confirmed the inhibitory effect of APS3 on mouse hemidiaphragm nerve-evoked muscle contraction with a median inhibitory concentration (IC(50)) of 20.3 µM, without affecting directly elicited muscle contraction. The compound inhibited also miniature endplate potentials and nerve-evoked endplate potentials with an IC(50) of 7.28 µM in mouse hemidiaphragm. Finally, APS3 efficiently blocked acetylcholine-activated membrane inward currents flowing through Torpedo nicotinic acetylcholine receptors (nAChRs) incorporated to Xenopus oocytes, with an IC(50) of 0.19 µM. In conclusion, our results strongly suggest that APS3 blocks muscle-type nAChRs, and show for the first time that in vivo toxicity of APS3 is likely to occur through an antagonist action of the compound on these receptors.

The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors.

APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC(50)=0.74 µM), without affecting directly-elicited twitch tension up to 2.72 µM. The compound (0.007-3.40 µM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68 µM, without affecting their frequency. Full size endplate potentials, recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC(50)=0.36 µM) without significant change in the resting membrane potential of the muscle fibers up to 3.40 µM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1(2)ß1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC(50)=0.0005 µM), indicating a higher affinity of the compound for Torpedo (α1(2)ß1γδ) than for the mouse (α1(2)ß1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction.

Binding and permeabilization of lipid bilayers by natural and synthetic 3-alkylpyridinium polymers.

Naturally occurring 3-alkylpyridinium polymers from the marine sponge Reniera sarai are membrane-active compounds exerting a selective cytotoxicity towards non small cell lung cancer cells, and stable transfection of nucleated mammalian cells. In view of their possible use as chemotherapeutics and/or transfection tools, three poly-APS based synthetic compounds were tested on their activity using natural and artificial lipid membranes. The tested compounds were found to be very stable over a wide range of temperature, ionic strength, and pH, and to prefer the solid-ordered membrane state. Their membrane-damaging activity increases with the length of their alkyl chains and the degree of polymerization.

In vivo toxic and lethal cardiovascular effects of a synthetic polymeric 1,3-dodecylpyridinium salt in rodents.

APS12-2 is one in a series of synthetic analogs of the polymeric alkylpyridinium salts isolated from the marine sponge Reniera sarai. As it is a potential candidate for treating non small cell lung cancer (NSCLC), we have studied its possible toxic and lethal effects in vivo. The median lethal dose (LD(50)) of APS12-2 in mice was determined to be 11.5mg/kg. Electrocardiograms, arterial blood pressure and respiratory activity were recorded under general anesthesia in untreated, pharmacologically vagotomized and artificially ventilated rats injected with APS12-2. In one group, the in vivo effects of APS12-2 were studied on nerve-evoked muscle contraction. Administration of APS12-2 at a dose of 8mg/kg caused a progressive reduction of arterial blood pressure to a mid-circulatory value, accompanied by bradycardia, myocardial ischemia, ventricular extrasystoles, and second degree atrio-ventricular block. Similar electrocardiogram and arterial blood pressure changes caused by APS12-2 (8mg/kg) were observed in animals pretreated with atropine and in artificially ventilated animals, indicating that hypoxia and cholinergic effects do not play a crucial role in the toxicity of APS12-2. Application of APS12-2 at sublethal doses (4 and 5.5mg/kg) caused a decrease of arterial blood pressure, followed by an increase slightly above control values. We found that APS12-2 causes lysis of rat erythrocytes in vitro, therefore it is reasonable to expect the same effect in vivo. Indeed, hyperkalemia was observed in the blood of experimental animals. Hyperkalemia probably plays an important role in APS12-2 cardiotoxicity since no evident changes in histopathology of the heart were found. However, acute lesions were observed in the pulmonary vessels of rats after application of 8mg/kg APS12-2. Predominant effects were dilation of interalveolar blood vessels and lysis of aggregated erythrocytes within their lumina.