(S)-Erypoegin K, an isoflavone isolated from Erythrina poeppigiana, is a novel inhibitor of topoisomerase IIα: Induction of G2 phase arrest in human gastric cancer cells.

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has a single chiral carbon in its structure and exists naturally as a racemic mixture. Our previous study showed (S)-erypoegin K selectively exhibits potent anti-proliferative and apoptosis-inducing activity against human leukemia HL-60 cells. To identify the target molecule of (S)-erypoegin K, we employed the human cancer cell panel analysis (termed JFCR39) coupled with a drug sensitivity database of pharmacologically well-characterized drugs for comparison using the COMPARE algorithm. (S)-erypoegin K exhibited a similar profile to that of etoposide, suggesting the molecular target for erypoegin K may be topoisomerase II (Topo II). Subsequent experiments using purified human Topo IIα established that the (S)-isomer selectively stabilizes the cleavage complex composed of double-stranded plasmid DNA and the enzyme. Moreover, (S)-erypoegin K inhibited decatenation of kinetoplast DNA. Molecular docking studies clearly indicated specific binding of the (S)-isomer to the active site of Topo IIα involving hydrogen bonds that help stabilize the cleavage complex. (S)-erypoegin K displayed potent cytotoxic activity against two human gastric cancer cells GCIY and MKN-1 with IC50 values of 0.270 and 0.327 µM, respectively, and induced enzyme activities of caspase 3 and 9. Cell cycle analysis showed marked cell cycle arrest at G2 phase in both cell lines. (S)-erypoegin K also displayed significant antitumor activity toward GCIY xenografted mice. The present study suggests (S)-erypoegin K acts as a Topo II inhibitor to block the G2/M transition of cancer cells.

Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption.

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+ /K+ -ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate-mediated excitotoxicity. Under a pyruvate-restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 µM. The OCR monitoring also revealed the contribution of the N-methyl-D-aspartate receptor and Na+ /K+ -ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte-dependent protection of neurons from glutamate-mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.

A mutant HCN4 channel in a family with bradycardia, left bundle branch block, and left ventricular noncompaction.

We found that a female infant presenting with left bundle branch block and left ventricular noncompaction carries uninvestigated gene mutations HCN4(G811E), SCN5A(L1988R), DMD(S2384Y), and EMD(R203H). Here, we explored the possible pathogenicity of HCN4(G811E), which results in a G811E substitution in hyperpolarization-activated cyclic nucleotide-gated channel 4, the main subunit of the cardiac pacemaker channel. Voltage-clamp measurements in a heterologous expression system of HEK293T cells showed that HCN4(G811E) slightly reduced whole-cell HCN4 channel conductance, whereas it did not affect the gating kinetics, unitary conductance, or cAMP-dependent modulation of voltage-dependence. Immunocytochemistry and immunoblot analysis showed that the G811E mutation did not impair the membrane trafficking of the channel subunit in the heterologous expression system. These findings indicate that HCN4(G811E) may not be a monogenic factor to cause the cardiac disorders.

Effect of Mechanical Agitation on Cationic Liposome Transport across an Unstirred Water Layer in Caco-2 Cells.

To develop an effective oral delivery system for plasmid DNA (pDNA) using cationic liposomes, it is necessary to clarify the characteristics of uptake and transport of cationic liposome/pDNA complexes into the intestinal epithelium. In particular, evaluation of the involvement of an unstirred water layer (UWL), which is a considerable permeability barrier, in cationic liposome transport is very important. Here, we investigated the effects of a UWL on the transfection efficiency of cationic liposome/pDNA complexes into a Caco-2 cell monolayer. When Caco-2 cells were transfected with cationic liposome/pDNA complexes in shaking cultures to reduce the thickness of the UWL, gene expression was significantly higher in Caco-2 cells compared with static cultures. We also found that this enhancement of gene expression by shaking was not attributable to activation of transcription factors such as activator protein-1 and nuclear factor-kappaB (NF-κB). In addition, the increase in gene expression by mechanical agitation was observed at all charge ratios (1.5, 2.3, 3.1, 4.5) of cationic liposome/pDNA complexes. Transport experiments using Transwells demonstrated that mechanical agitation increased the uptake of cationic liposome/pDNA complexes by Caco-2 cells, whereas transport of the complexes across a Caco-2 cell monolayer did not occurr. Moreover, the augmentation of the gene expression of cationic liposome/pDNA complexes by shaking was observed in Madin-Darby canine kidney cells. These results indicate that a UWL greatly affects the uptake and transfection efficiency of cationic liposome/pDNA complexes into an epithelial monolayer in vitro.

Lack of modulatory effect of the SCN5A R1193Q polymorphism on cardiac fast Na+ current at body temperature.

SCN5A encodes the main subunit of the NaV1.5 channel, which mediates the fast Na+ current responsible for generating cardiac action potentials. The single nucleotide polymorphism SCN5A(R1193Q), which results in an amino acid replacement in the subunit, is common in East Asia. SCN5A(R1193Q) is often identified in patients with type 3 long QT syndrome and Brugada syndrome. However, its linkage to arrhythmic disorders is under debate. Previous electrophysiological studies performed at room temperature inconsistently reported the gain- or loss-of-function effect of SCN5A(R1193Q) on the NaV1.5 channel. More recently, it was theoretically predicted that SCN5A(R1193Q) would exert a loss-of-function effect at body temperature. Here, we experimentally assessed whether SCN5A(R1193Q) modulates the NaV1.5 channel at various temperatures including normal and febrile body temperatures. We compared voltage-gated Na+ currents in SCN5A(R1193Q)-transfected and wild-type SCN5A-transfected HEK293T cells using a whole-cell voltage-clamp technique. First, we made comparisons at constant temperatures of 25°C, 36.5°C, and 38°C, and found no difference in the conductance density, voltage dependence of gating, or time dependence of gating. This suggested that SCN5A(R1193Q) does not modulate the NaV1.5 channel regardless of temperature. Second, we made comparisons while varying the temperature from 38°C to 26°C in 3 min, and again observed no difference in the time course of the amplitude or time dependence of gating during the temperature change. This also indicated that SCN5A(R1193Q) does not modulate the NaV1.5 channel in response to an acute body temperature change. Therefore, SCN5A(R1193Q) may not be a monogenic factor that triggers arrhythmic disorders.