Phaseoloideside E (PE), a new oleanane-type triterpene saponin, was isolated from the seed kernels of Entada phaseoloides (Linn.) Merr. PE had strong cytotoxic activity against an array of malignant cells. Typical morphological and biochemical features of apoptosis were observed in PE-treated Ec-109 cells. PE induced a dose-dependent increase in the sub-G1 fraction of the cell cycle and DNA fragmentation. Decreases in the mitochondrial membrane potential, SOD activity, and GSH content were also observed. Further investigations revealed that PE reduced the ratio of Bcl-2 to Bax and increased the activities of caspase-3 and -9, but this was prevented by Z-VAD-fmk. PE also induced a decrease of the sub-G1 fraction. Furthermore, PE-induced apoptosis was mediated by up-regulating cellular ROS, which was suppressed by cotreating the cells with N-acetylcysteine (NAC). NAC also attenuated the ratio of sub-G1, the generation of DNA fragmentation and the expression of Bcl-2, Bax, caspase-3, and caspase-9. Interestingly, PE did not up-regulate ROS or induce cell death in untransformed cells. These data showed that PE induces cell death through up-regulation of cellular ROS production. Our investigation provides the scientific basis for the traditional application of this herb and suggests the possibility that PE may be used for a treatment of esophageal carcinoma.
[Supplementary materials: available only at http://dx.doi.org/10.1254/jphs.12193FP]

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A growing body of evidence suggests that podocyte apoptosis is a major cause of decreased podocyte number, which leads to albuminuria and glomerular injury. The aim of this study was to clarify the molecular mechanisms of angiotensin II (Ang II)-induced apoptosis in cultured mouse podocytes. We examined the effects of Ang II (100 nmol/L) on apoptosis, superoxide anions, and cytosol pH in podocytes. For intracellular pH measurements, image analysis was conducted using confocal laser microscopy after incubation with carboxy-seminaphthorhodafluor-1. Superoxide anions and intracellular pH were elevated with Ang II treatment. Apoptotic cell numbers, as measured by TUNEL staining and caspase 3 activity, were also augmented in the Ang II–treated group. Pre-treatment with olmesartan (100 nmol/L, an Ang II type 1–receptor blocker), apocynin (50 μmol/L, NADPH oxidase inhibitor), or 5-N,N hexamethylene amiloride [30 μmol/L, Na⁺/H⁺ exchanger type 1 (NHE-1) inhibitor] abolished Ang II–induced podocyte apoptosis, whereas NHE-1 mRNA and protein expression was not affected by Ang II treatment. Moreover, Ang II increased NHE-1 phosphorylation. These results suggest that superoxide production, NHE-1 activation, and intracellular alkalization were early features prior to apoptosis in Ang II–treated mouse podocytes, and may offer new insights into the mechanisms responsible for Ang II–induced podocyte injury.

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The in vivo effectiveness of 4-dihydropyridine (bis-1,4-DHP), a new calcium-channel blocker, as a nephroprotector in isolated perfused kidney was evaluated by determining its effects on parameters associated with renal injury in diabetic rats. Diabetes in male Wistar rats, control, diabetic, control + bis-1,4-DHP, and diabetic + bis-1,4-DHP, was induced by a single administration of STZ (55 mg・kg⁻¹, i.p.). In the drug-treated groups, treatment with bis-1,4-DHP (10 mg・kg⁻¹・day⁻¹) started one week before diabetes induction; bis-1,4-DHP was dissolved in DMSO (0.3%) and suspended in drinking water with carboxymethyl cellulose (3%). Parameters evaluated were body weight, blood glucose, albuminuria, proteinuria, creatinine, urea excretion, kidney’s weight / body weight ratio, and kidney perfusion pressure in all rat groups at different times of diabetes (2, 4, 6, and 10 weeks). Kidney weight of diabetic rats significantly increased vs. control, control + bis-1,4-DHP, and diabetic + bis-1,4-DHP rats at different times of diabetes. The ratios % kidney weight / 100 g body weight were different between control, control + bis-1,4-DHP, and diabetic + bis-1,4-DHP rats vs. diabetic rats (P < 0.05). Kidney perfusion pressure was decreased by diabetes, while it was partially recovered by bis-1,4-DHP treatment in response to phenylephrine. Bis-1,4-DHP had a tendency to decrease hyperglycemia vs. diabetic rats, even though glycemia was too high as compared with controls, and it ameliorated albuminuria, creatinine, and urea excretion, suggesting a favorable effect on renal haemodynamics. Bis-1,4-DHP, by inhibiting Ca²⁺ entrance, induced vasodilation in renal vascular bed and thus may have a nephroprotective effect against diabetes-induced renal dysfunction, but does not have significant impact on hyperglycemia.

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Release of growth hormone (GH) from the somatotroph is regulated by binding GH-releasing hormone (GHRH) to its cognate receptor (GHRHR), one of the members of the G protein–coupled receptor (GPCR) superfamily. Proteins bound to the carboxy (C)-terminus of GPCR have been reported to regulate intracellular trafficking and function of the receptor; however, no functionally significant protein associated with GHRHR has been reported. We have identified a protein interacting with C-kinase 1 (PICK1) as a binding partner of GHRHR. In vitro binding assay revealed the PDZ-domain of PICK1 and the last four amino acid residues of GHRHR were prerequisite for the interaction. Further, in vivo association of these proteins was confirmed. Immunostaining data of a stable cell line expressing GHRHR with or without PICK1 suggested the C-terminus of GHRHR promoted cell surface expression of GHRHR and PICK1 affected the kinetics of the cell surface expression of GHRHR. Furthermore, cAMP production assay showed the C-terminus of GHRHR is involved in the regulation of receptor activation, and the interaction of GHRHR with PICK1 may influence intensities of the signal response after ligand stimulation. Thus, the interaction of the C-terminus of GHRHR with PICK1 has a profound role in regulating the trafficking and the signaling of GHRHR.
[Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.12287FP]

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The fluoroquinolone antibiotic ciprofloxacin has been reported to block delayed rectifier K⁺ channels at much higher concentrations than those at which it exerts its bactericidal activity. In this study using the halothane-anesthetized guinea pig, we assessed whether ciprofloxacin has a proarrhythmic activity. Ciprofloxacin at a clinically relevant dose of 3 mg/kg, i.v. did not affect any electrocardiographic parameters. At 10 mg/kg, it prolonged the QT interval and the duration of the monophasic action potential of the ventricle under sinus rhythm and constant ventricular pacing (n = 6). The extents of its effects on the ventricular repolarization phase were comparable to those of another fluoroquinolone antibiotic moxifloxacin at a clinically relevant dose of 3 mg/kg (n = 6). Meanwhile, the PR interval and QRS width were also increased by ciprofloxacin at 10 mg/kg, suggesting that the drug inhibited cardiac K⁺ channels as well as Na⁺ and Ca²⁺ channels in vivo. These results suggest that ciprofloxacin exerted a multi-ion channel–blocking action in the heart within the supra-therapeutic dose range. Therefore, careful observation may be necessary for patients with heart disease receiving a higher dose of ciprofloxacin in order to prevent the emergence of resistance.

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The proposed mechanism for proton pump inhibitors (PPIs) is that PPIs are activated at low pH to the sulfenamide form, which reacts with the sulfhydryl group of cysteine(s) at the active site of the proton pump, to produce reducible disulfide-bonded PPI-proton pump conjugates. However, this mechanism cannot explain the observations that some PPI-protein conjugates are irreducible. This study was designed to investigate the chemistry of the irreducible conjugates by mass spectrometry, using three PPIs and 17 cysteine-containing peptides. While some peptides favored the formation of reducible PPI-peptide adduct, the other peptides mainly produced irreducible adducts. Characterization of the irreducible adduct revealed that the irreducible bonding required the participation of both a sulfhydryl group and a nearby primary amino group. High resolution mass spectrometry suggested a molecular structure of the irreducible adduct. These results suggested a reaction mechanism in which the PPI pyridone form reacted with an amino group and a sulfhydryl group to form an irreducible adduct. The irreducible adduct becomes the dominant product over time because of the irreversible nature of the pyridone-mediated reaction. These findings may explain the irreducible inhibition of H/K-ATPase by PPIs and their relatively slow biological turnover in vivo.
[Supplementary materials: available only at http://dx.doi.org/10.1254/jphs.13058FP]

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Glioblastoma is the most lethal brain cancer. In spite of intensive therapy, the prognosis of patients with glioblastoma is very poor. To discover novel therapeutic agents, we screened a combinatorial compound library containing 372 thiazolidinone compounds using U87MG human glioblastoma cells. (2E,5Z)-5-(2-hydroxybenzylidene)-2-((4-phenoxyphenyl)imino) thiazolidin-4-one (HBPT) was identified as the most potent anti-glioblastoma compound. HBPT inhibits U87MG human glioblastoma cell proliferation with an IC₅₀ of 20 μM, which is almost 5-fold more potent than temozolomide (a widely used drug for treating malignant glioma in the clinic). Mechanistic investigation demonstrated that HBPT is a novel microtubule-depolymerizing agent, which arrests cancer cells at the G2/M phase of the cell cycle and induces cell apoptosis. In the mouse U87MG xenograft model, HBPT elicits a robust tumor inhibitory effect. More importantly, no obvious toxicity was observed for HBPT therapy in animal experiments. These findings indicate that HBPT has the potential to be developed as a novel agent for the treatment of glioblastoma.
[Supplementary Tables: available only at http://dx.doi.org/10.1254/jphs.13064FP]

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We have previously demonstrated that riluzole has anxiolytic-like effects in rats, without affecting spontaneous alternation performance in the Y-maze test. However, the effects of riluzole on hippocampal synaptic plasticity were still unclear. In this study, we showed that bath application of riluzole did not impair long-term potentiation and long-term depression, whereas a benzodiazepine anxiolytic, diazepam, significantly impaired them. Furthermore, the acquisition of spatial memory in the Morris water maze test was impaired in diazepam-treated but not riluzole-treated rats. We thus provide further evidence for the potential usefulness of riluzole as an anxiolytic that does not cause amnesia.

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