A novel cromakalim analogue induces cell cycle arrest and apoptosis in human cervical carcinoma HeLa cells through the caspase- and mitochondria-dependent pathway.

In the present study, a series of seven synthetic croma-kalim analogues were prepared and evaluated for cytotoxic effect on human cervical carcinoma HeLa cells using WST-8 assay. A preliminary screening of these cromakalim analogues showed that 1-[(3S,4R)-4-(2-ethoxy-4-methyl-1H-pyrrol-1-yl)-3-hydroxy- 2,2-dimethylchroman-6-yl-3-phenylurea (compound 6) had the highest cytotoxic effect (IC50 of 138 µM) and significantly inhibited HeLa cell proliferation after 36 h. In an effort to understand the cytotoxic mechanism of compound 6, we examined its effect on apoptosis and cell cycle distribution. Our results showed that compound 6 induced marked changes in apoptotic morphology and significantly increased early apoptosis of HeLa cells after 48 h by using Annexin V-FITC/PI dual staining assay. This apoptotic induction was associated with an increase in Bax expression, a decrease in Bcl-2 expression, release of cytochrome c and subsequent activation of caspase-9 and -3, which indicated that compound 6 induced apoptosis via caspase- and mitochondria-dependent pathway. By DNA content analysis and [3H]thymidine incorporation assay, compound 6 was found to induce an increase in the number of cells in G1 phase, accompanied by a decrease in the S phase to prevent DNA synthesis after 24 h of treatment. In addition, compound 6 caused significant DNA damage, as detected by the alkaline comet assay. Taken together, the data demonstrate that compound 6 induces apoptosis in HeLa cells through caspase- and mitochondria-dependent pathway and this apoptotic effect is associated with cell cycle arrest and DNA damage. These findings provide further understanding of the molecular mechanisms of compound 6 in cervical cancer.

Assessment of ECG interval and restitution parameters in the canine model of short QT syndrome.

INTRODUCTION: The short QT syndrome (SQTS) is characterized by a short QT interval resulting from accelerated ventricular repolarization, and may be associated with ventricular fibrillation but not torsades de pointes. There are abundant data on the adverse effects of long QT, but knowledge of SQTS is sparse. The aim of this study was to examine whether analyses of several ECG biomarkers (QT, QTcB, QTcF, QTcV, QT(btb), and QT(RR1000)) and dynamic restitution of the beat-to-beat QT-TQ relationship (TQ(min), %QT/TQ ratio>1, QT/TQ ratio(max)) can be used to assess ECG changes in conscious dogs. METHODS: Sling-trained dogs were infused with escalating concentration of levcromakalim (0, 1.0, 3.3, and 10.0 microg/kg/min), pinacidil (0, 3.3, 10.0, and 33.3 microg/kg/min), and nicorandil (0, 0.03, 0.1, and 0.3 mg/kg/min), drugs known to shorten QT. The RR, QT, QTcB, QTcF, QTcV, QT(RR1000), and TQ were measured before and after each concentration of the QT shortening test compounds. RESULTS: Levcromakalim, pinacidil, and nicorandil but not vehicle significantly shortened RR, QT, QT(btb), QT(RR1000), and TQ but not QTc(B,F,V). The QT-RR cloud also shifted to the lower bounds of the normal QT-RR boundary by the test compounds. The percentage of beats with a QT/TQ ratio>1 was significantly increased in a dose response manner with levcromakalim and pinacidil and the lower TQ interval boundary (5th percentile) was decreased when compared to baseline or vehicle. DISCUSSION: QT(btb), QT(RR1000), and dynamic beat-to-beat measurements of restitution constitute clinically applicable ECG biomarkers for assessment of changes associated with arrhythmogenic risk of ventricular fibrillation due to QT abbreviation.

A simple method to investigate the inhibitory effects of drugs on gastric emptying in the mouse in vivo.

INTRODUCTION: The aim was to develop a simple method to study modification of gastric motility in the mouse in vivo. METHODS: Mice were fed a hydrated diet in which the fluid content of standard laboratory chow was increased by adding water. Gastric emptying was assessed at specified times following a 1-h treatment period with orally administered pharmacological agents. RESULTS: We demonstrated consistent and progressive gastric emptying over a 4-h period, stomach content being decreased from 7.52+/-0.90 at time zero to 2.80+/-0.25 mg/g body weight after 4 h. Results demonstrated typical effects of inhibitory agents (atropine and morphine) and showed inhibitory effects of three potassium channel opening agents, pinacidil, cromakalim, and SDZ PCO400: the residue remaining in the stomach was increased by 3.66+/-0.84, 6.56+/-1.35, and 5.68+/-1.33 mg/g body weight respectively 1 h after treatment with 10 mg/kg of these agents, compared to vehicle controls. DISCUSSION: The inhibitory activity observed correlated well with previous studies on the effects of potassium channel opening agents on mouse gastrointestinal motility in vivo and in vitro. The present model may thus be of value in the pharmacological investigation of gastrointestinal motility owing to cost and convenience advantages, together with the possibility of its application to studies using transgenic animals.

Cromakalim: embryonic effects and reduction of tolbutamide-induced dysmorphogenesis in vitro.

Cromakalim is a K(+) channel opener that causes smooth muscle relaxation by activating ATP-sensitive K(+) (K(ATP)) channels and producing membrane hyperpolarization. Cromakalim counteracts sulfonylurea-induced K(ATP) channel inhibition in adult cells, but little is known regarding its embryonic effects, alone or in combination with sulfonylureas. K(ATP) channels have been demonstrated in the embryo, but their role in normal and abnormal development is unknown. Early-somite mouse embryos were exposed for 24 hr in vitro to cromakalim at concentrations of 0 (Cntl), 1, 10, 100, 200, or 500 microM in 0.125% DMSO. Embryos were also exposed for 24 hr in vitro to a dysmorphogenic tolbutamide concentration (110 microg/ml) combined with a subdysmorphogenic concentration of cromakalim (1 microM). Embryos were evaluated for somite number, heart rate, malformations, and embryonic and yolk sac protein content. Embryos exposed to 1 microM cromakalim were similar to controls. Cromakalim exposure increased malformation rates at concentrations >/=200 microM, decreased heart rates at >/=10 microM, and decreased somite and protein values at 500 microM. Defects involved cranial neural tube, optic vesicle, heart, and somites. A malformation rate of 59% in embryos exposed to 110 microg/ml tolbutamide was reduced to 13% by adding 1 microM cromakalim to the culture medium. Heart rate, somite number, and protein values were also improved by combined exposure to cromakalim and tolbutamide compared with exposure to tolbutamide alone. These results support previous findings with diazoxide (K(+) channel opener) and chlorpropamide (sulfonylurea) and further suggest a potential role for K(ATP) channel effects in sulfonylurea-induced dysmorphogenesis.