Effects of the hypoxia-inducible factor-1 inhibitor echinomycin on vascular endothelial growth factor production and apoptosis in human ectopic endometriotic stromal cells.

Recent evidence points to a possible role for hypoxia-inducible factor (HIF)-1 in the pathogenesis and development of endometriosis. The objectives of this study were to investigate the critical role of HIF-1 in endometriosis and the effect of the HIF-1 inhibitor echinomycin on human ectopic endometriotic stromal cells (eESCs). Ectopic endometriotic tissues were obtained from 20 patients, who received an operation for ovarian endometriomas. We examined vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) production, HIF-1 expression, cell proliferation and apoptosis of eESCs. Cobalt chloride (CoCl2) significantly induced expression of HIF-1α protein and VEGF production in a time-dependent manner in eESCs, but reduced SDF-1 production. VEGF production was significantly suppressed by treatment of 100 nM echinomycin without causing cell toxicity, but 0.1-10 nM echinomycin or 100 nM progestin had no significant effect. SDF-1 production was not affected by echinomycin treatment at any dose. Echinomycin inhibited cell proliferation and induced apoptotic cell death of the eESCs, and significantly inhibited expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Echinomycin inhibits VEGF production and induces apoptosis of eESCs by suppression of Bcl-2 and Bcl-xL. These findings suggest the unique therapeutic potential for echinomycin as an inhibitor of HIF-1 activation for endometriosis treatment.

Lipid-soluble fraction of Shakuyaku-kanzo-to inhibits myometrial contraction in pregnant women.

AIM: Shakuyaku-kanzo-to, a Kampo medicine composed equally of shakuyaku and kanzo, is an antispasmodic drug that can inhibit contraction of uterine smooth muscles in pregnant women and rats. We aimed to test the inhibitory effects of water- and lipid-soluble extracts of shakuyaku-kanzo-to, shakuyaku, and kanzo in order to identify the fraction responsible for inhibiting uterine smooth muscle contraction in pregnancy. MATERIAL AND METHODS: Myometrial tissues were obtained from pregnant women and rats. The water- and lipid-soluble fractions of shakuyaku-kanzo-to, shakuyaku, and kanzo were obtained using the method of Bligh and Dyer. Lipid-soluble fractions were also partially purified using thin-layer chromatography (TLC) with a chloroform : methanol : water (65:25:4 by volume) solvent system to yield four TLC fractions. The effect of each fraction on oxytocin-induced myometrial contraction was examined in vitro. RESULTS: Lipid-soluble fractions obtained from shakuyaku-kanzo-to and kanzo inhibited myometrial contraction; water-soluble fractions had no effect. Of the four TLC fractions, the inhibitory effect was greatest with TLC fraction 1 (0.75 < Rf value ≤ 1.0). Neither the water-soluble nor the lipid-soluble fraction from shakuyaku inhibited myometrial contraction. CONCLUSIONS: These results suggest that lipid-soluble substances with low polarity derived from kanzo are responsible for the inhibitory effect of shakuyaku-kanzo-to on myometrial contraction.

Two-step inhibitory effect of kanzo on oxytocin-induced and prostaglandin F2α-induced uterine myometrial contractions.

We previously reported that shakuyaku-kanzo-to, a kampo medicine consisting of shakuyaku and kanzo, has an inhibitory effect on myometrial contractions in pregnant women. In this study, we evaluated the effects of kanzo, glycyrrhizin (a major component of kanzo), glycyrrhetinic acid (GA; a major metabolite of glycyrrhizin), shakuyaku, and paeoniflorin (a major component of shakuyaku) on agonist-induced contractions of the uterus of pregnant humans and rats. We prepared myometrial strips from the uterus of pregnant humans and rats and induced contractions with oxytocin (50 µU/mL) or prostaglandin F2α (PGF2α) (10(-7) or 10(-6) M). Kanzo (250 µg/mL) and GA (5 × 10(-6) M) inhibited the oxytocin-induced and PGF2α-induced contractions in pregnant human and rat myometrium, but shakuyaku (250 µg/mL), paeoniflorin (10(-5) M), and glycyrrhizin (10(-5) M) did not inhibit contractions in either. Interestingly, kanzo and GA showed an inhibitory effect after temporarily enhancing the PGF2α-induced contractions in the rat myometrium, but not in the human myometrium. These results suggest that kanzo has at least a two-step inhibitory effect on the myometrial contractions that originate from the kanzo itself and a metabolite of glycyrrhizin in kanzo. Furthermore, kanzo was found to be safe for inhibiting PGF2α-induced contractions in humans because it did not temporarily enhance PGF2α-induced contractions.

Shakuyaku-kanzo-to inhibits smooth muscle contractions of human pregnant uterine tissue in vitro.

AIM: Shakuyaku-kanzo-to (SK) is a herbal medicine and is known to possess an antispasmodic effect on skeletal muscle and intestinal smooth muscle. However, it is unclear whether SK is effective in antagonizing uterine smooth muscle contractions. Herein, we investigated the effects of SK on smooth muscle contractions of human pregnant uterine samples. MATERIAL AND METHODS: We prepared myometrial strips from uterine tissues of pregnant women who underwent cesarean section for obstetrical indications, and examined the inhibitory effects of SK and its components, shakuyaku (S) and kanzo (K), on agonist-induced and spontaneous contractions in vitro. Oxytocin, prostaglandinF(2α) , and high KCl were utilized as agonists in this study. RESULTS: SK inhibited agonist-induced and spontaneous contractions in a dose-dependent manner. Inhibition of SK on oxytocin-induced contractions occurred at a concentration of 100 µg/mL and reached maximum effect at a concentration of more than 1000 µg/mL. The half max inhibitory concentration of SK was approximately 440 µg/mL in oxytocin-induced contractions. SK at 1000 µg/mL completely inhibited the oxytocin- and prostaglandinF(2α)-induced contractions but not the high KCl-induced contractions. The inhibitory effects on agonist-induced contractions of K, but not S, matched those of SK. CONCLUSION: These results suggest that the inhibitory effect of SK on smooth muscle contractions is due to K. The mechanism of the inhibitory effects of SK on oxytocin- and prostaglandinF(2α) -induced contractions may differ from that on KCl-induced contractions.

Apoptosis in cerebrum of macular mutant mouse.

Neuronal cell death in the brain of macular mutant mouse, a model of copper metabolism abnormality, has features of both apoptosis and necrosis. Apoptotic cells were morphologically identified by the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) method and electron microscopy. Numerous TUNEL-positive cells were identified in the cerebral cortex, hippocampus and thalamus of the hemizygotes after postnatal day 11. Ultramicroscopic studies confirmed that a number of cells had apoptotic features characterized by condensation and segregation of the nuclei. Furthermore, genomic DNA gel electrophoresis revealed a laddering pattern in the hemizygous brain. Starvation, which produced a low body weight in normal mice similar to that seen in the hemizygotes, did not result in an increase of TUNEL-positive cells. We also found that there was no increase of apoptotic cells in the brains of heterozygotes and copper-supplemented hemizygotes. Immunocytochemical analysis revealed that the distribution of copper/zinc superoxide dismutase-containing cells differed from that of TUNEL-positive cells. These findings suggest that copper deficiency is a key factor triggering apoptosis in the brain of macular mutant mouse through a mechanism different from suppression of antioxidant action of the dismutase. The improved survival period of the copper-supplemented hemizygotes may be attributed, in part, to inhibition of excessive neuronal apoptosis identified in the late stage of the disease.