The role of potassium channels in the proliferation and migration of endometrial adenocarcinoma HEC1-A cells.

BACKGROUND: Endometrial cancer is the most common gynecological cancer in developed countries. Potassium channels, which have many types, are suggested to play a major role in cancer progression. However, their role in endometrial cancer has not been fully investigated. We aimed to demonstrate whether the ATP-sensitive potassium channel blocker glibenclamide, voltage-sensitive potassium channel blocker 4-aminopyridine, non-selective (voltage-sensitive and calcium-activated) potassium channels blocker tetraethylammonium and potassium chloride (KCl) have any effect on the proliferation and migration of HEC1-A cells. METHODS AND RESULTS: Proliferation and migration were evaluated by real-time cell analysis (xCELLigence system) and wound healing assays, respectively. Proliferation was reduced by glibenclamide (0.1 and 0.2 mM, P < 0.05 and P < 0.01, respectively), 4-aminopyridine (10 and 20 mM, P < 0.001) and tetraethylammonium (10 and 20 mM, P < 0.01 and P < 0.001, respectively). However, KCl did not change the proliferation. Migration was reduced by glibenclamide (0.01, 0.1 and 0.2 mM, P < 0.001, P < 0.001 and P < 0.01, respectively) and 4-aminopyridine (10 and 20 mM, P < 0.05 and P < 0.01, respectively). Tetraethylammonium did not change migration. However, KCl reduced it (10, 25 and 50 mM, P < 0.05, P < 0.01 and P < 0.01, respectively). Both proliferation and migration were reduced by glibenclamide and 4-aminopyridine. However, tetraethylammonium only reduced proliferation and KCl only reduced migration. CONCLUSIONS: Potassium channels have an important role in HEC1-A cell proliferation and migration and potassium channel blockers needs to be further investigated for their therapeutic effect in endometrial cancer.

Effects of the Rho/Rho-Kinase Pathway on Perfusion Pressure in the Isolated-Perfused Rat Hind Limb Vascular Bed.

BACKGROUND: Rho/ROCK signaling has been demonstrated to be involved in the vascular reactivity of many arterial networks. However, RhoA expression and the contribution of Rho/ROCK pathway to the control of perfusion pressure have not been investigated in the rat hind limb vascular bed as a skeletal muscle vascular network. AIMS: To investigate the contribution of the Rho/ROCK pathway in the control of perfusion pressure in the isolated-perfused rat hind limb vascular bed. STUDY DESIGN: Animal experimentation. METHODS: Two Rho inhibitors (atorvastatin and C3 exoenzyme) and ROCK inhibitors (Y-27632 and fasudil) were tested on the phenylephrine-elevated perfusion pressure in the isolated-perfused rat hind limb vascular bed. Furthermore, we sought the expression of RhoA protein in the femoral, popliteal and saphenous arteries as well as quadriceps and gastrocnemius muscles by Western blotting. RESULTS: The ROCK inhibitors Y-27632 and fasudil (both 10-8 to 10-5 M) induced substantial vasodilatations. The maximum vasodilatations induced by Y-27632 and fasudil (both at 10-5 M) were 84.0 ± 6.9% and 76.9 ± 6.9%, respectively (P = .091). Y-27632 was not more potent than fasudil, as the EC50 values for Y-27632 and fasudil were 0.7 ± 2.1 µM and 2.5 ± 2.4 µM, respectively (P = .177). Atorvastatin (10-7 to 10-4 M) and C3 exoenzyme (3 × 10-8 M) also produced vasodilatation (maximum vasodilatation; 20.3 ± 1.7% and 13.7 ± 3.6%, respectively). The EC50 value for atorvastatin was 94.9 ± 1.2 µM. The western blot analysis showed that the femoral, saphenous, and popliteal arteries, as well as the gastrocnemius and quadriceps muscles, express RhoA protein. CONCLUSION: The Rho/ROCK pathway contributes significantly to the control of perfusion pressure in the rat hind limb vascular bed.

Effects of Vitamin D Analogs Alfacalcidol and Calcitriol on Cell Proliferation and Migration of HEC1A Endometrial Adenocarcinoma Cells.

Endometrial carcinoma is the most diagnosed among infiltrating tumor of the female genital tract. Vitamin D has antiproliferative and immunomodulatory properties besides its classical effect on calcium and phosphate. We aimed to demonstrate whether alfacalcidol and calcitriol have any effect on proliferation and migration. Endometrial adenocarcinoma HEC1A was used as a cancer cell line. The effect of alfacalcidol (1α-hydroxyvitamin D3) and calcitriol (1α,25-dihydroxyvitamin D3) on proliferation was demonstrated by real-time cell analysis device and migration was shown by a wound healing model. 10-11-10-9M alfacalcidol and calcitriol reduced both proliferation and migration. In contrast, the high concentration of alfacalcidol and calcitriol (10-8-10-6M) increased proliferation and migration. The proliferative effects of alfacalcidol (0-12 h) immediately started earlier than calcitriol (12-48 h). In contrast, the antiproliferative effects of calcitriol (12-24 h) have begun earlier than alfacalcidol (48-60 h). While the high concentrations of alfacalcidol and calcitriol increased the migration relatively earlier (12-24 h), low concentrations decreased the migration at late times (24-48 h). Lower concentrations of vitamin D prevent proliferation and migration in endometrial adenocarcinoma HEC1A cells. In contrast, high concentrations of vitamin D increase proliferation and migration.

The functional significance of the rho/rho-kinase pathway in human erythrocytes.

OBJECTIVE: Erythrocyte deformability, which can be influenced by various intracellular signaling mechanisms, such as nitric oxide, cAMP, cGMP, and protein kinases, is the most important physiological factor providing the blood flow in microcirculation. However, the functional significance of the Rho/Rho-kinase pathway, which contributes cell shape changes and the reorganization of the actin cytoskeleton, has yet to be explored in erythrocytes. Therefore, we examined the influence of several activators and inhibitors of Rho/Rho-kinase signaling on human erythrocyte deformability. MATERIALS AND METHODS: RhoA and ROCK-2 proteins were studied by western blotting. Influences of 2 Rho-kinase inhibitors, fasudil and Y-27632 (both 10-7 to 10-4 M), on erythrocyte deformability was determined by ektacytometer at various shear stresses (0-30 Pa) in the presence or absence of a known Rho activator, lysophosphatidic acid (LPA, 10-5 to 5x10-5 M, 1-15 min). RESULTS: LPA incubation reduced deformability with concomitant RhoA-GTP inhibition. Y-27632 and fasudil also decreased deformability, but had no effect on LPA-induced reduction of deformability. Rho inhibitor C3 had no effect on RhoA activation. Reduction in RhoA activation was induced by sub-hemolytic mechanical stress. CONCLUSION: Our findings may indicate that the Rho/Rho-kinase pathway could contribute to the regulation of deformability of human erythrocytes.

Nitric oxide does not downregulate Rho-kinase (ROCK-2) expression in rat coronary endothelial cells.

Rho kinase (ROCK) and nitric oxide (NO) are important targets in cardiovascular diseases. Therefore, we investigated the possible influence of NO on Rho kinase (ROCK-2 isoform) expressions in cultured rat coronary microvascular endothelial cells. The cells were isolated from Wistar rats on a Langendorff system, and were incubated overnight (approximately 16 h) with an NO generator, A-23187 (10 to 10 M), NO donors, such as sodium nitroprusside (10 to 10 M), glyceryl trinitrate (10 to 10 M), 2,2'-(hydroxynitrosohydrazono)bis-ethanimine (10 to 10 M), and NaNO2 (10 to 10 M) or a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methylester (2 x 10 M), or two ROCK inhibitors, (+)-(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10 M) and fasudil (10 M) in the absence or presence of thrombin (4 U/mL). ROCK-2 and endothelial NOS (eNOS) expressions were detected by Western blotting. Moreover, nitrite/nitrate levels were detected by Griess method in the presence of the ROCK inhibitors. The NO donors and the NO generator had no significant effects on ROCK-2 expression. Y-27632 and fasudil did not alter eNOS expression and NO production. Nitrite/nitrate levels were 4.4 +/- 0.32 microM in control and 4.0 +/- 0.93 microM and in Y-27632 group. These results demonstrate that prolong NO donation could not suppress the expression of ROCK-2 protein, and the ROCK inhibitor did not change e-NOS expression and NO production in the cultured rat coronary microvascular endothelial cells.

Rho-kinase inhibitor, Y-27632, has an antinociceptive effect in mice.

The possible antinociceptive effect of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), was investigated in mice by using the hot-plate and abdominal constriction response (writhing) tests. In addition, the expression of Rho-kinase protein (ROCK-2) was studied in the mouse brain and spinal cord by Western blotting. Male balb/c mice (n=8, for each group) were used in the experiment. Hot-plate latency and the number of writhes were recorded in control and in Y-27632-treated (1-5 mg/kg, i.p.) groups. Y-27632 (1 mg/kg) did not affect hot-plate latency; however, it considerably diminished the number of writhes, from 89+/-12 in control to 30+/-6 in the mice treated with 1 mg/kg Y-27632 (P=0.001). At a higher dose (5 mg/kg), Y-27632 prolonged the hot-plate latency from 8.7+/-1.0 s to 14.4+/-1.7 s (P=0.005) and decreased the number of writhes from 80+/-8 to 24+/-7 (P=0.002). Western blot analysis revealed that mouse spinal cord and brain homogenates expressed ROCK-2 protein. These results indicate that Rho-kinase may be involved in nociception and that its inhibitors, such as Y-27632, may represent a new type of antinociceptive drug.