Antiangiogenic effect of alpha-anordrin in vitro and in vivo.

AIM: To study the antiangiogenic effect of alpha-anordrin (alpha-Ano), a partial antagonist of estrogen receptor. METHODS: The in vivo inhibitory effect of alpha-Ano on angiogenesis was determined by microvascular density (MVD) in tumors and the chicken chorioallantoic membrane (CAM) model. The in vitro effects of alpha-Ano on proliferation, migration, and attachment of human umbilical vein endothelial cells (HUVEC) were assessed by trypan blue exclusion, wound-induced two-dimensional migration model, and their ability to adhere to type I collagen, respectively. The possible involvement of nitric oxide (NO) in alpha-Ano antiangiogenic effect was determined by measuring NO content using fluorescent assay. RESULTS: alpha-Ano significantly inhibited the MVD in Lewis lung carcinoma model and this effect was correlated with its inhibition of the tumor growth. alpha-Ano also showed an inhibitory effect on the angiogenesis of CAM with the inhibitory rate of 53% and such action of alpha-Ano could not be blocked by simultaneous administration of 17 beta-estrodiol, a typical agonist of estrogen receptor. In vitro studies showed that alpha-ANO obviously suppressed the proliferation and migration of HUVEC, but had no obvious effect on the attachment of HUVEC to the type I collagen. Moreover, alpha-Ano significantly reduced the level of NO released by HUVEC in a dose- and time-dependent manner. CONCLUSION: alpha-Ano possesses an antiangiogenic effect, and this effect is mediated, at least in part, by reducing the NO content and subsequently inhibiting the proliferation and migration of endothelial cells.

Evaluation of toxicity of some saponins on brine shrimp.

Toxicity of several types of saponins (1-11) against brine shrimp (Artemia salina) were evaluated. As a result, it was found that most tested compounds were not toxic to brine shrimp at high enough concentration. The most toxic saponin (1) to brine shrimp showed also cytotoxicity towards HL-60 tumor cell line using MTT assay. Brine shrimp model may thus be used as bench-top assay in finding cytotoxic components from saponin-containing fractions of plant extracts.

Nongenomic mechanisms of glucocorticoid inhibition of nicotine-induced calcium influx in PC12 cells: involvement of protein kinase C.

Nongenomic mechanisms of corticosterone (B) inhibition of nicotine (Nic)-induced calcium influx were investigated in PC12 cells. Corticosterone could rapidly inhibit the Ca2+ influx induced by Nic, and BSA-conjugated B had a similar inhibitory effect. The inhibition of Nic-induced Ca2+ influx by B could be mimicked by protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) and reversed by PKC inhibitors, chelerythrine chloride and Gö6976. When PC12 cells were pretreated with pertussis toxin, the inhibitory effect of B on Nic-induced Ca2+ influx was blocked. Both B and BSA-conjugated B could activate PKC activity, with the maximal responses at 10(-9) and 10(-7) M at 37 C, respectively. The dose-response curve was bell shaped. At 25 C, however, the dose-response curve considerably shifted to the right, and B was most potent at 10(-5) M. The time course showed that PKC activity was highest at 5 min of B's action. The results suggest that B might act via putative membrane receptors and inhibit the Ca2+ influx induced by Nic through the pertussis toxin-sensitive G protein-PKC pathway and that PKC plays an important role in the mechanisms of glucocorticoid nongenomic action.

N-Methyl-D-aspartate attenuates opioid receptor-mediated G protein activation and this process involves protein kinase C.

The effects of N-methyl-D-aspartate (NMDA) on opioid receptor-mediated G protein activation were explored in neuroblastoma X glioma hybrid (NG108-15) cells. Treatment of the cells with NMDA resulted in a remarkable attenuation of [35S]guanosine-5'-O-(3-thio)triphosphate binding stimulated by [D-Pen2,D-Pen5]-enkephalin (DPDPE), a delta-opioid receptor agonist. The effects of NMDA were dose and time dependent with an IC50 value of 5 nM and could be blocked by NMDA receptor antagonists. After NMDA treatment, the DPDPE dose-response curve shifted to the right (EC50 value increased approximately 7-fold, from 6 to 40 nM), and the maximal response induced by DPDPE was reduced by approximately 60%. The effects of NMDA were reversible, and the DPDPE response could recover within 60 min. The functional responses of delta-, mu-, and kappa-opioid receptors in primarily cultured neurons also were attenuated significantly by NMDA treatment. The inhibitory effects of NMDA on opioid receptor-mediated G protein activation could be blocked by coadministration of the protein kinase C (PKC) inhibitors or by elimination of the extracellular Ca2+. Correspondingly, NMDA treatment of NG108 cells significantly elevated cellular PKC activity and stimulated Gialpha2 phosphorylation. Transient transfection into NG108-15 cells of the wild-type Gialpha2 and a mutated Gialpha2 (Ser144Ala) resulted in a 2-fold increase in DPDPE-stimulated G protein activation. The DPDPE responses were greatly inhibited by NMDA treatment in the wild-type Gialpha2-transfected cells but much less affected in the mutant Gialpha2-transfected cells. In summary, NMDA attenuates opioid receptor/G protein coupling, and this process requires activation of PKC.

Nociceptin/orphanin FQ activates mitogen-activated protein kinase in Chinese hamster ovary cells expressing opioid receptor-like receptor.

The effect of nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for the newly identified opioid receptor-like (ORL1) receptor, on mitogen-activated protein kinase (MAPK) was investigated in Chinese hamster ovary cells stably expressing ORL1 receptor. N/OFQ rapidly stimulated phosphorylation and activity of MAPK (p42 and p44 isoforms) in a concentration-dependent manner. The p42 isoform was preferentially activated by N/OFQ. Maximal activation (5.4 +/- 1.2-fold of basal for p42 isoform) was achieved after a 1-min exposure of cells to 100 nM N/OFQ. The activation was blocked completely by pretreatment with pertussis toxin, but was not reversed by naloxone. U-73122, a phospholipase C-specific inhibitor, significantly inhibited phospholipase C activity, as well as MAPK activation stimulated by N/OFQ. Furthermore, N/OFQ-stimulated MAPK activation was suppressed by a protein kinase C-specific inhibitor, chelerythrine. The results demonstrate that N/OFQ can effectively stimulate MAPK by the activation of ORL1 receptor and pertussis toxin-sensitive G proteins, and that phospholipase C, as well as protein kinase C, is critically involved in these processes.

Nociceptin/orphanin FQ activates protein kinase C, and this effect is mediated through phospholipase C/Ca2+ pathway.

The effect of nociceptin/orphanin FQ (N/OFQ) on protein kinase C (PKC) was investigated in Chinese hamster ovary cells stably expressing opioid receptor-like (ORL1) receptor (CHO-ORL1 cells). N/OFQ significantly activated PKC in CHO-ORL1 cells with EC50 of 0.2 nM. This response was blocked by PKC inhibitors chelerythrine and Gö 6976, and by pretreatment of cells with pertussis toxin (PTX). The inhibition of PKC activation by N/OFQ was also achieved by use of Ca(2+)-chelators and phospholipase C (PLC) inhibitor U-73122. These results indicate that N/OFQ can effectively activate PKC via ORL1 receptor, and suggest the activation involve the PLC/Ca2+ system.

Modulation of protein kinase C and cAMP-dependent protein kinase by delta-opioid.

Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.

alpha-Anordrin-induced apoptosis of leukemia K562 cells is not prevented by cicloheximide.

AIM: To study effect of protein synthesis inhibitor cicloheximide (Cic) on the apoptosis induced by alpha-anordrin (Ano) in leukemia K562 cells. METHODS: Morphological changes were observed by fluorescent microscopy. DNA content was measured by flow cytometry. DNA fragmentation was analyzed by agarose gel electrophoresis. RESULTS: Exposure of K562 cells to Ano 50 mumol.L-1 for 24 h induced apoptotic cell death. Cic 1 mumol.L-1 did not abrogate or delay this effect. Indeed, Ano-induced apoptosis was augmented by Cic. Cic 100 mumol.L-1 itself stimulated 25% K562 cell apoptosis after 24-h culture. CONCLUSION: Ano-induced apoptosis was independent of de novo protein synthesis.

Induction of apoptosis in human leukemia K562 cells by alpha-anordrin.

AIM: To study antitumor action of alpha-anordrin (Ano). METHODS: Morphological assessment of apoptosis was performed with light microscope and electron microscope. Membrane integrity was determined by trypan blue exclusion method. Endonucleolysis was assessed by agarose gel electrophoresis and flow cytometric methods. RESULTS: Exposure of exponentially growing K562 cells to Ano 2.5-50 mumol.L-1 for 48 h resulted in growth arrest, Ano 50 mumol.L-1 inhibited the growth of K562 cells by 67%. Cells were mainly blocked to progress through S-phase and arrested at G1 phase. After treatment of K562 cells with Ano, marked morphological changes including condensed chromatin, nuclear fragmentation, and reduction in volume were observed. Agarose gel electrophoresis of DNA from cells treated with Ano for 24-48 h revealed "ladder" pattern, typical features of apoptosis, and near 70% of cells underwent apoptosis as determined by flow cytometry. The S-phase cells were more susceptible to apoptosis. Despite extensive cleavage of DNA and nuclear fragmentation, the cell membrane of Ano-treated cells remained intact, excluding trypan blue. Apoptotic cells were detected as early as 8 h after Ano (50 mumol.L-1) treatment. CONCLUSION: Ano induces apoptosis in K562 cells.