Noriko Yoshikawa

Antitumour activity of cordycepin in mice.

1. The antitumour effect of orally administered cordycepin, a component isolated from water extracts of Cordyceps sinensis, was examined in mice inoculated with B16 melanoma (B16‐BL6) cells.

Cordycepin (3′-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3β activation and cyclin D1 suppression

Cordyceps sinensis, a parasitic fungus on the larvae of Lepidoptera, has been used as a traditional Chinese medicine. We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells was inhibited by cordycepin (3′-deoxyadenosine), an active ingredient of C. sinensis, and its effect was antagonized by MRS1191, a selective adenosine A3 receptor antagonist. In this study, the radioligand binding assay using [125I]-AB-MECA (a selective adenosine A3 receptor agonist) has shown that B16-BL6 cells express adenosine A3 receptors and that cordycepin binds to these receptors. We also confirmed the involvement of adenosine A3 receptors in the action of cordycepin using MRS1523 and MRS1220, specific adenosine A3 receptor antagonists. Next, indirubin, a glycogen synthase kinase-3β (GSK-3β) inhibitor, antagonized the growth suppression induced by cordycepin. Furthermore, the level of cyclin D1 protein in B16-BL6 cells was decreased by cordycepin using Western blot analysis. In conclusion, this study demonstrated that cordycepin inhibits the proliferation of B16-BL6 cells by stimulating adenosine A3 receptors followed by the Wnt signaling pathway, including GSK-3β activation and cyclin D1 inhibition.

Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model

We characterized the metastatic ability and mortality of four different mouse melanoma cell lines, B16-F0, -F1, -F10 and -BL6. B16-F0 is the parent cell line. B16-F1 was obtained by a one-time selective procedure and B16-F10 by a ten-time selective procedure using Fidlers method. B16-BL6 derived from B16-F10 has much more invasive activity than B16-F10. To investigate the difference in mortal malignancy among B16-F0, -F1, -F10 and -BL6, we examined the survival time of syngeneic C57BL/6Cr mice intravenously inoculated with these cells. As a control, we used the C57BL/6J-embryo mouse fibroblast-like semi-normal cell line. The ability to form lung metastatic nodules in mice gradually increased in the order: B16-F0, -F1, and -F10 (=-BL6). C57BL/6J-embryo cell (1 x 10(5)/mouse)-inoculated mice survived for over 46 days. B16-F0, -F1, -F10 and -BL6 (1 x 10(5)/mouse)-inoculated mice survived 31.4+/-4.4 (7), 25.7+/-2.8 (7), 23.6+/-1.5 (7) and 25.3+/-2.3 (7) days [mean+/-S.D. (number of mice)], respectively. According to the Mann-Whitney test, the B16-F0 inoculated group versus -F1 inoculated group (P<0.05), -F0 inoculated group versus -BL6 inoculated group (P<0.05), and -F0 inoculated group versus -F10 inoculated group (P<0.01) were significantly different, but the B16-F1 group versus -F10 group, -F1 group versus -BL6 group, and -F10 group versus -BL6 group were not. These results suggest that mortal malignancy is not necessarily correlated with lung-colonizing potential and even only one-time selected B16-F0 mouse melanoma cells are useful as an experimental metastatic model in vivo.

CORDYCEPIN AND CORDYCEPS SINENSIS REDUCE THE GROWTH OF HUMAN PROMYELOCYTIC LEUKAEMIA CELLS THROUGH THE Wnt SIGNALLING PATHWAY

1 The mechanism of the antitumour effect of cordycepin (3¢‐deoxyadenosine) and water extracts of Cordyceps sinensis (WECS) was examined using human promyelocytic leukaemia (HL60) cells. 2 The growth of HL60 cells was inhibited by cordycepin (60 mmol/L) and WECS (30 mg/mL) and the inhibitory effects of these agents were significantly antagonized by MRS1191, a selective adenosine A3 receptor antagonist. 3 The growth inhibitory effects of cordycepin and WECS on HL60 cells were also significantly antagonized by indirubin, a glycogen synthase kinase (GSK)‐3b inhibitor. 4 These findings suggest that cordycepin and WECS display an antitumour effect by stimulating adenosine A3 receptors, followed by activation of GSK‐3b in the Wnt signalling pathway.

Disturbance of vasodilation via protease-activated receptor 2 in SHRSP.Z-Leprfa/IzmDmcr rats with metabolic syndrome

Protease-activated receptor-2 (PAR2) activation causes vascular inflammation and vasodilation, but its role in metabolic syndrome (MetS) remains uncertain. Therefore, we examined whether the PAR2-induced vasodilation of SHRSP.Z-Lepr(fa)/IzmDmcr rats (SHRSP.ZF) is impaired and if so, whether administering telmisartan is protective. PAR2-activating peptide, 2-furoyl-LIGRLO-amide (2fly), relaxed the isolated superior and first-order branches of mesenteric arteries (MAs) from Wistar-Kyoto rats (WKY) and SHRSP.ZF. Superior-MA relaxation by 2fly was less in SHRSP.ZF than in WKY. Relaxation of first-order MAs by 2fly was the same in SHRSP.ZF and WKY. NO synthase inhibitor partially reduced 2fly-induced relaxation of superior and first-order MAs in SHRSP.ZF and WKY; inhibition of relaxation was proportionately larger in SHRSP.ZF. In SHRSP.ZF, nitroprusside-induced relaxation and the expression of soluble guanylyl cyclase decreased. In SHRSP.ZF, telmisartan reversed these abnormalities, and decreased blood pressure and serum levels of thiobarbituric acid reactive substances, an index of oxidative stress. Vasodilation via PAR2 activation was preserved in small-caliber MAs, in contrast to large-caliber MAs, even when MetS reduced NO-dependent relaxation mechanisms. NO and non-NO relaxing factor(s) contributed to PAR2-mediated relaxation in MAs, and the balance between factors may be altered to preserve vasodilation in MetS. Telmisartan prevented vascular dysfunction in MetS by protecting arteries against oxidative stress.

Effect of PKC412, a selective inhibitor of protein kinase C, on lung metastasis in mice injected with B16 melanoma cells

PKC412, a selective inhibitor of protein kinase C (PKC), is currently in clinical trials as an anti-tumor drug. In the present study, we investigated the anti-metastatic effect of PKC412 using an experimental metastatic mouse model intravenously injected with melanoma cells. One-hour exposure to various concentrations of PKC412 (0.5, 5 and 50 microM) dose-dependently reduced the lung-metastatic potential of highly metastatic B16-F10 and -BL6 mouse melanoma cells in syngeneic mice. Following the exposure, PKC activities in B16-F10 and -BL6 cells were significantly decreased, but growth curves were not influenced. To elucidate the mechanism of the anti-metastatic effect of PKC412, we examined the activity to invade the extracellular matrix and the platelet-aggregating activity of the melanoma cells incubated with PKC412 (0.5, 5 and 50 microM) for 1 hour. PKC412 significantly reduced both the invasive and platelet-aggregating activities. These results suggest that PKC412 shows an anti-metastatic function through the inhibition of the invasive and/or platelet-aggregating activities of melanoma cells. PKC412 is potentially a promising candidate for an anti-metastatic agent.

Intracellular Metabolism of α,β-Unsaturated Carbonyl Compounds, Acrolein, Crotonaldehyde and Methyl Vinyl Ketone, Active Toxicants in Cigarette Smoke: Participation of Glutathione Conjugation Ability and Aldehyde-Ketone Sensitive Reductase Activity.

The major toxicants in cigarette smoke, α,β-unsaturated aldehydes, such as acrolein (ACR) and crotonaldehyde (CA), and α,β-unsaturated ketone, methyl vinyl ketone (MVK), are known to form Michael-type adducts with glutathione (GSH) and consequently cause intracellular GSH depletion, which is involved in cigarette smoke-induced cytotoxicity. We have previously clarified that exposure to cigarette smoke extract (CSE) of a mouse melanoma cell culture medium causes rapid reduction of intracellular GSH levels, and that the GSH-MVK adduct can be detected by LC/MS analysis while the GSH-CA adduct is hardly detected. In the present study, to clarify why the GSH-CA adduct is difficult to detect in the cell medium, we conducted detailed investigation of the structures of the reaction products of ACR, CA, MVK and CSE in the GSH solution or the cell culture medium. The mass spectra indicated that in the presence of the cells, the GSH-CA and GSH-ACR adducts were almost not detected while their corresponding alcohols were detected. On the other hand, both the GSH-MVK adducts and their reduced products were detected. In the absence of the cells, the reaction of GSH with all α,β-unsaturated carbonyls produced only their corresponding adducts. These results show that the GSH adducts of α,β-unsaturated aldehydes, CA and ACR, are quickly reduced by certain intracellular carbonyl reductase(s) and excreted from the cells, unlike the GSH adduct of α,β-unsaturated ketone, MVK. Such a difference in reactivity to the carbonyl reductase might be related to differences in the cytotoxicity of α,β-unsaturated aldehydes and ketones.

Effect of Cordyceps sinensis on TIMP-1 secretion from mouse melanoma cell

Cordyceps sinensis is a Chinese medicinal fungus traditionally used in cancer treatments. In a previous study, we investigated the antimetastatic activity of Cordyceps sinensis (WECS) extract using liver metastatic model mice injected with B16-F0 mouse melanoma cells into the spleen. WECS reduced the number of metastatic nodules of B16-F0 cells in the liver of C57BL/6 mice, and significantly prolonged survival of the mice. Furthermore, we examined the effects of WECS on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. WECS was shown to significantly reduce HGF-accelerated B16-F0 cell invasion. In the present study, we investigated the effect of WECS on Tissue Inhibitor of Metalloproteinase (TIMP)-1 secretion from B16-F0 cells in order to identify clues to the mechanism underlying the anti-invasive action of WECS. As a result, WECS significantly increased the secretion of TIMP-1 from B16-F0 cells. Moreover, we investigated the effect of cordycepin (3′-deoxyadenosine), a component of WECS, on TIMP-1 secretion from B16-F0 cells to potentially identify the pharmacologically active ingredient in WECS extract. Cordycepin was shown to significantly accelerate the release of TIMP-1 from cells. These findings suggest that WECS exerts anti-invasive activity, in part by increasing TIMP-1 secretion from melanoma cells, and that cordycepin potentially functions as the effective component.

Enhanced Nitric Oxide Synthase Activation via Protease-Activated Receptor 2 Is Involved in the Preserved Vasodilation in Aortas from Metabolic Syndrome Rats

Endothelium-dependent vasodilation via protease-activated receptor 2 (PAR2) is preserved in mesenteric arteries from SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF) with metabolic syndrome even though nitric oxide (NO)-mediated vasodilation is attenuated. Therefore, we examined the PAR2 mechanisms underlying metabolic syndrome-resistant vasodilation in SHRSP.ZF aortas with ageing. In isolated aortas, the PAR2 agonist 2-furoyl-LIGRLO-amide (2fly) caused vasodilation that was sustained in male SHRSP.ZF until 18 weeks of age, but was attenuated afterwards compared with age-matched Wistar-Kyoto rats (controls) at 23 weeks. In contrast, acetylcholine-induced vasodilation was impaired in SHRSP.ZF already at 18 weeks of age. Treatments of aortas with inhibitors of NO synthase and soluble guanylate cyclase abolished the sustained 2fly- and residual acetylcholine-induced vasodilation in SHRSP.ZF at 18 weeks of age. In the aortas of SHRSP.ZF, 8-bromo-cGMP-induced vasodilation, NO production and cGMP accumulation elicited by 2fly were not different from in the controls. PAR2 agonist increased phospho-Ser1177-eNOS protein content only in SHRSP.ZF aortas. These results indicate that vasodilation mediated by PAR2 is sustained even though NO-dependent relaxation is attenuated with ageing/exposure to metabolic disorders in large-caliber arteries from SHRSP.ZF. PAR2 stimulation of NO production via an additional pathway that targets phosphorylation of Ser1177-eNOS suggests a regulatory mechanism for sustaining agonist-mediated vasodilation in metabolic syndrome.

Age-related changes to vascular protease-activated receptor 2 in metabolic syndrome: A relationship between oxidative stress, receptor expression and endothelium-dependent vasodilation.

Protease-activated receptor 2 (PAR2) is expressed in vascular endothelium. Nitric oxide (NO) - cyclic GMP-mediated vasodilation in response to 2-furoyl-LIGRLO-amide (2fLIGRLO), a PAR2-activating peptide, is impaired in aortas from aged SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome. Here we investigated mechanisms linking PAR2s vascular effects to phenotypic characteristics of male SHRSP.ZF rats at 10, 20, and 30 weeks of age. We found vasodilation responses to either 2fLIGRLO or enzyme-mediated PAR2 activation by trypsin were sustained until 20 weeks and lessened at 30 weeks. PAR2 protein and mRNA levels were lower in aortas at 30 weeks than at 10 and 20 weeks. PAR2-mediated responses positively correlated with PAR2 protein and mRNA levels. Decreased cGMP accumulation in the presence of 2fLIGRLO paralleled the decreased relaxations elicited by nitroprusside and the cGMP analog 8-pCPT-cGMP, and the less soluble guanylyl cyclase protein at 30 weeks. 2fLIGRLO-induced relaxation was negatively correlated with serum thiobarbituric acid reactive substances, an index of oxidative stress, which increased with age. Forward stepwise data regression supported a model of age-related decreases in PAR2 function resulting from decreased PAR2 mRNA and increased oxidative stress. We conclude that decreased responsiveness of aortic smooth muscle to NO and downregulation of receptor expression impair PAR2 functions at later stages of metabolic syndrome in SHRSP.ZF rats.