Caryophyllene sesquiterpenes from the marine-derived fungus Ascotricha sp. ZJ-M-5 by the one strain-many compounds strategy.

The marine-derived fungus Ascotricha sp. ZJ-M-5, which can produce cyclonerodiol analogues, a 3,4-seco lanostane triterpenoid, and diketopiperazines in an eutrophic medium, was subjected to a one strain-many compounds (OSMAC) analysis. It was found to produce three new caryophyllene derivatives (1-3) and the known 1,3,6-trihydroxy-8-methylxanthone (4) in an oligotrophic medium, Czapek Dox broth with or without Mg(2+). (+)-6-O-Demethylpestalotiopsin A (1) and (+)-6-O-demethylpestalotiopsin C (2), which have a five-membered hemiacetal structural moiety, showed growth inhibitory effects against HL-60 and K562 leukemia cell lines with the lowest GI50 value of 6.9 ± 0.4 µM. It can be concluded that modification of the culture media is still effective in the discovery of novel bioactive fungal secondary metabolites.

Isolation and cytotoxic activity of selaginellin derivatives and biflavonoids from Selaginella tamariscina.

Five selaginellin derivatives, including two new selaginellins termed selaginellins M (1) and N (2), and three previously identified compounds, selaginellin (3), selaginellin A (4), and selaginellin C (5), were isolated from the Selaginella tamariscina (Beauv.) Spring plant. In addition, four known biflavonoids, namely neocryptomerin ( 6), hinokiflavone (7), pulvinatabiflavone (8), and 7''- O-methylamentoflavone (9), were also isolated. The structures of new compounds 1 and 2 were elucidated by spectroscopic analysis. The cytotoxic activity of compounds 1- 9 was evaluated against a small panel of human cancer cell lines, including U251 (human glioma cells), HeLa (human cervical carcinoma cells), and MCF-7 (human breast cancer cells). The two new selaginellins, selaginellins M (1) and N (2), showed medium activity against the human cancer cell lines.

Activation of ATM-Chk2 by 16-dehydropregnenolone induces G1 phase arrest and apoptosis in HeLa cells.

The natural pregnane steroid 16-dehydropregnenolone (16-DHP) showed the growth inhibitory activity against human tumor cells; however, the mechanisms of actions of 16-DHP were not clarified. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to investigate the growth inhibitory effect of 16-DHP. Cell cycle distribution was analyzed using flow cytometry. Hoechst 33258 staining and DNA agarose gel electrophoresis were used to detect apoptosis. The levels of proteins were probed by Western blotting, and caspase activities were analyzed using Caspase Activity Kit. We found that 16-DHP inhibited the growth of human cervical carcinoma cells (HeLa cells) in a time- and dose-dependent manner. The growth inhibitory effect of 16-DHP was associated with G1 arrest mediated by ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2)-p53 signaling, as demonstrated by induction of the phosphorylations of ATM, Chk2, and p53 proteins. Followed by G1 arrest, 16-DHP-treated HeLa cells underwent caspase-dependent apoptosis. The inhibitors of caspase-3 and caspase-9 but not caspase-8 inhibitor blocked 16-DHP-induced apoptosis. Moreover, 16-DHP increased the level of Bax protein and the release of cytochrome c from mitochondria, but had no effect on the level of Bcl-2. These results suggested that 16-DHP inhibited the growth of HeLa cells via inducing ATM-Chk2-p53 activation-mediated G1 arrest and mitochondrial cell apoptosis.

Antiangiogenic activity of beta-eudesmol in vitro and in vivo.

Abnormal angiogenesis is implicated in various diseases including cancer and diabetic retinopathy. In this study, we examined the effect of beta-eudesmol, a sesquiterpenoid alcohol isolated from Atractylodes lancea rhizome, on angiogenesis in vitro and in vivo. Proliferation of porcine brain microvascular endothelial cells and human umbilical vein endothelial cells (HUVEC) was inhibited by beta-eudesmol (50-100 microM). It also inhibited the HUVEC migration stimulated by basic fibroblast growth factor (bFGF) and the tube formation by HUVEC in Matrigel. beta-eudesmol (100 microM) blocked the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 induced by bFGF or vascular endothelial growth factor. Furthermore, beta-eudesmol significantly inhibited angiogenesis in subcutaneously implanted Matrigel plugs in mice and in adjuvant-induced granuloma in mice. These results indicate that beta-eudesmol inhibits angiogenesis, at least in part, through the blockade of the ERK signaling pathway. We considered that beta-eudesmol may aid the development of drugs to treat angiogenic diseases.

Beta-eudesmol suppresses tumour growth through inhibition of tumour neovascularisation and tumour cell proliferation.

In the present study, we investigated the potential anti-angiogenic mechanism and anti-tumour activity of beta-eudesmol using in vitro and in vivo experimental models. Proliferation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor (VEGF, 30 ng/ml) and basic fibroblast growth factor (bFGF, 30 ng/ml) was significantly inhibited by beta-eudesmol (50-100 microM). Beta-eudesmol (100 microM) also blocked the phosphorylation of cAMP response element binding protein (CREB) induced by VEGF (30 ng/ml) in HUVEC. Beta-eudesmol (10-100 microM) inhibited proliferation of HeLa, SGC-7901, and BEL-7402 tumour cells in a time- and dose-dependent manner. Moreover, beta-eudesmol treatment (2.5-5 mg/kg) significantly inhibited growth of H(22) and S(180) mouse tumour in vivo. These results indicated that beta-eudesmol inhibited angiogenesis by suppressing CREB activation in growth factor signalling pathway. This is the first study to demonstrate that beta-eudesmol is an inhibitor of tumour growth.

Synthesis and antiproliferative in-vitro activity of natural flavans and related compounds.

The total synthesis of natural flavan racemates (+/-) 1, (+/-) 2 and natural flavones 3, 4 had thus been achieved. A straightforward synthetic procedure of flavans via the Pd-C catalyzed hydrogenation/hydrogenolysis of corresponding flavones was developed. Furthermore, the antiproliferative activities of racemic flavans (+/-) 1, (+/-) 2, flavones 3, 4, and five synthetic intermediates toward human SGC-7901, BEL-7402, HeLa, and HL-60 cell lines in vitro were evaluated by MTT assay, and the racemic flavans (+/-) 1 were found to have significant antiproliferative activity against all four cell lines.

Nicotinic enhancement of proliferation in bovine and porcine cerebral microvascular endothelial cells.

Nicotinic acetylcholine receptors are found in microvascular endothelial cells. To reveal the functional role in cerebral angiogenic processes, we studied the nicotinic modulation of proliferation activity in cultured bovine and porcine cerebral microvascular endothelial cells. The proliferation activity was determined by an increase in the number of cells present in culture dishes. When the bovine cerebral endothelial cells at different passages were cultured in the presence of nicotine (10 nM), the proliferation activities were significantly increased in the cells at passage 1 and passage 3, but not at passage 4. Reverse transcriptase-polymerase chain reaction studies demonstrated that the expression of mRNAs coding for alpha3 nicotinic receptor subunit was significantly reduced in the bovine cerebral endothelial cells at passage 4, compared with that at passage 1. The proliferation of porcine cerebral endothelial cells (passage 1) was enhanced by acetylcholine (10 nM-100 microM) in the presence of atropine, a muscarinic antagonist, and this enhancing effect was inhibited by hexamethonium (100 microM, a nicotinic antagonist). The stimulation by acetylcholine (1 microM, with atropine) or nicotine (10 nM) induced the phosphorylation of a mitogen-activated protein (MAP) kinase (extracellular-signal regulated kinase: ERK) in the serum-starved endothelial cells. In the presence of PD98059 (2 microM, a MAP kinase kinase inhibitor) and atropine, acetylcholine (1 microM) failed to enhance the proliferation of porcine cerebral endothelial cells. These results demonstrate that nicotinic stimulation promotes the proliferation of bovine and porcine cerebral microvascular endothelial cells, at least in part, through the MAP kinase activation.