Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells

We investigated the effects of genistein and genistin on proliferation and apoptosis of human ovarian SK-OV-3 cells and explored the mechanism for these effects. SK-OV-3 cells were treated with genistein and genistin at various concentrations (ranging from 1 to 100 muM) either alone or in combination for 24 and 48 h. Cell proliferation was estimated using an MTT assay, and cell cycle arrest was evaluated using FACS. Caspase-3 activity and annexin-based cell cycle analysis were used as measures of apoptosis. In addition, genistein- and genistin-induced cytotoxicity was determined by measuring release of LDH. Genistein treatment for 24 or 48 h substantially inhibited SK-OV-3 cell proliferation in a dose-dependent manner, and genistin treatment for 48 h also inhibited cell proliferation. Genistein caused cell cycle arrest at G2/M phase in dose- and time-dependent manner, and genistin caused cell cycle arrest not only at G2/M phase but also at G1 phase. Genistein markedly induced apoptosis and significantly increased LDH release, whereas genistin did not affect LDH release. Moreover, exposure to both genistein and genistin in combination for 48 h induced apoptosis without increasing LDH release. Genistein and genistin inhibit cell proliferation by disrupting the cell cycle, which is strongly associated with the arrest induction of either G1 or G2/M phase and may induce apoptosis. Based on our findings, we speculate that both genistein and genistin may prove useful as anticancer drugs and that the combination of genistein and genistin may have further anticancer activity.     

ACAT inhibition of alkamides identified in the fruits of Piper nigrum

In this study, via a bioactivity-guided fractionation of MeOH extracts of the fruits of Piper nigrum, alkamide (5) and five previously-identified alkamides were isolated. Their structures were elucidated via spectroscopic analysis ((1)H, (13)C NMR and ESI-MS), as follows: retrofractamide A (1), pipercide (2), piperchabamide D (3), pellitorin (4), dehydroretrofractamide C (5) and dehydropipernonaline (6). The IC(50) values determined for the compounds were 24.5 (1), 3.7 (2), 13.5 (3), 40.5 (4), 60 (5) and 90 microM (6), according to the results of an ACAT enzyme assay system using rat liver microsomes. These compounds all inhibited cholesterol esterification in HepG2 cells.     

Genetic organization of the biosynthetic gene cluster for the indolocarbazole K-252a in <Emphasis Type="Italic">Nonomuraea longicatena</Emphasis> JCM 11136

Indolocarbazole metabolite K-252a is a natural product that was previously reported as a potent protein kinase C inhibitor with in vitro and in vivo potency. From a biosynthetic viewpoint, this compound possesses structurally interesting features such as an unusual furanosyl sugar moiety, which are absent in the well-studied staurosporine and rebeccamycin. A cosmid library from genomic DNA of Nonomuraea longicatena JCM 11136 was constructed and screened for the presence of genes to be involved in the biosynthesis of indolocarbazole K-252a. Using as a probe an internal fragment of vioB, a Chromobacterium violaceum gene encoding a multifunctional enzyme that catalyzes tryptophan decarboxylation and condensation reaction in violacein biosynthesis, we isolated a DNA region that directed the biosynthesis of K-252a when introduced into the heterologous expression host Streptomyces albus. Sequence analysis of 45 kb revealed genes for indolocarbazole core formation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes. The cloned genes should help to elucidate the molecular basis for indolocarbazole biosynthesis and generate new indolocarbazole analogues by genetic engineering.     

Hypoglycemic effects of exopolysaccharides produced by mycelial cultures of two different mushrooms Tremella fuciformis and Phellinus baumii in ob/ob mice

The anti-diabetic activities of the exopolysaccharides (EPS) produced by submerged mycelial culture of two different mushrooms, Tremella fuciformis and Phellinus baumii, in ob/ob mice were investigated. All the animals were randomly divided into three groups with seven animals in each group: The control group received 0.9% NaCl solution; the diabetic groups were treated with EPS from T. fuciformis (Tf EPS) and P. baumii (Pb EPS) at the level of 200 mg/kg body weight using an oral zoned daily for 52 days. The plasma glucose levels in the EPS-fed mice were substantially reduced by about 52% (Tf EPS) and 32% (Pb EPS), respectively, as compared to control mice. The results of oral glucose tolerance test (OGTT) revealed that both EPS-fed groups significantly increased the glucose disposal after 52 days of EPS treatments. Furthermore, higher food efficiency ratios and reduced blood triglyceride levels were observed in the EPS-treated groups. Because peroxisome proliferator-activated receptor gamma (PPAR-γ) is indeed a key regulator of insulin action, we investigated the expression pattern of adipose tissue PPAR-γ messenger RNA (mRNA) and plasma levels of PPAR-γ. It was revealed that PPAR-γ was significantly activated in response to EPS treatments. The results suggested that both EPS exhibited considerable hypoglycemic effect and improved insulin sensitivity possibly through regulating PPAR-γ-mediated lipid metabolism. Our results indicated that two mushroom-derived EPS might be developed as potential oral hypoglycemic agents or functional foods for the management of non-insulin-dependent diabetes mellitus.     

Abdominal obesity in BTBR male mice is associated with peripheral but not hepatic insulin resistance

Insulin resistance is a common feature of obesity. BTBR mice have more fat mass than most other inbred mouse strains. On a chow diet, BTBR mice have elevated insulin levels relative to the C57BL/6J (B6) strain. Male F1 progeny of a B6 x BTBR cross are insulin resistant. Previously, we reported insulin resistance in isolated muscle and in isolated adipocytes in this strain. Whereas the muscle insulin resistance was observed only in male F1 mice, adipocyte insulin resistance was also present in male BTBR mice. We examined in vivo mechanisms of insulin resistance with the hyperinsulinemic euglycemic clamp technique. At 10 wk of age, BTBR and F1 mice had a >30% reduction in whole body glucose disposal primarily due to insulin resistance in heart, soleus muscle, and adipose tissue. The increased adipose tissue mass and decreased muscle mass in BTBR and F1 mice were negatively and positively correlated with whole body glucose disposal, respectively. Genes involved in focal adhesion, actin cytoskeleton, and inflammation were more highly expressed in BTBR and F1 than in B6 adipose tissue. The BTBR and F1 mice have higher levels of testosterone, which may be related to the pathological changes in adipose tissue that lead to systemic insulin resistance. Despite profound peripheral insulin resistance, BTBR and F1 mice retained hepatic insulin sensitivity. These studies reveal a genetic difference in body composition that correlates with large differences in peripheral insulin sensitivity.     

Dampening of IFN-gamma-inducible gene expression in human choriocarcinoma cells is due to phosphatase-mediated inhibition of the JAK/STAT-1 pathway

Trophoblast cells (TBCs) form the blastocyst-derived component of the placenta and play essential roles in fetal maintenance. The proinflammatory cytokine IFN-gamma plays a central role in activating cellular immunity, controlling cell proliferation, and inducing apoptosis. IFN-gamma is secreted by uterine NK cells in the placenta during pregnancy and in mice is required for proper formation of the decidual layer and remodeling of the uterine vasculature. Despite the presence of IFN-gamma in the placenta, TBCs do not express either MHC class Ia or class II Ags, and are resistant to IFN-gamma-mediated apoptosis. In this study, we demonstrate that IFN-gamma-induced expression of multiple genes is significantly reduced in human trophoblast-derived choriocarcinoma cells relative to HeLa epithelial or fibroblast cells. These results prompted us to investigate the integrity of the JAK/STAT-1 pathway in these cells. Choriocarcinoma cells and HeLa cells express comparable levels of the IFN-gamma receptor. However, tyrosine phosphorylation of JAK-2 is compromised in IFN-gamma-treated choriocarcinoma cells. Moreover, phosphorylation of STAT-1 at tyrosine 701 is substantially reduced in both IFN-gamma-treated human choriocarcinoma and primary TBCs compared with HeLa cells or primary foreskin fibroblasts. A corresponding reduction of both IFN regulatory factor 1 mRNA and protein expression was observed in IFN-gamma-treated TBCs. Treatment of choriocarcinoma cells with the tyrosine phosphatase inhibitor pervanadate significantly enhanced IFN-gamma-inducible JAK and STAT-1 tyrosine phosphorylation and select IFN-gamma-inducible gene expression. We propose that phosphatase-mediated suppression of IFN-gamma signaling in TBCs contributes to fetal maintenance by inhibiting expression of genes that could be detrimental to successful pregnancy.