Luteolin induces apoptotic cell death through AIF nuclear translocation mediated by activation of ERK and p38 in human breast cancer cell lines

The flavonoid, luteolin, has been shown to have anticancer activity in various cancer cells; however, the precise molecular mechanism of its action is not completely understood, and studies were conducted to find out how it induces apoptosis in breast cancer cells. Luteolin induced a reduction of viability in a dose- and time-dependent manner. The pro-apoptotic effect of luteolin was demonstrated by cell cycle measurement and Hoechst 3325 staining. Western blot analysis showed that luteolin activates ERK (extracellular-signal-regulated kinase) and p38. Pharmacological inhibition or knockdown of ERK and p38 protected against luteolin-induced cell death; however, the caspase-3-specific inhibitor had no effect. Immunocytochemical examination indicated that luteolin induced nuclear translocation of AIF (apoptosis-inducing factor), which was mediated by activation of ERK and p38. Transfection of a vector expressing the miRNA (microRNA) of AIF prevented luteolin-induced apoptosis. The data suggest that luteolin induces a caspase-dependent and -independent apoptosis involving AIF nuclear translocation mediated by activation of ERK and p38 in breast cancer cells.     

鹤望兰类黄酮3′,5′-羟化酶基因SrF3′5′H的克隆及表达分析

采用RT-PCR和RACE方法从鹤望兰黄色花萼中克隆到类黄酮生物合成途径关键基因SrF3′5′H。该cDNA全长1 766 bp,具有完整的开放阅读框（ORF）,共1 509个碱基,编码503个氨基酸。氨基酸同源性分析表明,SrF3′5′H编码的氨基酸序列与已报道的其他植物的F3′5′H蛋白具有很高的同源性。系统进化树分析显示,鹤望兰SrF3′5′H与非洲紫罗兰蛋白亲缘关系较近。应用半定量PCR分析表明,SrF3′5′H在始花期转录水平达到最高,且在蓝色花瓣中表达最高,在黄色花萼中几乎没有表达。     

铺地锦竹草水提取物主要成分及抗氧化活性研究

以市售铺地锦竹草为材料制备其水提取物,采用分光光度法测定总黄酮、总花色苷、总糖等主要活性成分含量,采用ICP-MS质谱仪法测定提取物中18种金属元素的组成和含量,最后采用分光光度法研究提取物的体外抗氧化活性。结果显示,铺地锦竹草提取物中黄酮含量为2.04%,总蛋白含量1.83%,总糖含量55%,总花色苷含量7.2%,Ca、Mn、Mg等有益微量元素含量较高,Pb、Hg、Ag、Co、Ge等有害重金属微量检出或未检出。提取物清除DPPH·的IC50达0.265mg·mL^-1,清除·OH自由基IC50为1.16mg·mL^-1,1mg提取物的总还原力与39μgVc相当,对亚铁离子未表现出有规律的螯合力。以上说明铺地锦竹草提取物中天然活性成分含量较高,有害重金属极微,具有良好的抗氧化能力,其抗氧化活性是由多种活性因子通过协同作用机制起作用的,具有开发为功能性食品的良好潜力。     

Kaempferol promotes primordial follicle activation through the phosphatidylinositol 3‐kinase/protein kinase B signaling pathway and reduces DNA fragmentation of sheep preantral follicles cultured in vitro

The aim of this study was to evaluate the effects of kaempferol on the morphology, follicular activation, growth, and DNA fragmentation of ovine preantral follicles cultured in situ, and the effects of a phosphatidylinositol 3 kinase (PI3K) inhibitor and the expression of phosphorylated protein kinase B (pAKT) after culture. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) analyses (fresh control) or cultured in α‐MEM+ alone (control) or with different concentrations of kaempferol (0.1, 1, 10, or 100 μM) for 7 days. Follicles were classified as normal or atretic, primordial or growing, and the oocyte and follicle diameters were measured. Proliferating cells were analyzed and DNA fragmentation was evaluated by the TUNEL assay. Inhibition of PI3K activity was performed through pretreatment in media added with 50 µM LY294002 for 1 hr and pAKT immunohistochemistry was performed after culture in the absence or presence of LY294002. After culture, the percentage of normal follicles was similar among the treatments (p > 0.05), except for 100 µM kaempferol, which had less normal follicles (p < 0.05). Moreover, kaempferol at 10 μM showed a higher percentage of follicular activation and cell proliferation than the other treatments (p < 0.05) and a percentage of TUNEL‐positive cells similar to that in the fresh control and lower than other treatments (p < 0.05). LY294002 significantly inhibited primordial follicle activation stimulated by α‐MEM+ and 10 μM kaempferol and reduced pAKT expression in those follicles. In conclusion, 10 μM kaempferol promotes primordial follicle activation and cell proliferation through the PI3K/AKT pathway and reduces DNA fragmentation of ovine preantral follicles cultured in vitro.     

Glycan expression as a tool for a deeper understanding of a reproductive gland in a skate of economic importance

Atlantoraja platana is an oviparous skate endemic to the south-west Atlantic Ocean, and is one of the skate species most exploited by local industrial bottom trawl fisheries. Oviparous elasmobranchs encapsulate their eggs in complex egg cases produced by the oviductal gland (OG). This organ is exclusively present in these fishes and comprises four distinct zones: club, baffle, papillary and terminal. The relative size and structural complexity of these zones correlate with mode of reproduction. Glycans are known to play major roles in reproduction so their distribution in each zone of the OG could explain the functional multiplicity of the gland in skates, but this topic has not been previously investigated. In this study, morphological, histochemical and lectin-histochemical analysis revealed various novel aspects of A. platana's OG. The club, papillary and terminal zones positively stained for periodic acid Schiff's reagent (PAS) and Alcian Blue (AB), indicating the presence of neutral and acid mucopolysaccharides. However, the buffle zone was negative for PAS and AB stains, but was positive for all the lectins used. Each zone of the OG had a characteristic pattern of glycan expression. Finally, we confirmed the presence of sperm but not sperm storage. This is the first lectin-histochemical study of the OG in chondrichtyan fish and it has proven to be an important tool to understand some of the mechanisms of fertility and reproductive success in economic important species such as A. platana.     

Enantiospecific Analysis of 8‐Prenylnaringenin in Biological Fluids by Liquid‐Chromatography‐Electrospray Ionization Mass Spectrometry: Application to Preclinical Pharmacokinetic Investigations

8‐Prenylnaringenin (8PN) is a naturally occurring bioactive chiral prenylflavonoid found most commonly in the female flowers of hops (Humulus lupulus L.). A stereospecific method of analysis for 8PN in biological fluids is necessary to study the pharmacokinetic disposition of each enantiomer. A novel and simple liquid chromatographic‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS) method was developed for the simultaneous determination of R‐ and S‐8PN in rat serum and urine. Carbamazepine was used as the internal standard (IS). Enantiomeric resolution of 8PN was achieved on a Chiralpak^® AD‐RH column with an isocratic mobile phase consisting of 2‐propanol and 10 mM ammonium formate (pH 8.5) (40:60, v/v) and a flow rate of 0.7 mL/min. Detection was achieved using negative selective ion monitoring (SIM) of 8PN at m/z 339.15 for both enantiomers and positive SIM m/z at 237.15 for the IS. The calibration curves for urine were linear over a range of 0.01–75 µg/mL and 0.05–75 µg/mL for serum with a limit of quantification of 0.05 µg/mL in serum and 0.01 µg/mL in urine. The method was successfully validated showing that it was sensitive, reproducible, and accurate for enantiospecific quantification of 8PN in biological matrices. The assay was successfully applied to a preliminary study of 8PN enantiomers in rat. Chirality 26:419–426, 2014. © 2014 Wiley Periodicals, Inc.     

Flavonoids Modulate the Proliferation of Neospora caninum in Glial Cell Primary Cultures

Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3'',4''-dihydroxyflavone, 3'',4'',5,7-tetrahydroxyflavone (luteolin), and 3,3'',4'',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation.