Antioxidant activity, cytotoxicity, and DNA information of Glossogyne tenuifolia

This study investigates the antioxidant activity and cytotoxicity of Glossogyne tenuifolia extract on various cancer cell lines. The 5.8s DNA of G. tenuifolia was isolated, and the species of this plant was confirmed by NCBI's DNA database. G. tenuifolia was then extracted with ethanol and separated into several fractions using the partition procedure with water, n-butanol, and ethyl acetate (EA). Among these, the EA fraction most significantly affected the activity of DPPH(*) and superoxide anion scavenging. Additionally, only the EA fraction exhibited cytotoxicity on breast cancer cells (MCF-7 and MDA-MB-231) and liver cancer cells (Hep G2 and Hep 3B). Next, the EA fraction was further separated by column chromatography, and 15 fractions were obtained. Three effective components were isolated and identified separately from the active fractions: oleanolic acid (OA) from fraction 6, luteolin from fractions 8-10, and luteolin-7-glucoside from fraction 12. The test of these three compounds on scavenging activity of DPPH(*) and superoxide anion indicates that luteolin had the highest antioxidant activity, whereas the effect of OA was negligible. Additionally, a synergistic effect between luteolin and luteolin-7-glucoside was observed. Kick-out experiments showed that the activities were vanished or decreased. Especially on MDA-MB-231 and MCF-7 cells, the cytotoxicity completely disappeared when luteolin was eliminated from fractions 8-10. These findings demonstrate that luteolin plays a crucial role in the inhibition of the growth of hepatoma cancer cell lines. Fraction 3, which did not contain luteolin, luteolin-7-glucoside, and oleanolic acid, had cytotoxicity on MDA-MB-231, MCF-7, Hep G2, Hep 3B, and A549, which implies that this fraction contained some other effective ingredients and requires further study. The investigation is currently underway in our laboratory.     

Evaluation of the estrogenic effects of legume extracts containing phytoestrogens

Seven legume extracts containing phytoestrogens were analyzed for estrogenic activity. Methanol extracts were prepared from soybean (Glycine max L.), green bean (Phaseolus vulgaris L.), alfalfa sprout (Medicago sativa L.), mung bean sprout (Vigna radiata L.), kudzu root (Pueraria lobata L.), and red clover blossom and red clover sprout (Trifolium pratense L.). Extracts of kudzu root and red clover blossom showed significant competitive binding to estrogen receptor beta (ERbeta). Estrogenic activity was determined using an estrogen-dependent MCF-7 breast cancer cell proliferation assay. Kudzu root, red clover blossom and sprout, mung bean sprout, and alfalfa sprout extracts displayed increased cell proliferation above levels observed with estradiol. The pure estrogen antagonist, ICI 182,780, suppressed cell proliferation induced by the extracts, suggesting an ER-related signaling pathway was involved. The ER subtype-selective activities of legume extracts were examined using transiently transfected human embryonic kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist activity toward ERbeta. Using HPLC to collect fractions and MCF-7 cell proliferation, the active components in kudzu root extract were determined to be the isoflavones puerarin, daidzin, genistin, daidzein, and genistein. These results show that several legumes are a source of phytoestrogens with high levels of estrogenic activity.     

Greek plant extracts exhibit selective estrogen receptor modulator (SERM)-like properties

To prevent bone loss that occurs with increasing age, nutritional and pharmacological factors are needed. Traditional therapeutic agents (selective estrogen receptor modulators or SERMs, biphosphonates, calcitonin) may have serious side effects or contraindications. In an attempt to find food components potentially acting as SERMs, we submitted four plant aqueous extracts derived from Greek flora (Sideritis euboea, Sideritis clandestina, Marticaria chamomilla, and Pimpinella anisum) in a series of in vitro biological assays reflective of SERM profile. We examined their ability (a) to stimulate the differentiation and mineralization of osteoblastic cell culture by histochemical staining for alkaline phosphatase and Alizarin Red-S staining, (b) to induce, like antiestrogens, the insulin growth factor binding protein 3 (IGFBP3) in MCF-7 breast cancer cells, and (c) to proliferate cervical adenocarcinoma (HeLa) cells by use of MTT assay. Our data reveal that all the plant extracts studied at a concentration range 10-100 microg/mL stimulate osteoblastic cell differentiation and exhibit antiestrogenic effect on breast cancer cells without proliferative effects on cervical adenocarcinoma cells. The presence of estradiol inhibited the antiestrogenic effect induced by the extracts on MCF-7 cells, suggesting an estrogen receptor-related mechanism. In conclusion, the aqueous extracts derived from Sideritis euboea, Sideritis clandestina, Marticaria chamomilla, and Pimpinella anisum may form the basis to design "functional foods" for the prevention of osteoporosis.     

Antiproliferative activity of pomiferin in normal (MCF-10A) and transformed (MCF-7) breast epithelial cells

Pomiferin and osajin are prenylated isoflavones from Osage orange fruit that both have potent antioxidant activity in a variety of assays. Pomiferin, in particular, has strong activity against the superoxide anion in a photochemiluminescence (PCL) assay system. In vitro, pomiferin, but not osajin, demonstrated selective antiproliferative activity against the tumorigenic breast epithelial cell line MCF-7 (IC(50) = 5.2 μM) with limited toxicity toward nontumorigenic breast epithelial cells (MCF-10A). The differential sensitivity of normal and tumorigenic cells to the antiproliferative action of pomiferin was examined further by using cDNA microarrays. With a stringent cutoff of p < 0.01, a total of 94 genes were significantly differentially expressed between MCF-7 and MCF-10A cells; 80 up-regulated and 14 down-regulated when cells were exposed to 5 μM pomiferin for 24 h. Fold changes by microarray analysis were confirmed using RT-qPCR, and the most significant changes were found with genes related to antioxidant enzymes. Genes involved in mitotic inhibition and apoptotic regulations were also found to be up-regulated. Pomiferin is therefore a good anticancer candidate agent that may be useful either alone or in combination with other therapeutic agents and, because of its selectivity toward tumor cells, likely to have fewer side effects that classic chemotherapy drugs.     

Antiproliferative terpenoids from almond hulls (Prunus dulcis): identification and structure-activity relationships

Bioassay-guided fractionation of the EtOAc crude extract from Sicilian almond hulls, a waste material from Prunus dulcis crop, allowed identification of 10 constituents, isolated as pure compounds (1-5, 7, and 10) or unseparable mixtures (5 + 6 and 8 + 9). All compounds were subjected to spectroscopic analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide bioassay on MCF-7 human breast cancer cells. In addition to the main components oleanolic (1), ursolic (2), and betulinic (3) acids, the 2-hydroxy analogues alphitolic (4), corosolic (5), and maslinic (6) acids, as well as the related aldehydes, namely, betulinic (7), oleanolic (8), and ursolic (9), were identified. From a more polar fraction, the beta-sitosterol 3-O-glucoside (10) was also identified. A sample of commercially available betulin (11) was also included in bioassays as further support to a structure-activity relationship study. Betulinic acid showed antiproliferative activity toward MCF-7 cells (GI50 = 0.27 microM), higher than the anticancer drug 5-fluorouracil.     

Evaluation of the phytoestrogenic activity of Cyclopia genistoides (honeybush) methanol extracts and relevant polyphenols

Unfermented C. genistoides methanol extracts of different harvestings and selected polyphenols were evaluated for phytoestrogenic activity by comparing binding to both ER subtypes, transactivation of an ERE-containing promoter reporter, proliferation of MCF-7-BUS and MDA-MB-231 breast cancer cells, and binding to SHBG. The extracts from one harvesting of C. genistoides (P104) bound to both ER subtypes. All extracts transactivated ERE-containing promoter reporters via ERbeta but not via ERalpha. All extracts, except P122, caused proliferation of the estrogen-sensitive MCF-7-BUS cells. Proliferation of MCF-7-BUS cells was ER-dependent as ICI 182,780 reversed proliferation. Physiologically more relevant, extracts antagonized E2-induced MCF-7-BUS cell proliferation. Furthermore, all extracts, except P122, induced proliferation of the estrogen-insensitive MDA-MB-231 cells, suggesting that the extracts are able to induce ER-dependent and ER-independent cell proliferation. Binding to SHBG by extracts was also demonstrated. These results clearly show that C. genistoides methanol extracts display phytoestrogenic activity and act predominantly via ERbeta. HPLC and LC-MS analysis, however, suggests that the observed phytoestrogenic activity cannot be ascribed to polyphenols known to be present in other Cyclopia species.     

Protection by polyphenols of postprandial human plasma and low-density lipoprotein modification: the stomach as a bioreactor

Recent studies dramatically showed that the removal of circulating modified low-density lipoprotein (LDL) results in complete prevention of atherosclerosis. The gastrointestinal tract is constantly exposed to food, some of it containing oxidized compounds. Lipid oxidation in the stomach was demonstrated by ingesting heated red meat in rats. Red wine polyphenols added to the rats' meat diet prevented lipid peroxidation in the stomach and absorption of malondialdehyde (MDA) in rat plasma. In humans, postprandial plasma MDA levels rose by 3-fold after a meal of red meat cutlets. MDA derived from meat consumption caused postprandial plasma LDL modification in human. The levels of plasma MDA showed a 75% reduction by consumption of red wine polyphenols during the meat meal. Locating the main biological site of action of polyphenols in the stomach led to a revision in the understanding of how antioxidants work in vivo and may help to elucidate the mechanism involved in the protective effects of polyphenols in human health.     

In vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

Both selenium and phycocyanin have been reported to show potent cancer chemopreventive activities. In this study, we investigated the in vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin (Se-PC) purified from selenium-enriched Spirulina platensis. The antioxidant activity of Se-PC was evaluated by using four different free radical scavenging assays, namely, the 2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid (ABTS) assay, 1,1-diphenyl-2-picryhydrazyl (DPPH) assay, superoxide anion scavenging assay, and erythrocyte hemolysis assay. The results indicated that Se-PC exhibited stronger antioxidant activity than phycocyanin by scavenging ABTS, DPPH, superoxide anion, and 2,2'-azobis-(2-amidinopropane)dihydrochloride free radicals. Se-PC also showed dose-dependent protective effects on erythrocytes against H 2O 2-induced oxidative DNA damage as evaluated by the Comet assay. Moreover, Se-PC was identified as a potent antiproliferative agent against human melanoma A375 cells and human breast adenocarcinoma MCF-7 cells. Induction of apoptosis in both A375 and MCF-7 cells by Se-PC was evidenced by accumulation of sub-G1 cell populations, DNA fragmentation, and nuclear condensation. Further investigation on intracellular mechanisms indicated that depletion of mitochondrial membrane potential (DeltaPsi m) was involved in Se-PC-induced cell apoptosis. Our findings suggest that Se-PC is a promising organic Se species with potential applications in cancer chemoprevention.     

Multivariate analysis of antioxidant power and polyphenolic composition in red wines

It has been demonstrated that the dietary intake of compounds having antioxidant activity is very important, and various chemical, biological, and electrochemical methods have been proposed to evaluate the antioxidant power of compounds such as polyphenols. Wine, although nonessential, has a high polyphenol content up to 2-3 g/L in red wines obtained by traditional maceration. The polyphenol content of wines is usually evaluated by the Folin reagent, which provides an appropriate response to the requirements of wine manufacturers. Because the presence of individual polyphenols may be evaluated by HPLC, more or less selective methods toward the various chemical classes of polyphenols have been developed. An HPLC method set up recently was applied to evaluate how individual polyphenols contributed to the overall antioxidant power (AOP) of 60 Italian red wines, trying to identify the effect that individual compounds may have on the total AOP. Application of the multivariate analysis allowed us to detect some determining compounds such as gallic acid and some flavonols. On the basis of the correlation between two traditional chemical methods, namely the total polyphenol determination by the Folin reagent and the flavanol determination by the condensation reaction with p-(dimethylamino)-cinnamaldehyde, it was shown that the use of these two merely chemical methods is well correlated (r = 0.83 and 0.87) to an AOP evaluation of red wines.     

Interactions between yeast lees and wine polyphenols during simulation of wine aging: I. Analysis of remnant polyphenolic compounds in the resulting wines

Wine aging on yeast lees is a traditional enological practice used during the manufacture of wines. This technique has increased in popularity in recent years for the aging of red wines. Although wine polyphenols interact with yeast lees to a limited extent, such interactions have a large effect on the reactivity toward oxygen of wine polyphenolic compounds and yeast lees. Various domains of the yeast cell wall are protected by wine polyphenols from the action of extracellular hydrolytic enzymatic activities. Polysaccharides released during autolysis are thought to exert a significant effect on the sensory qualities of wine. We studied the chemical composition of polyphenolic compounds remaining in solution or adsorbed on yeast lees after various contact times during the simulation of wine aging. The analysis of the remnant polyphenols in the wine indicated that wine polyphenols adsorption on yeast lees follows biphasic kinetics. An initial and rapid fixation is followed by a slow, constant, and saturating fixation that reaches its maximum after about 1 week. Only very few monomeric phenolic compounds remained adsorbed on yeast lees, and no preferential adsorption of low or high polymeric size tannins occurred. The remnant condensed tannins in the wine contained fewer epigallocatechin units than the initial tannins, indicating that polar condensed tannins were preferentially adsorbed on yeast lees. Conversely, the efficiency of anthocyanin adsorption on yeast lees was unrelated to its polarity.     

Purification and characterization of a rhamnose-binding chinook salmon roe lectin with antiproliferative activity toward tumor cells and nitric oxide-inducing activity toward murine macrophages

In this study, a rhamnose-binding lectin from the roe of chinook salmon (Oncorhynchus tshawytscha) was purified and characterized, and its biological activities were examined in several model systems. Chinook salmon roe lectin had a molecular mass of 30 kDa and agglutinated rabbit and bovine erythrocytes. The hemagglutination activity of the lectin was not affected by metal ions. The lectin was stable up to 70 °C and between pH 4 and pH 11. Chinook salmon roe lectin did not exert antifungal activity toward the fungal species tested and did not exhibit mitogenic response toward mouse splenocytes up to a concentration of 5 mg/mL. The lectin had selective antiproliferative activity toward human breast cancer MCF-7 cells and hepatoma Hep G2 cells. It also induced the production of nitric oxide from mouse peritoneal macrophages. This is the first report that demonstrates these biological activities from chinook salmon roe lectin.     

Effects of tea polyphenols on the invasion and matrix metalloproteinases activities of human fibrosarcoma HT1080 cells

The effects of tea polyphenols on the invasion of highly metastatic human fibrosarcoma HT1080 cells through a monolayer of human umbilical vein endothelial cells (HUVECs) and the accompanying basal membrane were investigated. Among the tea polyphenols tested, epicatechin gallate (ECg), epigallocatechin gallate (EGCg), and theaflavin strongly suppressed the invasion of HT1080 cells into the monolayer of HUVECs/gelatin membrane, whereas epicatechin, epigallocatechin, tea flavonols, tea flavones, and gallate derivatives had no effect. Both theaflavin-digallate and theasinensin D showed a weak invasion inhibitory effect. ECg significantly inhibited the invasion without cytotoxicity against cancer cells and HUVECs. Ester-type catechins (ECg and EGCg) and theaflavin strongly suppressed the gelatin degradation mediated by matrix metalloproteinase (MMP) 2 and MMP-9, which were secreted into the conditioned medium of HT1080 cells. In conclusion, among the tea polyphenols tested, ECg was considered to be the agent with the most potential antimetastasis activity because it inhibited invasion in the absence of cytotoxicity.     

Effect of 2alpha-hydroxyursolic acid on NF-kappaB activation induced by TNF-alpha in human breast cancer MCF-7 cells

Apples are one of the largest contributors of fruit phenolics of all fruits consumed by Americans and contain a variety of bioactive compounds, which have health benefits. Consumption of apples has been linked to reduced risk of chronic diseases such as cancer and cardiovascular disease. Apple extracts have been shown to have the capabilities of inhibiting NF-kappaB activation in human breast cancer MCF-7 cells. 2Alpha-hydroxyursolic acid is one of the major triterpenoids isolated from apple peels, and its effects on cell proliferation and TNF-alpha-induced NF-kappaB activation in MCF-7 cells were examined. 2Alpha-hydroxyursolic acid significantly inhibited MCF-7 cell proliferation at doses of 20 microM (p < 0.05). Preincubation with 2alpha-hydroxyursolic acid suppressed TNF-alpha-induced NF-kappaB activation in a dose-dependent manner and significantly inhibited the activation at a dose of 20 microM of 2alpha-hydroxyursolic acid (p < 0.05). 2Alpha-hydroxyursolic acid treatment did not affect the phosphorylation level of NF-kappaB inhibitor (IkappaB-alpha), but proteasome activity in MCF-7 cells was inhibited significantly at doses of 10 and 20 microM ( p < 0.05). These results suggest that 2alpha-hydroxyursolic acid has antiproliferative activities against MCF-7 cells and capabilities inhibiting NF-kappaB activation induced by TNF-alpha partially by suppressing proteasome activities.     

Cranberry phytochemicals: Isolation, structure elucidation, and their antiproliferative and antioxidant activities

Bioactivity-guided fractionation of cranberries was used to determine the chemical identity of bioactive constituents. Twenty compounds were isolated using gradient solvent fractionation, silica gel and ODS columns, and preparative RP-HPLC. Their chemical structures were identified using HR-MS, 1D and 2D NMR, and X-ray diffraction analysis. Antiproliferative activities of isolated compounds against HepG2 human liver cancer and MCF-7 human breast cancer cells were evaluated. Among the compounds isolated, ursolic acid, quercetin, and 3,5,7,3',4'-pentahydroxyflavonol-3-O-beta-D-glucopyranoside showed potent antiproliferative activities against HepG2 cell growth, with EC50 values of 87.4 +/- 2.7, 40.9 +/- 1.1, and 49.2 +/- 4.9 microM, respectively. Ursolic acid, quercetin, and 3,5,7,3',4'-pentahydroxyflavonol-3-O-beta-D-glucopyranoside showed potent inhibitory activity toward the proliferation of MCF-7 cells, with EC50 values of 11.7 +/- 0.1, 137.5 +/- 2.6, and 23.9 +/- 3.9 microM, respectively. Quercetin, 3,5,7,3',4'-pentahydroxyflavonol-3-O-beta-D-glucopyranoside, 3,5,7,3',4'-pentahydroxyflavonol-3-O-beta-D-galactopyranoside, and 3,5,7,3',4'-pentahydroxyflavonol-3-O-alpha-l-arabinofuranoside showed potent antioxidant activities, with EC50 values of approximately 10 microM. These results showed cranberry phytochemical extracts have potent antioxidant and antiproliferative activities.     

Identification of triterpene hydroxycinnamates with in vitro antitumor activity from whole cranberry fruit (Vaccinium macrocarpon)

Bioactivity-guided fractionation of cranberry fruit was used to determine the identity of triterpenoid esters from Vaccinium macrocarpon, which inhibit tumor cell growth and may play a role in cancer prevention. In our previous study, a fraction from whole fruit exhibited tumor cell growth inhibition in vitro. The major components of this fraction were isolated by chromatographic separation of ethyl acetate extracts, purified by semipreparative HPLC, and identified by NMR as cis- (1) and trans- (2) isomers of 3-O-p-hydroxycinnamoyl ursolic acid. These triterpenoid esters have not been previously reported in Vaccinium fruit. Bioassay of the purified triterpene cinnamates in tumor cell lines in vitro showed slightly greater activity of compound 1 in most cell lines, with GI(50) values of approximately 20 microM in MCF-7 breast, ME180 cervical and PC3 prostate tumor cell lines. Quercetin was slightly less active than 1, while cyanidin-3-galactoside exhibited much lower cytotoxicity, with GI(50) greater than 250 microM in all cell lines. Phenylboronic acid (3) was also isolated from the fruit but showed insignificant antitumor activity.     

Inhibition of cancer cell proliferation in vitro by fruit and berry extracts and correlations with antioxidant levels

The effects of 10 different extracts of fruits and berries on cell proliferation of colon cancer cells HT29 and breast cancer cells MCF-7 were investigated. The fruits and berries used were rosehips, blueberries, black currant, black chokeberries, apple, sea buckthorn, plum, lingonberries, cherries, and raspberries. The extracts decreased the proliferation of both colon cancer cells HT29 and breast cancer cells MCF-7, and the effect was concentration dependent. The inhibition effect for the highest concentration of the extracts varied 2-3-fold among the species, and it was in the ranges of 46-74% (average = 62%) for the HT29 cells and 24-68% (average = 52%) for the MCF-7 cells. There were great differences in the content of the analyzed antioxidants in the extracts. The level of the vitamin C content varied almost 100-fold, and the content of total carotenoids varied almost 150-fold among the species. Also in the composition and content of flavonols, hydroxycinnamic acids, anthocyanins, and phenolics were found great differences among the 10 species. The inhibition of cancer cell proliferation seen in these experiments correlated with levels of some carotenoids and with vitamin C levels, present at levels that can be found in human tissues. The same inhibition of cell proliferation could not be found by ascorbate standard alone. This correlation might indicate a synergistic effect of vitamin C and other substances. In MCF-7 cells, the anthocyanins may contribute to the inhibition of proliferation.     

Triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for apple's anticancer activity

Bioactivity-guided fractionation of apple peels was used to determine the chemical identity of bioactive constituents. Thirteen triterpenoids were isolated, and their chemical structures were identified. Antiproliferative activities of the triterpenoids against human HepG2 liver cancer cells, MCF-7 breast cancer cells, and Caco-2 colon cancer cells were evaluated. Most of the triterpenoids showed high potential anticancer activities against the three human cancer cell lines. Among the compounds isolated, 2alpha-hydroxyursolic acid, 2alpha-hydroxy-3beta-{[(2E)-3-phenyl-1-oxo-2-propenyl]oxy}olean-12-en-28-oic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid showed higher antiproliferative activity toward HepG2 cancer cells. Ursolic acid, 2alpha-hydroxyursolic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid exhibited higher antiproliferative activity against MCF-7 cancer cells. All triterpenoids tested showed antiproliferative activity against Caco-2 cancer cells, especially 2alpha-hydroxyursolic acid, maslinic acid, 2alpha-hydroxy-3beta-{[(2E)-3-phenyl-1-oxo-2-propenyl]oxy}olean-12-en-28-oic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid, which displayed much higher antiproliferative activities. These results showed the triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for the anticancer activities of whole apples.     

红曲中非酿酒酵母的分离鉴定及其对黄酒风味成分的影响

为探究红曲中非酿酒酵母对黄酒风味成分的影响,首先对红曲中的酵母菌株进行分离纯化,并采用分子生物学方法进行鉴定,然后将分离的非酿酒酵母菌株分别与红曲霉相结合,以纯种发酵的方式酿造红曲黄酒,分析各酒样中有机酸、氨基酸、挥发性物质等风味成分的组成及含量。结果表明,分离获得的4株非酿酒酵母CKS-2为威克汉姆酵母(Wickerhamomyces sp.)、CKS-3为季也蒙毕赤酵母(Meyerozyma guilliermondii)、CKS-4为胶红酵母(Rhodotorula mucilaginosa)、CKS-7为德尔布有孢酵母(Torulaspora delbrueckii),其中,CKS-7具有较好的产乙醇和苹果酸能力,发酵酒样中丝氨酸、组氨酸、色氨酸和亮氨酸的含量也较高,并且具有良好的合成2-苯乙醇、异戊醇、棕榈酸乙酯等挥发性风味成分的能力。本研究对红曲黄酒品质提升,以及筛选潜在可应用于红曲黄酒酿造的优良非酿酒酵母菌株具有一定作用。     

Tea polyphenol (-)-epigallocatechin 3-gallate suppresses heregulin-beta1-induced fatty acid synthase expression in human breast cancer cells by inhibiting phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase cascade signaling

Tumor-associated fatty acid synthase (FAS) is implicated in tumorigenesis and connected to HER2 (human epidermal growth factor receptor 2) by systemic analyses. Suppression of FAS in cancer cells may lead to growth inhibition and cell apoptosis. Our previous study demonstrated that (-)-epigallocatechin 3-gallate (EGCG), the green tea catechin, could down-regulate FAS expression by suppressing EGFR (epidermal growth factor receptor) signaling and downstream phosphatidylinositol 3-kinase (PI3K)/Akt activation in the MCF-7 breast cancer cell line. Herein, we examined the effects of EGCG on FAS expression modulated by another member of the erbB family, that is, HER2 or HER3. We identified that heregulin-beta1 (HRG-beta1), a HER3 ligand, stimulated dose-dependent FAS expression in breast cancer cell lines MCF-7 and AU565, but not MDA-MB-453. The time-dependent increase in FAS expression after HRG-beta1 stimulation was also observed in MCF-7 cells, and this up-regulation was de novo RNA synthesis dependent. Treatment of MCF-7 cells with EGCG markedly inhibited HRG-beta1-dependent induction of mRNA and protein of FAS. EGCG also decreased the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 that were demonstrated as selected downstream HRG-beta1-responsive kinases required for FAS expression using dominant-negative Akt, PI3K inhibitors (LY294002 and wortmannin), or MEK inhibitor (PD98059). FAS induction by HRG-beta1 was also blocked by AG825, a selective HER2 inhibitor, and by genistein, a selective tyrosine kinase inhibitor, indicating the formation of a heterodimer between HER2 and HER3, and their tyrosine kinase activities are essential for HRG-beta1-mediated elevation of FAS. Additionally, growth inhibition of HRG-beta1-treated cells was parallel to suppression of FAS by EGCG. Taken together, these findings extend our previous study to indicate that EGCG may be useful in the chemoprevention of breast carcinoma in which FAS overexpression results from HER2 or/and HER3 signaling.     

乳腺癌细胞中Rab7基因的克隆及序列分析

本实验主要是从人乳腺癌细胞中克隆Rab7基因并进行基因序列比对和分析。通过培养乳腺癌细胞MCF-7和MDA-MB231,提取细胞总RNA,逆转录为cDNA,以此cDNA为模板,根据GenBank公布的人Rab7全长序列设计引物,扩增Rab7基因的开放阅读框。将其与真核表达载体pc-DNA3.1连接,构建pcDNARab7重组质粒。测序后发现MCF-7细胞中克隆的Rab7序列与GenBank序列的同源性为91%,发生了多处突变,其中94.4%为同义突变,5.5%为错义突变。但是MDA-MB231细胞中克隆的Rab7基因序列与GenBank中的Rab7序列的同源性为100%。由于发现Rab7在MCF-7乳腺癌细胞中发生突变,而在MDA-MB-231乳腺癌细胞中基因未发生突变,我们推测Rab7基因的突变是否与乳腺癌的产生有关系,这个发现为以后研究Rab7与乳腺癌的关系奠定了基础。