Oleuropein,a Secoiridoid Derived from Olive Tree,Inhibits the Proliferation of Human Colorectal Cancer Cell Through Downregulation of HIF-1α

Oleuropein (OL) is the most prominent phenolic compound in the fruit of olive tree. Although OL has shown powerful anticancer activity the underlying action mechanism remains largely unknown. The present study evaluated the effects of OL on hydroxityrosol (HT)-29 human colon adenocarcinoma cells in comparison to hydroxytyrosol, its hydrolysis product, and to elucidate the underlying anticancer molecular mechanisms involved. Cell proliferation was determined using SRB assay. Cell cycle and apoptosis were assessed by flow cytometry and changes in MAPK cascade protein expression, HIF-1α, p53, PPARγ, and NFKβ signaling pathways by Western blot. Although OL showed less potency than HT, in terms of cell growth inhibition, induced significant changes in cell cycle analysis and caused a significant increase in the apoptotic population. Both compounds produced a remarkable decrease in HIF-1α protein and an upregulation of p53 protein expression. However, no significant changes in IkB-α and MAPK cascade protein expressions were observed. HT produced a significant upregulation in peroxisome proliferator-activated receptor gamma (PPARγ) expression whereas OL failed. PPARγ upregulation may be one of the principal mechanisms of the tumor shrinkage by HT. Our novel findings demonstrate that OL limits the growth and induces apoptosis in HT-29 cells via p53 pathway activation adapting the HIF-1α response to hypoxia.     

Methyl selenium-induced vascular endothelial apoptosis is executed by caspases and principally mediated by p38 MAPK pathway

The induction of vascular endothelial cell apoptosis and inhibition of tumor-associated angiogenesis by selenium may contribute to its cancer chemopreventive effects. Here we examined the stress-activated/mitogen-activated protein kinases (p38 MAPK, ERK1/2) and protein kinase B/AKT as potential signaling mediators for apoptosis induction by a methylselenol precursor methylseleninic acid (MSeA) in human umbilical vein endothelial cells (HUVEC). Time course experiments showed that p38 MAPK hyperphosphorylation and ERK1/2 dephosphorylation occurred before the cleavage of procaspase-3 and poly(ADP-ribose) polymerase (PARP), whereas AKT dephosphorylation occurred after caspase activation. The p38 MAPK inhibitor SB202190 attenuated the MSeA-induced morphological changes and decreased DNA fragmentation and the cleavage of procaspase-3 and PARP in concordant proportions. The general caspase inhibitor zVADfmk completely blocked the MSeA-induced PARP cleavage and DNA fragmentation, whereas zDEVDfmk, an inhibitor for caspase-3-like activities, was nearly as effective for inhibiting apoptosis. In comparison, apoptosis induced by selenite in HUVECs was observed in the complete absence of an activation of the major caspases. Taken together, the data support p38 MAPK as a key upstream mediator for the methylselenol-specific induction of vascular endothelial caspase-dependent apoptosis, which is principally executed by caspase-3-like activities.     

Estimated intake of milk fat is negatively associated with cardiovascular risk factors and does not increase the risk of a first acute myocardial infarction. A prospective case-control study

'Orujo' olive oil is obtained by chemical processes from the waste resulting from the mechanical extraction of virgin olive oil. The aim of the present study was to evaluate a new pharmacological property of two natural triterpenoids contained in olive oil, as vasodilatory agents, and to determine their mechanism of action. The two compounds studied were oleanolic acid and erythrodiol. The vasorelaxant effect induced by these pentacyclic triterpenoids was studied in isolated thoracic rat aorta. Oleanolic acid and erythrodiol, accumulatively added, showed vasorelaxant activities in aortic rings with endothelium pre-contracted by 10(-6) m-phenylephrine (maximum percentage of relaxation 86.38 (sem 2.89) and 73.53 (sem 6.01), respectively). They had almost no relaxant effect on depolarised or endothelium-denuded aortic segments. The relaxation was significantly attenuated by pre-treatment with the NO synthase inhibitor N(omega)-nitro-L-arginine-methylester (L-NAME; 3x10(-4) m). To characterise the involvement of endothelial factors, in addition to NO, arteries with endothelium were exposed to 10(-5) m-indomethacin (INDO), a cyclo-oxygenase inhibitor, or INDO plus L-NAME. INDO did not have any significant effect on the relaxant response of both compounds. The combination of L-NAME plus INDO only abolished the oleanolic acid-induced relaxation. The present results suggest that the mechanism of relaxation seems to be mainly mediated by the endothelial production of NO; however, other mechanisms cannot be excluded. It can be concluded that oleanolic acid and erythrodiol may have interesting therapeutic potential as new vasodilator drugs, thus protecting the cardiovascular system. Therefore, the intake of 'orujo' olive oil, as a source of these compounds, might be beneficial in this regard.     

Antiproliferative and apoptosis-inducing effects of maslinic and oleanolic acids, two pentacyclic triterpenes from olives, on HT-29 colon cancer cells

We have previously reported the anticarcinogenic effects of an olive fruit extract composed of pentacyclic triterpenes, the main components of which are maslinic acid (73.25%) and oleanolic acid (25.75%). Here we examined the effects of the individual components on proliferation, necrosis and apoptosis rates by fluorescence-based techniques in human HT-29 colon cancer cells. Oleanolic acid showed moderate antiproliferative activity, with an ec50 of 160.6 (se 10.6) micromol/l, and moderate cytotoxicity at high concentrations ( > or = 250 micromol/l). On the other hand, maslinic acid inhibited cell growth with an ec50 of 101.2 (se 7.8) micromol/l, without necrotic effects. Oleanolic acid, which lacks a hydroxyl group at the carbon 2 position, failed to activate caspase-3 as a prime apoptosis protease. In contrast, maslinic acid increased caspase-3-like activity at 10, 25 and 50 micromol/l by 3-, 3.5- and 5-fold over control cells, respectively. The detection of ROS in the mitochondria, which serve as pro-apoptotic signal, evidenced the different bioactivity of the two triterpenes. Confocal microscopy analysis revealed that maslinic acid generated superoxide anions while oleanolic acid-treated cells did not differ from the control. Completion of apoptosis by maslinic acid was confirmed microscopically by the increase in plasma membrane permeability, and detection of DNA fragmentation. In conclusion, the anticancer activity observed for olive fruit extracts seems to originate from maslinic acid but not from oleanolic acid. Maslinic acid therefore is a promising new compound for the chemoprevention of colon cancers.     

Oleanolic Acid: Extraction,Characterization and Biological Activity

Oleanolic acid, a pentacyclic triterpenoid ubiquitously present in the plant kingdom, is receiving outstanding attention from the scientific community due to its biological activity against multiple diseases. Oleanolic acid is endowed with a wide range of biological activities with therapeutic potential by means of complex and multifactorial mechanisms. There is evidence suggesting that oleanolic acid might be effective against dyslipidemia, diabetes and metabolic syndrome, through enhancing insulin response, preserving the functionality and survival of β-cells and protecting against diabetes complications. In addition, several other functions have been proposed, including antiviral, anti-HIV, antibacterial, antifungal, anticarcinogenic, anti-inflammatory, hepatoprotective, gastroprotective, hypolipidemic and anti-atherosclerotic activities, as well as interfering in several stages of the development of different types of cancer; however, due to its hydrophobic nature, oleanolic acid is almost insoluble in water, which has led to a number of approaches to enhance its biopharmaceutical properties. In this scenario, the present review aimed to summarize the current knowledge and the research progress made in the last years on the extraction and characterization of oleanolic acid and its biological activities and the underlying mechanisms of action.     

Activation of MEK 1/2 and p42/44 MAPK by angiotensin II in hepatocyte nucleus and their potentiation by ethanol

Hepato-subcellular effect of angiotensin II (Ang II) and ethanol on the p42/44 mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK 1/2) was investigated in the nucleus of rat hepatocytes. Hepatocytes were treated with ethanol (100 mM) for 24 h and stimulated with Ang II (100 nM, 5 min). The levels of p42/44 MAPK and MEK 1/2 were monitored in the nuclear fraction using antibodies. Ang II itself caused significant accumulation of phosphorylated p42/44 MAPK (phospho-p42/44 MAPK) in the nucleus without any significant translocation of p42/44 MAPK protein thereby suggesting activation of p42/44 MAPK in the nucleus. Ang II caused marked accumulation of phosphorylated MEK 1/2 (phospho-MEK 1/2) in the nucleus without any significant accumulation of MEK 1/2 protein. Ratio of phospho-MEK 1/2 to MEK 1/2 protein in the nucleus after Ang II treatment was 2.4 times greater than control suggesting phosphorylation of MEK 1/2 inside the nucleus. Ethanol had no effect on the protein level or the activation of p42/44 MAPK in the nucleus. Ethanol treatment potentiated nuclear activation of p42/44 MAPK by Ang II but not translocation of p42/44 MAPK protein. This was accompanied by potentiation of Ang II-stimulated accumulation of phospho-MEK 1/2 in the nucleus by ethanol. MEK 1/2 inhibitor, U-0126 inhibited Ang II response and its potentiation by ethanol. These results suggest that Ang II-mediated accumulation of phospho-p42/44 MAPK in the hepatocyte nucleus involves MEK 1/2-dependent activation and this effect is potentiated by ethanol.     

(-)Epigallocatechin gallate and quercetin enhance survival signaling in response to oxidant-induced human endothelial apoptosis

We reported recently that (-)epigallocatechin gallate and quercetin inhibited H2O2-induced apoptosis through modulation of the expression of apoptosis-related Bcl-2 and Bax in endothelial cells. This study attempted to identify possible regulatory sites and mechanisms of antiapoptotic flavonoids, focusing on ROS-mediated signaling in HUVEC. The effects of apigenin on the signaling pathway downstream were compared. Submillimolar H2O2 caused >30% cell killing with intracellular oxidant generation. H2O2-induced oxidant generation markedly decreased total intracellular glutathione (GSH) levels. Micromolar (-)epigallocatechin gallate and quercetin partially eliminated the dichlorodihydrofluorescein (DCF) and phospho-p53 staining, suggesting that these flavonoids inhibited the accumulation of intracellular oxidants and nuclear transactivation of p53 in H2O2-exposed cells. In contrast, cells treated with apigenin remained DCF and phospho-p53 staining positive in response to H2O2. (-)Epigallocatechin gallate significantly raised the total GSH level that had been depleted by H2O2. Caspase-3 activity was enhanced by H2O2, and this increase was inhibited by (-)epigallocatechin gallate and quercetin. Additionally, the upregulation of caspase-3 activation was reversed by these flavonoids at > or =10 micromol/L; these inhibitory effects were dose dependent. Western blot data revealed that H2O2 upregulated phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which was rapidly reversed by quercetin within 30 min; H2O2 activation of c-Jun was downregulated. (-)Epigallocatechin gallate inhibited H2O2-induced phosphorylation of JNK and p38 MAPK after 60 min. These results reveal that quercetin blocks JNK- and p38 MAPK-related signaling triggered by the oxidant and may regulate expression of apoptotic downstream genes, preventing apoptosis and promoting cell survival. (-)Epigallocatechin gallate may function as an antiapoptotic agent through other antiapoptotic pathways.     

石英诱导细胞cyclin D1-CDK4蛋白表达的降低及其影响因子

目的 探讨石英暴露的人胚肺成纤维细胞(human embryonic lung fibroblast,HELF)中细胞周期蛋白D1(cyclin D1)-细胞周期依赖蛋白依赖激酶4(CDK4)蛋白的表达水平,同时探讨细胞外调节蛋白激酶(ERK)、JNK、p38和核转录因子(AP-1)等信号蛋白在石英诱导eyelin D1-CDK4蛋白表达改变中的作用.方法 石英刺激HELF后,收获细胞,检测cyclin D1和CDK4蛋白表达.选用特异化学抑制剂或分子抑制剂抑制ERK、JNK、p38或AP-1的活性后,分别采用免疫细胞化学和免疫蛋白印迹方法检测HELF中cyclin D1和CDK4蛋白表达变化.结果 HELF暴露于石英粉尘2h后,cyclin D1和CDK4蛋白表达水平分别为(7.91±0.29)x103和(5.17±0.28)x104,均明显低于HELF组,差异有统计学意义(P＜0.05).用ERK的化学抑制剂或分子抑制剂抑制ERK的活力后,能够防止石英诱导的cyclin D1和CDK4蛋白表达降低.用SP600125抑制JNK的活力后,可以防止cyclin DI和CDK4蛋白表达降低.但是抑制p38的活力对石英诱导的cyclin D1和CDK4蛋白表达降低均没有作用.用姜黄素抑制AP-1的活性后,只能防止石英诱导的CDK4的表达降低,而对cyefin D1表达降低没有影响.结论 石英诱导HELF中cyclin D1和CDK4蛋白表达降低与ERK和JNK蛋白激酶有关.AP-1与石英诱导的CDK4蛋白表达降低有关.     

槲皮素通过促进Nrf2转位拮抗人肝细胞酒精性氧化损伤

目的探讨槲皮素诱导Ⅰ型血红素氧化酶(HO-1)对肝细胞酒精性氧化损伤的保护效应及相关信号通路。方法乙醇(100 mmol/L)孵育的人原代肝细胞经槲皮素(100μmol/L)处理24h后,测定细胞HO-1活性、Nrf2转位表达及细胞相应的氧化损伤程度;在此基础上,联合应用HO-1及各(MAPK)通路抑制剂,观察上述指标的变化。结果槲皮素处理使HO-1进一步升高,并明显减轻乙醇孵育所导致的细胞GSH耗竭、MDA升高及胞内门冬氨酸转氨酶(AST)与乳酸脱氢酶(LDH)的释放;HO-1诱导剂血红素也具有类似的效应,而HO-1抑制剂锌原卟啉Ⅸ及p38与细胞外信号调节激酶(extracellular regulated protein kinase,ERK)抑制剂(SB203580与PD98059)则明显抑制了Nrf2的转位活化及槲皮素的保护效应。结论槲皮素通过诱导HO-1保护肝细胞免受酒精性氧化损伤,其信号通路与p38及ERK活化促使Nrf2转位入核启动HO-1表达有关。     

The unsaponifiable fraction of virgin olive oil in chylomicrons from men improves the balance between vasoprotective and prothrombotic factors released by endothelial cells

Minor components of virgin olive oil (VOO) may play a key role in the beneficial effects of VOO on atherosclerosis. In the present study we evaluated the influence of the unsaponifiable fraction of VOO on the production of eicosanoids and nitric oxide (NO) by endothelial cells (HUVECs). Triglyceride-rich lipoprotein (TRLs) were isolated from human serum after the intake of meals enriched in 3 high-oleic acid oils, i.e., high-oleic sunflower (HOSO), VOO, or enriched-virgin olive (EVO) oils, the last-mentioned containing 2.4% of unsaponifiable matter. HOSO induced a greater accumulation of triglycerides (TGs) in the postprandial serum than VOO or EVO, as measured by calculating the area under the curve. The incubation with TRLs increased NO release by endothelial cells compared with untreated control cells, but the effects of the various TRLs did not differ. EVO-derived TRLs reduced the production of prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TxB(2)) (the stable metabolite of TxA(2)) compared with VOO- or HOSO-derived TRLs. The release of PGI(2) (as 6-keto PGF(1alpha)) was similarly diminished by all TRLs compared with the control. In conclusion, the unsaponifiable fraction of VOO does not affect postprandial triglyceridemia, but it has favorable effects on endothelial function, mainly by reducing proinflammatory and vasoconstrictor eicosanoid synthesis (PGE(2) and TxB(2)).     

Total fat and (n-3):(n-6) fat ratios influence eicosanoid production in mice.

Previous studies have not addressed the effect of differing fat intake on the effectiveness of varying (n-3) polyunsaturated fatty acid (PUFA) ingestion in altering tissue composition and eicosanoid production. This study examined (n-3):(n-6) PUFA ratios of 0, 0.1:1, 0.2:1, 0.4:1, and 1:1 with total fat at 5, 10, 15, and 20 g/100 g of diet and (n-6) PUFA fixed at 1.5 g/100 g of diet on tissue composition and peritoneal cell eicosanoid response to an in vivo inflammatory stimulus in 240 mice. Both (n-3) PUFA and total fat intake influenced tissue composition and eicosanoid biosynthesis. Increased (n-3) PUFA intake was associated with an increase in tissue (n-3) PUFA and a decrease in long-chain (n-6) PUFA. Although hepatic tissue linoleic acid (LA) was not altered by (n-3) PUFA intake or changes in total fat, peritoneal cell LA increased in response to increasing total fat but was unaffected by changes in dietary (n-3) PUFA. Four-series leukotrienes (LT) decreased progressively with increased (n-3) PUFA at all fat intake levels. In addition, four-series LT decreased with increased total fat at low (n-3):(n-6) ratios (0 and 0.1). At high (n-3):(n-6) ratios (0.4 and 1.0) increasing dietary fat between the 5 and 15 g/100 g diets increased four-series LT synthesis, which reached a plateau between 15 and 20 g fat/100 g diets. Five-series LT production generally rose with increased (n-3) PUFA intake; this effect was most evident in mice fed the 5 g fat/100 g diet. Increasing total dietary fat at the three highest (n-3):(n-6) ratios (0.2, 0.4, 1.0) decreased five-series LT production. Elevated (n-3) PUFA and total fat intake exerted an additive effect with respect to prostacyclin (PGI(2)) production because it was reduced with increasing intakes of both. Compared with the mice consuming the no (n-3) 5 g/100 g diets, PGI(2) levels were reduced by 88% in mice consuming the highest total fat and (n-3) PUFA diets. At low fat intake (5 and 10 g/100 g diet), increasing the (n-3) PUFA intake was associated with a decrease in PGE(2) synthesis. However, unlike PGI(2), high fat intake reduced PGE(2) to basal levels with no further reduction induced by increased (n-3) PUFA intake.     

Differential effects of dietary vitamin E and antioxidants on eicosanoid synthesis in young rabbits

This study was designed to evaluate the effects of dietary vitamin E and synthetic antioxidants on prostacyclin (PGI2) synthesis in isolated aorta segments and perfused hearts as well as thromboxane (TxA2) synthesis in thrombin-stimulated washed platelets. Weanling male New Zealand rabbits were fed a vitamin E-deficient basal diet or the basal diet supplemented with either all-rac-alpha-tocopherol acetate or propyl gallate or DPPD (N,N'-diphenyl-p-phenylenediamine). After 30 days on the diet, plasma tocopherol level, pyruvate kinase and liver microsomal NADPH oxidase were determined. DPPD but not propyl gallate prevented the development of myopathy. None of the synthetic antioxidants could substitute for vitamin E in decreasing enzymatic lipid peroxidation. PGI2 release by the aorta was lowered in vitamin E deficiency and was highest with DPPD supplementation. In the Langendorff perfused heart, however, PGI2 release was highest in the vitamin E-deficient group, possibly due to cardiomyopathy. TxA2 synthesis by washed platelets challenged with thrombin was independent of the antioxidant status of the animal. The data showed that dietary antioxidants selectively affect eicosanoid synthesis in different tissues.     

Mammary cancer promotion and MAPK activation associated with consumption of a corn oil-based high-fat diet

Our earlier work has shown a selective promotional effect on the genesis of mammary carcinomas bearing a wild-type, but not mutant, Ha-ras codon 12 in a 1-methyl-1-nitrosourea (MNU)-induced carcinogenesis model by high-fat diets (Nutr Cancer 23, 283-290, 1995). To test the hypothesis that activation of the mitogen-activated protein kinase (MAPK) pathway is associated with this promotional effect, we compared the in vivo MAPK phosphorylation state of carcinomas from rats consuming a low-fat (5% corn oil, modified AIN-93G) with that from rats consuming a high-fat (25% corn oil) diet. Specifically, 21-day-old female Sprague-Dawley rats were given an intraperitoneal injection of MNU and one week later were randomized to the two diets for six weeks. The number of mammary carcinomas per rat was 68% greater in the high-fat group, and Ha-ras mutation was rare in this short-term model. The levels of the phosphorylated MAPK2 (active) and of proliferating cell nuclear antigen (PCNA) were significantly higher in carcinomas from the high-fat group, and the two parameters were substantially correlated (r2 = 0.43, p < 0.01). The expression level of c-Raf was fourfold higher in the high-fat group but was only modestly associated with MAPK activation (r2 = 0.35, p < 0.05). The levels of the total MAPK1 and MAPK2, guanosine triphosphatase-activating protein, Ha-Ras, and MAPK kinase did not change. These results suggest that an upregulation of c-Raf expression by high fat may in part account for the in vivo MAPK activation, which in turn may enhance cell proliferation and mammary carcinogenesis.