Low concentrations of quercetin and ellagic acid synergistically influence proliferation,cytotoxicity and apoptosis in MOLT-4 human leukemia cells

Little information is available regarding possible synergistic or antagonistic biochemical interactions among polyphenols contained in fruits and vegetables. Identifying potential interactions among these compounds may help to define the efficiency of polyphenol-containing foods in cancer prevention as related to structure-function activity of the compounds. The objective of this study was to investigate interactions between quercetin and ellagic acid, two polyphenolics that are present predominantly in small fruits, on cell death and proliferation-related variables in the MOLT-4 human leukemia cell line. Assays were performed to determine cell cycle kinetics, proliferation, apoptotic DNA-fragmentation and caspase-3-activity after 12, 24 and 48 h. Ellagic acid significantly potentiated the effects of quercetin (at 5 and 10 micro mol/L each) in the reduction of proliferation and viability and the induction of apoptosis. Significant alterations in cell cycle kinetics were also observed. The synergy was confirmed by an isobolographic analysis of the cell proliferation data. The interaction of ellagic acid and quercetin demonstrated an enhanced anticarcinogenic potential of polyphenol combinations, which was not based solely on the additive effect of individual compounds, but rather on synergistic biochemical interactions.     

Ellagic Acid Induces Novel and Atypical PKC Isoforms and Promotes Caspase-3 Dependent Apoptosis by Blocking Energy Metabolism

Antioxidant ellagic acid is a herbal polyphenolic compound shown to possess growth-inhibiting and apoptotic activities in cancer. Protein kinase C (PKC) plays an important role in cell proliferation, apoptosis, and differentiation. Apoptosis of tumor cells is induced by inactivation of glycolytic enzyme of anaerobic metabolism, lactate dehydrogenase (LDH)-A, and by activating apoptotic protein caspase-3 via PKCδ. The present study aims to analyze the role of ellagic acid on regulation of novel and atypical isozymes of PKC to modulate apoptosis and anaerobic metabolism to prevent lymphoma growth as its role on classical PKCs is reported earlier. Expression of novel and atypical isozymes of PKC, activity of PKCδ, expression and activity of caspase-3, and LDH-A have been analyzed. Expression is measured by RT-PCR, activities of PKCδ as level of its catalytic fragment, caspase-3 as level of its p17 fragment, and LDH-A by specific staining. Lymphoma bearing mice were treated with 3 different doses of ellagic acid. The treatment enhanced expression of all novel and atypical PKCs, activity and expression of caspase-3, and activity of PKCδ but decreased activity and expression of LDH-A. Our results suggest that ellagic acid induces apoptosis via novel and atypical PKCs in association with caspase-3 and induces cancer cell death by blocking the energy metabolism.     

Soybean phytochemicals inhibit the growth of transplantable human prostate carcinoma and tumor angiogenesis in mice.

The objectives of our studies are to characterize the ability of dietary soybean components to inhibit the growth of prostate cancer in mice and alter tumor biomarkers associated with angiogenesis. Soy isoflavones (genistein or daidzein) or soy phytochemical concentrate inhibit the growth of prostate cancer cells LNCaP, DU 145 and PC-3 in vitro, but only at supraphysiologic concentrations, i.e., 50% inhibitory concentration (IC(50)) > 50 micromol/L. G2-M arrest and DNA fragmentation consistent with apoptosis of prostate cancer cells are also observed at concentrations causing growth inhibition. In contrast, the in vitro proliferation of vascular endothelial cells was inhibited by soy phytochemcials at much lower concentrations. We evaluated the ability of dietary soy phytochemical concentrate and soy protein isolate to inhibit the growth of the LNCaP human prostate cancer in severe combined immune-deficient mice. Mice inoculated subcutaneously with LNCaP cells (2 x 10(6)) were randomly assigned to one of the six dietary groups based on the AIN-76A formulation for 3 wk. A 2 x 3 factorial design was employed with two protein sources (20%, casein vs. soy protein) and three levels of soy phytochemical concentrate (0, 0.2 and 1.0% of the diet). Soy components did not alter body weight gain or food intake. Compared with casein-fed controls, the tumor volumes after 3 wk were reduced by 11% (P = 0.45) by soy protein, 19% (P = 0.17) by 0.2% soy phytochemical concentrate, 28% by soy protein with 0.2% soy phytochemical concentrate (P < 0.05), 30% by 1.0% soy phytochemical concentrate (P < 0.05) and 40% by soy protein with 1.0% soy phytochemical concentrate (P < 0.005). Histologic examination of tumor tissue showed that consumption of soy products significantly reduced tumor cell proliferation, increased apoptosis and reduced microvessel density. The angiogenic protein insulin-like growth factor-I was reduced in the circulation of mice fed soy protein and phytochemical concentrate. Our data suggest that dietary soy products may inhibit experimental prostate tumor growth through a combination of direct effects on tumor cells and indirect effects on tumor neovasculature.     

Ellagic acid suppresses oxidised low-density lipoprotein-induced aortic smooth muscle cell proliferation: studies on the activation of extracellular signal-regulated kinase 1/2 and proliferating cell nuclear antigen expression

Proliferation of intimal vascular smooth muscle cells is an important component in the development of atherosclerosis. Ellagic acid is a phenolic compound present in fruits (raspberries, blueberries, strawberries) and walnuts. The present study investigated the effect of ellagic acid on the oxidised LDL (ox-LDL)-induced proliferation of rat aortic smooth muscle cells (RASMC). The study found that ellagic acid significantly inhibited ox-LDL-induced proliferation of RASMC and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Furthermore, ellagic acid also blocked the ox-LDL-induced (inducible) cell-cycle progression and down regulation of the expression of proliferating cell nuclear antigen (PCNA) in RASMC. Therefore, ellagic acid reduced the amount of ox-LDL-induced proliferation of RASMC via inactivation of the ERK pathway and suppression of PCNA expression. These results may significantly advance the understanding of the role that antioxidants play in the prevention of atherosclerosis.     

Extraction, stability, and quantitation of ellagic acid in various fruits and nuts

This study was undertaken to determine the amount of ellagic acid, a naturally occurring inhibitor of carcinogenesis, in various fruits and nuts. Ellagic acid was extracted from freeze-dried berries, pears, peaches, plums, grapes, apples, kiwi, and several nuts using either acetone/water or methanol. The extracts were treated with trifluoroacetic acid to hydrolyze the ellagic acid glucosides, and analyzed by high-performance liquid chromatography (HPLC). Both acetone/water and continuous soxhlet extraction with methanol were equally effective in extracting ellagic acid from strawberries. In raspberries, the amount of ellagic acid liberated by the two extraction methods was not significantly different after hydrolysis with trifluoroacetic acid. The extracts were stable over 90 days at −20, 4, and 25°C. Analysis showed that every food sample tested contained ellagic acid, but only the amounts present in strawberries (630 μg), raspberries (1500 μg), blackberries (1500 μg), walnuts (590 μg), pecans (330 μg), and cranberries (120 μg ellagic acid/g dry wt) were within the calibration range of the assay. In strawberries, 95.7% of the ellagic acid was found in the pulp while 4.3% was contained in the seeds. The seeds of raspberries contained 87.8% of the ellagic acid, and 12.2% was present in the pulp. The juice of both fruits contained negligible amounts of ellagic acid.     

Beware of pomegranates bearing 40% ellagic Acid

A recent profusion of pomegranate nutraceutical products, "standardized to 40% ellagic acid," has appeared in the marketplace. This Perspective reviews the chemical and functional studies of pomegranate as well as the virtues and dangers of ellagic acid, and concludes that synergy among the various pomegranate fractions and phytochemicals is the most important factor for assessing strength of pomegranate nutraceutical preparations, and not simply the concentration of ellagic acid. Ellagic acid concentration in final products is likely to have an optimal therapeutic range, which very likely is less than 40%. The wisdom of designing and engineering pomegranate nutraceutical products to maximize therapeutic or chemopreventive synergy is suggested, as opposed to preparations that are designed and engineered simply to maximize the concentration of a single phytochemical. The implications of this strategy may be generalized for the optimization of nutraceutical preparations from other medicinal plants as well.     

Ellagic acid attenuates high-carbohydrate, high-fat diet-induced metabolic syndrome in rats

Background

Fruits and nuts may prevent or reverse common human health conditions such as obesity, diabetes and hypertension; together, these conditions are referred to as metabolic syndrome, an increasing problem. This study has investigated the responses to ellagic acid, present in many fruits and nuts, in a diet-induced rat model of metabolic syndrome.

Methods

Eight- to nine-week-old male Wistar rats were divided into four groups for 16-week feeding with cornstarch diet (C), cornstarch diet supplemented with ellagic acid (CE), high-carbohydrate, high-fat diet (H) and high-carbohydrate, high-fat diet supplemented with ellagic acid (HE). CE and HE rats were given 0.8 g/kg ellagic acid in food from week 8 to 16 only. At the end of 16 weeks, cardiovascular, hepatic and metabolic parameters along with protein levels of Nrf2, NF-κB and CPT1 in the heart and the liver were characterised.

Results

High-carbohydrate, high-fat diet-fed rats developed cardiovascular remodelling, impaired ventricular function, impaired glucose tolerance, non-alcoholic fatty liver disease with increased protein levels of NF-κB and decreased protein levels of Nrf2 and CPT1 in the heart and the liver. Ellagic acid attenuated these diet-induced symptoms of metabolic syndrome with normalisation of protein levels of Nrf2, NF-κB and CPT1.

Conclusions

Ellagic acid derived from nuts and fruits such as raspberries and pomegranates may provide a useful dietary supplement to decrease the characteristic changes in metabolism and in cardiac and hepatic structure and function induced by a high-carbohydrate, high-fat diet by suppressing oxidative stress and inflammation.     

二十二碳六烯酸对人胰腺癌细胞增殖的抑制

目的探讨二十二碳六烯酸（DHA）对人胰腺癌细胞株Patu8988和SW1990生长的作用。方法采用MTT法检测DHA作用后Patu8988和SW1990细胞的增殖,流式细胞术检测DHA作用后Patu8988和SWl990细胞的凋亡、细胞周期和肿瘤相关蛋白环氧合酶2（COX-2）的表达量。结果DHA作用人胰腺癌细胞株24、48、72h后,细胞的增殖受到明显抑制（P〈0．01）,同时DHA能诱导细胞凋亡,随着作用时间延长和作用剂量增加,效果越明显。50μg／ml DHA作用24h后,胰腺癌细胞的COX-2表达量下降（P〈0．05）。结论DHA能有效地抑制胰腺癌细胞增殖,同时诱导细胞凋亡,可能与COX-2在胰腺癌细胞中的表达下调有关。     

Vitamin E Analog α -TEA,Methylseleninic Acid,and Trans-Resveratrol in Combination Synergistically Inhibit Human Breast Cancer Cell Growth

Alpha-tocopherol ether-linked acetic acid analog [2,5,7,8-tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid (α -TEA)] is a novel form of vitamin E effective at killing cancer cells but not normal cells. α -TEA alone and together with methylseleninic acid (MSA) and trans-resveratrol (t-RES) were investigated for ability to induce apoptosis, DNA synthesis arrest, and cellular differentiation and inhibit colony formation in human MDA-MB-435-F-L breast cancer cells in culture. The 3 agents alone were effective in inhibiting cell growth by each of the 4 different assays, and 3-way combination treatments synergistically inhibited cell proliferation in each assay in comparison to individual treatments. Furthermore, combinations of α -TEA, t-RES, and MSA significantly enhanced levels of apoptosis in human breast (MDA-MB-231, MCF7, and T47D) and prostate (LnCaP, PC-3, and DU-145) cancer cell lines as well as in immortalized but nontumorigenic MCF10A cells but not primary cultures of human mammary epithelial cells. Western immunoblotting confirmed the induction of apoptosis in that the 3 agents induced poly(adenosine diphosphate-ribose) polymerase cleavage, with earlier detection and more complete cleavage seen in the combination treatment. Mechanistic studies showed combination treatments to inhibit cell proliferation via downregulation of cyclin D1 and induce apoptosis via activation of caspases 8 and 9 and downregulation of prosurvival proteins FLIP and survivin. In summary, the combination of α -TEA, MSA, and t-RES is more effective than single treatments for inhibiting cell proliferation, inducing cellular differentiation, and inducing cell death by apoptosis in human cancer cells in culture.     

Ellagic acid and natural sources of ellagitannins as possible chemopreventive agents against intestinal tumorigenesis in the Min mouse

Ellagic acid has been shown to have chemopreventive effects in various experimental cancer models. We wanted to see whether pure ellagic acid and natural ellagitannins from cloudberry (Rubus chamaemorus) seed and pulp have any effect on adenoma formation in Apc-mutated Min mice. From the age of 5 wk, the mice were fed either a control diet, a diet containing pure ellagic acid at 1,564 mg/kg, or diets containing 4.7% (wt/wt) cloudberry seeds or 5.3% cloudberry pulp. The concentrations of ellagitannins and free ellagic acid in the seed diet were 807 and 42 mg/kg and in the pulp diet 820 and 34 mg/kg, respectively. After the 10-wk feeding period, ellagic acid had no effect on the number or size of adenomas in the distal or total small intestine, but it increased adenoma size in the duodenum when compared with the control diet (1.50+/-0.29 vs. 1.16+/-0.31 mm; P=0.029). Neither cloudberry seed nor pulp diets had any effect on the adenoma formation. Chemopreventive effects and mechanisms of whole cloudberry and other similar sources of phenolic compounds should, however, be studied, further taking into account food matrix and interactions with other dietary constituents that may be involved in the bioavailability and metabolism of ellagitannins.     

槐定碱通过PI3K/Akt/mTOR信号通路诱导自噬抑制胰腺癌细胞增殖

目的 探讨槐定碱对胰腺癌细胞增殖及自噬的影响,并分析其机制。方法 MTT法分析Sw1990细胞增殖, MDC染色法检测细胞自噬水平,western blot检测细胞自噬相关蛋白、PI3K/Akt/mTOR信号通路相关蛋白表达水平,运用自噬抑制剂（3 - MA）研究自噬对细胞增殖的影响;裸鼠成瘤实验检测体内胰腺癌细胞增殖情况,并分析瘤组织中LC3 II、p - mTOR蛋白水平。结果 槐定碱抑制胰腺癌Sw1990细胞的增长,促进自噬小泡的形成,上调LC3 II/ LC3 I、Beclin - 1水平,下调p - PI3K、p - AKT、p - mTOR水平（P＜0.05）;与槐定碱40 μmol/L组比较,槐定碱40 μmol/L + 3 - MA 5 μmol/L组细胞抑制率升高,LC3 II/ LC3 I降低,p - mTOR蛋白水平升高（P＜0.05）; 40 mg/kg槐定碱下调裸鼠瘤体体积、瘤体质量,上调LC3 II/ LC3 I水平,下调p - mTOR蛋白水平（P＜0.05）。结论 槐定碱能抑制Sw1990细胞增殖,与调控PI3K/Akt/mTOR信号通路影响自噬有关。     

Ellagic acid potentiates the effect of quercetin on p21waf1/cip1, p53, and MAP-kinases without affecting intracellular generation of reactive oxygen species in vitro

Anticarcinogenic effects attributed to polyphenols in fruits may be based on synergistic, additive, or antagonistic interactions of many compounds. In a previous study, it was demonstrated that quercetin and ellagic acid interacted synergistically in the induction of apoptosis in the human leukemia cell line, MOLT-4. To investigate possible cellular mechanisms, this study evaluated whether synergistic effects might be detectable within proapoptotic or antiproliferative signal transduction pathways. We found that quercetin and combinations of quercetin and ellagic acid nonsynergistically increased p53 protein levels. In contrast, ellagic acid potentiated the effects of quercetin for p21(cip1/waf1) protein levels and p53 phosphorylation at serine 15, possibly explaining the synergistic effect observed in apoptosis induction. Phosphorylation of the mitogen-activated protein (MAP) kinases, c-jun N-terminal (JNK)1,2 and p38, was also increased by the combination of ellagic acid and quercetin, whereas quercetin alone induced only p38. We further evaluated whether the generation of reactive oxygen species (ROS) and/or quercetin stability were influenced by interactions of ellagic acid with quercetin. Quercetin increased the generation of ROS, which was neither potentiated nor inhibited by ellagic acid. The stability of intracellular and extracellular quercetin was not influenced by the presence of ellagic acid. In summary, quercetin and ellagic acid combined increase the activation of p53 and p21(cip1/waf1) and the MAP kinases, JNK1,2 and p38, in a more than additive manner, suggesting a mechanism by which quercetin and ellagic acid synergistically induce apoptosis in cancer cells.     

Vitamin E analog alpha-TEA, methylseleninic acid, and trans-resveratrol in combination synergistically inhibit human breast cancer cell growth

Alpha-tocopherol ether-linked acetic acid analog [2,5,7,8-tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid (alpha-TEA)] is a novel form of vitamin E effective at killing cancer cells but not normal cells. alpha -TEA alone and together with methylseleninic acid (MSA) and trans-resveratrol (t-RES) were investigated for ability to induce apoptosis, DNA synthesis arrest, and cellular differentiation and inhibit colony formation in human MDA-MB-435-F-L breast cancer cells in culture. The 3 agents alone were effective in inhibiting cell growth by each of the 4 different assays, and 3-way combination treatments synergistically inhibited cell proliferation in each assay in comparison to individual treatments. Furthermore, combinations of alpha -TEA, t-RES, and MSA significantly enhanced levels of apoptosis in human breast (MDA-MB-231, MCF7, and T47D) and prostate (LnCaP, PC-3, and DU-145) cancer cell lines as well as in immortalized but nontumorigenic MCF10A cells but not primary cultures of human mammary epithelial cells. Western immunoblotting confirmed the induction of apoptosis in that the 3 agents induced poly(adenosine diphosphate-ribose) polymerase cleavage, with earlier detection and more complete cleavage seen in the combination treatment. Mechanistic studies showed combination treatments to inhibit cell proliferation via downregulation of cyclin D1 and induce apoptosis via activation of caspases 8 and 9 and downregulation of prosurvival proteins FLIP and survivin. In summary, the combination of alpha-TEA, MSA, and t-RES is more effective than single treatments for inhibiting cell proliferation, inducing cellular differentiation, and inducing cell death by apoptosis in human cancer cells in culture.     

Dietary Berries and Ellagic Acid Diminish Estrogen-Mediated Mammary Tumorigenesis &isin; ACI rats

Estrogen acts as a complete mammary carcinogen in ACI rats. Prevention studies in this model allowed us to identify agents that are effective against estrogen-induced mammary carcinogenesis. In this study, we investigated efficacy of dietary berries and ellagic acid to reduce estrogen-mediated mammary tumorigenesis. Female ACI rats (8–9 wk) were fed either AIN-93M diet (n = 25) or diet supplemented with either powdered blueberry (n = 19) and black raspberry (n = 19) at 2.5% wt/wt each or ellagic acid (n = 22) at 400 ppm. Animals received implants of 17β-estradiol 2 wk later, were palpated periodically for mammary tumors, and were euthanized after 24 wk. No differences were found in tumor incidence at 24 wk; however, tumor volume and multiplicity were reduced significantly after intervention. Compared with the control group (average tumor volume = 685 ± 240 mm3 and tumor multiplicity = 8.0 ± 1.3), ellagic acid reduced the tumor volume by 75% (P < 0.005) and tumor multiplicity by 44% (P < 0.05). Black raspberry followed closely, with tumor volume diminished by > 69% (P < 0.005) and tumor multiplicity by 37% (P = 0.07). Blueberry showed a reduction (40%) only in tumor volume. This is the first report showing the significant efficacy of both ellagic acid and berries in the prevention of solely estrogen-induced mammary tumors.     

COX-2及凋亡基因表达在二十二碳六烯酸抑制人胰腺癌细胞体外增殖中的变化

目的研究COX-2和凋亡相关基因Bcl-2、Bax在二十二碳六烯酸(DHA)抑制人胰腺癌细胞体外增殖中表达的变化,探讨DHA抑制肿瘤细胞的作用机制。方法采用四甲基偶氮唑蓝(MTT)实验、流式细胞技术对细胞增殖、细胞凋亡、细胞周期以及肿瘤相关蛋白COX-2和凋亡基因Bcl-2、Bax进行分析。结果DHA能以时间-剂量依赖关系抑制人胰腺癌细胞增殖,同时诱导细胞凋亡。DHA对正常人成纤维细胞的增殖无明显影响。流式细胞仪检测显示:G0-G1期细胞比例增高,S期细胞比例下降。DHA作用24h后,胰腺癌细胞的COX-2、Bcl-2、Bax蛋白表达下降。结论DHA下凋COX-2、Bcl-2、Bax的表达可能是其抑制胰腺癌细胞增殖的作用机制之一。     

Ellagic acid,phenolic acids,and flavonoids in Malaysian honey extracts demonstrate in vitro anti-inflammatory activity

Natural honey has been used in traditional medicine of different cultures throughout the world. This study looked into the extraction of Malaysian honey and the evaluation of the anti-inflammatory activity of these extracts. It was hypothesized that honey extracts contain varying amounts of phenolic compounds and that they possess different in vitro anti-inflammatory activities. Honey extracts were analyzed using liquid chromatography–mass spectrometry to identify and compare phenolic compounds, whereas high-performance liquid chromatography was used for their quantification. Subsequently, honey methanol extract (HME) and honey ethyl acetate extract (HEAE) were tested in vitro for their effect on nitric oxide production in stimulated macrophages. The extracts were also tested for their effects on tumor necrosis factor–α (TNF) cytotoxicity in L929 cells. The major phenolics in the extracts were ellagic, gallic, and ferulic acids; myricetin; chlorogenic acid; and caffeic acid. Other compounds found in lower concentrations were hesperetin, p-coumaric acid, chrysin, quercetin, luteolin, and kaempferol. Ellagic acid was the most abundant of the phenolic compounds recorded, with mean concentrations of 3295.83 and 626.74 μg/100 g of honey in HME and HEAE, respectively. The median maximal effective concentrations for in vitro nitric oxide inhibition by HEAE and HME were calculated to be 37.5 and 271.7 μg/mL, respectively. The median maximal effective concentrations for protection from TNF cytotoxicity by HEAE and HME were 168.1 and 235.4 μg/mL, respectively. In conclusion, HEAE exhibited greater activity in vitro, whereas HME contained a higher concentration of phenolic compounds per 100 g of honey.     

Icariside II Induces Apoptosis of Melanoma Cells Through the Downregulation of Survival Pathways

This study evaluated the antitumor effects of icariside II (IS), isolated from Herba Epimedii, on in vitro and in vivo models of melanoma and determined its mechanism of apoptosis. Mouse (B16) and human (A375, SK-MEL-5) melanoma cell lines were treated with IS at different concentrations (0–100 μM). Cell viability and proliferation was detected by WST-1 assay and with the xCELLigence system, respectively. Apoptosis was measured by the annexin-V/PI flow cytometric assay. Western blot was used to measure cleaved caspase 3, survivin, P-STAT3, P-ERK and P-AKT. B16 and A375 cells were injected subcutaneously into C57BL/6J and BALB/c-nu mice, respectively. After 1 wk, IS solution at (50 mg/kg, 100 mg/kg) was administered by intraperitoneal injection 3 times for a week. Tumor size was measured with an electronic digital caliper. IS inhibited the proliferation of melanoma cells in a dose- and time-dependent manner. Treatment of A375 cells with IS resulted in an increased number of apoptotic cells ranging from 5.6% to 26.3% mirrored by increases in cleaved caspase-3 and a decrease in survivin expression. IS significantly inhibited the activation of the JAK-STAT3 and MAPK pathways but promoted an unsustained activation peak of the PI3K-AKT pathway. IS administration (50 mg/kg) resulted in a 47.5% decreased tumor volume in A375 bearing mice. Furthermore, IS administration (50 mg/kg, 100 mg/kg) resulted in 41% and 49% decreased tumor volume in B16 bearing mice, respectively. IS dramatically inhibited the proliferation of melanoma cells in vivo and in vitro through the regulation of apoptosis. These effects demonstrate the ability of IS to effectively overcome the survival signals of tumor cells, which support further preclinical evaluation of IS in cancer as a new potential chemotherapeutic agent.