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Pomegranate Date Written 2007
Author Joe Holmes Date Revised 6-17-09

by Joe Holmes Research shows it aids in type 2 diabetes, obesity and fatty liver.

1. "Pomegranate flower ameliorates fatty liver in an animal model of type 2 diabetes and obesity. Xu KZ, Zhu C, Kim MS, Yamahara J, Li Y. Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia.

AIMS OF THE STUDY: Fatty liver is the most common cause of abnormal liver function tests. We investigated the effect and its underlying mechanism of pomegranate flower (PGF), a traditional antidiabetic medicine, on fatty liver. MATERIALS AND METHODS: At the endpoint of treatment of male Zucker diabetic fatty (ZDF) rats with PGF extract (500 mg/kg, p.o. x 6 weeks), liver weight index, hepatic lipid contents (enzymatic colorimetric methods) and droplet accumulation (Oil Red O staining) were determined. Gene profiles (RT-PCR) were analyzed in the liver of ZDF rats and in human liver-derived HepG2 cell line. RESULTS: PGF-treated ZDF rats showed reduced ratio of liver weight to tibia length, hepatic triglyceride contents and lipid droplets. These effects were accompanied by enhanced hepatic gene expression of peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase (ACO), and reduced stearoyl-CoA desaturase-1. In contrast, PGF showed minimal effects on expression of genes responsible for synthesis, hydrolysis or uptake of fatty acid and triglycerides. PGF treatment also increased PPAR-alpha and ACO mRNA levels in HepG2 cells. CONCLUSION: Our findings suggest that this Unani medicine ameliorates diabetes and obesity-associated fatty liver, at least in part, by activating hepatic expression of genes responsible for fatty acid oxidation." PMID: 19429373

Pomegranate (Punica granatum) and Cancer

Introduction; Pomegranate contains useable amounts of a list of chemicals that reads like an all-star list of cancer killers. They all work well together, are easily absorbed, nontoxic to normal cells and deadly to most cancers including cancers that are heavily defended. The list includes, ellagic acid, caffeic acid (that produces caffeic acid phenyl ester in the body), luteolin, punicic acid (or trichosanic acid), palmitic acid, stearic acid, oleic acid, linoleic acid, and conjugated linolenic acids. 1/10

1: J Nutr Biochem. 2005 Jun;16(6):360-7. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice.
Seeram NP, Adams LS, Henning SM, Niu Y, Zhang Y, Nair MG, Heber D. Center for Human Nutrition, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.

Pomegranate (Punica granatum L.) fruits are widely consumed as juice (PJ). The potent antioxidant and anti-atherosclerotic activities of PJ are attributed to its polyphenols including punicalagin, the major fruit ellagitannin, and ellagic acid (EA). Punicalagin is the major antioxidant polyphenol ingredient in PJ. Punicalagin, EA, a standardized total pomegranate tannin (TPT) extract and PJ were evaluated for in vitro antiproliferative, apoptotic and antioxidant activities. Punicalagin, EA and TPT were evaluated for antiproliferative activity at 12.5-100 microg/ml on human oral (KB, CAL27), colon (HT-29, HCT116, SW480, SW620) and prostate (RWPE-1, 22Rv1) tumor cells. Punicalagin, EA and TPT were evaluated at 100 microg/ml concentrations for apoptotic effects and at 10 microg/ml concentrations for antioxidant properties. However, to evaluate the synergistic and/or additive contributions from other PJ phytochemicals, PJ was tested at concentrations normalized to deliver equivalent amounts of punicalagin (w/w). Apoptotic effects were evaluated against the HT-29 and HCT116 colon cancer cell lines. Antioxidant effects were evaluated using inhibition of lipid peroxidation and Trolox equivalent antioxidant capacity (TEAC) assays. Pomegranate juice showed greatest antiproliferative activity against all cell lines by inhibiting proliferation from 30% to 100%. At 100 microg/ml, PJ, EA, punicalagin and TPT induced apoptosis in HT-29 colon cells. However, in the HCT116 colon cells, EA, punicalagin and TPT but not PJ induced apoptosis. The trend in antioxidant activity was PJ>TPT>punicalagin>EA. The superior bioactivity of PJ compared to its purified polyphenols illustrated the multifactorial effects and chemical synergy of the action of multiple compounds compared to single purified active ingredients. PMID: 15936648

2: Invest New Drugs. 2005 Mar;23(2):121-2. Pomegranate (Punica granatum) pure chemicals show possible synergistic inhibition of human PC-3 prostate cancer cell invasion across Matrigel. Lansky EP, Harrison G, Froom P, Jiang WG. Rimonest Ltd., P.O.B. 9945, Haifa, Israel.

Four pure chemicals, ellagic acid (E), caffeic acid (C), luteolin (L) and punicic acid (P), all important components of the aqueous compartments or oily compartment of pomegranate fruit (Punica granatum), and each belonging to different representative chemical classes and showing known anticancer activities, were tested as potential inhibitors of in vitro invasion of human PC-3 prostate cancer cells in an assay employing Matrigel artificial membranes. All compounds significantly inhibited invasion when employed individually. When C, P, and L were equally combined at the same gross dosage (4 microg/ml) as when the compounds were tested individually, a supradditive inhibition of invasion was observed, measured by the Kruskal-Wallis non-parametric test. PMID: 15744587

3: J Ethnopharmacol. 1999 Jul;66(1):11-7. Antioxidant and eicosanoid enzyme inhibition properties of pomegranate seed oil and fermented juice flavonoids. Schubert SY, Lansky EP, Neeman I. Laboratories of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa.

The antioxidant and eicosanoid enzyme inhibition properties of pomegranate (Punica granatum) fermented juice and seed oil flavonoids were studied. The pomegranate fermented juice (pfj) and cold pressed seed oil (pcpso) showed strong antioxidant activity close to that of butylated hydroxyanisole (BHA) and green tea (Thea sinensis), and significantly greater than that of red wine (Vitis vitifera). Flavonoids extracted from pcpso showed 31-44% inhibition of sheep cyclooxygenase and 69-81% inhibition of soybean lipoxygenase. Flavonoids extracted from pfj showed 21-30% inhibition of soybean lipoxygenase though no significant inhibition of sheep cyclooxygenase. The pcpso was analyzed for its polyphenol content and fatty acid composition. Total polyphenols in pcpso showed a concentration by weight of approximately 0.015%. Pcpso fatty acid composition showed punicic acid (65.3%) along with palmitic acid (4.8%), stearic acid (2.3%), oleic acid (6.3%), linoleic acid (6.6%) and three unidentified peaks from which two (14.2%) are probably isomers of punicic acid (El-Shaarawy, M.I., Nahpetian, A., 1983). Studies on pomegranate seed oil. Fette Seifen Anstrichmittel 83(3), 123-126). PMID: 10432202 [PubMed - indexed for MEDLINE]

4: J Biol Chem. 2003 Feb 14;278(7):4603-10. Epub 2002 Dec 2. Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds. Iwabuchi M, Kohno-Murase J, Imamura J. Plantech Research Institute, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan.

Conjugated linolenic acids are present as major seed oils in several plant species. Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containing cis-delta9, trans-delta11, cis-delta13 double bonds in the C(18) carbon chain. Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-delta11, cis-delta13 double bonds. Expression of TkFac and PgFac in Arabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to approximately 10% (w/w) of the total seed oils. In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter. In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2delta(9cis, 12cis) and 18:2delta(9cis, 12cis) production. Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and delta12 oleate desaturase activity. Furthermore, we demonstrate that 16:2delta(9cis, 12cis) and 18:3delta(9cis, 12cis, 15cis) as well as 18:2delta(9cis, 12cis) are potential substrates for the conjugases to form trans-delta11 and cis-delta13 double bonds. PMID: 12464604

5: Cancer Sci. 2004 Jun;95(6):481-6. Pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats. Kohno H, Suzuki R, Yasui Y, Hosokawa M, Miyashita K, Tanaka T. Department of Pathology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan.

Pomegranate (Punica granatum L.) seed oil (PGO) contains more than 70% cis(c)9,trans(t)11,c13-18:3 as conjugated linolenic acids (CLN). Our previous short-term experiment demonstrated that seed oil from bitter melon (Momordica charantia) (BMO), which is rich in c9,t11,t13-CLN, inhibited the occurrence of colonic aberrant crypt foci (ACF) induced by azoxymethane (AOM). In this study, we investigated the effect of dietary PGO on the development of AOM-induced colonic malignancies and compared it with that of conjugated linoleic acid (CLA). To induce colonic tumors, 6-week old male F344 rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for 2 weeks. One week before the AOM treatment they were started on diet containing 0.01%, 0.1%, or 1% PGO or 1% CLA for 32 weeks. Upon termination of the bioassay (32 weeks) colon tumors were evaluated histopathologically. AOM exposure produced colonic adenocarcinoma with an incidence of 81% and multiplicity of 1.88 +/- 1.54 at week 32. Administration of PGO in the diet significantly inhibited the incidence (AOM + 0.01% PGO, 44%, P < 0.05; AOM + 0.1% PGO, 38%, P < 0.01; AOM + 1% PGO, 56%) and the multiplicity (AOM + 0.01% PGO, 0.56 +/- 0.73, P < 0.01; AOM + 0.1% PGO, 0.50 +/- 0.73, P < 0.005; AOM + 1% PGO, 0.88 +/- 0.96, P < 0.05) of colonic adenocarcinomas, although a clear dose-response relationship was not observed at these dose levels. CLA feeding also slightly, but not significantly, reduced the incidence and multiplicity of colonic adenocarcinomas. The inhibition of colonic tumors by PGO was associated with an increased content of CLA (c9,t11-18:2) in the lipid fraction of colonic mucosa and liver. Also, administration of PGO in the diet elevated expression of peroxisome proliferator-activated receptor (PPAR) gamma protein in the non-tumor mucosa. These results suggest that PGO rich in c9,t11,c13-CLN can suppress AOM-induced colon carcinogenesis, and the inhibition is associated in part with the increased content of CLA in the colon and liver and/or increased expression of PPARgamma protein in the colon mucosa. PMID: 15182427 [PubMed - indexed for MEDLINE]

6: Eur J Cancer Prev. 2004 Aug;13(4):345-8. Breast cancer chemopreventive properties of pomegranate (Punica granatum) fruit extracts in a mouse mammary organ culture. Mehta R, Lansky EP. Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA.

We previously reported anticancer effects of pomegranate extracts in human breast cancer cells in vitro and also chemopreventive activity of pomegranate fermented juice polyphenols (W) in a mouse mammary organ culture (MMOC). In the present study we decided to expand the MMOC investigations to also include an evaluation of the potential chemopreventive efficacy of a purified chromatographic peak of W (Peak B), and also of whole pomegranate seed oil. In brief, an MMOC was established according to a known method. For the first 10 days of culture, the glands were treated with pomegranate fermented juice polyphenols (W), a high-performance liquid chromatographic (HPLC) peak separated from W (peak B), or pomegranate seed oil (Oil, and on day 3, exposed to the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), and for 10 days treated with the putative pomegranate chemopreventive. The glands were subsequently harvested and tumours counted by visual inspection. While W effected a 42% reduction in the number of lesions compared with control, peak B and pomegranate seed oil each effected an 87% reduction. The results highlight enhanced breast cancer preventive potential both for the purified compound peak B and for pomegranate seed oil, both greater than that previously reported for pomegranate fermented juice polyphenols. PMID: 15554563

7: J Med Food. 2004 Fall;7(3):274-83. Pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells. Albrecht M, Jiang W, Kumi-Diaka J, Lansky EP, Gommersall LM, Patel A, Mansel RE, Neeman I, Geldof AA, Campbell MJ. Institute of Anatomy and Cell Biology, Philipps University, Marburg, Germany.

We completed a multicenter study of the effects of pomegranate cold-pressed (Oil) or supercritical CO(2)-extracted (S) seed oil, fermented juice polyphenols (W), and pericarp polyphenols (P) on human prostate cancer cell xenograft growth in vivo, and/or proliferation, cell cycle distribution, apoptosis, gene expression, and invasion across Matrigel, in vitro. Oil, W, and P each acutely inhibited in vitro proliferation of LNCaP, PC-3, and DU 145 human cancer cell lines. The dose of P required to inhibit cell proliferation of the prostate cancer cell line LNCaP by 50% (ED(50)) was 70 microg/mL, whereas normal prostate epithelial cells (hPrEC) were significantly less affected (ED(50) = 250 g/mL). These effects were mediated by changes in both cell cycle distribution and induction of apoptosis. For example, the androgen-independent cell line DU 145 showed a significant increase from 11% to 22% in G(2)/M cells (P <.05) by treatment with Oil (35 microg/mL) with a modest induction of apoptosis. In other cell lines/treatments, the apoptotic response predominated, for example, in PC-3 cells treated with P, at least partially through a caspase 3-mediated pathway. These cellular effects coincided with rapid changes in mRNA levels of gene targets. Thus, 4-hour treatment of DU 145 cells with Oil (35 microg/mL) resulted in significant 2.3 +/- 0.001-fold (mean +/- SEM) up-regulation of the cyclin-dependent kinase inhibitor p21((waf1/cip1)) (P <.01) and 0.6 +/- 0.14-fold down-regulation of c-myc (P <.05). In parallel, all agents potently suppressed PC-3 invasion through Matrigel, and furthermore P and S demonstrated potent inhibition of PC-3 xenograft growth in athymic mice. Overall, this study demonstrates significant antitumor activity of pomegranate-derived materials against human prostate cancer.
Publication Types: Multicenter Study PMID: 15383219

8: J Med Food. 2004 Spring;7(1):13-8. Differentiation-promoting activity of pomegranate (Punica granatum) fruit extracts in HL-60 human promyelocytic leukemia cells. Kawaii S, Lansky EP. Laboratory of Bio-Organic Chemistry, Tokyo Denki University, Saitama, Japan.

Differentiation refers to the ability of cancer cells to revert to their normal counterparts, and its induction represents an important noncytotoxic therapy for leukemia, and also breast, prostate, and other solid malignancies. Flavonoids are a group of differentiation-inducing chemicals with a potentially lower toxicology profile than retinoids. Flavonoid-rich polyphenol fractions from the pomegranate (Punica granatum) fruit exert anti-proliferative, anti-invasive, anti-eicosanoid, and pro-apoptotic actions in breast and prostate cancer cells and anti-angiogenic activities in vitro and in vivo. Here we tested flavonoid-rich fractions from fresh (J) and fermented (W) pomegranate juice and from an aqueous extraction of pomegranate pericarps (P) as potential differentiation-promoting agents of human HL-60 promyelocytic leukemia cells. Four assays were used to assess differentiation: nitro blue tetrazolium reducing activity, nonspecific esterase activity, specific esterase activity, and phagocytic activity. In addition, the effect of these extracts on HL-60 proliferation was evaluated. Extracts W and P were strong promoters of differentiation in all settings, with extract J showing only a relatively mild differentiation-promoting effect. The extracts had proportional inhibitory effects on HL-60 cell proliferation. The results highlight an important, previously unknown, mechanism of the cancer preventive and suppressive potential of pomegranate fermented juice and pericarp extracts. PMID: 15117547

9: Angiogenesis. 2003;6(2):121-8. Preliminary studies on the anti-angiogenic potential of pomegranate fractions in vitro and in vivo.
Toi M, Bando H, Ramachandran C, Melnick SJ, Imai A, Fife RS, Carr RE, Oikawa T, Lansky EP. Department of Surgery, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan.

We previously showed pomegranate seed oil and fermented juice polyphenols to retard oxidation and prostaglandin synthesis, to inhibit breast cancer cell proliferation and invasion, and to promote breast cancer cell apoptosis. Here we evaluated the anti-angiogenic potential of these materials in several ways. We checked a possible effect on angiogenic regulation by measuring vascular endothelial growth factor (VEGF), interleukin-4 (IL-4) and migration inhibitory factor (MIF) in the conditioned media of estrogen sensitive (MCF-7) or estrogen resistant (MDA-MB-231) human breast cancer cells, or immortalized normal human breast epithelial cells (MCF-10A), grown in the presence or absence of pomegranate seed oil (SESCO) or fermented juice polyphenols (W). VEGF was strongly downregulated in MCF-10A and MCF-7, and MIF upregulated in MDA-MB-231, overall showing significant potential for downregulation of angiogenesis by pomegranate fractions. An anti-proliferative effect on angiogenic cells was shown in human umbilical vein endothelial cell (HUVEC) and in myometrial and amniotic fluid fibroblasts, and inhibition of HUVEC tubule formation demonstrated in an in vitro model employing glass carrier beads. Finally, we showed a significant decrease in new blood vessel formation using the chicken chorioallantoic membrane (CAM) model in vivo. 'In sum, these varied studies employing different models in different laboratories overall demonstrate for the first time an anti-angiogenic potential of pomegranate fractions, suggesting further in vivo and clinical investigations (for updates:
PMID: 14739618

10:Am J Chin Med. 2003;31(1):37-46. Antileukemic activity of selected natural products in Taiwan. Chiang LC, Chiang W, Chang MY, Ng LT, Lin CC. Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC.

The aim of the present study was to evaluate the antileukemic activity of six chemical classes of pure compounds present in commonly used medicinal plants in Taiwan--such as the genus Plantago. Studies were conducted on a series of human leukemia and lymphoma cell lines. Results showed that water soluble compounds (aucubin, caffeic acid, chlorogenic acid, ferulic acid, p-coumaric acid and vanillic acid) exhibited a weak antileukemic activity (IC50: 26-56 microg/ml, SI: 2-11). On the other hand, water insoluble compounds such as triterpenoids (oleanolic acid and ursolic acid), monoterpene (linalool) and flavonoid (luteolin) possessed strong activity against human leukemia and lymphoma cell lines. Among them, linalool showed the strongest activity against histiocytic lymphoma cells U937 (IC50: 3.51 microg/ml, SI: 592.6) and Burkitt lymphoma cells P3HR1 (IC50: 4.21 microg/ml, SI: 494.1). Ursolic acid was effective against P3HR1 cells (IC50: 2.5 microg/ml, SI: 262.6) and chronic myelogenous leukemia cells K562 (IC50: 17.79 microg/ml, SI: 36.91), whereas oleanolic acid inhibited the growth of P3HR1 cells (IC50: 26.74 microg/ml, SI: 11.37). Luteolin exhibited effective activity against K562 cells (IC50 18.96 microg/ml, SI: 5.14) and P3HR1 cells (IC50: 18.99 microg/ml, SI: 5.13). We conclude that terpenes and flavonoid in commonly used medicinal plants possess strong activity against lymphoma and leukemia cells, especially human lymphoma cells, suggesting the potential use of these compounds for treatment of lymphoma.
PMID: 12723753

11:Ellagic acid 11/14 11: Anticancer Res. 2005 Mar-Apr;25(2A):971-9. Ellagic acid induced p53/p21 expression, G1 arrest and apoptosis in human bladder cancer T24 cells. Li TM, Chen GW, Su CC, Lin JG, Yeh CC, Cheng KC, Chung JG. School of Chinese Medicine, China Medical University, Taichung City 400, Taiwan, ROC.

It is well known that dietary phenolic compounds can elicit vital cellular responses such as cytotoxicity, cell cycle arrest and apoptosis by activating a cascade of molecular events. Ellagic acid is one of these phenolic compounds, but the exact mechanism of its action is still unclear. The objective of this study was to investigate ellagic acid-induced cell cycle arrest and apoptosis in T24 human bladder cancer cells in vitro. Assays were performed to determine cell viability, cell cycle arrest, apoptosis, caspases-3 activity and gene expression, measured by flow cytometric assay, polymerase chain reaction (PCR) and determination of caspase-3 activity. Ellagic acid significantly reduced the viable cells, induced G0/G1-phase arrest of the cell cycle and apoptosis. Ellagic acid also increased p53 and p21 and decreased CDK2 gene expression, that may lead to the G0/G1 arrest of T24 cells. Ellagic acid also promoted caspase-3 activity after exposure for 1, 3, 6, 12 and 24 h, which led to induction of apoptosis. Furthermore, the ellagic acid-induced apoptosis on T24 cells was blocked by the broad-spectrum caspase inhibitor (z-VAD-fmk). PMID: 15868936

12: Carcinogenesis. 2005 Apr;26(4):821-6. Epub 2005 Jan 20. Combined inhibition of PDGF and VEGF receptors by ellagic acid, a dietary-derived phenolic compound. Labrecque L, Lamy S, Chapus A, Mihoubi S, Durocher Y, Cass B, Bojanowski MW, Gingras D, Beliveau R. Laboratoire de Medecine Moleculaire, Hopital Ste-Justine-Universite du Quebec a Montreal, Centre de Cancerologie Charles-Bruneau, 3175 Chemin Cote-Ste-Catherine, Montreal, Quebec, Canada H3T 1C5.

The vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors play essential and complementary roles in angiogenesis and combined inhibition of these receptors has been shown to result in potent antitumor activity in vivo. In this study, we report that ellagic acid (EA), a natural polyphenol found in fruits and nuts, inhibits VEGF-induced phosphorylation of VEGFR-2 in endothelial cell (EC) as well as PDGF-induced phosphorylation of PDGFR in smooth muscle cells, leading to the inhibition of downstream signaling triggered by these receptors. EA also specifically inhibited VEGF-induced migration of ECs as well as their differentiation into capillary-like tubular structures and abolished PDGF-dependent smooth muscle cell migration. Interestingly, EA presents a greater selectivity for normal cells than for tumor cells since the migration of the U87 and HT1080 cell lines were much less affected by this molecule. The identification of EA as a naturally occurring dual inhibitor of VEGF and PDGF receptors suggests that this molecule possesses important antiangiogenic properties that may be helpful for the prevention and treatment of cancer. PMID: 15661805 [PubMed - indexed for MEDLINE]

13: J Nutr Biochem. 2004 Nov;15(11):672-8. In vitro anti-proliferative activities of ellagic acid. Losso JN, Bansode RR, Trappey A 2nd, Bawadi HA, Truax R. Department of Food Science, Louisiana State University, Agricultural Center, Baton Rouge, LA 70803, USA.

The potential cytotoxic and anti-proliferative activities of ellagic acid (a naturally occurring bioactive compound in berries, grapes, and nuts) was evaluated using human umbilical vein endothelial cells (HUVEC), normal human lung fibroblast cells HEL 299, Caco-2 colon, MCF-7 breast, Hs 578T breast, and DU 145 human prostatic cancer cells. Ellagic acid at concentration in the range 10-100 micromol/L did not affect the viability of normal fibroblast cells during a 24-hour incubation. An increase in adenosine triphosphate (ATP) bioluminescence of approximately 18-21% was observed in normal cells incubated with ellagic acid. In contrast, ellagic acid at 1-100 micromol/L dose-dependently inhibited HUVEC tube formation and proliferation on a reconstituted extracellular matrix and showed strong anti-proliferative activity against the colon, breast, and prostatic cancer cell lines investigated. The most sensitive cells were the Caco-2, and the most resistant were the breast cancer cells. Ellagic acid induced cancer cell death by apoptosis as shown by the microscopic examination of cell gross morphology. Ellagic acid induced reduced cancer cell viability as shown by decreased ATP levels of the cancer cells. After 24 hours incubation of 100 micromol/L of ellagic acid with Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells, ellagic acid suppressed fetal bovine serum (FBS) stimulation of cell migration. The apoptosis induction was
accompanied by a decreased in the levels of pro-matrix metalloproteinase-2 (pro-MMP-2 or gelatinase A), pro-matrix metalloproteinase-9 (pro-MMP-9 or gelatinase B), and vascular endothelial growth factor (VEGF(165)) in conditioned media. The results suggest that ellagic acid expressed a selective cytotoxicity and anti-proliferative activity, and induced apoptosis in Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells without any toxic effect on the viability of normal human lung fibroblast cells. It was also observed that the mechanism of apoptosis induction in ellagic acid-treated cancer cells was associated with decreased ATP production, which is crucial for the viability of cancer cells. PMID: 15590271

14: Life Sci. 2002 Mar 1;70(15):1821-39. Interactive gene expression pattern in prostate cancer cells exposed to phenolic antioxidants. Narayanan BA, Narayanan NK, Stoner GD, Bullock BP. Microarray Systems Laboratory, American Health Foundation, Valhalla, NY 10595, USA.

Dietary phenolic compounds are known to elicite vital cellular responses such as cell cycle arrest, apoptosis and differentiation by activating a cascade of molecular events. As there is an increasing interest to improve the efficacy of these compounds for use as potential chemopreventive agents, we wanted to understand the impact of phenolic compounds on target genes in prostate cancer. In this study we used human cDNA microarrays with 2400 clones consisting of 17 prosite motifs to characterize alterations in gene expression pattern in response to the phenolic antioxidants ellagic acid (EA) and resveratrol (RE). Over a 48-hr exposure of androgen - sensitive LNCaP cells to EA and RE, a total of 593 and 555 genes respectively, showed more than a two fold difference in expression. A distinct set of genes in both EA-and RE-treated cells may represent the signature profile of phenolic antioxidant-induced gene expression in LNCaP cells. Although extensive similarity was found between effects of EA - and RE - responsive genes in prostate cancer cells, out of 246 genes with overlapping responses, 25 genes showed an opposite effect. Quantitative RT-PCR was used to verify and validate the differential expression of selected genes identified from cDNA microarrays. In-depth analysis of the data from this study provided insight into the alterations in the p53 - responsive genes, p300, Apaf-1, NF-kBp50 and p65 and PPAR families of genes, suggesting the activation of multiple signaling pathways that leads to growth inhibition of LNCaP cells. This is a first study to look for changes in a large number of human genes in response to dietary compounds. PMID: 12002526

15: Caffeic acid 15/22 15: Clin Chim Acta. 2005 Jul 5; [Epub ahead of print] Caffeic acid phenyl ester in propolis is a strong inhibitor of matrix metalloproteinase-9 and invasion inhibitor: Isolation and identification. Jin UH, Chung TW, Kang SK, Suh SJ, Kim JK, Chung KH, Gu YH, Suzuki I, Kim CH. Department of Biochemistry and Molecular Biology, Dongguk University College of Oriental Medicine and National Research Laboratory for Glycobiology, Sukjang-Dong 707 Kyungju City, Kyungbuk 780-714, Korea.

BACKGROUND: Propolis has been used as a folk medicine and has several proven biological activities. Herbal remedies recommended for cancer therapies in Korea. METHODS: Matrix metalloproteinase (MMP)-9-inhibitory activity of propolis has been assessed. CAPE as an acting compound was isolated and molecular structure was determined. Anti-invasion activity of CAPE was assayed using hepatocarcinoma cells. RESULTS: Propolis ethanol extracts showed a strong inhibitory effect of MMP-9 activity, which is known to be involved in tumor cell invasion and metastasis in a concentration-dependent manner on zymography. Assay guided fractionation led to the isolation of a caffeic acid phenyl ester CAPE) as the compound responsible for the anti-MMP-9 activity. CAPE was obtained by reversed-phase HPLC, and its structure was elucidated by fast atom bombardment mass spectrometry and tandem mass spectrometry. The purified CAPE inhibited MMP-9 activity with the IC(50) of 1.0-2.0 nmol/l. CONCLUSIONS: CAPE possesses selective antiproliferative activity toward hepatocaricoma cell line Hep3B, but not primary cultured mouse hepatocytes. PMID: 16004979

16: Toxicology. 2005 Feb 28;207(3):383-90. A new matrix metalloproteinase-9 inhibitor 3,4-dihydroxycinnamic acid (caffeic acid) from methanol extract of Euonymus alatus: isolation and structure determination. Park WH, Kim SH, Kim CH. Department of Biochemistry, Molecular Biology and Diagnostics, Dongguk University, College of Oriental Medicine, Sukjang-Dong 707, Kyungju City, Kyungju, Kyungbuk 780-714, Republic of Korea.

In a previous paper [Cha, B.Y., Park, C.J., Lee, D.G., Lee, Y.C., Kim, D.W., Kim, J.D., Seo, W.G., Moon, S.K., Kim, C.H., 2003. Inhibitory effect of methanol extract from Euonymus alatus on matrix metalloproteinase-9, J. Ethnopharm. 85, 163-167], methanol extracts prepared from stems of Euonymus alatus showed a strong inhibitory effect of matrix metalloproteinase (MMP)-9 activity, which is known to be involved in tumor cell invasion and metastasis, in a concentration-dependent manner on zymography. Assay guided fractionation led to the isolation of a caffeic acid (CA) as the compound responsible for the anti-MMP-9 activity. CA was finally obtained by reversed-phase HPLC, and its structure was elucidated by fast atom bombardment mass spectrometry and tandem mass spectrometry. The purified CA inhibited MMP-9 activity with the IC50 of 10-20 nM. PMID: 15664266

17: Ann N Y Acad Sci. 2004 Dec;1030:501-7. Inhibition of cyclooxygenase-2 expression and restoration of gap junction intercellular communication in H-ras-transformed rat liver epithelial cells by caffeic acid phenethyl ester. Lee KW, Chun KS, Lee JS, Kang KS, Surh YJ, Lee HJ. Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea.

One of the most frequent defects in human cancers is the uncontrolled activation of the ras signaling pathways. Increased expression of cyclooxygenase-2 (COX-2) and inhibition of gap junction intercellular communication (GJIC) have been frequently observed in several forms of human malignancies. The present study investigated the effects of caffeic acid phenethyl ester (CAPE), a chemopreventive phytochemical derived from honey propolis, on COX-2 expression and GJIC in Harvey-ras-transformed WB-F344 rat liver epithelial cells (H-ras WB cells). H-ras induced COX-2 expression in WB-F344 rat liver epithelial cells (WB cells). H-ras WB cells also exhibited complete inhibition of GJIC and predominant unphosphorylation of connexin 43 (Cx43), a major protein modulating GJIC. CAPE significantly inhibited the constitutive expression of COX-2 and restored the disrupted GJIC through the phosphorylation of Cx43 at a concentration of 12.5 microM in H-ras WB cells. Although the molecular basis for the cancer chemopreventive activity of CAPE is not completely understood, several studies suggest that CAPE is a potent and specific inhibitor of the transcription factor nuclear factor kappaB (NF-kappaB) activation. We also found that CAPE significantly inhibited H-ras-induced NF-kappaB DNA-binding activity without affecting the activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, which are major intracellular molecules involved in the Ras signaling pathways. In conclusion, CAPE may exert cancer chemopreventive effects through the inhibition of COX-2 expression and the restoration of disrupted GJIC induced by H-ras, possibly by targeting NF-kappaB. PMID: 15659835

18: FASEB J. 2004 Nov;18(14):1670-81. Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism. Chung TW, Moon SK, Chang YC, Ko JH, Lee YC, Cho G, Kim SH, Kim JG, Kim CH. National Research Laboratory for Glycobiology and Department of Biochemistry and Molecular Biology, Dongguk University College of Oriental Medicine, Kyungju, Kyungbuk, Korea.

Our previous studies have clearly shown that the angiogenic enzymes, matrix metalloproteinase (MMP) -2/9, are directly involved in human hepatic tumorigenesis and metastasis and suggest that the MMP-2/9 inhibitors, which have
dual inhibitory activities on enzyme activity and transcription, represent the best candidates for achieving tumor regression. Many anti-cancer drugs have strong cellular cytotoxicity and side effects, indicating that strong anti-cancer drugs that have no or minimal cytotoxicity and side effects need to be developed. The specific aim of the present study was to develop powerful anti-cancer drugs with specific tumor regression and anti-metastatic potential having the dual inhibitory activities of specific MMP-2 and -9 enzyme activities and gene transcription at the molecular level. Caffeic acid (CA), a strong and selective MMP-9 activity and transcription inhibitor, was isolated from the plant Euonymus alatus and its derivative, caffeic acid phenethyl ester (CAPE), was synthesized. CA and CAPE selectively inhibited MMP-2 and -9 but not -1, -3, -7, or cathepsin K. Treatment of HepG2 cells with CA (100 microg/mL) and CAPE (5 microg/mL) suppressed phorbol 12-myristate 13-acetate (PMA) -induced MMP-9 expression by inhibiting the function of NF-kappaB, but not AP-1. We confirmed that CA and CAPE suppressed the growth of HepG2 tumor xenografts in nude mice in vivo. The subcutaneous and oral administrations of CA and CAPE significantly reduced the liver metastasis. These results confirm the therapeutic potential of the compounds and suggest that the anti-metastatic and anti-tumor effects of CA and CAPE are mediated through the selective suppression of MMP-9 enzyme activity and transcriptional down-regulation by the dual inhibition of NF-kappaB as well as MMP-9 catalytic activity. PMID: 15522912

19: J Radiat Res (Tokyo). 2004 Jun;45(2):253-60. Cell killing and radiosensitization by caffeic acid phenethyl ester (CAPE) in lung cancer cells. Chen MF, Wu CT, Chen YJ, Keng PC, Chen WC. Department of Radiation Oncology, Chang Gung Memorial Hospital, Chia-yi, Taiwan.

Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of honeybee propoplis. The cytotoxicity and radiation sensitization effects of CAPE were evaluated in human lung cancer A549 cells and normal lung fibroblast WI-38 cells. A549 cells treated with 6 microg/ml CAPE showed marked growth inhibition (60%) at 48 hr after treatments. During the same time, the number of viable cells decreased to 46% of the control value. In contrast, WI-38 cells showed 20% growth inhibition with no change in the number of viable cells under the same treatment conditions. At 72 hr after CAPE treatment (6 microg/ml), the percentage of apoptotic cells in A549 cultures increased significantly to 67% and an S/G2 arrest was also detected in the culture. Furthermore, there was a significant decrease in the level of intracellular glutathione and hydrogen peroxide contents within one hr after CAPE treatment, and the expression of cyclin B1 was reduced 6 hr after treatment. The radiation sensitization effect of CAPE on A549 cells was determined from the clonogenic survival curves, and the results showed a small but significant difference in radiation survival between cells treated with or without CAPE. Taken together, our results suggest that the effects of CAPE on differential cytotoxicity, apoptosis, and radiosensitization are associated with glutathione depletion that occurred shortly after treatments. PMID: 15304968

20: BJU Int. 2004 Aug;94(3):402-6. Caffeic acid phenethyl ester-induced PC-3 cell apoptosis is caspase-dependent and mediated through the loss of inhibitors of apoptosis proteins. McEleny K, Coffey R, Morrissey C, Fitzpatrick JM, Watson RW. Department of Surgery, Mater Misericordiae University Hospital, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.

OBJECTIVE: To investigate the effects of a novel agent, caffeic acid phethyl ester (CAPE) on nuclear factor (NF)-kappaB activation and apoptosis in the androgen-independent PC3 prostate cancer cell line. MATERIALS AND METHODS: PC-3 cells were assessed for NF-kappaB activation induced by paclitaxel and tumour necrosis factor-alpha (TNF-alpha), using a p65 enzyme-linked immunosorbent assay, with or without CAPE treatment. The corresponding apoptosis was assessed with propidium iodide DNA staining using flow cytometry. The pan-caspase inhibitor Z-AD-FMK was used to investigate the mechanism of apoptosis. Alterations in the expression of inhibitor of apoptosis proteins IAP), cIAP-1, cIAP-2 and XIAP, were detected using western blot analysis. RESULTS: CAPE prevented paclitaxel and NFalpha-mediated NF-kappaB activation. Its ability to induce apoptosis in a dose-dependent manner was associated with the loss of cIAP-1, cIAP-2 and XIAP expression. Pretreatment with Z-VAD-FMK prevented CAPE-induced apoptosis and the loss of the IAPs. CONCLUSIONS: CAPE is an effective inhibitor of NF-kappaB activation in PC-3 cells, but the mechanism of apoptosis, and the corresponding loss of IAP expression, is caspase-dependent. PMID: 15291876

21: J Agric Food Chem. 2003 Dec 31;51(27):7907-12. Inhibitory effect of caffeic acid phenethyl ester on angiogenesis, tumor invasion, and metastasis. Liao HF, Chen YY, Liu JJ, Hsu ML, Shieh HJ, Liao HJ, Shieh CJ, Shiao MS, Chen YJ. Department of Medical Research and Radiation Oncology, Mackay Memorial Hospital, Taipei 104, Taiwan.

Caffeic acid phenethyl ester (CAPE) derived from honeybee propolis has been used as a folk medicine and has several proven biological activities. The present study investigated the effect of CAPE on angiogenesis, tumor invasion, and metastasis. A cytotoxicity assay of CAPE in CT26 colon adenocarcinoma cells showed a dose-dependent decrease in cell viability but no significant influence on the growth of human umbilical vein epithelial cells (HUVEC). A low concentration of CAPE (1.5 microg/mL) inhibited 52.7% of capillary-like tube formation in HUVEC culture on Matrigel. CAPE (6 microg/mL)-treated CT26 cells showed not only inhibited cell invasion by 47.8% but also decreased expression of matrix metalloproteinase (MMP)-2 and -9. Vascular endothelial growth factor
(VEGF) production from CT26 cells was also inhibited by treatment with CAPE (6 microg/mL). Intraperitoneal injection of CAPE (10 mg/kg/day) in BALB/c mice reduced the pulmonary metastatic capacity of CT26 cells accompanied with a decreased plasma VEGF level. CAPE treatment also prolonged the survival of mice implanted with CT26 cells. These results indicate that CAPE has potential as an antimetastatic agent. PMID: 14690372

22: Biochem Pharmacol. 2003 Dec 15;66(12):2281-9. Involvement of tumor suppressor protein p53 and p38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells. Lee YJ, Kuo HC, Chu CY, Wang CJ, Lin WC, Tseng TH. Department of Chemistry, National Changhua University of Education, Changhua, Taiwan, ROC.

Caffeic acid phenethyl ester (CAPE), an active component of propolis, has many biological and pharmacological activities including antioxidant, anti-inflammation, antiviral action, and anticancer effect. Our previous studies showed that CAPE exhibited significant cytotoxicity in oral cancer cells. Herein we further investigated the cytotoxicity potential of CAPE and the mechanism of its action in C6 glioma cells. The data exhibited that C6 glioma cells underwent internucleosomal DNA fragmentation 24 hr after the treatment of CAPE (50 microM). The proportion of C6 glioma cells with hypodiploid nuclei was increased to 24% at 36 hr after the exposure. Further results showed that CAPE induced the release of cytochrome c from mitochondria into cytosol, and the activation of CPP32. CAPE application also enhanced the expression of p53, Bax, and Bak. Finally, the potential signaling components underlying CAPE induction of apoptosis were elucidated. We found that CAPE activated extracellular signal-regulated kinase (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in C6 glioma cells. More importantly, p38 kinase formed a complex with p53 after the treatment of CAPE for 0.5 hr. The expression of p53, phospho-serine 15 of p53, and Bax, and inactivate form of CPP32 was suppressed by a pretreatment of a specific p38 MAPK inhibitor, SB203580. The resultant data suggest that p38 MAPK mediated the CAPE-induced p53-dependent apoptosis in C6 glioma cells. PMID: 14637186 [PubMed - indexed for MEDLINE]

23:Luteolin 23/30 23: J Asian Nat Prod Res. 2005 Aug;7(4):615-26. Flavonoids from Vitex trifolia L. inhibit cell cycle progression at G2/M phase and induce apoptosis in mammalian cancer cells. Li WX, Cui CB, Cai B, Wang HY, Yao XS. Tianjin Institute for Biomedicinal Research, Tianjin 300384, China.

Six flavonoids, persicogenin (1), artemetin (2), luteolin (3), penduletin (4), vitexicarpin (5) and chrysosplenol-D (6), have been isolated for the first time as new cell cycle inhibitors from Vitex trifolia L., a Chinese folk medicine used to treat cancers, through a bioassay-guided separation procedure. They were identified by spectroscopic methods. The inhibitory effects of 1-6 on the proliferation of mammalian cancer cells have been evaluated by the SRB (sulforhodamine B) method and their effects on cell cycle and apoptosis investigated by flow cytometry with the morphological observation under light microscope and by agarose-gel electrophoresis to detect internucleosomal DNA fragmentation. Compounds 1-6 inhibited the proliferation of mouse tsFT210 cancer cells with the IC50s (microg ml(-1)) > 100 (inhibition rate at 100 microg ml(-1), 47.9%) for 1, >100 (inhibition rate at 100 microg ml(-1), 49.6 %) for 2, 10.7 for 3, 19.8 for 4, 0.3 for 5, and 3.5 for 6. Flow cytometric investigations for 1-6 demonstrated that 1-5 mainly inhibited cell cycle at the G2/M phase in a dose-dependent manner with a weak induction of apoptosis on the tsFT210 cells, while 6 induced mainly apoptosis of the same tsFT210 cells also in a dose-dependent manner together with a weak inhibition of the cell cycle at the G0/G1 and G2/M phases, demonstrating that 1-6 exert their anti-proliferative effect on tsFT210 cells through inhibiting cell cycle and inducing apoptosis. In contrast to the cell cycle G2/M phase inhibitory main effect on tsFT210 cells, 5 induced mainly apoptosis on human myeloid leukemia K562 cells with a weak inhibition of the cell cycle at the G2/M phase. The present result provides flavonoids 1-6 as new cell cycle inhibitors and 1 and 4 as new anticancer flavonoids, which not only provide the first example of cell cycle G2/M phase inhibitory and apoptosis-inducing constituents of V. trifolia L. but also explain the use of Vitex trifolia L. by Chinese people to treat cancers. PMID: 16087636

24: Oncogene. 2005 Jul 11; [Epub ahead of print] Luteolin induces apoptosis via death receptor 5 upregulation in human malignant tumor cells. Horinaka M, Yoshida T, Shiraishi T, Nakata S, Wakada M, Nakanishi R, Nishino H, Matsui H, Sakai T. [1] 1Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan [2] 2Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.

Luteolin, a naturally occurring flavonoid, induces apoptosis in various cancer cells. Little is known however concerning the underlying molecular mechanisms responsible for this activity. In this report, we reveal a novel mechanism by which luteolin-induced apoptosis occurs, and show for the first time that the apoptosis by luteolin is mediated through death receptor 5 (DR5) upregulation. Luteolin markedly induced the expression of DR5, along with Bcl-2-interacting domain cleavage and the activation of caspase-8, -10, -9 and -3. In addition, suppression of DR5 expression with siRNA efficiently reduced luteolin-induced caspase activation and apoptosis. Human recombinant DR5/Fc also inhibited luteolin-induced apoptosis. On the other hand, luteolin induced neither DR5 protein expression nor apoptosis in normal human peripheral blood mononuclear cells. These results suggest that DR5 induced by luteolin plays a role in luteolin-induced apoptosis, and raises the possibility that treatment with luteolin might be promising as a new therapy against cancer.Oncogene advance online publication, 11 July 2005; doi:10.1038/sj.onc.1208874. PMID: 16007131

25: Life Sci. 2005 Mar 4;76(16):1883-93. Epub 2005 Jan 20. Increase of Bax/ Bcl-XL ratio and arrest of cell cycle by luteolin in immortalized human hepatoma cell line. Chang J, Hsu Y, Kuo P, Kuo Y, Chiang L, Lin C. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, No. 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan.

Luteolin is a common constituent of many kinds of fruits and vegetables. It possesses the anti-neoplastic activities against several human cancers, but its activity against hepatocellular carcinoma (HCC) is seldom mentioned. To evaluate the activity against HCC and to provide information about the mechanism, we tested luteolin against five human hepatoma cell lines, namely HepG2, SK-Hep-1, PLC/PRF/5, Hep3B, and HA22T/VGH, with XTT assay and flow cytometry. The results showed that luteolin inhibited PLC/PRF/5, Hep3B and HA22T/VGH at a concentration of 1 microg/ml, but it needed 5 microg/ml to inhibit HepG2 and 10 microg/ml for SK-Hep1 (P <0.05). The inhibitive concentrations of 50% (IC50) of luteolin were between 7.29 microg/ml and 32.59 microg/ml, which were comparable with those of 5-FU (15.35 microg/ml to 32.84 microg/ml). The least effective cell line as affected by luteolin (SK-Hep1) was the most effective one when treating with 5-FU. The least effective cell line as affected by 5-FU (HA22T/VGH) was effectively affected by luteolin. It seemed that luteolin had some complementary activity to 5-FU against these HCC cell lines. The luteolin-treated PLC/PRF/5 cells exhibited typical changes of apoptosis with a characteristic DNA laddering pattern on gel electrophoresis. Luteolin also activated casepase-3, increased Bax protein with a concomitant decrease in Bcl-XL level. Increase in Bax/ Bcl-XL ratio and activation of caspase-3 supported the apoptotic finding on gel electrophoresis. Luteolin also induced cell cycle arrest at G0/G1 phase. We suggested that luteolin might exhibit anti-HCC activity as efficient as 5-FU by the mechanism of not only cell cycle arrest but also apoptosis. PMID: 15698865

26: Cancer Res. 2004 Nov 1;64(21):7936-46. Luteolin inhibits vascular endothelial growth factor-induced angiogenesis; inhibition of endothelial cell survival and proliferation by targeting phosphatidylinositol 3'-kinase activity. Bagli E, Stefaniotou M, Morbidelli L, Ziche M, Psillas K, Murphy C, Fotsis T. Laboratory of Biological Chemistry and Department of Ophthalmology, Medical School, University of Ioannina, Ioannina, Greece.

In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have previously shown that certain flavonoids inhibit in vitro angiogenesis. Here, we show that the flavonoid luteolin inhibited tumor growth and angiogenesis in a murine xenograft model. Furthermore, luteolin inhibited vascular endothelial growth factor (VEGF)-induced in vivo angiogenesis in the rabbit corneal assay. In agreement, luteolin inhibited both VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVECs) with an IC(50) of about 5 mumol/L. Luteolin inhibited VEGF-induced phosphatidylinositol 3'-kinase (PI3K) activity in HUVECs, and this inhibition was critical for both the antisurvival and antimitotic affects of the compound. Indeed, luteolin abolished VEGF-induced activation of Akt, a downstream target of PI3K conveying both survival and mitotic downstream signals. Because overexpression of a constitutively active form of Akt rescued HUVECs only from the antisurvival effects of luteolin, the result indicated that luteolin targeted mainly the survival signals of the PI3K/Akt pathway. With regard to its antimitotic activity, luteolin inhibited VEGF-induced phosphorylation of p70 S6 kinase (S6K), a downstream effector of PI3K responsible for G(1) progression. Indeed, VEGF-induced proliferation of HUVECs was sensitive to rapamycin, an inhibitor of p70 S6K activation. Surprisingly, luteolin did not affect VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases, a pathway that is considered important for the mitotic effects of VEGF. Thus, blockade of PI3K by luteolin was responsible for the inhibitory effects of the compound on VEGF-induced survival and proliferation of HUVECs. The antisurvival effects of luteolin were mediated via blockage of PI3K/Akt-dependent pathways, whereas inhibition of the PI3K/p70 S6K pathway mediated the antimitotic effects of the compound. PMID: 15520200

27: Oncogene. 2004 Oct 7;23(46):7712-21. Luteolin sensitizes tumor necrosis factor-alpha-induced apoptosis in human tumor cells.
Shi RX, Ong CN, Shen HM. Department of Community, Occupational and Family Medicine, Faculty of Medicine, National University of Singapore, 16 Medical Drive, Singapore 117597, Republic of Singapore.

Tumor necrosis factor-alpha (TNFalpha) activates both cell death and cell vcsurvival pathways, which render most cancer cells resistant to its cytotoxicity. In this study, we found that pretreatment with luteolin, a plant flavonoid, greatly sensitized TNFalpha-induced apoptotic cell death in a number of human cancer cell lines; including colorectal cancer COLO205, HCT116 cells and cervical cancer HeLa cells. In the search of the molecular mechanisms responsible for the sensitization effect of luteolin, we discovered that luteolin inhibited TNFalpha-induced activation of nuclear transcription factor-kappa B (NF-kappaB), the main survival factor in TNFalpha signaling. As a result, luteolin suppressed the expression of NF-kappaB-targeted antiapoptotic genes, including A20 and cellular inhibitor of apoptosis protein-1 (c-IAP1). The role of A20 and c-IAP1 was further confirmed by ectopic expression of these two genes, which significantly protected cell death induced by luteolin followed by TNFalpha. In addition, inhibition of NF-kappaB by luteolin led to augmentation and prolongation of c-Jun N-terminal kinase (JNK) activation induced by TNFalpha. Suppression of JNK activation, either by a synthetic JNK inhibitor (SP600125) or by overexpression of the dominant negative forms of JNK kinase 1 (JNKK1) and JNK kinase 2 (JNKK2), conferred significant protection against apoptotic cell death induced by luteolin and TNFalpha, suggesting that NF-kappaB and JNK are closely associated with the sensitization effect of luteolin. Data from this study reveal a novel function of luteolin and enhance the value of luteolin as an anticancer agent. PMID: 15334063

28: Biochem Pharmacol. 2004 Jun 1;67(11):2103-14. Transinactivation of the epidermal growth factor receptor tyrosine kinase and focal adhesion kinase phosphorylation by dietary flavonoids: effect on invasive potential of human carcinoma cells. Lee LT, Huang YT, Hwang JJ, Lee AY, Ke FC, Huang CJ, Kandaswami C, Lee PP, Lee MT. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

Focal adhesion kinase (FAK), a member of a growing family of structurally distinct protein tyrosine kinases (PTK), has been linked to specific phosphorylation events, and the elevation of FAK activity in human carcinoma cells correlated with increased invasive potential. Transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase activity is proposed to stimulate cell migration and the subsequent activation of downstream signaling pathways. Quercetin (Qu) and luteolin (Lu), are potent PTK inhibitors as well as putative chemopreventive agents. The present work, we demonstrate that Qu and Lu at concentration of 20 microM transinactivated EGFR tyrosine kinase activity with marked reduction in phosphotyrosyl level of 170, 125, 65, 60 and 42 kDa cellular proteins, and induced apoptosis in MiaPaCa-2 cells. The 125 kDa protein was further identified as a FAK by immunoprecipitation and immunoblotting analyses. Tumor cells treated with Lu or Qu dampened the phosphorylation of FAK. In addition, our data clearly demonstrated that tumor cells responded to Qu and Lu by parallel reductions in the levels of phosphorylated FAK and the secreted matrix metalloproteinase (MMP) that may lead to the suppression of invasive potential and cell migration in vitro. While the molecular mechanism of FAK regulation of MMP secretion in tumor cells remains unclear, our results suggested that blockade of the EGFR-signaling pathway may contributed to the net effect. As suggested in the current study, targeting EGFR and FAK with the objective of modulating their regulatory pathways could offer prospects for the treatment of EGFR-responsive cancers in the future. PMID: 15135307

29: Biochem Pharmacol. 2004 Apr 15;67(8):1607-17.
Structural requirements for the flavonoid-mediated modulation of glutathione S-transferase P1-1 and GS-X pump activity in MCF7 breast cancer cells. van Zanden JJ, Geraets L, Wortelboer HM, van Bladeren PJ, Rietjens IM, Cnubben NH. Division of Toxicology, Wageningen University, PO Box 8000, 6700 EA, Wageningen, The Netherlands.

The objective of this study was to investigate the structural requirements necessary for inhibition of glutathione S-transferase P1-1 (GSTP1-1) and GS-X pump (MRP1 and MRP2) activity by structurally related flavonoids, in GSTP1-1 transfected MCF7 cells (pMTG5). The results reveal that GSTP1-1 activity in MCF7 pMTG5 cells can be inhibited by some flavonoids. Especially galangin was able to inhibit almost all cellular GSTP1-1 activity upon exposure of the cells to a concentration of 25microM. Other flavonoids like kaempferol, eriodictyol and quercetin showed a moderate GSTP1-1 inhibitory potential. For GSTP1-1 inhibition, no specific structural requirements necessary for potent inhibition could be defined. Most flavonoids appeared to be potent GS-X transport inhibitors with IC(50) values ranging between 0.8 and 8microM. Luteolin and quercetin were the strongest inhibitors with IC(50) values of 0.8 and 1.3microM, respectively. Flavonoids without a C2-C3 double bond like eriodictyol, taxifolin and catechin did not inhibit GS-X pump activity. The results of this study demonstrate that the structural features necessary for high potency GS-X pump inhibition by flavonoids are (1) the presence of hydroxyl groups, especially two of them generating the 3',4'-catechol moiety; and (2) a planar molecule due to the presence of a C2-C3 double bond. Other factors, like lipophilicity and the total number of hydroxyl groups do not seem to be dominating the flavonoid-mediated GS-X pump inhibition. To identify the GS-X pump responsible for the DNP-SG efflux in MCF7 cells, the effects of three characteristic flavonoids quercetin, flavone and taxifolin on MRP1 and MRP2 activity were studied using transfected MDCKII cells. All three flavonoids as well as the typical MRP inhibitor (MK571) affected MRP1-mediated transport activity in a similar way as observed in the MCF7 cells. In addition, the most potent GS-X pump inhibitor in the MCF7 cells, quercetin, did not affect MRP2-mediated transport activity. These observations clearly indicate that the GS-X pump activity in the MCF7 cells is likely to be the result of flavonoid-mediated inhibition of MRP1 and not MRP2. Altogether, the present study reveals that a major site for flavonoid interaction with GSH-dependent toxicokinetics is the GS-X pump MRP1 rather than the conjugating GSTP1-1 activity itself. Of the flavonoids shown to be most active especially quercetin is frequently marketed in functional food supplements. Given the physiological levels expected to be reached upon supplement intake, the IC(50) values of the present study point at possible flavonoid-drug and/or flavonoid-xenobiotic interactions especially regarding transport processes involved in toxicokinetics.
PMID: 15041478

30: J Ethnopharmacol. 2004 Mar;91(1):65-8. Cytotoxic activities of flavonoids from two Scutellaria plants in Chinese medicine.
Sonoda M, Nishiyama T, Matsukawa Y, Moriyasu M. Department of Natural Medicinal Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.

The effects of 17 flavonoids, isolated from two flavonoid-rich Scutellaria species (Scutellaria baicalensis Georgi and Scutellaria rivularis Wall) used in traditional Chinese medicine, on HL-60 cells were assessed by WST-8. Ten of the
flavonoids inhibited the proliferation of HL-60, as shown by IC50 values used as indexes of the inhibition. 2',3',5,7-tetrahydroxy flavone (IC50=9.5 microM), apigenin (15.0 microM), viscidulin III (17.4 microM), wogonin (17.4 microM) and luteolin (18.4 microM) were more effective than baicalein (23.0 microM) which reportedly inhibits the proliferation of some cancer cell lines. Others were less effective, and oroxylin A stimulated the proliferation. Scutellaria rivularis, used for the treatment of tumors in the clinic, contained flavonoids that were more inhibitive than those in Scutellaria baicalensis. These results are demonstrative of some reasons for the use of Scutellaria rivularis as a crude antitumor drug. PMID: 15036470


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