Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Select Dietary Phytochemicals Function as Inhibitors of COX-1 but Not COX-2

  • Haitao Li,

    Affiliation Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America

  • Feng Zhu,

    Affiliation Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America

  • Yanwen Sun,

    Affiliation Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America

  • Bing Li,

    Affiliation Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America

  • Naomi Oi,

    Affiliation Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America

  • Hanyong Chen,

    Affiliation Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America

  • Ronald A. Lubet,

    Affiliation National Institutes of Health, National Cancer Institute, Bethesda, Maryland, United States of America

  • Ann M. Bode,

    Affiliation Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America

  • Zigang Dong

    zgdong@hi.umn.edu

    Affiliation Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America

Select Dietary Phytochemicals Function as Inhibitors of COX-1 but Not COX-2

  • Haitao Li, 
  • Feng Zhu, 
  • Yanwen Sun, 
  • Bing Li, 
  • Naomi Oi, 
  • Hanyong Chen, 
  • Ronald A. Lubet, 
  • Ann M. Bode, 
  • Zigang Dong
PLOS
x

Abstract

Recent clinical trials raised concerns regarding the cardiovascular toxicity of selective cyclooxygenase-2 (COX-2) inhibitors. Many active dietary factors are reported to suppress carcinogenesis by targeting COX-2. A major question was accordingly raised: why has the lifelong use of phytochemicals that likely inhibit COX-2 presumably not been associated with adverse cardiovascular side effects. To answer this question, we selected a library of dietary-derived phytochemicals and evaluated their potential cardiovascular toxicity in human umbilical vein endothelial cells. Our data indicated that the possibility of cardiovascular toxicity of these dietary phytochemicals was low. Further mechanistic studies revealed that the actions of these phytochemicals were similar to aspirin in that they mainly inhibited COX-1 rather than COX-2, especially at low doses.

Introduction

Consistent clinical studies have indicated that long-term administration of COX-2 inhibitors is associated with an enhanced risk of experiencing adverse cardiovascular events [1,2]. Although the exact mechanism still remains unclear, accumulating evidence supports the idea that COX-2 plays a cardioprotective role after cardiac injury [3-5]. Functional recovery after induced cardiac injury was improved in COX-2 transgenic mouse, but was greatly reduced by deficiency of COX-2. In the search to identify promising cancer chemopreventive agents, dietary phytochemicals have emerged as potential agents based on their observed anticancer activities as well as perceived safety [6]. Some mechanistic studies revealed that active dietary factors, such as EGCG, curcumin or resveratrol, might suppress carcinogenesis by targeting COX-2 [7-13]. One question that has been raised is why the lifelong use of phytochemicals that likely inhibit COX-2 has reportedly not been associated with adverse cardiovascular side effects. We hypothesize that those naturally occurring compounds might share a similar mechanism of action with aspirin, and might preferentially target COX-1 rather than COX-2. To test this idea, we selected a library of well-known active dietary factors and evaluated their potential cardiovascular toxicity as well as their effects on COX activity.

Materials and Methods

Reagents and chemicals

The COX-1 (#4841) and COX-2 (#12282) antibodies were purchased from Cell Signaling Technology (Beverley, MA). The antibody against β-actin (SC47778) was from Santa Cruz Biotechnology (Santa Cruz, CA). Interleukin-1 beta (IL-1β) was from Millipore (Billerica, MA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

Cell culture

COX-2 Wild-type (COX-2+/+) and COX-2 knockout (COX-2-/-) mouse embryonic fibroblasts (MEFs) were kind gifts from Drs. Jeff Reese and Sudhansu K. Dey (University of Kansas Medical Center) [14]. The cells were derived from COX-2 knockout mice supplied by Drs. Joseph E. Dinchuk and James M. Trzaskos (DuPont Merck Pharmaceutical Co.) [15]. The cells were cultured in monolayers at 37 °C, 5% CO2 using Dulbecco’s modified Eagle’s medium containing 10% FBS, 1% penicillin/streptomycin and 2mM L-glutamine. All other cell lines used in this study were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained following their instructions. Cells were cytogenetically tested and authenticated before the cells were frozen. The passage number was routinely limited to approximately 20 and morphology monitored with each passage.

Cell viability assay

Cells were seeded in 96-well-plate at a density of 5000 cells per well and allowed to incubate at 37 °C for 24 h for attachment. After drug treatment for 8 h, 20 µL CellTiter96 Aqueous One Solution (Invitrogen, Carlsbad, CA) were added, and cells were further incubated for 1 h at 37 °C. Finally, the optical density was determined at 492 nm.

Measurement of NO

HUVEC cells (6×105) were seeded in a six-well plate in the presence of 10% FBS. At 70-80% confluence, cells were pretreated with vehicle or individual compounds in 1 mL fresh medium for 2 h. After that, cells were incubated or not with IL-1β (17.5 ng/mL) for another 8 h. Supernatant fractions were collected for measurement of nitric oxide (NO). NO concentration was measured as nitrite using the Nitrate/Nitrite colorimetric assay kit (Cayman Chemicals, Ann Arbor, MI).

In vitro COX enzyme assay

COX activity was evaluated using a COX Inhibitor Screening Kit from Cayman Chemical Company (Ann Arbor, MI) according to the manufacturer’s instructions.

Thromboxane B2 (TXB2) and 6-keto prostaglandin F (6-keto PGF) assay

Cells (6×105) were seeded in a six-well plate in the presence of 10% FBS. At 70-80% confluence, cells were pretreated with vehicle or individual compounds in 1 mL fresh medium for 2 h. After that, cells were or were not incubated with IL-1β (17.5 ng/mL) for another 8 h. Supernatant fractions were collected for prostaglandin measurement using enzyme immunoassay kits (Cayman Chemical Company).

Western blot analysis

Protein samples (20 µg) were resolved by SDS-PAGE and transferred to Hybond C nitrocellulose membranes (Amersham Corporation, Arlington Heights, IL). After blocking, the membranes were probed with primary antibodies (1:1000) overnight at 4 °C. The targeted protein bands were visualized using an enhanced chemiluminescence reagent (Amersham Corporation) after hybridization with a secondary antibody conjugated with horseradish peroxidase.

Statistical analysis

All experiments were performed at least three times independently. Statistical analysis was performed using the Prism statistical package. Turkey’s t-test was used to compare data between two groups. One-way ANOVA and the Bonferroni correction were used to compare data between three or more groups. Values are expressed as means ± S.E.M. and a p < 0.05 was considered statistically significant.

Results

Evaluation of potential cardiovascular toxicity

The imbalance between COX-1-derived pro-thrombotic thromboxane A2 (TXA2) and COX-2-relateded anti-thrombotic prostacyclin (PGI2) production has long been suspected to contribute to cardiovascular side effects of COX-2 inhibitors [16,17]. COX-2-/- mice are more prone to cardiovascular risk than wild type mice, evidenced by increased cardiac ischemia and/or reperfusion injury [18]. Therfore, we firstly examined this idea in wildtype (COX2+/+) or knockout (COX-/-) mouse embryonic fibroblasts (MEFs, Figure 1A). COX-2 deficiency enhanced the ratio of thromboxane B2 (TXB2, the stable breakdown product of TXA2) to 6-keto prostaglandin F (6-keto PGF, the hydrolysis product of PGI2) by 22-fold. We further tested such hypothesis in an in vitro model using human umbilical vein endothelial cells (HUVECs). Again, the ratio of TXB2/6-keto-PGF1α was dramatically increased by the selective COX-2 inhibitor celecoxib, but not by aspirin, which is known to target COX-1 rather than COX-2 (Figure 1B). All of these findings support the reliability of the ratio of thromboxane B2 to 6-keto prostaglandin F as a biomarker for COX-2 inhibition-related cardiovascular toxicity.

thumbnail
Figure 1. Effect of COX-2 inactivation on TXB2 and 6-keto-PGF in murine embryo fibroblasts.

A. COX-2 deficiency enhanced the ratio of thromboxane B2 to 6-keto prostaglandin F. Western blot analysis of murine embryo fibroblasts (MEFs). Cells (6×105) were seeded in a six-well plate in the presence of 10% FBS. When cell reached 70-80% confluence, fresh culture medium (1 mL/well) was added. After further incubation for 24 h, supernatant fractions were collected for prostaglandin measurement. Data are presented as means ± S.E.M (n = 4) and the asterisk(s) indicate a significant (*, p < 0.05; ***, p < 0.001) difference versus the COX-2 wildtype group. B. COX-2 inhibition enhanced the ratio of thromboxane B2 to 6-keto prostaglandin F. Human umbilical vein endothelial cells (HUVECs) were seeded in a six-well plate in the presence of 10% FBS. At 70-80% confluence, cells were pretreated with 1 mL fresh medium containing DMSO or each individual compound for 2 h, and then IL-1β (17.5 ng/mL) was added together with each individual compound for another 8 h incubation. Supernatant fractions were collected for prostaglandin measurement. Data are presented as means ± S.E.M. (n = 4) and the asterisk(s) indicate a significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001) difference versus Control.

https://doi.org/10.1371/journal.pone.0076452.g001

The potential cardiovascular toxicity of dietary phytochemicals was then evaluated in this in vitro model. Considering their clinically achievable serum concentrations, all dietary factors were administrated at 3 µM [19-27]. Compared with celecoxib, all of dietary factors only weakly disturbed the ratio of TXB2/6-keto-PGF1α (Figure 2).

thumbnail
Figure 2. Effects of dietary phytochemicals on (A) TXB2 and (B) 6-keto-PGF1α under physiological conditions.

HUVECs were seeded in a six-well-plate (6×105 cells per well). At 70-80% confluence, 1 mL fresh medium containing DMSO or 3 µM of each individual compound was added and cells were further incubated for 8 h. Supernatant fractions were collected for prostaglandin measurement. (C) The ratio of TXB2/6-keto-PGF1α. Data are presented as means ± S.E.M (n = 4) and the asterisk(s) indicate a significant (*, p < 0.05; ***, p < 0.001) difference versus the vehicle control group.

https://doi.org/10.1371/journal.pone.0076452.g002

Considering the fact that people usually take selective COX-2 inhibitors to relieve pain and reduce inflammation, HUVECs were treated with IL-1β, an inflammatory cytokine implicated in vascular diseases, to mimic pro-inflammatory conditions. Stimulation of cells with IL-1β resulted in a remarkable increase in COX-2 expression as well as 6-keto-PGF1α synthesis. Similar to physiological conditions, the ratio of TXB2/6-keto-PGF1α was significantly enhanced by celecoxib, whereas was only weakly affected by aspirin as well as by dietary factors (Figure 3).

thumbnail
Figure 3. Effects of dietary phytochemicals on (A) TXB2 and (B) 6-keto-PGF1α under pro-inflammatory conditions.

HUVECs were seeded in a six-well-plate (6×105 cells per well). At 70-80% confluence, cells were pretreated with 1 mL fresh medium containing DMSO or 3 µM of each individual compound for 2 h and then IL-1β (17.5 ng/mL) was added together with each individual compound for another 8 h incubation. Supernatant fractions were collected for prostaglandin measurement. (C) The ratio of TXB2/6-keto-PGF1α. Data are presented as means ± S.E.M. (n = 4) and the asterisk(s) indicate a significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001) difference versus Group 1 (IL-1β).

https://doi.org/10.1371/journal.pone.0076452.g003

In addition to COX-2-related PGI2, endothelial-derived nitric oxide (NO) also acted as an endogenous vasodilator and protected the blood vessel wall by inhibiting platelet aggregation. In this study, we observed that IL-1β treatment caused a 2.6 fold increase in NO production compared with the control group. More importantly, most dietary phytochemicals had no effect on NO release (Figure 4A). We also excluded generalized cytotoxicity by examining the effects of dietary phytochemicals on cell viability and found that they had little effect on HUVEC cell viability after 8 hours of treatment (Figure 4B). Taken together, these findings suggested that the possible cardiovascular toxicity of dietary phytochemicals is low.

thumbnail
Figure 4. Effects of dietary phytochemicals on (A) nitric oxide (NO) production and (B) cell viability.

NO concentration was measured as nitrite using the Nitrate/Nitrite colorimetric assay kit as described in “Materials and Methods”. Data are presented as means ± S.E.M. (n = 3) and the asterisk(s) indicate a significant (**, p < 0.01) difference versus IL-1β group. Cell viability was tested as described in “Materials and Methods”. Data are presented as means ± S.E.M. (n = 3) and the asterisk(s) indicate a significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001) difference versus control (DMSO).

https://doi.org/10.1371/journal.pone.0076452.g004

Determination of the effect of phytochemicals on COX activity in vitro

The differential effects between dietary factors and celecoxib on cardiovascular toxicity biomarkers indicated that these dietary phytochemicals might not be specific COX-2 inhibitors. Accordingly, we determined their effect on COXs activity using a COX inhibitor screening assay kit. Results clearly indicated that most of the compounds are COX-1 inhibitors with a mild to moderate COX-1 selectivity index (Table 1). Among eight dietary phytochemicals, six selectively inhibited COX-1 activity rather than COX-2. Another two flavonols (naringenin and quercetin) are likely not COX1/2 inhibitors because their 50% inhibitory concentrations (IC50) against COX1/2 activity was higher than 400 µM.

CompoundNatural SourceCOX-1 IC50 (µM)COX-2 IC50 (µM)
Celecoxib95.4±12.70.02±0.009
AspirinWhite willow4.7±1.218.1±4.3
ApigeninCelery94.1±12.3146.4±16.5
CurcuminCurry330.1±34.3NA
GenisteinSoybean9.9±2.3256.2±35.7
EGCGGreen tea17.9±4.228.6±3.8
KaempferolBroccoli110.6±7.5235.8±19.7
NaringeninOrangeNANA
QuercetinBlack teaNANA
ResveratrolGrape3.4±1.18.5±2.3

Table 1. Inhibition of COX activity by dietary phytochemicals.

The effect of selected dietary factors on COX activity was evaluated using a COX Inhibitor Screening Kit (Cayman Chemical) according to the manufacturer’s instructions. IC50 values were calculated from a plot of percent inhibition versus the logarithm of concentration. Data are presented as means ± S.E.M. of 3 independent experiments.
CSV
Download CSV

Discussion

In this study, the cardiovascular safety of selected dietary factors was systemically evaluated for the first time. Our data indicated that the possible cardiovascular toxicity of dietary phytochemicals was low because the compounds tested might share a mechanism of action similar to aspirin and most appeared to preferentially target COX-1 rather than COX-2.

During the course of this study, EGCG, an active ingredient in green tea, exhibited an unexpected cardioprotective property and might merit further investigation. Among dietary factors studied, EGCG exhibited the most potent inhibitory effect against the ratio of TXB2/6-keto-PGF1α either under physiological or pathological conditions. This finding was consistent with several recent epidemiologic studies, which suggested regular consumption of green tea might provide cardioprotective effects [28,29]. This unanticipated finding provides critical insight into the potential application of green tea for cardioprotection.

Aspirin at low dose (81 mg per day) is widely accepted to be able to provide both cardioprotective and chemopreventive effects [17,23,30]. However, pharmacokinetic data analysis revealed that at this dose, aspirin might mainly targets COX-1 rather than COX-2, because the maximal serum concentration achieved was well below the reported whole blood COX-2 IC50 values [31,32]. In this study, we confirmed that most natural product-based compounds were COX-1, rather than COX-2 selective inhibitors. This raised the question of whether those natural occurring compounds exert their chemopreventive activity, at least in part, by targeting COX-1. Although no conclusion can be drawn due to insufficient data at this time, accumulating evidence suggests that COX-1 is involved in carcinogenesis [33-36]. For example, overexpression of COX-1 leads to tumorigenic transformation, whereas genetic disruption of ptgs-1 greatly reduced cancer incidence both in skin and colon. Although COX-1 is now becoming a target to be reconsidered for cancer prevention or treatment, selective COX-1 inhibition is still a controversial issue. For example, inhibition COX-1 has been strongly implicated in the gastric ulceration and bleeding induced by non-steroidal anti-inflammatory drugs (NSAIDs) because people believe that COX-1 is responsible for the prostaglandins essential for normal mucosal physiology in gut. As no gastrointestinal toxicity data were collected in this study, whether these phytochemicals cause gastrointestinal bleeding is still unknown and further study in these areas is required.

Author Contributions

Conceived and designed the experiments: HL FZ RAL AMB ZD. Performed the experiments: HL NO HC YS BL. Analyzed the data: HL FZ RAL ZD. Contributed reagents/materials/analysis tools: HC RAL ZD. Wrote the manuscript: HL AMB.

References

  1. 1. Bresalier RS, Sandler RS, Quan H, Bolognese JA, Oxenius B et al. (2005) Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N Engl J Med 352: 1092-1102. doi:10.1056/NEJMoa050493. PubMed: 15713943.
  2. 2. Solomon SD, McMurray JJ, Pfeffer MA, Wittes J, Fowler R et al. (2005) Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med 352: 1071-1080. doi:10.1056/NEJMoa050405. PubMed: 15713944.
  3. 3. Inserte J, Molla B, Aguilar R, Través PG, Barba I et al. (2009) Constitutive COX-2 activity in cardiomyocytes confers permanent cardioprotection Constitutive COX-2 expression and cardioprotection. J Mol Cell Cardiol 46: 160-168. doi:10.1016/j.yjmcc.2008.11.011. PubMed: 19084534.
  4. 4. Shinmura K, Tang XL, Wang Y, Xuan YT, Liu SQ et al. (2000) Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits. Proc Natl Acad Sci U S A 97: 10197-10202. doi:10.1073/pnas.97.18.10197. PubMed: 10963682.
  5. 5. Wang D, Patel VV, Ricciotti E, Zhou R, Levin MD et al. (2009) Cardiomyocyte cyclooxygenase-2 influences cardiac rhythm and function. Proc Natl Acad Sci U S A 106: 7548-7552. doi:10.1073/pnas.0805806106. PubMed: 19376970.
  6. 6. Lee KW, Bode AM, Dong Z (2011) Molecular targets of phytochemicals for cancer prevention. Nat Rev Cancer 11: 211-218. doi:10.1038/nrc3017. PubMed: 21326325.
  7. 7. Chun KS, Keum YS, Han SS, Song YS, Kim SH et al. (2003) Curcumin inhibits phorbol ester-induced expression of cyclooxygenase-2 in mouse skin through suppression of extracellular signal-regulated kinase activity and NF-kappaB activation. Carcinogenesis 24: 1515-1524. doi:10.1093/carcin/bgg107. PubMed: 12844482.
  8. 8. Chun KS, Kundu JK, Park KK, Chung WY, Surh YJ (2006) Inhibition of phorbol ester-induced mouse skin tumor promotion and COX-2 expression by celecoxib: C/EBP as a potential molecular target. Cancer Res Treat 38: 152-158. doi:10.4143/crt.2006.38.3.152. PubMed: 19771276.
  9. 9. Lee KW, Kim JH, Lee HJ, Surh YJ (2005) Curcumin inhibits phorbol ester-induced up-regulation of cyclooxygenase-2 and matrix metalloproteinase-9 by blocking ERK1/2 phosphorylation and NF-kappaB transcriptional activity in MCF10A human breast epithelial cells. Antioxid Redox Signal 7: 1612-1620. doi:10.1089/ars.2005.7.1612. PubMed: 16356124.
  10. 10. Mutoh M, Takahashi M, Fukuda K, Komatsu H, Enya T et al. (2000) Suppression by flavonoids of cyclooxygenase-2 promoter-dependent transcriptional activity in colon cancer cells: structure-activity relationship. Jpn J Cancer Res 91: 686-691. doi:10.1111/j.1349-7006.2000.tb01000.x. PubMed: 10920275.
  11. 11. Na HK, Mossanda KS, Lee JY, Surh YJ (2004) Inhibition of phorbol ester-induced COX-2 expression by some edible African plants. Biofactors 21: 149-153. doi:10.1002/biof.552210130. PubMed: 15630188.
  12. 12. Subbaramaiah K, Chung WJ, Michaluart P, Telang N, Tanabe T et al. (1998) Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Biol Chem 273: 21875-21882. doi:10.1074/jbc.273.34.21875. PubMed: 9705326.
  13. 13. Zhang F, Altorki NK, Mestre JR, Subbaramaiah K, Dannenberg AJ (1999) Curcumin inhibits cyclooxygenase-2 transcription in bile acid- and phorbol ester-treated human gastrointestinal epithelial cells. Carcinogenesis 20: 445-451. doi:10.1093/carcin/20.3.445. PubMed: 10190560.
  14. 14. Reese J, Paria BC, Brown N, Zhao X, Morrow JD et al. (2000) Coordinated regulation of fetal and maternal prostaglandins directs successful birth and postnatal adaptation in the mouse. Proc Natl Acad Sci U S A 97: 9759-9764. doi:10.1073/pnas.97.17.9759. PubMed: 10944235.
  15. 15. Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD et al. (1995) Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature 378: 406-409. doi:10.1038/378406a0. PubMed: 7477380.
  16. 16. McAdam BF, Catella-Lawson F, Mardini IA, Kapoor S, Lawson JA et al. (1999) Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2. Proc Natl Acad Sci U S A 96: 272-277. doi:10.1073/pnas.96.1.272. PubMed: 9874808.
  17. 17. Ulrich CM, Bigler J, Potter JD (2006) Non-steroidal anti-inflammatory drugs for cancer prevention: promise, perils and pharmacogenetics. Nat Rev Cancer 6: 130-140. doi:10.1038/nrc1801. PubMed: 16491072.
  18. 18. Camitta MG, Gabel SA, Chulada P, Bradbury JA, Langenbach R et al. (2001) Cyclooxygenase-1 and -2 knockout mice demonstrate increased cardiac ischemia/reperfusion injury but are protected by acute preconditioning. Circulation 104: 2453-2458. doi:10.1161/hc4401.098429. PubMed: 11705824.
  19. 19. Chen ZP, Sun J, Chen HX, Xiao YY, Liu D et al. (2010) Comparative pharmacokinetics and bioavailability studies of quercetin, kaempferol and isorhamnetin after oral administration of Ginkgo biloba extracts, Ginkgo biloba extract phospholipid complexes and Ginkgo biloba extract solid dispersions in rats. Fitoterapia 81: 1045-1052. doi:10.1016/j.fitote.2010.06.028. PubMed: 20603197.
  20. 20. Chow HH, Cai Y, Alberts DS, Hakim I, Dorr R et al. (2001) Phase I pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E. Cancer Epidemiol Biomarkers Prev 10: 53-58. PubMed: 11205489.
  21. 21. Gradolatto A, Basly JP, Berges R, Teyssier C, Chagnon MC et al. (2005) Pharmacokinetics and metabolism of apigenin in female and male rats after a single oral administration. Drug Metab Dispos 33: 49-54. PubMed: 15466493.
  22. 22. Graefe EU, Derendorf H, Veit M (1999) Pharmacokinetics and bioavailability of the flavonol quercetin in humans. Int J Clin Pharmacol Ther 37: 219-233. PubMed: 10363620.
  23. 23. Hall MN, Campos H, Li H, Sesso HD, Stampfer MJ et al. (2007) Blood levels of long-chain polyunsaturated fatty acids, aspirin, and the risk of colorectal cancer. Cancer Epidemiol Biomarkers Prev 16: 314-321. doi:10.1158/1055-9965.EPI-06-0346. PubMed: 17301265.
  24. 24. Kanaze FI, Bounartzi MI, Georgarakis M, Niopas I (2007) Pharmacokinetics of the citrus flavanone aglycones hesperetin and naringenin after single oral administration in human subjects. Eur J Clin Nutr 61: 472-477. PubMed: 17047689.
  25. 25. Moon YJ, Morris ME (2007) Pharmacokinetics and bioavailability of the bioflavonoid biochanin A: effects of quercetin and EGCG on biochanin A disposition in rats. Mol Pharm 4: 865-872. doi:10.1021/mp7000928. PubMed: 17970592.
  26. 26. Xu X, Harris KS, Wang HJ, Murphy PA, Hendrich S (1995) Bioavailability of soybean isoflavones depends upon gut microflora in women. J Nutr 125: 2307-2315. PubMed: 7666247.
  27. 27. Zubik L, Meydani M (2003) Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women. Am J Clin Nutr 77: 1459-1465. PubMed: 12791624.
  28. 28. Aneja R, Hake PW, Burroughs TJ, Denenberg AG, Wong HR et al. (2004) Epigallocatechin, a green tea polyphenol, attenuates myocardial ischemia reperfusion injury in rats. Mol Med 10: 55-62. doi:10.1007/s00894-003-0166-5. PubMed: 15502883.
  29. 29. Kuriyama S, Shimazu T, Ohmori K, Kikuchi N, Nakaya N et al. (2006) Green tea consumption and mortality due to cardiovascular disease, cancer, and all causes in Japan: the Ohsaki study. JAMA 296: 1255-1265. doi:10.1001/jama.296.10.1255. PubMed: 16968850.
  30. 30. Ridker PM, Cook NR, Lee IM, Gordon D, Gaziano JM et al. (2005) A randomized trial of low-dose aspirin in the primary prevention of cardiovascular disease in women. N Engl J Med 352: 1293-1304. doi:10.1056/NEJMoa050613. PubMed: 15753114.
  31. 31. Pedersen AK, FitzGerald GA (1984) Dose-related kinetics of aspirin. Presystemic acetylation of platelet cyclooxygenase. N Engl J Med 311: 1206-1211. doi:10.1056/NEJM198411083111902. PubMed: 6436696.
  32. 32. Gray PA, Warner TD, Vojnovic I, Del Soldato P, Parikh A et al. (2002) Effects of non-steroidal anti-inflammatory drugs on cyclo-oxygenase and lipoxygenase activity in whole blood from aspirin-sensitive asthmatics vs healthy donors. Br J Pharmacol 137: 1031-1038. doi:10.1038/sj.bjp.0704927. PubMed: 12429575.
  33. 33. Chulada PC, Thompson MB, Mahler JF, Doyle CM, Gaul BW et al. (2000) Genetic disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in Min mice. Cancer Res 60: 4705-4708. PubMed: 10987272.
  34. 34. Narko K, Ristimäki A, MacPhee M, Smith E, Haudenschild CC et al. (1997) Tumorigenic transformation of immortalized ECV endothelial cells by cyclooxygenase-1 overexpression. J Biol Chem 272: 21455-21460. doi:10.1074/jbc.272.34.21455. PubMed: 9261162.
  35. 35. Perrone MG, Scilimati A, Simone L, Vitale P (2010) Selective COX-1 inhibition: A therapeutic target to be reconsidered. Curr Med Chem 17: 3769-3805. doi:10.2174/092986710793205408. PubMed: 20858219.
  36. 36. Takeda H, Sonoshita M, Oshima H, Sugihara K, Chulada PC et al. (2003) Cooperation of cyclooxygenase 1 and cyclooxygenase 2 in intestinal polyposis. Cancer Res 63: 4872-4877. PubMed: 12941808.