Vitexin inhibits APEX1 to counteract the flow-induced endothelial inflammation.

Vascular endothelial cells are exposed to shear stresses with disturbed vs. laminar flow patterns, which lead to proinflammatory vs. antiinflammatory phenotypes, respectively. Effective treatment against endothelial inflammation and the consequent atherogenesis requires the identification of new therapeutic molecules and the development of drugs targeting these molecules. Using Connectivity Map, we have identified vitexin, a natural flavonoid, as a compound that evokes the gene-expression changes caused by pulsatile shear, which mimics laminar flow with a clear direction, vs. oscillatory shear (OS), which mimics disturbed flow without a clear direction. Treatment with vitexin suppressed the endothelial inflammation induced by OS or tumor necrosis factor-α. Administration of vitexin to mice subjected to carotid partial ligation blocked the disturbed flow-induced endothelial inflammation and neointimal formation. In hyperlipidemic mice, treatment with vitexin ameliorated atherosclerosis. Using SuperPred, we predicted that apurinic/apyrimidinic endonuclease1 (APEX1) may directly interact with vitexin, and we experimentally verified their physical interactions. OS induced APEX1 nuclear translocation, which was inhibited by vitexin. OS promoted the binding of acetyltransferase p300 to APEX1, leading to its acetylation and nuclear translocation. Functionally, knocking down APEX1 with siRNA reversed the OS-induced proinflammatory phenotype, suggesting that APEX1 promotes inflammation by orchestrating the NF-κB pathway. Animal experiments with the partial ligation model indicated that overexpression of APEX1 negated the action of vitexin against endothelial inflammation, and that endothelial-specific deletion of APEX1 ameliorated atherogenesis. We thus propose targeting APEX1 with vitexin as a potential therapeutic strategy to alleviate atherosclerosis.

The regulation of protein kinase casein kinase II by apigenin is involved in the inhibition of ultraviolet B-induced macrophage migration inhibitory factor-mediated hyperpigmentation.

Ultraviolet (UV) radiation elicits melanogenesis and pigmentation in the skin. Apigenin (4',5,7-trihydroxyflavone [AGN]) is a plant flavone contained in various herbs, fruits, and vegetables. We herein investigated antimelanogenic properties of AGN and the molecular mechanisms of the action of AGN. In UVB-treated mice, AGN inhibited cutaneous hyperpigmentation and macrophage migration inhibitory factor (MIF) expression as a melanogenesis-related key factor. In mouse keratinocytes, AGN inhibited the expression of MIF and also the related factors (e.g., stem cell factor and proteinase-activated receptor 2) induced by MIF. In addition to ellagic acid as a casein kinase II (CK2) inhibitor, AGN suppressed CK2 enzymatic activity and UVB-induced CK2 expression and subsequent phosphorylation of IκB and MIF expression. These results suggest that AGN inhibits UVB-induced hyperpigmentation through the regulation of CK2-mediated MIF expression in keratinocytes.

Apigenin produced by maize flavone synthase I and II protects plants against UV-B-induced damage.

Flavones, one of the largest groups of flavonoids, have beneficial effects on human health and are considered of high nutritional value. Previously, we demonstrated that maize type I flavone synthase (ZmFNSI) is one of the enzymes responsible for the synthesis of O-glycosyl flavones in floral tissues. However, in related species such as rice and sorghum, type II FNS enzymes also contribute to flavone biosynthesis. In this work, we provide evidence that maize has both one FNSI and one FNSII flavone synthases. Arabidopsis transgenic plants expressing each FNS enzyme were generated to validate the role of flavones in protecting plants against UV-B radiation. Here, we demostrate that ZmCYP93G7 (FNSII) has flavone synthase activity and is able to complement the Arabidopsis dmr6 mutant, restoring the susceptibility to Pseudomonas syringae. ZmFNSII expression is controlled by the C1/PL1 + R/B anthocyanin transcriptional complexes, and both ZmFNSI and ZmFNSII are regulated by UV-B. Arabidopsis transgenic plants expressing ZmFNSI or ZmFNSII that accumulate apigenin exhibit less UV-B-induced damage than wild-type plants. Together, we show that maize has two FNS-type enzymes that participate in the synthesis of apigenin, conferring protection against UV-B radiation.

Influence of the flavonoids apigenin, kaempferol, and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1.

OATP1A2 and OATP2B1 are uptake transporters of the human organic anion transporting polypeptide (OATP) family with a broad substrate spectrum including several endogenous compounds as well as drugs such as the antihistaminic drug fexofenadine and HMG-CoA reductase inhibitors. Both transporters are localized in the apical membrane of human enterocytes. Flavonoids, abundantly occurring in plants, have previously been shown to interact with drug metabolizing enzymes and transporters. However, the impact of flavonoids on OATP1A2 and OATP2B1 transport function has not been analyzed in detail. Therefore, HEK293 cell lines stably expressing OATP1A2 and OATP2B1 were used to investigate the influence of the Ginkgo flavonoids apigenin, kaempferol, and quercetin on the transport activity of OATP1A2 and OATP2B1. K(i) values of all three flavonoids determined from Dixon plot analyses using BSP as substrate indicated a competitive inhibition with quercetin as the most potent inhibitor of OATP1A2 (22.0µM) and OATP2B1 (8.7µM) followed by kaempferol (OATP1A2: 25.2µM, OATP2B1: 15.1µM) and apigenin (OATP1A2: 32.4µM OATP2B1: 20.8µM). Apigenin, kaempferol, and quercetin led to a concentration-dependent decrease of the OATP1A2-mediated fexofenadine transport with IC(50) values of 4.3µM, 12.0µM, and 12.6µM, respectively. The OATP1A2- and OATP2B1-mediated transport of atorvastatin was also efficiently inhibited by apigenin (IC(50) for OATP1A2: 9.3µM, OATP2B1: 13.9µM), kaempferol (IC(50) for OATP1A2: 37.3µM, OATP2B1: 20.7µM) and quercetin (IC(50) for OATP1A2: 13.5µM, OATP2B1: 14.1µM). These data indicate that modification of OATP1A2 and OATP2B1 transport activity by apigenin, kaempferol, and quercetin may be a mechanism for food-drug or drug-drug interactions in humans.

Inhibition of interleukin-4 production in activated T cells via down-regulation of NF-AT DNA binding activity by apigenin, a flavonoid present in dietary plants.

Apigenin, a flavonoid present in many dietary plants, exhibits anti-allergic activity and inhibits IL-4 production by basophils and mast cells. However, the mechanism by which apigenin suppresses IL-4 production especially in T cells remains unclear. In this study, the effect of apigenin and its underlying mechanism on IL-4 production were investigated in activated T cells. Apigenin significantly inhibited IL-4 production in both EL4 T thymoma cells and primary lymph node cells. Apigenin also inhibited IL-4 gene promoter activity in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this inhibitory effect was impaired in EL4 cells transfected with an IL-4 promoter construct deleted of NF-AT binding sites. In addition, apigenin inhibited NF-AT DNA binding activities, indicating that apigenin may inhibit IL-4 production in T cells via down-regulation of NF-AT DNA binding activity.

Up-regulation of insulin-like growth factor binding protein-3 by apigenin leads to growth inhibition and apoptosis of 22Rv1 xenograft in athymic nude mice.

Epidemiological studies suggest that increased intake of fruits and vegetables may be associated with a reduced risk of prostate cancer. Apigenin (4', 5, 7,-trihydroxyflavone), a common dietary flavonoid abundantly present in fruits and vegetables, has shown remarkable anti-proliferative effects against various malignant cell lines. However, the mechanisms underlying these effects remain to be elucidated. We investigated the in vivo growth inhibitory effects of apigenin on androgen-sensitive human prostate carcinoma 22Rv1 tumor xenograft subcutaneously implanted in athymic male nude mice. Apigenin was administered to mice by gavage at doses of 20 and 50 mug/mouse/day in 0.2 ml of a vehicle containing 0.5% methyl cellulose and 0.025% Tween 20 in two different protocols. In the first protocol, apigenin was administered for 2 wk before inoculation of tumor and was continued for 8 wk, resulting in significant inhibition of tumor volume by 44 and 59% (P<0.002 and 0.0001), and wet weight of tumor by 41 and 53% (P<0.05), respectively. In the second protocol, administration of apigenin began 2 wk after tumor inoculation and continued for 8 wk; tumor volume and wet weights of tumor were reduced by 39 and 53% (P<0.01 and 0.002) and 31 and 42% (P<0.05), respectively. The tumor inhibitory effect of apigenin was more pronounced in the first protocol of extended treatment, which was associated with increased accumulation of human IGFBP-3 in mouse serum along with significant increase in IGFBP-3 mRNA and protein expression in tumor xenograft. Apigenin intake by these mice also resulted in simultaneous decrease in serum IGF-I levels and induction of apoptosis in tumor xenograft. Importantly, tumor growth inhibition, induction of apoptosis, and accumulation of IGFBP-3 correlated with increasing serum and tumor apigenin levels. In both studies, animals did not exhibit any signs of toxicity or reduced food consumption. In cell culture studies, apigenin treatment resulted in cell growth inhibition and induction of apoptosis, which correlated with increased accumulation of IGFBP-3 in culture medium and cell lysate. These effects were associated with significant reduction in IGF-I secretion; inhibition of IGF-I-induced cell cycle progression and insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, along with an increase in sub-G1 peak by apigenin. Further, treatment of cells with IGFBP-3 antisense oligonucleotide reversed these effects and attenuated apigenin-mediated inhibition of IRS-1 phosphorylation conferring inhibitory effects of apigenin on IGF-signaling. This study presents the first evidence that the in vitro and in vivo growth inhibitory effects of apigenin involve modulation of IGF-axis signaling in prostate cancer.

Apigenin decreases hemin-mediated heme oxygenase-1 induction.

Hemin is a strong inducer of heme oxygenase-1 (HO-1) expression in vitro and in vivo. Whereas moderate overexpression of HO-1 is protective against oxidative stress, uncontrolled levels of HO-1 can be detrimental. Therefore, we evaluated the effects of apigenin (APG), a flavonoid involved in a number of phosphorylation pathways and also known to inhibit inducible genes, such as iNOS and COX-2, on HO-1 expression. Incubation of mouse embryonic fibroblasts with APG (5--40 microM) decreased hemin-induced HO-1 protein and mRNA expression. APG also reduced the induction of HO-1 promoter activity, as assessed by bioluminescence imaging, in NIH3T3 cells transfected with the 15-kb HO-1 promoter fused with the reporter gene luciferase (HO-1-luc). Furthermore, through the use of specific inhibitors, APG's effect was found to be unrelated to its PKC, CK 2, PI 3 K, p38, or ERK inhibitory activities. Quercetin (10--40 microM), also a flavonoid, also inhibited hemin-induced HO-1 expression. Additionally, in vivo studies using HO-1-luc transgenic mice showed that APG (50 mg/kg) decreased hemin-induced HO activity and HO-1 protein expression in the liver. These results suggest that hemin-induced HO-1 expression can be attenuated by flavonoids, such as APG.