The inhibitory potential of Broussochalcone A for the human cytochrome P450 2J2 isoform and its anti-cancer effects via FOXO3 activation.

BACKGROUND: Broussonetia papyrifera (L.) Ventenat, a traditional medicinal herb, has been applied as a folk medicine to treat various diseases. Broussochalcone A (BCA), a chalcone compound isolated from the cortex of Broussonetia papyrifera (L.) Ventenat, exhibits several biological activities including potent anti-oxidant, antiplatelet, and cytotoxic effects. PURPOSE: The purpose of this study is to elucidate the inhibitory effect of BCA against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines. STUDY DESIGN: The inhibitory effect of BCA on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its anti-cancer effect against human hepatoma HepG2 cells was also evaluated. METHODS: Two representative CYP2J2-specific probe substrates, astemizole and ebastine, were incubated in HLMs with BCA. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. To investigate the binding model between BCA and CYP2J2, we carried out structure-based docking simulations by using software and scripts written in-house. RESULTS: BCA inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activities in a concentration dependent manner with Ki values of 2.3 and 3.7 µM, respectively. It also showed cytotoxic effects against human hepatoma HepG2 cells in a dose-dependent manner with activation of apoptosis related proteins. CONCLUSION: Overall, this was the first report of the inhibitory effects of BCA on CYP2J2 in HLMs. The present data suggest that BCA is a potential candidate for further evaluation for its CYP2J2 targeting anti-cancer activities.

Selective inhibitory effects of machilin A isolated from Machilus thunbergii on human cytochrome P450 1A and 2B6.

BACKGROUND: The bark of Machilus thunbergii (Lauraceae) has been used as a folk medicine to treat abdominal pain and distension, and leg edema in Korea. Machilin A (MA), a lignan isolated from Machilus thunbergii, exhibits several biological activities including anti-oxidant and stimulatory effects on cell differentiation and proliferation. PURPOSE: Potential drug-interactions with MA via inhibition of cytochrome P450 (CYP) activity in human liver microsomes (HLMs), have not been investigated. STUDY DESIGN: The inhibitory effects of MA on the activities of CYPs were investigated using cocktail probe substrates in pooled HLMs and on human recombinant cDNA-expressed CYP isoforms. METHODS: The nine CYP-specific substrates were incubated in HLM or recombinant cDNA-expressed CYP 1A1, 1A2 and 2B6 with MA. After incubation, the samples were injected onto a C18 column for liquid chromatography-tandem mass spectrometry analysis. To investigate the binding poses between MA and CYP, we carried out structure-based docking simulations by using software and scripts written in-house (ALIS-DOCK; Automatic pLatform for Iterative Structure-based DOCKing). RESULTS: MA strongly inhibited CYP1A2-mediated phenacetin O-deethylation and CYP2B6-mediated bupropion hydroxylation with IC50 values of 3.0 and 3.9 µM, respectively, while it did not significantly inhibit other CYPs. A Dixon plot indicated that MA competitively inhibits CYP1A2 and CYP2B6 with Ki values of 0.71 and 4.1 µM, respectively. CONCLUSION: Overall, this was the first investigation of the inhibitory effects of MA on CYP1A2 and CYP2B6 in HLMs, and it has identified that MA acts via competitive inhibition.

Vascular barrier protective effects of pellitorine in LPS-induced inflammation in vitro and in vivo.

Pellitorine (PT), an active amide compound, is well known to possess insecticidal, antibacterial and anticancer properties. In this study, we first investigated the possible barrier protective effects of pellitorine against pro-inflammatory responses induced by lipopolysaccharide (LPS) and the associated signaling pathways in vitro and in vivo. The barrier protective activities of PT were determined by measuring permeability, monocyte adhesion and migration, and activation of pro-inflammatory proteins in LPS-activated human umbilical vein endothelial cells (HUVECs) and in mice. We found that PT inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs) and adhesion/transendothelial migration of monocytes to human endothelial cells. PT also suppressed LPS-induced hyperpermeability and leukocyte migration in vivo. Further studies revealed that PT suppressed the production of tumor necrosis factor-α (TNF-α) or Interleukin (IL)-6 and activation of nuclear factor-κB (NF-κB) or extracellular regulated kinases (ERK) 1/2 by LPS. Moreover, treatment with PT resulted in reduced LPS-induced lethal endotoxemia. These results suggest that PT protects vascular barrier integrity by inhibiting hyperpermeability, expression of CAMs, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases.

In vitro inhibitory effect of piperlonguminine isolated from Piper longum on human cytochrome P450 1A2.

Piperlonguminine (PL), a major alkaloid isolated from Piper longum fruits, shows several biological activities including anti-tumor, anti-hyperlipidemic and anti-inflammatory effects. Although there have been studies of the biological effects of PL, the potential drug-interaction effect of PL following evaluation of inhibitory effects of cytochrome P450 (CYP) activities was not investigated. Here, to investigate the inhibitory effects of PL on the activities of CYP isoforms, CYP inhibition assays were conducted using a cocktail of probe substrates in pooled human liver microsome (HLMs) and human recombinant cDNA-expressed CYP. PL strongly inhibited CYP1A2-mediated phenacetin O-deethylation with an IC50 value of 8.8 µM, as NADPH-independent inhibition, while other CYPs were not significantly inhibited. A Lineweaver-Burk plot resulted in the inhibition mechanism of PL being divided into two different modes, reversible competitive inhibition in a low concentration range of 0-16 µM with a Ki value of 1.39 µM and uncompetitive inhibitory behavior at a high concentration range of 16-40 µM. In addition, PL only decreased CYP 1A2-catalyzed phenacetin O-deethylase activity with IC50 values of 10.0 µM in human recombinant cDNA-expressed 1A2, not 1A1. Overall, this is the first investigation of potential herb-drug interactions associated with PL conducted by identifying the competitive inhibitory effects of PL on CYP1A2 in HLMs.

Selective inhibition of the cytochrome P450 isoform by hyperoside and its potent inhibition of CYP2D6.

Hyperoside, quercetin-3-O-galactoside, is a flavonoid isolated from Oenanthe javanica. In the present study, we investigated potential herb-drug inhibitory effects of hyperoside on nine cytochrome P450 (CYP) isoforms in pooled human liver microsomes (HLMs) and human recombinant cDNA expressed CYP using a cocktail probe assay. Hyperoside strongly inhibited CYP2D6-catalyzed dextromethorphan O-demethylation, with IC50 values of 1.2 and 0.81 µM after 0 and 15 min of preincubation, and a Ki value of 2.01 µM in HLMs, respectively. Hyperoside strongly decreased CYP2D6 activity dose-, but not time-, dependently in HLMs. In addition, the Lineweaver-Burk and Secondary plots for the inhibition of CYP2D6 in HLMs fitted a competitive inhibition mode. Furthermore, hyperoside decreased CYP2D6-catalyzed dextromethorphan O-demethylation activity of human recombinant cDNA-expressed CYP2D6, with an IC50 value of 3.87 µM. However, other CYPs were not inhibited significantly by hyperoside. In conclusion, our data demonstrate that hyperoside is a potent selective CYP2D6 inhibitor in HLMs, and suggest that hyperoside might cause herb-drug interactions when co-administrated with CYP2D substrates.

Inhibitory effect of glyceollins on vasculogenesis through suppression of endothelial progenitor cell function.

SCOPE: Endothelial progenitor cells (EPCs) are derived from hematopoietic stem cells, and have the ability to differentiate into mature endothelial cells and contribute to neovascularization. Glyceollins are a type of phytoalexin produced in soybeans under stress conditions. The aim of this study is to determine the effect of glyceollin treatment on EPCs during early tumor vasculogenesis. METHODS AND RESULTS: We found that glyceollin treatment significantly decreased the number of EPC colony-forming units in human cord blood-derived AC133⁺ cells and mouse bone-marrow-derived c-Kit⁺/Sca-1⁺/Lin⁻ cells. Glyceollin treatment diminished the number of lineage-committed EPC cells in a dose-dependent manner (1-20 µM). Glyceollin treatment inhibited EPC migration, tube formation and the mRNA expression of angiopoietin-1 (Ang-1), Tie-2, stromal-derived factor-1 (SDF-1), C-X-C-chemokine receptor-4 (CXCR4), and endothelial nitric oxide synthase (eNOS) in cultured EPCs. Glyceollin treatment suppressed activation of Akt, Erk, and eNOS induced by SDF-1α or vascular endothelial growth factor (VEGF). Treatment with 10 mg/kg glyceollins significantly reduced the number of tumor-induced circulating EPCs and the incorporation of EPCs into neovessels in bone marrow transplanted mice. CONCLUSION: These results suggest that glyceollins inhibit the function of EPCs in tumor neovascularization. Glyceollins from soybean elicitation could be beneficial in prevention of cancer development via vasculogenesis.