Austin M. Guo

Activation of Vascular Endothelial Growth Factor through Reactive Oxygen Species Mediates 20-Hydroxyeicosatetraenoic Acid-Induced Endothelial Cell Proliferation

20-Hydroxyeicosatetraenoic acid (20-HETE) is formed by the ω-hydroxylation of arachidonic acid by cytochrome P450 4A and 4F enzymes, and it induces angiogenic responses in vivo. To test the hypothesis that 20-HETE increases endothelial cell (EC) proliferation via vascular endothelial growth factor (VEGF), we studied the effects of WIT003 [20-hydroxyeicosa-5(Z),14(Z)-dienoic acid], a 20-HETE analog on human macrovascular or microvascular EC. WIT003, as well as pure 20-HETE, stimulated EC proliferation by ∼40%. These proliferative effects were accompanied by increased VEGF expression and release that were observed as early as 4 h after 20-HETE agonist addition. This was accompanied by increased phosphorylation of the VEGF receptor 2. The proliferative effects of 20-HETE were markedly inhibited by a VEGF-neutralizing antibody. Polyethylene glycol-superoxide dismutase (PEG-SOD) markedly inhibited both the increases in VEGF expression and the proliferative effects of 20-HETE. In contrast, administration of the NAD(P)H oxidase inhibitor apocynin had no effect to the proliferative response to 20-HETE. The 20-HETE agonist markedly increased superoxide formation as reflected by an increase in dihydroethidium staining of EC, and this increase was inhibited by PEG-SOD but not by apocynin. 20-HETE also increased the phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) in EC, whereas an inhibitor of MAPK [U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] suppressed the proliferative and the VEGF changes but not the pro-oxidant effects of 20-HETE. These data suggest that 20-HETE stimulates superoxide formation by pathways other than apocynin-sensitive NAD(P)H oxidase, thereby activating MAPK and then enhancing VEGF synthesis that drives EC proliferation. Thus, 20-HETE may be involved in the regulation of EC functions, such as angiogenesis.

Cytochrome P450 ω-hydroxylase promotes angiogenesis and metastasis by upregulation of VEGF and MMP-9 in non-small cell lung cancer

PurposeCytochrome P450 (CYP) ω-hydroxylase, mainly consisting of CYP4A and CYP4F, converts arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) that induces angiogenic responses in vivo and in vitro. The present study examined the role of CYP ω-hydroxylase in angiogenesis and metastasis of human non-small cell lung cancer (NSCLC).MethodsThe effect of WIT003, a stable 20-HETE analog, on invasion was evaluated using a modified Boyden chamber in three NSCLC cell lines. A549 cells were transfected with CYP4A11 expression vector or exposed to CYP ω-hydroxylase inhibitor (HET0016) or 20-HETE antagonist (WIT002), and then ω-hydroxylation activity toward arachidonic acid and the levels of matrix metalloproteinases (MMPs) and VEGF were detected. The in vivo effects of CYP ω-hydroxylase were tested in established tumor xenografts and an experimental metastasis model in athymic mice.ResultsAddition of WIT003 or overexpression of CYP4A11 with an associated increase in 20-HETE production significantly induced invasion and expression of VEGF and MMP-9. Treatment of A549 cells with HET0016 or WIT002 inhibited invasion with reduction in VEGF and MMP-9. The PI3 K or ERK inhibitors also attenuated expression of VEGF and MMP-9. Compared with control, CYP4A11 transfection significantly increased tumor weight, microvessel density (MVD), and lung metastasis by 2.5-fold, 2-fold, and 3-fold, respectively. In contrast, WIT002 or HET0016 decreased tumor volume, MVD, and spontaneous pulmonary metastasis occurrences.ConclusionCYP ω-hydroxylase promotes tumor angiogenesis and metastasis by upregulation of VEGF and MMP-9 via PI3 K and ERK1/2 signaling in human NSCLC cells.

Downregulation of COX-2 and CYP 4A signaling by isoliquiritigenin inhibits human breast cancer metastasis through preventing anoikis resistance, migration and invasion

Flavonoids exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Anoikis resistance occurs at multiple key stages of the metastatic cascade. Here, we demonstrate that isoliquiritigenin (ISL), a flavonoid from Glycyrrhiza glabra, inhibits human breast cancer metastasis by preventing anoikis resistance, migration and invasion through downregulating cyclooxygenase (COX)-2 and cytochrome P450 (CYP) 4A signaling. ISL induced anoikis in MDA-MB-231 and BT-549 human breast cancer cells as evidenced by flow cytometry and the detection of caspase cleavage. Moreover, ISL inhibited the mRNA expression of phospholipase A2, COX-2 and CYP 4A and decreased the secretion of prostaglandin E2 (PGE2) and 20-hydroxyeicosatetraenoic acid (20-HETE) in detached MDA-MB-231 cells. In addition, it decreased the levels of phospho-PI3K (Tyr(458)), phospho-PDK (Ser(241)) and phospho-Akt (Thr(308)). Conversely, the exogenous addition of PGE2, WIT003 (a 20-HETE analog) and an EP4 agonist (CAY10580) or overexpression of constitutively active Akt reversed ISL-induced anoikis. ISL exerted the in vitro anti-migratory and anti-invasive activities, whereas the addition of PGE2, WIT003 and CAY10580 or overexpression of constitutively active Akt reversed the in vitro anti-migratory and anti-invasive activities of ISL in MDA-MB-231 cells. Notably, ISL inhibited the in vivo lung metastasis of MDA-MB-231 cells, together with decreased intratumoral levels of PGE2, 20-HETE and phospho-Akt (Thr(308)). In conclusion, ISL inhibits breast cancer metastasis by preventing anoikis resistance, migration and invasion via downregulating COX-2 and CYP 4A signaling. It suggests that ISL could be a promising multi-target agent for preventing breast cancer metastasis, and anoikis could represent a novel mechanism through which flavonoids may exert the anti-metastatic activities.

20-HETE Regulates the Angiogenic Functions of Human Endothelial Progenitor Cells and Contributes to Angiogenesis In Vivo

Circulating endothelial progenitor cells (EPC) contribute to postnatal neovascularization. We identified the cytochrome P450 4A/F–20-hydroxyeicosatetraenoic acid (CYP4A/F–20-HETE) system as a novel regulator of EPC functions associated with angiogenesis in vitro. Here, we explored cellular mechanisms by which 20-HETE regulates EPC angiogenic functions and assessed its contribution to EPC-mediated angiogenesis in vivo. Results showed that both hypoxia and vascular endothelial growth factor (VEGF) induce CYP4A11 gene and protein expression (the predominant 20-HETE synthases in human EPC), and this is accompanied by an increase in 20-HETE production by ∼1.4- and 1.8-fold, respectively, compared with the control levels. Additional studies demonstrated that 20-HETE and VEGF have a synergistic effect on EPC proliferation, whereas 20-HETE antagonist 20-HEDGE or VEGF-neutralizing antibody negated 20-HETE- or VEGF-induced proliferation, respectively. These findings are consistent with the presence of a positive feedback regulation on EPC proliferation between the 20-HETE and the VEGF pathways. Furthermore, we found that 20-HETE induced EPC adhesion to fibronectin and endothelial cell monolayer by 40 ± 5.6 and 67 ± 10%, respectively, which was accompanied by a rapid induction of very late antigen-4 and chemokine receptor type 4 mRNA and protein expression. Basal and 20-HETE-stimulated increases in adhesion were negated by the inhibition of the CYP4A–20-HETE system. Lastly, EPC increased angiogenesis in vivo by 3.6 ± 0.2-fold using the Matrigel plug angiogenesis assay, and these increases were markedly reduced by the local inhibition of 20-HETE system. These results strengthened the notion that 20-HETE regulates the angiogenic functions of EPC in vitro and EPC-mediated angiogenesis in vivo.

Expression of CYP4A1 in U251 human glioma cell induces hyperproliferative phenotype in vitro and rapidly growing tumors in vivo.

Exogenous 20-hydroxyeicosatetraenoic acid (20-HETE) increases the growth of human glioma cells in vitro. However, glioma cells in culture show negligible 20-HETE synthesis. We examined whether inducing the expression of a 20-HETE synthase in a human glioma U251 cell line would increase proliferation. U251 cells transfected with CYP4A1 cDNA (termed U251 O) increased the formation of 20-HETE from less than 1 to over 60 pmol/min/mg proteins and increased their proliferation rate by 2-fold (p < 0.01). Compared with control U251, U251 O cells were rounded, smaller, showed a disorganized cytoskeleton, exhibited reduced vinculin staining, and were easily detached from the growing surface. They showed a marked increase in dihydroethidium staining, suggesting increased oxidative stress. The expression of phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1/2, and vascular endothelial growth factor was markedly elevated in U251 O. The hyperproliferative and signaling effects seen in U251 O cells are abolished by selective CYP4A inhibition of 20-HETE formation with HET0016 [N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine], by small interfering RNA against the enzyme, and by the putative 20-HETE antagonist, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid. In vivo, implantation of U251O cells in the brain of nude rats resulted in a ∼10-fold larger tumor volume (10 days postimplantation) compared with animals receiving mock-transfected U251 cells. These data show that elevations in 20-HETE synthesis in U251 cells lead to an increased growth both in vitro and in vivo. This suggests that 20-HETE may have proto-oncogenic properties in U251 human gliomas. Further studies are needed to determine whether 20-HETE plays a role promoting growth of some human gliomas.

20-HETE can act as a nonhypoxic regulator of HIF-1α in human microvascular endothelial cells

20-HETE increases the expression of VEGF in human dermal microvascular endothelial cells (ECs). Since VEGF is regulated by hypoxia inducible factor (HIF)-1, we studied whether 20-HETE also upregulates HIF-1alpha using the stable 20-HETE analog 20-hydroxyeicosa-5(Z),14(Z)dienoic acid (WIT003; 1-10 microM) and found that it induced a marked increase in HIF-1alpha protein levels. The increases in VEGF after the addition of WIT003 preceded the changes in HIF-1alpha, and the increases in HIF-1alpha were prevented by a VEGF neutralizing antibody. This suggests that 20-HETE first causes increases in VEGF, which then, in turn, cause the upregulation of HIF-1alpha. Stimulation with exogenously added VEGF also led to an upregulation of HIF-1alpha. Incubation with the MEK1/ERK1/2 inhibitor U-0126 (10 microM) completely abolished the increases in VEGF and thus HIF-1alpha, suggesting the involvement of ERK1/2 activation. The addition of WIT003 resulted in a rapid and sustained increase in superoxide formation. When WIT003 was added in the presence of the nitric oxide (NO) synthase (NOS) inhibitor N-nitro-L-arginine, no changes in superoxide, VEGF, or HIF-1alpha were observed. This suggests that NOS is responsible for the early changes in superoxide induced by WIT003. Furthermore, WIT003 induced the expression of the NADPH oxidase subunit p47(phox) in ECs before the increases in HIF-1alpha. Incubation with polyethylene glycol-superoxide dismutase (400 U/ml), apocynin (100 microM), diphenylene iodonium (10 microM), or p47(phox) downregulation with small interfering (si)RNA all inhibited the increases in HIF-1alpha expression. This indicates that the early changes in superoxide lead to VEGF increases and thereby NADPH oxidase-dependent superoxide production, which is required for HIF-1alpha upregulation. We also found that the higher HIF-1alpha expression induced by WIT003 was accompanied by higher expression of erythropoietin receptor and angiopoietin-2 proteins. These increases were caused by HIF-1alpha because their levels were markedly decreased by siRNA downregulation of HIF-1alpha. 20-HETE may be a novel nonhypoxic regulator of HIF-1alpha and HIF-1alpha-regulated genes in ECs.

Isoliquiritigenin, a flavonoid from licorice, blocks M2 macrophage polarization in colitis-associated tumorigenesis through downregulating PGE2 and IL-6.

M2 macrophage polarization is implicated in colorectal cancer development. Isoliquiritigenin (ISL), a flavonoid from licorice, has been reported to prevent azoxymethane (AOM) induced colon carcinogenesis in animal models. Here, in a mouse model of colitis-associated tumorigenesis induced by AOM/dextran sodium sulfate (DSS), we investigated the chemopreventive effects of ISL and its mechanisms of action. Mice were treated with AOM/DSS and randomized to receive either vehicle or ISL (3, 15 and 75 mg/kg). Tumor load, histology, immunohistochemistry, and gene and protein expressions were determined. Intragastric administration of ISL for 12 weeks significantly decreased colon cancer incidence, multiplicity and tumor size by 60%, 55.4% and 42.6%, respectively. Moreover, ISL inhibited M2 macrophage polarization. Such changes were accompanied by downregulation of PGE2 and IL-6 signaling. Importantly, depletion of macrophages by clodronate (Clod) or zoledronic acid (ZA) reversed the effects of ISL. In parallel, in vitro studies also demonstrated that ISL limited the M2 polarization of RAW264.7 cells and mouse peritoneal macrophages with concomitant inactivation of PGE2/PPARδ and IL-6/STAT3 signaling. Conversely, exogenous addition of PGE2 or IL-6, or overexpression of constitutively active STAT3 reversed ISL-mediated inhibition of M2 macrophage polarization. In summary, dietary flavonoid ISL effectively inhibits colitis-associated tumorigenesis through hampering M2 macrophage polarization mediated by the interplay between PGE2 and IL-6. Thus, inhibition of M2 macrophage polarization is likely to represent a promising strategy for chemoprevention of colorectal cancer.

The Cytochrome P450 4A/F-20-Hydroxyeicosatetraenoic Acid System: A Regulator of Endothelial Precursor Cells Derived from Human Umbilical Cord Blood

Endothelial progenitor cells (EPCs) contribute to physiological and pathological neovascularization. Previous data have suggested that the cytochrome P450 4A/F (CYP4A/F)-20-hydroxyeicosatetraenoic acid (20-HETE) system regulates neovascularization. Therefore, we studied whether the angiogenic effects of the CYP4A/F-20-HETE system involve regulation of EPC function. We extracted human umbilical cord blood and isolated EPCs, which express AC133+CD34+ and kinase insert domain receptor (KDR) surface markers and contain mRNA and protein for CYP4A11 and CYP4A22 enzymes, as opposed to mesenchymal stem cells, which only express negligible amounts of CYP4A11/22. When EPCs were incubated with arachidonic acid, they produced 20-HETE, which stimulated the cells to proliferate and migrate, as did vascular endothelial growth factor. Incubation with 1 μM N-hydroxy-N′-(4-butyl-2-methylphenyl)formamidine (HET0016), a selective inhibitor of 20-HETE synthesis, reduced the proliferative and migratory effects of vascular endothelial growth factor and also significantly abolished EPC migration mediated by stroma-derived factor-1α, as did (6,15) 20-hydroxyeicosadienoic acid. Coculturing EPCs and endothelial cells on a Matrigel matrix led to tube formation, which in turn was inhibited by both HET0016 and 20-hydroxyeicosadienoic acid. We concluded that the CYP4A/F-20-HETE system is expressed in EPCs and can act as both an autocrine and a paracrine regulatory factor.

20-HETE in neovascularization.

Cytochrome P450 4A/F (CYP4A/F) converts arachidonic acid (AA) to 20-HETE by ω-hydroxylation. The contribution of 20-HETE to the regulation of myogenic response, blood pressure, and mitogenic actions has been well summarized. This review focuses on the emerging role of 20-HETE in physiological and pathological vascularization. 20-HETE has been shown to regulate vascular smooth muscle cells (VSMC) and endothelial cells (EC) by affecting their proliferation, migration, survival, and tube formation. Furthermore, the proliferation, migration, secretion of proangiogenic molecules (such as HIF-1α, VEGF, SDF-1α), and tube formation of endothelial progenitor cells (EPC) are stimulated by 20-HETE. These effects are mediated through c-Src- and EGFR-mediated downstream signaling pathways, including MAPK and PI3K/Akt pathways, eNOS uncoupling, and NOX/ROS system activation. Therefore, the CYP4A/F-20-HETE system may be a therapeutic target for the treatment of abnormal angiogenic diseases.

Role of 12-lipoxygenase in regulation of ovarian cancer cell proliferation and survival

PurposeEicosanoid-related enzymes have been implicated in the pathogenesis of various cancers. Little is known about the relevance of lipoxygenase pathway to ovarian cancer growth. In this study, we examined the role of 12-lipoxygenase (12-LOX), the main human 12-HETE generating enzyme, in the regulation of proliferation and survival in epithelial ovarian cancer.MethodsImmunohistological analysis of 12-LOX expression in high-grade serous ovarian carcinoma and normal ovarian epithelium tissues was performed. The presence of 12-LOX-12-HETE system was confirmed in two epithelial ovarian cancer (EOC) cell lines, OVCAR-3 and SK-OV-3, using RT–PCR, Western blot and LC/MS analysis. The effects of N-benzyl-N-hydroxy-5-phenyl-pentanamide (BMD-122), a specific 12-LOX inhibitor, on cell growth, survival, apoptosis, and cell signaling were determined.ResultsWe found that a significantly higher level of 12-LOX expression in high-grade serous ovarian carcinoma compared to normal ovarian epithelium. OVCAR-3 and SK-OV-3 were found to express high level of 12-LOX mRNA and protein. Both EOC increased their 12-HETE production when incubated with arachidonic acid. BMD-122 inhibited the EOC growth in a dose-dependent fashion. Purified 12-HETE significantly reversed such inhibitory effects of BMD-122. In addition, BMD-122 blocked the MAPK signaling pathway by inhibiting the phosphorylation of ERK and induced a ~20–30% increase in the EOC apoptosis. Down-regulation of the 12-LOX expression using 12-LOX siRNA also resulted in markedly reduction in cell growth.ConclusionsThese data suggest that 12-LOX is involved in the regulation of ovarian cancer cell growth and survival and is a potential new therapeutic target.