Yunjin Jung

IL-1β-mediated up-regulation of HIF-1α via an NFκB/COX-2 pathway identifies HIF-1 as a critical link between inflammation and oncogenesis

Growing evidence indicates that inflammation is a contributing factor leading to cancer development. However, pathways involved in this progression are not well understood. To examine whether HIF‐1α is a factor linking inflammation and tumorigenesis, we investigated whether the HIF‐1 signaling pathway was stimulated by the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) in A549 cells. We find that IL‐1β up‐regulated HIF‐1α protein under normoxia and activated the HIF‐1‐responsive gene vascular endothelial growth factor (VEGF) via a pathway dependent on nuclear factor κB (NFkB). Interestingly, although this pathway is stimulated by upstream signaling via AKT and mTOR and requires new transcription, IL‐1 mediated HIF‐1α induction also utilizes a post‐transcriptional mechanism that involves antagonism of VHL‐dependent HIF‐1α degradation, which results in increased HIF‐1α protein stability. IL‐1 mediated NFkB‐dependent cyclooxygenases‐2 (COX‐2) expression served as a positive effector for HIF‐1α induction. Although COX‐2 inhibitors attenuated IL‐1 mediated HIF‐1α induction, prostaglandin E2 (PGE2), a physiological product of COX‐2, induced HIF‐1α protein in a dose‐dependent manner. Our data, therefore, demonstrate that IL‐1β up‐regulates functional HIF‐1α protein through a classical inflammatory signaling pathway involving NFkB and COX‐2, culminating in up‐regulation of VEGF, a potent angiogenic factor required for tumor growth and metastasis. Thus, HIF‐1 is identified as a pivotal transcription factor linking the inflammatory and oncogenic pathways.

Curcumin is an inhibitor of p300 histone acetylatransferase.

Histone acetyltransferases (HATs), and p300/CBP in particular, have been implicated in cancer cell growth and survival, and as such, HATs represent novel, therapeutically relevant molecular targets for drug development. In this study, we demonstrate that the small molecule natural product curcumin, whose medicinal properties have long been recognized in India and Southeast Asia, is a selective HAT inhibitor. Furthermore the data indicate that alpha, beta unsaturated carbonyl groups in the curcumin side chain function as Michael reaction sites and that the Michael reaction acceptor functionality of curcumin is required for its HAT-inhibitory activity. In cells, curcumin promoted proteasome-dependent degradation of p300 and the closely related CBP protein without affecting the HATs PCAF or GCN5. In addition to inducing p300 degradation curcumin inhibited the acetyltransferase activity of purified p300 as assessed using either histone H3 or p53 as substrate. Radiolabeled curcumin formed a covalent association with p300, and tetrahydrocurcumin displayed no p300 inhibitory activity, consistent with a Michael reaction-dependent mechanism. Finally, curcumin was able to effectively block histone hyperacetylation in both PC3-M prostate cancer cells and peripheral blood lymphocytes induced by the histone deacetylase inhibitor MS-275. These data thus identify the medicinal natural product curcumin as a novel lead compound for development of possibly therapeutic, p300/CBP-specific HAT inhibitors.

Hypoxia-inducible factor induction by tumour necrosis factor in normoxic cells requires receptor-interacting protein-dependent nuclear factor kappa B activation.

Tumour necrosis factor alpha (TNF-alpha) binds to its receptor (TNFR1) and activates both death- and inflammation/survival-related signalling pathways. The inflammation and survival-related signalling cascade results in the activation of the transcription factor, nuclear factor kappa B (NF-kappa B) and requires recruitment of receptor-interacting protein (RIP) to TNFR1. The indispensable role of RIP in TNF-induced NF-kappa B activation has been demonstrated in RIP(-/-) mice and in cell lines derived from such mice. In the present study, we show that the TNF-alpha-induced accumulation of hypoxia-inducible factor 1 alpha (HIF-1 alpha) protein in normoxic cells is RIP-dependent. Exposing fibroblasts derived from RIP(-/-) mice to either cobalt or PMA resulted in an equivalent HIF-1 alpha induction to that seen in RIP(+/+) fibroblasts. In contrast, RIP(-/-) cells were unable to induce HIF-1 alpha in response to TNF-alpha. Further, transient transfection of NIH 3T3 cells with an NF-kappa B super-repressor plasmid (an inhibitor of NF-kappa B activation) also prevented HIF-1 alpha induction by TNF-alpha. Surprisingly, although HIF-1 alpha mRNA levels remained unchanged after induction by TNF, induction of HIF-1 alpha protein by the cytokine was completely blocked by pretreatment with the transcription inhibitors actinomycin D and 5,6-dichlorobenzimidazole riboside. Finally, TNF failed to induce both HIF-1 alpha, made resistant to von Hippel-Lindau (VHL), and wild-type HIF-1 alpha transfected into VHL(-/-) cells. These results indicate that HIF-1 alpha induction by TNF-alpha in normoxic cells is mediated by protein stabilization but is nonetheless uniquely dependent on NF-kappa B-driven transcription. Thus the results describe a novel mechanism of HIF-1 alpha up-regulation and they identify HIF-1 alpha as a unique component of the NF-kappa B-mediated inflammatory/survival response.

NRF2 blockade suppresses colon tumor angiogenesis by inhibiting hypoxia-induced activation of HIF-1α

Transcription factor NRF2 is an important modifier of cellular responses to oxidative stress. Although its cytoprotective effects are firmly established, recent evidence suggesting important roles in cancer pathobiology has yet to be mechanistically developed. In the current study, we investigated the role of NRF2 in colon tumor angiogenesis. Stable RNAi-mediated knockdown of NRF2 in human colon cancer cells suppressed tumor growth in mouse xenograft settings with a concomitant reduction in blood vessel formation and VEGF expression. Similar antiangiogenic effects of NRF2 knockdown were documented in chick chorioallantoic membrane assays and endothelial tube formation assays. Notably, NRF2-inhibited cancer cells failed to accumulate HIF-1α protein under hypoxic conditions, limiting expression of VEGF and other HIF-1α target genes. In these cells, HIF-1α was hydroxylated but pharmacological inhibition of PHD domain-containing prolyl hydroxylases was sufficient to restore hypoxia-induced accumulation of HIF-1α. Mechanistic investigations demonstrated that reduced mitochondrial O(2) consumption in NRF2-inhibited cells was probably responsible for HIF-1α degradation during hypoxia; cellular O(2) consumption and ATP production were lower in NRF2 knockdown cells than in control cells. Our findings offer novel insights into how cellular responses to O(2) and oxidative stress are integrated in cancer cells, and they highlight NRF2 as a candidate molecular target to control tumor angiogenesis by imposing a blockade to HIF-1α signaling.

Metabolic and pharmacological properties of rutin, a dietary quercetin glycoside, for treatment of inflammatory bowel disease.

PurposeOrally administered rutin reportedly ameliorates 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis of rats. We investigated the metabolic and pharmacological properties of rutin underlying the rutin-mediated amelioration of the rat colitis.MethodsApparent partition coefficients of rutin and its aglycone quercetin were compared. The biochemical/chemical stability of rutin was examined in the contents of various segments of gastrointestinal tracts of rats. Inflammatory indices were determined in the colitis rats after oral administration of rutin or rectal administration of quercetin. In human colon epithelial cells, the effect of quercetin on tumor necrosis factor-alpha (TNF-α)-induced nuclear factor kappa B (NFκB) activation was examined.ResultsThe sugar residue in rutin greatly lowered the apparent partition coefficient and was rapidly deglycosylated to liberate quercetin in the cecal contents, whereas it was stable in the contents of the upper intestine. Not only oral administration of rutin but also rectal administration of quercetin remarkably ameliorated TNBS-induced colitis rats, indicating that quercetin liberated from rutin is therapeutically active. Furthermore, quercetin dose-dependently inhibited an inflammatory signal TNF-α-dependent NFκB activation.ConclusionsOur data suggest that rutin acted as a quercetin deliverer to the large intestine and its anti-inflammatory action in TNBS-induced colitis rats may be through quercetin-mediated inhibition of TNF-α-induced NFκB activation.

Caffeic acid phenethyl ester-mediated Nrf2 activation and IκB kinase inhibition are involved in NFκB inhibitory effect: Structural analysis for NFκB inhibition

Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated potential molecular mechanisms underlying CAPE-mediated nuclear factor kappa beta (NFkappaB) inhibition and analyzed structure of CAPE for its biological effect. CAPE attenuated expression of NFkappaB dependent luciferase stimulated with TNF-alpha or LPS and suppressed LPS-mediated induction of iNOS, a target gene product of NFkappaB. In HCT116 cells, CAPE interfered with TNF-alpha dependent IkappaBalpha degradation and subsequent nuclear accumulation of p65, which occurred by direct inhibition of inhibitory protein kappaB kinase (IKK). CAPE increased the expression of Nrf2-dependent luciferase and heme oxygenase-1, a target gene of Nrf2, and elevated the nuclear level of Nrf2 protein, indicating that CAPE activated the Nrf2 pathway. In HCT116 cells with stable expression of Nrf2 shRNA, CAPE elicited a reduced inhibitory effect on TNF-alpha-activated NFsmall ka, CyrillicB compared to scramble RNA expressing control cells. On the other hand, the NFkappaB inhibitory effect of CAPE was diminished by removal or modification of the Michael reaction acceptor, catechol or phenethyl moiety in CAPE. These data suggest that CAPE inhibits TNF-alpha-dependent NFkappaB activation via direct inhibition of IKK as well as activation of Nrf2 pathway, in which the functional groups in CAPE may be involved.

NC post-processor for 5-axis milling machine of table-rotating/tilting type

Abstract The free surface machining with more than three axes motion needs CAD/CAM system for the cutter location and orientation data. Since these data are defined with respect to the coordinate system of workpiece, they need converting for machine control commands in machine coordinate system, through processing so called a NC post-processing. Most commercial CAD/CAM software vendors provide these post-processors for each type of 5-axis machines. However, they may cause some erroneous operation during phase reverse process that is due to unsymmetrical operation limits about rotating axes. In this work, efficient algorithms for NC post-processing have been presented for typical 5-axis milling machine of table-rotating/tilting type. The first algorithm is for avoiding the interference between a workpiece and the machine tool during the process of phase reverse. The second is for better strategy of machine path, which needs fewer number of phase reverse processes.

Quercetin Activates an Angiogenic Pathway, Hypoxia Inducible Factor (HIF)-1-Vascular Endothelial Growth Factor, by Inhibiting HIF-Prolyl Hydroxylase: a Structural Analysis of Quercetin for Inhibiting HIF-Prolyl Hydroxylase

We investigated a molecular mechanism underlying quercetin-mediated amelioration of colonic mucosal injury and analyzed chemical structure contributing to the quercetins effect. Quercetin up-regulated vascular endothelial growth factor (VEGF), an ulcer healing factor, not only in colon epithelial cell lines but also in the inflamed colonic tissue. VEGF derived from quercetin-treated colon epithelial cells promoted tube formation. The VEGF induction was dependent on quercetin-mediated hypoxia-inducible factor-1 (HIF-1) activation. Quercetin delayed HIF-1α protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1α hydroxylation and subsequent von Hippel Lindau-dependent HIF-1α degradation. HPH inhibition by quercetin was neutralized significantly by an elevated dose of iron. Consistent with this, cellular induction of HIF-1α by quercetin was abolished by pretreatment with iron. Two iron-chelating moieties in quercetin, -OH at position 3 of the C ring and/or -OH at positions 3′ and 4′ of the B ring, enabled the flavonoid to inhibit HPH and subsequently induce HIF-1α. Our data suggest that the clinical effect of quercetin may be partly attributed to the activation of an angiogenic pathway HIF-1-VEGF via inhibiting HPH and the chelating moieties of quercetin were required for inhibiting HPH.

Nitric oxide-releasing poly(lactic-co-glycolic acid)-polyethylenimine nanoparticles for prolonged nitric oxide release, antibacterial efficacy, and in vivo wound healing activity

Nitric oxide (NO)-releasing nanoparticles (NPs) have emerged as a wound healing enhancer and a novel antibacterial agent that can circumvent antibiotic resistance. However, the NO release from NPs over extended periods of time is still inadequate for clinical application. In this study, we developed NO-releasing poly(lactic-co-glycolic acid)-polyethylenimine (PEI) NPs (NO/PPNPs) composed of poly(lactic-co-glycolic acid) and PEI/diazeniumdiolate (PEI/NONOate) for prolonged NO release, antibacterial efficacy, and wound healing activity. Successful preparation of PEI/NONOate was confirmed by proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and ultraviolet/visible spectrophotometry. NO/PPNPs were characterized by particle size, surface charge, and NO loading. The NO/PPNPs showed a prolonged NO release profile over 6 days without any burst release. The NO/PPNPs exhibited potent bactericidal efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa concentration-dependently and showed the ability to bind on the surface of the bacteria. We also found that the NO released from the NO/PPNPs mediates bactericidal efficacy and is not toxic to healthy fibroblast cells. Furthermore, NO/PPNPs accelerated wound healing and epithelialization in a mouse model of a MRSA-infected wound. Therefore, our results suggest that the NO/PPNPs presented in this study could be a suitable approach for treating wounds and various skin infections.

What should be considered on design of a colon-specific prodrug?

Importance of the field: Generally, a prodrug, a pharmacologically inactive derivative of an active drug, is designed to modulate pharmacokinetic properties of the parent drug. Targeted distribution of an orally administered drug at the large intestine confers therapeutic advantages on treatment of colonic diseases, peptide and protein therapy and chronotherapy. Areas covered in this review: To achieve such distribution control in the gastrointestinal tract, the adoption of the prodrug concept gives birth to a colon-specific prodrug. The requirement for a prodrug to be colon-specific is described along with the necessary and sufficient conditions of drugs for conversion to a colon-specific prodrug. The known and previously unnoticed factors that negatively influence therapeutic activity and reproducibility of a colon-specific prodrug are presented with suggestions to minimize the negative influence. What the reader will gain: This review provides tactics to satisfy the requirements for being colon-specific and the potential strategies to circumvent obstacles in developing an efficient colon-specific prodrug. Take home message: On design of a colon-specific prodrug, one should take into consideration not only delivery of a drug to the target site, but also the therapeutic effectiveness there.