Curcumin induces radiosensitivity of in vitro and in vivo cancer models by modulating pre-mRNA processing factor 4 (Prp4)

Radiation therapy plays a central role in adjuvant strategies for the treatment of both pre- and post-operative human cancers. However, radiation therapy has low efficacy against cancer cells displaying radio-resistant phenotypes. Ionizing radiation has been shown to enhance ROS generation, which mediates apoptotic cell death. Further, concomitant use of sensitizers with radiation improves the efficiency of radiotherapy against a variety of human cancers. Here, the radio-sensitizing effect of curcumin (a derivative of turmeric) was investigated against growth of HCT-15 cells and tumor induction in C57BL/6J mice. Ionizing radiation induced apoptosis through ROS generation and down-regulation of Prp4K, which was further potentiated by curcumin treatment. Flow cytometry revealed a dose-dependent response for radiation-induced cell death, which was remarkably reversed by transfection of cells with Prp4K clone. Over-expression of Prp4K resulted in a significant decrease in ROS production possibly through activation of an anti-oxidant enzyme system. To elucidate an integrated mechanism, Prp4K knockdown by siRNA ultimately restored radiation-induced ROS generation. Furthermore, B16F10 xenografts in C57BL/6J mice were established in order to investigate the radio-sensitizing effect of curcumin in vivo. Curcumin significantly enhanced the efficacy of radiation therapy and reduced tumor growth as compared to control or radiation alone. Collectively, these results suggest a novel mechanism for curcumin-mediated radio-sensitization of cancer based on ROS generation and down-regulation of Prp4K.     

Schistosoma mansoni: Egg-induced downregulation of hepatic stellate cell activation and fibrogenesis

Eggs of Schistosoma mansoni trapped in human liver can lead to fibrosis. Since liver fibrosis requires activation of hepatic stellate cells (HSC) from a quiescent to a myofibroblastic phenotype, we investigated the effects of S. mansoni eggs on this process using in vitro co-cultures with human HSC and evaluated established biomarkers for activation and fibrosis. HSC demonstrate significantly reduced expression of α-smooth muscle actin (p < 0.001), connective tissue growth factor (p < 0.01) and type I collagen (p < 0.001) but significantly increased expression of peroxisome proliferator-activated receptor-γ (p < 0.01). Morphologically, HSC exhibited elongated fine cellular processes and reduced size, increased accumulation of lipid droplets and reduced expression and organization of α-smooth muscle actin and F-actin stress fibres. Additionally, schistosome eggs prevented the HSC fibrogenic response to exogenous transforming growth factor-β. In summary, schistosome eggs blocked fibrogenesis in HSC, a finding which may have implications for our understanding of the fibrotic pathology in S. mansoni infections.     

Protection of NAD(P)H:quinone oxidoreductase 1 against renal ischemia/reperfusion injury in mice

Ischemia/reperfusion (I/R) is the most common cause of acute renal injury. I/R-induced reactive oxygen species (ROS) are thought to be a major factor in the development of acute renal injury by promoting the initial tubular damage. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a well-known antioxidant protein that regulates ROS generation. The purpose of this study was to investigate whether NQO1 modulates the renal I/R injury (IRI) associated with NADPH oxidase (NOX)-derived ROS production in an animal model. We analyzed renal function, oxidative stress, and tubular apoptosis after IRI. NQO1^−/− mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In the kidneys of NQO1^−/− mice, the cellular NADPH/NADP⁺ ratio was significantly higher and NOX activity was markedly higher than in those of NQO1^+/+ mice. The activation of NQO1 by β-lapachone (βL) significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis by renal I/R. Moreover, the βL treatment significantly lowered the cellular NADPH/NADP⁺ ratio and dramatically reduced NOX activity in the kidneys after IRI. From these results, it was concluded that NQO1 has a protective role against renal injury induced by I/R and that this effect appears to be mediated by decreased NOX activity via cellular NADPH/NADP⁺ modulation. These results provide convincing evidence that NQO1 activation might be beneficial for ameliorating renal injury induced by I/R.     

NAD(P)H oxidase inhibitor prevents blood pressure elevation and cardiovascular hypertrophy in aldosterone-infused rats

Increased bioavailability of reactive oxygen species (ROS) has been implicated in the pathogenesis of mineralocorticoid hypertension. To find out the source of ROS, we evaluated the role of NAD(P)H oxidase in blood pressure (BP) elevation, cardiovascular hypertrophy, and fibrosis in aldosterone-salt rats. Aldosterone infusion (0.75 microg/h) significantly increased BP, which is attenuated by apocynin (1.5 mmol/L). Cardiac hypertrophy developed by aldosterone infusion was also normalized with apocynin. Greater mRNA for p22phox and NAD(P)H oxidase activity (more than twofold) in aorta of aldosterone-infused rats was reduced in apocynin-treated rats. Aldosterone infusion increased marginally procollagen I and III expression in LV compared to controls and apocynin decreased procollagen. Masson's Trichrome stain showed increased cardiac perivascular fibrosis, which was reduced by apocynin. These results suggest that NAD(P)H oxidase plays an important role in cardiovascular damage associated with mineralocorticoid hypertension.     

Triaza-based amphiphilic chelators: Synthetic route, in vitro characterization and in vivo studies of their Ga(III) and Al(III) chelates

Radiogallium chelates are important for diagnostic imaging in nuclear medicine (PET (positron emission tomography) and γ-scintigraphy). Micelles are adequate colloidal vehicles for the delivery of therapeutic and diagnostic agents to organs and tissues. In this paper we describe the synthesis and in vitro and in vivo studies of a series of micelles-forming Ga(III) chelates targeted for the liver. The amphiphilic ligands are based on NOTA (NOTA = 1,4,7-triazacyclonoane-N,N′N″-triacetic acid) and bear a α-alkyl chain in one of the pendant acetate arms (the size of the chain changes from four to fourteen carbon atoms). A multinuclear NMR study (¹H, ¹³C, ²⁷Al and ⁷¹Ga) gave some insights into the structure and dynamics of the metal chelates in solution, consistent with their rigidity and octahedral or pseudo-octahedral geometry. The critical micellar concentration of the chelates was determined using a fluorescence method and ²⁷Al NMR spectroscopy (Al(III) was used as a surrogate of Ga(III)), both showing similar results and suggesting that the chelates of NOTAC6 form pre-micellar aggregates. The logP (octanol-water) determination showed enhancement of the lipophilic character of the Ga(III) chelates with the increase of the number of carbons in the α-alkyl chain. Biodistribution and γ-scintigraphic studies of the ⁶⁷Ga(III) labeled chelates were performed on Wistar rats, showing higher liver uptake for [⁶⁷Ga](NOTAC8) in comparison to [⁶⁷Ga](NOTAC6), consistent with a longer α-alkyl chain and a higher lipophilicity. After 24 h both chelates were completely cleared off from the tissues and organs with no deposition in the bones and liver/spleen. [⁶⁷Ga](NOTAC8) showed high kinetic stability in blood serum.     

Rapid fluorescent visualization of multiple NAD(P)H oxidoreductases in homogenate,permeabilized cells,and tissue slices

Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed: (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices. The method allows one to assess, visualize, and localize, using fluorescent markers of cellular compartments, multiple NADH and NADPH oxidoreductase activities. The application of selective inhibitors (e.g., VAS2870, a NOX2 inhibitor; plumbagin, a NOX4 inhibitor) allows one to distinguish and compare specific NAD(P)H oxidoreductase activities in cells and tissues and to attribute them to known enzymes. The method is simple, rapid, and flexible. It can be easily adapted to a variety of tasks. It will be useful for investigations of the role of various NAD(P)H oxidoreductases in a number of physiological and pathophysiological processes.     

Natural products as sources of new fungicides (V): Design and synthesis of acetophenone derivatives against phytopathogenic fungi in vitro and in vivo

A series of acetophenone derivatives (10a–10i, 11, 12a–12g, 13a–13g, 14a–14d and 15a–15l) were designed, synthesized and evaluated for antifungal activities in vitro and in vivo. The antifungal activities of 53 compounds were tested against several plant pathogens, and their structure–activity relationship was summarized. Compounds 10a–10f displayed better antifungal effects than two reference fungicides. Interestingly, the most potent compound 10d exhibited antifungal properties against Cytospora sp., Botrytis cinerea, Magnaporthe grisea, with IC₅₀ values of 6.0–22.6?µg/mL, especially Cytospora sp. (IC₅₀?=?6.0?µg/mL). In the in vivo antifungal assays, 10d displayed the significant protective efficacy of 55.3% to Botrytis cinerea and 73.1% to Cytospora sp. The findings indicated that 10d may act as a potential pesticide lead compound that merits further investigation.     

Suppression of histone deacetylase 3 (HDAC3) enhances apoptosis induced by paclitaxel in human maxillary cancer cells in vitro and in vivo

Inclusion of chemotherapeutic drugs in treatment of patients with newly diagnosed head and neck cancer has improved response rates and prolonged median survival. Nevertheless, most patients with advanced head and neck cancer are destined to relapse and to develop resistance to initially used drugs such as paclitaxel. Consequently, it has been more important in cancer therapy to determine the molecular mechanisms that are related to cell-killing effects of anti-cancer agents or cancer resistance against them. Consequently, we examined whether abrogation of histone deacetylase 3 (HDAC3) expression by anti-sense oligonucleotides (ASOs) potentiates the efficacy of paclitaxel in human maxillary cancer IMC-3 cells. Here, we showed that paclitaxel-induced apoptosis was enhanced significantly by addition of ASOs for HDAC3 in cultured cells. Furthermore, paclitaxel-induced apoptosis in IMC-3 tumors transplanted in nude mice was enhanced significantly by administration of ASOs for HDAC3, thereby suppressing tumor growth. We provide new evidence that HDAC3 is a novel molecular target whose inactivation can potentiate the efficacy of anti-cancer drugs disrupting microtubules such as paclitaxel.     

Dinuclear copper(II) complexes containing 6-(benzylamino)purines as bridging ligands: Synthesis, characterization, and in vitro and in vivo antioxidant activities

A series of dinuclear copper(II) complexes involving 6-(benzylamino)purine derivatives, (HL_n), as bridging ligands were synthesized, characterized and tested for both their in vitro and in vivo antioxidant activities. Based on results of elemental analyses, temperature dependence of magnetic susceptibility measurements, UV-vis, FTIR, EPR, NMR and MALDI-TOF mass spectroscopy, conductivity measurements and thermal analyses, the complexes with general compositions of [Cu₂(μ-HL_n)₄Cl₂]Cl₂ · 2H₂O (1-4) and [Cu₂(μ-HL_n)₂(μ-Cl)₂Cl₂] (5-7) were prepared {where n = 1-4; HL₁ = 6-[(2-methoxybenzyl)amino]purine, HL₂ = 6-[(4-methoxybenzyl)amino]purine, HL₃ = 6-[(2,3-dimethoxybenzyl)amino]purine and HL₄ = 6-[(3,4-dimethoxybenzyl)amino]purine}. In the case of complexes 2, 3, 5 and 7, the antioxidant activities were studied by both in vitro {superoxide dismutase-mimic (SOD-mimic) activity} and in vivo {cytoprotective effect against the alloxan-induced diabetes (antidiabetic activity)} methods. The obtained IC₅₀ value of the SOD-mimic activity for the complex 5 (IC₅₀ = 0.253 μM) was shown to be even better than that of the native bovine Cu,Zn-SOD enzyme (IC₅₀ = 0.480 μM), used as a standard. As for the antidiabetic activity, the pretreatment of mice with complexes 3 and 7 led to the complete elimination of cytotoxic attack of alloxan and its free radical metabolites, used as a diabetogenic agent. The cytoprotective effect of these compounds was proved by the preservation of the initial blood glucose levels of the pretreated animals, as against the untreated control group.     

Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase

Ca(2+)-activated K(+) channels (K(Ca)) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of K(Ca) with NO production in isolated arterial mesenteric beds of the rat. In phenylephrine-contracted mesenteries, acetylcholine (ACh)-induced vasodilation was reduced by NO synthase (NOS) inhibition with N(ω)-nitro-L-arginine (L-NA), but in the presence of tetraethylammonium, L-NA did not further affect the response. In KCl-contracted mesenteries, the relaxation elicited by 100 nM ACh or 1 μM ionomycin was abolished by L-NA, tetraethylammonium, or simultaneous blockade of small-conductance K(Ca) (SK(Ca)) channels with apamin and intermediate-conductance K(Ca) (IK(Ca)) channels with triarylmethane-34 (TRAM-34). Apamin-TRAM-34 treatment also abolished 100 nM ACh-activated NO production, which was associated with an increase in superoxide formation. Endothelial cell Ca(2+) buffering with BAPTA elicited a similar increment in superoxide. Apamin-TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOS(Thr495)). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOS(Thr495) observed during apamin and TRAM-34 application. Our results indicate that blockade of SK(Ca) and IK(Ca) activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOS(Thr495). These findings disclose a novel mechanism involved in the control of NO production.     

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)α-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)α and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARα ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARα activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARα ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARα knockout (KO) mice compared with its wild type (WT) litter mates (130 ± 10 mmHg versus 107 ± 4 mmHg). l-NAME (100 mg/L p.o.), the inhibitor of NO production abolished the difference between PPARα KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8 ± 1.4 pM/mg versus 8.3 ± 0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46 ± 6%, p < 0.05) and a 3 fold greater Ca²⁺-dependent NOS activity in kidney homogenates of untreated PPARα WT compared with the KO mice. Clofibrate, a PPARα ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19 ± 4%, p < 0.05), increased urinary NO excretion (4.06 ± 0.53–7.07 ± 1.59 μM/24 h; p < 0.05) and reduced plasma 8-isoprostane level (45.8 ± 15 μM versus 31.4 ± 8 μM), and NADP(H) oxidase activity (16 ± 5%). Implantation of DOCA pellet (20 mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193 ± 13 mmHg versus 130 ± 12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARα activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.     

Homocysteine induces VCAM-1 gene expression through NF-kappaB and NAD(P)H oxidase activation: protective role of Mediterranean diet polyphenolic antioxidants

Hyperhomocysteinemia is a recognized risk factor for vascular disease, but pathogenetic mechanisms involved in its vascular actions are largely unknown. Because VCAM-1 expression is crucial in monocyte adhesion and early atherogenesis, we evaluated the NF-kappaB-related induction of VCAM-1 by homocysteine (Hcy) and the possible inhibitory effect of dietary polyphenolic antioxidants, such as trans-resveratrol (RSV) and hydroxytyrosol (HT), which are known inhibitors of NF-kappaB-mediated VCAM-1 induction. In human umbilical vein endothelial cells (HUVEC), Hcy, at 100 micromol/l, but not cysteine, induced VCAM-1 expression at the protein and mRNA levels, as shown by enzyme immunoassay and Northern analysis, respectively. Transfection studies with deletional VCAM-1 promoter constructs demonstrated that the two tandem NF-kappaB motifs in the VCAM-1 promoter are necessary for Hcy-induced VCAM-1 gene expression. Hcy-induced NF-kappaB activation was confirmed by EMSA, as shown by the nuclear translocation of its p65 (RelA) subunit and the degradation of the inhibitors IkappaB-alpha and IkappaB-beta by Western analysis. Hcy also increased intracellular reactive oxygen species by NAD(P)H oxidase activation, as shown by the membrane translocation of its p47(phox) subunit. NF-kappaB inhibitors decreased Hcy-induced intracellular reactive oxygen species and VCAM-1 expression. Finally, we found that nutritionally relevant concentrations of RSV and HT, but not folate and vitamin B6, reduce (by >60% at 10(-6) mol/l) Hcy-induced VCAM-1 expression and monocytoid cell adhesion to the endothelium. These data indicate that pathophysiologically relevant Hcy concentrations induce VCAM-1 expression through a prooxidant mechanism involving NF-kappaB. Natural Mediterranean diet antioxidants can inhibit such activation, suggesting their possible therapeutic role in Hcy-induced vascular damage.     

NAD(P)H oxidase and eNOS play differential roles in cytomegalovirus infection-induced microvascular dysfunction

Primary cytomegalovirus (CMV) infection promotes oxidative stress and reduces nitric oxide (NO) bioavailability in endothelial cells. These events are among the earliest vascular responses to cardiovascular risk factors. We assessed the roles of NAD(P)H oxidase and NO bioavailability in microvascular responses to persistent CMV infection alone or with hypercholesterolemia. Wild-type (WT) or gp91^phox (NAD(P)H oxidase subunit) knockout mice received mock inoculum or 3 × 10⁴ PFU murine CMV (mCMV) ip 5 weeks before placement on a normal or high-cholesterol diet (HC) for 4 weeks before assessment of arteriolar function and venular blood cell recruitment using intravital microscopy. Some WT groups received sepiapterin (a precursor of the nitric oxide synthase cofactor tetrahydrobiopterin) or apocynin (NAD(P)H oxidase inhibitor/antioxidant). Endothelium-dependent vasodilation was impaired in mCMV vs mock WT, regardless of diet. This was not affected by sepiapterin, and pharmacological inhibition of nitric oxide synthase reduced dilation similarly in mock and mCMV mice. Apocynin or deficiency of total, but not blood cell or vascular wall only (tested using bone marrow chimeras), gp91^phox protected against arteriolar dysfunction. Blood cell recruitment was induced by mCMV–HC. Sepiapterin, but not NAD(P)H oxidase deficiency/apocynin, reduced leukocyte accumulation, whereas platelet adhesion was reduced by sepiapterin, apocynin, or total, platelet-specific, or vascular wall gp91^phox deficiency. These data implicate activation of both hematopoietic and vessel wall NAD(P)H oxidase in mCMV-induced arteriolar dysfunction and platelet and vascular NAD(P)H oxidase in the thrombogenic phenotype induced by mCMV–HC. In contrast, findings with sepiapterin suggest that eNOS dysfunction, perhaps uncoupling, mediates venular, but not arteriolar, responses to mCMV–HC, thus indicating that NAD(P)H oxidase and eNOS differentially regulate microvascular responses to mCMV.     

Long‐term ammonium nutrition of Arabidopsis increases the extrachloroplastic NAD(P)H/NAD(P)+ ratio and mitochondrial reactive oxygen species level in leaves but does not impair photosynthetic capacity

Ammonium nutrition has been suggested to be associated with alterations in the oxidation‐reduction state of leaf cells. Herein, we show that ammonium nutrition in Arabidopsis thaliana increases leaf NAD(P)H/NAD(P)⁺ ratio, reactive oxygen species content and accumulation of biomolecules oxidized by free radicals. We used the method of rapid fractionation of protoplasts to analyse which cellular compartments were over‐reduced under ammonium supply and revealed that observed changes in NAD(P)H/NAD(P)⁺ ratio involved only the extrachloroplastic fraction. We also showed that ammonium nutrition changes mitochondrial electron transport chain activity, increasing mitochondrial reactive oxygen species production. Our results indicate that the functional impairment associated with ammonium nutrition is mainly associated with redox reactions outside the chloroplast.