Nordihydroguaiaretic Acid Treatment Research Articles

Nordihydroguaiaretic acid (NDGA) maintains the postharvest quality of fresh daylily flower buds (Hemerocallis citrina) by enhancing antioxidant metabolism and regulating phytohormone equilibrium

Daylily (Hemerocallis citrina) bud is a functional vegetable rich in various nutrients and many bioactive compounds. However, fresh daylily buds are very susceptible to senescence and quality deterioration occurs rapidly after harvest. The effects of nordihydroguaiaretic acid (NDGA) on postharvest quality, reactive oxygen species (ROS) metabolism and phytohormone alterations in daylily buds were investigated. The results showed that NDGA treatments delayed the loss of overall quality, and alleviated yellowing and disaggregation. The NDGA treatment inhibited the production of hydrogen peroxide and malondialdehyde, enhanced activities of ROS-scavenging enzymes and delayed the decrease in total antioxidant capacity. The biosynthesis of abscisic acid was decreased, while the contents of indole-3-acetic acid and brassinosteroids were increased by NDGA treatment, and therefore equilibrium involving several phytohormones was altered by NDGA. Transcriptomic profiling of genes associated with the signal transduction pathways of the main phytohormones was performed to reveal alterations in the regulation of postharvest senescence of daylily buds in response to NDGA. Collectively, NDGA treatment is a potential approach for the preservation of fresh daylily flower buds.

PaPYL9 is involved in the regulation of apricot fruit ripening through ABA signaling pathway

Abscisic acid (ABA) is a major regulator of non-climacteric fruit ripening; however, the role of ABA in the ripening of climacteric fruit is not clear. Here, as a typical climacteric fruit, apricots were used to investigate the role of ABA in fruit ripening. Based on weighted gene co-expression network analysis (WGCNA) of our previous transcriptome data, we treated ‘Danxing’ fruit with exogenous ABA and obtained ABA receptor genes, genes related to ABA biosynthesis and signal transduction, and analyzed the response of these candidate genes to exogenous ABA during fruit ripening. Subsequently, the full length of candidate PYLs genes were cloned, and their putative function were analyzed by phylogenetic analysis and protein structure domain analysis. And then the function of one candidate gene PaPYL9 was verified by using transgenic tomato. Furthermore, the response genes in transgenic tomato were screened by transcriptome sequencing, and ultimately the related regulatory network was proposed. The results showed that the injection of exogenous 1.89 mmol·L-1 ABA remarkably promoted fruit coloration, and increased the color index for red grapes (CIRG) and the total soluble solids (TSS) content, but significantly decreased the firmness and titratable acid (TA) content (p < 0.01). Nordihydroguaiaretic acid (NDGA), the inhibitor of ABA, appeared to have the converse role in TA, TSS, CIRG and firmness, during the ripening process. One NCED (9-cis-epoxycarotenoiddioxygenase) and five ABA receptor genes related to signal transduction were mined from the transcriptome data of apricot fruit through WGCNA. Compared with the control, the expression levels of NCED1, PYL9 (PYR/PYL/RCAR), SnRK2 (SUCROSE NON-FERMENTING1 (SNF1)-RELATED PROTEIN KINASE 2S), and ABF2 (ABRE-binding bZIP transcription) were induced dramatically by ABA treatment (p < 0.01), while NDGA treatment significantly inhibited their expression. Based on gene expression and protein domain analysis, we inferred that PaPYL9 is putatively involved in apricot fruit ripening. Overexpression of PaPYL9 in Micro-TOM tomatoes resulted in the promotion of early ripening. Simultaneously, the expression levels of genes related ethylene biosynthesis, chlorophyll degradation, fruit softening, flavor formation, pigment synthesis, and metabolism were all significantly induced in overexpression of PaPYL9 tomatoes. This indicates the central role of ABA in climacteric fruit ripening. A regulatory network was tentatively proposed, laying the foundation to unveil the molecular mechanism of the regulatory role of PaPYL9 in fruit ripening.

Effects of abscisic acid (ABA) on ethanol fermentation during postharvest ripening of Nanguo pear fruit (Pyrus ussuriensis Maxim.)

The aim of this study was to explore the role of abscisic acid (ABA) in ethanol fermentation during postharvest ripening of Nanguo pear fruit (Pyrus ussuriensis Maxim.). Pears harvest at commercial maturity were treated with 100 μmol L−1 ABA or its biosynthesis inhibitor nordihydroguaiaretic acid (NDGA). Our data indicated that ABA treatment promoted respiration rate and ethylene production during ripening compared with controls, while NDGA treatment had the opposite effects. During the early ripening stage, flesh firmness in ABA treatment group was lower than in controls; while in NDGA treatment group, it was higher than in controls. The contents of glucose, ethanol and acetaldehyde were increased by ABA treatment while reduced by NDGA treatment during ripening compared with the controls. ABA treatment promoted pyruvate and acetyl-CoA contents, whereas application of NDGA reduced pyruvate content and delayed the peak of acetyl-CoA content compared with the controls. The oxaloacetic acid (OA) and citric acid (CA) contents were reduced by ABA treatment while increased by NDGA treatment during ripening. ABA treatment promoted pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) activities, whereas NDGA treatment reduced their activities mainly at the early ripening stage. Quantitative real-time polymerase chain reaction (qPCR) showed that expression of PDC1, ADH1 and ADH2 was differentially up-regulated by ABA treatment during ripening while down-regulated by NDGA treatment compared with the controls. PDC2 expression was enhanced after application of NDGA during ripening while reduced by ABA treatment at the early ripening stage. Our data indicated that ABA had a promoting effect on ethanol fermentation during ripening of Nanguo pear fruit.

Nordihydroguaiaretic acid reduces secondary organ injury in septic rats after cecal ligation and puncture.

BackgroundNordihydroguaiaretic acid (NDGA) is a plant extract that has been shown to act as a free radical scavenger and pluripotent inhibitor of pro-inflammatory cytokines, two major cellular processes involved in the pathophysiology of sepsis. We investigated whether NDGA would improve markers of organ injury as well as survival in a rodent model of sepsis.MethodsAbdominal sepsis was induced by cecal ligation and double puncture (CLP) in male Sprague-Dawley rats. NDGA was administered either at the time of injury (pre-) or 6 hours later (post-treatment). A sham surgery group and a vehicle only group were also followed as controls. Blood and lung tissue were collected 24 h after CLP. Lung tissue was used for histopathologic analysis and to measure pulmonary edema. Arterial oxygenation was measured directly to generate PaO2/FiO2, and markers of renal injury (blood urea nitrogen), liver injury (alanine aminotransferase), and tissue hypoxia (lactate) were measured. In a separate set of animals consisting of the same treatment groups, animals were followed for up to 36 hours for survival.ResultsNDGA pre-treatment resulted in improved oxygenation, less lung edema, lower lactate, lower BUN, and reduced histologic lung injury. NDGA post-treatment resulted in less lung edema, lower lactate, lower BUN, and less histologic lung injury, but did not significantly change oxygenation. None of the NDGA treatment groups statistically affected ALT or creatinine. NDGA pre-treatment showed improved survival compared with control CLP animals at 36 hours, while post-treatment did not.ConclusionsNDGA represents a novel pleiotropic anti-inflammatory agent with potential clinical utility for modulation of organ injury secondary to sepsis.

Anthocyanin accumulation is initiated by abscisic acid to enhance fruit color during fig (Ficus carica L.) ripening

Fig fruit is well-known for its attractive flavor, color, and nutritional and medicinal value. Anthocyanin contributes to the fruit's color and constitutes a high percentage of the total antioxidant content of the fig fruit. We quantified the major anthocyanins and characterized the expression levels of anthocyanin-biosynthesis and transcription factor genes in fruit treated on-tree with exogenous abscisic acid (ABA) or ethephon, or the ABA inhibitors nordihydroguaiaretic acid (NDGA) or fluridone. The major anthocyanins cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside were found in significantly higher quantities in exogenous ABA- and ethephon-treated fruit, with early dark purple color compared to the controls. On the other hand, NDGA- and fluridone-treated fruit had significantly lower amounts of anthocyanins, with less purple color coverage than controls. Expression levels of the anthocyanin-biosynthesis genes FcPAL, FcCHS2, FcCHI, FcF3H, FcDFR, FcANS, FcUFGT and Fc3RT were upregulated by exogenous ABA and ethephon treatment, and downregulated by NDGA and fluridone treatment. The MYB–bHLH–WD40 complex-related genes of ripe fig fruit were identified. In particular, FcMYB113 was strongly upregulated by exogenous ABA and ethephon, and strongly downregulated by NDGA and fluridone. In addition, moderate upregulation of FcGL3 and FcWD40 was observed with exogenous ABA and ethephon treatment, and moderate downregulation in NDGA- and fluridone-treated fruit. These results indicate that ABA can initiate anthocyanin biosynthesis, which ultimately improves the color and nutritional value of fig fruit, enhancing their attractiveness to consumers.

Effect of nordihydroguaiaretic acid on grape berry cracking

Fruit cracking is a phenomenon involving the splitting of the fruit peel, which causes considerable economic losses. The objective of the present study was to determine whether a nordihydroguaiaretic acid (NDGA) treatment can decrease the incidence of grape berry cracking in the cracking-susceptible Vitis vinifera cultivar ‘Aishen Meigui’. The results indicated that NDGA can significantly decrease the fruit-cracking rate. There may be two reasons for this observation. Firstly, the NDGA treatment decreased the synthesis of endogenous abscisic acid (ABA) as well as the soluble sugar and total soluble solid contents, which ultimately lowered the osmotic pressure, water absorption capacity, and peel turgor pressure. Second, the decrease in the endogenous ABA content also decreased the pectin methylesterase and polygalacturonase activities, delayed the degradation of pectin, increased the peel protopectin content, enhanced the peel mechanical properties, and increased the resistance to fruit cracking.

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a target of NDGA and provide mechanistic insight into its role in longevity.

Nordihydroguaiaretic Acid, a Lignan from Larrea tridentata (Creosote Bush), Protects Against American Lifestyle-Induced Obesity Syndrome Diet-Induced Metabolic Dysfunction in Mice.

To determine the effects of nordihydroguaiaretic acid (NDGA) on metabolic and molecular changes in response to feeding a typical American fast food or Western diet, mice were fed an American lifestyle-induced obesity syndrome (ALIOS) diet and subjected to metabolic analysis. Male C57BL/6J mice were randomly assigned to the ALIOS diet, the ALIOS diet supplemented with NDGA (NDGA+ALIOS), or a control diet and were maintained on the specific diet for 8 weeks. Mice fed the ALIOS diet showed increased body, liver, and epididymal fat pad weight as well as increased plasma alanine transaminase (ALT) and aspartate aminotransferase (AST) levels (a measure of liver injury) and liver triglyceride content. Coadministration of NDGA normalized body and epididymal fat pad weight, ALT and AST levels, and liver triglycerides. NDGA treatment also improved insulin sensitivity but not glucose intolerance in mice fed the ALIOS diet. In mice fed the NDGA+ALIOS diet, NDGA supplementation induced peroxisome proliferator-activated receptor α (PPARα; the master regulator of fatty acid oxidation) and mRNA levels of carnitine palmitoyltransferases Cpt1c and Cpt2, key genes involved in fatty acid oxidation, compared with the ALIOS diet. NDGA significantly reduced liver endoplasmic reticulum (ER) stress response C/EBP homologous protein, compared with chow or the ALIOS diet, and also ameliorated ALIOS diet-induced elevation of apoptosis signaling protein, caspase 3. Likewise, NDGA downregulated the ALIOS diet-induced mRNA levels of Pparg, fatty acid synthase Fasn, and diacylglycerol acyltransferase Dgat2 NDGA treatment of ALIOS-fed mice upregulated the hepatic expression of antioxidant enzymes, glutathione peroxidase 4, and peroxiredoxin 3 proteins. In conclusion, we provide evidence that NDGA improves metabolic dysregulation by simultaneously modulating the PPARα transcription factor and key genes involved in fatty acid oxidation, key antioxidant and lipogenic enzymes, and apoptosis and ER stress signaling pathways.

Effect of Creosote Bush-Derived NDGA on Expression of Genes Involved in Lipid Metabolism in Liver of High-Fructose Fed Rats: Relevance to NDGA Amelioration of Hypertriglyceridemia and Hepatic Steatosis.

Nordihydroguaiaretic acid (NDGA), the main metabolite of Creosote bush, has been shown to have profound effects on the core components of the metabolic syndrome (MetS), lowering blood glucose, free fatty acids (FFA) and triglyceride (TG) levels in several models of dyslipidemia, as well as improving body weight (obesity), insulin resistance, diabetes and hypertension, and ameliorating hepatic steatosis. In the present study, a high-fructose diet (HFrD) fed rat model of hypertriglyceridemia was employed to further delineate the underlying mechanism by which NDGA exerts its anti-hypertriglyceridemic action. In the HFrD treatment group, NDGA administration by oral gavage decreased plasma levels of TG, glucose, FFA, and insulin, increased hepatic mitochondrial fatty acid oxidation and attenuated hepatic TG accumulation. qRT-PCR measurements indicated that NDGA treatment increased the mRNA expression of key fatty acid transport (L-FABP, CD36), and fatty acid oxidation (ACOX1, CPT-2, and PPARα transcription factor) genes and decreased the gene expression of enzymes involved in lipogenesis (FASN, ACC1, SCD1, L-PK and ChREBP and SREBP-1c transcription factors). Western blot analysis indicated that NDGA administration upregulated hepatic insulin signaling (P-Akt), AMPK activity (P-AMPK), MLYCD, and PPARα protein levels, but decreased SCD1, ACC1 and ACC2 protein content and also inactivated ACC1 activity (increased P-ACC1). These findings suggest that NDGA ameliorates hypertriglyceridemia and hepatic steatosis primarily by interfering with lipogenesis and promoting increased channeling of fatty acids towards their oxidation.

Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly.

Abstract 064: The Lipoxigenase Inhibitor Nordihydroguaiaretic Acid Prevents the Development of Hypoxia-Induced Pulmonary Hypertension in Mice

Pulmonary hypertension (PH) is a chronic lung disease characterized by progressively elevated pulmonary arterial pressures and severe pulmonary vascular remodeling resulting from interactions between oxidized lipoprotein deposition and increased endothelial proliferation. Previously we have shown increased plasma levels of biological oxidation products such as hydroxyoctadecadienoic acids (HODEs) and hydroxyeicosatetraenoic acids (HETEs) in the rat monocrotaline model of PH. Here we investigated the role of HETEs and HODEs in the development of PH and whether their inhibition with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) attenuates the progression of PH. Mice were placed in a hypoxic chamber with O2 concentrations of ≤10% for 21 days and either left untreated to develop PH (n=7) or treated with NDGA daily (10mg/kg/day, i.p., n=4) from day 1. Direct RV catheterization was terminally performed to record RV pressure (RVP). Pulmonary arteriolar thickening and oxidized lipid deposition were assessed by staining lung sections with Masson’s Trichrome or with α-smooth muscle actin and E-06 (marker for oxidized low-density lipoproteins). In vitro, human pulmonary artery smooth muscle cell (hPASMC) proliferation was assessed by MTT assays in the absence or presence of 12-HETE (100ng/ml), 9-HODE (1µg/ml) and 13-HODE (1µg/ml) alone or together with NDGA (10, 25 and 50µM). In-vitro, HETE/HODE treatment increased hPASMC proliferation ~ 2-fold when compared to untreated cells and NDGA significantly inhibited the proliferative effects of all three oxidized lipids. In-vivo, NDGA treatment prevented the development of PH. RVP was lower in the NDGA-treated group vs. the PH group (24.01±1.39mmHg vs. 36.91±5.74mmHg, p&lt;0.05) and was comparable to control normoxic mice (20.93±2.52mmHg). RV hypertrophy index was significantly elevated in the PH mice versus control mice (0.38±0.03 vs. 0.28±0.02 (p&lt;0.001), while NDGA treatment completely prevented the development of RV hypertrophy (0.28±0.04). Lung sections demonstrated arteriolar thickening and E-06 positive deposits in the PH group, which was prevented by NDGA therapy. We conclude that oxidized fatty acid deposition and accumulation might play a role in the development of PH.

NDGA reduces secondary damage after spinal cord injury in rats via anti-inflammatory effects

After spinal cord injury (SCI), a series of complex pathophysiological processes follows the initial injury. Because inflammation plays a key role in this secondary pathology damage, anti-inflammatory drug treatment may reduce secondary damage and protect neurons after SCI. Though nordihydroguaiaretic acid (NDGA) can inhibit inflammatory responses, its potential roles in neuroprotection and anti- inflammation in an SCI model have not been studied. In this study, we investigated the anti-inflammatory effects of NDGA in SCI. First, histopathological alterations were evaluated with hematoxylin/eosin (HE) and Nissl staining, showing an increased number of neurons after NDGA administration. Additionally, the extent of secondary damage was assessed by TUNEL assay and measurement of astrocyte proliferation. The data showed that the numbers of apoptotic cells and the proliferative extent of astrocytes were significantly decreased by the use of NDGA. The anti-inflammatory effect of NDGA was evaluated by measuring myeloperoxidase (MPO) levels as an indicator of neutrophil activity, macrophage/microglia numbers, and expression of inflammatory cytokines including IL-1β and TNF-α. NDGA treatment significantly decreased the MPO level and the number of macrophages/microglia. In addition, NDGA also suppressed the expression of IL-1β and TNF-α after SCI. These data suggest that anti-inflammatory action by NDGA can reduce secondary damage after SCI

Nordihydroguaiaretic acid inhibition of NFATc1 suppresses osteoclastogenesis and arthritis bone destruction in rats

Nordihydroguaiaretic acid (NDGA) is known to have prominent anticancer activity against several cancers, and is also known to be an inhibitor of 5-lipoxygenase (5-LO). In this study, we investigated the regulatory function of NDGA on inflammatory bone destruction mediated by osteoclasts. NDGA markedly inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced formation of osteoclasts in cultures of murine osteoclast precursor cell line RAW-D cells and primary bone marrow-derived macrophages culture systems. The inhibitory effect of NDGA on osteoclastogenesis did not arise from the inhibition of 5-LO activity. NDGA did not affect MAPKs, such as p38, JNK, and NF-κB, but significantly inhibited the induction of NFATc1, a key transcription factor for osteoclastogenesis. NDGA also suppressed activation of ERK in osteoclast precursors. RANKL-induced calcium oscillation observed in osteoclast precursors was completely diminished by the addition of NDGA. In mature osteoclasts, RANKL-induced nuclear translocation of NFATc1 was clearly inhibited by NDGA treatment. Finally, in vivo studies demonstrated that administration of NDGA significantly reduced severe bone destruction and osteoclast recruitment in the ankle joint of rats with adjuvant-induced arthritis. These results indicate the potential utility of NDGA as a therapeutic agent for ameliorating inflammatory bone destruction in rheumatoid arthritis.

Nordihydroguaiaretic acid improves metabolic dysregulation and aberrant hepatic lipid metabolism in mice by both PPARα-dependent and -independent pathways

Creosote bush-derived nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, possesses antioxidant properties and functions as a potent antihyperlipidemic agent in rodent models. Here, we examined the effect of chronic NDGA treatment of ob/ob mice on plasma dyslipidemia, hepatic steatosis, and changes in hepatic gene expression. Feeding ob/ob mice a chow diet supplemented with either low (0.83 g/kg diet) or high-dose (2.5 g/kg diet) NDGA for 16 wk significantly improved plasma triglyceride (TG), inflammatory chemokine levels, hyperinsulinemia, insulin sensitivity, and glucose intolerance. NDGA treatment caused a marked reduction in liver weight and TG content, while enhancing rates of fatty acid oxidation. Microarray analysis of hepatic gene expression demonstrated that NDGA treatment altered genes for lipid metabolism, with genes involved in fatty acid catabolism most significantly increased. NDGA upregulated the mRNA and nuclear protein levels of peroxisome proliferator-activated receptor α (PPARα), and the activated (phosphorylated) form of AMP-activated kinase. NDGA increased PPARα promoter activity in AML12 hepatocytes and also prevented the fatty acid suppression of PPARα expression. In contrast, PPARα siRNA abrogated the stimulatory effect of NDGA on fatty acid catabolism. Likewise, no stimulatory effect of NDGA on hepatic fatty acid oxidation was observed in the livers of PPARα-deficient mice, but the ability of NDGA to reverse fatty liver conditions was unaffected. In conclusion, the beneficial actions of NDGA on dyslipidemia and hepatic steatosis in ob/ob mice are exerted primarily through enhanced fatty acid oxidation via PPARα-dependent pathways. However, PPARα-independent pathways also contribute to NDGA's action to ameliorate hepatic steatosis.

Mode of action of abscisic acid in triggering ethylene biosynthesis and softening during ripening in mango fruit

The role of abscisic acid (ABA) in triggering ethylene biosynthesis and ripening of mango fruit was investigated by applying ABA [S-(+)-cis,trans-abscisic acid] and an inhibitor of its biosynthesis [nordihydroguaiaretic acid (NDGA)]. Application of 1mM ABA accelerated ethylene biosynthesis through promoting the activities of ethylene biosynthesis enzymes (1-aminocyclopropane-1-carboxylic acid synthase, ACS; 1-aminocyclopropane-1-carboxylic acid oxidase, ACO) and accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC), enhanced fruit softening and activity of endo-polygalacturonase and reduced pectin esterase activity in the pulp. The activities of ethylene biosynthesis and softening enzymes were significantly delayed and/or suppressed in the pulp of NDGA-treated fruit. The ABA-treated fruit had higher total sugars and sucrose as well as degradation of total organic acids, and citric and fumaric acids compared with NDGA treatment. These results suggest that ABA is involved in regulating mango fruit ripening and its effects are, at least in part, mediated by changes in ethylene production.

Transcriptional Regulation of Genes Encoding Key Enzymes of Abscisic Acid Metabolism During Melon (Cucumis melo L.) Fruit Development and Ripening

The ‘Elizabeth’ melon (Cucumis melo L. cv. Elizabeth) is regarded as a climacteric fruit, but it has characteristics of both climacteric and nonclimacteric fruits during the ripening stage. To clarify whether the ethylene-independent pathways during ripening were related to abscisic acid (ABA), four cDNAs that encode for key enzymes of ABA metabolism (CMNCEDs, CMCYP707A1, and CMBG1) were cloned and their transcription expression levels were analyzed. The levels of endogenous ABA and ethylene production in fruit ripening and the effect of exogenous ABA or nordihydroguaiaretic acid (NDGA) treatment on fruit senescence post-harvest were also investigated. The results showed that the ABA levels in young fruit were initially high then gradually decreased. At 20 days after fruit setting (DAFS) there was a rapid increase that peaked at 30 DAFS. Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) content, and ACC oxidase (ACO) activity were all at low levels at the early stages of fruit setting, and their peaks were observed 5 days after the ABA peak. The CMNCED3 gene and the β-glucosidase gene CMBG1 had similar expression patterns, with expression peaks that occurred at 30 DAFS, which was consistent with the accumulation of ABA. The expression of CMNCED2 was relatively high during the early stages and no peak value occurred in the ripening stage. The CMCYP707A1 gene was expressed mainly at the late ripening stage. The exogenous ABA treatment promoted fruit ripening and softening by the upregulation and expression of the ethylene synthesis genes CmACS1 and CmACO1 and the cell wall catabolic enzyme gene CmPG1. The effect was proportional to the concentration of ABA, but too much ABA (>500 μmol/L) had a negative effect on fruit development. The NDGA treatment also had a negative effect on fruit ripening and development. ABA and ethylene have a synergistic effect on the regulation of melon fruit ripening. ABA mainly acted in the early stages of development, whereas ethylene played a major role in later stages of ripening. The endogenous ABA levels were maintained in a dynamic balance of biosynthesis and catabolism, which were regulated by CMNCEDs, CMBG1, and CMCYP707A1.

Nordihydroguaiaretic Acid from Creosote Bush (Larrea tridentata) Mitigates 12‐O‐Tetradecanoylphorbol‐13‐Acetate‐Induced Inflammatory and Oxidative Stress Responses of Tumor Promotion Cascade in Mouse Skin

Nordihydroguaiaretic acid (NDGA) is a phenolic antioxidant found in the leaves and twigs of the evergreen desert shrub, Larrea tridentata (Sesse and Moc. ex DC) Coville (creosote bush). It has a long history of traditional medicinal use by the Native Americans and Mexicans. The modulatory effects of topically applied NDGA was studied on acute inflammatory and oxidative stress responses in mouse skin induced by stage I tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). Double TPA treatment adversely altered many of the marker responses of stage I skin tumor promotion cascade. Pretreatment of NDGA in TPA-treated mice mitigated cutaneous lipid peroxidation and inhibited production of hydrogen peroxide. NDGA treatment also restored reduced glutathione level and activities of antioxidant enzymes. Elevated activities of myeloperoxidase, xanthine oxidase and skin edema formation in TPA-treated mice were also lowered by NDGA indicating a restrained inflammatory response. Furthermore, results of histological study demonstrated inhibitory effect of NDGA on cellular inflammatory responses. This study provides a direct evidence of antioxidative and anti-inflammatory properties of NDGA against TPA-induced cutaneous inflammation and oxidative stress corroborating its chemopreventive potential against skin cancer.

Removal of the toxic effects of chlormadinon acetate on the development of Drosophila melanogaster via the use of nordihydroguaiaretic acid

In this study, the effects of chlormadinon acetate (CMA) and CMA + nordihydroguaiaretic acid (NDGA) on various developmental stages of Drosophila melanogaster were investigated. Different concentrations of CMA (1.0; 3.0; 5.0 and 10.0 μM/100 mL medium) and CMA + NDGA as the concentrations of CMA (1.0 + 1.0; 3.0 + 3.0; 5.0 + 5.0 and 10.0 + 10.0 μM/100 mL medium) were carried out during the developmental periods of the flies. When F(1) progeny of control and application groups were compared, CMA was found to extend the process of metamorphosis and decrease the total offspring numbers. However, these negative effects were inhibited by NDGA treatment at different concentrations. These results suggest that NDGA could effectively inhibit CMA-induced abnormalities in developmental stages of D. melanogaster. It was found that the difference between the groups was significantly important (p < 0.05).

Nordihydroguaiaretic acid inhibits transforming growth factor β type 1 receptor activity and downstream signaling

It has been well documented that nordihydroguaiaretic acid (NDGA), a phenolic lignan isolated from the creosote bush, Larrea tridentate, has anti-cancer activity in vitro and in vivo. Several mechanisms have been identified that could contribute to these actions, as NDGA directly inhibits metabolic enzymes and receptor tyrosine kinases that are established anti-cancer targets. In the present study, we show that NDGA inhibits the transforming growth factor β (TGF-β) type I receptor, a serine threonine kinase receptor. In cultured cells, NDGA treatment repressed Smad2 phosphorylation induced by TGF-β treatment and by a constitutively active mutant of TGF-β type I receptor (T202D). NDGA also inhibited downstream transcriptional activation mediated by both TGF-β treatment and the constitutively active mutant receptor. In vitro, NDGA inhibited TGF-β type I receptor mediated Smad2 phosphorylation in crude cell lysates and in a purified preparation. Importantly, screening select analogs demonstrated that modification of NDGA's structure resulted in altered potency against the receptor. These results indicated that the structure of NDGA can be modified to achieve increased potency. Together our data provide a novel mechanism for NDGA activity which could help explain its anti-cancer activity, and suggest that NDGA could serve as a structural motif for developing serine/threonine kinase inhibitors with selectivity for TGF-β type I receptor.

Reactive oxygen species and lipoxygenases regulate the oncogenicity of NPM-ALK-positive anaplastic large cell lymphomas

The chimera nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the tyrosine kinase activity of which is constitutively upregulated, is the causative agent of 75% of the anaplastic large-cell lymphomas (ALCLs). We have demonstrated that NPM-ALK induces the production of reactive oxygen species (ROS) by a pathway involving the arachidonic acid-metabolizing enzymes of the lipoxygenase (LOX) family. The use of the LOX inhibitor nordihydroguaiaretic acid (NDGA) and of the anti-oxidant N-acetylcysteine (NAC) demonstrated that ROS are important in maintaining the ALK kinase active. Consistent with this, NDGA treatment resulted in the inhibition of key pathways, such as Akt, signal transducer and activator of transcription factor 3 (STAT3) and extracellular signal-regulated kinase (ERK), which are involved in NPM-ALK antiapoptotic and pro-mitogenic functions. Conversely, the stress-activated kinase p38, described in some instances as a mediator of apoptosis, was activated. Interestingly, 5-LOX, an isoform involved in many cancers, was found to be activated in NPM-ALK(+) cells. Functional studies have shown that transforming properties, namely proliferation and resistance to apoptosis, were abrogated by treatment with either NDGA or the 5-LOX inhibitor (N-(3-phenoxycinnamyl)-acetohydroxamic acid) (BW A4C). Together, these data point to the ROS/LOX pathway as a potential new target for therapy in NPM-ALK-positive tumors.