ß-Cryptoxanthin Attenuates Cigarette-Smoke-Induced Lung Lesions in the Absence of Carotenoid Cleavage Enzymes (BCO1/BCO2) in Mice.

High dietary intake of ß-cryptoxanthin (BCX, an oxygenated provitamin A carotenoid) is associated with a lower risk of lung disease in smokers. BCX can be cleaved by ß-carotene-15,15'-oxygenase (BCO1) and ß-carotene-9',10'-oxygenase (BCO2) to produce retinol and apo-10'-carotenoids. We investigated whether BCX has protective effects against cigarette smoke (CS)-induced lung injury, dependent or independent of BCO1/BCO2 and their metabolites. Both BCO1-/-/BCO2-/- double knockout mice (DKO) and wild type (WT) littermates were supplemented with BCX 14 days and then exposed to CS for an additional 14 days. CS exposure significantly induced macrophage and neutrophil infiltration in the lung tissues of mice, regardless of genotypes, compared to the non-exposed littermates. BCX treatment significantly inhibited CS-induced inflammatory cell infiltration, hyperplasia in the bronchial epithelium, and enlarged alveolar airspaces in both WT and DKO mice, regardless of sex. The protective effects of BCX were associated with lower expression of IL-6, TNF-α, and matrix metalloproteinases-2 and -9. BCX treatment led to a significant increase in hepatic BCX levels in DKO mice, but not in WT mice, which had significant increase in hepatic retinol concentration. No apo-10'-carotenoids were detected in any of the groups. In vitro BCX, at comparable doses of 3-OH-ß-apo-10'-carotenal, was effective at inhibiting the lipopolysaccharide-induced inflammatory response in a human bronchial epithelial cell line. These data indicate that BCX can serve as an effective protective agent against CS-induced lung lesions in the absence of carotenoid cleavage enzymes.

Dietary ß-Cryptoxanthin Inhibits High-Refined Carbohydrate Diet-Induced Fatty Liver via Differential Protective Mechanisms Depending on Carotenoid Cleavage Enzymes in Male Mice.

BACKGROUND: ß-Cryptoxanthin (BCX), a provitamin A carotenoid shown to protect against nonalcoholic fatty liver disease (NAFLD), can be cleaved by ß-carotene-15,15'-oxygenase (BCO1) to generate vitamin A, and by ß-carotene-9',10'-oxygenase (BCO2) to produce bioactive apo-carotenoids. BCO1/BCO2 polymorphisms have been associated with variations in plasma carotenoid amounts in both humans and animals. OBJECTIVES: We investigated whether BCX feeding inhibits high refined-carbohydrate diet (HRCD)-induced NAFLD, dependent or independent of BCO1/BCO2. METHODS: Six-week-old male wild-type (WT) and BCO1-/-/BCO2-/- double knockout (DKO) mice were randomly fed HRCD (66.5% of energy from carbohydrate) with or without BCX (10 mg/kg diet) for 24 wk. Pathological and biochemical variables were analyzed in the liver and mesenteric adipose tissues (MATs). Data were analyzed by 2-factor ANOVA. RESULTS: Compared to their respective HRCD controls, BCX reduced hepatic steatosis severity by 33‒43% and hepatic total cholesterol by 43‒70% in both WT and DKO mice (P < 0.01). Hepatic concentrations of BCX, but not retinol and retinyl palmitate, were 33-fold higher in DKO mice than in WT mice (P < 0.001). BCX feeding increased the hepatic fatty acid oxidation protein peroxisome proliferator-activated receptor-α, and the cholesterol efflux gene ATP-binding cassette transporter5, and suppressed the lipogenesis gene acetyl-CoA carboxylase 1 (Acc1) in the MAT of WT mice but not DKO mice (P < 0.05). BCX feeding decreased the hepatic lipogenesis proteins ACC and stearoyl-CoA desaturase-1 (3-fold and 5-fold) and the cholesterol synthesis genes 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and HMG-CoA synthase 1 (2.7-fold and 1.8-fold) and increased the cholesterol catabolism gene cholesterol 7α-hydroxylase (1.9-fold) in the DKO but not WT mice (P < 0.05). BCX feeding increased hepatic protein sirtuin1 (2.5-fold) and AMP-activated protein kinase (9-fold) and decreased hepatic farnesoid X receptor protein (80%) and the inflammatory cytokine gene Il6 (6-fold) in the MAT of DKO mice but not WT mice (P < 0.05). CONCLUSION: BCX feeding mitigates HRCD-induced NAFLD in both WT and DKO mice through different mechanisms in the liver-MAT axis, depending on the presence or absence of BCO1/BCO2.

Ablation of carotenoid cleavage enzymes (BCO1 and BCO2) induced hepatic steatosis by altering the farnesoid X receptor/miR-34a/sirtuin 1 pathway.

ß-Carotene-15, 15'-oxygenase (BCO1) and ß-carotene-9', 10'-oxygenase (BCO2) are essential enzymes in carotenoid metabolism. While BCO1/BCO2 polymorphisms have been associated with alterations to human and animal carotenoid levels, experimental studies have suggested that BCO1 and BCO2 may have specific physiological functions beyond the cleavage of carotenoids. In the present study, we investigated the effect of ablation of both BCO1/BCO2 in the development of non-alcoholic fatty liver disease (NAFLD) and its underlying molecular mechanism(s). BCO1/BCO2 double knock out (DKO) mice developed hepatic steatosis (8/8) and had significantly higher levels of hepatic and plasma triglyceride and total cholesterol compared to WT (0/8). Hepatic changes in the BCO1/BCO2 DKO mice were associated with significant: 1) increases in lipogenesis markers, and decreases in fatty acid ß-oxidation markers; 2) upregulation of cholesterol metabolism markers; 3) alterations to microRNAs related to TG accumulation and cholesterol metabolism; 4) increases in an hepatic oxidative stress marker (HO-1) but decreases in anti-oxidant enzymes; and 5) decreases in farnesoid X receptor (FXR), small heterodimer partner (SHP), and sirtuin 1 (SIRT1). The present study provided novel experimental evidence that BCO1 and BCO2 could play a significant role in maintaining normal hepatic lipid and cholesterol homeostasis, potentially through the regulation of the FXR/miR-34a/SIRT1 pathway.

Tobacco carcinogen induces both lung cancer and non-alcoholic steatohepatitis and hepatocellular carcinomas in ferrets which can be attenuated by lycopene supplementation.

Early epidemiologic studies have reported that tobacco smoking, which is causally associated with liver cancer, is an independent risk factor for non-alcoholic fatty liver diseases (NAFLD). Lycopene from tomatoes has been shown to be a potential preventive agent against NAFLD and hepatocellular carcinoma (HCC). In the present study, we investigated whether the tobacco carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lesions in both lungs and livers of ferrets with or without lycopene intervention. Male ferrets (6 groups, n = 8-10) were treated either with NNK (50 mg/kg BW, i.p., once a month for four consecutive months) or saline with or without dietary lycopene supplementation (2.2 and 6.6 mg/kg BW/day, respectively) for 26 weeks. Results demonstrate that NNK exposure results in higher incidences of lung tumors, HCC and steatohepatitis (which is characterized by severe inflammatory cell infiltration with concurrent fat accumulation in liver, hepatocellular ballooning degeneration and increased NF-κB expression), as well as elevations in bilirubin and AST levels in ferrets. Lycopene supplementation at two doses prevented NNK-induced expressions of α7 nicotinic acetylcholine receptor in the lung and NF-κB and CYP2E1 in the liver and attenuated the NNK-induced mortality and pathological lesions in both the lungs and livers of ferrets. The present study provided strong experimental evidence that the tobacco carcinogen NNK can induce both HCC and steatohepatitis in the ferrets and can be a useful model for studying tobacco carcinogen-associated NAFLD and liver cancer. Furthermore, lycopene could provide potential benefits against smoke carcinogen-induced pulmonary and hepatic injury.

Apo-10'-lycopenoic acid, a lycopene metabolite, increases sirtuin 1 mRNA and protein levels and decreases hepatic fat accumulation in ob/ob mice.

Lycopene has been shown to be beneficial in protecting against high-fat diet-induced fatty liver. The recent demonstration that lycopene can be converted by carotene 9',10'-oxygenase into a biologically active metabolite, ALA, led us to propose that the function of lycopene can be mediated by ALA. In the present study, male ob/ob mice were fed a liquid high-fat diet (60% energy from fat) with ALA supplementation (ALA group, 240 µg · kg body weight(-1) · d(-1)) or without ALA supplementation as the control (C group) for 16 wk. Steatosis, SIRT1 expression and activity, genes involved in lipid metabolism, and ALA concentrations in the livers of mice were examined. The results showed that ALA supplementation resulted in a significant accumulation of ALA in the liver and markedly decreased the steatosis in the ALA group without altering body and liver weights compared to the C group. The mRNA and protein levels of hepatic SIRT1 were higher in the ALA group compared to the C group. SIRT1 activity also was higher in the ALA group, as indicated by the lower levels of acetylated forkhead box class O1 protein levels. In addition, the mRNA level of acetyl CoA carboxylase 1 was significantly lower in the ALA group than in the C group. Because SIRT1 plays a key role in lipid homeostasis, the present study suggests that the lycopene metabolite, ALA, protects against the development of steatosis in ob/ob mice by upregulating SIRT1 gene expression and activity.

Extracellular Nicotinamide Phosphoribosyltransferase Is a Component of the Senescence-Associated Secretory Phenotype.

Cellular senescence is a stress or damage response by which a cell adopts of state of essentially permanent proliferative arrest, coupled to the secretion of a number of biologically active molecules. This senescence-associated secretory phenotype (SASP) underlies many of the degenerative and regenerative aspects of cellular senescence - including promoting wound healing and development, but also driving diabetes and multiple age-associated diseases. We find that nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in nicotinamide adenine dinucleotide (NAD) biosynthesis, is elevated in senescent cells without a commensurate increase in NAD levels. This elevation is distinct from the acute DNA damage response, in which NAD is depleted, and recovery of NAD by NAMPT elevation is AMPK-activated protein kinase (AMPK)-dependent. Instead, we find that senescent cells release extracellular NAMPT (eNAMPT) as part of the SASP. eNAMPT has been reported to be released as a catalytically active extracellular vesicle-contained dimer that promotes NAD increases in other cells and extends lifespan, and also as free monomer that acts as a damage-associated molecular pattern and promotes conditions such as diabetes and fibrosis. Senescent cells released eNAMPT as dimer, but surprisingly eNAMPT appeared in the soluble secretome while being depleted from exosomes. Finally, diabetic mice showed elevated levels of eNAMPT, and this was lowered by treatment with the senolytic drug, ABT-263. Together, these data reveal a new SASP factor with implications for NAD metabolism.

Xanthophyll ß-Cryptoxanthin Inhibits Highly Refined Carbohydrate Diet-Promoted Hepatocellular Carcinoma Progression in Mice.

SCOPE: ß-Cryptoxanthin (BCX) can be cleaved by both ß-carotene 15,15'-oxygenase (BCO1) and ß-carotene 9',10'-oxygenase (BCO2), generating biological active vitamin A and apocarotenoids. We examined whether BCX feeding could inhibit diethylnitrosamine (DEN)-initiated, highly refined carbohydrate diet (HRCD)-promoted hepatocellular carcinoma (HCC) development, dependent or independent of BCO1/BCO2 activity. METHODS AND RESULTS: Two-week-old male wild-type (WT) and BCO1-/- /BCO2-/- double knockout (DKO) mice are given a single intraperitoneal injection of DEN (25 mg kg-1 body weight) to initiate hepatic carcinogenesis. At 6 weeks of age, all animals are fed HRCD (66.5% of energy from carbohydrate) with or without BCX for 24 weeks. BCX feeding increases hepatic vitamin A levels in WT mice, but not in DKO mice that shows a significant accumulation of hepatic BCX. Compared to their respective HRCD littermates, both WT and DKO fed BCX have significantly lower HCC multiplicity, average tumor size, and total tumor volume, and the steatosis scores. The chemopreventive effects of BCX are associated with increased p53 protein acetylation and decreased protein levels of lactate dehydrogenase and hypoxia-inducible factor-1α in tumors. CONCLUSION: This study suggests that BCX feeding may alleviate HRCD-promoted HCC progression by modulating the acetylation of p53, hypoxic tumor microenvironment, and glucose metabolism, independent of BCO1/BCO2.

Modulation of CREB activity by the Rho GTPase regulates cell and organism size during mouse embryonic development.

Rho GTPases regulate several aspects of tissue morphogenesis during animal development. We found that mice lacking the Rho-inhibitory protein, p190-B RhoGAP, are 30% reduced in size and exhibit developmental defects strikingly similar to those seen in mice lacking the CREB transcription factor. In p190-B RhoGAP-deficient mice, CREB phosphorylation is substantially reduced in embryonic tissues. Embryo-derived cells contain abnormally high levels of active Rho protein, are reduced in size, and exhibit defects in CREB activation upon exposure to insulin or IGF-1. The cell size defect is rescued by expression of constitutively activated CREB, and in wild-type cells, expression of activated Rho or dominant-negative CREB results in reduced cell size. Together, these results suggest that activity of the Rho GTPase modulates a signal from insulin/IGFs to CREB that determines cell size and animal size during embryogenesis.

Apo-10'-lycopenoic acid inhibits cancer cell migration and angiogenesis and induces peroxisome proliferator-activated receptor γ.

SCOPE: We have previously shown that apo-10'-lycopenoic acid (ALA), a derivative of lycopene through cleavage by carotene-9',10'-oxygenase, inhibits tumor progression and metastasis in both liver and lung cancer animal models. The underlying mechanism remains unknown. We hypothesized that ALA inhibits cancer cell motility and angiogenesis by up-regulating peroxisome proliferator-activated receptor γ (PPARγ) which is involved in controlling angiogenesis, tumor progression and metastasis. METHODS AND RESULTS: ALA treatment, in dose-dependent manner, was effective at inhibiting migration and invasion of liver and lung cancer cells (HuH7 and A549) in both Transwell and wound-healing models, as well as suppressing actin remodeling and ruffling/lamellipodia formation in HuH7 and immortalized lung BEAS-2B cells. ALA treatment resulted in suppression of angiogenesis in both tube formation and aortic ring assays and inhibition of matrix metalloproteinase-2 expression and activation in both HuH7 and A549 cells. Additionally, ALA dose-dependently increased the mRNA expression and protein levels of PPARγ in human THLE-2 liver cells. CONCLUSION: ALA inhibits cancer cell motility and angiogenesis and induces PPARγ expression, which could be one of the potential mechanisms for ALA protecting against tumor progression.

Dietary Tomato Powder Inhibits High-Fat Diet-Promoted Hepatocellular Carcinoma with Alteration of Gut Microbiota in Mice Lacking Carotenoid Cleavage Enzymes.

Both incidence and death rate due to liver cancer have increased in the United States. Higher consumption of lycopene-rich tomato and tomato products is associated with a decreased risk of cancers. ß-Carotene-15, 15'-oxygenase (BCO1), and ß-carotene-9', 10'-oxygenase (BCO2) cleave lycopene to produce bioactive apo-lycopenoids. Although BCO1/BCO2 polymorphisms affect human and animal lycopene levels, whether dietary tomato consumption can inhibit high-fat diet (HFD)-promoted hepatocellular carcinoma (HCC) development and affect gut microbiota in the absence of BCO1/BCO2 is unclear. BCO1/BCO2 double knockout mice were initiated with a hepatic carcinogen (diethylnitrosamine) at 2 weeks of age. At 6 weeks of age, the mice were randomly assigned to an HFD (60% of energy as fat) with or without tomato powder (TP) feeding for 24 weeks. Results showed that TP feeding significantly decreased HCC development (67%, 83%, and 95% reduction in incidence, multiplicity, and tumor volume, respectively, P < 0.05). Protective effects of TP feeding were associated with (1) decreased hepatic inflammatory foci development and mRNA expression of proinflammatory biomarkers (IL1ß, IL6, IL12α, monocyte chemoattractant protein-1, and inducible NO synthase); (2) increased mRNA expression of deacetylase sirtuin 1 and nicotinamide phosphoribosyltransferase involving NAD+ production; and (3) increased hepatic circadian clock genes (circadian locomotor output cycles kaput, period 2, and cryptochrome-2, Wee1). Furthermore, TP feeding increased gut microbial richness and diversity, and significantly decreased the relative abundance of the genus Clostridium and Mucispirillum, respectively. The present study demonstrates that dietary tomato feeding independent of carotenoid cleavage enzymes prevents HFD-induced inflammation with potential modulating gut microbiota and inhibits HFD-promoted HCC development.

Tomato Powder Inhibits Hepatic Steatosis and Inflammation Potentially Through Restoring SIRT1 Activity and Adiponectin Function Independent of Carotenoid Cleavage Enzymes in Mice.

SCOPE: Beta-carotene-15,15'-oxygenase (BCO1) and beta-carotene-9',10'-oxygenase (BCO2) metabolize lycopene to biologically active metabolites, which can ameliorate nonalcoholic fatty liver disease (NAFLD). We investigate the effects of tomato powder (TP containing substantial lycopene (2.3 mg/g)) on NAFLD development and gut microbiome in the absence of both BCO1 and BCO2 in mice. METHOD AND RESULTS: BCO1-/- /BCO2-/- double knockout mice were fed a high fat diet (HFD) alone (n = 9) or with TP feeding (n = 9) for 24 weeks. TP feeding significantly reduced pathological severity of steatosis and hepatic triglyceride levels in BCO1-/- /BCO2-/- mice (p < 0.04 vs HFD alone). This was associated with increased SIRT1 activity, nicotinamide phosphoribosyltransferase expression and AMP-activated protein kinase phosphorylation, and subsequently decreased lipogenesis, hepatic fatty acid uptake, and increasing fatty acid ß-oxidation (p < 0.05). TP feeding significantly decreased mRNA expression of proinflammatory genes (tnf-α, il-1ß, and il-6) in both liver and mesenteric adipose tissue, which were associated with increased plasma adiponectin and hepatic adiponectin receptor-2. Multiplexed 16S rRNA gene sequencing was performed using DNA extracted from cecum fecal samples. TP feeding increased microbial richness and decreased relative abundance of the genus Clostridium. CONCLUSION: Dietary TP can inhibit NAFLD independent of carotenoid cleavage enzymes, potentially through increasing SIRT1 activity and adiponectin production and decreasing Clostridium abundance.

A functional screen in human cells identifies UBF2 as an RNA polymerase II transcription factor that enhances the beta-catenin signaling pathway.

beta-Catenin signaling plays an important role in the development of many organisms and has a key part in driving the malignant transformation of epithelial cells comprising a variety of cancers. beta-Catenin can activate gene expression through its association with transcription factors of the lymphoid enhancer factor 1 (LEF-1)/T-cell factor (TCF) family. We designed a screen in human cells to identify novel genes that activate a beta-catenin-LEF/TCF-responsive promoter and isolated the high-mobility group box transcription factor, UBF2. UBF1 and UBF2 are splice variants of a common precursor RNA. Although UBF1 has been shown to activate RNA polymerase I-regulated genes, the function of UBF2 has remained obscure. Here, we show for the first time that both UBF1 and UBF2 activate RNA polymerase II-regulated promoters. UBF2 associates with LEF-1, as shown by coimmunoprecipitation experiments, and potentiates transcriptional activation stimulated by LEF-1/beta-catenin from a synthetic promoter with multimerized LEF/TCF binding sites and a natural cyclin D1 promoter with consensus LEF/TCF binding sites. Downregulation of endogenous UBF expression using an RNA interference approach reduces transcriptional activation of a beta-catenin-LEF/TCF-responsive promoter by means of overexpressed beta-catenin, further implicating UBF as a transcriptional enhancer of the beta-catenin pathway.

ß-Cryptoxanthin Reduced Lung Tumor Multiplicity and Inhibited Lung Cancer Cell Motility by Downregulating Nicotinic Acetylcholine Receptor α7 Signaling.

Despite the consistent association between a higher intake of the provitamin A carotenoid ß-cryptoxanthin (BCX) and a lower risk of lung cancer among smokers, potential mechanisms supporting BCX as a chemopreventive agent are needed. We first examined the effects of BCX on 4-[methyl nitrosamino]-1-[3-pyridyl]-1-butanone (NNK)-induced lung tumorigenesis in A/J mice. BCX supplementation was given daily to the mice starting 2 weeks prior to the injection of NNK and continued 16 weeks after NNK injection. BCX supplementation resulted in a dose-dependent increase of BCX concentration in both serum and lungs of the mice without a significant alteration of vitamin A (retinol and retinyl palmitate) concentration. BCX significantly reduced the multiplicity of the NNK-induced lung tumor by 52% to 63% compared with the NNK-treated mice without BCX supplementation. The protective effect of BCX in the lungs was associated with reductions of both mRNA and protein of the homopentameric neuronal nicotinic acetylcholine receptor α7 (α7-nAChR), which has been implicated in lung tumorigenesis. We then conducted an in vitro cell culture study and found that BCX treatment suppressed α7-nAChR expression and inhibited the migration and invasion of α7-nAChR-positive lung cancer cells but not in cells lacking α7-nAChR. The activities of BCX were significantly attenuated by activators of α7-nAChR/PI3K signaling or by overexpression of constitutively active PI3K. Collectively, the results suggest that BCX inhibits lung tumorigenesis and cancer cell motility through the downregulation of α7-nAChR/PI3K signaling, independent of its provitamin A activity. Therefore, BCX can be used as a chemopreventive agent or a chemotherapeutic compound against lung cancer. Cancer Prev Res; 9(11); 875-86. ©2016 AACR.

Isocaloric Pair-Fed High-Carbohydrate Diet Induced More Hepatic Steatosis and Inflammation than High-Fat Diet Mediated by miR-34a/SIRT1 Axis in Mice.

To investigate the different effects of isocaloric high-fat diet (HFD) and high-carbohydrate diet (HCD) on hepatic steatosis and the underlying mechanisms, especially the role of microRNA-34a/silent information regulator T1 (SIRT1) axis, C57BL/6J mice (n = 12/group) were isocaloric pair-fed with Lieber-DeCarli liquid diet containing either high fat (HFLD) or high carbohydrate (HCLD) for 16 weeks. As compared to the HFLD fed mice, despite the similar final body weights, HCLD feeding: (1) induced more severe hepatic steatosis; (2) up-regulated hepatic expression of miR-34a accompanied with significant decrease of SIRT1 and nicotinamide phosphoribosyltransferase (NAMPT), SIRT1 activity and phosphorylation of AMPK; (3) up-regulated de novo lipogenesis (DNL) related proteins expression (ACC, SCD1), and down-regulated expressions of miR-122, miR-370 and miR-33; (4) decreased mRNA expressions of genes Cpt1, Pparα and Pgc1α related to fatty acid oxidation; (5) increased hepatic total cholesterol concentration and decreased expression of cholesterol metabolism related genes Abcg5, Abcg8, Abcg11, Cyp7a1 and Cyp8b1; and (6) induced higher hepatic inflammatory response accompanied with significant increased mRNA expressions of Il1ß, Tnfα and Mcp1. Thus, isocaloric HCLD feeding induced greater severity in hepatic steatosis and inflammatory response than HFLD feeding, potentially through miR-34a/SIRT1 axis mediated promotion of DNL, inhibition of fatty acid oxidation and cholesterol metabolism.

Tumor progression locus 2 ablation suppressed hepatocellular carcinoma development by inhibiting hepatic inflammation and steatosis in mice.

BACKGROUND: Tumor progression locus 2 (TPL2), a serine-threonine kinase, functions as a critical regulator of inflammatory pathways and mediates oncogenic events. The potential role of Tpl2 in nonalcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) development remains unknown. METHODS: Both wild-type and Tpl2 knockout male mice were initiated by a hepatic carcinogen (diethylnitrosamine, i.p. with a single dose of 25 mg.kg(-1))at 2 weeks of age, and then were given the high carbohydrate diet feeding to induce hepatic steatosis, inflammation, adenoma and HCC for 24 weeks. RESULTS: Tpl2 knockout mice had significantly lower incidences of liver tumor and developed hepatocellular adenoma only, which is contrast to wild-type mice where they all developed HCC. Tpl2 knockout mice had significantly down-regulated phosphorylation of JNK and ERK, and levels of mRNA expression of pro-inflammatory cytokines (Il-1ß, Il-18, Mcp-1 and Nalp3), which correlated with the reduced incidence and number of hepatic inflammatory foci. Furthermore, Tpl2 ablation resulted in decreased hepatic steatosis and expression of de novo lipogenesis related markers (ACC, SCD1, SREBP1C and AKT phosphorylation), as well as reduction of endoplasmic reticulum stress biomarkers PERK and eIF-2a. CONCLUSION: The study revealed for the first time that Tpl2 plays a significant role in promoting HCC development by its pro-inflammatory effect, which suggested that Tpl2 could be a molecular target for HCC prevention.

Development of ferret as a human lung cancer model by injecting 4-(Nmethyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

OBJECTIVE: Development of new animal lung cancer models that are relevant to human lung carcino-genesis is important for lung cancer research. Previously we have shown the induction of lung tumor in ferrets (Mustela putorius furo) exposed to both tobacco smoke and a tobacco carcinogen (4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK). In the present study, we investigated whether NNK treatment alone induces both preneoplastic and neoplastic lesions in the lungs of ferrets. METHODS: We exposed ferrets to NNK by i.p. injection of NNK (50 mg/kg BW) once a month for four consecutive months and then followed up for 24, 26 and 32 weeks. The incidences of pulmonary pre-neoplastic and neoplastic lesions were assessed by histopathological examination. The expressions of 7 nicotinic acetylcholine receptor ( 7 nAChR, which has been shown to promote lung carcinogenesis)and its related molecular biomarkers in lungs were examined by immunohistochemistry and/or Western blotting analysis. RESULTS: Ferrets exposed to NNK alone developed both preneoplastic lesions (squamous metaplasia, dysplasia and atypical adenomatous hyperplasia) and tumors (squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma), which are commonly seen in humans. The incidence of tumor induced by NNK was time-dependent in the ferrets (16.7%, 40.0% and 66.7% for 24, 26 and 32 weeks, respectively). 7 nAChR is highly expressed in the ferret bronchial/bronchiolar epithelial cells, and alveolar macrophages in ferrets exposed to NNK, and in both squamous cell carcinoma and adenocarcinoma of the ferrets. In addition, we observed the tendency for an increase in phospho-ERK and cyclin D1 protein levels (p = 0.081 and 0.080, respectively) in the lungs of ferrets exposed to NNK. CONCLUSION: The development of both preneoplastic and neoplastic lesions in ferret lungs by injecting NNK alone provides a simple and highly relevant non-rodent model for studying biomarkers/molecular targets for the prevention, detection and treatment of lung carcinogenesis in humans.

ß-cryptoxanthin restores nicotine-reduced lung SIRT1 to normal levels and inhibits nicotine-promoted lung tumorigenesis and emphysema in A/J mice.

Nicotine, a large constituent of cigarette smoke, is associated with an increased risk of lung cancer, but the data supporting this relationship are inconsistent. Here, we found that nicotine treatment not only induced emphysema but also increased both lung tumor multiplicity and volume in 4-nitrosamino-1-(3-pyridyl)-1-butanone (NNK)-initiated lung cancer in A/J mice. This tumor-promoting effect of nicotine was accompanied by significant reductions in survival probability and lung Sirtuin 1 (SIRT1) expression, which has been proposed as a tumor suppressor. The decreased level of SIRT1 was associated with increased levels of AKT phosphorylation and interleukin (il)-6 mRNA but decreased tumor suppressor p53 and retinoic acid receptor (RAR)-ß mRNA levels in the lungs. Using this mouse model, we then determined whether ß-cryptoxanthin (BCX), a xanthophyll that is strongly associated with a reduced risk of lung cancer in several cohort studies, can inhibit nicotine-induced emphysema and lung tumorigenesis. We found that BCX supplementation at two different doses was associated with reductions of the nicotine-promoted lung tumor multiplicity and volume, as well as emphysema in mice treated with both NNK and nicotine. Moreover, BCX supplementation restored the nicotine-suppressed expression of lung SIRT1, p53, and RAR-ß to that of the control group, increased survival probability, and decreased the levels of lung il-6 mRNA and phosphorylation of AKT. The present study indicates that BCX is a preventive agent against emphysema and lung cancer with SIRT1 as a potential target. In addition, our study establishes a relevant animal lung cancer model for studying tumor growth within emphysematous microenvironments.

Lycopene metabolite, apo-10'-lycopenoic acid, inhibits diethylnitrosamine-initiated, high fat diet-promoted hepatic inflammation and tumorigenesis in mice.

Obesity is associated with increased risk in hepatocellular carcinoma (HCC) development and mortality. An important disease control strategy is the prevention of obesity-related hepatic inflammation and tumorigenesis by dietary means. Here, we report that apo-10'-lycopenoic acid (APO10LA), a cleavage metabolite of lycopene at its 9',10'-double bond by carotene-9',10'-oxygenase, functions as an effective chemopreventative agent against hepatic tumorigenesis and inflammation. APO10LA treatment on human liver THLE-2 and HuH7 cells dose dependently inhibited cell growth and upregulated sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase that may suppress hepatic carcinogenesis. This observed SIRT1 induction was associated with decreased cyclin D1 protein, increased cyclin-dependent kinase inhibitor p21 protein expression, and induced apoptosis. APO10LA supplementation (10 mg/kg diet) for 24 weeks significantly reduced diethylnitrosamine-initiated, high fat diet (HFD)-promoted hepatic tumorigenesis (50% reduction in tumor multiplicity; 65% in volume) and lung tumor incidence (85% reduction) in C57Bl/6J mice. The chemopreventative effects of APO10LA were associated with increased hepatic SIRT1 protein and deacetylation of SIRT1 targets, as well as with decreased caspase-1 activation and SIRT1 protein cleavage. APO10LA supplementation in diet improved glucose intolerance and reduced hepatic inflammation [decreased inflammatory foci, TNFα, interleukin (IL)-6, NF-κB p65 protein expression, and STAT3 activation] in HFD-fed mice. Furthermore, APO10LA suppressed Akt activation, cyclin D1 gene, and protein expression and promoted PARP protein cleavage in transformed cells within liver tumors. Taken together, these data indicate that APO10LA can effectively inhibit HFD-promoted hepatic tumorigenesis by stimulating SIRT1 signaling while reducing hepatic inflammation.

Beta-cryptoxanthin suppresses the growth of immortalized human bronchial epithelial cells and non-small-cell lung cancer cells and up-regulates retinoic acid receptor beta expression.

Recent findings of an inverse association between beta-cryptoxanthin and lung cancer risk in several observational epidemiologic studies suggest that beta-cryptoxanthin could potentially act as a chemopreventive agent against lung cancer. However, the biological activity of beta-cryptoxanthin and molecular mechanism(s) by which beta-cryptoxanthin affects lung tumourigenesis have not been studied. In the present study, we found that beta-cryptoxanthin inhibited the growth of A549 cells, a non-small-cell lung cancer cell line and BEAS-2B cells, an immortalized human bronchial epithelial cell line in a dose-dependent manner. beta-Cryptoxanthin suppressed the protein levels of cyclin D1 and cyclin E, up-regulated the cell cycle inhibitor p21, increased the number of lung cancer cells in the G1/G0 phase and decreased those in the S phase of the cell cycle. Consistent with inhibition of the lung cancer cell growth, beta-cryptoxanthin induced the mRNA levels of retinoic acid receptor beta (RARbeta) in BEAS-2B cells, although this effect was less pronounced in A549 cells. Furthermore, beta-cryptoxanthin transactivated RAR-mediated transcription activity of the retinoic acid response element. These findings suggest a mechanism of anti-proliferative action of beta-cryptoxanthin and indicate that beta-cryptoxanthin may be a promising chemopreventive agent against lung cancer.

Distinct but overlapping functions for the closely related p190 RhoGAPs in neural development.

The p190 RhoGAPs, p190A and p190B, are highly related GTPase-activating proteins for the Rho GTPases. Rho GTPases and p190A reportedly control various aspects of brain development, and we hypothesized that p190B would be likewise involved in neuronal development. We find that like p190A, p190B is prominently expressed in the developing and adult brain. Unlike p190A, p190B is not abundantly tyrosine phosphorylated. We further demonstrate, using p190B-deficient mice, that p190B is required for normal brain development. Mice lacking p190B display several major defects, including (1) deficits in the formation of major forebrain commissures, including the corpus callosum and anterior commissure, (2) dilation of the lateral ventricles, suggesting inhibition of neurogenesis and/or survival, (3) thinning of the neocortical intermediate zone, suggesting defects in neuronal differentiation and/or axonal outgrowth, and (4) impaired neuronal differentiation. These defects are similar to, but distinct from, those described in p190A-deficient mice. RNA interference-mediated knockdown of neither p190 protein results in significant inhibition of neurite outgrowth in neuroblastoma cells, despite an apparent increase in RhoA activity. We conclude that p190 RhoGAPs control pivotal aspects of neural development, including neuronal differentiation and process outgrowth, and that these effects are mediated by signaling systems that include, but are not limited to, RhoA.