A Basic ApoE-Based Peptide Mediator to Deliver Proteins across the Blood-Brain Barrier: Long-Term Efficacy, Toxicity, and Mechanism.

We have investigated delivery of protein therapeutics from the bloodstream into the brain using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a lysosomal disease due to deficiencies in tripeptidyl peptidase 1 (TPP1). Supraphysiological levels of TPP1 are delivered to the mouse brain by acute intravenous injection when co-administered with K16ApoE, a peptide that in trans mediates passage across the blood-brain barrier (BBB). Chronic treatment of LINCL mice with TPP1 and K16ApoE extended the lifespan from 126 to >294 days, diminished pathology, and slowed locomotor dysfunction. K16ApoE enhanced uptake of a fixable biotin tracer by brain endothelial cells in a dose-dependent manner, suggesting that its mechanism involves stimulation of endocytosis. Pharmacokinetic experiments indicated that K16ApoE functions without disrupting the BBB, with minimal effects on overall clearance or uptake by the liver and kidney. K16ApoE has a narrow therapeutic index, with toxicity manifested as lethargy and/or death in mice. To address this, we evaluated variant peptides but found that efficacy and toxicity are associated, suggesting that desired and adverse effects are mechanistically related. Toxicity currently precludes direct clinical application of peptide-mediated delivery in its present form but it remains a useful approach to proof-of-principle studies for biologic therapies to the brain in animal models.

δ- and γ-tocopherols inhibit phIP/DSS-induced colon carcinogenesis by protection against early cellular and DNA damages.

Tocopherols, the major forms of vitamin E, are a family of fat-soluble compounds that exist in alpha (α-T), beta (ß-T), gamma (γ-T), and delta (δ-T) variants. A cancer preventive effect of vitamin E is suggested by epidemiological studies. However, past animal studies and human intervention trials with α-T, the most active vitamin E form, have yielded disappointing results. A possible explanation is that the cancer preventive activity of α-T is weak compared to other tocopherol forms. In the present study, we investigated the effects of δ-T, γ-T, and α-T (0.2% in diet) in a novel colon cancer model induced by the meat-derived dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by dextran sodium sulfate (DSS)-induced colitis in CYP1A-humanized (hCYP1A) mice. PhIP/DSS treatments induced multiple polypoid tumors, mainly tubular adenocarcinomas, in the middle to distal colon of the hCYP1A mice after 10 wk. Dietary supplementation with δ-T and γ-T significantly reduced colon tumor formation and suppressed markers of oxidative and nitrosative stress (i.e., 8-oxo-dG and nitrotyrosine) as well as pro-inflammatory mediators (i.e., NF-κB p65 and p-STAT3) in tumors and adjacent tissues. By administering δ-T at different time periods, we obtained results suggesting that the inhibitory effect of δ-T against colon carcinogenesis is mainly due to protection against early cellular and DNA damages caused by PhIP. α-T was found to be ineffective in inhibiting colon tumors and less effective in attenuating the molecular changes. Altogether, we demonstrated strong cancer preventive effects of δ-T and γ-T in a physiologically relevant model of human colon cancer. © 2016 Wiley Periodicals, Inc.

Combinatorial treatment of idiopathic pulmonary fibrosis using nanoparticles with prostaglandin E and siRNA(s).

Inhalation delivery of prostaglandin E (PGE2) in combination with selected siRNA(s) was proposed for the efficient treatment of idiopathic pulmonary fibrosis (IPF). Nanostructured lipid carriers (NLC) were used as a delivery system for PGE2 with and without siRNAs targeted to MMP3, CCL12, and HIF1Alpha mRNAs. The model of IPF was developed in SKH1 mice by intratracheal administration of bleomycin at a dose of 1.5U/kg. Results showed that NLC-PGE2 in combination with three siRNAs delivered locally to the lungs by inhalation markedly reduced mouse body mass, substantially limited hydroxyproline content in the lungs and disturbances of the mRNAs and protein expression, restricted lung tissue damage and prevented animal mortality. Our data provide evidence that IPF can be effectively treated by inhalation of the NLC-PGE2 in combination with siRNAs delivered locally into the lungs. This effect could not be achieved by using NLC containing just PGE2 or siRNA(s) alone.

Metabotropic glutamate receptor 1 disrupts mammary acinar architecture and initiates malignant transformation of mammary epithelial cells.

Metabotropic glutamate receptor 1 (mGluR1/Grm1) is a member of the G-protein-coupled receptor superfamily, which was once thought to only participate in synaptic transmission and neuronal excitability, but has more recently been implicated in non-neuronal tissue functions. We previously described the oncogenic properties of Grm1 in cultured melanocytes in vitro and in spontaneous melanoma development with 100 % penetrance in vivo. Aberrant mGluR1 expression was detected in 60-80 % of human melanoma cell lines and biopsy samples. As most human cancers are of epithelial origin, we utilized immortalized mouse mammary epithelial cells (iMMECs) as a model system to study the transformative properties of Grm1. We introduced Grm1 into iMMECs and isolated several stable mGluR1-expressing clones. Phenotypic alterations in mammary acinar architecture were assessed using three-dimensional morphogenesis assays. We found that mGluR1-expressing iMMECs exhibited delayed lumen formation in association with decreased central acinar cell death, disrupted cell polarity, and a dramatic increase in the activation of the mitogen-activated protein kinase pathway. Orthotopic implantation of mGluR1-expressing iMMEC clones into mammary fat pads of immunodeficient nude mice resulted in mammary tumor formation in vivo. Persistent mGluR1 expression was required for the maintenance of the tumorigenic phenotypes in vitro and in vivo, as demonstrated by an inducible Grm1-silencing RNA system. Furthermore, mGluR1 was found be expressed in human breast cancer cell lines and breast tumor biopsies. Elevated levels of extracellular glutamate were observed in mGluR1-expressing breast cancer cell lines and concurrent treatment of MCF7 xenografts with glutamate release inhibitor, riluzole, and an AKT inhibitor led to suppression of tumor progression. Our results are likely relevant to human breast cancer, highlighting a putative role of mGluR1 in the pathophysiology of breast cancer and the potential of mGluR1 as a novel therapeutic target.

Effective intravenous therapy for neurodegenerative disease with a therapeutic enzyme and a peptide that mediates delivery to the brain.

The blood-brain barrier (BBB) presents a major challenge to effective treatment of neurological disorders, including lysosomal storage diseases (LSDs), which frequently present with life-shortening and untreatable neurodegeneration. There is considerable interest in methods for intravenous delivery of lysosomal proteins across the BBB but for the most part, levels achievable in the brain of mouse models are modest and increased lifespan remains to be demonstrated. In this study, we have investigated delivery across the BBB using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a neurodegenerative LSD caused by loss of tripeptidyl peptidase I (TPP1). We have achieved supraphysiological levels of TPP1 throughout the brain of LINCL mice by intravenous (IV) coadministration of recombinant TPP1 with a 36-residue peptide that contains polylysine and a low-density lipoprotein receptor binding sequence from apolipoprotein E. Importantly, IV administration of TPP1 with the peptide significantly reduces brain lysosomal storage, increases lifespan and improves neurological function. This simple "mix and inject" method is immediately applicable towards evaluation of enzyme replacement therapy to the brain in preclinical models and further exploration of its clinical potential is warranted.

Critical role of the endogenous interferon ligand-receptors in type I and type II interferons response.

Separate ligand-receptor paradigms are commonly used for each type of interferon (IFN). However, accumulating evidence suggests that type I and type II IFNs may not be restricted to independent pathways. Using different cell types deficient in IFNAR1, IFNAR2, IFNGR1, IFNGR2 and IFN-γ, we evaluated the contribution of each element of the IFN system to the activity of type I and type II IFNs. We show that deficiency in IFNAR1 or IFNAR2 is associated with impairment of type II IFN activity. This impairment, presumably resulting from the disruption of the ligand-receptor complex, is obtained in all cell types tested. However, deficiency of IFNGR1, IFNGR2 or IFN-γ was associated with an impairment of type I IFN activity in spleen cells only, correlating with the constitutive expression of type II IFN (IFN-γ) observed on those cells. Therefore, in vitro the constitutive expression of both the receptors and the ligands of type I or type II IFN is critical for the enhancement of the IFN activity. Any IFN deficiency can totally or partially impair IFN activity, suggesting the importance of type I and type II IFN interactions. Taken together, our results suggest that type I and type II IFNs may regulate biological activities through distinct as well as common IFN receptor complexes.

Mutagenicity and tumorigenicity of the four enantiopure bay-region 3,4-diol-1,2-epoxide isomers of dibenz[a,h]anthracene.

Each enantiomer of the diastereomeric pair of bay-region dibenz[a,h]anthracene 3,4-diol-1,2-epoxides in which the benzylic 4-hydroxyl group and epoxide oxygen are either cis (isomer 1) or trans (isomer 2) were evaluated for mutagenic activity. In strains TA 98 and TA 100 of Salmonella typhimurium, the diol epoxide with (1S,2R,3S,4R) absolute configuration [(-)-diol epoxide-1] had the highest mutagenic activity. In Chinese hamster V-79 cells, the diol epoxide with (1R,2S,3S,4R) absolute configuration [(+)-diol epoxide-2] had the highest mutagenic activity. The (1R,2S,3R,4S) diol epoxide [(+)-diol epoxide-1] also had appreciable activity, whereas the other two bay-region diol epoxide enantiomers had very low activity. In tumor studies, the (1R,2S,3S,4R) enantiomer was the only diol epoxide isomer tested that had strong activity as a tumor initiator on mouse skin and in causing lung and liver tumors when injected into newborn mice. This stereoisomer was about one-third as active as the parent hydrocarbon, dibenz[a,h]anthracene as a tumor initiator on mouse skin; it was several-fold more active than dibenz[a,h]anthracene as a lung and liver carcinogen when injected into newborn mice. (-)-(3R,4R)-3ß,4α-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(-)-3,4-dihydrodiol] was slightly more active than dibenz[a,h]anthracene as a tumor initiator on mouse skin, whereas (+)-(3S,4S)-3α,4ß-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(+)-3,4-dihydrodiol] had only very weak activity. The present investigation and previous studies with the corresponding four possible enantiopure bay-region diol epoxide enantiomers/diastereomers of benzo[a]pyrene, benz[a]anthracene, chrysene, benzo[c]phenanthrene, dibenz[c,h]acridine, dibenz[a,h]acridine and dibenz[a,h]anthracene indicate that the bay-region diol epoxide enantiomer with [R,S,S,R] absolute stereochemistry has high tumorigenic activity on mouse skin and in newborn mice.

Dietary tocopherols inhibit cell proliferation, regulate expression of ERα, PPARγ, and Nrf2, and decrease serum inflammatory markers during the development of mammary hyperplasia.

Previous clinical and epidemiological studies of vitamin E have used primarily α-tocopherol for the prevention of cancer. However, γ-tocopherol has demonstrated greater anti-inflammatory and anti-tumor activity than α-tocopherol in several animal models of cancer. This study assessed the potential chemopreventive activities of a tocopherol mixture containing 58% γ-tocopherol (γ-TmT) in an established rodent model of mammary carcinogenesis. Female ACI rats were utilized due to their sensitivity to 17ß-estradiol (E2 ) to induce mammary hyperplasia and neoplasia. The rats were implanted subcutaneously with sustained release E2 pellets and given dietary 0.3% or 0.5% γ-TmT for 2 or 10 wk. Serum E2 levels were significantly reduced by the treatment with 0.5% γ-TmT. Serum levels of inflammatory markers, prostaglandin E2 and 8-isoprostane, were suppressed by γ-TmT treatment. Histology of mammary glands showed evidence of epithelial hyperplasia in E2 -treated rats. Immunohistochemical analysis of the mammary glands revealed a decrease in proliferating cell nuclear antigen (PCNA), cyclooxygenase-2 (COX-2), and estrogen receptor α (ERα), while there was an increase in cleaved-caspase 3, peroxisome proliferator-activated receptor γ (PPARγ), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in γ-TmT-treated rats. In addition, treatment with γ-TmT resulted in a decrease in the expression of ERα mRNA, whereas mRNA levels of ERß and PPARγ were increased. In conclusion, γ-TmT was shown to suppress inflammatory markers, inhibit E2 -induced cell proliferation, and upregulate PPARγ and Nrf2 expression in mammary hyperplasia, suggesting that γ-TmT may be a promising agent for human breast cancer prevention.

Inhibition of lung tumor growth by complex pulmonary delivery of drugs with oligonucleotides as suppressors of cellular resistance.

Development of cancer cell resistance, low accumulation of therapeutic drug in the lungs, and severe adverse treatment side effects represent main obstacles to efficient chemotherapy of lung cancer. To overcome these difficulties, we propose inhalation local delivery of anticancer drugs in combination with suppressors of pump and nonpump cellular resistance. To test this approach, nanoscale-based delivery systems containing doxorubicin as a cell death inducer, antisense oligonucleotides targeted to MRP1 mRNA as a suppressor of pump resistance and to BCL2 mRNA as a suppressor of nonpump resistance, were developed and examined on an orthotopic murine model of human lung carcinoma. The experimental results show high antitumor activity and low adverse side effects of proposed complex inhalatory treatment that cannot be achieved by individual components applied separately. The present work potentially contributes to the treatment of lung cancer by describing a unique combinatorial local inhalation delivery of drugs and suppressors of pump and nonpump cellular resistance.

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia.

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

A Spontaneous Melanoma Mouse Model Applicable for a Longitudinal Chemotherapy and Immunotherapy Study.

Mouse models that reflect human disorders provide invaluable tools for the translation of basic science discoveries to clinical therapies. However, many of these in vivo therapeutic studies are short term and do not accurately mimic patient conditions. In this study, we used a fully immunocompetent, transgenic mouse model, TGS, in which the spontaneous development of metastatic melanoma is driven by the ectopic expression of a normal neuronal receptor, mGluR1, as a model to assess longitudinal treatment response (up to 8 months) with an inhibitor of glutamatergic signaling, troriluzole, which is a prodrug of riluzole, plus an antibody against PD-1, an immune checkpoint inhibitor. Our results reveal a sex-biased treatment response that led to improved survival in troriluzole and/or anti-PD-1-treated male mice that correlated with differential CD8+ T cells and CD11b+ myeloid cell populations in the tumor-stromal interface, supporting the notion that this model is a responsive and tractable system for evaluating therapeutic regimens for melanoma in an immunocompetent setting.

Cyclic adenosine monophosphate differentiated beta-endorphin neurons promote immune function and prevent prostate cancer growth.

Pituitary adenylate cyclase-activating peptide (PACAP), a cAMP-activating agent, is highly expressed in the hypothalamus during the period when many neuroendocrine cells become differentiated from the neural stem cells (NSCs). Activation of the cAMP system in rat hypothalamic NSCs differentiated these cells into beta-endorphin (BEP)-producing neurons in culture. When these in vitro differentiated neurons were transplanted into the paraventricular nucleus (PVN) of the hypothalamus of an adult rat, they integrated well with the surrounding cells and produced BEP and its precursor gene product, proopiomelanocortin (POMC). Animals with BEP cell transplants demonstrated remarkable protection against carcinogen induction of prostate cancer. Unlike carcinogen-treated animals with control cell transplants, rats with BEP cell transplants showed rare development of glandular hyperplasia, prostatic intraepithelial neoplasia (PIN), or well differentiated adenocarcinoma with invasion after N-methyl-N-nitrosourea (MNU) and testosterone treatments. Rats with the BEP neuron transplants showed increased natural killer (NK) cell cytolytic function in the spleens and peripheral blood mononuclear cells (PBMCs), elevated levels of antiinflammatory cytokine IFN-gamma, and decreased levels of inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in plasma. These results identified a critical role for cAMP in the differentiation of BEP neurons and revealed a previously undescribed role of these neurons in combating the growth and progression of neoplastic conditions like prostate cancer, possibly by increasing the innate immune function and reducing the inflammatory milieu.

Antitumor activity of type I and type III interferons in BNL hepatoma model.

Hepatocellular carcinoma (HCC) occurs most commonly secondary to cirrhosis due to chronic hepatitis C or B virus (HCV/HBV) infections. Type I interferon (IFN-alpha) treatment of chronic HCV/HBV infections reduces the incidence of HCC in cirrhotic patients. However, IFN-alpha toxicity limits its tolerability and efficacy highlighting a need for better therapeutic treatments. A recently discovered type III IFN (IFN-lambda) has been shown to possess antiviral properties against HCV and HBV in vitro. In phase I clinical trials, IFN-lambda treatment did not cause significant adverse reactions. Using a gene therapy approach, we compared the antitumor properties of IFN-alpha and IFN-lambda in a transplantable hepatoma model of HCC. BALB/c mice were inoculated with syngeneic BNL hepatoma cells, or BNL cells expressing IFN-lambda (BNL.IFN-lambda cells) or IFN-alpha (BNL.IFN-alpha cells). Despite the lack of antiproliferative activity of IFNs on BNL cells, both BNL.IFN-lambda and BNL.IFN-alpha cells displayed retarded growth kinetics in vivo. Depletion of NK cells from splenocytes inhibited splenocyte-mediated cytotoxicity, demonstrating that NK cells play a role in IFN-induced antitumor responses. However, isolated NK cells did not respond directly to IFN-lambda. There was also a marked NK cell infiltration in IFN-lambda producing tumors. In addition, IFN-lambda and, to a lesser extent, IFN-alpha enhanced immunocytotoxicity of splenocytes primed with irradiated BNL cells. Splenocyte cytotoxicity against BNL cells was dependent on IL-12 and IFN-gamma, and mediated by dendritic cells. In contrast to NK cells, isolated from spleen CD11c+ and mPDCA+ dendritic cells responded directly to IFN-lambda. The antitumor activities of IFN-lambda against hepatoma, in combination with HCV and HBV antiviral activities warrant further investigation into the clinical use of IFN-lambda to prevent HCC in HCV/HBV-infected cirrhotic patients, as well as to treat liver cancer.

Mixed tocotrienols inhibit prostate carcinogenesis in TRAMP mice.

The biological activities of tocotrienols are receiving increasing attention. Herein, we report the efficacy of a mixed-tocotrienol diet against prostate tumorigenesis in the transgenic adenocarcinoma mouse prostate (TRAMP) mouse model. Male TRAMP mice, 8 wk old, were fed 0.1%, 0.3%, or 1% mixed tocotrienols in AIN-76A diet up to 24 wk old. Likewise, a positive control group consisting of male TRAMP mice and a negative control group consisting of wild-type nontransgenic mice were fed regular AIN-76A diet up to 24 wk old. Our results show that mixed-tocotrienol-fed groups had a lower incidence of tumor formation along with a significant reduction in the average wet weight of genitourinary apparatus. Furthermore, mixed tocotrienols significantly reduced the levels of high-grade neoplastic lesions as compared to the positive controls. This decrease in levels of high-grade neoplastic lesions was found to be associated with increased expression of proapoptotic proteins BAD (Bcl(2) antagonist of cell death) and cleaved caspase-3 and cell cycle regulatory proteins cyclin dependent kinase inhibitors p21 and p27. In contrast, the expression of cyclins A and E were found to be decreased in mixed-tocotrienol groups. Taken together, our results show that by modulating cell cycle regulatory proteins and increasing expression of proapoptotic proteins, mixed tocotrienols suppress prostate tumorigenesis in the TRAMP mice.

Fetal alcohol exposure increases mammary tumor susceptibility and alters tumor phenotype in rats.

BACKGROUND: Altered fetal programming because of a suboptimal in utero environment has been shown to increase susceptibility to many diseases later in life. This study examined the effect of alcohol exposure in utero on N-nitroso-N-methylurea (NMU)-induced mammary cancer risk during adulthood. METHODS: Study 1: Pregnant Sprague Dawley rats were fed a liquid diet containing 6.7% ethanol (alcohol-fed), an isocaloric liquid diet (pair-fed), or rat chow ad libitum (ad lib-fed) from day 11 to 21 of gestation. At birth, female pups were cross-fostered to ad lib-fed control dams. Adult offspring were given an I.P. injection of NMU at a dose of 50 mg/kg body weight. Mammary glands were palpated for tumors twice a week, and rats were euthanized at 23 weeks postinjection. Study 2: To investigate the role of estradiol (E2), animals were exposed to the same in utero treatments but were not given NMU. Serum was collected during the preovulatory phase of the estrous cycle. RESULTS: At 16 weeks postinjection, overall tumor multiplicity was greater in the offspring from the alcohol-fed group compared to the control groups, indicating a decrease in tumor latency. At study termination, 70% of all animals possessed tumors. Alcohol-exposed animals developed more malignant tumors and more estrogen receptor-α-negative tumors relative to the control groups. In addition, IGF-binding protein-5 (IGFBP-5) mRNA and protein were decreased in tumors of alcohol-exposed animals. Study 2 showed that alcohol-fed animals had significantly increased circulating E2 when compared to either control group. CONCLUSIONS: These data indicate that alcohol exposure in utero increases susceptibility to mammary tumorigenesis in adulthood and suggest that alterations in the IGF and E2 systems may play a role in the underlying mechanism.

Gamma-tocopherol-enriched mixed tocopherol diet inhibits prostate carcinogenesis in TRAMP mice.

Gamma-tocopherol (gamma-T) alone or in combination with alpha-tocopherol has been shown to suppress biomarkers of oxidative stress in asthamatics and human subjects with metabolic syndrome. Oxidative stress has been implicated as a key event in prostate carcinogenesis. Hence, the purpose of this study was to examine the effects of gamma-tocopherol-enriched mixed tocopherol diet on prostate carcinogenesis in a murine prostate cancer model (TRAMP). 8 week old TRAMP males were fed 0.1% gamma-T-enriched mixed tocopherol diet that contained 20-fold higher levels of gamma-tocopherol, and roughly 3-fold higher levels of alpha-tocopherol. The effect of such diet on tumor and PIN development was observed. The expression of phase II detoxifying, antioxidant enzymes and Nrf2 mRNA and protein were determined by RT-PCR, immunohistochemistry and western blotting techniques. Treatment with gamma-T-enriched mixed tocopherols significantly suppressed the incidence of palpable tumor and Prostate Intraepithelial Neoplasia (PIN) development without affecting the expression of the transgene (SV-40). Tumor progression occurred with a significant suppression of antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase, heme-oxygenase-1 and phase II detoxifying enzymes. Treatment with gamma-T-enriched mixed tocopherol diet upregulated the expression of most detoxifying and antioxidant enzymes. Nrf2-a redox sensitive transcription factor known to mediate the expression of phase II detoxifying enzymes, was also significantly upregulated following treatment with gamma-T-enriched mixed tocopherol diet. Gamma-T-enriched mixed tocopherols significantly up-regulated the expression of Nrf2 and its related detoxifying and antioxidant enzymes thereby suppressing PIN and tumor development.

Effect of alpha-tocopherol, N-acetylcysteine and omeprazole on esophageal adenocarcinoma formation in a rat surgical model.

We previously demonstrated that oxidative stress subsequent to gastroesophageal reflux is an important driving force of esophageal adenocarcinoma (EAC) formation in the esophagogastroduodenal anastomosis (EGDA) rat model. This study investigated the possible tumor inhibitory effects of 2 antioxidants, alpha-tocopherol (389 and 778 ppm), N-acetylcysteine (NAC, 500 and 1,000 ppm), and their combination (389 and 500 ppm, respectively), as well as an antacid therapeutic agent, omeprazole (1,400 ppm). The rats were fed experimental diets 2 weeks after EGDA. All the animals were sacrificed 40 weeks after EGDA and the esophagi were harvested for histopathological examination. alpha-Tocopherol dose-dependently decreased the incidence of EAC (p = 0.03), with 778 ppm alpha-tocopherol reducing the incidence of EAC to 59% (16/27) in comparison with 84% (26/31) in the control group (p = 0.04). Supplementation of alpha-tocopherol also increased the serum concentration of alpha-tocopherol. NAC at 500 and 1,000 ppm did not significantly decrease EAC incidence; however, the combination of alpha-tocopherol 389 ppm and NAC 500 ppm significantly reduced the incidence of EAC to 55% (15/27) (p = 0.02). alpha-Tocopherol alone or in combination with NAC significantly reduced the number of infiltrating cells positively stained for 4-hydroxynonenal. Omeprazole showed only a slight nonsignificant inhibitory effect at the dose given. Our results suggest that supplementation with alpha-tocopherol inhibits the development of EAC in the rat EGDA model and similar inhibitory effect can be achieved when a lower dose of alpha-tocopherol is used in combination with NAC.

Green Tea Polyphenols Modify the Gut Microbiome in db/db Mice as Co-Abundance Groups Correlating with the Blood Glucose Lowering Effect.

SCOPE: The effects of green tea polyphenols, Polyphenon E (PPE), and black tea polyphenols, theaflavins (TFs), on gut microbiota and development of diabetes in db/db mice are investigated and compared. METHODS AND RESULTS: Supplementation of PPE (0.1%) in the diet of female db/db mice for 7 weeks decreases fasting blood glucose levels and mesenteric fat while increasing the serum level of insulin, possibly through protection against ß-cell damage. However, TFs are less or not effective. Microbiome analysis through 16S rRNA gene sequencing shows that PPE and TFs treatments significantly alter the bacterial community structure in the cecum and colon, but not in the ileum. The key bacterial phylotypes responding to the treatments are then clustered into 11 co-abundance groups (CAGs). CAGs 6 and 7, significantly increased by PPE but not by TFs, are negatively associated with blood glucose levels. The operational taxonomic units in these CAGs are from two different phyla, Firmicutes and Bacteroidetes. CAG 10, decreased by PPE and TFs, is positively associated with blood glucose levels. CONCLUSION: Gut microbiota respond to tea polyphenol treatments as CAGs instead of taxa. Some of the CAGs associated with the blood glucose lowering effect are enriched by PPE, but not TFs.

The major green tea polyphenol, (-)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice.

In this study, we investigated the effects of the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), on high-fat-induced obesity, symptoms of the metabolic syndrome, and fatty liver in mice. In mice fed a high-fat diet (60% energy as fat), supplementation with dietary EGCG treatment (3.2 g/kg diet) for 16 wk reduced body weight (BW) gain, percent body fat, and visceral fat weight (P < 0.05) compared with mice without EGCG treatment. The BW decrease was associated with increased fecal lipids in the high-fat-fed groups (r(2) = 0.521; P < 0.05). EGCG treatment attenuated insulin resistance, plasma cholesterol, and monocyte chemoattractant protein concentrations in high-fat-fed mice (P < 0.05). EGCG treatment also decreased liver weight, liver triglycerides, and plasma alanine aminotransferase concentrations in high-fat-fed mice (P < 0.05). Histological analyses of liver samples revealed decreased lipid accumulation in hepatocytes in mice treated with EGCG compared with high-fat diet-fed mice without EGCG treatment. In another experiment, 3-mo-old high-fat-induced obese mice receiving short-term EGCG treatment (3.2 g/kg diet, 4 wk) had decreased mesenteric fat weight and blood glucose compared with high-fat-fed control mice (P < 0.05). Our results indicate that long-term EGCG treatment attenuated the development of obesity, symptoms associated with the metabolic syndrome, and fatty liver. Short-term EGCG treatment appeared to reverse preexisting high-fat-induced metabolic pathologies in obese mice. These effects may be mediated by decreased lipid absorption, decreased inflammation, and other mechanisms.

VPA-induced apoptosis and behavioral deficits in neonatal mice.

Sodium valproate (VPA) administered to neonatal mice causes cognitive and motor deficits similar to those observed in humans with autism. In an effort to further evaluate similarities between early VPA exposure and autism, the present study examined treated mice for deficits in social behavior and neuronal damage. BALB/c mice injected on P14 with 400 mg/kg VPA engaged in fewer social interactions (including ano-genital sniffs, allogrooming, and crawl-under/over behaviors) than control mice. Treated mice also exhibited reduced motor activity in a social context but were not significantly different from controls when motor activity was assessed in non-social settings. A second set of BALB/c mice were treated with VPA on P14 and sacrificed at different times thereafter for histopathological analysis. At time-points 12 and 24 h following VPA, treated mice had up to a 30-fold increase in the number of TUNEL-positive cells in the external granule cell layer of the cerebellum and a 10-fold increase in TUNEL-positive cells in the dentate gyrus of the hippocampus. These observations may provide a histopathological correlate for the social deficits observed following post-natal VPA exposure and supports the use of early VPA administration as an animal model for the study of autism.