Eat4Genes: a bioinformatic rational gene targeting app and prototype model for improving human health.

Introduction and aims: Dietary Rational Gene Targeting (DRGT) is a therapeutic dietary strategy that uses healthy dietary agents to modulate the expression of disease-causing genes back toward the normal. Here we use the DRGT approach to (1) identify human studies assessing gene expression after ingestion of healthy dietary agents with an emphasis on whole foods, and (2) use this data to construct an online dietary guide app prototype toward eventually aiding patients, healthcare providers, community and researchers in treating and preventing numerous health conditions. Methods: We used the keywords "human", "gene expression" and separately, 51 different dietary agents with reported health benefits to search GEO, PubMed, Google Scholar, Clinical trials, Cochrane library, and EMBL-EBI databases for related studies. Studies meeting qualifying criteria were assessed for gene modulations. The R-Shiny platform was utilized to construct an interactive app called "Eat4Genes". Results: Fifty-one human ingestion studies (37 whole food related) and 96 key risk genes were identified. Human gene expression studies were found for 18 of 41 searched whole foods or extracts. App construction included the option to select either specific conditions/diseases or genes followed by food guide suggestions, key target genes, data sources and links, dietary suggestion rankings, bar chart or bubble chart visualization, optional full report, and nutrient categories. We also present user scenarios from physician and researcher perspectives. Conclusion: In conclusion, an interactive dietary guide app prototype has been constructed as a first step towards eventually translating our DRGT strategy into an innovative, low-cost, healthy, and readily translatable public resource to improve health.

Thyroid Hormone Induces Oral Cancer Growth via the PD-L1-Dependent Signaling Pathway.

Oral cancer is a fatal disease, and its incidence in Taiwan is increasing. Thyroid hormone as L-thyroxine (T4) stimulates cancer cell proliferation via a receptor on integrin αvß3 of plasma membranes. It also induces the expression of programmed death-ligand 1 (PD-L1) and cell proliferation in cancer cells. Thyroid hormone also activates ß-catenin-dependent cell proliferation in cancer cells. However, the relationship between PD-L1 and cancer proliferation is not fully understood. In the current study, we investigated the role of inducible thyroid hormone-induced PD-L1-regulated gene expression and proliferation in oral cancer cells. Thyroxine bound to integrin αvß3 to induce PD-L1 expressions via activation of ERK1/2 and signal transducer and activator of transcription 3 (STAT3). Inactivated STAT3 inhibited PD-L1 expression and nuclear PD-L1 accumulation. Inhibition of PD-L1 expression reduced ß-catenin accumulation. Furthermore, nuclear PD-L1 formed a complex with nuclear proteins such as p300. Suppression PD-L1 expression by shRNA blocked not only expression of PD-L1 and ß-catenin but also signal transduction, proliferative gene expressions, and cancer cell growth. In summary, thyroxine via integrin αvß3 activated ERK1/2 and STAT3 to stimulate the PD-L1-dependent and ß-catenin-related growth in oral cancer cells.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-Glucoside improves female ovarian aging.

Reduced fertility associated with normal aging may reflect the over-maturity of oocytes. It is increasingly important to reduce aging-induced infertility since recent trends show people marrying at later ages. 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside (THSG), a polyphenol extracted from Polygonum multiflorum, has been reported to have anti-inflammatory and anti-aging properties. To evaluate whether THSG can reduce aging-related ovarian damage in a female mouse model of aging, THSG was administered by gavage at a dose of 10 mg/kg twice weekly, starting at 4 weeks of age in a group of young mice. In addition, the effect of THSG in a group of aged mice was also studied in mice starting at 24 weeks of age. The number of oocytes in the THSG-fed group was higher than in the untreated control group. Although the percentage of secondary polar bodies (PB2) decreased during aging in the THSG-fed group, it decreased much more slowly than in the age-matched control group. THSG administration increased the quality of ovaries in young mice becoming aged. Western blotting analyses also indicated that CYP19, PR-B, and ER-ß expressions were significantly increased in 36-week-old mice. THSG also increased oocyte numbers in aged mice compared to mice without THSG fed. Studies of qPCR and immunohistochemistry (IHC) analyses of ovaries in the aged mice groups were conducted. THSG increased gene expression of anti-Müllerian hormone (AMH), a biomarker of oocyte number, and protein accumulation in 40-week-old mice. THSG increased the expression of pgc1α and atp6, mitochondrial biogenesis-related genes, and their protein expression. THSG also attenuated the fading rate of CYP11a and CYP19 associated with sex hormone synthesis. And THSG maintains a high level of ER-ß expression, thereby enhancing the sensitivity of estrogen. Our findings indicated that THSG increased or extended gene expression involved in ovarian maintenance and rejuvenation in young and aged mice. On the other hand, THSG treatments significantly maintained oocyte quantity and quality in both groups of young and aged mice compared to each age-matched control group. In conclusion, THSG can delay aging-related menopause, and the antioxidant properties of THSG may make it suitable for preventing aging-induced infertility.

Heteronemin and Tetrac Induce Anti-Proliferation by Blocking EGFR-Mediated Signaling in Colorectal Cancer Cells.

Overexpressed EGFR and mutant K-Ras play vital roles in therapeutic resistance in colorectal cancer patients. To search for an effective therapeutic protocol is an urgent task. A secondary metabolite in the sponge Hippospongia sp., Heteronemin, has been shown to induce anti-proliferation in several types of cancers. A thyroxine-deaminated analogue, tetrac, binds to integrin αvß3 to induce anti-proliferation in different cancers. Heteronemin- and in combination with tetrac-induced antiproliferative effects were evaluated. Tetrac enhanced heteronemin-induced anti-proliferation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC). Heteronemin and tetrac arrested cell cycle in different phases. Combined treatment increased the cell accumulation in sub-G1 and S phases. The combined treatment also induced the inactivation of EGFR signaling and downregulated the phosphorylated ERK1/2 protein in both cell lines. Heteronemin and the combination showed the downregulation of the phosphorylated and total PI3K protein in HT-29 cells (KRAS WT CRC). Results by NanoString technology and RT-qPCR revealed that heteronemin and combined treatment suppressed the expression of EGFR and downstream genes in HCT-116 cells (KRAS MT CRC). Heteronemin or combined treatment downregulated genes associated with cancer progression and decreased cell motility. Heteronemin or the combined treatment suppressed PD-L1 expression in both cancer cell lines. However, only tetrac and the combined treatment inhibited PD-L1 protein accumulation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC), respectively. In summary, heteronemin induced anti-proliferation in colorectal cancer cells by blocking the EGFR-dependent signal transduction pathway. The combined treatment further enhanced the anti-proliferative effect via PD-L1 suppression. It can be an alternative strategy to suppress mutant KRAS resistance for anti-EGFR therapy.

Dietary rational targeting of redox-regulated genes.

Nutrigenomics is the study of how food and associated nutrients affect gene expression. This field sits at the intersection of diet, the genome and health with the ultimate goal of exploiting its understanding to design a precision nutrition strategy for humans. We have studied diet and nutrigenomics in the context of something we call "dietary rational gene targeting." Here, healthy diet is used to alter disease-causing gene expression back toward the normal to treat various diseases and conditions while lowering treatment cost and toxicity. In this paper, we discuss the use of this strategy to modulate the expression of redox-associated genes to improve human health. Most human disorders are associated, at least to some extent, with oxidative stress and so treatments (including diet) that target redox-related genes have major potential clinical significance. Healthy dietary options here are wide-ranging and include whole foods and botanical-based beverages. In some cases, botanical supplements may also be useful gene modulators although their health benefits are less clear. Key redox gene targets for these dietary agents include antioxidant genes, related transcription factors, detoxification genes, and DNA repair genes. Other important considerations include bioavailability, the contribution of the microbiome, and advancing technologies. In this review, specific examples of redox associated genes and pathologies and their potential treatment with healthy diet are presented to illustrate our approach. This will also serve as a foundation for the design of future clinical studies to improve diet-related health.

Compassion and Empathy in Basic Medical Science Teaching: A Suggested Model.

Medical school education typically consists of two main student bodies: medical students and biomedical graduate students. For both groups, compassion and empathy represent a major component of future professional roles. For medical students, this takes the form of the all-important doctor-patient relationship and adherence to the Hippocratic Oath. For biomedical students, future research and teaching are often driven by the opportunity to contribute to treatments to help pain and suffering for those in need. For both groups, such positive contributions further extend to families, who often suffer emotional distress watching the health struggles of a loved one. Given the key role that compassion and empathy play here, including them as part of student educational development is important. Such focus, however, is limited - especially during the initial academic classroom years - with most time here dedicated to the learning of facts and foundational material. Given its importance in the future professional roles of these students, we posit that more can be done to introduce and reinforce the concept of compassion and empathy during the initial didactic course years. Modest but viable options exist for the introduction of these concepts as a part of basic teaching that will provide additional reinforcement of this all-important sensitivity for others. Here we present a model providing suggestions and recommendations for the integration of compassion and empathy in otherwise basic scientific teaching, and in a way that also includes progressive equality positions on social issues. While the focus here is medical school education since it represents this author's expertise as well as a field where young trainees graduate to professional careers requiring compassion, it can potentially be applied to many other disciplines.

Lower concentrations of curcumin inhibit Her2-Akt pathway components in human breast cancer cells, and other dietary botanicals potentiate this and lapatinib inhibition.

Her2-dependent breast cancer is treated with pharmacological drugs (eg, Herceptin, lapatinib) that target Her2 signaling. Curcumin has emerged as a potential co-treatment for this and other cancers, but prior studies have focused on non-attainable concentrations. Here we test the hypothesis that attainable in vivo levels of dietary curcumin can reduce Her2 signaling. Consistent with previous studies, higher dose curcumin (18 µmol/L) inhibits Her2-Akt pathway signaling (pHer2, total Her2 and pAkt levels) and cell growth using AU565 human breast cancer cells. We then examined lower, more physiologically relevant concentrations of curcumin, alone and in combination with other dietary botanicals (quercetin and OptiBerry fruit extract). At 4 µmol/L, curcumin reduced Her2 signaling, and even more when combined with quercetin or OptiBerry. At 1.5 µmol/L curcumin, pHer2 and Her2 (but not pAkt) were reduced, with all three pathway markers reduced more in the presence of quercetin. We also found that 1.5 µmol/L curcumin strongly potentiated lapatinib inhibition of Her2-Akt pathway signaling, and more so for pAkt, when combined with quercetin plus OptiBerry (CQO). Parallel analyses revealed cell growth inhibition at 18 and 4 µmol/L but not 1.5 µmol/L curcumin, and potentiation of 1.5 µmol/L curcumin growth arrest with other botanicals +/- lapatinib. These studies demonstrate that a physiological attainable level of curcumin (1.5 µmol/L) can reduce some components of the critical Her2-Akt pathway; that even more complete inhibition can be achieved by combination with other dietary botanicals; and that curcumin and other botanicals can potentiate the action of the Her2-cancer metastatic drug lapatinib, in turn suggesting the potential anti-cancer clinical use of these botanicals.

NDAT suppresses pro-inflammatory gene expression to enhance resveratrol-induced anti-proliferation in oral cancer cells.

Nano-diamino-tetrac (NDAT), a tetraiodothyroxine deaminated nano-particulated analog, has shown to inhibit expression of pro-inflammatory genes. NDAT inhibits expression of programmed death-ligand 1 (PD-L1). On the other hand, in addition to inhibiting inflammatory effect, the stilbene, resveratrol induces expression of cyclooxygenase-2 (COX-2) and its accumulation. Sequentially, inducible COX-2 complexes with p53 and induces p53-dependent anti-proliferation. In current study, we investigated mechanisms involved in combined treatment of NDAT and resveratrol on anti-proliferation in human oral cancer cells. Both resveratrol and NDAT inhibited expression of pro-inflammatory IL-1ß and TNF-α. They also inhibited expression of CCND1 and PD-L1. Both resveratrol and NDAT induced BAD expression but only resveratrol induced COX-2 expression in both OEC-M1 and SCC-25 cells. Combined treatment attenuated gene expression significantly compared with resveratrol treatment in both cancer cell lines. Resveratrol reduced nuclear PD-L1 accumulation which was enhanced by a STAT3 inhibitor, S31-201 or NDAT suggesting that NDAT may inactivate STAT3 to inhibit PD-L1 accumulation. In the presence of T4, NDAT further enhanced resveratrol-induced anti-proliferation in both cancer cell lines. These findings provide a novel understanding of the inhibition of NDAT in thyroxine-induced pro-inflammatory effect on resveratrol-induced anticancer properties.

Leptin-derived peptides block leptin-induced proliferation by reducing expression of pro-inflammatory genes in hepatocellular carcinoma cells.

The obesity-regulated gene, leptin, is essential for diet. Leptin resistance causes obesity and related diseases. Certain types of diet are able to decrease leptin resistance. However, leptin has been shown to be correlated with inflammation and stimulate proliferation of various cancers. Two synthetic leptin derivatives (mimetics), OB3 and [D-Leu-4]-OB3, show more effective than leptin in reducing obesity and diabetes in mouse models. OB3 inhibits leptin-induced proliferation in ovarian cancer cells. However, effects of these mimetics in hepatocellular carcinoma (HCC) have not been investigated. In the present study, we examined the effects of OB3 and [D-Leu-4]-OB3 on cell proliferation and gene expressions in human HCC cell cultures. In contrast to what was reported for leptin, OB3 and [D-Leu-4]-OB3 reduced cell proliferation in hepatomas. Both OB3 and [D-Leu-4]-OB3 stimulated expression of pro-apoptotic genes. Both compounds also inhibited expressions of pro-inflammatory, proliferative and metastatic genes and PD-L1 expression. In combination with leptin, OB3 inhibited leptin-induced cell proliferation and expressions of pro-inflammation-, and proliferation-related genes. Furthermore, the OB3 peptide inhibited phosphoinositide 3-kinase (PI3K) activation which is essential for leptin-induced proliferation in HCC. These results indicate that OB3 and [D-Leu-4]-OB3 may have the potential to reduce leptin-related inflammation and proliferation in HCC cells.

Control (Native) and oxidized (DeMP) mitochondrial RNA are proinflammatory regulators in human.

Inflammation is implicated in a wide range of disorders, and thought to be involved in most leading causes of death today in the United States with high associated costs. New insights into better understanding its etiology, detection and prevention are thus of major importance in health care. One emerging field providing such insights has been the identification of DAMPs, or damage-associated molecular patterns. We have studied DAMPs within the context of degraded and oxidized mitochondrial DNA and RNA ("DeMP"), most recently demonstrating potent mitochondrial RNA (mtRNA) immunogenic response in mouse macrophages. Here, we extend these studies to assess the proinflammatory role of mitochondrial control (native) and oxidized RNA using human RNA and cells. THP-1 macrophage mtRNA triggered a proinflammatory response (induction of IL-6 and TNFα) when transfected into the same cells. Modestly oxidized mtRNA (DeMP RNA) but not cytoplasmic RNA induced a similar response, in contrast to attenuated immunogenicity previously observed with more oxidized DeMP RNA. This DeMP RNA may also cause a mild prooxidant stress. The proinflammatory effects of mtRNA was significantly reduced following pretreatment with RNases specific for single and double stranded RNA, implicating these forms of mtRNA in proinflammatory response. The natural nucleic acid-encapsulating peptide LL-37 also triggered a proinflammatory effect in the presence of control mtRNA and DeMP RNA. Finally, human blood plasma RNA exhibits proinflammatory activity. These results provide new insights into the immunostimulation of mitochondrial RNA including its activity in human cells; identify human plasma RNA as proinflammatory; and provide further evidence that oxidized DeMP mtRNA acts as a sensitive and broad-spectrum sensor and regulator of mitochondrial oxidative stress.

Participation of liver stem cells in cholangiocarcinogenesis after aflatoxin B1 exposure of glutathione S-transferase A3 knockout mice.

Aflatoxin B1, arguably the most potent human carcinogen, induces liver cancer in humans, rats, trout, ducks, and so on, but adult mice are totally resistant. This resistance is because of a detoxifying enzyme, mouse glutathione S-transferase A3, which binds to and inactivates aflatoxin B1 epoxide, preventing the epoxide from binding to DNA and causing mutations. Glutathione S-transferase A3 or its analog has not been detected in any of the sensitive species, including humans. The generation of a glutathione S-transferase A3 knockout (represented as KO or -/-) mice has allowed us to study the induction of liver cancer in mice by aflatoxin B1. In contrast to the induction of hepatocellular carcinomas in other species, aflatoxin B1 induces cholangiocarcinomas in GSTA3-/- mice. In other species and in knockout mice, the induction of liver cancer is preceded by extensive proliferation of small oval cells, providing additional evidence that oval cells are bipolar stem cells and may give rise to either hepatocellular carcinoma or cholangiocarcinoma depending on the nature of the hepatocarcinogen and the species of animal. The recent development of mouse oval cell lines in our laboratory from aflatoxin B1-treated GSTA3-/- mice should provide a new venue for study of the properties and potential of putative mouse liver stem cells.

Characterization of liver injury, oval cell proliferation and cholangiocarcinogenesis in glutathione S-transferase A3 knockout mice.

We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time.

Oxidized and Original article degraded mitochondrial polynucleotides (DeMPs), especially RNA, are potent immunogenic regulators in primary mouse macrophages.

Certain mitochondrial components can act as damage-associated molecular patterns (DAMPs) or danger signals, triggering a proinflammatory response in target (usually immune) cells. We previously reported the selective degradation of mitochondrial DNA and RNA in response to cellular oxidative stress, and the immunogenic effect of this DNA in primary mouse astrocytes. Here, we extend these studies to assess the immunogenic role of both mitochondrial DNA and RNA isolated from hydrogen peroxide (HP) treated HA1 cells (designated "DeMPs" for degraded mitochondrial polynucleotides) using mouse bone marrow derived macrophages (BMDMs), a conventional immune cell type. DeMPs and control mitochondrial DNA (cont mtDNA) and RNA (cont mtRNA) were transfected into BMDMs and cell-free media analyzed for the presence of proinflammatory cytokines (IL-6, MCP-1, and TNFα) and Type I interferon (IFN-α and IFN-ß). Cont mtDNA induced IL-6 and MCP-1 production, and this effect was even greater with DeMP DNA. A similar response was observed for Type I interferons. An even stronger induction of proinflammatory cytokine and type 1 interferons was observed for cont mtRNA. However, contrary to DeMP DNA, DeMP RNA attenuated rather than potentiated the cont mtRNA cytokine inductions. This attenuation effect was not accompanied by an IL-10 or TGFß anti-inflammatory response. All DeMP effects were observed at multiple oxidant concentrations. Finally, DeMP production and immunogenicity overlaps with cellular adaptive response and so may contribute to cellular oxidant protection. These results provide new insight into the immunogenicity of mitochondrial polynucleotides, and identify new roles and selective consequences of cellular oxidation.

Novel leptin OB3 peptide-induced signaling and progression in thyroid cancers: Comparison with leptin.

Obesity results in increased secretion of cytokines from adipose tissue and is a risk factor for various cancers. Leptin is largely produced by adipose tissue and cancer cells. It induces cell proliferation and may serve to induce various cancers. OB3-leptin peptide (OB3) is a new class of functional leptin peptide. However, its mitogenic effect has not been determined. In the present study, because of a close link between leptin and the hypothalamic-pituitary-thyroid axis, OB3 was compared with leptin in different thyroid cancer cells for gene expression, proliferation and invasion. Neither agent stimulated cell proliferation. Leptin stimulated cell invasion, but reduced adhesion in anaplastic thyroid cancer cells. Activated ERK1/2 and STAT3 contributed to leptin-induced invasion. In contrast, OB3 did not affect expression of genes involved in proliferation and invasion. In vivo studies in the mouse showed that leptin, but not OB3, significantly increased circulating levels of thyrotropin (TSH), a growth factor for thyroid cancer. In summary, OB3 is a derivative of leptin that importantly lacks the mitogenic effects of leptin on thyroid cancer cells.

Fish oil improves gene targets of Down syndrome in C57BL and BALB/c mice.

We have considered a novel gene targeting approach for treating pathologies and conditions whose genetic bases are defined using diet and nutrition. One such condition is Down syndrome, which is linked to overexpression of RCAN1 on human chromosome 21 for some phenotypes. We hypothesize that a decrease in RCAN1 expression with dietary supplements in individuals with Down syndrome represents a potential treatment. Toward this, we used in vivo studies and bioinformatic analysis to identify potential healthy dietary RCAN1 expression modulators. We observed Rcan1 isoform 1 (Rcan1-1) protein reduction in mice pup hippocampus after a 4-week curcumin and fish oil supplementation, with only fish oil reduction being statistically significant. Focusing on fish oil, we observed a 17% Rcan1-1 messenger RNA (mRNA) and 19% Rcan1-1 protein reduction in BALB/c mice after 5 weeks of fish oil supplementation. Fish oil supplementation starting at conception and in a different mouse strain (C57BL) led to a 27% reduction in hippocampal Rcan1-1 mRNA and a 34% reduction in spleen Rcan1-1 mRNA at 6 weeks of age. Hippocampal protein results revealed a modest 11% reduction in RCAN1-1, suggesting translational compensation. Bioinformatic mining of human fish oil studies also revealed reduced RCAN1 mRNA expression, consistent with the above studies. These results suggest the potential use of fish oil in treating Down syndrome and support our strategy of using select healthy dietary agents to treat genetically defined pathologies, an approach that we believe is simple, healthy, and cost-effective.

Genetic or pharmacologic activation of Nrf2 signaling fails to protect against aflatoxin genotoxicity in hypersensitive GSTA3 knockout mice.

Mice are resistant to aflatoxin hepatotoxicity, primarily due to high expression of glutathione S-transferases (GSTs), and in particular the GSTA3 subunit. Nuclear factor erythroid 2 related factor 2 (Nrf2) signaling, which controls a broad-based cytoprotective response, was activated either genetically or pharmacologically in an attempt to rescue GSTA3 knockout mice from aflatoxin genotoxicity. Genetic activation of Nrf2 signaling was attained in a GSTA3: hepatocyte-specific Keap1 double knockout (DKO) mouse whereas pharmacologic activation of Nrf2 was achieved through pretreatment of mice with the triterpenoid 1-[2-cyano-3-,12-dioxoleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) prior to aflatoxin B1 exposure. Following oral treatment with aflatoxin, urine was collected from mice for 24 h and hepatic and urinary aflatoxin metabolites then quantified using isotope dilution-mass spectrometry. Although Nrf2 was successfully activated genetically and pharmacologically, neither means affected the response of GSTA3 knockout mice to chemical insult with aflatoxin. Hepatic aflatoxin B1-N(7)-guanine levels were elevated 120-fold in GSTA3 knockout mice compared with wild-type and levels were not attenuated by the interventions. This lack of effect was mirrored in the urinary excretion of aflatoxin B1-N(7)-guanine. By contrast, urinary excretion of aflatoxin B1-N-acetylcysteine was >200-fold higher in wild-type mice compared with the single GSTA3 knockout or DKO mouse. The inability to rescue GSTA3 knockout mice from aflatoxin genotoxicity through the Nrf2 transcriptional program indicates that Gsta3 is unilaterally responsible for the detoxication of aflatoxin in mice.

Mechanisms of ceramide-induced COX-2-dependent apoptosis in human ovarian cancer OVCAR-3 cells partially overlapped with resveratrol.

Ceramide is a member of the sphingolipid family of bioactive molecules demonstrated to have profound, diverse biological activities. Ceramide is a potential chemotherapeutic agent via the induction of apoptosis. Exposure to ceramide activates extracellular-signal-regulated kinases (ERK)1/2- and p38 kinase-dependent apoptosis in human ovarian cancer OVCAR-3 cells, concomitant with an increase in the expression of COX-2 and p53 phosphorylation. Blockade of cyclooxygenase-2 (COX-2) activity by siRNA or NS398 correspondingly inhibited ceramide-induced p53 Ser-15 phosphorylation and apoptosis; thus COX-2 appears at the apex of the p38 kinase-mediated signaling cascade induced by ceramide. Induction of apoptosis by ceramide or resveratrol was inhibited by the endocytosis inhibitor, cytochalasin D (CytD); however, cells exposed to resveratrol showed greater sensitivity than ceramide-treated cells. Ceramide-treated cells underwent a dose-dependent reduction in trans-membrane potential. Although both ceramide and resveratrol induced the expressions of caspase-3 and -7, the effect of inducible COX-2 was different in caspase-7 expression induced by ceramide compared to resveratrol. In summary, resveratrol and ceramide converge on an endocytosis-requiring, ERK1/2-dependent signal transduction pathway and induction of COX-expression as an essential molecular antecedent for subsequent p53-dependent apoptosis. In addition, expressions of caspase-3 and -7 are observed. However, a p38 kinase-dependent signal transduction pathway and change in mitochondrial potential are also involved in ceramide-induced apoptosis.