Stimulation of melanogenesis by the citrus flavonoid naringenin in mouse B16 melanoma cells

Naringenin is a naturally occurring citrus flavanone. In this study, we examined the effect of naringenin on melanogenesis in mouse B16 melanoma cells. Melanin contents and tyrosinase activities were strongly increased by naringenin. Naringenin was found to cause marked increases in the expression levels of melanogenic enzymes.     

The flavonoid naringenin stimulates the intercellular colonization of wheat roots by Azorhizobium caulinodans

We have studied intercellular colonization of wheat roots by Azorhizobium caulinodans and other diazotrophic bacteria, using strains marked with the lacZ reporter gene to facilitate their detection and identification. A. caulinodans was observed by light and electron microscopy to enter the roots of wheat at high frequency at the points of emergence of lateral roots (lateral root cracks). After lateral root crack colonization, bacteria moved into intercellular spaces within the cortical cell layer of roots. The flavonoid naringenin at 10 and 100 mmol m^–3 significantly stimulated root colonization. The roles of the structural nodABC genes and the regulatory nodD gene were also studied; lateral root crack colonization of wheat was shown to be Nod factor- and NodD-independent. Similar frequencies of lateral root crack colonization were observed following inoculation of wheat with Azospirillum brasilense. Colonization by A. brasilense was stimulated by naringenin and also by other flavonoid molecules.     

Targeting the DNA double strand break repair machinery in prostate cancer

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways.     

A 1,4-dihydropyridine derivative reduces DNA damage and stimulates DNA repair in human cells in vitro

Compounds of the 1,4-dihydropyridine (1,4-DHP) series have been shown to reduce spontaneous, alkylation- and radiation-induced mutation rates in animal test systems. Here we report studies using AV-153, the 1,4-DHP derivative that showed the highest antimutagenic activity in those tests, to examine if it modulates DNA repair in human peripheral blood lymphocytes and in two human lymphoblastoid cell lines, Raji and HL-60. AV-153 caused a 50% inhibition of growth (IC50) of Raji and HL-60 cells at 14.9+/-1.2 and 10.3+/-0.8mM, respectively, but did not show a cytotoxic effect at concentrations <100 microM. Alkaline single-cell gel electrophoresis (comet) assays showed that AV-153 reduced the number of DNA strand breaks in untreated cells and also in cells exposed to 2 Gy of gamma-radiation, 100 microM ethylmethane sulfonate (EMS), or 100 microM H2O2. DNA damage was reduced by up to 87% at AV-153 concentrations between 1 and 10nM, and a positive dose-effect relationship was seen between 0.01 and 1 nM. Comparison of the kinetics of DNA strand-break rejoining in the presence and absence of AV-153 revealed a considerable influence on the rate of repair. In view of the resemblance of this compound's structure to that of dihydronicotinamide, a substrate for poly(ADP-rybose)polymerase, the modulation of DNA repair by AV-153 could involve an influence on poly(ADP)ribosylation.     

Citrus flavonoid naringenin inhibits TLR2 expression in adipocytes

Toll-like receptors (TLRs) were recently shown to be involved in obesity-induced inflammation in adipose tissue, which contributes to the development of insulin resistance and type 2 diabetes. Thus, the appropriate regulation of TLR expression or activation is an important strategy for improving obesity-related diseases. In this report, we show that naringenin, a citrus flavonoid, inhibits TLR2 expression during adipocyte differentiation. This effect is mediated in part through peroxisome proliferator-activated receptor γ activation. In addition, naringenin suppresses TLR2 expression induced by the co-culture of differentiated adipocytes and macrophages and also inhibits tumor necrosis factor-α (TNF-α)-induced TLR2 expression by inhibiting the activation of nuclear factor-κB and c-Jun NH₂-terminal kinase pathways in differentiated adipocytes. Furthermore, naringenin decreases TLR2 expression in adipose tissue of high-fat diet-fed mice. These results are correlated with the improvement of hyperglycemia and the suppression of inflammatory mediators, including TNF-α and monocyte chemotactic protein-1. Taken together, these data suggest that naringenin exhibits anti-inflammatory properties, presumably by inhibiting TLR2 expression in adipocytes. Our findings suggest a molecular mechanism by which naringenin exerts beneficial effects against obesity-related diseases.     

Low doses of selenium specifically stimulate the repair of oxidative DNA damage in LNCaP prostate cancer cells

Epidemiological studies have demonstrated an inverse relationship between selenium (Se) intake and cancer incidence and/or mortality. However, the molecular mechanisms underlying the cancer chemopreventive activity of Se compounds remain largely unknown. The objective of this study was to investigate the effect of low doses of Se on the stimulation of DNA repair systems in response to four different qualities of DNA damage. P53-proficient LNCaP human prostate adenocarcinoma cells were grown either untreated or in the presence of low concentrations of two Se compounds (30° nM sodium selenite, or 10 μM selenomethionine) and exposed to UVA, H2O2, methylmethane sulfonate (MMS) or UVC. Cell viability as well as DNA damage induction and repair were evaluated by the alkaline Comet assay. Overall, Se was shown to be a very potent protector against cell toxicity and genotoxicity induced by oxidative stress (UVA or H2O2) but not from the agents that induce other types of deleterious lesions (MMS or UVC). Furthermore, Se-treated cells exhibited increased oxidative DNA repair activity, indicating a novel mechanism of Se action. Therefore, the benefits of Se could be explained by a combination of antioxidant activity, the reduction in DNA damage and the enhancement of oxidative DNA repair capacity.     

Nicotinamide stimulates repair of DNA damage in human lymphocytes

Nicotinamide stimulates the amount of DNA repair synthesis that occurs when freshly isolated, normal human lymphocytes are treated with UV irradiation, N-methyl-N′-nitro-N-nitroso guanidine, or dimethyl sulfate. Stimulation of DNA repair synthesis is concentration dependent and reaches a maximum between 2 to 5 mM nicotinamide. In contrast, DNA synthesis in cells that have not been subjected to DNA damage is not affected by nicotinamide at concentrations below 2 mM and is inhibited by concentrations between 2 to 5 mM. In the same concentration range, nicotinic acid has no effect on the rate of DNA synthesis in the presence or absence of DNA damage.     

RecA stimulates AlkB-mediated direct repair of DNA adducts

The Escherichia coli AlkB protein is a 2-oxoglutarate/Fe(II)-dependent demethylase that repairs alkylated single stranded and double stranded DNA. Immunoaffinity chromatography coupled with mass spectrometry identified RecA, a key factor in homologous recombination, as an AlkB-associated protein. The interaction between AlkB and RecA was validated by yeast two-hybrid assay; size-exclusion chromatography and standard pull down experiment and was shown to be direct and mediated by the N-terminal domain of RecA. RecA binding results AlkB–RecA heterodimer formation and RecA–AlkB repairs alkylated DNA with higher efficiency than AlkB alone.     

The differential tissue distribution of the citrus flavanone naringenin following gastric instillation

Citrus flavonoids have been investigated for their biological activity, with both anti-inflammatory and -carcinogenic effects being reported. However, little information is known on the bioavailability of these compounds in vivo. The objectives of this study were to determine the tissue distribution of naringenin after gastric gavage of [³H]-naringenin to rats. Unlabelled naringenin was also used to quantify the levels of naringenin and its major metabolites in tissues and eliminated in the urine and faeces. Significant radioactivity was detected in the plasma as well as all tissues examined 2 h post-gavage. After 18 h, higher levels of radioactivity were retained in plasma and tissues (55% of the administered radioactivity). Investigation of the nature of metabolites, using unlabelled naringenin, revealed that the glucuronides were the major components in plasma, tissues and urine, in addition to the colonic metabolite 3-(4-hydroxyphenyl) propionic acid, detected in the urine. The aglycone was the form extensively retained in tissues after 18 h post-gavage. Total identified metabolites detected after 18 h in most tissues were only 1-5% of the levels detected after 2 h. However, the brain, lungs and heart retained 27, 20 and 11%, respectively, relative to the total metabolites detected at 2 h. While radioactive detection suggests increased levels of breakdown products of naringenin after 18 h versus 2 h, the products identified using unlabelled naringenin are not consistent with this, suggesting that a predominant proportion of the naringenin breakdown products at 18 h are retained as smaller decomposition molecules which cannot yet be identified.     

The prostate cancer risk locus at 10q11 is associated with DNA repair capacity

Genome-wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) that mildly predict prostate cancer risk. These SNPs are local tagging markers for causal gene alterations. Consideration of candidate genes in the tagged regions would be facilitated by additional information on the particular pathomechanisms which contribute to the observed risk increase. In this study we test for an association of prostate cancer tagging SNPs with alterations in DNA repair capacity, a phenotype that is frequently involved in cancer predisposition. DNA repair capacity was assessed on blood lymphocytes from 128 healthy probands after ionizing irradiation. We used the micronucleus (MN) assay to determine the cellular DNA double-strand break repair capacity and flow cytometry to measure damage induced mitotic delay (MD). Probands were genotyped for a panel of 14 SNPs, each representing an independent prostate cancer risk locus previously identified by GWAS. Associations between germline variants and DNA repair capacity were found for the SNPs rs1512268 (8p21), rs6983267 (8q24) and rs10993994 (10q11). The most significant finding was an association of homozygous rs10993994 T-allele carriers with a lower MN frequency (p=0.0003) and also a decreased MD index (p=0.0353). Cells with prostate cancer risk alleles at rs10993994 seem to cope more efficiently with DNA double strand breaks (less MN) in a shorter time (decreased MD index). This intriguing finding imposes concern about the accuracy of repair, with respect to the cancer risk that is mediated by T genotypes. To date, MSMB (microseminoprotein β) is favored as the causal gene at the 10q11 risk locus, since it was the first candidate gene known to be expressionally altered by rs10993994. Based on the present observation, candidate genes from the contexts of DNA repair and apoptosis may be more promising targets for expression studies with respect to the rs10993994 genotype.     

De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

Background

Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background.

Results

Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type.

Conclusions

The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality.

     

DNA methylation in prostate cancer

Prostate cancer is the most common malignancy and the second leading cause of cancer death among men in the United States. There are three well-established risk factors for prostate cancer: age, race and family history. The molecular bases for these risk factors are unclear; however, they may be influenced by epigenetic events. Epigenetic events covalently modify chromatin and alter gene expression. Methylation of cytosine residues within CpG islands on gene promoters is a primary epigenetic event that acts to suppress gene expression. In tumorigenesis, the normal functioning of the epigenetic-regulatory system is disrupted leading to inappropriate CpG island hypermethylation and aberrant expression of a battery of genes involved in critical cellular processes. Cancer-dependent epigenetic regulation of genes involved in DNA damage repair, hormone response, cell cycle control and tumor-cell adhesion/metastasis can contribute significantly to tumor initiation, progression and metastasis and, thereby, increase prostate cancer susceptibility and risk. In this review, we will discuss current research on genes that are hypermethylated in human prostate cancer. We will also discuss the potential involvement of DNA methylation in age-related, race-related and hereditary prostate cancer, and the potential use of hypermethylated genes as biomarkers to detect prostate cancer and assess its risk.