Adiponectin Upregulates Filaggrin Expression via SIRT1-Mediated Signaling in Human Normal Keratinocytes

BACKGROUND: Filaggrin (FLG) is the major component of the epidermal granular layer and binds to and condenses the keratin cytoskeleton. FLG thus contributes to cell compaction and serves as a natural moisturizing factor by promoting unfolding and degradation into hygroscopic amino acids. Loss or downregulation of FLG has been shown to result in a weak stratum corneum, which causes water loss and increases the possibility of skin barrier-related seizure. Adiponectin (Acrp30) contributes to the functional recovery of somatic cells, including human normal epidermal keratinocytes (NHEKs). OBJECTIVE: To investigate the effect of Acrp30 in FLG expression and identifying its signal transduction mechanism. METHODS: Normal human keratinocytes were treated with Acrp30 and the levels of FLG were examined. Silent mating type information regulation 2 homolog (SIRT)-targeting siRNA and aryl hydrocarbon receptor nuclear translocator (ARNT)-targeting siRNA were used to identify the role of various signal transduction pathway components. RESULTS: Acrp30 upregulated SIRT1 and ARNT expression in NHEKs, resulting in increased FLG expression. Treatment with both SIRT1-targeting siRNA and ARNT-targeting siRNA blocked Acrp30 stimulation and silenced FLG expression. CONCLUSION: Adiponectin upregulates FLG expression through a SIRT1-mediated pathway. Our results suggest that Acrp30 is a promising agent for skin barrier permeability improvement.

Influência dos alelos R e S do gene Slc11a1 na suscetibilidade à carcinogênese de pele induzida por 7, 12-dimetilbenzantraceno / Influence of R and S alleles of Slc11a1 gene in the susceptibility to skin cancer induced by 7 12-Dimethylbenz[a]anthracene

Camundongos AIRmax e AIRmin diferem na sensibilidade à carcinogênese, sendo os AIRmin mais sensíveis à carcinogênese de pele devido ao seu fundo genético e um polimorfismo no gene Ahr. Mais que isso, estas linhagens possuem um desequilíbrio de frequência dos alelos do gene Slc11a1. Para estudar a interação dos alelos de resistência (R) ou suscetibilidade (S) do gene Slc11a1 com os loci de resposta inflamatória aguda dos animais AIRmax e AIRmin, foram produzidas sublinhagens homozigotas para estes alelos:AIRmaxRR, AIRmaxSS, AIRminRR e AIRminSS. Nosso objetivo foi investigar a diferença de sensibilidade à carcinogênese de pele induzida por DMBA nestas sublinhagens. A incidência de câncer de pele foi de 7% nos animais AIRminRR e de 13% nos animais AIRminSS.Os animais AIRmaxSS não apresentaram câncer de pele, mas a incidência de câncer em órgãos internos foi 100% nesta sublinhagem.Esses dados mostraram que os camundongos AIRmaxSS tem maior suscetibilidade à carcinogênese, sugerindo que o alelo S, no fundo genético AIRmax, pode influenciar na suscetibilidade ao câncer.

Mice AIRmax and AIRmin differ on sensibility to carcinogenesis. AIRmin mice are significantly more sensitive to skin carcinogenesis than AIRmax mice due to the genetic background and the polymorphism of aryl hydrocarbon receptor (Ahr) gene. Furthermore,these mice have an imbalance of frequency of Slc11a1 gene alleles.To study the interaction of resistant (R) or susceptible (S) Slc11a1 alleles with acute inflammatory reaction loci found in AIRmax and AIRmin mice, homozygous sublines for these alleles were produced: AIRmaxRR, AIRmaxSS, AIRminRR and AIRminSS. The objective of this study was to investigate the difference in skin carcinogenesis sensibility induced by DMBA agent in these sublines.The incidence of skin cancer was 7% in AIRminRR mice and 13% in AIRminSS mice.AIRmaxRR and AIRmaxSS mice did not show skin cancer, but the incidence of internal organs cancer was 100% only in AIRmaxSS mice. These data showed that AIRmaxSS animals have higher susceptibility, suggesting that the S allele in the AIRmax background could influence susceptible to cancer.

Eupatilin Inhibits Gastric Cancer Cell Growth by Blocking STAT3-Mediated VEGF Expression

PURPOSE: Eupatilin is an antioxidative flavone and a phytopharmaceutical derived from Artemisia asiatica. It has been reported to possess anti-tumor activity in some types of cancer including gastric cancer. Eupatilin may modulate the angiogenesis pathway which is part of anti-inflammatory effect demonstrated in gastric mucosal injury models. Here we investigated the anti-tumor effects of eupatilin on gastric cancer cells and elucidated the potential underlying mechanism whereby eupatilin suppresses angiogenesis and tumor growth. MATERIALS AND METHODS: The impact of eupatilin on the expression of angiogenesis pathway proteins was assessed using western blots in MKN45 cells. Using a chromatin immunoprecipitation assay, we tested whether eupatilin affects the recruitment of signal transducer and activator of transcription 3 (STAT3), aryl hydrocarbon receptor nuclear translocator (ARNT) and hypoxia-inducible factor-1alpha (HIF-1alpha) to the human VEGF promoter. To investigate the effect of eupatilin on vasculogenesis, tube formation assays were conducted using human umbilical vein endothelial cells (HUVECs). The effect of eupatilin on tumor suppression in mouse xenografts was assessed. RESULTS: Eupatilin significantly reduced VEGF, ARNT and STAT3 expression prominently under hypoxic conditions. The recruitment of STAT3, ARNT and HIF-1alpha to the VEGF promoter was inhibited by eupatilin treatment. HUVECs produced much foreshortened and severely broken tubes with eupatilin treatment. In addition, eupatilin effectively reduced tumor growth in a mouse xenograft model. CONCLUSIONS: Our results indicate that eupatilin inhibits angiogenesis in gastric cancer cells by blocking STAT3 and VEGF expression, suggesting its therapeutic potential in the treatment of gastric cancer.

No association of AhR gene 1661G/A and ARNT gene 567G/C polymorphisms with endometriosis in southern Han Chinese women / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the association between the arylhydrocarbon receptor gene (AhR) 1661G/A or arylhydrocarbon nuclear translocatorgene (ARNT) 567G/C polymorphism and endometriosis in southern Han Chinese women.</p><p><b>METHODS</b>The polymorphisms of AhR gene 1661G/Aand ARNT gene 567G/C in 431 cases of endometriosis and 499 healthy women were genotyped by fluorescence quantitative PCR-based high resolution melting.</p><p><b>RESULTS</b>The frequencies of genotypes AA, AG, GG and alleles A and G in controls were 12.0%, 41.9%, 46.1%, 33.0% and 67.0%, respectively, which were not significantly different from those in patients with endometriosis (9.7%, 44.6%, 45.7%, 32.0% and 68.0%, respectively). The genotype frequencies of GG, GC, CC and alleles C and G in controls (15.6 %, 51.7%, 32.7%, 58.5%, 41.5%) were not significantly different from those in patients with endometriosis (13.5%, 47.8%, 38.7%, 62.6%, 37.4%), either. And no interaction of AhR 1661G/A and ARNT 567G/C on endometriosis was found.</p><p><b>CONCLUSION</b>No association between AhR 1661G/A and ARNT 567G/C genetic polymorphisms and endometriosis was found in the southern Han Chinese women in this study.</p>

Significance of p27(kip1) as potential biomarker for intracellular oxidative status

Our previous proteomic study demonstrated that oxidative stress and antioxidant delphinidin regulated the cellular level of p27(kip1) (referred to as p27) as well as some heat shock proteins in human colon cancer HT 29 cells. Current study was conducted to validate and confirm the regulation of these proteins using both in vitro and in vivo systems. The level of p27 was decreased by hydrogen peroxide in a dose-dependent manner in human colon carcinoma HCT 116 (p53-positive) cells while it was increased upon exposure to hydrogen peroxide in HT 29 (p53-negative) cells. However, high concentration of hydrogen peroxide (100 micrometer) downregulated p27 in both cell lines, but delphindin, one of antioxidative anthocyanins, enhanced the level of p27 suppressed by 100 micrometer hydrogen peroxide. ICR mice were injected with varying concentrations of hydrogen peroxide, delphinidin and both. Western blot analysis for the mouse large intestinal tissue showed that the expression of p27 was upregulated by 25 mg/kg BW hydrogen peroxide. To investigate the association of p27 regulation with hypoxia-inducible factor 1-beta (HIF-1beta), the level of p27 was analyzed in wild-type mouse hepatoma hepa1c1c7 and Aryl Hydrocarbon Nuclear Translocator (arnt, HIF-1beta)-defective mutant BPRc1 cells in the absence and presence of hydrogen peroxide and delphinidin. While the level of p27 was responsive to hydrogen peroxide and delphinidin, it remained unchanged in BPRc1, suggesting that the regulation of p27 requires functional HIF-1beta. We also found that hydrogen peroxide and delphinidin affected PI3K/Akt/mTOR signaling pathway which is one of upstream regulators of HIFs. In conclusion, hydrogen peroxide and antioxidant delphinidin seem to regulate intracellular level of p27 through regulating HIF-1 level which is, in turn, governed by its upstream regulators comprising of PI3K/Akt/mTOR signaling pathway. The results should also encourage further study for the potential of p27 as a biomarker for intracellular oxidative or antioxidant status.

Effects of ARNT2 gene on invasion and migration of human hepatocellular carcinoma HCCLM6 cell line / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of ARNT2 on invasion and migration of HCCLM6 cells.</p><p><b>METHODS</b>Four short hairpin oligos targeting to ARNT2 were s cloned into the pLVTHM vector. Lentiviral vectors shRNA-ARNT2i, pCMV-dR8.74 and pMD2G were cotransfected into 293T cells using Lipofectamine 2000. HCCLM6 was infected with virus supernatant. ARNT2 mRNA and protein expressions were detected using quantitative Real time-PCR and Western blot, respectively. The invasion and migration of HCCLM6 cells were evaluated using wound healing assay and cell invasion assay in vitro. Statistical analysis was performed with SPSS 16.0.</p><p><b>RESULTS</b>The relative mRNA levels of ARNT2 were 0.154+/-0.024, 0.860+/-0.145, 1.004+/-0.009 in shRNA-ARNT2i virus infected HCCLM6 cells, mock-infected cells and control vector virus infected cells (F = 113.14, P more than 0.01). The expression of ARNT2 at protein level was 16.45+/-1.6, 44.56+/-2.07 in the HCCLM6 cells infected with shRNA-ARNT2i virus and negative control vector virus, respectively (t = 18.58, P less than 0.01). The scrape wound of HCCLM6 cells infected with shRNA-ARNT2i virus healed faster than cells infected with control vector virus or mock-infected cells. The number of cells invading through Matrigel was higher in the HCCLM6 cells infected with shRNA-ARNT2i virus (13.25+/-1.04) than that in mock-infected HCCLM6 cells and the HCCLM6 cells infected with negative control vector virus (6.50+/-2.56, 6.75+/-2.05) (F = 29.645, P less than 0.01).</p><p><b>CONCLUSION</b>Inhibition of ARNT2 gene promotes the invasion and migration of HCCLM6 cells.</p>

Regulation of glucose metabolism-related genes and VEGF by HIF-1alpha and HIF-1beta, but not HIF-2alpha, in gastric cancer

Hypoxia-inducible factors (HIFs) are transcription factors that activate the transcription of target genes involved in crucial aspects of cancer development. This study investigated the expression of HIFs and their contribution to the regulation of target genes related to angiogenesis and glucose metabolism in gastric cancer. The data showed that HIFs were over-expressed in gastric cancer and that activation of the target genes was observed mainly in the early stages. Moreover, the results of the present study revealed that only HIF-1alpha, but not HIF-2alpha dimerizes with HIF-1beta and then regulates expression of target genes in response to hypoxia. The results of the present study demonstrate that HIF-1alpha and HIF-1beta enhances expression of VEGF and glucose metabolism-related genes in response to hypoxia in gastric cancer. These data offer important information regarding HIF pathways in the development of gastric cancer.

Estudo comparativo da atividade catalítica e expressão protéica do citocromo P4501A (CYP1A) em cascudos (Loricariidae) e tilápias (Cichlidae) / Comparative study of the catalytic activity and proteinic expression of cytochrome P4501A (CYP1A) in suckermouth armored catfishes (Loricariidae) and tilapias (Cichlidae)

Os citocromos P450 (CYP) desempenham importantes papéis na biotransformação de xenobióticos e no metabolismo de substâncias endógenas. A subfamília CYP1A foi bem conservada ao longo da evolução dos vertebrados, tendo sido encontrada em todas as espécies de peixes mandibulados estudadas até o momento. Em trabalho anterior, verificamos que algumas espécies de cascudos da família Loricariidae não apresentavam atividade da etoxiresorufina-O-desetilase (EROD) em microssomos hepáticos. Como EROD é atividade catalisada predominantemente por CYP1A em diversos vertebrados, esse achado nos motivou a investigar em detalhe os CYP, em especial da subfamília 1A, em cascudos. Este trabalho é um estudo comparativo da capacidade de cascudos (Hyposthomus luetkeni e H. affinis), tilápias do Nilo (Oreochromis niloticus; Cichlidae) e camundongos, controles e tratados com indutores conhecidos de CYP1A [50 mg/kg ip; beta-naftoflavona (BNF), ou 7-12 dimetil-benzoantraceno (DMBA)] de: i - catalisar diversas reações químicas sabidamente mediadas por CYP, ii - expressar a proteína CYP1A no tecido hepático, e iii - ativar o pró-mutágeno DMBA. Nos microssomos hepáticos das tilápias e dos camundongos, detectamos atividades constitutivas de EROD e também de desalquilação de outros ésteres da resorufina (MROD, PROD e BROD). Nessas duas espécies, as atividades dessas monooxigenases foram induzidas pelos tratamentos com BNF e DMBA. Nos cascudos, controles e tratados com os indutores de CYP1A, as atividades das alcoxi-resorufina-O-desalquilases não foram detectadas. A atividade da etoxicumarina desetilase (ECOD) foi cerca de cinco vezes maior no figado de cascudos e de camundongos do que no das tilápias. O CYP1A não parece ter papel importante na catálise de ECOD nessas duas espécies de peixes. Os resultados também mostraram que dois anticorpos anti-CYP1A de peixe reconheceram proteínas com massa molecular compatível com o de CYPs nos microssomos hepáticos de tilápias e camundongos...

Detection of interaction of binding affinity of aromatic hydrocarbon receptor to the specific DNA by exonuclease protection polymerase chain reaction assay / 中华预防医学杂志

<p><b>OBJECTIVE</b>To establish an exonuclease protection mediated polymerase chain reaction (PCR) assay for the non-radioactive, sensitive detection of the binding of protein and DNA.</p><p><b>METHODS</b>The 1 pmol/L-10 nmol/L 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dissolved in dimethyl sulphoxide (DMSO), was added into 100 microl SD rat hepatic cytosol in vitro, which contained different amount of aromatic hydrocarbon receptors (AhR) and relative proteins, and ligand-AhR-DRE complex were formed in addition to 1 fmol/L-100 nmol/L DNAs probes containing the sequence of DRE. With the digestion of Exonuclease III and S1 nuclease, free DNAs were digested to oligonucleotide and binding DNA remained due to protein (AhR) protection and be amplified by PCR. The results of PCRs were shown by loading on 2% agarose electrophoresis. DMSO was used as negative control and blank control was set up.</p><p><b>RESULTS</b>Target DNA (285 bp) could be observed in the ligand groups, but not in the control group. The minimal amount of receptor was 2.5 fmol/L and the minimal amount of DNA probes was 2 fmol.</p><p><b>CONCLUSIONS</b>Exonuclease protection mediated PCR assay should be a good non-radioactive tool to quantify the interaction of protein and DNA with high sensitivity and simplicity.</p>