Chemopreventive effects and anti-tumorigenic mechanisms of Actinidia arguta, known as sarunashi in Japan toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- induced lung tumorigenesis in a/J mouse

Background Previously, we reported the inhibitory effect of Actinidia arguta juice, known as sarunashi juice (sar-j) in Japan, on mutagenesis, inflammation, and mouse skin tumorigenesis. The components of A. arguta responsible for the anti-mutagenic effects were identified to be water-soluble, heat-labile phenolic compounds. We proposed isoquercetin (isoQ) as a candidate anticarcinogenic component. In this study, we sought to investigate the chemopreventive effects of A. arguta juice and isoQ on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice, and identify the possible mechanisms underlying the anti-tumorigenic effects of A. arguta. Results The number of tumor nodules per mouse lung in the group injected with NNK and administered A. arguta juice orally was significantly lower than that in the group injected with NNK only. Oral administration of isoQ also reduced the number of nodules in the mouse lungs. As expected, the mutagenicity of NNK and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) detected using S. typhimurium TA1535 decreased in the presence of sar-j. However, NNK and MNNG mutagenicity detected using S. typhimurium YG7108, a strain lacking the O6-methylguanine DNA methyltransferases (ogtST and adaST) did not decrease in the presence of sar-j suggesting that sar-j may mediate its antimutagenic effect by enhancing the DNA damage repair by ogtST and adaST. Phosphorylation of Akt, with or without epidermal growth factor stimulation, in A549 cells was significantly decreased following sar-j and isoQ treatment, indicating that components in sar-j including isoQ suppressed the PI3K/AKT signaling pathways. Conclusions Sar-j and isoQ reduced NNK-induced lung tumorigenesis. Sar-j targets both the initiation and growth/progression steps during carcinogenesis, specifically via anti-mutagenesis, stimulation of alkyl DNA adduct repair, and suppression of Akt-mediated growth signaling. IsoQ might contribute in part to the biological effects of sar-j via suppression of Akt phosphorylation, but it may not be the main active ingredient

Les normes, comment?

La description normalisée des ressources d’enseignement et d’apprentissage requiert l’utilisation d’outils d’implantation conformes aux conventions d’un standard ou d’une norme internationale et un réseau d’entraide et d’accompagnement

Vinculin and Rab5 complex is required [correction of requited]for uptake of Staphylococcus aureus and interleukin-6 expression.

Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response

Effects of vinculin and Rab5 knockdown on <i>S. aureus</i> infection in murine lung.

<p>(A and B) Mouse lungs were transfected with vinculin and Rab5 siRNA. After 3 days, lungs were homogenized and treated with anti-vinculin, anti-Rab5, and anti-GAPDH (internal control) antibodies. (C and D) Murine lungs in which vinculin or Rab5 had been knocked down were infected with <i>S</i>. <i>aureus</i>. Lung tissues were analyzed for colony formation. The graph shows the mean ± S.E. of five independent experiments. (E–F) Murine lung in which vinculin (E and G) or Rab5 (F and H) had been knocked down was infected with <i>S</i>. <i>aureus</i>. The murine lung homogenates were assayed with anti-JNK, anti-phosphor-JNK, anti-ERK, anti-phosphor-Erk, anti-p38, anti-phosphor-p38, anti-IL-6, and anti-GAPDH (internal control) antibodies by western blotting.</p

Effect of vinculin and Rab5 knockdown on <i>S. aureus</i>-induced IL-6 expression.

<p>(A and B) <i>S. aureus</i> was added to the medium of HeLa cells with vinculin (A) or Rab5 (B) knockdown and incubated for 60 min at 37°C. The cell lysates were assayed with anti-JNK, anti-phosphor-JNK, anti-ERK, anti-phosphor-Erk, anti-p38, and anti-phosphor-p38 antibodies by western blotting. (C and D) <i>S. aureus</i> was added to the medium of HeLa cells with vinculin or Rab5 knockdown and incubated for 16 h. The cell lysate (C) and conditioned medium (D) were assayed with anti-IL-6 antibody by western blotting. GAPDH and albumin were internal controls.</p

Direct binding of vinculin and Rab5.

<p>(A) Endogenous vinculin and Rab5 were immunoprecipitated from Cos-7 lysates with anti-Rab5 or anti-vinculin antibodies. Vinculin and Rab5 were assayed by western blotting using specific antibodies to vinculin and Rab5. (B) HA-Rab5 (S34N), HA-Rab5 (Q79L) and HA-Rab5 (WT) were transiently expressed in Cos-7 cells and subjected to immunoprecipitation with anti-HA antibody. Note that immunoprecipitation of HA-Rab5 (WT) was carried out with GTPγS and GDP. Proteins were assayed by western blotting. The graph shows mean ± S.E. values of three independent experiments (C) A pull-down assay from a Cos-7 lysate was performed using GST, GST-Rab5 (S34N), and GST-Rab5 (Q79L). Vinculin bound to the beads was assayed by western blotting, and GST, GST-Rab5 (S34N), and GST-Rab5 (Q79L) on a PVDF membrane were stained with Ponceau S. The graph shows mean ± S.E. values of three independent experiments (D) GST, GST-Rab5 (S34N), and GST-Rab5 (Q79L) were incubated with purified His-vinculin, and a pull-down assay was performed using glutathione beads. His-Vinculin bound to the beads was assayed by western blotting, and GST, GST-Rab5 (S34N), and GST-Rab5 (Q79L) on a PVDF membrane were stained with Ponceau S. The graph shows mean ± S.E. values of three independent experiments.</p

Determination of the Rab5-binding domain of vinculin.

<p>(A) Schematic of vinculin deletion mutants. (B) GST-Rab5 (Q79L) was incubated with purified His-vinculin deletion mutants, and a His-pull-down assay was performed. The beads were assayed by western blotting. (C) GFP, GFP-Vin1-258, GFP-Vin1-880, GFP-Vin258-880, GFP-Vin881-1066 and GFP-vinculin (full length) were coexpressed with HA-Rab5 in Cos-7 cells and immunostained with anti-HA antibody. (D) pHrodo red-labeled <i>S. aureus</i> was added to the medium of Cos-7 cells expressing each vinculin deletion mutant and incubated for 2 h at 37°C. The graph shows mean ± S.E. values of six independent experiments, **<i>p</i><0.01. (E) Cos-7 cells were transfected with HA-Rab5 (WT) and vinculin deletion mutants. <i>S. aureus</i> was added to the medium of transfected cells and incubated for 60 min at 37°C. The cells were lysed and subjected to a GST-R5BD pull-down assay. GST-R5BD-bound beads and lysates were assayed by western blotting with anti-HA antibody.</p

Effects of vinculin and Rab5 knockdown on cellular uptake.

<p>(A) siRNAs for vinculin were transfected into HeLa cells, and cell lysates were assayed by western blotting using anti-vinculin, anti-Rab5, and anti-GAPDH antibodies (internal control). (B–F) Various uptake indicators were added to the media of vinculin knockdown cells and incubated at 37°C for 2 h. Uptake of pHrodo red-labeled <i>S. aureus</i> decreased, but that of transferrin Alexa 555, albumin Alexa 555, Lucifer yellow, and FM 4-64 did not decrease. Error bar: n = 3−6±SE, **<i>p</i><0.01. (G) siRNAs for Rab5 were transfected into HeLa cells and cell lysates were assayed by western blotting with anti-Rab5, anti-vinculin, and anti-GAPDH antibodies. (H–L) HeLa cells transfected with Rab5 siRNA. Various uptake indicators were added to the media of Rab5 knockdown cells and incubated at 37°C for 2 h. Uptake of pHrodo red-labeled <i>S. aureus</i>, transferrin Alexa 555, albumin Alexa 555, Lucifer yellow, and FM 4-64 was decreased. Error bar: n = 3−6±SE, **<i>p</i><0.01.</p