26 research outputs found

    HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

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    <p>HSPA5/GRP78/BiP plays an important role in cell survival or tumor progression. For these reasons, HSPA5 is an emerging therapeutic target in cancer development. Here we report that HSPA5 contributes to head and neck cancer (HNC) survival via maintenance of lysosomal activity; however, a nonthermal plasma (NTP, considered as a next-generation cancer therapy)-treated solution (NTS) inhibits HNC progression through HSPA5-dependent alteration of lysosomal activity. HSPA5 prevents NTS-induced lysosome inhibition through lysosomal-related proteins or regulation of gene expression. However, NTS-induced MUL1/MULAN/GIDE/MAPL (mitochondrial ubiquitin ligase activator of NFKB 1) leads to downregulation of HSPA5 via K48-linked ubiquitination at the lysine 446 (K446) residue. <i>MUL1</i> knockdown hinders NTS-induced lysosome inhibition or cytotoxicity through the reduction of HSPA5 ubiquitination in HNC cells. While MUL1 was suppressed, HSPA5 was overexpressed in tissues of HNC patients. NTS strongly inhibited HNC progression via alterations of expression of MUL1 and HSPA5, in vivo in a xenograft model. However, NTS did not induce inhibition of tumor progression or HSPA5 reduction in <i>MUL1</i> knockout (KO) HNC cells which were generated by CRISPR/Cas9 system. The data provide compelling evidence to support the idea that the regulation of the MUL1-HSPA5 axis can be a novel strategy for the treatment of HNC.</p

    TTP deficiency blocks the anti-inflammatory function of CO in DSS-induced colitis.

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    <p>After treatment with 2% (w/v) DSS for 7 days, WT and TTP KO mice were exposed to CO at a concentration of 250 ppm for 5 days and analyzed for colitis. A. Clinical scores were assessed as described in Materials and Methods. Data are mean ± SD for 5 mice (**, p<0.01; ***, p<0.005). ns, not significant. B and C. Colon length. B. Representative images of 5 tests conducted in each group. C. Data are mean ± SD for 5 mice (*, p<0.05; **, p<0.01). D. Representative H&E sections of each group from colons of WT and TTP KO mice. E. Neutrophil infiltration into the colon, quantified by measuring MPO activity. Data are mean ± SD for 5 mice (*, p<0.05; **, p<0.01). F. Colonic cytokine/chemokine mRNA levels, analyzed by semi-quantitative RT-PCR. Data represent 1 of 3 independent experiments with similar results. The band densities in the agarose gel were quantified by PhosphorImager, normalized to the internal control GAPDH and expressed as percentage (%) of the value of untreated control cells. Data shown are mean ± SD (n = 3). **, p<0.01; ***, p<0.005. ns, not significant.</p

    CORM-2 induces TTP promoter activity and <i>TTP</i> mRNA in macrophages.

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    <p>A. RAW264.7 cells were incubated with different concentration of CORM-2 or iCORM-2 for 4 h. <i>TTP</i> mRNA expression was determined by semi-quantitative RT-PCR. B. RAW264.7 cells were transfected with pGL3/mTTP P-1309 containing the mouse <i>TTP</i> promoter. After 24 h, cells were treated with different concentrations of CORM-2 for 4 h, and luciferase activity was determined. The level of firefly luciferase activity was normalized using <i>Renilla</i> luciferase activity. The relative luciferase activity is presented as a fold increase over untreated cells. Values are mean ± SD (n = 3). *, <i>p</i><0.05; **, <i>p<</i>0.01.</p

    TTP deficiency blocks the anti-inflammatory function of CORM-2 in macrophages.

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    <p>Peritoneal macrophages were harvested from WT and TTP KO mice. Cells were treated with 1 µg/ml LPS in the presence of 10 µM CORM-2 for 4 h. A. TNF-α in the supernatant of cells was determined by ELISA. Data shown are mean ± SD (n = 3). **, <i>p<</i>0.01. B. The expression levels of <i>TTP</i> and <i>TNF-α</i> mRNA were determined by semi-quantitative RT-PCR. C. Expression of <i>TNF</i> mRNA in macrophages was determined by quantitative real-time PCR at indicated times after the addition of 5 µg/ml actinomycin D. Values are mean ± SD (n = 3). ***, <i>p<</i>0.001. The band densities in the agarose gel were quantified by PhosphorImager, normalized to the internal control GAPDH and expressed as percentage (%) of the value of untreated control cells. Data shown are mean ± SD (n = 3). ***, p<0.005. ns, not significant.</p

    The frequencies of candidate genetic loci.

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    <p>The most common allele for each locus is underlined.</p><p>Abbreviation: HWE, Hardy–Weinberg equilibrium.</p>a<p>Comparison between our data and Western data with pediatric ALL. Data for 15 loci was adopted from Ref. 10 and data of <i>ITPA</i> 94 C>A was from Ref. 9.</p>b<p>Comparison between our data and normal Japanese data. Reference data was adopted by SNP searching on NCBI reference assembly (<a href="http://www.ncbi.nlm.nih.gov/snp/" target="_blank">http://www.ncbi.nlm.nih.gov/snp/</a>). Data for <i>CYP3A4*1B</i> was from Coriell Cell Repository samples and all the others were from Japanese data of the Hapmap project.</p>c<p>Hardy-Weinberg equilibrium was reached after the A variant was excluded (<i>P</i> = 0.38).</p>d<p>Variant alleles : <i>TPMT*1</i> (Wild type), <i>TPMT*2</i> (238 G>C), <i>TPMT*3A</i> (460 G>A and 719 A>G), <i>TPMT*3B</i> (460 G>A), <i>TPMT*3C</i> (719 A>G).</p

    Correlation between the dose percent of 6-MP and MTX in each patient and <i>TPMP</i> genotype.

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    <p>The correlation analysis between the dose percent of 6-MP and MTX in each patient showed a statistically significant linear relationship (<i>R</i><sup>2</sup> = 0.628, <i>P</i> = 0.00).</p
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