Additional file 1: of Suberoylanilide hydroxamic acid represses glioma stem-like cells

Supplementary information includes Figures S1 to S3 and supplementary methods. (DOCX 654 kb

Increased expression levels of pIKKβ(S181), pS6K1(T389), and VEGF-A in liver tissues of HBx transgenic mice.

<p>(A). A gross view of representative liver tumors (T1, T2, T3) developed in HBx transgenic mice in several months of breeding. The ALT values are shown. (B). The H&E staining of non-tumor and tumor parts in HBx transgenic mice. (C). The expression levels of pIKKβ (S181), IKKβ, pS6K1 (T389), S6K1, VEGF-A, HBx, and β-actin detected by Western blotting in non-tumor and tumor parts of liver tissues of three HBx transgenic mice (#824, #825, and #826) were compared to the normal liver tissues of the wild-type age-matched mouse. The HBx mRNA levels were also measured by RT-PCR and the GAPDH mRNA levels were used as an internal control. The relative levels of pIKKβ (S181), pS6K1 (T389), and VEGF-A were quantified by densitometry and normalized with total IKKβ, total S6K1, and actin. Results are shown as ratios of average levels of pIKKβ (S181) pS6K1 (T389), and VEGF-A in non-tumor and tumor parts of liver tissues of three HBx transgenic mice (#824, #825, and #826) relative to that in the normal liver tissues of the wild-type age-matched mouse (set as 1). Data are shown as means ± S.D. of measurements of three mice. (D). Immunohistochemistry analyses show expression levels of pIKKβ (S181), pS6K1 (T389), VEGF-A, and CD31 in normal liver tissues of the wild-type mouse, and non-tumor and tumor parts of liver tissues of HBx transgenic mice. One representative data are shown. N = 3.</p

The IKKβ/TSC1/mTOR signaling pathway is activated by HBx.

<p>(A). Expression of HBx mRNA in Hep3Bx, HepG2x, and parental Hep3B and HepG2 cells was detected using semi-quantitative RT-PCR. Levels of GAPDH mRNA were used as an internal control. RNAs of a HBV-positive patient’s serum (P) and RNAs of a control HBV-negative serum (N) were used as controls. (B). Levels of HBx protein were detected in lysates of Hep3Bx, HepG2x, and parental Hep3B and HepG2 cells using Western blotting by antibody specific against HBx protein and β-actin. (C). Levels of pIKKβ(S181), pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin were assessed in lysates of Hep3Bx, HepG2x, and parental Hep3B and HepG2 cells using Western blotting by specific antibody as indicated. (D). Data shown are ratios of viable cells in Hep3Bx and HepG2x cells relative to that in Hep3B and HepG2 cells (set as 1), respectively, at 24 h after seeding using MTT assay. (E). Levels of pIKKβ (S181), pS6K1 (T389), total IKKβ, total S6K1, HBx, and β-actin were assessed in lysates of Huh7 cells transfected with empty vector alone, payw1.2WT, or payw*7. (F). Levels of pIKKβ (S181), pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin were assessed in lysates of Hep3B and Hep3Bx with or without TNF-α treatment using Western blotting as described earlier.</p

Expression of VEGF is increased in Hep3Bx and HepG2x cells and is further enhanced by TNF-α and blocked by IKKβ inhibitor Bay 11-7082 or the mTOR inhibitor rapamycin.

<p>(A). The expression levels of secreted VEGF in the culture medium of Hep3B, Hep3Bx, HepG2, and HepG2x cells were measured by ELISA assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041931#s2" target="_blank">Methods</a>. (B). The expression levels of VEGFA mRNA were assessed in Hep3B, Hep3Bx, HepG2, and HepG2x cells using semi-quantitative RT-PCR (left) or real-time RT-PCR (right) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041931#s2" target="_blank">Methods</a>. (C). The amounts of secreted VEGF in the culture medium of Hep3B, Hep3Bx, HepG2, or HepG2x cells treated with or without TNF-α in the presence or absence of Bay11-7082 or rapamycin were measured by ELISA assay. Data are shown as means ± S.D. of three experiments. Comparisons were made between different groups as indicated. *<i>P</i><0.001 is determined by X test.</p

TNF-α-stimulated increases of pTSC1 (S511), pS6K1 (T389) and cell proliferation in Hep3Bx and HepG2x cells are blocked by the IKKβ inhibitor Bay 11-7082, siRNA specific for IKKβ, and the mTOR inhibitor rapamycin.

<p>(A). Lysates of Hep3Bx and HepG2x cells treated with or without TNF-α in the presence or absence of Bay 11-7082 were analyzed for levels of pIKKβ (S181), pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin using Western blotting as described earlier. (B). Lysates of Hep3Bx and HepG2x cells with transfection of IKKβsiRNAs or control siRNAs were assessed for levels of pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin. (C). Lysates of Hep3Bx and HepG2x cells treated with or without TNF-α in the presence or absence of rapamycin were analyzed for levels of pIKKβ (S181), pS6K1 (T389), total IKKβ, and total S6K1. (D). Data shown are ratios of viable cells in Hep3B, Hep3Bx, HepG2, and HepG2x cells treated with or without TNF-α in the presence or absence of Bay11-7082 or rapamycin relative to that in Hep3B and HepG2 cells without any treatment (set as 1), at 24 h after seeding using MTT assay. Data are shown as means ± S.D. of three experiments. Comparisons were made between different groups as indicated. *P<0.001 is determined by X test.</p

Positive association between pIKKβ(S181), pTSC1(S511), and pS6K1(T389) in HBV-associated human HCC specimens.

<p>(A). Immunohistochemistry analysis of pIKKβ (S181), pTSC1(S511) and pS6K1(T389) in tumor tissues of 95 human HBV-associated HCC specimens. Results of one representative specimens stained by specific antibodies are shown. (B). Upper graph shows percentages of specimens with low or high pIKKβ (S181) expression in which pS6K1 (T389) expression was high or was not observed (low). Lower graph shows percentages of specimens with low or high pIKKβ (S181) expression in which pTSC1 (S511) expression was high or was not observed (low). Positive correlations was noted between pIKKβ (S181) and pS6K1 (T389) (*<i>P</i><0.01) and between pIKKβ (S181) and pTSC1 (S511) (*<i>P</i><0.01)<b>.</b> (C) The Kaplan-Meier disease-free survival curves show that expression of pIKKβ (S181) (<i>p</i> = 0.003), pTSC1 (S511) (<i>p</i> = 0.048), or pS6K1 (T389) (<i>p</i> = 0.0027) is associated with early tumor recurrence. Co-expression of pIKKβ (S181) and pS6K1 (T389) (<i>p</i> = 0.0013) was a better predictor of patients’ recurrence-free time survival in HCC patients who received curative surgery for up to 48-month investigation.</p

Allergies and Risk of Head and Neck Cancer: An Original Study plus Meta-Analysis

<div><h3>Background</h3><p>Although the relationship between allergy and cancer has been investigated extensively, the role of allergy in head and neck cancer (HNC) appears less consistent. It is not clear whether allergies can independently influence the risk of HNC in the presence of known strong environmental risk factors, including consumption of alcohol, betel quid, and cigarette.</p> <h3>Methods</h3><p>The current paper reports results from: 1) an original hospital-based case-control study, which included 252 incident cases of HNC and 236 controls frequency-matched to cases on sex and age; and 2) a meta-analysis combining the results of the current case-control study and 13 previously published studies (9 cohort studies with 727,569 subjects and 550 HNC outcomes and 5 case-control studies with 4,017 HNC cases and 10,928 controls).</p> <h3>Results</h3><p>In the original case-control study, we observed a strong inverse association between allergies and HNC [odds ratio = 0.41, 95% confidence interval (CI): 0.27–0.62]. The meta-analysis also indicated a statistically significant inverse association between HNC and allergies [meta-relative risk (RR) = 0.76, 95% CI: 0.63–0.91], particularly strong for allergic rhinitis (meta-RR = 0.55, 95% CI: 0.40–0.76). In addition, the inverse association between allergies and HNC was observed only among men (meta-RR = 0.67, 95% CI: 0.54–0.84) but not among women (meta-RR = 0.98, 95% CI: 0.81–1.18).</p> <h3>Conclusions</h3><p>These findings suggest that immunity plays an influential role in the risk of HNC. Future studies investigating immune biomarkers, including cytokine profiles and genetic polymorphisms, are warranted to further delineate the relationship between allergies and HNC. Understanding the relationship between allergies and HNC may help devise effective strategies to reduce and treat HNC.</p> </div

The association between lifestyle factors and head and neck cancer by allergy status.

*<p>OR was adjusted for age, sex, education, alcohol drinking (frequency), betel quid chewing (pack-years), and cigarette smoking (pack-years).</p><p>Abbreviations: CI, confidence interval; OR, odds ratio.</p

Funnel plot of the 14 studies assessing the association between allergies and head and neck cancer risk.

<p>Funnel plot of the 14 studies assessing the association between allergies and head and neck cancer risk.</p

The association between allergies and head and neck cancer.

*<p>OR was adjusted for age, sex, education, alcohol drinking (frequency), betel quid chewing (pack-years), and cigarette smoking (pack-years).</p><p>Abbreviations: CI, confidence interval; N, number; OR, odds ratio.</p