食中毒細菌を防御する植物抽出液を用いたハードルテクノロジーの開発

内容の要約広島大学(Hiroshima University)博士(学術)Doctor of Philosophydoctora

Down-Regulation of Neogenin Accelerated Glioma Progression through Promoter Methylation and Its Overexpression in SHG-44 Induced Apoptosis

Dependence receptors have been proved to act as tumor suppressors in tumorigenesis. Neogenin, a DCC homologue, well known for its fundamental role in axon guidance and cellular differentiation, is also a dependence receptor functioning to control apoptosis. However, loss of neogenin has been reported in several kinds of cancers, but its role in glioma remains to be further investigated.Western blot analysis showed that neogenin level was lower in glioma tissues than in their matching surrounding non-neoplastic tissues (n = 13, p<0.01). By immunohistochemical analysis of 69 primary and 16 paired initial and recurrent glioma sections, we found that the loss of neogenin did not only correlate negatively with glioma malignancy (n = 69, p<0.01), but also glioma recurrence (n = 16, p<0.05). Kaplan-Meier plot and Cox proportional hazards modelling showed that over-expressive neogenin could prolong the tumor latency (n = 69, p<0.001, 1187.6 ± 162.6 days versus 687.4 ± 254.2 days) and restrain high-grade glioma development (n = 69, p<0.01, HR: 0.264, 95% CI: 0.102 to 0.687). By Methylation specific polymerase chain reaction (MSP), we reported that neogenin promoter was methylated in 31.0% (9/29) gliomas, but absent in 3 kinds of glioma cell lines. Interestingly, the prevalence of methylation in high-grade gliomas was higher than low-grade gliomas and non-neoplastic brain tissues (n = 33, p<0.05) and overall methylation rate increased as glioma malignancy advanced. Furthermore, when cells were over-expressed by neogenin, the apoptotic rate in SHG-44 was increased to 39.7% compared with 8.1% in the blank control (p<0.01) and 9.3% in the negative control (p<0.01).These observations recapitulated the proposed role of neogenin as a tumor suppressor in gliomas and we suggest its down-regulation owing to promoter methylation is a selective advantage for glioma genesis, progression and recurrence. Furthermore, the induction of apoptosis in SHG-44 cells after overexpression of neogenin, indicated that neogenin could be a novel target for glioma therapy

Expression of neogenin in 16 paired primary and recurrent glioma sections.

<p>(A-B) Representative immunohistochemical pictures (200× magnification): (A) primary glioma, (B) matching recurrent glioma. (C) statistical graph of mean neogenin expression in primary and recurrent gliomas. Paired t-test, n = 16, * <i>p</i><0.05, error bars indicate standard error means.</p

Primary and recurrent clinicopathologic features of 16 patients (January, 2001 – January, 2011).

<p>IOD: integral optical density;</p><p>IOD values were supplied as Mean±Standard Deviation.</p>*<p>: paired <i>t</i>-test;</p>†<p>: undefined grade gliomas.</p

Immunohistochemical analysis of neogenin in primary gliomas.

<p>(A–D) Representative immunohistochemical pictures of grade I-IV glioma respectively, (A’–D’) Magnification of the red squares in (A–D), (A–D) 200× magnification; (A’–D’) 400× magnification. (E) Scatter diagram of neogenin expression in 69 primary gliomas, blue bars are the means of four grades, n = 69, One-Way ANOVA was used in statistical analysis. (F) Histogram of mean neogenin expression in low-grade gliomas and high-grade gliomas, statistical analysis was performed with independent <i>t</i>-test. *<i>p</i><0.05, **<i>p</i><0.01, error bars indicate standard error means.</p

The 69 patients’ classified statistic according to clinical and pathological features (January, 2006 – January, 2011).

<p>IOD: integral optical density;</p>*<p>: independent <i>t</i>-test,</p>†<p>: One-Way ANOVA. All tumor sizes were measured by CT or MRI except the data from visual inspection in the operation (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038074#pone.0038074.s002" target="_blank">Table S2</a>),</p>‡<p>: n = 57. IOD values were supplied as Mean±Standard Deviation.</p

Overexpression of neogenin in SHG-44 cell line.

<p>(A) Western blot results showed neogenin expression in the blank control which was untreated (Con), the negative control which was transfected by empty vector (Vec) and the over-expressive group which was transfected by neogenin expression plasmid (Neo) in SHG-44 cell line at 48 hours after transfection; (B-D) Status of cells in the blank, negative and over-expressive group of neogenin respectively at 48 h after transfection. (E) Apoptotic distribution maps of cells in the blank, negative and over-expressive group were drew by flow cytometry assay respectively. (F) Average apoptotic rate of cells in the blanck, negative and over-expressive group in the flow cytometry assay, One-Way ANOVA, n = 3, **<i>p</i><0.01, error bars indicate standard error means.</p

Clinicopathologic features and relative neogenin level profile in 13 paired surrounding and glioma tissues (January – November, 2011).

<p>SD: standard deviation;</p>*<p>: paired <i>t</i>-test.</p

The difference of neogenin expression between gliomas and their homologous surrounding tissues.

<p>(A–B) Representative immunohistochemical pictures (200× magnification): (A) The surrounding tissue of the tumor, the arrow points to a positive normal cell; (B) The typical glioblastoma area, the arrow points to positive tumor cell. (C) Western blot showed neogenin expression in normal brains (N), surrounding non-neoplastic tissues (S) and homologous gliomas (T). (D) Statistical graph of neogenin expression in surrounding non-neoplastic tissues and gliomas. Paired <i>t</i> -test, n = 13, ** <i>p</i><0.01, error bars indicate standard error means.</p