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Down-regulation of neogenin accelerated glioma progression through promoter Methylation and its overexpression in SHG-44 Induced Apoptosis.

Wu X, Li Y, Wan X, Kayira TM, Cao R, Ju X, Zhu X, Zhao G - PLoS ONE (2012)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Cytology and Genetics, Northeast Normal University, Changchun, China.

ABSTRACT

Background: 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.

Methodology/principal findings: 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).

Conclusions/significance: 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.

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The difference of neogenin expression between gliomas and their homologous surrounding tissues.(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 t -test, n = 13, ** p<0.01, error bars indicate standard error means.
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pone-0038074-g001: The difference of neogenin expression between gliomas and their homologous surrounding tissues.(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 t -test, n = 13, ** p<0.01, error bars indicate standard error means.

Mentions: In this study, a clear pattern of neogenin expression in gliomas was the first priority. Two normal brain tissues and thirteen paired surrounding and glioma tissues (Table S1) were analysed (each glioma tissue was paired with its neighbouring non-neoplastic tissue). Immunohistochemistry results showed that cells in the surrounding tissues were sparse, well distributed, rarely polynuclear and karyokinetic, normal glial cell shape though seldom hyperplasia (Fig. 1A), whereas in the typical tumor tissues, cells were more heterogenous: ranging from small to giant, disordered, polynuclear to karyokinetic (Fig. 1B). Western blot analysis showed that neogenin in tumor tissues was lower relative to that in the normal brain tissues and surrounding non-neoplastic tissues, using β-actin as an internal control for electrophoresis efficiency (paired t-test, n = 13, p<0.01, Fig. 1C, D). Strikingly, the down-expression of neogenin was more clear in high-grade gliomas (n = 7) and female patients (n = 7) than low-grade gliomas and male patients (Table 1, paired t-test, n = 7, p<0.01). These results signified that the expression of neogenin was down-regulated in gliomas and more pronounced in high-grade gliomas.


Down-regulation of neogenin accelerated glioma progression through promoter Methylation and its overexpression in SHG-44 Induced Apoptosis.

Wu X, Li Y, Wan X, Kayira TM, Cao R, Ju X, Zhu X, Zhao G - PLoS ONE (2012)

The difference of neogenin expression between gliomas and their homologous surrounding tissues.(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 t -test, n = 13, ** p<0.01, error bars indicate standard error means.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3362578&req=5

pone-0038074-g001: The difference of neogenin expression between gliomas and their homologous surrounding tissues.(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 t -test, n = 13, ** p<0.01, error bars indicate standard error means.
Mentions: In this study, a clear pattern of neogenin expression in gliomas was the first priority. Two normal brain tissues and thirteen paired surrounding and glioma tissues (Table S1) were analysed (each glioma tissue was paired with its neighbouring non-neoplastic tissue). Immunohistochemistry results showed that cells in the surrounding tissues were sparse, well distributed, rarely polynuclear and karyokinetic, normal glial cell shape though seldom hyperplasia (Fig. 1A), whereas in the typical tumor tissues, cells were more heterogenous: ranging from small to giant, disordered, polynuclear to karyokinetic (Fig. 1B). Western blot analysis showed that neogenin in tumor tissues was lower relative to that in the normal brain tissues and surrounding non-neoplastic tissues, using β-actin as an internal control for electrophoresis efficiency (paired t-test, n = 13, p<0.01, Fig. 1C, D). Strikingly, the down-expression of neogenin was more clear in high-grade gliomas (n = 7) and female patients (n = 7) than low-grade gliomas and male patients (Table 1, paired t-test, n = 7, p<0.01). These results signified that the expression of neogenin was down-regulated in gliomas and more pronounced in high-grade gliomas.

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Cytology and Genetics, Northeast Normal University, Changchun, China.

ABSTRACT

Background: 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.

Methodology/principal findings: 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).

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus