The roles of ING5 in gliomas: a good marker for tumorigenesis and a potential target for gene therapy

Abstract

To elucidate the anti-tumor effects and molecular mechanisms of ING5 on glioma cells, we overexpressed it in U87 cells, and examined the phenotypes and their relevant molecules. It was found that ING5 overexpression suppressed proliferation, energy metabolism, migration, invasion, and induced G₂/M arrest, apoptosis, dedifferentiation, senescence, mesenchymal- epithelial transition and chemoresistance to cisplatin, MG132, paclitaxel and SAHA in U87 cells. There appeared a lower expression of N-cadherin, Twist, Slug, Zeb1, Zeb2, Snail, Ac-H3, Ac-H4, Cdc2, Cdk4 and XIAP, but a higher expression of Claudin 1, Histones 3 and 4, p21, p53, Bax, β-catenin, PI₃K, Akt, and p-Akt in ING5 transfectants. ING5 overexpression suppressed tumor growth of U87 cells in nude mice by inhibiting proliferation and inducing apoptosis. Down-regulated ING5 expression was closely linked to the tumorigenesis and histogenesis of glioma. These data indicated that ING5 expression might be considered as a good marker for the tumorigenesis and histogenesis of gliomas. It might be employed as a potential target for gene therapy of glioma. PI₃K/Akt or β-catenin/TCF-4 activation might be positively linked to chemotherapeutic resistance, mediated by ING5.

Keywords: ING5; chemotherapy; gene therapy; glioma; tumorigenesis.

Figure 1. Effect of ING5 on aggressive phenotypes of glioma cells

After transfection of pEGFP-N1-ING5, ING5-overexpressing clones were selected by realtime PCR (A) and western blot (B). ING5 overexpression suppressed proliferation by MTT assay (C), glucolysis and mitochondrial respiration by cellular energy metabolism assay (D), differentiation by ALP activity (E), and induced G₂/M arrest by PI staining (F), apoptosis by Annexin-V staining (G) in U87 cells. ING5 decreased the ability of U87 cells to migrate and invade, evidenced by wound healing (H) and transwell (I) assays. ING5 overexpression also promoted the U87 cell senescence by β-galactosidase staining (J), * p < 0.05.

Figure 2. ING5 attenuated the sensitivity of U87 to chemotherapeutic agents

After exposed to cisplatin (DDP), MG132, paclitaxel, and SAHA, ING5 transfectants showed a higher proliferation (A) and a lower apoptotic level (B) than the control in both concentration- and time-dependent manners.

Figure 3. ING5 altered the expression and transcriptional activity of phenotypes-related proteins in U87 cells

The phenotype-related molecules were screebed by western blot analysis (A). Dual luciferase gene assay demonstrated that ING5 increased TCF-4 promoter activity and TCF-4-mediated gene transcription activity (B).*p < 0.05.

Figure 4. ING5 suppresses the tumor growth of glioma cells in vivo

The growth of U87 and clone cells were revealed by measurement of tumor size (A) and growth curve (B) volume and weight (C). Immunohistochemistry showed weaker Ki-67 staining and stronger TUNEL staining in clone cells (D). *p < 0.05.

Figure 5. ING5 expression in gliomas

According to immunohistochemistry, the positivity to ING5 protein was detectable in the nucleus and cytoplasm of normal brain tissues, astrocytoma, anaplastic astrocytoma and glioblastoma (A). Lee’s, Bredel’s, Sun's and Murat's datasets were employed for bioinformatical analysis to compare ING5 mRNA expression between brain normal tissues and glioblastomas (B).