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Silencing platelet-derived growth factor receptor-β enhances the radiosensitivity of C6 glioma cells in vitro and in vivo.

ABSTRACT: Platelet-derived growth factor receptor (PDGFR)-β is an important tyrosine kinase and its downregulation has been reported to alter the radiosensitivity of glioma cells, although the underlying mechanism is unclear. In order to investigate the effect of PDGFR-β on the radiosensitivity of glioblastoma, the present study transfected C6 glioma cells with a PDGFR-β-specific small interfering (si)RNA expression plasmid, and downregulation of the expression of PDGFR-β in C6 glioma cells was confirmed by western blotting and immunohistochemical analysis. Clone formation assays and xenograft growth curves demonstrated that PDGFR-β-siRNA enhanced the radiosensitivity of C6 glioma cells in vitro and in vivo. Furthermore, MTT and xenograft growth curves demonstrated that PDGFR-β-siRNA inhibited the proliferation of C6 glioma cells in vitro and in vivo, and terminal deoxynucleotidyl transferase dUTP nick end-labeling and immunohistochemical analyses demonstrated that PDGFR-β-siRNA induced apoptosis and inhibited the expression of Ki-67, cyclin B1 and vascular endothelial growth factor in C6 glioma cell xenografts. Taken together, these results suggested that PDGFR-β may be used as a target for the radiosensitization of glioblastoma.

SUBMITTER: Hong JD

PROVIDER: S-EPMC5494875 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Project description:Paediatric high-grade gliomas (HGGs) account for the most brain tumour-related deaths in children and have a median survival of 12-15 months. One promising avenue of research is the development of novel therapies targeting the properties of non-neoplastic cell-types within the tumour such as tumour associated macrophages (TAMs). TAMs are immunosuppressive and promote tumour malignancy in adult HGG; however, in paediatric medulloblastoma, TAMs exhibit anti-tumour properties. Much is known about TAMs in adult HGG, yet little is known about them in the paediatric setting. This raises the question of whether paediatric HGGs possess a distinct constituency of TAMs because of their unique genetic landscapes. Using human paediatric HGG tissue samples and murine models of paediatric HGG, we demonstrate diffuse midline gliomas possess a greater inflammatory gene expression profile compared to hemispheric paediatric HGGs. We also show despite possessing sparse T-cell infiltration, human paediatric HGGs possess high infiltration of IBA1+ TAMs. CD31, PDGFRβ, and PDGFB all strongly correlate with IBA1+ TAM infiltration. To investigate the TAM population, we used the RCAS/tv-a system to recapitulate paediatric HGG in newborn immunocompetent mice. Tumours are induced in Nestin-positive brain cells by PDGFA or PDGFB overexpression with Cdkn2a or Tp53 co-mutations. Tumours driven by PDGFB have a significantly lower median survival compared to PDGFA-driven tumours and have increased TAM infiltration. NanoString and quantitative PCR analysis indicates PDGFB-driven tumours have a highly inflammatory microenvironment characterized by high chemokine expression. In vitro bone marrow-derived monocyte and microglial cultures demonstrate bone marrow-derived monocytes are most responsible for the production of inflammatory signals in the tumour microenvironment in response to PDGFB stimulation. Lastly, using knockout mice deficient for individual chemokines, we demonstrate the feasibility of reducing TAM infiltration and prolonging survival in both PDGFA and PDGFB-driven tumours. We identify CCL3 as a potential key chemokine in these processes in both humans and mice. Together, these studies provide evidence for the potent inflammatory effects PDGFB has in paediatric HGGs.

Project description:BackgroundUltrasound-triggered microbubble destruction (UTMD) is a widely used noninvasive technology in both military and civilian medicine, which could enhance radiosensitivity of various tumors. However, little information is available regarding the effects of UTMD on radiotherapy for glioblastoma or the underlying mechanism. This study aimed to delineate the effect of UTMD on the radiosensitivity of glioblastoma and the potential involvement of autophagy.MethodsGL261, U251 cells and orthotopic glioblastoma-bearing mice were treated with ionizing radiation (IR) or IR plus UTMD. Autophagy was observed by confocal microscopy and transmission electron microscopy. Western blotting and immunofluorescence analysis were used to detect progesterone receptor membrane component 1 (PGRMC1), light chain 3 beta 2 (LC3B2) and sequestosome 1 (SQSTM1/p62) levels. Lentiviral vectors or siRNAs transfection, and fluorescent probes staining were used to explore the underlying mechanism.ResultsUTMD enhanced the radiosensitivity of glioblastoma in vitro and in vivo (P < 0.01). UTMD inhibited autophagic flux by disrupting autophagosome-lysosome fusion without impairing lysosomal function or autophagosome synthesis in IR-treated glioblastoma cells. Suppression of autophagy by 3-methyladenine, bafilomycin A1 or ATG5 siRNA had no significant effect on UTMD-induced radiosensitization in glioblastoma cells (P < 0.05). Similar results were found when autophagy was induced by rapamycin or ATG5 overexpression (P > 0.05). Furthermore, UTMD inhibited PGRMC1 expression and binding with LC3B2 in IR-exposed glioblastoma cells (P < 0.01). PGRMC1 inhibitor AG-205 or PGRMC1 siRNA pretreatment enhanced UTMD-induced LC3B2 and p62 accumulation in IR-exposed glioblastoma cells, thereby promoting UTMD-mediated radiosensitization (P < 0.05). Moreover, PGRMC1 overexpression abolished UTMD-caused blockade of autophagic degradation, subsequently inhibiting UTMD-induced radiosensitization of glioblastoma cells. Finally, compared with IR plus UTMD group, PGRMC1 overexpression significantly increased tumor size [(3.8 ± 1.1) mm2 vs. (8.0 ± 1.9) mm2, P < 0.05] and decreased survival time [(67.2 ± 2.6) d vs. (40.0 ± 1.2) d, P = 0.0026] in glioblastoma-bearing mice.ConclusionUTMD enhanced the radiosensitivity of glioblastoma partially by disrupting PGRMC1-mediated autophagy.

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Silencing platelet-derived growth factor receptor-β enhances the radiosensitivity of C6 glioma cells in vitro and in vivo.

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