Abstract
Glioblastoma is a malignant brain tumor with poor prognosis that rapidly acquires resistance to available clinical treatments. The herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system produces the selective elimination of HSVtk-positive cells and is a candidate for preclinical testing against glioblastoma via its ability to regulate proliferation and differentiation. Therefore, in this study, we aimed to establish a plasmid encoding the HSVtk/GCV system driven by a glial fibrillary acidic protein (GFAP) promoter and verify its possibility of neural differentiation of glioblastoma cell line under the GCV challenge. Four stable clones—N2A-pCMV-HSVtk, N2A-pGFAP-HSVtk, U251-pCMV-HSVtk, and U251-pGFAP-HSVtk—were established from neuronal N2A and glioblastoma U251 cell lines. In vitro GCV sensitivity was assessed by MTT assay for monitoring time- and dosage-dependent cytotoxicity. The capability for neural differentiation in stable glioblastoma clones during GCV treatment was assessed by performing immunocytochemistry for nestin, GFAP, and βIII-tubulin. Under GFAP promoter control, the U251 stable clone exhibited GCV sensitivity, while the neuronal N2A clones were nonreactive. During GCV treatment, cells underwent apoptosis on day 3 and dying cells were identified after day 5. Nestin was increasingly expressed in surviving cells, indicating that the population of neural stem-like cells was enriched. Lower levels of GFAP expression were detected in surviving cells. Furthermore, βIII-tubulin-positive neuron-like cells were identified after GCV treatment. This study established pGFAP-HSVtk-P2A-EGFP plasmids that successfully ablated GFAP-positive glioblastoma cells, but left neuronal N2A cells intact. These data suggest that the neural differentiation of glioblastoma cells can be promoted by treatment with the HSVtk/GCV system.
Highlights
Glioblastoma is a devastating, uniformly lethal primary brain tumor
This study constructed a pGFAP-herpes simplex virus thymidine kinase (HSVtk)-P2A-EGFP plasmid bearing the HSVtk/GCV system controlled by the glial fibrillary acidic protein (GFAP) promoter to establish a strategy for cancer management in vitro
We demonstrated that this system could selectively ablate GFAP-positive glioblastoma cell lines, while it left the neuronal N2A cells intact and promoted neural differentiation after challenge with the HSVtk/GCV system (Fig 6C)
Summary
Glioblastoma is a devastating, uniformly lethal primary brain tumor. The standard treatment for glioblastoma is surgery followed by radiotherapy and chemotherapy, but tumor recurrence is observed in most patients [1]. A contributor to the tumor reoccurrence is a cell population with cancer stem cell properties present in glioblastoma. Previous studies revealed that malignant tumors are initiated by a population of tumor cells sharing similar biological properties with normal adult stem cells [3, 4]. These tumor cells, known as cancer stem cells, have self-renewal phenotypes, which may be the cause of resistance to the current standard care of concomitant chemoradiotherapy [5]. Regulating the differentiation of glioma cells has been suggested as a strategy for controlling glioblastoma [11]. Autophagy was suggested to play an essential role in the regulation of selfrenewal, differentiation, and tumorigenicity of GIC [13]
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