Abstract
A novel suicide gene therapy approach was tested in U87 MG glioblastoma multiforme cells. A 26nt G-rich double-stranded DNA aptamer (AS1411) was integrated into a vector at the 5′ of a mammalian codon-optimized saporin gene, under CMV promoter. With this plasmid termed “APTSAP”, the gene encoding ribosome-inactivating protein saporin is driven intracellularly by the glioma-specific aptamer that binds to cell surface-exposed nucleolin and efficiently kills target cells, more effectively as a polyethyleneimine (PEI)-polyplex. Cells that do not expose nucleolin at the cell surface such as 3T3 cells, used as a control, remain unaffected. Suicide gene-induced cell killing was not observed when the inactive saporin mutant SAPKQ DNA was used in the (PEI)-polyplex, indicating that saporin catalytic activity mediates the cytotoxic effect. Rather than apoptosis, cell death has features resembling autophagic or methuosis-like mechanisms. These main findings support the proof-of-concept of using PEI-polyplexed APTSAP for local delivery in rat glioblastoma models.
Highlights
Gliomas and Glioblastoma multiforme (GBM) are incurable diseases with a low life expectancy for affected patients, whose treatment is currently limited to surgery and chemotherapy (Stupp and Weber, 2005)
We describe the use of the aptamer AS1411 (APT) DNA sequence embedded in a plasmid to deliver the gene of the plant toxin saporin into glioblastoma cancer cells
We further show that APTSAP enters target cells presumably by forming a G-quadruplex structure, as revealed by microscopy in the presence of protoporphyrin IX, confirming that this structure can act as a delivery guide for saporin-DNA to reach the surface of U87 cells where it can be recognized by nucleolin
Summary
Gliomas and Glioblastoma multiforme (GBM) are incurable diseases with a low life expectancy for affected patients, whose treatment is currently limited to surgery and chemotherapy (Stupp and Weber, 2005). These aggressive tumors generally undergo recurrences within 12–18 months. The first line treatment for GBM patients is temozolomide. This DNA damaging agent is useful in a subclass of glioblastoma cells, those possessing a methylated promoter for the gene of O6-Methylguanine-DNA Methyltransferase (MGMT) that is involved in damage repair induced by alkylating agents. The intrinsic heterogeneity of glioblastoma cells defines an urgent need to find new targets available for an efficient multiple drug-targeting approach
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