Introduction

Glioblastoma multiforme (GBM), a brain cancer, is one of the deadliest human diseases, and cannot be cured by any therapy available today. The localization of GBM in the central nervous system and the very solid structure of this tumor renders it almost impermeable to large particles, such as viral vectors [1]. A major challenge in the treatment of GBM is to kill the accessible cancer cells on the surface of the tumor more rapidly than the rate of replication of the cells. Otherwise, the unexposed, internal cells can replicate and compensate for the cells that have just been eliminated. Thus, an effective treatment for GBM must incorporate the following features: (a) high selectivity and safety, to avoid damage to non-cancerous brain tissue; (b) rapid and efficient cell killing, preferably by simultaneous activation of multiple killing mechanisms. The simultaneous activation of multiple killing pathways will ensure tumor cell death, even if one or several pathways are inactive; and, (c) inhibition of the growth or killing of neighboring, unexposed tumor cells. This ‘‘bystander effect’’ should assist in eliminating the tumor before it can re-grow. It should also inhibit the growth of any tumor cells that may have a different phenotype from the targeted cells and are not themselves targeted by the treatment, including cancer stem cells.

In an attempt to meet all these demands in one treatment, we have taken advantage of the frequent (50%–70%) overexpression of epidermal growth factor receptor (EGFR) in GBM [2]. We have attached synthetic, double-stranded RNA (dsRNA) to a non-viral vector that can home in on EGFR. The dsRNA is selectively introduced into the cancer cells via receptor-mediated endocytosis. Double-stranded RNA, frequently expressed in cells infected with viruses, activates a number of pro-apoptotic processes simultaneously. These include the dsRNA dependent protein kinase (PKR) and 2,5- oligo-A synthetase, both of which turn off protein synthesis [3]. Double-stranded RNA also activates p38 and JNK, and stimulates the synthesis of pro-apoptotic proteins, such as IRF3-DRAF1 and NFjB [3–5]. These dsRNA-induced mechanisms efficiently kill infected cells and induce expression of anti-proliferative cytokines from the interferon family, thereby preventing spread of the virus [4].

In order to specifically introduce poly IC into EGFR overexpressing cells, we utilized polyethylenimine (25 kDa)-polyethylene- glycol-mEGF (PEI25-PEG-EGF) complexes [6,7]. We expected this approach to be highly selective, because the number of EGFRs on tumor cells is 10–100 times higher than that on non-tumor cells [2]. PEI25-PEG-EGF conjugates are significantly safer than replication-deficient or replicationcompetent viruses, in terms of immunotoxic reactions, inadvertent recombination and viral replication in healthy cells. Cell death was expected to be fast, because dsRNA activates cell killing mechanisms within minutes of entering the cell. Finally, induction of interferons, clinically used against GBM, was expected to exert a bystander effect and inhibit the growth of adjacent, untransfected tumor cells.

Methods

Reagents and Assays

Poly IC was obtained from Sigma (Rehovot, Israel). It was dissolved in DEPC-treated double-distilled H2O. The polyethylenimine (PEI), PEI25, branched and succinimidyl 3-(2- pyridyldithio) propionate (SPDP) were purchased from Sigma-Aldrich (Munich, Germany). NHS-PEG-MAL (MW ¼ 3400) was obtained from Nektar Therapeutics (Huntsville, Alabama, United States) and the recombinant mouse EGF (mEGF) from Pepro Tech EC Ltd. (London, United Kingdom). The PEI content of the conjugate was determined spectrophotometrically by TNBS assay at 405 nm. The amount of dithiopyridine linkers in PEI was determined after reduction of an aliquot with dithiothreitol (DTT) followed by absorption measurement of released pyridine-2-thione at 343 nm.

The molar ratio of mEGF: dithiopyridine was determined spectrophotometrically at 280 and 340 nm. The amount of dithiopyridine was determined as described [6,7]. The yield of mEGF (mg) was calculated in two equations. Equation 1: A280 (a) ¼ A340 with DTT 3 5.1/8.1. Equation 2: A280 revised ¼ A280A280 (a). The result of equation 2 was the amount of mEGF in mg.

The Ellman assay was used for the determination of the mercapto groups in mEGF-SH. Liquid chromatography of conjugates was performed with the A¨ KTA basic system from Amersham Biosciences (Little Chalfont, United Kingdom).

Melittin (Mel) (D-Mel-SH; e280 ¼ 5570, MW ¼ 2893.6) was purchased from IRIS Biotech GmbH (Marktredwitz, Germany).

All other chemicals were purchased from Sigma-Aldrich.

Fluorescence Microscopy

Poly IC was labeled with the Fluorescein ULS labeling kit (Fermentas, Hanover, Maryland, United States) at 1 unit of fluorescein per 1 lg of poly IC according to the manufacturer’s instructions and then condensed with the appropriate PEI conjugate. Cells were incubated with the complexes in DMEM/FCS for 4 h at 37 8C and washed twice with PBS. Cells were viewed on a Zeiss confocal microscope. Green fluorescence was viewed with filter sets appropriate for fluorescein.

In Vitro Apoptosis

Detection Cells were seeded into 24-well plates at a density of 10,000 in 1 ml of medium per well and grown overnight. After appropriate treatment cells were washed, fixed and stained using the Annexin-V-Biotin kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. In addition apoptotic death was also determined by TUNEL assay using the In situ Cell Death Detection kit (Roche). The brown colored apoptotic cells were visualized in a microscope, counted (6 fields per sample), and photographed using digital camera.

In Vitro Bystander Effect

For this assay, 500,000 U87MGwtEGFR cells were seeded onto 6-cm plates, grown overnight in 2 ml of medium, and transfected with 1 lg/ml poly IC using PEI-PEG-EGFþPEI-Mel complexes. Medium was collected at 24 h after transfection. U87MG and U87MGDEGFR ‘‘indicator’’ cells were seeded in duplicate in 96-well plates (4,000 cells/well) and grown overnight in 200 ll of medium. Then, 100 ll of medium was then replaced by the medium collected from the transfected (þpoly IC) or untransfected (poly IC) U87MGwtEGFR cells. Where indicated the medium was pre-incubated for 1 h at room temperature, with neutralizing polyclonal anti-IFN-a antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, California, United States). In NT samples, medium was not replaced. Growth inhibition was examined 48 h after medium exchange.