Inhibitory effect of antisense epidermal growth factor receptor RNA on theproliferation of rat C6 glioma cells in vitro and in vivo

Object. The goal of this study was to evaluate the effect of antisense epid ermal growth factor receptor (EGFR) RNA on the growth of rat glioma cells i n vitro and in vivo and to determine the feasibility of targeting the EGFR gene for gene therapy in gliomas. Methods. Antisense EGFR complementary (c)DNA was transfected into C6 glioma cells by using lipofectamine. In vitro studies, Southern and Northern blot analyses, in situ hybridization, and immunohistochemical staining were des igned to examine the integration and expression of antisense EGER construct s. The 3'(4,5-dimethylthiazol-2-yl)1,5-diphenyl tetrazolium bromide (MTT) a ssay and the average number of argyrophilic nuclear organizer regions (Ag-N ORs) were used to evaluate cell proliferation, whereas the terminal deoxynu cleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method and microscopy were used to observe cell apoptosis. As part of the in vivo studies, parental C6 cells and C6 cells transfected with EGF R antisense cDNA were implanted stereotactically into the right caudate nuc leus of Wistar rats (C6-injected animals and transfected C6-injected animal s). Rats with well-established cerebral C6 glioma foci were treated intratu morally with either antisense EGFR cDNA or empty-vector DNA by using lipofe ctamine (treated-C6 and control treated group). The general behavior and su rvival of the rats, findings on magnetic resonance images of their brains, histopathological changes, proliferation activity, and apoptosis of the cer ebral gliomas in each group of rats were examined. Exogenous antisense EGFR cDNA was integrated into the genome of C6 cells an d expressed. Ln clones with a high expression of the antisense construct, t here was a dramatic decrease in endogenous EGFR messenger RNA and protein l evers, reduced proliferation activity, and induction of apoptosis in vitro. The mean survival time of rats injected with C6 cells was 17.3 days. The m ean survival time of rats injected with C6 cells followed by treatment with empty vector in lipofectamine was 15.4 days. Survival time was significant ly prolonged in 100% of the rats injected with antisense-transfected C6 cel ls and in two thirds of the rats injected with C6 cells followed by antisen se EGFR cDNA. Magnetic resonance imaging revealed distinct cerebral tumor f oci in C6-injected rats and in control rats of the treated group, but none were found in the mts injected with transfected C6 cells. Furthermore, tumo r foci disappeared completely in C6-injected rats treated with antisense EG FR cDNA. The cerebral gliomas of the rats treated by injection of antisense EGFR RNA were characterized by reduced proliferation activity and the indu ction of apoptosis. Conclusions. The results of this study indicate that EGFR plays an importan t role in the genesis of malignant gliomas. It may, therefore, be an effect ive target of antisense gene therapy in patients with gliomas.