Treatment of the T98G Glioblastoma Cell Line with Antisense Oligonucleotides Directed Toward mRNA Encoding Transforming Growth Factor-α and the Epidermal Growth Factor Receptor

Abstract

Antisense oligonucleotides (oligos) complementary to mRNA encoding transforming growth factor-α (TGF-α) and its target, the epidermal growth factor receptor (EGFR), are efficacious against human prostate and breast cancers carried in athymic nude mice. Glioblastomas, also regulated by EGFR expression, would appear to be similarly susceptible, and we now employ them against the T98G tumor model. T98G cells were distributed into wells and allowed to adhere prior to addition of oligos (12.5 µM) directed against TGF-α and/or EGFR for 6 d of treatment before thymidine radiolabeling. Supplemental media and oligos (25 µM final concentration) were added after d 3. Statistically significant inhibition by oligos directed against TGF-α, EGFR, and their combination was 13.8%, 26.3%, and 18.1%, respectively. In a subsequent experiment cells were incubated with increasing amounts of each oligo and their combination for 3 d prior to radiolabeling. Statistically significant inhibition of growth for either oligo at every concentration was found. Cells incubated with 6.25, 12.5, 25, and 50 µM antisense directed against TGF-α had a mean inhibition of 29.3%, 33.3%, 21.7%, and 46.6%, respectively. Cells similarly treated with oligos against EGFR had a mean inhibition of 77.9%, 80.3%, 82.0%, and 83.7%, respectively, and cells incubated with 6.25, 12.5, 25 and 50 µM of each oligo had a mean inhibition of 74.7%, 70.6%, 70.8%, and 76.3%, respectively. Lastly, in a paired experiment, cells treated with 0, 0.39, 0.78, 1.56, 3.125, and 6.25 µM of oligos, either specifically directed against EGFR or a random control, for 3 d were evaluated for both thymidine incorporation and EGFR expression. Statistically significant inhibition of ³H-thymidine incorporation was seen in cells with the oligo specifically directed against EGFR at 3.125 µM and 6.25 µM when compared to non-oligo containing controls. This was accompanied by a comparable significantly decreased expression of a low-MW reactive derivative of EGFR at 3.125 µM and 6.25 µM in Western blots, and of a high-MW reactive EGFR at 6.25 µM. The significant effect against high-MW EGFR was observed vs both the non-oligo containing control and the random sequence. Oligo concentrations between 0.78 and 1.5 µM also resulted in decreased expression of the low-MW form, but not significant differences in thymidine radiolabeling. In recovery experiments, cells treated initially with greater oligo concentrations required significantly increased time to recover, particularly in cells treated with EGFR directed oligos. Intracellular uptake and nuclear localization was demonstrated with FITC tagged oligos. In summary, even at relatively low oligo concentrations and short exposure, oligos against TGF-α, and particularly EGFR, significantly inhibit in vitro growth of the T98G glioblastoma, possibly mediated by decreased EGFR expression.