Radio-resistance of glioblastoma cells is a major challenge for developing an efficient therapy. Previous studies have established that constitutive activity of the PI3K-Akt pathway and radio-induction of cellular senescence are involved in radio-resistance of U87G cells. Herein, using a rational molecular design, we identified a new potential anti-tumor DPT-peptide, named DPT-E4orf4_23-38, that combines the HIV-1 Tat shuttle plus the canine adenoviral type 2 E4orf4_23-38 PP2A₁-binding sequence. This new chimeric sequence is a helical peptide that inhibits the survival of U87G cell line. In addition we found that DPT-E4orf4_23-38 inhibited Akt-phosphorylation (ser473), and decreased the growth of highly radio-resistant U87G cells. Finally DPT-E4orf4_23-38 also decreased the growth of X-irradiated (2Gy) U87G senescent cells. Together our results suggest that DPT-E4orf4_23-38 alone or combined with irradiation might be clinically evaluated as a potential therapeutic molecule against PI3K-dependent and human radio-resistant and radio-senescent glioblastomas.