Cell-penetrating d-Isomer Peptides of p53 C-terminus: Long-term Inhibitory Effect on the Growth of Bladder Cancer

Objectives: To investigate whether a single application of the membrane-permeable d-isomer of the p53 C-terminus connected with a retro-inverso version of the NH₂-terminal 20-amino acid peptide of the influenza virus hemagglutinin-2 protein (riHA2) inhibited the growth of bladder cancer cells. The transduction of p53 using poly-arginine is useful for targeting and suppressing the growth of bladder cancer cells. However, the protein's intracellular half-life is short, and repeated application is necessary to achieve an anti-tumor effect. Methods: The p53 carboxyl-terminal peptides covalently coupled with cell-penetrating peptides were synthesized with d- or l-amino acids. Moreover, the peptides were connected with riHA2 by a disulfide bridge. Human bladder cancer cell lines were incubated with each peptide and cell viability was assessed with the WST assay. Apoptotic cells were confirmed by Hoechst and active capase-3 staining. The p53 peptides were injected into severe combined immunodeficiency disease mice transplanted with J82 cells to investigate their anti-tumor effect on bladder tumors. A survival curve was plotted using the Kaplan-Meier method. Results: A single application of cell-penetrating d-isomer peptides of the p53 C-terminus connected with riHA2 (d11R-p53C′-riHA2 and dFHV-p53C′-riHA2) inhibited the growth and induced the apoptosis of bladder cancer cells. The tumor-bearing mice treated only with vehicle had a mean survival time of 12 days, whereas treatment with d11R-p53C′-riHA2 resulted in a long-term survival rate of 50%. Conclusions: Peptide transduction therapy using the d-isomer p53 C-terminal peptide with riHA2 may be an innovative method for the treatment of bladder cancer.