A cell penetrating peptide-integrated and enediyne-energized fusion protein shows potent antitumor activity

Arginine-rich peptides belong to a subclass of cell penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytotic mode of uptake and a subsequent release from vesicles or a direct membrane penetration. Lidamycin is an antitumor antibiotic, which consists of an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). In the present study, a fusion protein (Arg)9–LDP composed of cell penetrating peptide (Arg)9 and LDP was prepared by DNA recombination, and the enediyne-energized fusion protein (Arg)9–LDP–AE was prepared by molecular reconstitution. The data in fixed cells demonstrated that (Arg)9–LDP could rapidly enter cells, and the results based on fluorescence activated cell sorting indicated that the major route for (Arg)9-mediated cellular uptake of protein molecules was endocytosis. (Arg)9–LDP–AE demonstrated more potent cytotoxicity against different carcinoma cell lines than lidamycin in vitro. In the mouse hepatoma 22 model, (Arg)9–LDP–AE (0.3 mg/kg) suppressed the tumor growth by 89.2%, whereas lidamycin (0.05 mg/kg) by 74.6%. Furthermore, in the glioma U87 xenograft model in nude mice, (Arg)9–LDP–AE at 0.2 mg/kg suppressed tumor growth by 88.8%, compared with that of lidamycin by 62.9% at 0.05 mg/kg. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein (Arg)9–LDP–AE was more effective than lidamycin and would be a promising candidate for glioma therapy. In addition, this approach to manufacturing fusion proteins might serve as a technology platform for the development of new cell penetrating peptides-based drugs.