PTD4‐apoptin protein therapy inhibits tumor growth in vivo

Abstract

Apoptin protein harbors tumor-selective cell death activity, which makes it a potential anticancer therapy candidate. This study reports an apoptin therapy approach based on protein transduction domain 4 (PTD4)-mediated transduction of recombinant apoptin protein. In vitro, the PTD4-apoptin fusion protein is located in the nucleus and induces cell death in, e.g., human hepatocarcinoma HepG2 cells. In normal human L-02 hepatocytes, PTD4-apoptin protein retained mainly cytoplasmic and did not induce detectable levels of cell death, illustrating that the PTD4 domain does not affect apoptin's tumor-selective characteristics. In vivo, liver, cervix and gastric carcinoma xenografts treated with PTD4-apoptin protein for 6 days via the tumor epidermis exhibited a significant tumor growth inhibition because of apoptin-mediated cell death. In addition, treatment of human hepatocarcinoma xenografts during 3 weeks showed that PTD4-apoptin protein has significant anticancer activity, whereas control treatment with PTD4-enhanced green fluorescence protein or saline did not. Cell death and disruption of the tumor integrity were apparent in the PTD4-apoptin transduced xenografted tumors. As important, although PTD4-apoptin protein could be detected in the epidermal tissue covering the subcutaneous tumor tissue and in several organs, such as liver and brain, of the treated mice, no tissue disruption or signs of cell death could be detected. Our in vivo data reveal that apoptin protein delivery constitutes a novel powerful and safe anticancer therapy. ' UICCKey words: cell death; cervix tumor; gastric tumor; hepatocarcinoma; PTD4-mediated apoptin protein delivery Anticancer therapies are severely limited by, e.g., side toxicity to normal tissues. 1,2 The chicken anemia virus-derived apoptin protein harbors unique characteristics with potentials to become a novel anticancer therapy. 3,4 In vivo and in vitro data indicate that apoptin can induce selectively cell death in tumor cells and transformed cells. In fact, the absence of cell death induction in normal cells seems to be correlated with apoptin's normal-cell-specific neutralization. 5,6 Thus far, only vehicles have been developed for the in vivo delivery of the gene encoding apoptin. [7][8][9][10] To avoid the potential limitations related to exogenous gene transduction 11,12 and to expand apoptin's antitumor therapy potential, we have developed an apoptin protein therapy. Recently, Guelen et al. fused apoptin to the HIV-TAT protein transduction domain and delivered in vitro TAT-apoptin protein to human tumor and to normal cells. Strikingly, TAT-apoptin migrated from the cytoplasm to the nucleus of human tumor cells resulting in cell death, whereas in normal cells it remained in the cytoplasm and did not kill these normal cells. 13 In our study, we have fused apoptin to the transduction domain named protein transduction domain 4 (PTD4), which has shown to deliver proteins across the cellular membrane in a very efficient way. 14 The PTD4 domain consists of the amino acids YA...

pH-sensitive degradable nanoparticles for highly efficient intracellular delivery of exogenous protein

“…However it also resulted in the other problems in the therapeutic process such as the poor expression efficiency of heterologous gene. Researchers have also used cell-penetrating peptides, such as TAT 28 and PTD4 29 , to improve the efficiency of cellular uptake and localization. But the exposed heterologous protein may result in the body's immune response and be rapidly eliminated by the cells of the reticuloendothelial system (RES) from our body in blood circulation.…”

Towards novel paradigms for cancer therapy

“…Finally, apoptin induces apoptosis in a wide variety of human cancer cell lines via classical apoptotic pathways (Rohn and Noteborn, 2004;Maddika et al, 2005Maddika et al, , 2006Backendorf et al, 2008). Several independent in vivo analyses using human xenografted tumors have proven that administration of apoptin, as well as its recombinant version protein transduction domain 4 (PTD4)-apoptin, results in a significant reduction of tumor growth without major side effects (Peng et al, 2007;Sun et al, 2009). An important feature shared by these three viral-derived anticancer proteins is the fact that they do induce cell death in a p53-independent manner and that the tumoricidal activity is not affected by the overexpression of Bcl-2 (Table 1).…”

Mitofusin-2 Triggers Cervical Carcinoma Cell Hela Apoptosis via Mitochondrial Pathway in Mouse Model

“…Protein concentrations were determined using the BCA protein assay kit (Pierce, 23227) according to the manufacturer’s instructions. The proteins were fractionated on a 12% and 15% SDS-polyacrylamide gel and electroblotted onto Immobilon-P PVDF transfer membranes (Millipore, IPVH08130), as recently reported [28]. The blots were incubated with Mfn-2, anti-p53, cleaved caspase-3, cleaved caspase-9, cyto c, Bax, Bcl-2, Bid and p-Bad antibodies.…”