Secretory TAT-VP3 Fusion Protein Induces Apoptosis In Hepatocellular Carcinoma Cells

Viral protein 3 (VP3), a protein of 13.6 kDa derived from the Chicken Anaemia Virus (CAV), represents a new anti-cancer tool with great potentials . It appears to have innate tumor-specific, p53-independent, Bcl-2-enhanced proapoptotic activity and hence is of considerable interest to achieve efficient targeting and specific elimination of cancer cells. The antitumor activity of VP3 appears to be linked to its ability to localize in the nuclei of transformed cells, but not in those of primary or non-transformed cells. Therefore, VP3 has been explored to achieve efficient targeting and specific elimination of cancer cells.To use VP3 in cancer therapy, efficient delivery of VP3 to cancer cells or expression of VP3 in these cells is required. The HIV Tat-derived protein transduction peptide is a small basic peptide that has been successfully shown to deliver a large variety of materials, from small particles to proteins, peptides and nucleic acids across the cell membrane. The region conveying the cell penetrating properties appears to be confined to a small (11 amino acids) stretch of basic amino acids (aa 47-57; YGRKKRRQRRR). This Tat transduction domain has been successfully used to deliver VP3 into cancer cells.In this study, we designed a secretory Tat-VP3 fusion protein by adding a secretory signal to the N-terminal of the recombinant molecule to gain an additive by-stander effect as an anti-cancer therapy. Secreted Tat-VP3 from transformed cells would enter un-transformed cancer cells and cause apoptosis in them. We employed an artificial human secretory signal peptide whose amino acid sequence and corresponding cDNA sequences was generated by SP-HMM model (signal peptide hidden Markov model). The results showing both the expression of the secretory fusion protein (SP-Tat-VP3) and induction of apoptosis by the secreted Tat-VP3 in HepG2 cells are presented. The major results:1. In this study, we generated a cDNA construct of SP-Tat-Apoptin fusion.2. SP-Tat-VP3 induces apoptosis only in malignant cells.3. SP-Tat alone does not have cytotoxicity for HepG2 cells.4. The secretory Tat-VP3 has an additive by-stander effect as an anti-cancer therapy in vitro.5. In a preliminary experiment, a single describe the generation and characterization of an aden- intratumoral injection of Lipofectamine^? 2000- SP-Tat-VP3/ plasmid into a xenogeneic tumor (HepG2 cells in BALB/C nude mice) resulted in a significant reduction of tumor growth.Taken together, our data demonstrate that Secretory Tat-VP3 may be a powerful tool for the treatment of solid tumors .