Application Of Apoptin In Anti-tumor Therapy And Tumorigenesis

Apoptin, which is also named as VP3, is a small viral protein encoded by chicken anemia virus (CAV). The early study on Apoptin has shown that Apoptin induces apoptosis in a great variety of tumors (>70), as well as transformed cells. However, Apoptin does not induce significant apoptosis in human normal cells, including primary human hematopoietic stem CD34+ cells, primary human mesenchymal stem cells, or human primary hepatocytes. Human normal cells expressing Apoptin proliferate and divide regularly without any potential of transformation or toxicity. The normal development and growth of transgenic mice also prove that Apoptin is non-toxic to normal cells in vivo. Thus, Apoptin is a promising anti-tumor agent, which induces apoptosis in tumor or transformed cells but not in normal cells. Moreover, Apoptin can also be activated by the transient transforming signals conferred by SV40 LT (large tumor antigen). Under the transient transformation situation established by SV40 LT, one can investigate the tumor-related pathway involved in tumorigenesis by using Apoptin as a sensor. This study mainly focuses on the application of Apoptin in anti-tumor therapy and tumorigenesis in which Apoptin is involved.Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and among the most fatal of human neoplasms. To improve the potential of a therapy against hepatocarcinoma based on Apoptin gene expression, one needs to develop systemic delivery vehicles, which can selectively deliver the Apoptin gene to liver (tumor) cells. The asialoglycoprotein receptor (ASGPR) is specially and abundantly expressed on the surface of normal hepatocytes as well as hepatocarcinoma cells. DNA plasmids, which have been coupled to the ASGPR ligand asialoglycoprotein (Asor) via poly-L-lysine linkers (PLL), can be selectively taken up by hepatocytes and hepatocarcinoma cells both in vitro. In this study, we have constructed a novel hepatic systemic delivery vehicle, Asor–Apoptin, which consists of Asor, PLL, and the plasmid pcDNA-vp3 encoding Apoptin. Particularly, in vivo studies demonstrate that the Asor–Apoptin complexes deliver the Apoptin gene specifically to hepatocytes and to in situ hepatocarcinoma, resulting in tumor regression without side effects on normal hepatocytes, which thus strongly suggests the effectiveness and perspectives of systemic delivery of Asor–Apoptin for therapeutic treatment of HCC-derived tumors. Our results have significant contribution to the further clinic application of Apoptin.On the other hand, Apoptin can also be applied in the study on tumor-related pathway as a tumor specific death inducer. Recently it's shown that Apoptin is phosphorylated at Thr108 specifically in human tumor and transformed cells, but not in human normal cells both in vivo and in vitro. Phosphorylated Apoptin translocates to nucleus rapidly and induces apoptosis subsequently specifically in tumor or transformed cells, whereas it remains unphosphorylated state and localizes mainly in cytoplasm without apoptosis inducing. Therefore, it has been thought that in tumor or transformed cells there exists a common tumor related phosphatase which phosphorylates Apoptin.Moreover, Apoptin can also be activated by the transient transformation signals conferred by SV40 large T antigen (LT), which rapidly induces Apoptin's three tumor-specific properties, phosphorylation, nuclear translocation and apoptosis induction. Thus, under the transient transformation situation established by SV40 LT, one can investigate the tumor-related pathway by using Apoptin as a sensor. Previous study demonstrated that the truncated N-terminal determinant of LT (LT136/st, encoding N-terminal of LT as well as small t antigen) was sufficient to confer Apoptin three of its tumor-specific properties, phosphorylation, nuclear translocation and apoptosis induction. In this study, we reported the role of related domains of N terminal determinant of SV40 T antigen in the activation of Apoptin. First, the PP2A binding domain of SV40 small tumor antigen (st) plays a vital role in the activation of tumor-related pathway sensed by Apoptin. However, st lacking its own nuclear localization signal has to be translocated to nucleus for Apoptin's activation.In addition, J domain shared by both LT136 and st is structurally needed. The first 1-82 amino acids (Hsc70 binding domain, that is, J domain) of LT136 and st are identical each other. It has been reported that Hsc70 (Heat shock protein cognate 70) can self-associate into dimmers or oligomers via their COOH-terminal peptide binding domain. Therefore, it's possible that the two Hsc70 molecule which binds to J domain of LT136 and st respectively can self-associate into the compounds which contains two Hsc70 molecule and its binding proteins, LT136 and st. By the nuclear localization signal (NLS) of LT136 or the NLS of pRb which binds to LT136, these compounds containing st were targeted to nucleus, where the activity of PP2A was inhibited by its association with st and in turn altered or activated tumor-related pathway. Subsequently, the tumor-related kinase was activated rapidly, which phosphorylated and activated Apoptin. Taken together, our data first reveal that st is targeted to nucleus and inactivates PP2A by its association with st, which consequently activates tumor-related pathway sense by Apoptin. This tumor-related pathway is thought to be activated in the early stage of tumorigenesis and exists during the whole process of tumor formation. Our data also indicate that PP2A plays a crucial role in the early stage of cellular transformation and tumorigenesis.Taken together, this study investigated the application of Apoptin in cancer gene therapy and tumorigenesis. We established a systemic Apoptin gene delivery vehicle, Asor-Apoptin, which was specifically targeted to hepatocytes as well as hepatocarcinoma cells and subsequently induced apoptosis of tumor cells without any toxicity to normal cells or normal tissues both in vivo and in vitro. These results established determinate foundation for further application in clinic. We also studied on tumor-related pathway by using Apoptin as a sensor and found the key molecule involved in the early stage of celluar transformation or tumorigenesis, which thus contributes to further research on tumorigenesis. The results from this study have been first reported by us, which provided valuable theory reference and experimental data for further study on Apoptin.