A Study On The Fusion Protein VP3/TAT-apoptin Induced Apoptosis Of Tumor And It's Anti-tumor Effect In Node Mice Bearing Human Hepatocarcinoma

VP3, a small protein of 121 amino acids, is encoded by chicken anemia virus(CAV). As it can selectively induce the apoptosis of tumor cells, it is named as Apoptin by Noteborn et al. From the researches on various tumor cells and normal cells, it was found that VP3/ Apoptin can only induce apoptosis of cells with tumorigenesis phenotype or transformed ones but does not kill normal cells, so Apoptin is a protein which can selectively induce the apoptosis of tumor cells.By the sequencing of VP3/Apoptin, it was found that VP3/Apoptin contains two nuclear localization signal (NLS) domains, one is in positions 82 to 88, the other is in positions 111 to 121, which may be closely related with it's specific apoptosis effect on tumor cells. VP3/Apoptin has high affinity with DNA, testified by biophysics. Recombinant MBP-Apoptin can bind with exposed DNA fragment in vitro in high affinity, conforming a polymer by 20 MBP-Apoptin, the diameter of which is 200 nm. The result above showed that VP3/Apoptin may move into nucleus mediated by NLS, and bind to DNA to interfere the transcription, thus to induce the apoptosis of tumor cells.For the study on the relation between VP3/Apoptin's location, Subcellular distribution and VP3/Apoptin's apoptosis effect, enhanced green fluorescent protein(EGFP)was used as tracer which was connected with Apoptin at Apoptin's N end by the genetic engineering, thus the eukaryotic expression vector of EGFP-Apoptin was constructed. The EGFP and VP3/Apoptin was expressed as fusion protein. EGFP was connected with Apoptin at Apoptin's N end in order to decrease the effect of EGFP on VP3/Apoptin's location and apoptosis effect.The plasmid pEGFP-Apoptin was transfected into human hepatocarcinoma HepG2 and 24h later, fusion protein EGFP-Apoptin got a high expression and move from cytoplasm into nucleus gradually, and located in it. 48 later, fusion protein EGFP-Apoptin accumulated in nucleus making nucleus showing great green fluorescence. The outline of nucleus is clear but cytoplasm has no fluorescence. The distribution of green fluorescence in nucleus is not average but regional accumulation which are like dots or macular ones. The cytoplasm and nucleus of some transfected cells were contracted showing the occurrence of apoptosis. In the control group transfected with plasmid pEGFP, the distribution of EGFP is average, both cytoplasm and nucleus showing great green fluorescence with no obvious discrepancy. 4-5D after HepG2 was transfected with pEGFP-Apoptin, the apoptosis of some tumors were observed by TUNEL assay, while in the control group transfected with plasmid pEGFP, the apoptosis were not observed, which indicated that fusion protein EGFP-Apoptin with EGFP as tracer in it's N end can induce the apoptosis of tumor cells.Although the mechanism of VP3/Apoptin induced apoptosis of tumor is not very clear, lots of researches have indicated that the transfection of vp3 gene or the introduction of VP3/Apoptin protein into tumor cells can induce the apoptosis of the tumor. It is worth researching on how to use VP3/Apoptin to induce the apoptosis of the tumor selectively and safely in vivo for the clinical application of it.Protein transduction technology becomes a newly emerging macromolecule transduction strategy in recent years: Direct delivery therapeutic active biomacromolecule into cells to show biological effect making use of the proteins and peptides with protein transduction domain. These polypeptides, with the ability to deliver materials across the cell membrane are named as cell-penetrating peptides, CPPs, such as TAT(11 peptides, 13 peptides), Antp(16 peptides), VP22(34 peptides), Transportan (28 peptides) and MAP9(18 peptides). Many kinds of substances can be delivered by CPP, there are proteins, DNA, antibodies, imaging agent, toxin, nanometer chemicals granules and liposome, etc. TAT, of which transduction sequence origins from HIV-1, can mediate the proteins bond with it, crossing cell membranes and playing biological role of corresponding proteins. Therefore it could be a kind of effective method to treat cancers using VP3, by constructing TAT-VP3 fusion protein, delivering VP3 using of the protein transduction domain of TAT and carrying out its direct therapeutic effect across cell membranes.Smearing on the skin was adopted to observe the anti-tumor effect in the bearing subcutaneous hepatocarcinoma nude mice model, which afford a experimental data for the clinical application, we prepared fusion protein TAT-VP3, which can make VP3 protein move into cells by TAT to suppress the proliferation of the tumors, as VP3 can induce the apoptosis of tumor cells. In the bearing tumor animal model, we found that protein TAT-VP3 induced the apoptosis of tumor cells, and suppress the proliferation of the tumors. Considering the safety of method for medication and control of dose, smearing on the skin was adopted to avoid the immune reaction by injection of vein. The preparation of bearing subcutaneous tumor nude mice model is accorded with reference 7. When tumors grew for 7 days, they were divided as sample group and control group randomly. P is 0.05445 (P>0.05) showed no discrepancy. The volume of tumors in sample group and control group were evaluated on third day and 7^th day to observe the anti-tumor effect. The results were analyzed by statistics. As P is 0.011272 (P<0.05) in sample group and P is 0.002143 (P<0.01) in control group, they are striking difference, which indicated fusion protein TAT-VP3 can suppress the growing of HepG₂ under the skin of nude mice. Because Apoptin can be hydrolyzed by proteases, meanwhile the therapy was applied when the diameter of the tumor reach about 8mm for only a week, in the future research, the therapy should be applied in the early time, frequency of the therapy per day should be increased and the time for therapy should be extended in order to enhance the anti-tumor effect. The research of therapy in vivo by Apoptin/VP3 carried by adenovirus indicates that Apoptin/VP3 can reach all the tissues of the animals, with no apoptosis effect on the normal tissues in our researches smearing drugs on the skin was adopted for the considering of high safety.Our investigation shows that EGFP-Apoptin fusion protein has the effect of nuclear location and inducing the apoptosis of the tumor cells, which further testified the assumption of Noteborn. Therefore, pEGFP-Apoptin can be used as a tool for the research on the VP3/Apoptin's distribution and subcellular location in tumor cells, for the further study on the mechanism of VP3/Apoptin' induced apoptosis on tumor cells.In the other hand, we study the anti-tumor effect of penetrating VP3/Apoptin. Smearing on the skin was adopted to observe the anti-tumor effect in the bearing subcutaneous hepatocarcinoma nude mice model. The growth of tumors in the therapy group was suppressed obviously, the inhibition rate of tumor weight is 56.9%, which testified TAT-VP3 has the ability of skin-penetration and of inhibition of tumor growth.With the advance of cell biology and molecular biology, protein or peptide will widely used in the clinic therapy of many diseases. By change the conformation of biomacromolecule to develop it's penetrating role will decrease it's activity. By injection or gene transfection, the process is too complicated and cost is too high for widely application. By TAT confusion protein to develop the penetrating of aim protein, the method is simply and the activity of biomacromolecule will not change, which provided a model for penetrating of biomacromolecule such as peptide or protein and bring a bright future for the clinic therapy of tumor by biomacromolecule.