Targeted Antitumor Effect Of A Novel P53 Protein Fused With TAT And ODD Domain

Cancer is the major public problem around the world. Increasing clinical reports suggested that over 50% tumors contain the mutant P53 gene. P53 gene is one of the most important tumor suppression genes and its encoding product, p53, plays a central role to inhibit the tumorigenesis, such as sustaining the genomic stability, repairing the DNA damage, and protecting the malignant transformation via inducing apoptosis of cells which are suffered the irreversible injury. Therefore, p53 is called as"Genome guard". However, p53 is highly susceptible to various mutations leading to abnormality. Mutant p53 not only fails to protect the normal cells undergoing the tumorigenesis, but also triggers the over expression of a set of oncogenes to promote the cellular carcinogenesis and accelerate the tumor progression. Hence, p53 has been a promising therapeutic target of tumor treatment. Although various reagents have been developed for restoration of inactive p53, their utility extensively limited because of their poor cell permeability and low targeting delivery.Moreover, over 90% tumors are solid tumors. Inadequate oxygenation, known as hypoxia, is the predominant pathophysiological feature in the solid tumors. The unequal balance between extensive cell proliferation and angiogenesis is the leading cause to the insufficient supplement of oxygen in tale. Malignant cells under this microenvironment are hypoxic tumor cells which are extremely resistant to traditional chemo- and radiotherapy and result in the metastasis in the advanced stage of cancers. Hypoxia-inducible factors (HIFs) play a pivotal role in these biological processes. HIFs family consists of three members, including HIF-1, HIF-2 and HIF-3. HIF-1 is well-studied. HIF-1 is a heterodimer, containingαandβsubunits. HIF-1βis constitutive, but HIF-1αis the regulatory subunit. Under normoxia, HIF-1αis unstable. Its oxygen-dependent degradation domain (ODD) could induce its degradation via unbiquitin-proteasome pathway mediated by Von-Hipple Lindau protein (pVHL). In the hypoxia, however, oxygen is deprived and HIF-1 prolyhydroxylase (HIF PH) become inactivated, fail to degrade HIF-1αvia pVHL dependent pathway. The stable HIF-1αcould combine withβsubunit, functioning as transcriptional factor to regulate a set of genes expression for the hypoxic adaption of tumor cells. Therefore, how to thoroughly clean the hypoxic tumor cells is the key step for the regime of solid tumors. Moreover, it is suggested that hypoxia could select the subpopulation of cancer cells harboring the mutant p53, which are more easily to prone to have aggressive phenotype to promote the progression, invasion and metastasis of tumors.Protein transduction domains, shorted as PTDs, are series of polypeptides which are capable to deliver bio-macromolecules into cells via cross-membrane transduction, including TAT peptide of HIV-1, VP22 from HSV, antennapedia homeoprotein of drosophila and a set of synthetic polypeptides with cell permeability. The predominant feature of these peptides is rich of basic amino acid in the sequence, resulting in the interaction with some bio-molecules on the cell surface to induce the internalization of the macromolecules with poor cell permeability, such as DNA, siRNA, proteins with big molecular weight and even nano-particles. This put the promising insight on the development of therapeutic protein drugs. TAT is one of the most well-studied and widely used PTDs. Its minimum functional domain is the residue 47~57. Many repots displayed that various of TAT fused proteins were developed for research and/or therapeutic utilities, such as TAT-EGFP, TAT-HT and TAT-p27, which could play their bio-functions delivered into cells and/or tissues via TAT transportation. These results support the fundamental information for further creation of TAT-based biological reagents.In this study, aiming at restoration of dysfunctional p53 and scavenging the hypoxic cancer cells in solid tumor, a novel p53 fusion protein, TAT-ODD-p53, was designed for targeting therapy of solid tumors. This protein was fused with TAT47-57, protein transduction peptide, the minimum functional motif of ODD (ODD557-574) and human wild-type p53 protein. This protein should have higher cell permeability and selectively localizes in the hypoxic regions of solid tumor to induce hypoxic tumor cells apoptosis. First, the encoding genes of p53, TAT-p53 and TAT-ODD-p53 were constructed and cloned into pET28a prokaryotic expression vector, respectively. Second, these p53 fusion proteins were expressed in E.coli, BL21 (DE3) engineering bacteria. The highly purified proteins were prepared via the procedure of extraction, denaturation, purification and renaturation. The transmembrane delivery and targeting stability of TAT-ODD-p53 were assessed by fluorescent immunocytochemistry and Western blotting in vitro. MTT assay, Annexin V & PI staining combined with cytometry analysis were used for evaluation of the cytotoxicity of this fusion protein; TAT-ODD-p53 induced cell-cycle arrest was analyzed using cytometry with PI staining. To investigate the mechanism of anti-tumor affect of TAT-ODD-p53, a set of p53 downstream genes expression was analyzed by Western blotting in vitro. On the other hand, tumor-bearing mode was established with Balb/c nude mice by subcutaneous injection of H1299, human non-small lung cancer cell line, to investigate the distribution and tumor suppression activity of TAT-ODD-p53. The protein's accumulation in tumor and normal tissues was determined after the ip injection of p53 fusion protein at the dosage of 5 mg/kg. To assess the tumor suppression activity of TAT-ODD-p53, the proteins were i.p. injected to mice at the dosage of 5 mg/kg/time for 12 days with one day's intermission. Immunochemical staining was used to analysis the existence of TAT-ODD-p53 in different tissues and its anti-tumor mechanism in vivo. Our experimental results demonstrated that: 1. TAT-ODD-p53 fusion gene was constructed, cloned into pET28a vector, and successfully expressed; 2. The fusion protein was purified via affinity chromatography; 3. TAT-ODD-p53 could be effectively delivered into tumor cells and accumulated in the cytoplasm and nucleus, the protein was more stable in hypoxia than that under normoxia, the half-life time significantly prolonged. 4. MTT assay displayed that TAT-ODD-p53 had predominant growth inhibition on various tumor cell lines under hypoxia, but had not significant function in the normal oxygen tension condition. 5. Cytometry analysis suggested that under hypoxia the apoptosis percentage of tumor cells increased significantly when the cells were treated with TAT-ODD-p53 at the dose of 30μg/ml with the prolonged time and the cell cycle was blocked in the G1 phase. 6. Western blotting displayed that TAT-ODD-p53 could up-regulate the p21 expression and activate the caspase-3 pathway to suppress the tumor cell growth in vitro. 7. In vivo, TAT-ODD-p53 could selectively accumulate in the hypoxic regions of solid tumor of mice, but not be detected in the normal tissue, such as liver. The co-localization assessment showed that TAT-ODD-p53 could scatter the area where HIF-1αwas highly expressed. 8. The weight and volume of tumor in mice treated with TAT-ODD-p53 were lower than those of other treatment groups, and there were no any severe side-effects observed; 9. The immunochemical staining showed that the p53-associated gene expression was up-regulated after treatment with TAT-ODD-p53, and some proteins involved in tumor locaregional spread, distant metastasis and angiogenesis were down-regulated in the tumor tissue.In conclusion, TAT-ODD-p53 has good cell permeability and selectively accumulated in the hypoxic regions of solid tumor. It effectively cleans the hypoxic tumor cells and suppresses the tumor growth via inducing tumor cells undergoing apoptosis via up-regulation of a set of p53 down-stream genes expression.