The Expression And Antitumor Effect Of RTA Fusion Proteins

VP22 is the tegument protein of herpes simplex virus type I. Being a cell-penetrating peptide, or protein transduction domain, it could intra- or intercellular deliver large molecules to the interior of surrounding cells. And another cell-penetrating peptide, TAT is a peptide from human immunodeficiency virus with eleven amino acid residues. It has been widely used to transform the peptides, oligonucleotides and proteins into cells via cross-membrane transduction. GnRH is a 10-amino acid hormone which is secreted by the hypothalamus tissue. It had the function to make anterior pituitary to facilitate release of gonadotrophin. People found that GnRH receptors (GnRH-R) were expressed in cell membrane of tumors derived from the breast, prostate, colon and liver. The oxygen-dependent degradation domain (ODD) is a key domain of HIF-1α, and it could control stability of HIF-1α. In normal oxygen level, the degradation of HIF-1αcould be accelerated by ODD, whereas, its fusion protein could be stabilized in the cell under hypoxia which is the typical pathological result of solid tumor. Ricin toxin A chain can exert RNA N-glycosidase activity to inhibit ribosome, causing protein synthesis inhibition and killing the cells.By using GnRH,TAT,VP22 to deliver RTA, and ODD to target solid tumor cells, we aimed to evaluate the anti-tumor effect of RTA series fusion proteins. The plasmids pET28a-RTA, pET28a-VP22-RTA, pET28a-GnRH-RTA, pET28a-TAT-RTA and pET28a-VP22-ODD-RTA were constructed. The RTA fusion proteins were expressed in E.coli, BL21 (DE3) engineering bacteria. The fusion proteins were purified by affinity chromatography and their bioactive funtionss were tested. The viability of tumor cells treated with RTA series fusion proteins was measured by the SRB method. Immunofluorescence analysis and Immunocytochemistry analysis were employed to test the location of RTA fusion proteins in different kinds of tumor cells. The flow cytometry was also used to test the induced apoptosis in A549 cells treated with RTA fusion proteins. The mice with xenograft tumor of A549 cells were used to determine the anti-tumor effect of saline buffer, RTA, GnRH-RTA and TAT-RTA in vivo. The serum biochemical parameters of tumor-bearing mice were also tested.RTA fusion proteins, RTA,GnRH-RTA,TAT-RTA,VP22-RTA and VP22-ODD-RTA, were expressed and purified. Immunocytochemistry analysis, as well as immunofluorescence analysis, indicated that GnRH-RTA and TAT-RTA could permeate into the A549,MDA-MB-231 and H1299 cells. The cell-free protein synthesis inhibition assay showed that all fusion proteins could inhibit protein synthesis apparently at molecular level. In order of the inhibition activity from high to low, the proteins were listed as: VP22-ODD-RTA,VP22-RTA,RTA,TAT-RTA, GnRH-RTA. Cell viability experiments showed TAT-RTA,GnRH-RTA,VP22-RTA and VP22-ODD-RTA had obvious functions to inhibit cell proliferation in A549,MDA-MB-231,H1299,HepG2 and Hela cells. Generally, TAT-RTA had a greater effect than other fusion proteins. The result also indicated that there is no significant difference between the cell proliferation inhibitions induced by VP22-ODD-RTA under normal and low oxygen tensions. Flow cytometry experiment showed us that RTA series fusion protein could induce early apoptosis. And percentage of early apoptosis cells was the time dependent. Compared with control, cells treated with TAT-RTA at 48 h had the highest percentage of early apoptosis. Tumor-bearing mode was established with Balb/c nude mice by subcutaneous injection of A549, human lung adenocarcinoma cell line, to investigate the tumor suppression activity of RTA, TAT-RTA and GnRH-RTA. Mice were injected (i.p.) with fusion proteins of at 3 mg/kg/day, and the assay reduced xenograft tumor in volume. However, weight analysis of these experimental animals showed that animals treated with RTA and TAT-RTA had a great loss in their weight, indicating that these two fusion proteins seemed noxious to animals. The plasma biochemical parameters of tumor-bearing mice showed RTA,GnRH-RTA and TAT-RTA caused damage to liver and kidney in different scales.In conclusion, fusion proteins GnRH-RTA and TAT-RTA could suppress tumor growth in vitro and in vivo. However, TAT-RTA seemed noxious to animals because it could not distinguish normal cells from tumor cells. GnRH-RTA and TAT-RTA could be a novel protein drug candidate for anti-tumor therapy if they could be transducted into tumor cells specifically.