Construction Of Engineered Fusion Protein Consisting Of Cell Penetrating Peptide (Arg)₉ And Lidamycin And The Study Of Its Antitumor Activity

Lidamycin (LDM, C-1027), a macromolecular peptide antibiotic produced by Streptomyces globisporus, showed extremely potent cytotoxicity against tumor cell lines, anti-angiogenic activity and marked growth inhibition of transplantable tumors in mice. LDM consists of an active enedinye chomophore (AE) and an apoprotein (LDP), which can be separated and reconstituted without losing its activity. The primary role of LDP is as a packing carrier protein for the chemically unstable chromophore, however, the antitumor activity of LDP remains unidentified.Glioma, the most common primary brain tumor, is highly vascularized and angiogenesis-dependent. Current reports show that the vascular microenvironment of glioma plays a central role in glioma progression, invasion, diagnosis and the effectiveness of clinic treatment. Because of blood-brain barrier, the application of antiangiogenesis drugs in glioma therapy is limited.Cell-penetrating peptides (CPPs) are short peptides of less than 30 amino acids that are able to penetrate cell membranes and translocate different cargoes into cells. The mechanism of cell translocation is not known but it is apparently receptor and energy independent, although, in certain cases, translocation can be partially mediated by endocytosis. Cargoes that are successfully internalized by CPPs range from small molecules, oligonucleotide to proteins, bangosome and supramolecular particles. CPPs are novel vehicles for the translocation of cargo into cells, their properties make them potential drug delivery agents of interest for future use.In this study, recombinant lidamycin apoprotein rLDP was prepared and recombinant lidamycin rLDM was reconstituted, the affinity activity of rLDP to tumor cells and the antitumor activity of rLDP and rLDM was observed. Fusion protein (Arg)9-LDP was prepared and energized fusion protein (Arg)9-LDP-AE was reconstituted. The membrane translocation ability of fusion protein and the potent cytotoxicity of fusion protein and its energized protein were observed.1. Preparation of recombinant lidamycin and the study of its activityE.coli BL21 (DE3) starTM cells which has the recombinant plasmid pET30-sldp were preserved in our laboratory. The recombinant lidamycin apoprotein rLDP with His-tag was successfully secreted into the culture medium and periplasmic space of E.coli after IPTG inducing. rLDP was purified by Ni2+ affinity chromatography and the purity of fusion proteins was all over 98%as determined by SDS-PAGE. Finally about 23 mg of purified protein was obtained from 1 L of culture medium. The immunofluorescent cytochemical staining and FACS-based fluorescence assay showed that rLDP has strong binding activity to cancer cell lines, such as human lung carcinoma A549 cells and H460 cells, human ovarian carcinoma OVCAR3 cells, human glioma U87 cells, and human hepatoma Bel-7402 cells. However, rLDP has no binding activity to L02 cells came from normal hepatic tissue. The results of MTT assay proved that rLDP displayed cytotoxicity to Bel-7402 cells with IC50 value of 7.05×10-5 mol/L. FACS analysis of cell cycle showed that the cells were arrested in G2/M phase after rLDP treatment, and the degree of arrest was concentration dependent. Transplantable hepatoma 22 (H22) in Kunming mice was used to investigate the inhibitory effects of rLDP. Evidently, rLDP suppressed H22 tumor growth. The inhibition rates of rLDP at dose of 15 mg/kg,30 mg/kg and 60 mg/kg were 44.6%,57.1%and 50.1%respectively for intravenous injection. The inhibition rates of rLDP at dose of 30 mg/kg,60 mg/kg and 120 mg/kg were 52.1%,49.3%and 59.5%respectively for intraperitoneal injection. The inhibition rates of rLDP at dose of 30 mg/kg,60 mg/kg and 120 mg/kg were 34.3%, 30.2%and 60.3%respectively for intragastric administration. The recombinant lidamycin rLDM was prepared by the reconstitution of rLDP and AE. The results of MTT assay showed that the IC50 values of rLDM for SKOV3 cells, U87 cells, MCF-7 cells and Bel-7402 cells were close to those of LDM. FACS analysis of cell cycle showed that rLDM and LDM induced similar cell cycle arrest in SKOV3 cells and U87cells. Cells were both arrested in G2/M phase and S phase after rLDM and LDM treatment at 1 nM, SKOV3 cells were arrested in G2/M phase and U87cells were arrested in G1 phase after treatment at 0.001 nM.2. Construction of fusion protein (Arg)9LDP and the study of its activity(arg)9ldp gene was ligated into the plasmid pET-30a(+), then the recombinant plasmid pET30-(arg)yldp was transformed into competent E.coli BL21 (DE3) starTM cells. Fusion protein (Arg)9-LDP with His-tag was produced in the form of inclusion after IPTG inducing. (Arg)9-LDP was purified by Ni2+ affinity chromatography and the purity of fusion proteins was all over 95%as determined by SDS-PAGE. Finally about 15 mg of purified protein was obtained from 1 L of culture medium. The result of fluorescent cytochemical staining showed that (Arg)9-LDP could be transferred into tumor cells and distributed in cytoplasm for 2 h incubation. The FACS-based fluorescence assay revealed that the translocation of (Arg)9-LDP was influenced by temperature. The results of MTT assay proved that (Arg)9-LDP had moderate cytotoxicity to U87 cells with IC50 value of 5.25×10-5 mol/L. FACS ananlysis of cell cycle showed that the cells were arrested in G2/M phase and S phase after (Arg)9-LDP treatment at 200μM and G1 phase at 20μM. 3. Preparation of energized fusion protein (Arg)9-LDP-AE and the study of its antitumor activityThe energized fusion protein (Arg)9-LDP-AE was prepared by the reconstitution of (Arg)9-LDP and AE. The results of MTT assay proved that (Arg)9-LDP-AE had stronger cytotoxicity than LDM. The IC50 values of (Arg)9-LDP-AE for U87 cells and MCF-7 cells were close to those of LDM, the IC50 values of (Arg)9-LDP-AE was only 1/20 of that of LDM for OVCAR3 cells and 1/10 for Bel-7402 cells respectively. FACS ananlysis of cell cycle showed that the U87 cells were arrested in G2/M phase and S phase after (Arg)9-LDP-AE treatment, and the degree of arrest was concentration dependent. The results of Annexin V-FITC/PI assay indicated the potent and dose-dependent proapoptotic effect of (Arg)9-LDP-AE on U87 cells. In vivo, the energized fusion protein (Arg)9-LDP-AE showed stronger antitumor activity than LDM. The inhibition rates of (Arg)9-LDP-AE against murine hepatoma H22 at dose of 0.2 mg/kg and 0.3 mg/kg were 85.3%and 89.2%respectively, while that of LDM at 0.05 mg/kg was 74.6%. (Arg)9-LDP-AE at the dose of 0.1 mg/kg and 0.2 mg/kg inhibited the growth of human glioma U87 xenografts by 79.2%and 88.8%, while that of LDM at 0.05 mg/kg was 62.9%.