The Therapeutic Effect Of C225/p5HRE-cfosp-iNOS-IFNG Based On Magnetic Albumin Nanospheres For Lung Cancer

Lung cancer is a malignant tumor that displays one of the highest incidence and mortality rates, and these rates are significantly increasing. Latest tumor epidemiology investigation shows that the morbidity and mortality of lung cancer is the first in malignant tumor of the male and the mortality of lung cancer is the second in malignant tumor of the female. Lung cancer has become one of the greatest hazards to human health and life. In this study, we designed the gene-loaded targeted magnetic albumin nanospheres (GTMANS) based oh the gene circuit which contains the IFN-y gene, PEI-Fe3O4 as a gene vector and the cetuximab. We explored the efficacy of this novel combination therapy consisting of radiation therapy, immune-related gene therapy and molecular targeted therapy for lung cancer and the main contents are as follows:Part I Construction and identification of the recombined eukaryotic expression plasmid (p5HRE-cfosp-iNOS-IFNG) and its inducible expression in cells.[Objective]:To construct and identify the recombined eukaryotic expression plasmid p5HRE-cfosp-iNOS-IFNG for further gene therapy and evaluate the expression efficiency of IFN-y gene after different means of induction.[Methods]:① The C-fos promoter sequence was excised form the pUC57-Simple-cfosp and inserted into the pYr-adshuttle-8 to construct the pYr-ads-8-cfosp plasmid.② The oligomers of primers based on pDONR223-BFNG plasmid, and cloned into the pYr-adshuttle-8 vector to get pYr-ads-8-IFNG plasmid. ③ The C-fos promoter sequence was excised form the pUC57-Simple-cfosp and inserted into pYr-ads-8-IFNG plasmid to get the pYr-ads-8-cfosp-IFNG.④ The 5HRE (5 copies of HREs) region was excised from the pUC57-5HRE plasmid, this region was inserted into the pUC57-Simple-cfosp, creating the pUC57-Simple-5HRE-cfosp recombinant plasmid. And the 5HRE-cfosp region was excised and inserted into the pYr-adshuttle-8 and pYr-ads-8-IFNG to get pYr-ads-8-5HRE-cfosp and pYr-ads-8-5HRE-cfosp-IFNG plasmid. ⑤ The oligomers of primers based on pIRES2-ZsGreen 1 plasmid, and cloned into the pYr-ads-1-iNOS vector to get pYr-ads-iNOS-IRES plasmid. The oligomers of primers based on pDONR223-IFNG plasmid, and cloned into the pYr-ads-iNOS-IRES to get pYr-ads-iNOS-IFNG. ⑥ The oligomers of primers based on pIRES2-ZsGreenl plasmid pUC57-Simple-cfosp,and cloned into the pYr-ads-iNOS-IFNG vector to get pYr-ads-cfosp-iNOS-IFNG. ⑦ The 5HRE-cfosp region was inserted into the pYr-ads-iNOS-IFNG vector to get pYr-ads-5HRE-cfosp-iNOS-IFNG plasmid (p5HRE-cfosp-iNOS-IFNG). ⑧ The recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. At last, the recombinant plasmids were extracted and purified. ⑨ GLC-82 cells were respectively trasfected with pYr-ads-8-cfosp, pYr-ads-8-IFNG, pYr-ads-8-cfosp-IFNG, pYr-ads-8-5HRE-cfosp-IFNG,pYr-ads-cfosp-iNOS-IFNG,pYr-ads-iNOS-IFNG, p5HRE-cfosp-iNOS-IFNG and induced by 2Gy X ray to detect the IFN-y and iNOS gene expression by QT-PCR. ⑩ GLC-82 cells were trasfected with p5HRE-cfosp-iNOS-IFNG. First, the transfected cells were radiated with 2Gy X ray and harvested at different time points (6h,12h,24h,48h and 72h). Then the trasnfected cells were radiated with different dose of X rays (OGy, 1Gy,2Gy,4Gy, and 8Gy) and harvested at 24h. At last, detected the IFN-y and iNOS gene expression by QT-PCR.[Results]: ① Recombinant p5HRE-cfosp-iNOS-IFNG plasmid was gained. The target gene obtained by PCR amplification had the same molecular size as predicted and showed that recombined plasmid contained correct recombinant sequences. The forepart interface between the vector and the insert was sequenced, which confirmed that the insert was in frame.②QT-PCR assay was used to detect IFN-y and iNOS gene expression in GLC-82 cells after induced. The IFN-y gene expression of the p5HRE-cfosp-iNOS-IFNG plasmid was 1.46 times higher than that in ⑥ group and the IFN-y gene expression was 2.11 times higher than that in ⑤ group.(p<0.05) ③ QT-PCR assay was used to detect IFN-y and iNOS gene expression in GLC-82 cells after 2Gy X ray irradiation at 6h,12h,24h,48h and 72h. IFN-y and iNOS gene expression first increased and then decreased and the highest expression was at 24 hour time point.④ After OGy, 1Gy,2Gy,4Gy and 8Gy irradiation, the IFN-y and iNOS gene expression were detected at 24h. The IFN-y and iNOS gene expression of 2Gy group was highest. The IFN-y gene expression of 2Gy group was 4.37 times higher than 1Gy group,1.52 times than 4Gy group and 2.05 times than 8Gy group.(p<0.05) The iNOS gene expression of 2Gy group was higher than 4.96 times than 1Gy group,1.56 times than 4Gy group and 2.17 times than 8Gy group.(p<0.05)[Conclusions]:① p5HRE-cfosp-iNOS-BFNG plamid had been constructed successfully. ② Evaluated the effectiveness of the gene circute, the gene expression is highest after 2Gy irradiation at 24 h. Verified the enhancement of HRE in hypoxic environment.Part II Preparation of gene-loaded targeted magnetic albumin nanospheres (GTMANS) and targeting evaluation of the nanospheres in vitro and in vivo[Objective]: ① To study the preparation and characterization of Fe3O4 magnetic nanoparticles. To study the preparation and characterization of PEI-coated Fe3O4, and to evaluate the feasibility of using magnetic nanoparticles PEI-Fe3O4 as gene vector. ② To study the preparation of gene-loaded magnetic albumin nanospheres (GMANS) and gene-loaded targeted magnetic albumin nanospheres (GTMANS).③ To identify the immunological properties (targeting) of targeted magnetic albumin nanospheres and to observe its transfection efficiency.④ To explore the targeting effect of the nanospheres on human lung adenocarcinoma cell line GLC-82 cells in vitro and in vivo.[Methods]: ① Fe3O4 magnetic nanoparticles were prepared by the technique of chemical co-precipitation, then the surface of Fe3O4 particles was modified by PEI. ②Their characteristics were observed by means of transmission electron microscope (TEM), ZETA SIZER3000 laser particle size analyzer, Fourier transform infrared spectroscopy (FTIR) was used to confirm the existence of PEI. ③ The adsorbing amount of PEI-Fe3O4 and DNA binding efficiency were observrd by electrophoresis experiment. Transfection was determined by delivering reporter gene, pEGFP-Cl, to GLC-82 cell lines using PEI-Fe3O4 as vector.④ GMANS were prepared by desolvation-crosslinking method and GTMANS were prepared through using heterobifunctional crosslinker SPDP to couple nanospheres and cetuximab monoclonal antibody (C225). TEM and ZETA SIZER3000 laser particle size analyzer were used to characterize the nanospheres. To asses the stability of the GTMANS, the hydrated size of the GTMANS in PBS and in RPMI 1640 was analyzed by DLS within 24 hours, And the particle size was measured for 32 days with storage at 4℃ and ambient humidity. Plasmid release rate in vitro was dynamically evaluated. Iron content were measured by thiocyanate spectrophotometric.⑤ The transfection efficiency of GTMANS was observed by using it transfect the GLC-82 cells.⑥ To identify the immune characteristics of the nanospheres through slide agglutination test and the immunofluorescence staining. ⑦ To observe the specific binding ability of the nanospheres with human lung cancer cells in vitro though prussian blue staining, immunofluorescence experiments and MRI experiment.⑧ To establish subcutaneously transplanted tumor in nude mice. MRI was performed when tumor diameter reached about 0.5 cm. Mice were randomly divided into C225 targeting groups which were injected C225 magnetic albumin nanospheres through tail vein, non-C225 targeting groups injected magnetic albumin nanospheres and C225 suppressing groups injected C225 magnetic albumin nanospheres with C225. MRI imaging were performed to measure T2WI and T2*WI signal strength of the regions of interest (tumor and muscle tissue) before injection and after injection at different time points (2 h,8 h,24 h,72 h) respectively and calculate the signal change rate. SPSS 18.0 statistical software finally was used for statistical analysis. Histopathological examination included HE staining and Prussian blue staining.[Results]:① The prepared Fe3O4 and PEI-Fe3O4 nanoparticles were high electron-dense, uniform in size observed by TEM, their morphology and size were not obvious differences; while the analysis results of ZETA SIZER3000 analyzer demonstrated that the surface charge of Fe3O4 in pH 7 was 0±0.5 mV and turned into 36.3±0.6mV after modified with PEI. The results of FTIR that showed the characteristic peaks of PEI demonstrated the successful adsorption of PEI. ② PEI-Fe3O4 had good binding ability with DNA, and could deliver foreign gene to GLC-82 cell line. And the gene could express with high efficiency. ③ TEM analysis showed that the ANS and the GTMANS were approximately spherical and uniform in size. It was clearly demonstrated that the magnetic materials with high electron density were well-incorporated in the core of albumin nanospheres. DLS showed the hydratcd particle size of the GMANS was 189.5±2.4 nm and the hydrated particle size of the GTMANS was 211.9±3.1 nm. The surface charge of GMANS was-37.9±0.4 mV and slightly reduced to-40.2±0.7 mV for GTMANS. To assess the stability of the GTMANS, the hydrated size of the GTMANS was analyzed by DLS. The GTMANS showed limited variation (<10%) in volume size after 32 days of storage in PBS at 4℃, indicating excellent stability in aqueous medium. ④ The nanospheres cumulative release rate was 96.45%by calculating dynamic release rate of 15 hours in vitro and the curve was flat. Iron content was about 2.2 mg/ml measured by thiocyanate spectrophotometric method. The GTMANS can carry the plasmid to GLC-82 cells and its transfection efficiency is higher than the the GMANS. ⑤ Slide agglutination experiment observed the agglutination clearly after the targeted magnetic albumin nanospheres incubated with antiserum compared with the control group; immunofluorescence staining showed that bright green fluorescence was observed on the surface of targeted magnetic nanospheres, while not observed in the control group.⑥ Prussian blue staining demonstrated that after the monoclonal antibody targeting group incubated with GLC-82 cells, a large number of blue-stained iron particles were observed in the cells, while little in the cells of the non-targeting group. Immunofluorescence staining showed that the green fluorescence was observed on the surface of the cells of the monoclonal antibody targeting group, while not observed in the non-targeting group. The MRI T2 signal intensity of GLC-82 cells decreased significantly after incubating with C225 targeting groups, but did not show obvious decrease after incubating with non-C225 targeting groups and C225 suppressing groups. ⑦ In vivo experiment:As in vivo MRI imaging, the T2 and T2* signal intensity and signal change rate of the tumor tissue of C225 targeted groups decreased at different time points after injection and the change had significant difference. The signal intensity of non-C225 targeting groups and C225 suppressing groups also slightly decreased immediately, at 8h and at 24 h after injection, but the MRI signal intensity decreased marginally and duration time of signal was shorter than C225 targeted groups.⑧ Pathological examination:prussian blue staining showed that much more intracellular iron particles existed in C225-targeted tumor tissue, while intracellular iron particles of the non-C225 targeting groups and C225 suppressing groups were almost invisible.[Conclusion]: ⑨ Fe3O4 magnetic nanoparticles can be successfully prepared through improved co-precipitation process, PEI has been coated on nanoparticles Fe3O4. ② After modified, the PEI-Fe3O4 nanoparticles present favorable dispensability and and transfect capability. ③ Albumin nanospheres and gene-loaded magnetic albumin nanospheres were prepared successfully; GTMANS were coupled with cetuximab monoclonal antibody successfully. ④ The GTMANS can transfect GLC-82 cells with high efficiency and slowly release the plasmid. ⑤ The targeted magnetic albumin nanospheres had good targeting effection on human lung cancer cell line GLC-82 in vitro and in vivo.Part III The Therapeutic effect of GTMANS for lung cancer in vitro and in vivo[Objective]: ① To explore the therapeutic effect of GTMANS combined with radiotherapy on human lung cancer in vitro. ② To explore the therapeutic effect of the GTMANS combined with radiotherapy to GLC-82 xenograft tumor in nude mice in vivo level.[Methods]:① To study the effect of treatment of the GTMANS on lung cancer in vitro by the CCK8 assay and flow cytometry. And observed the changes of the ultrastructure of cells by transmission electron microscope. ② Established lung cancer xenograft model in nude mice, and injected the GTMANS into the mice via tail vein. 6 weeks after treatment, the tumor volume, quality inhibition rate and tumor tissue pathological changes were detected.③ The safety of targeting therapy, radiation therapy combined with gene therapy was evaluated by serum biochemical examination, blood routine test and histopathological examination of internal organs.[Results]:④ CCK8 experimental results showed that the GTMANS combined with radiotherapy could significantly inhibit the proliferation of human lung adenocarcinoma cell line GLC-82 and the FCM detection displayed that it could induce apoptosis more effectively compared with other group. The cells of each treatment group were observed apoptosis and nano-magnetic materials could be observed in cells under TEM. ⑤ The result of anti-tumor experiment in vivo showed that the quality inhibition rate and volume inhibition rate of GTMANS combined with radiotherapy group were 82.54% and 85.72, which were significantly higher than that C225 therapy group(36.51% and 34.33%), radiotherapy group(42.06% and 40.89%), gene therapy combined with radiotherapy group (64.29% and 65.64%) and C225 combined with radiotherapy group (70.63% and 72.57%).⑥ The tumor tissues of the treatment groups displayed different degrees of necrosis, and we observed the infiltration of inflammatory cells surrounding areas of partial necrosis in the tumors. Tumor necrosis was the most extensive in group VI (the necrotic areas are stained red), in which massive necrosis was observed, as demonstrated by the disintegration of tumor cells and the disappearance of nuclei. The internal organs of all groups were not observed any histopathological change.④ No significant abnomity was observed on blood parameters in the experimental group mice compared with the control group mice.[Conclusions]: ① The GTMANS combined with radiotherapy could significantly inhibit the proliferation of the cultured human lung adenocarcinoma cell line GLC-82 and induce apoptosis.② The therapy by combination molecular targeted therapy, radiotherapy and gene therapy on lung cancer has obvious complementary and synergistic effects and can effectively inhibit the growth of lung cancer, and its effect was better than any treatment alone.Part IV The study of the molecular mechanism of GTMANS for lung cancer[Objective]:To explore the therapeutic molecular mechanism of GTMANS combined with radiotherapy for lung cancer in molecular field.[Methods]: ① To explore molecular mechanisms of GTMANS combined with radiotherapy on lung cancer,the expression of Bcl-2, Bax, survivin, VEGF, EGFR, PCNA, p53, Ki67 in tumor tissue were detected using immunohistochemistry.② Using western bloting to detect the expression of factors such as Bcl-2, Ki67, survivin, caspase-3 and caspase-8. And the Gel-pro32 software was used to gray analysis.[Results]:① The immunohistochemistry showed that each treatment could increase Bax expression and reduce Bcl-2, Ki67, p53, PCNA, survivin, EGFR and VEGF expression. But the GTMANS combined with radiotherapy was the most effective. ② Western blot showed that the protein expression of Bcl-2, Ki67, survivin was significantly decreased in treated groups compared to the normal control group, but the expression of casepase-3 and casepase-8 was increased. The changes were most obvious in GTMANS combined with radiotherapy group.[Conclusions]:The molecular mechanism of the GTMANS combined with radiotherapy for lung cancer was involved in down-regulation of Ki67 and PCNA expression leading to repressing of tumor cell proliferation, inhibition of Bcl-2, p53 and survivin protein expression and up-regulation of Bax protein expression inducing tumor cells apoptosis, inhibiton of tumor angiogenesis. The expression of EGFR decreased after treatment.