Character Of Bifidobacterium Breve Carrying Endostatin And IFNγ And The Role Of Anti-mouse Lung Cancer

Cancer is a severe disease that threats the human health. Common methods to cure cancer include operation, radiotherapy and chemotherapy, as well as endocrine therapy, traditional Chinese medicine therapy, hyperthermia and radiofrequency ablation therapy, whose effects are quite limited. Gene therapy provides a new way to cancer treatment, which makes the combination of traditional methods and gene therapy a new idea for cancer therapy. Cancer gene therapy is to interfere with tumor occurrence, development and processes through operating genetic material, includes the correction of the own gene structural and functional disorders, the kill of broken cells or the enhancement of the body's ability to remove diseased cells. The most important features of cancer gene therapy includes the selection of target genes and efficient stable targeted-cell vector system, so that the therapeutic genes highly expressed in the target cells will achieve the therapeutic purposes. Bifidobacteria are obligate anaerobic bacteria, which are useful and non-toxic to the human body and have the growth properties targeting anaerobic conditions in vivo. There are hypoxic areas inside of the tumor. Bifidobacterium as a carrier can overcome the difficulties in penetrating the blood vessel wall and organism matrix. On the contrary, the non-viral vectors and viral vectors have the fatal disadvantage because of tumor blood flow derangement, and will be cleared by the human immune system. Endostatin gene has a strong inhibitory function of angiogenesis, inhibits the reborn tumor vessel; and interferon-y has anti-virus, regulation immune function, anti-tumor and other biological functions. We select Bifidobacterium breve as a targeted vector system, carrying two therapeutic genes: Endostatin and IFNγto reveal the inhibitory effect on lung graft tumor in mice and the anti-tumor mechanism. The ideas and results will bring hopes to cancer treatment.1 Construction of prokaryotic expression vector carrying Endostatin and IFNy geneIn this study, prokaryotic expression vector pNZ44-ssEndostatin and pNZ44-IFNγmediated Lactobacillus promoter were constructed. The target gene secretion Endostatin and IFN7γwere got by PCR amplification using pcDNA3.1-ssEndostatin and pcDNA3.1-IFNγplasmid as template. The primer carried Noc I and Xba I. Xho I and Xba I enzyme digestion site at both ends. PCR products were ligated to pNZ44 vector double-digested by Noc I and Xba I, Xho I and Xba I restriction enzymes, to construct prokaryotic expression vector. After amplification in E. coli DH5a, plasmid was extracted. The plasmid was identified by PCR and restriction enzyme digestion. After PCR, two fragments of 630 bp and 500 bp could be seen, after double-digestion, the fragments of 630 bp and 3400 bp,500 bp, and 3500 bp could be found, PCR identification and restriction enzyme digestion were consistent with prediction, which suggested that the construction was right.2 Endostatin and IFNy protein expression in Bifidobacterium breveThe prokaryotic expression vector carrying secretory Endostatin and IFNy gene pNZ44-ssEndostatin and pNZ44-IFNy were transformed into Bifidobacterium breve by electrotransformation to construct B-b-pNZ44-ssEndostatin and B-b-pNZ44-IFNy. The positive clones in MRS medium plate would be cultured for 20 and 48 h under normoxic and hypoxic condition. Endostatin and IFNy protein expression in culture supernatants and Bifidobacterium cells were detected by ELISA, which showed that under normoxic or hypoxic condition, Endostation did not express in the supernatant, while the IFNy protein expression could be seen. And IFNy expression was medium in Bifidobacterium breve (B-b) and Bifidobacterium breve transferred pNZ-44 (B-b-pNZ44) at 20 h and 48 h, and the difference was not large. But expression was high in B-b-pNZ44-IFNy, and the expression was higher under hypoxic condition than those under normoxic condition, and the expression was higher at 20 h than those at 48 h. Both Endostatin and IFNy expressed in B-b, B-b-pNZ44, B-b-pNZ44-ssEndostatin under normoxic and hypoxic condition, Endostatin expressed little in B-b and B-b-pNZ44, but expressed highly in B-b-pNZ44-ssEndostatin, the expression was higher under normoxic condition at 48 h than those under hypoxic condition at 20 h. IFNy protein expressed little in B-b and B-b-pNZ44, but expressed highly in B-b-pNZ44-Endostatin, and it was highest at 20 h under hypoxic condition. These results showed Endostatin and IFN protein expressed highly in Bifidobacterium breve carrying secretory Endostatin and IFNy gene prokaryotic expression vector under hypoxic condition.3 The distribution of Bifidobacterium breve in mice bearing lung cancerBifidobacterium breve cultured in medium contained 3H-TdR 12 h under hypoxic condition were injected into normal and bearing tumor mice by Caudal vein injection, which indicate the distribution of Bifidobacterium breve in mice by detecting radioactivity in heart, liver, lung, kidney and tumor. The results showed that the radioactivity in normal mouse various organs decreased gradually at 1,3,5 and 7 d, indicating that Bifidobacterium breve were removed gradually; and the radioactivity in bearing lung tumor mouse various organs decreased gradually at 1,3,5 and 7 d, but radioactivity in tumor increased gradually, indicating that Bifidobacterium breve in the organ were removed, but accumulated in the tumor, this suggested that Bifidobacterium breve had a trend to anaerobic environment.4 The inhibitory effects on lung cancer in mice by Bifidobacterium breve carrying Endostatin and IFNyBifidobacterium breve B-b-pNZ44-ssEndostatin and B-b-pNZ44-IFNy were given to mice bearing lung cancer by gavage and caudal vein injection and the inhibitory effects on mouse lung cancer were measured by observation of tumor volume, tumor weigh and average survival time of lung cancer in mice. Results showed that tumor volume increased rapidly, it increased 50 times 21 days later, but the tumor volume in mice given B-b and B-b-pNZ44 by gavage and caudal vein injection decreased slightly, it had no difference compared with control group; the tumor volume in mice which given B-b-pNZ44-ssEndostatin, B-b-pNZ44-IFN and B-b-pNZ44-ssEndostatin+B-b-pNZ44-IFNy reduced significantly, the inhibitory effect of B-b-pNZ44-ssEndostatin and B-b-pNZ44-IFNγwas similar, the combination of two was more strong; the anti-tumor effect by caudal vein injection was more obvious than by gavage, it might relate closely to the number of Bifidobacterium breve reaching target.21 days later, tumor weight was weighed, the results showed that the tumor weight in mice given by B-b and B-b-pNZ44 decreased slightly, but was not obvious; and the tumor weight decreased obviously in mice given by B-b-pNZ44-ssEndostatin, B-b-pNZ44-IFNy and B-b-pNZ44-ssEndostatin +B-b-pNZ44-IFNy, and the combination of two was more obvious; and tumor weight decreased more significantly in mice given by caudal vein injection than by gavage. The average survival time of mice showed that B-b-pNZ44-ssEndostatin and B-b-pNZ44-IFNy could prolong the average survival time and combination of the two genes could be better. Comparing the administration, the effect by caudal vein injection was stronger than by gavage. These results showed that Bifidobacterium breve carrying pNZ44-ssEndostatin and pNZ44-IFNy had significant inhibition on mice lung cancer xenograft and the combination was better, the anti-tumor effect by caudal vein injection was more significant, it might relate closely to the number of Bifidobacterium breve reaching tumor.5 The inhibitory mechanism of lung cancer in mice of Bifidobacterium breve carrying Endostatin and IFNy21 days later, transplanted lung cancer in mice treated by B-b-pNZ44-IFNγand B-b-pNZ44-ssEndostatin were obtained and the Endostatin and IFNy expression were measured in tumor. Spleen and peritoneal macrophage were got to detect some immunological indicators, such as CTL cell activity, NK cell activity and TNFa secretion activity of peritoneal macrophage. Tumor micro-vessel density changes were observed by immunohistochemistry. The results showed that Endostatin and IFNy expressed highly in tumor in mice by given B-b-pNZ44-ssEndostatin and B-b-pNZ44-IFNy both by gavage and caudal vein injection.The expression was higher in tumor by caudal vein injection administration. CTL activity and NK activity in mouse spleen and TNFa secretion activity in peritoneal macrophage increased slightly after B-b,B-b-pNZ44 and B-b-pNZ44-ssEndostatin, but increased obviously after B-b-pNZ44-IFNγand B-b-pNZ44-ssEndostatin+B-b-pNZ44-IFNy.They had no obvious difference but the immune ability by vein injection improved more than by gavage which indicated that the immunological parameters increase was mainly the result of IFNy gene transfer. Little change of the intratumoral micro-vessel density had been found in the group of B-b,B-b-pNZ44 and B-b-pNZ44-IFNy, while the micro-vessel density decreased significantly in mice given B-b-pNZ44-ssEndostatin and B-b-pNZ44-ssEndostatin+B-b-pNZ44-IFNγ. It decreased more obviously by caudal vein injection than gavage, which suggested that Endostatin gene could inhibit tumor blood vessel regeneration, but the function of IFNy inhibit angiogenesis was not shown in this experiment.