Escherichia coli Nissle 1917 engineered to express Tum-5 can restrain murine melanoma growth
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Lian He1, Huijun Yang1, Fei Liu1, Yiyan Chen1, Sijia Tang1, Wei Ji1, Jianli Tang1, Zhudong Liu1, Yunjun Sun1, Shengbiao Hu1, Youming Zhang1, Xiong Liu1, Weitao Huang1, Xuezhi Ding1 and Liqiu Xia1
1Hunan Provincial Key Laboratory of Microbial Molecular Biology, State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha 410081, Hunan, China
Liqiu Xia, email: [email protected]
Keywords: Tum-5, E. coli Nissle 1917, lux operon, anti-angiogenesis, murine melanoma
Received: May 16, 2017 Accepted: July 25, 2017 Published: August 24, 2017
Tumor growth and metastasis depend on angiogenesis. Thus, inhibiting tumor angiogenesis has become promising cancer therapeutic strategy in recent years. Tumstatin is a more powerful angiogenesis inhibitor than endostatin. Anti-angiogenic active fragment encoding amino acids 45–132 (Tum-5) of tumstatin was subcloned into four different inducible expression vectors and successfully solubly expressed in Escherichia coli BL21 (DE3) in this study. Subsequently, an anaerobic inducible expression vector was constructed under Vitreoscilla hemoglobin gene promoter Pvhb in E. coli Nissle 1917 (EcN). The secretory expression of Tum-5 in the engineered bacterium was determined in vitro and in vivo by Western blot or immunochemistry. The anti-tumor effect detection demonstrated that EcN could specifically colonize the tumor, and B16 melanoma tumor growth was remarkably restrained by EcN (Tum-5) in mice bearing B16 melanoma tumor. Abundant infiltrating inflammatory cells were observed in tumor areas of the EcN-treated group through hematoxylin and eosin staining, with a relatively reduced expression of endothelial marker platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) by immunofluorescence in tumor sections of EcN (Tum-5)-treated mice. No significant morphological differences were observed in the liver, kidney and spleen between EcN-treated mice and the control group, indicating that EcN was cleared by the immune system and did not cause systemic toxicity in mice. These findings demonstrated that the gene delivery of Tum-5 to solid tumors could be an effective strategy for cancer therapy.
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