Anti-tumor effect of a novel soluble recombinant human endostatin: administered as a single agent or in combination with chemotherapy agents in mouse tumor models.

Zhihua Ren,Yongping Jiang,Wei Dai,Wenhong Jiang,Yanan Wang,Irina V Lebedeva

doi:10.1371/journal.pone.0107823

Abstract

BackgroundAngiogenesis has become an attractive target in cancer treatment. Endostatin is one of the potent anti-angiogenesis agents. Its recombinant form expressed in the yeast system is currently under clinical trials. Endostatin suppresses tumor formation through the inhibition of blood vessel growth. It is anticipated that combined therapy using endostatin and cytotoxic compounds may exert an additive effect. In the present study, we expressed and purified recombinant human endostatin (rhEndostatin) that contained 3 additional amino acid residues (arginine, glycine, and serine) at the amino-terminus and 6 histidine residues in its carboxyl terminus. The recombinant protein was expressed in E. Coli and refolded into a soluble form in a large scale purification process. The protein exhibited a potent anti-tumor activity in bioassays. Furthermore, rhEndostatin showed an additive effect with chemotherapy agents including cyclophosphamide (CTX) and cisplatin (DDP).MethodsrhEndostatin cDNA was cloned into PQE vector and expressed in E. Coli. The protein was refolded through dialysis with an optimized protocol. To establish tumor models, nude mice were subcutaneously injected with human cancer cells (lung carcinoma A549, hepatocellular carcinoma QGY-7703, or breast cancer Bcap37). rhEndostatin and/or DDP was administered peritumorally to evaluate the rate of growth inhibition of A549 tumors. For the tumor metastasis model, mice were injected intravenously with mouse melanoma B16 cells. One day after tumor cell injection, a single dose of rhEndostatin, or in combination with CTX, was administered intravenously or at a site close to the tumor.ResultsrhEndostatin reduced the growth of A549, QGY-7703, and Bcap37 xenograft tumors in a dose dependent manner. When it was administered peritumorally, rhEndostatin exhibited a more potent inhibitory activity. Furthermore, rhEndostatin displayed an additive effect with CTX or DDP on the inhibition of metastasis of B16 tumors or growth of A549 tumors.ConclusionSoluble rhEndostatin exhibits a potent anti-tumor activity in mouse xenograft models and it also has an additive effect with CTX and DDP, implying possible applications in clinical settings.

Highlights

Angiogenesis plays a key role in promoting growth, invasion and metastasis of tumors [1,2,3]
Modified rhEndostatin was expressed in E. coli using a pQE vector expression system
The rhEndostatin was purified by affinity purification with nickel (Ni) resin followed by size-exclusion chromatography, and the denatured protein underwent an optimized refolding procedure to gain a refolding rate of over 80%

Summary

Introduction

Angiogenesis plays a key role in promoting growth, invasion and metastasis of tumors [1,2,3]. It has become an attractive target for tumor therapy. It was reported that endostatin inhibits endothelial cell proliferation and migration both in vivo and in vitro, inhibiting tumor growth [4,5]. Its recombinant form expressed in the yeast system is currently under clinical trials. We expressed and purified recombinant human endostatin (rhEndostatin) that contained 3 additional amino acid residues (arginine, glycine, and serine) at the amino-terminus and 6 histidine residues in its carboxyl terminus. RhEndostatin showed an additive effect with chemotherapy agents including cyclophosphamide (CTX) and cisplatin (DDP)

Methods

Results

Discussion

Conclusion