Molecular Cloning, Recombinant Expression and Functional Characterization of the Soluble Tumor Necrosis Factor-related Apoptosis-inducing Ligand for the <i>Macaca mulatta</i>_Journal of Biomedical Engineering

Authors：

FANMiaomiao , JIADianlong , YANGHao , WanLin ,  LUXiaofeng

Key Lab of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, China;

Corresponding author：

LUXiaofeng, Email: xiaofenglu@yahoo.com

Keywords：

tumor necrosis factor-related apoptosis-inducing ligand; apoptosis; cancer-targeted therapy

DOI：

10.7507/1001-5515.20150110

Video：

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Human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) might be developed as a novel anti-tumor drug due to its selective cytotoxicity in tumor cells. The predicted Macaca mulatta TRAIL (mmTRAIL) is highly homologous to hTRAIL in nucleotide acid as well as amino acid sequence, suggesting that mmTRAIL might induce apoptosis of human cancer cells. However, the cytotoxicity of mmTRAIL in human cancer cells has not been investigated. In this paper, it is reported that the gene encoding mmTRAIL has been cloned by using reverse-transcriptase polymerase chain reaction (RT-PCR) from monkey peripheral blood mononuclear cells (PBMCs) in our laboratory. Subsequently, an expression plasmid was constructed by inserting mmTRAIL gene into pQE30 plasmid. After induction by addition of Isopropylβ-D-1-Thiogalactopyranoside (IPTG), mmTRAIL was expressed. MmTRAIL was recovered from supernatant of sonicated bacteria by Ni-NTA agarose affinity chromatography. SDS-PAGE and gel filtration chromatography demonstrated that mmTRAIL forms trimer in solution. In vitro assays indicated that mmTRAIL was cytotoxic to human COLO205 tumor cells but not to normal cells at low concentration of nanomole. In addition, antitumor effect of mmTRAIL was evaluated in mice bearing human COLO205 tumor xenografts. Intratumorally injected mmTRAIL significantly inhibited growth of tumor grafts. These results suggested that mmTRAIL was valuable as candidate drug for cancer-targeted therapy.

Citation： FANMiaomiao, JIADianlong, YANGHao, WanLin, LUXiaofeng. Molecular Cloning, Recombinant Expression and Functional Characterization of the Soluble Tumor Necrosis Factor-related Apoptosis-inducing Ligand for the Macaca mulatta. Journal of Biomedical Engineering, 2015, 32(3): 605-611. doi: 10.7507/1001-5515.20150110 Copy

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13.	LI J F, AI H X, ZHANG J, et al. Molecular cloning, functional characterization and phylogenetic analysis of TRAIL in Japanese pufferfish Takifugu rubripes[J]. J Fish Biol, 2011, 79(3):747-760.
14.	ZOR T. SELINGER Z. Linerarization of the Bradford protein assay increases its sensitivity:theoretical and experimental studies[J]. Anal Biochem, 1996, 236(2):302-308.
15.	CAI H, CHEN L, WAN L, et al. High-level expression of a functional humanized anti-CTLA4 single-chain variable fragment antibody in Pichia pastoris[J]. Appl Microbiol Biotechnol, 2009, 82(1):41-48.
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17.	WANG H Z, DAVIS J S. WU X W. Prolongs its plasma half-life and enhances its anti-tumor activity immunoglobulin Fc domain fusion to TRAIL significantly[J]. Mol Cancer Ther, 2014, 13(3):643-650.

1. AGGARWAL S. Targeted cancer therapies[J]. Nat Rev Drug Discov, 2010, 9:427-428.
2. SCOTT A M, WOLCHOK J D, OLD L J. Antibody therapy of cancer[J]. Nat Rev Cancer, 2012, 12:278-287.
3. HOLOCH P A, GRIFFITH T S. TNF-related apoptosis-inducing ligand(TRAIL):a new path to anti-cancer therapies[J]. Eur J Pharmacol, 2009, 625(1-3):63-72.
4. MCGRATH E E. The tumor necrosis factor-related apoptosis-inducing ligand and lung cancer:still following the right TRAIL?[J]. J Thorac Oncol, 2011, 6(6):983-987.
5. TAYLOR D J, PARSONS C E, HAN H A, et al. Parallel screening of FDA-approved antineoplastic drugs for identifying sensitizers of TRAIL-induced apoptosis in cancer cells[J]. BMC Cancer, 2011, 11:470.
6. PICARDA G, TRICHET V, TÉLETCHÉA S, et al. TRAIL receptor signaling and therapeutic option in bone tumors:the trap of the bone microenvironment[J]. Am J Cancer Res, 2012, 2(1):45-64.
7. ASHKENAZI A, HOLLAND P, ECKHARDT S G. Ligand-based targeting of apoptosis in cancer:the potential of recombinant human apoptosis ligand 2/Tumor necrosis factor-related apoptosis-inducing ligand (rhApo2L/TRAIL)[J]. J Clin Oncol, 2008, 26(21):3621-3630.
8. SORIA J, SMIT E, KHAYAT E, et al. Phase 1b study of dulanermin(recombinant human Apo2L/TRAIL)in combination with paclitaxel, carboplatin, and bevacizumab in patients with advanced non-squamous non-small-cell lung cancer[J]. J Clin Oncol, 2010, 28:1527-1533.
9. HERBST R S, ECKHARDT S G, KURZROCK R, et al. Phase I dose-escalation study of recombinant human Apo2L/TRAIL, a dual proapoptotic receptor agonist, in patients with advanced cancer[J]. J Clin Oncol, 2010, 28(17):2839-2846.
10. HOLLAND P M, MILLER R, JONES J, et al. Combined therapy with the RANKL inhibitor RANK-Fc and rhApo2L/TRAIL/dulanermin reduces bone lesions and skeletal tumor burden in a model of breast cancer skeletal metastasis[J]. Cancer Biol Ther, 2010, 9:539-550.
11. DE WILT L H, KROON J, JANSEN G, et al. Bortezomib and TRAIL:a perfect match for apoptotic elimination of tumor cells?[J]. Crit Rev Oncol Hematol, 2013, 85(3):363-372.
12. RONG S, CAI J H, ANDREWS J. Cloning and apoptosis-inducing activities of canine and feline TRAIL[J]. Mol Cancer Ther, 2008, 7(7):2181-2191.
13. LI J F, AI H X, ZHANG J, et al. Molecular cloning, functional characterization and phylogenetic analysis of TRAIL in Japanese pufferfish Takifugu rubripes[J]. J Fish Biol, 2011, 79(3):747-760.
14. ZOR T. SELINGER Z. Linerarization of the Bradford protein assay increases its sensitivity:theoretical and experimental studies[J]. Anal Biochem, 1996, 236(2):302-308.
15. CAI H, CHEN L, WAN L, et al. High-level expression of a functional humanized anti-CTLA4 single-chain variable fragment antibody in Pichia pastoris[J]. Appl Microbiol Biotechnol, 2009, 82(1):41-48.
16. BERLEC A. ŠTRUKELJ B. Current state and recent advances in biopharmaceutical production in escherichia coli, yeasts and mammalian cells[J]. J Ind Microbiol Boit, 2013, 40:257-274.
17. WANG H Z, DAVIS J S. WU X W. Prolongs its plasma half-life and enhances its anti-tumor activity immunoglobulin Fc domain fusion to TRAIL significantly[J]. Mol Cancer Ther, 2014, 13(3):643-650.

Journal of Biomedical Engineering

Molecular Cloning, Recombinant Expression and Functional Characterization of the Soluble Tumor Necrosis Factor-related Apoptosis-inducing Ligand for the Macaca mulatta

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