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Induction of potent TRAIL-mediated tumoricidal activity by hFLEX/Furin/TRAIL recombinant DNA construct
1Gene Vector Laboratory, Division of Cellular and Molecular Research, National Cancer Center, 11 Hospital Drive, Singapore 169610, Singapore.
Abstract:
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert selectively cytotoxic activity against many tumor cells but not normal cells. On the other hand, the ligand for the receptor tyrosine kinase Fms-like tyrosine kinase 3 (Flt3L) is a growth factor for hematopoietic progenitors and is a potent stimulating factor for dendritic and NK cells. Previously, we have demonstrated that it is possible to inhibit the outgrowth of primary tumors by the administration of an hFlex (the extracellular domain of the Flt3L) and TRAIL (amino acid residues 95-281) secreted fusion protein. Here, we report that by the insertion of a linker sequence encoding the cleavage site for the Golgi-expressed endoprotease furin between the DNA sequences encoding hFlex and TRAIL, the tumoricidal activity of the cleaved TRAIL protein generated was greatly enhanced in comparison to the hFlex/TRAIL fusion protein. Furthermore, we demonstrate that intratumoral injection of the hFlex/furin/TRAIL DNA, in conjunction with cationic liposomes, significantly suppressed the outgrowth of the human CNE-2 nasopharyngeal tumor xenografts in SCID mice. In situ histological examinations confirmed the expression of TRAIL in the treated tumor nodules and the induction of apoptosis was also evidenced by the presence of numerous pyknotic nuclei.
Insights
This study enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) efficacy by incorporating a furin cleavage site into a fusion protein. This improved TRAIL delivery suppressed tumor growth in mice.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells.
- Fms-like tyrosine kinase 3 ligand (Flt3L) promotes hematopoietic progenitor growth and stimulates immune cells.
- Previous work fused hFlex (extracellular Flt3L domain) with TRAIL to inhibit tumor outgrowth.
Purpose of the Study:
- To enhance the tumoricidal activity of TRAIL through protein engineering.
- To investigate the efficacy of a novel hFlex/furin/TRAIL fusion protein in vivo.
- To assess the impact of a furin cleavage site on TRAIL-mediated apoptosis.
Main Methods:
- Constructed a fusion protein encoding hFlex, a furin cleavage site, and TRAIL.
- Administered hFlex/furin/TRAIL DNA via intratumoral injection with cationic liposomes in SCID mice xenograft models.
- Performed histological examinations to confirm TRAIL expression and apoptosis induction.
Main Results:
- The hFlex/furin/TRAIL fusion protein demonstrated significantly enhanced tumoricidal activity compared to the original hFlex/TRAIL fusion.
- Intratumoral delivery of hFlex/furin/TRAIL DNA suppressed the growth of human CNE-2 nasopharyngeal tumor xenografts.
- Histological analysis confirmed TRAIL expression and apoptosis induction in treated tumors.
Conclusions:
- Incorporating a furin cleavage site into the hFlex/TRAIL fusion protein potentiates TRAIL's tumoricidal efficacy.
- The engineered hFlex/furin/TRAIL DNA holds promise as a therapeutic strategy for suppressing tumor growth.
- This approach offers a novel method for targeted delivery and activation of TRAIL for cancer therapy.
