Preparation and Characterization of Gelonin-Melittin Fusion Biotoxin for Synergistically Enhanced Anti-Tumor Activity

Abstract

Purpose To investigate the applicability of fusion biotoxins combining pore-forming toxins (PFTs) and ribosome-inactivating proteins (RIPs) for the anti-cancer treatment. Methods Membrane active PFTs tend to destabilize cell membranes of tumor cells, but lack a warhead inducing significant cause of cell death. Cell-impermeable RIPs possess a powerful warhead, yet not able to enter the tumor cells. To address these challenges for anti-tumor effects, we introduced a fusion strategy of conjugating melittin (a PFT) and gelonin (a type 1 RIP) via chemical and recombinant methods, followed by in vitro assays and in vivo animal studies. Results In vitro characterization results confirmed that the chimeric gelonin-melittin fusion proteins retained equivalent intrinsic activity to that of unmodified gelonin in inhibiting protein translation. However, chemically conjugated gelonin-melittin (cGel-Mel) and recombinant chimeric gelonin-melittin fusion (rGel-Mel) exhibited greater cell uptake, yielding a significantly enhanced cytotoxic activity over treatment of gelonin, melittin or physical mixture of gelonin and melittin. Remarkably, cGel-Mel and rGel-Mel displayed 32- and 10-fold lower IC50 than gelonin in the cell lines. The superior anti-tumor efficacy of multivalent cGel-Mel to monovalent rGel-Mel suggested that valency could be a crucial factor for the extent of melittin-mediated cell uptake. Tumoricidal effects observed from animal studies were in good accordance with our findings from the cellular assays. Conclusions This study successfully demonstrated that fusion of biotoxins could provide a simple yet effective way to synergistically augment their anti-tumor activity.

Molecular tumor targeting of gelonin by fusion with F3 peptide

“…Nevertheless, the data obtained using a cell-free translational system (a rabbit reticulocyte lysate assay) confirmed that the F3-Gel product possessed protein synthesis inhibition activity equivalent to that of unmodified gelonin (Figure 2). This result appears consistent with our previous reports that gelonin is quite stable in fusions due to its structure [26,32] , which consequently contributes to the retention of its activity even after fusion with F3 peptide. To elucidate any anti-cancer targeting effects of F3-Gel or gelonin alone, for the in vitro cell experiments, we used HeLa, LnCaP, 9L, and U87 MG cancer cells because these cell lines were fully examined in other previous studies related to the F3 peptide [18,33] .…”

“…However, despite the high potency of gelonin, its clinical translation as an anticancer drug remains a challenge because of its poor cellular uptake. To overcome this challenge of intracellular delivery, we previously synthesized various gelonin fusion proteins with different membrane-active peptides (eg, low molecular weight protamine, TAT peptide and melittin) and tested the constructs in different tumor cell lines [24][25][26] . Despite achieving significantly improved cellular uptake, these fusion proteins lacked cell selectivity for their activity, and the toxicity concern remained a great obstacle for clinical application of these fusion proteins.…”

Melittin, a major peptide component of bee venom, and its conjugates in cancer therapy

“…The conjugate exhibited higher cellular uptake and significantly enhanced cytotoxic activity in Hela, colon CT26 and LS174T and malignant glioma 9L and U87 cancer cells over each agent alone or their physical mixture. Further, it also exhibited superior anti-tumor efficacy in HeLa tumor implanted on athymic nudes [88]. …”

Production of Recombinant Gelonin Using an Automated Liquid Chromatography System

“…When introduced to living cells, gelonin has, however, limited cytotoxic potential due to entrapment and subsequent degradation in endosomes and lysosomes [13]. Several attempts have been made to circumvent challenges related to intracellular delivery of gelonin including various membrane-active and/or pore-forming peptides [11,[14][15][16] and toxic gene therapy [17,18]. However, although these methods improve the cellular potency of gelonin considerably, subsequent toxicity to normal cells arises as a major hurdle which must be overcome for therapeutic applicability.…”