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

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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.

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Abbreviations

cGel-Mel:

Chemically conjugated gelonin-melittin

DMEM:

Dulbecco’s modified eagle medium

EDTA:

Ethylenediaminetetraacetic acid

F.I.:

Fluorescence intensity

FBS:

Fetal bovine serum

G/M:

Mixture of gelonin and melittin

Gelonin-SH:

Thiol activated-gelonin

HPLC:

High performance liquid chromatography

IPTG:

Isopropyl-β-D-thiogalactopyranoside

MWCO:

Molecular weight cut-off

NHS-PEG-PDP:

Pyridyldithio polyethylene glycol succinimidylpropionate

PFTs:

Pore-forming toxins

rGel-Mel:

Recombinant gelonin-melittin fusion chimera

RIPs:

Ribosome-inactivating proteins

TRITC:

Rhodamine B isothiocyanate

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ACKNOWLEDGMENTS AND DISCLOSURES

This work was partially supported by the NSFC 2013 A3 Foresight Program (81361140344), National Key Basic Research Program of China (2013CB932502) and National Natural Science Foundation of China (NSFC, 81402856). In addition, this work was also supported in part by National Institutes of Health R01 Grants CA114612 and grants from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2015R1A6A3A01020598 & NRF-2015R1C1A1A02036781). We thank Dr. Wolfgang E. Trommer (University of Kaiserslautern, Germany) for the gelonin expression vector (pET28a-Gel).

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    Authors and Affiliations

    1. College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, 501 Jinju Daero, Jinju, Gyeongnam, 52828, Republic of Korea

      Meong Cheol Shin & Kyoung Ah Min

    2. Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church St., Ann Arbor, Michigan, 48109-1065, USA

      Meong Cheol Shin, Kyoung Ah Min & Victor C. Yang

    3. College of Pharmacy, Inje University, 197 Injero, Gimhae, Gyeongnam, 50834, Republic of Korea

      Kyoung Ah Min

    4. Comparative Biomedicine Research Branch, Division of Cancer Biology, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang, Gyeonggi-do, 10408, Republic of Korea

      Heesun Cheong

    5. College of Pharmacy, Sunchon National University, 255 Jungang-ro, Suncheon, Jeonnam, 57922, Republic of Korea

      Cheol Moon

    6. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd., Shanghai, 201203, China

      Yongzhuo Huang

    7. Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin, 300070, China

      Huining He & Victor C. Yang

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    1. Meong Cheol Shin
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    Correspondence to Victor C. Yang.

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    Meong Cheol Shin and Kyoung Ah Min contributed equally to this work.

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    Shin, M.C., Min, K.A., Cheong, H. et al. Preparation and Characterization of Gelonin-Melittin Fusion Biotoxin for Synergistically Enhanced Anti-Tumor Activity. Pharm Res 33, 2218–2228 (2016). https://doi.org/10.1007/s11095-016-1959-4

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