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01.12.2014 | PRECLINICAL STUDIES | Ausgabe 6/2014

Investigational New Drugs 6/2014

AD-O53.2—a novel recombinant fusion protein combining the activities of TRAIL/Apo2L and Smac/Diablo, overcomes resistance of human cancer cells to TRAIL/Apo2L

Zeitschrift:
Investigational New Drugs > Ausgabe 6/2014
Autoren:
Jerzy S. Pieczykolan, Konrad Kubiński, Maciej Masłyk, Sebastian D. Pawlak, Anna Pieczykolan, Piotr K. Rózga, Michał Szymanik, Marlena Gałązka, Małgorzata Teska-Kamińska, Bartłomiej Żerek, Katarzyna Bukato, Katarzyna Poleszak, Albert Jaworski, Wojciech Strożek, Robert Świder, Rafał Zieliński
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s10637-014-0153-y) contains supplementary material, which is available to authorized users.

Summary

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors became promising molecules for selective targeting of tumor cells without affecting normal tissue. Unfortunately, cancer cells have developed a number of mechanisms that confer resistance to TRAIL\Apo2L-induced apoptosis, which substantiates the need for development of alternative therapeutic strategies. Here we present a recombinant variant of TRAIL\Apo2L peptide, named AD-O53.2, fused to the peptide-derived from Smac/Diablo protein—the natural inhibitor of the apoptotic X-linked IAP (XIAP) protein considered as a pro-apoptotic agent. The proposed mechanism of action for this construct involves specific targeting of the tumor by TRAIL\Apo2L followed by activation and internalization of pro-apoptotic peptide into the cancer cells. While in the cytoplasm , the Smac\Diablo peptide inhibits activity of X-linked IAP (XIAP) proteins and promotes caspase-mediated apoptosis. AD-O53.2 construct was expressed in E.coli and purified by Ion Exchange Chromatography (IEC). Derived protein was initially characterized by circular dichroism spectroscopy (CD), HPLC-SEC chromatography, surface plasmon resonance, protease activation and cell proliferation assays. Our Smac/Diablo-TRAIL fusion variant was tested against a panel of cancer cells (including lung, colorectal, pancreatic, liver, kidney and uterine) and showed a potent cytotoxic effect with the IC50 values in femtomolar range for the most sensitive cell lines, while it remained ineffective against non-transformed HUVEC cells as well as isolated normal human and rat hepatocytes. Importantly, the construct was well tolerated by animals and significantly reduced the rate of the tumor growth in colon and lung adenocarcinoma animal models.

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Zusatzmaterial
Supplemental materials Figure S1 (DOC 67 kb)
10637_2014_153_MOESM1_ESM.doc
Supplemental materials Figure S2 (DOC 90 kb)
10637_2014_153_MOESM2_ESM.doc
Supplemental materials Figure S3 (DOC 178 kb)
10637_2014_153_MOESM3_ESM.doc
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