Abstract 5488: Fusion protein capable to initiate two apoptotic pathways in cancer cells resistant to classical PARA's.

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising antitumor agent, but its clinical utility in human malignancies is limited by the resistance mechanism evolved in many cancers types, including overexpresion of antiapoptotic Bcl-2 like proteins and lack or disfunction of proapoptotic agents. Bid is well known proapoptotic factor able to inhibit Bcl-2 proteins after activation by caspases 8 or 10. Bid may also promote apoptosis by forming selective voltage dependent anion-channels in mitochondrial membrane through interaction with Bax protein. AD-O57.4 is a fusion protein consisting of a short peptide derived from Bid linked to an amino terminus of soluble TRAIL domain. The separating sequence contains additional motif recognized by MMPs and uPa proteases that enables cutting-off and releasing the proapoptotic peptide in tumor environment. Activity of AD-O57.4 was examined using a MTT assay on NCI panel and primal, patient derived cancer cell lines. Apoptosis was analyzed by caspase 3 activation and mitochondrial membrane potential change. The expression of Bid was determined by Western Blot analysis. Various of tested cell lines were very sensitive to AD-O57.4 protein with IC50 value between 0.06-173 ng/ml, whereas 5 cell lines showed weak sensitivity or remained resistant. Cytotoxic activity of TRAIL was significantly lower in comparison with AD-O57.4 protein. AD-O57.4 induced relatively strong mitochondrial depolarization both in moderate sensitive cell line (NCI-H460) and TRAIL-resistant cell line (A549). Analysis of caspase 3 activation and expression of Bid protein showed very fast induction of apoptosis by AD-O57.4 in comparison to TRAIL protein. Strong antitumor activity of AD-O57.4 fusion protein was analyzed on the xenograft models of series of human cancer cell lines regarding TRAIL sensitive human colorectal carcinoma Colo205, TRAIL resistant, hepatocellular carcinoma HepG2 and human uterine sarcoma (MES-SA/Dx5) - a multidrug resistant cell line where it caused almost complete tumor regression and showed much higher efficacy than TRAIL alone or standard chemotherapeutic agent. We demonstrated that AD-O57.4 protein has broad in vitro cytotoxic activity against a panel of cancer cell lines and in vivo antitumor activity on xenograft model. Sensitization by AD-O57.4 was dependent on short peptide derived from Bid protein indicating its role for mitochondrial signal amplification in the proapoptotic TRAIL activity. The use of a peptide derived from Bid protein causes a significant increase in potency of TRAIL activity and therefore TRAIL resistant cancer cells can be sensitized by the fusion peptide which can connect extrinsic and intrinsic pathways of apoptosis. Citation Format: Jerzy S. Pieczykolan, Sebastian D. Pawlak, Bartlomiej M. Zerek, Piotr K. Rozga, Anna Pieczykolan, Michal Szymanik, Albert Jaworski, Marlena Galazka, Wojciech Strozek, Katarzyna Wiciejowska, Malgorzata Teska-Kaminska, Lukasz Kutner. Fusion protein capable to initiate two apoptotic pathways in cancer cells resistant to classical PARA's. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5488. doi:10.1158/1538-7445.AM2013-5488