Abstract 2071: SW43-DOX - a potential new targeted drug for the treatment of liver malignancies - An in vitro evaluation of receptor affinity and tumor internalization
Authors: Ludwig Johannes M. , Gai Yongkang , Lingyi Sun , Zeng Dexing , Kim Hyun S. 
Published: July 2016

Abstract
Purpose: The σ2-receptor is known to be overexpressed on many rapidly growing cancer cells, thus represents a tumor biomarker and is potentially attractive for targeted tumor therapy. We have synthesized the conjugate SW43-DOX consisting of the σ2-receptor agonist SW43 and the anticancer agent Doxorubicin (DOX). The purpose of this study was to compare the half maximal effective concentration (EC50) of SW43-DOX and DOX and to evaluate the receptor affinity and internalization of SW43-DOX in hepatocellular carcinoma and other cancer cell lines in vitro.
Material & Methods: SW43 was synthesized and conjugated with Doxorubicin (A Chemtek Inc.) For saturation binding & internalization assays, the chelator L-NETA was attached & loaded with Lu177. Hep G2, Hep 3B, Panc-1 & HT-29 cell lines were evaluated. EC50 was assessed by treatment with various concentrations ranging from 0.5-700 μM of SW43-DOX/Doxorubicin (CellTiter-Glo®; Promega). Saturation binding assay: σ2-receptor blocking was achieved with 10-100 μM SW43 for 15 min. at RT prior to adding 1-300 μM SW43-DOX-Lu177 in HBSS buffer (+0.1% BSA) incubating for 3.5h on ice. Internalization assay: After receptor blocking (7.5 μM SW43; 15 min.), cells were incubated with 75 nM SW43-DOX-Lu177 for .5, 1 & 2h at 37°C/5% CO2. The cell surface bound fraction from the internalization assay was scavenged by incubation with HBSS (+20 mM NaOAc; pH4.0; 10 min; 37°C/5% CO2). Cells were lysed with 0.5% SDS, radioactivity measured (Cobra -counter, Packard Bell) & normalized to whole cell protein. Statistics: EC50: Dose response non-linear fitting model; Internalization: ANOVA + Bonferroni post-hoc test, Saturation binding: One site saturation non-linear regression fit model. Prism V6.0.
Results: EC50 showed lower needed concentration (μM) for SW43-DOX compared to Doxorubicin: Hep G2 (12.0 (95%CI: 10.3-13.9) vs. 81.5 (95%CI: 66.7-99.5); p&lt;0.001), Hep 3B (8.0 (95%CI: 7.2-9) vs. 17.6 (95%CI: 14.1-22); p&lt;0.001), Panc-1 (21.59 (95%CI: 15.44-30.2) vs. 64.5 (95%CI: 50.3-82.6); p = 0.31), and HT-29 (20.7 (95%CI: 18.3-23.3) vs. 179.8 (95%CI: 65.9-593.5); p&lt;0.001). Maximal specific cell surface binding capacity (Bmax; pmol/mg): Hep G2 (21.5; 95%CI: 14.3-28.6), Hep 3B (35.2; 95%CI: 27.9-42.5), Panc-1 (66.5; 95%CI: 51.6-78.2) & HT-29 (33.4; 95%CI: 8.43-58.5). Specific binding affinity (KD) in nM: Hep G2 (49.2; 95%CI: 2.2-96.3), Hep 3B (40.8; 95%CI: 13.4-68.1), Panc-1 (96.4; 95%CI: 51.4-141.5), HT-29 (31.3; 95%CI: -40.5-84.4). Specific internalization was evident at all time points for all cell lines (p&lt;0.05). Specific internalization (pmol/mg ±SD) after 2h was 29.5 ±6.6 (Panc-1), 10.3 ±1.6 (HT-29), 18.2 ±2.36 (Hep G2) & 30.7 ±8.2 (Hep 3B).
Conclusion: SW43-DOX exerts a higher antitumoral effect than Doxorubicin & binds specifically onto the cell surface with high affinity & subsequently specific uptake via the σ2-receptor.
Citation Format: Johannes M. Ludwig, Yongkang Gai, Sun Lingyi, Dexing Zeng, Hyun S. Kim. SW43-DOX - a potential new targeted drug for the treatment of liver malignancies - An in vitro evaluation of receptor affinity and tumor internalization. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2071.