The novel sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in combination with gemcitabine - PubMed (original) (raw)

The novel sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in combination with gemcitabine

John R Hornick et al. Mol Cancer. 2010.

Abstract

Background: Sigma-2 receptors are over-expressed in proliferating cancer cells, making an attractive target for the targeted treatment of pancreatic cancer. In this study, we investigated the role of the novel sigma-2 receptor ligand SW43 to induce apoptosis and augment standard chemotherapy.

Results: The binding affinity for sigma-2 ligands is high in pancreas cancer, and they induce apoptosis with a rank order of SV119 < SW43 < SRM in vitro. Combining these compounds with gemcitabine further increased apoptosis and decreased viability. Our in vivo model showed that sigma-2 ligand treatment decreased tumor volume to the same extent as gemcitabine. However, SW43 combination treatment with gemcitabine was superior to the other compounds and resulted in stabilization of tumor volume during treatment, with minimal toxicities.

Conclusions: This study shows that the sigma-2 ligand SW43 has the greatest capacity to augment gemcitabine in a pre-clinical model of pancreas cancer and has provided us with the rationale to move this compound forward with clinical investigations for patients with pancreatic cancer.

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Figures

Figure 1

Figure 1

Sigma-2 ligands have high affinity for pancreas cancer and decrease viability. A. Chemical structures of sigma-2 ligands. B. Representative competitive binding data for inhibition of [125I]-ISO-2 binding to sigma-2 receptors in Panc02 tumor membrane homogenates. IC50 for binding affinities are pentazocine 1381 ± 33 nM, siramesine 1.9 ± 0.1 nM, SV119 7.8 ± 1.7 nM, and SW43 18 ± 2.1 nM. C. Pancreas cancer cell lines were treated with escalating doses of the sigma-2 ligands SV119, SW43, and siramesine (SRM) or DMSO vehicle for 18 hours and viability relative to vehicle determined by CellTiter-Glo Luminescent Viability Assay (Promega).

Figure 2

Figure 2

Sigma-2 ligands induce caspase-3 activation in pancreas cancer. Panc02 cells were pre-treated with caspase-3 inhibitor (DEVD-FMK 1 μM) or DMSO vehicle for one hour prior to exposure to sigma-2 ligands (25 μM) SV119, SW43, siramesine (SRM) or DMSO vehicle for 18 hours. A. Caspase-3 activation was quantified by cleavage of the fluorogenic substrate Ac-DEVD-AMC and expressed relative to vehicle. The caspase-3 specific inhibitor DEVD-FMK abrogates caspase-3 dependent cleavage. B. Caspase-3 inhibition does not protect cells from sigma-2 ligand induced cell death. C. Flow cytometry for Annexin-V verifies apoptosis by sigma-2 ligand. D. Representative flow cytometry dot plot showing cells moving from Annexin-V negative quadrant (upper panel) with vehicle towards Annexin-V positive quadrant (lower panel) with SW43 treatment. (Means ± SEM), n ≥ 3, *p < 0.05, **p > 0.05.

Figure 3

Figure 3

Sigma-2 ligands induce generation of reactive oxygen species in Panc02 cells. A. Sigma-2 ligands induce oxidation of 5-(and-6)-carboxy-2',7'-dichlorodihydro-fluorescein diacetate (carboxy-H2DCFDA) to 2',7'-dichlorofluorescein (DCF) and quantified by fluorescence of DCF. B. Viability was partially rescued for SW43 and siramesine (SRM) in the presence of alpha-tocopherol (200 μg/mL), but not for SV119. C. Sigma-2 ligand induced caspase-3 activity was partially reduced for SW43 in the presence of lipophilic α-toco, but completely for SRM. D. Flow cytometry for Annexin-V verifies that caspase-3 activity correlates with apoptosis. (Means ± SEM), n ≥ 3, *p < 0.05, **p > 0.05.

Figure 4

Figure 4

Sigma-2 ligands enhance gemcitabine induced cell death in pancreas cancer. Multiple pancreatic cancer cell lines, including Cfpac, Panc1, BxPC3 (Table 1), and Panc02 (shown) were treated with DMSO vehicle or sigma-2 ligands (25 μM) SV119, SW43, or siramesine (SRM) following 24 hour pre-treatment with gemcitabine (0.5 μM). Eighteen hours later cell viability was determined using the CellTiter-Glo Luminescent Viability Assay (Promega), (mean ± SEM), n ≥ 3.

Figure 5

Figure 5

Sigma-2 ligands decrease Panc02 tumor burden in C57BL/6. One million Panc02 cells were inoculated subcutaneously into female, 10 week old C57BL/6 mice and when tumors had reached a mean diameter of 5 mm, daily sigma-2 ligand treatment with or without weekly gemcitabine began by IP injection. All treatments decreased tumor volume compared to DMSO vehicle alone, p < 0.001. A. Siramesine (SRM) treatment did not confer a significantly different tumor volume than gemcitabine alone, *p > 0.05. Single agent sigma-2 ligands SV119 B. and SW43 C. decreased tumor volume similar to gemcitabine, *p > 0.05, while combination therapy produced much smaller tumors, **p < 0.001.

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