Sigma-2 receptor ligands potentiate conventional chemotherapies and improve survival in models of pancreatic adenocarcinoma

Abstract

Background: We have previously reported that the sigma-2 receptor is highly expressed in pancreas cancer. Furthermore, we have demonstrated that sigma-2 receptor specific ligands induce apoptosis in a dose-dependent fashion. Here, we examined whether sigma-2 receptor ligands potentiate conventional chemotherapies such as gemcitabine and paclitaxel.

Methods: Mouse (Panc-02) and human (CFPAC-1, Panc-1, AsPC-1) pancreas cancer cell lines were used in this study. Apoptosis was determined by FACS or immunohistochemical analysis after TUNEL and Caspase-3 staining. Combination therapy with the sigma-2 ligand SV119 and the conventional chemotherapies gemcitabine and paclitaxel was evaluated in an allogenic animal model of pancreas cancer.

Results: SV119, gemcitabine, and paclitaxel induced apoptosis in a dose-dependent fashion in all pancreas cancer cell lines tested. Combinations demonstrated increases in apoptosis. Mice were treated with SV119 (1 mg/day) which was administered in combination with paclitaxel (300 microg/day) over 7 days to mice with established tumors. A survival benefit was observed with combination therapy (p = 0.0002). Every other day treatment of SV119 (1 mg/day) in combination with weekly treatment of gemcitabine (1.5 mg/week) for 2 weeks also showed a survival benefit (p = 0.046). Animals tolerated the combination therapy and no gross toxicity was noted in serum biochemistry data or on necropsy.

Conclusion: SV119 augments tumoricidal activity of paclitaxel and gemcitabine without major side effects. These results highlight the potential utility of the sigma-2 ligand as an adjuvant treatment in pancreas cancer.

Figure 1

Sigma-2 ligands have a high affinity for pancreatic adenocarcinoma cell lines compared to normal cell lines. Representative FACS analysis of human (A.) and murine (B.) pancreatic adenocarcinoma cell lines treated with the FITC-conjugated Sigma-2 ligand, SW120. In Panel A, HPDE (immortalized pancreatic ductal epithelial cells) were used as a control. In Panel B, competitive inhibition of SW120 binding was demonstrated by preincubation with the Sigma-2 ligand, SW95. Pentazocine, a Sigma-1 receptor ligand, was also used as a control and did not demonstrate competitive inhibition. Experiments were performed in triplicate with comparable results.

Figure 2

The apoptotic effect of the sigma-2 ligand, SV119, is enhanced by conventional chemotherapy in vitro. Model pancreatic adenocarcinoma cell lines were treated with escalating doses of SV119, SV119 and gemcitabine, or SV119 and paclitaxel. After 24 hours of treatment, percent caspase-3 positive cells were determined by flow cytometry. Results are expressed as the mean, with bars representing standard error of the mean. Experiments were performed in triplicate with comparable results. Where indicated, * = P < 0.01 for SV119+gemcitabine or SV119+paclitaxel vs. SV119-only control.

Figure 3

The sigma-2 ligand SV119 induces moderate apoptosis in both G0 and G1 to G2/S phase of pancreatic cancer cells in vitro. The murine pancreatic adenocarcinoma cell, Panc02, was treated with SV119 alone or in combination with gemcitabine or paclitaxel. After 24 hours of treatment, samples were stained for cleaved caspase-3 and Ki67. Representative histograms are shown from an experiment performed in triplicate.

Figure 4

The pro-apoptotic activity of the sigma-2 ligand, SV119, is enhanced by conventional chemotherapy in vivo. C57BL/6 mice bearing implanted tumor allografts were treated with a single dose of SV119 and conventional chemotherapy (gemcitabine or paclitaxel). Twenty-four hours after treatment, tumors were harvested and single cell suspensions were generated. Percent active caspase-3 was then measured in tumor cells by flow cytometry. Each experimental group represents an n = 3. Results are expressed as the mean, with bars representing standard error of the mean.

Figure 5

The sigma-2 ligand, SV119, combined with gemcitabine suppresses tumor growth and increases survival in model pancreatic adenocarcinoma in vivo. C57BL/6 mice bearing established tumor allografts were treated with every other day SV119 (1 mg/mouse, i.p. for 7 days) and weekly gemcitabine (3 mg/mouse, i.p. for two weeks). Mean tumor diameter (Panel A) and survival (Panel B) were measured. * = vs. control.

Figure 6

The sigma-2 ligand, SV119, combined with paclitaxel suppresses tumor growth and increases survival in model pancreatic adenocarcinoma in vivo. C57BL/6 mice bearing established tumor allografts were treated with daily SV119 (1 mg/mouse, i.p. for 7 days) and daily paclitaxel (0.3 mg/mouse, i.p. for 7 days). Mean tumor diameter (Panel A) and survival (Panel B) were measured. * = vs. control.