Pancreatic cancer is an aggressive disease and current therapy is disappointing. Many tumors are inoperable at the time of diagnosis, and recurrence is high in those deemed operable. Therefore, many patients in clinical trials have received chemotherapy or radiation therapy in different treatment combinations, as neo-adjuvants or as an adjuvant following surgery. While the technology of therapeutic options has increased over the years, the long-term survival rate has changed little over the past few decades, stressing the need for more effective treatments. We have shown that sigma-2 ligands increase survival of tumor bearing mice and are additive to combination regimens with chemotherapies. In this study, we identified a novel sigma-2 ligand that has superior efficacy in pancreatic cancer with limited offsite toxicity.
Chemotherapies and radiation are common in that they disregulate the cell cycle directly or indirectly, and activate apoptosis of tumor cells. Without specific targeting of these treatments, systemic toxicities occur, decreasing the therapeutic window. Compounds which target sigma-2 receptors appear to be selective to cancers. While, sigma-1 and sigma-2 receptors are located in many normal tissues, sigma-2 receptors are highly expressed in tumor cells, allowing selective targeting. We have also shown sigma-2 receptor ligands specifically taken up in a mouse model of pancreas cancer by micro PET/CT imaging [8
]. This specificity may also allow targeted therapy by the delivery of pro-apoptotic molecules specifically to pancreatic cancer cells.
In this study, we identified a novel sigma-2 receptor ligand that has superior efficacy in pancreatic cancer. Multiple human and mouse pancreatic adenocarcinoma cell lines were tested in vitro to asses the effect of our compounds on viability. We showed that SW43 had a greater capacity to decrease viability compared with SV119 in all pancreas cancer cell lines tested, with an IC50 value at least half that of SV119. Sigma-2 ligands also had an additive effect with gemcitabine to induce cell death. Cell death by SW43 occurs through a mechanism consistent with apoptosis in pancreatic cancer cell lines and induces caspase-3 activity even greater than our previous compound SV119, though caspase-3 inhibitor could not prevent cell death. The SW43 effect in vitro was similar to that of siramesine, a widely studied sigma-2 ligand. We verified that increased caspase-3 activity correlated with induction of apoptosis by flow cytometry for Annexin-V binding and the apoptotic effect was even further increased by pre-treatment with gemcitabine.
We chose siramesine for comparison in our study because it is an established sigma-2 ligand that has been used in a phase I clinical trial as an anxiolytic [33
]. Although siramesine did not meet the objective clinical response in this trial, it was shown to be well tolerated in 200 healthy volunteers. Following that trial, siramesine has been tested in mice for the treatment of breast cancer and was found to induce cytotoxicity mediated in part by oxidative stress [28
]. It was not determined whether this effect was mediated by sigma-2 receptors or by siramesine interactions in the membranes, though siramesine has been recently shown to form high-affinity complexes with phosphatidic acid [34
The generation of reactive oxygen species (ROS) has been well established to be a by-product as well as an initiator of apoptosis and necrosis. Intracellular transmission of apoptotic signals occurs through production of ceramide or directly acting on the mitochondria [35
]. Depending on cell type, ROS have been shown to increase mRNA and protein expression of pro-apoptotic molecules such as FasL, FasR, Bax, and caspases-2 and -3, modulation of MAPK pathways, as well as release of cytochrome c from the mitochondria [36
]. Mechanisms of apoptosis by ROS are varied and may be cell-type dependent.
We observed that SW43 induces ROS in pancreas cancer cells and that apoptosis can be partially relieved by treatment with the antioxidant alpha-tocopherol. In contrast, apoptosis by siramesine induced ROS was completely blocked by alpha-tocopherol, while SV119 generated little ROS. This suggests that the mechanism of SW43 induced apoptosis is at least partially through the generation of ROS. Based on the similar structure and binding characteristics of SW43 and SV119, it is plausible that they share an apoptotic pathway independent of siramesine.
In Panc02 cells, binding affinity is high for sigma-2 and low for sigma-1, with the relative affinity for sigma-2 ligands being SRM > SW43 = SV119. Increased binding affinity of siramesine supports our data showing a lower IC50
for viability than SW43 and SV119 and suggests that sigma-2 receptor binding is important for initiation of apoptosis. Binding affinity does not explain the lower IC50
for SW43 versus SV119 though. We speculate that the extended aminoakyl chain of SW43 increases lipophilicity and enhances membrane diffusion into the cell. Evidence for membrane diffusion is supported by our previous studies showing that sigma-2 receptor ligand internalization is only partially blocked with the well studied endocytosis inhibitor phenylarsine oxide [38
To verify that these promising in vitro results translate into an anti-tumorigenic effect in vivo, we utilized the aggressive Panc02 cell line in C57BL/6 mice. We have shown that this treatment schedule resulted in minimal toxicity in vivo that was well tolerated. Daily treatment of sigma-2 ligands produced an effect statistically similar to gemcitabine alone. Though SRM showed superior efficacy in vitro, the results did not translate in our in vivo model. SW43 combination therapy with gemcitabine resulted in the smallest tumor volume which had stabilized during the two week treatment period. As well, we demonstrate a therapeutic drug combination that completely eradicated an established Panc02 tumor in a C57BL/6 mouse, which we have not observed in over a decade of our own experience. Survival was increased for the combination of SW43 with gemcitabine from this treatment course. We expect that based on the stabilization of tumor volume during the limited treatment period (2 weeks), survival would be increased if the treatment duration was lengthened.