Currently available chemotherapeutics have poor efficacy in pancreatic cancer patients. Chemotherapeutic drugs enter normal and cancer tissues with similar kinetics, resulting in toxic effects on normal tissues often leading to changes in the therapeutic plan such as dose reductions that leads to less effective drug combinations and discontinuation of therapy. Without specific targeting of these treatments, off-site toxicities can occur and the therapeutic window can be reduced. Sigma-2 receptors ligands appear to be selective to cancers: sigma-2 receptors are highly expressed in tumor cells and allow selective targeting of cancers [
38]. Sigma-2 ligands have been demonstrated to image not only pancreatic tumors in animal models [
21] but, more importantly, in clinical imaging studies [
39].
PB28 and PB282 compounds previously showed encouraging results in vitro and in vivo on the viability of BxPC3 pancreatic cancer cells [
24]. Therefore, herein, we selected five different sigma-2 ligands, that were tested on a wide panel of human and mouse pancreatic adenocarcinoma cell lines in vitro.
Most of the compounds displayed high sigma-2 affinity and exerted appreciable cytotoxic activity in some pancreatic cancer cell lines. We showed that, in particular, F281 had a greater capacity to decrease viability in the pancreas cancer cell lines tested, except for Panc1. The mechanism by which sigma-2 ligands, at high doses, induce cell death is not completely understood. We do know that sigma-2 ligands induce apoptosis by caspase-3 dependent and independent mechanisms [
16,
22,
40]. The generation of reactive oxygen species (ROS) has been well demonstrated to be both a by-product and a promoter of apoptosis and necrosis. of The apoptotic signals is intracellularly transmitted through production of ceramide or through direct effect on the mitochondria [
41]. Diverse mechanisms of apoptosis by ROS are known and may be cell-type dependent, while antioxidants or intracellular enzymes such as superoxide dismutase, catalase, and glutathione peroxidase have been shown to protect against ROS. We observed that the five sigma-2 agonists induced ROS in Panc02 cells and that apoptosis could be partially ameliorated by treatment with the antioxidant α-tocopherol. On the other hand, in this study, the small diffusible hydrophilic antioxidant NAC, a precursor of glutathione, did not protect against cell death by sigma-2 ligands, resulting in an even higher toxicity in co-administration assays with PB183 and PB282. Since at low concentrations (100 μM), there are chances that NAC behaves as an oxidant (rather than acting as an antioxidant or a radical scavenger), higher concentrations of NAC (10 mM) were also used but results did not change. NAC is a general reducing agent whereas α-tocopherol protects against oxidative stress is by preventing membrane lipid peroxidation, so that we believe that our results indicate crucial differences in the intracellular sites exposed to oxidative stress by sigma-2 receptor ligands. By contrast, PB282 did not show cytotoxic activity in Panc02 cell lines. However, only PB282 and PB221, at the concentration and time point used, were able to generate a strong caspase-3 activation, which was completely blocked by α-tocopherol and by caspase-3 inhibitor Z-DEVD-FMK. Since PB28, PB183, PB221 and F281 showed cytotoxicity in Panc02 cells, these results suggest that their cytotoxic activity is caspase-3 independent, except for PB221 that strongly activated caspase-3, but both cytotoxity and caspase-3 activation involved ROS generation. In addition, all the compounds strongly increased superoxide mitochondrial radical production except for PB282. This result suggests that superoxide mitochondrial radical production is one of the factors responsible for the cytotoxic effect of these ligands. In line with this result, in other pancreatic cancer cell lines where these sigma-2 ligands do not induce appreciable cytotoxicity (AsPC1, BxPC3, KP-2), much less superoxide radical formation in the mitochondria was detected. Only F281 generated some mitochondrial superoxide in AsPC1 and BxPC3, where a moderate cytotoxicity was noticed, in line with the hypothesis that mitochondrial superoxide pathway is at least partially responsible for the cytotoxic activity caused by our compounds in pancreatic cancer cells. These data indicate that structurally diverse sigma-2 ligands can activate different pathways in a panel of diverse cancer cell lines, and for the first time mitochondrial superoxide pathway was demonstrated for sigma-2 ligands. To verify that these in vitro results could potentially translate into an anti-tumorigenic effects in vivo, we utilized the aggressive cell line Panc02 in a syngeneic model of pancreatic cancer using C57BL/6 mice. We have shown that our treatment schedule resulted in minimal off-target toxicities in vivo (measured by serum studies and body weight) and was well tolerated. Although PB282 did not show cytotoxicity in Panc02 cells in vitro, daily treatment with PB28 and PB282 produced an effect statistically similar to gemcitabine alone, while other compounds such as PB221 and F281, that showed good EC
50 values in this cell line in vitro, did not perform as well in vivo. This could be due to metabolic effects observed in in vivo experiment. In particular, PB282 could produce active metabolites, in contrast to PB221, PB183 and F281 that are less effective in vivo despite their higher toxicity in the same tumor in vitro. We would also consider that survival would have likely been further increased if the treatment duration would have been extended since tumor volume was stabilized during the limited treatment period of only two weeks, without anticipating additional off-site toxicities. Along these lines, necropsy and laboratory evaluations demonstrated that PB28, PB221, PB183, F281 and PB282 had no major off-target effects even in naïve mice during continuous treatment as they well tolerated the drugs without signs of weight loss and no casualties as a result of drug treatments.