Collateral Sensitivity of sigma-2 Receptor ligands: Potentials in the Treatment of Multidrug Resistant Tumors

  Tumors remain one of the main causes of human illnesses and death with MultiDrug Resistance (MDR) being the most severe limitation to the success of chemotherapy. MDR is mainly due to the overexpression of drug efflux transporters, such as P-glycoprotein (P-gp), but attempts to inhibit P-gp have not been clinically successful so far. Lately, some agents were found to be more effective against P-gp overexpressing cells, showing a property termed “collateral sensitivity” (CS). The molecular bases of CS are poorly understood and hypersensitivity to reactive oxygen species (ROS) is one of the hypotheses that accounts for it. We recently identified a few sigma-2 (s2) receptors ligands endowed with CS, likely because of their interaction with P-gp. In fact, a number of CS agents are P-gp substrates: they are actively effluxed by P-gp, and it is believed that they activate a futile ATP cycle, increase oxidative phosphorylation leading to higher ROS production and oxidative stress. Therefore, we verified ROS involvement to study the CS properties of s2 receptors ligands/P-gp substrates (F408 and siramesine) whose activity was measured in three parental and Pg-p-overexpressing cell line pairs. We also demonstrated the major consumption of ATP induced by these compounds in P-gp overexpressing vs the parental cells. We analyzed the effects of siramesine and F408 on mitochondrial respiratory chain (source of ROS and intracellular ATP). Siramesine and F408 decreased both the electron flux rate and the ATP levels, with MDR cells undergoing a much more pronounced decrease than parent cells. Therefore, we demonstrated depletion of mitochondrial ATP supply by siramesine and F408 as the mechanism by which these s2 ligands likely induce CS. In conclusion, CS and s2 ligands-mediated actions warrant further investigation as a way to face MDR

Multifunctional thiosemicarbazones and deconstructed analogues as a strategy to study the involvement of metal chelation, Sigma-2 (Ï\u832) receptor and P-gp protein in the cytotoxic action: In vitro and in vivo activity in pancreatic tumors

The aggressiveness of pancreatic cancer urgently requires more efficient treatment options. Because the sigma-2 (Ï\u832) receptor was recently proposed as a promising target for pancreatic cancer therapy, we explored our previously developed multifunctional thiosemicarbazones, designed to synergistically impair cell energy levels, by targeting Ï\u832and P-gp proteins and chelating Iron. A deconstruction approach was herein applied by removing one function at a time from the potent multifunctional thiosemicarbazones 1 and 2, to investigate the contribution to cytotoxicity of each target involved. The results from in vitro (panel of pancreatic tumor cells) and in vivo experiments (C57BL/6 bearing KP02 tumor), suggest that while the multifunctional activity was not required for the antitumor activity of these thiosemicarbazones, Ï\u832-targeting appeared to allow alternative tumor cell death mechanisms, leading to potent and less toxic off-targets toxicities compared to other thiosemicarbazones devoid of Ï\u832-targeting

Cytotoxic pathways activated by multifunctional thiosemicarbazones targeting sigma-2 receptors in breast and lung carcinoma cells

Background: Multifunctional thiosemicarbazones (TSCs) able to bind sigma receptors and chelate metals are considered as a promising avenue for the treatment of pancreatic cancer due to the encouraging results obtained on in vitro and in vivo models. Here, we assessed the biochemical mechanism of these TSCs also on lung (A549) and breast (MCF7) cancer cells. Methods: The density of sigma-2 receptors in normal (BEAS-2B and MCF10A) and in lung and breast (A549 and MCF7) cancer cells was evaluated by flow cytometry. In these cells, cytotoxicity (MTT assay) and activation of ER- and mitochondria-dependent cell death pathways (by spectrofluorimetric assays to measure Caspases 3/7/9; qRT-PCR detection of GRP78, ATF6, IRE1, PERK; MitoSOX, DCFDA-AM and JC-1 staining), induced by the TSCs FA4, MLP44, PS3 and ACThio1, were evaluated. Results: FA4 and PS3 exerted more potent cytotoxicity than MLP44 and ACThio1 in all cancer cell lines, where the density of sigma-2 receptors was higher than in normal cells. Remarkably, FA4 promoted ER- and mitochondria-dependent cell death pathways in both cell models, whereas the other TSCs had variable, cell-dependent effects on the activation of the two proapoptotic pathways. Conclusions: Our data suggest that FA4 is a promising compound that deserves to be further studied for lung and breast cancer treatment. However, the other multifunctional TSCs also hold promise for the development of therapies towards a personalized medicine approach. Indeed, the presence of the sigma-2 receptor-targeting moiety would lead to a more specific tumor delivery embracing the characteristics of individual tumor types

New Pharmacological Strategies against Pancreatic Adenocarcinoma: The Multifunctional Thiosemicarbazone FA4

A new sigma-2 (σ2) receptor ligand (FA4) was efficiently synthesized and evaluated for cytotoxic, proapoptotic, and antimigratory activity on pancreatic ductal adenocarcinoma (PDAC) primary cell cultures, which restrained the aggressive and chemoresistant behavior of PDAC. This compound showed relevant antiproliferative activity with half maximal inhibitory concentration (IC50) values ranging from 0.701 to 0.825 μM. The cytotoxic activity was associated with induction of apoptosis, resulting in apoptotic indexes higher than those observed after exposure to a clinically relevant concentration of the gemcitabine, the first-line drug used against PDAC. Interestingly, FA4 was also able to significantly inhibit the migration rate of both PDAC-1 and PDAC-2 cells in the scratch wound-healing assay. In conclusion, our results support further studies to improve the library of thiosemicarbazones targeting the σ-2 receptor for a deeper understanding of the relationship between the biological activity of these compounds and the development of more efficient anticancer compounds against PDAC

Sigma-2 receptor and progesterone receptor membrane component 1 (PGRMC1) are two different proteins: Proofs by fluorescent labeling and binding of sigma-2 receptor ligands to PGRMC1

A controversial relationship between sigma-2 and progesterone receptor membrane component 1 (PGRMC1) proteins, both representing promising targets for the therapy and diagnosis of tumors, exists since 2011, when the sigma-2 receptor was reported to be identical to PGRMC1. Because a misidentification of these proteins will lead to biased future research hampering the possible diagnostic and therapeutic exploitation of the two targets, there is the need to solve the debate on their identity. With this aim, we have herein investigated uptake and distribution of structurally different fluorescent sigma-2 receptor ligands by flow cytometry and confocal microscopy in MCF7 cells, where together with intrinsic sigma-2 receptors, PGRMC1 was constitutively present or alternatively silenced or overexpressed. HCT116 cells, with constitutive or silenced PGRMC1, were also studied. These experiments showed that the fluorescent sigma-2 ligands bind to their receptor irrespective of PGRMC1 expression. Furthermore, isothermal titration calorimetry was conducted to examine if DTG and PB28, two structurally distinct nanomolar affinity sigma-2 ligands, bind to purified PGRMC1 proteins that have recently been revealed to form both apo-monomeric and heme-mediated dimeric forms. While no binding to apo-PGRMC1 monomer was detected, a micromolar affinity to heme-mediated dimerized PGRMC1 was demonstrated in DTG but not in PB28. The current data provide evidence that sigma-2 receptor and PGRMC1 are not identical, paving the pathway for future unbiased research in which these two attractive targets are treated as different proteins while the identification of the true sigma-2 protein further needs to be pursued

Development of N-(1-Adamantyl)benzamides as Novel Anti-Inflammatory Multitarget Agents Acting as Dual Modulators of the Cannabinoid CB2 Receptor and Fatty Acid Amide Hydrolase

Cannabinoid type 2 receptor (CB2R), belonging to the endocannabinoid system, is overexpressed in pathologies characterized by inflammation, and its activation counteracts inflammatory states. Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the degradation of the main endocannabinoid anandamide; thus, the simultaneous CB2R activation and FAAH inhibition may be a synergistic anti-inflammatory strategy. Encouraged by principal component analysis (PCA) data identifying a wide chemical space shared by CB2R and FAAH ligands, we designed a small library of adamantyl-benzamides, as potential dual agents, CB2R agonists, and FAAH inhibitors. The new compounds were tested for their CB2R affinity/selectivity and CB2R and FAAH activity. Derivatives 13, 26, and 27, displaying the best pharmacodynamic profile as CB2R full agonists and FAAH inhibitors, decreased pro-inflammatory and increased anti-inflammatory cytokines production. Molecular docking simulations complemented the experimental findings by providing a molecular rationale behind the observed activities. These multitarget ligands constitute promising anti-inflammatory agents