Multifunctional thiosemicarbazones targeting sigma receptors: in vitro and in vivo antitumor activities in pancreatic cancer models

Abstract

Purpose Association of the metal chelating portion of thiosemicarbazone with the cytotoxic activity of sigma-2 receptors appears a promising strategy for the treatment of pancreatic tumors. Here, we developed a novel sigma-2 receptor targeting thiosemicarbazone (FA4) that incorporates a moiety associated with lysosome destabilization and ROS increase in order to design more efficient antitumor agents. Methods The density of sigma receptors in pancreatic cancer cells was evaluated by flow cytometry. In these cells, cytotoxicity (MTT assay) and activation of ER- and mitochondria-dependent cell death pathways (mRNA expression of GRP78, ATF6, IRE1, PERK; ROS levels by MitoSOX and DCFDA-AM; JC-1 staining) induced by the thiosemicarbazones FA4, MLP44, PS3 and ACthio-1, were evaluated. The expression of autophagic proteins (ATG5, ATG7, ATG12, beclin, p62 and LC3-I) was also studied. In addition, the in vivo effect of FA4 in xenograft models with and without gemcitabine challenge was investigated. Results We found that FA4 exerted a more potent cytotoxicity than previously studied thiosemicarbazones (MLP44, PS3 and ACthio-1), which were found to display variable effects on the ER or the mitochondria-dependent pro-apoptotic axis. By contrast, FA4 activated pro-apoptotic pathways and decreased autophagy, except in MiaPaCa2 cells, in which autophagic proteins were expressed at lower levels and remained unmodified by FA4. FA4 treatment of PANC-1 xenografted mouse models, poorly responsive to conventional chemotherapy, significantly reduced tumor volumes and increased intratumor apoptosis compared to gemcitabine, with no signs of toxicity. Conclusions Our data indicate that FA4 exhibits encouraging activity in pancreatic cancer cells unresponsive to gemcitabine. These results warrant further investigation in patient-derived pancreatic cancers, and hold promise for the development of therapies that can more efficiently target the specific characteristics of individual tumor types.

New Pharmacological Strategies against Pancreatic Adenocarcinoma: The Multifunctional Thiosemicarbazone FA4

“…While the affinity for the σ2 receptor was subnanomolar (Ki = 0.43 nM, Table 1), for MT8, the affinity for the σ-1 receptor was in the two-digit nanomolar range (Ki = 27.3 nM, Table 1): this compound was a moderately selective high-affinity σ-2 receptor ligand, with an improved profile compared to siramesine that, from our perspective, did not discriminate between the two σ subtypes (Ki = 10.5 nM for σ1 and Ki = 12.6 nM for σ2 receptors, respectively) [22]. a Values represent the mean of n ≥ 3 separate experiments in duplicate ±SEM; b value obtained by flow cytometry assay with a fluorescent ligand as described in [10]; c value obtained from [10]; d value obtained from [22].…”

“…FA4, which was designed based on the σ2 reference agonist siramesine, demonstrated a superior anticancer activity in a panel of PDAC cells in comparison with its congeners MLP44 and PS3, that only differ in the basic moiety, with the (Z)-N,Ndimethyl-2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamide unchanged (Figure 2). The results obtained in commercial PDAC cell lines [10] highlighted how the basic moiety 3H-spiro[isobenzofuran-1,4′-piperidine] of FA4, which differs from the 6,7dimethoxytetrahydroisoquinoline in MLP44 or the 1-cyclohexylpiperazine in PS3, may drive this increased activity. While MLP44 and PS3 underwent a deconstruction approach in order to evaluate how the chelating moiety impacts on the σ receptor affinity and overall activity, this was not investigated for FA4.…”

“…While MLP44 and PS3 underwent a deconstruction approach in order to evaluate how the chelating moiety impacts on the σ receptor affinity and overall activity, this was not investigated for FA4. Indeed, the removal of the thiosemicarbazone moiety importantly reduced the cytotoxic activity of MLP44 and PS3 [10]. Thus, FA4 was deconstructed by removing the thiosemicarbazone moiety, resulting…”

“…While MLP44 and PS3 underwent a deconstruction approach in order to evaluate how the chelating moiety impacts on the σ receptor affinity and overall activity, this was not investigated for FA4. Indeed, the removal of the thiosemicarbazone moiety importantly reduced the cytotoxic activity of MLP44 and PS3 [10]. Thus, FA4 was deconstructed by removing the thiosemicarbazone moiety, resulting The results obtained in commercial PDAC cell lines [10] highlighted how the basic moiety 3H-spiro[isobenzofuran-1,4 -piperidine] of FA4, which differs from the 6,7dimethoxytetrahydroisoquinoline in MLP44 or the 1-cyclohexylpiperazine in PS3, may drive this increased activity.…”

“…Indeed, the removal of the thiosemicarbazone moiety importantly reduced the cytotoxic activity of MLP44 and PS3 [10]. Thus, FA4 was deconstructed by removing the thiosemicarbazone moiety, resulting The results obtained in commercial PDAC cell lines [10] highlighted how the basic moiety 3H-spiro[isobenzofuran-1,4 -piperidine] of FA4, which differs from the 6,7dimethoxytetrahydroisoquinoline in MLP44 or the 1-cyclohexylpiperazine in PS3, may drive this increased activity. While MLP44 and PS3 underwent a deconstruction approach in order to evaluate how the chelating moiety impacts on the σ receptor affinity and overall activity, this was not investigated for FA4.…”