Giuseppe Gasparre

F281, synthetic agonist of the sigma-2 receptor, induces Ca2+ efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells

We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca(2+)] ([Ca(2+)](i)) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca(2+)](i). In particular, CB-64D induces a Ca(2+) response while PB28 supresses Ca(2+) signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24h incubation) was more cytotoxic than PB28 (EC(50) values 65.4nM and 8.13 microM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca(2+) from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca(2+)-entry through the channels activated by store depletion was also observed. After the increase in [Ca(2+)](i) and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death.

Effect of prostaglandin E2 on the proliferation, Ca2+ mobilization and cAMP in HT-29 human colon adenocarcinoma cells

Abstract Several lines of evidence suggest that non-steroidal antiinflammatory drugs (NSAIDs) have anticarcinogenic effects. The causal relationship linking the preventive effect of NSAIDs on colon cancer and the inhibition of prostaglandin synthesis is questioned by the contrasting results obtained by many laboratories. The experiments reported in this paper demonstrate that prostaglandin E 2 (PGE 2 ) did not stimulate the proliferation in HT-29 human colon adenocarcinoma cells under several experimental conditions. Moreover, PGE 2 and 17-phenyl trinor prostaglandin E 2 (a specific agonist of EP1 receptors) did not increase intracellular Ca 2+ concentration. Finally, PGE 2 did not affect the intracellular cAMP and did not reduce the isoproterenol dependent increase in cAMP. These results indicate that in HT-29 cells: (1) proliferation is not directly sensitive to PGE 2 ; and (2) PGE 2 does not stimulate a signal transduction pathway leading to intracellular increase in cAMP or Ca 2+ mobilization. Therefore, other cell lines should be used to assess the direct role played by prostanoids in promoting cell proliferation in colon cancer.

The proliferative response of HT-29 human colon adenocarcinoma cells to bombesin-like peptides.

Bombesin-like peptides (BLP) and their receptors are widely distributed throughout the intestine and are potential mitogens for gastrointestinal cancers. In this study we characterized the proliferation induced by BLP in the human adenocarcinoma cell line HT-29. The number of HT-29 cells, partially serum deprived (1% fetal bovine serum) for 48 h, was increased after 24 h of stimulation with bombesin, GRP, neuromedin B (NMB) and neuromedin C (NMC) ranging from 0.1 nM up to 1 microM. Reverse transcription polymerase chain reaction studies, revealed the presence of mRNA for NMB and for the GRP preferring receptor (GRP-R). mRNA for GRP, NMB preferring receptor (NMB-R) and bombesin receptor subtype 3 (BRS-3) were not detected. [D-Phe(6)]bombesin-(6-13)methyl ester (A1) and BIM-23127 (A2), are considered as inhibitors of binding to GRP-R and NMB-R, respectively. Surprisingly, A1 and A2 stimulated the proliferation of HT-29 cells. Moreover, in the simultaneous presence of 1 microM A1 and 0.1 microM GRP or 0.1 nM or 0.1 microM bombesin, inhibition of the proliferation was observed. Our data demonstrate that the proliferation induced by BLP in HT-29 cells is due to interaction with the GRP-R.

Naphthalenyl derivatives for hitting P-gp/MRP1/BCRP transporters.

Substituted naphthalenyl derivatives bearing oxazole, or thiazole or furyl heteronuclei have been carried out as bioisosters of aryl-oxazoles and -thiazoles derivatives previously reported in order to investigate the role of the hindrance on the activity towards P-gp/BCRP/and MRP1 transporters. In addition, the role of naphthalenyl group to modulate P-gp intrinsic activity of these compounds was ascertained. The results demonstrated that all naphthalenyl derivatives displayed comparable P-gp activity with respect to lead compounds previously characterized in our SAR studies but were less active towards BCRP and MRP1 pumps. In terms of intrinsic activity, the replacement of aryl with naphthalenyl moiety led to P-gp inhibitors, unambiguous or ambiguous substrates on the base of the heteronucleus and the substituent on the naphthalenyl fragment. Indeed, oxazole derivatives were: inhibitors (R=H, F, OH), unambiguous substrates (R=OCH(3)), or ambiguous substrate (R=Br); thiazole derivatives were: unambiguous substrates (R=OCH(3), Br), or ambiguous substrates (R=H, F). Finally furyl derivatives were ambiguous substrates.

The sigma-1 receptor antagonist PB212 reduces the Ca2+-release through the inositol (1, 4, 5)-trisphosphate receptor in SK-N-SH cells

Sigma-1 receptors are specifically located at the endoplasmic reticulum-mitochondrion interface, but upon stimulation by ligands or under prolonged cellular stress, they translocate to other areas of the cell. Sigma-1 receptors are involved in the regulation of intracellular [Ca(2+)] by affecting the Ca(2+)-influx or the release from intracellular stores. In SK-N-SH cells, we measured the affinity of 4-methyl-1-[4-(6-methoxynaphthalen-1-yl)butyl]piperidine (PB212) at sigma-1 receptor by using a competition binding assay with specific radioligand; we obtained a K(i) value=316 ± 19 nM. PB212 also showed an antiproliferative effect in SK-N-SH cells (EC(50)=32 ± 4 μM) but had no effect in MCF7 cells, which only express sigma-2 receptor; these findings suggest that PB212 behaves as a sigma-1 receptor antagonist. We have studied the effect of PB212 on Ca(2+) homeostasis of the SK-N-SH cell line with the fluorescent probe Fura-2. 100 μM PB212 induced a Ca(2+)-efflux from the endoplasmic reticulum through the inositol (1, 4, 5)-trisphosphate (IP(3)) receptor. Moreover, [PB212] ranging from 1 to 100μM reduced the Ca(2+)-response, triggered by carbachol or bradykinin that engage the phospholipase C/IP(3) pathway; such a response is generally increased by sigma-1 receptor agonists. On the other hand, PB212 did not reduce the Ca(2+)-response mediated by IP(3) in LoVo cells, which do not express neither sigma-1 nor sigma-2 receptors, and in MCF7 cells. The fact that the activity of the sigma-1 receptor can be experimentally modulated by agonists and antagonists supports the intriguing hypothesis that some endogenous molecules, unknown at the moment, modulate the sigma-1 receptor and its cellular targets.

The effects of new sigma (σ) receptor ligands, PB190 and PB212, in the models predictive of antidepressant activity.

BACKGROUND A number of σ receptor ligands have been demonstrated to possess antidepressant-like effect in some experimental paradigms (e.g. forced swim test, tail suspension test, olfactory bulbectomy model, conditioned fear stress). The objective of the present study was to find out whether PB190 and PB212, new σ1 receptor ligands, show the effects in some models predictive of antidepressant activity. METHODS The impact of PB190 and PB212 on the immobility time in the forced swim test (FST) and tail suspension test (TST) was assessed in C57BL/6J male mice. Extracellular bradykinin triggers a transient increase in intracellular calcium concentration by activating the phospholipase C/IP3 pathway. The intracellular calcium concentration was estimated with the dual wavelength ratiometric probe Fura-2. RESULTS In the FST model, PB190 showed a moderate antidepressant-like effect (only in the dose of 3mg/kg) which was enhanced by joint treatment with amantadine (AMA), 10mg/kg (inactive per se). The decrease in the immobility time induced by the combined treatment with PB190 and AMA was counteracted by PB212 and by BD1047, a σ1-receptor antagonist. The in vitro studies indicated that Ca(2+)-response was increased by 1μM PB190, like by the σ1-agonist (+)-pentazocine, while 1 μM PB212 behaved line σ1-antagonist, BD1063. On the other hand, 100 μM PB190 negatively affected the Ca(2+)-response after bradykinin. CONCLUSIONS The obtained results: 1/indicated that in the in vivo conditions PB190 behaved as a σ1-receptor agonist while PB212 counteracted its effect, confirming the in vitro data; 2/gave support to the hypothesis that σ1-receptors might be one of possible mechanisms by which drugs induce antidepressant-like activity; 3/revealed that this effect may be potentiated by NMDA receptor antagonists, e.g. AMA.

MC70 potentiates doxorubicin efficacy in colon and breast cancer in vitro treatment

A major limitation of cancer treatment is the ability of cancer cells to develop resistance to chemotherapeutic drugs, by the establishment of multidrug resistance. Here, we characterize MC70 as ABC transporters inhibitor and anticancer agent, alone or with chemotherapy. MC70 was analyzed for its interaction with ABCB1, ABCG2 and ABCC1 by specific transport assays. In breast and colon cancer cell lines, cell growth and apoptosis were measured by MTT assay and DNA laddering Elisa kit, respectively. Cell cycle perturbation and cellular targets modulation were analyzed by Flow-cytometry and Western blotting, respectively. MC70 interacted with ABC transporters. In breast cancer cells, MC70 slightly inhibited cell proliferation strongly enhancing doxorubicin effectiveness. By contrast, MC70 was found to inhibit cell growth in colon cancer cells without affecting doxorubicin efficacy and in combination with topoisomerase I inhibitors it could be a promising therapeutic approach. What is more, it was also observed that MC70 induced apoptosis, canceled in favor of necrosis when given in combination with high doses of doxorubicin. MC70 inhibited cell migration probably through its interaction with sigma-1 receptor. Modulations of i) cell cycle, ii) pAkt and the phosphorylation of the three MAPKs were highlighted, while any activity was excluded at transcription level, thus accounting for the phenotypic effects observed. MC70 might be considered as a new potential anticancer agent capable to i) enhance chemotherapy effectiveness and ii) to play a contributory role in the treatment of chemotherapy resistant tumors.

Investigation of σ receptors agonist/antagonist activity through N-(6-methoxytetralin-1-yl)- and N-(6-methoxynaphthalen-1-yl)alkyl derivatives of polymethylpiperidines.

A series of polymethyl-substituted piperidines linked to either a 6-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl or a 6-methoxynaphthalen-1-yl moiety was generated with the aim of verifying a previously generated hypothesis: tetralin and naphthalene nuclei confer opposite activity at the σ1 receptor. Compounds 6, 9 and 10 displayed appreciable affinity at both σ subtypes, but none of the novel compounds displayed significant antiproliferative activity in MCF7wt and MCF7σ1 cell lines. The effect on bradikynin-triggered Ca(2+) mobilization was studied as a methodology to suggest σ receptors mediated activity.