Mauro Niso

Antiproliferative and cytotoxic effects of some σ2 agonists and σ1 antagonists in tumour cell lines

To establish the activity of σ ligands at σ1 and σ2 receptor, we chose two tumour cell lines, the human SK-N-SH neuroblastoma and the rat C6 glioma lines, which express σ2 receptors at a high density and σ1 receptors in their high-affinity or low-affinity state. We tested the σ2 receptor agonist PB28 and the σ2 antagonist AC927, and (+)-pentazocine and NE100 as agonist and antagonist, respectively, at σ1 receptors, with regard to antiproliferative and cytotoxic effects. In addition, 1,3-di(2-tolyl)guanidine (DTG) and haloperidol were tested as reference compounds displaying nearly equipotent σ affinity (σ2>σ1 and σ1>σ2, respectively). In both SK-N-SH and C6 cells, PB28 and NE100 displayed the most potent results both in antiproliferative and cytotoxic assay while AC927 and (+)-pentazocine were inactive in both assays. The cytotoxic and antiproliferative effects of DTG and haloperidol reflected their σ1 antagonist activity and σ2 agonist activity. Moreover, our results in the tumour cell lines correlated well with those for σ2 activity found previously in a functional assay in the guinea-pig bladder. These findings establish a new model for evaluating both σ2 and σ1 receptor activity of σ ligands, which could be useful for developing new ligands having mixed σ2 agonist/σ1 antagonist activity as potential antineoplastic agents.

Perspectives of P-Glycoprotein Modulating Agents in Oncology and Neurodegenerative Diseases: Pharmaceutical, Biological, and Diagnostic Potentials

Human ATP binding cassette (ABC) transporters belong to a family of 49 genes classified into seven subfamilies: ABCA, ABC-B, ABC-C, ABC-D, ABC-E, ABC-F, ABC-G. Some of these transporters are involved in multidrug resistance (MDR), in particular ABC-B1, better known as Pglycoprotein (P-gp), ABC-G2, better known as breast cancer resistance protein (BCRP), and ABC-C1-6, also known as multidrug resistance associated proteins (MRP1-6). 3 These transporters are overexpressed in several tumor cell lines and are responsible for drug efflux out of the cells. They use the energyofATPhydrolysis to extrude compoundsbya complex translocation process. Three models for P-gp translocation have been suggested: (1) pore, (2) flippase, and (3) hydrophobic vacuum cleaner models. In the pore model, drugs binding P-gp to the cytosol are transported out through a protein channel. In the flippase model, P-gp flips drugs that are transported from the inner to the outer compartment of the plasmamembrane against a concentration gradient. In the hydrophobic vacuum cleaner model, molecules recognized by P-gp in the lipid bilayer enter the protein from the membranous site and exit through the central cavity. P-gp contains 12 transmembrane helices organized in two membrane spanning domains (MSDs), each containing six transmembrane helices, and two nucleotide binding domains (NBDs) responsible for ATP binding. BCRP is a “half transporter” because it is formed by only one MSD and one NBD although it dimerizes to be fully active. MRPs differ from P-gp because they display three MSDs, and the additional domain contains five transmembrane domains. This review will focus on the physiological and pathological role of P-gp and will highlight the involvement of this protein both in MDR of tumors and in the physiological function of several barriers. P-gp overexpression is a significant factor in chemotherapy failure due to the ability of this pump to limit the cell accumulation of antineoplastic drugs. Moreover, P-gp is expressed in barriers such as the blood-brain barrier (BBB), bloodcerebro spinal fluid (B-CSF) barrier, and blood-testis barrier (BTB). It modulates the absorption and excretion of xenobiotics across these barriers. P-gp is localized at the apical membranes of liver, kidney, placenta, and the villus tip of enterocytes in the gut. In the gut, P-gp displays a strategic activity modulating access of drugs to the CYP3A4 enzyme, thereby regulating drug metabolism and absorption. 1.1. P-gp Involvement in Cancer and CNS Diseases.MDR is a complex phenomenon that is caused by tumor microenvironment changes or cancer cell-specific factors. Cancer cell-specific factors can occur at different levels: (i) increased drug efflux or decreased drug influx; (ii) drug inactivation; (iii) drug target modification; (iv) apoptosis evasion. The first of these mechanisms can be mediated by plasma membrane transporters such as P-gp. Asmentioned above, high levels of P-gp are reported in the luminal membrane of the endothelial cells constituting the BBB, B-CSF, and BTB. This strategic localization gives P-gp a crucial physiologically role in keeping drugs in the blood. P-gp exerts a protective function in the BBB; indeed, recent studies have reported a potential correlation between P-gp activity and/or expression in CNS disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and epilepsy. AD, a neurodegenerative disorder characterized by a progressive loss of cognitive function, evolves as several forms of dementia that display insoluble β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs). Aβ secretion is *To whom correspondence should be addressed. Phone: þ39-0805442727. Fax þ39-080-5442231. E-mail: colabufo@farmchim.uniba.it. Abbreviations: Aβ, β-amyloid; AB, apical-basolateral; ABC, ATP binding cassette; AD, Alzheimer’s disease; AML, acute myelogenous leukemia; ATP, adenosine 50-triphosphate; BA, basolateral-apical; Bmax, maximal bound; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; B-CSF, blood-cerebro spinal fluid; BTB, blood-testis barrier; Caco-2, human colonic carcinoma; calcein-AM, calcein acethoxymethyl ester; CD56þ, peripheral blood mononuclear cells; CHOP, cyclophosphamide, hydroxydaunorubicin (adriamycin), oncovin (vincristine), prednisone/prednisolone; CNS, central nervous system; C-PIB, C-Pittsburgh compound B; CYP3A4, cytochrome P-4503A4; DV, distribution volume; FDA, Food and Drug Administration; F-FDDNP, F(2-(1-{6-[(2-[F]fluoroethyl(methyl)amino]-2-naphthyl}ethylidene)malononitrile; FMZ, flumazenil; GI, gastrointestinal; HCT-8, human colorectale adenocarcinoma; ISF, interstitial fluid; Kd, dissociation constant; LBs, Lewy bodies; LLC-PK1, porcine kidney cell line; LRP1, low density lipoprotein receptor-related protein; MDCK, Madin-Darby canine kidney; MDE, multidrug efflux; MDR, multidrug resistance;MPPþ, 1-methyl-4-phenylpyridinium;MRP,multidrug resistance associated proteins;MSA, multisystem atrophy;MSD, membrane spanning domain; MTD, maximum tolerated dose; MTL, medial temporal lobe; NBD, nucleotide binding domain; NFTs, neurofibrillary tangles; NSCLC, non-small-cell lung cancer; PAHG, hippocampus, parahippocampal, ambient gyrus; Papp, apparent permeability; PD, Parkinson’s disease; PET, positron emission tomography; P-gp, Pglycoprotein; PSP, progressive supranuclear palsy; SCLC, small-cell lung cancer; SNP, single nucleotide polymorphism; SPECT, single photon emission computed tomography; T8, T-suppressor cells; TLE, temporal lobe epilepsy.

Cyclohexylpiperazine derivative PB28, a σ2 agonist and σ1 antagonist receptor, inhibits cell growth, modulates P-glycoprotein, and synergizes with anthracyclines in breast cancer

σ Ligands have recently been shown to have cytotoxic activity, to induce ceramide-dependent/caspase-independent apoptosis, and to down-regulate P-glycoprotein (P-gp) mRNA levels in some mouse and human models. In this study, we verified whether a mixed σ2 agonist/σ1 antagonist, PB28, was able to have antitumor activity and to enhance anthracycline efficacy in two human breast cancer cell lines, MCF7 and MCF7 ADR, both characterized by significant σ2 receptor expression, by high and low σ1 receptor expression, and low and high P-gp expression, respectively. In both cell lines, PB28 showed high σ2 receptor affinity and low σ1 receptor affinity; furthermore, it inhibited cell growth with a clear effect at 48 hours (IC50 in nanomolar range), a consistent time exposure-independent increase of G0-G1-phase fraction (of ∼20% of both cell lines) and caspase-independent apoptosis (15% increased after 1-day drug exposure). PB28 also reduced P-gp expression in a concentration- and time-dependent manner (∼60% in MCF7 and 90% in MCF7 ADR). We showed also a strong synergism between PB28 and doxorubicin by adopting either simultaneous or sequential schedules of the two drugs. We suggest that this synergism could depend on PB28-induced increase of intracellular accumulation of doxorubicin (∼50% in MCF7 and 75% in MCF7 ADR by flow cytometry analysis). In conclusion, we suggest that the σ2 agonist PB28 could be an interesting antitumor agent either in monotherapy or in combination with conventional drugs. [Mol Cancer Ther 2006;5(7):1807–16]

4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline derivatives as potent P-gp modulators

Starting from lead compound 1 (EC(50)=1.64 microM), its non-basic nucleus has been conformationally restricted by 4-biphenyl and 2-naphthyl moieties. In each series we investigated if the presence of H-bond donor or acceptor substituents, the basicity and the lipophilicity (clogP) were correlated with the P-gp inhibiting activity of tested compounds. In the biphenyl series, derivative 4d displayed the best results (EC(50)=0.05 microM). The corresponding amide 3d was found less active (EC(50)=3.5 microM) ascertaining the importance of basicity in this series whilst the presence of hydroxy or methoxy substituents seems to be negligible. In the naphthyl series, both the basicity and the presence of H-bond donor or acceptor groups seem to be negligible. Moreover, the lipophilicity did not influence the P-gp inhibition activity of each series. Specific biological assays have been carried out to establish the P-gp interacting mechanism of tested compounds discriminating between substrates and inhibitors. Moreover, compound 4d displayed a potent P-gp inhibition activity with good selectivity towards BCRP pump.

ABC Pumps and Their Role in Active Drug Transport

Pharmacokinetic limitations affect drug bioavailability determining the loss or the reduction of the pharmacological effects. The Gastro Intestinal tract (GI) and the Blood Brain Barrier (BBB) are the most important restrictive and selective physiological lines of defense of the organism. Although several parameters such as LogP, LogD and K(a) have been extensively employed for determining drug bioavailability, the active transports, present in these biological barriers, play an important role for dosing and limiting cell drugs concentration. In particular, ATP Binding Cassette (ABC) transporters are involved in the active transport both in GI and BBB. Their strategic activity and biochemical and pharmacological role are herein treated.

Elements in support of the ‘non-identity’ of the PGRMC1 protein with the σ2 receptor

σ2 Receptor subtype is overexpressed in a variety of human tumors, with σ2 agonists showing antiproliferative effects towards tumor cells through multiple pathways that depend both on the tumor cell type and on the molecule type. Therefore, σ2 receptor is an intriguing target for tumor diagnosis and treatment despite the fact that that it has not yet been cloned. One of the last attempts to characterize σ2 receptors led to identify it as the progesterone receptor membrane component 1 (PGRMC1). Although still controversial, such identity appears to have been accepted. We the aim of contributing to solve this controversy, in this work we stably silenced or overexpressed PGRMC1 protein in human MCF7 adenocarcinoma cells. Western blotting analyses were performed to quantify the presence of PGRMC1 protein on each of the three MCF7 cell lines variants, while scatchard analyses with radioligand were performed in order to determine the expression of the σ2 receptors. In order to correlate the antiproliferative effect of σ2 receptor agonist with PGRMC1 density, some σ2 ligands were administered to each of the three MCF7 cells variants. The results suggested that PGRMC1 and σ2 receptors are two different molecular entities.

Phenylsulfonylfuroxans as Modulators of Multidrug-Resistance-Associated Protein-1 and P-Glycoprotein

A series of furoxan derivatives were studied for their ability to interact with P-gp and MRP1 transporters in MDCK cells overexpressing these proteins. 3-Phenylsulfonyl substituted furoxans emerged as the most interesting compounds. All of them were capable of inhibiting P-gp, and a few also were capable of inhibiting MRP1. Substituents at the 4-position of 3-phenylsulfonylfuroxan scaffold were able to modulate the selectivity and the intensity of inhibition. In some cases, they reverted MRP1 inhibitor activity, namely, they were capable of potentiating MRP1 dependent efflux. When compounds 16 and 17 were coadministered with doxorubicin, they restored a high degree of the activity of the antibiotic. Preliminary immunoblotting studies carried out on these two compounds indicate that they are capable of nitrating P-gp, which in this form is likely unable to efflux the antibiotic.

Multi-drug-resistance-reverting agents: 2-aryloxazole and 2-arylthiazole derivatives as potent BCRP or MRP1 inhibitors.

The 2‐aryloxazole and 2‐arylthiazole scaffolds were used for generating compounds that we characterized for their inhibitory activity toward ATP binding cassette transporters involved in multi‐drug resistance, such as BCRP and MRP1, by using tumor cell lines overexpressing each transporter. These SAR studies are a significant step toward improving the inhibitory potency against P‐glycoprotein, BCRP, and MRP1.

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.

Bicalutamide failure in prostate cancer treatment: Involvement of Multi Drug Resistance proteins

Prolonged bicalutamide treatment induced pathology regression although relapses with a more aggressive form of prostate cancer have been observed. This failure could be due to androgen receptor mutation. In the present work we hypothesized an alternative mechanism responsible for bicalutamide failure involving activity of ATP-binding cassette (ABC) pumps such as P-glycoprotein, Breast Cancer Receptor Protein (BCRP), and Multi Resistant Proteins (MRPs) that extrude the androgen antagonist from the cell membrane. As experimental models androgen-dependent (LnCap) and androgen-independent (PC-3) prostate cancer cell lines have been employed. Bicalutamide has been tested in the cell lines mentioned above in the absence and in the presence of MC18, our potent P-glycoprotein/BCRP/MRP1 inhibitor. The results displayed that bicalutamide antiproliferative effect at 72 h was ameliorated in LnCap cells (EC(50) from 51.9+/-6.1 microM to 17.8+/-2.6 microM in the absence and in the presence of MC18, respectively) and restored in PC-3 cells (EC(50) from 150+/-2.4 microM to 60+/-3.5 microM in the absence and in the presence of MC18, respectively). Moreover, we established the contribution of each transporter employing stable transfected cells (MDCK) overexpressing P-glycoprotein or BCRP or MRP1 pump. The results displayed that P-glycoprotein and BCRP were involved in bicalutamide efflux while MRP1 was unable to bind the antiandrogen drug.