Cyclohexylpiperazine derivative PB28, a S 2 agonist and S 1 antagonistreceptor,inhibitscellgrowth,modulates P-glycoprotein,andsynergizeswithanthracyclines inbreastcancer Amalia Azzariti, 1 Nicola A. Colabufo, 2 FrancescoBerardi, 2 LetiziaPorcelli, 1 MauroNiso, 2 Grazia M. Simone, 1 Roberto Perrone, 2 and Angelo Paradiso 1 1 Clinical Experimental Oncology Laboratory, National Cancer Institute; and 2 Dipartimento Farmaco-Chimico, University of Bari, Bari, Italy Abstract S Ligands have recently been shown to have cytotoxic activity, to induce ceramide-dependent/caspase-inde- pendent 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 S 2 agonist/S 1 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 character- ized by significant S 2 receptor expression, by high and low S 1 receptor expression, and low and high P-gp expression, respectively. In both cell lines, PB28 showed high S 2 receptor affinity and low S 1 receptor affinity; furthermore, it inhibited cell growth with a clear effect at 48 hours (IC 50 in nanomolar range), a consistent time exposure-indepen- dent increase of G 0 -G 1 -phase fraction (of f20% 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 (f60% 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 (f50% in MCF7 and 75% in MCF7 ADR by flow cytometry analysis). In conclusion, we suggest that the S 2 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] Introduction j Receptors have been described in human cells by Martin et al. (1) and classified into two distinct class, j 1 and j 2 . These receptors have been recognized in the central nervous system, in endocrine tissues, in the liver and kidneys, and in immune system cells; now, most of their physiologic functions have been studied and elucidated (2–4). High levels of j 1 receptors have also been found in embryonic stem cells (5). At subcellular level, j 1 receptors seems to be localized in the plasma membrane, mitochon- dria, and endoplasmic reticulum (6–9). By contrast, the localization of j 2 receptors is still unknown. The j 1 receptor has been isolated and its cDNA has been cloned (10). The receptor protein (25 kDa) consists of 223 amino acids and displays 30% homology with the D 8 -D 7 isomerase even if the j 1 receptor has no enzyme activity. The characteristics of the j 2 receptor are less known because of the lack of potent and selective ligands, which makes its purification and characterization very difficult (11). In the central nervous system, the j 1 subtype is involved in the modulation of K + and Ca 2+ channels and in N-methyl-D- aspartate, serotonergic, dopaminergic, and muscarinic neurotransmission (12–14). Both j receptor subtypes are expressed in human tumor cells; furthermore, recent experimental results have shown that the differential expression of each subtype could be of prognostic value (15–17). It has been also reported that j 2 expression is related to tumor growth rate, supporting the idea that it could be a reliable tumor cell proliferation biomarker (18, 19). The high expression of j 2 receptors by a variety of cancer cell lines has also suggested that they may be used as targets for antitumor agents. Preliminary studies have confirmed that j receptor ligands are able to kill glioma cells and the cytotoxic effects of these drugs have been found to be mediated specifically by the j 2 receptor subtype (20–22). Pharmacologic studies have shown that j 2 receptor agonists induce the modulation of different cell processes by promoting Ca 2+ depletion from endoplasmic and mitochondrial stores and inducing caspase-indepen- dent apoptosis (23–25). These compounds were also able to decrease P-glycoprotein (P-gp or MultiDrug Resistance-1; ref. 26), which is one of the three major groups of ATP- binding cassette transporters involved in multidrug resis- tance (MDR; ref. 27) together with other MRP proteins and ABCG2. Many cytotoxic drugs, such as anthracyclines (28), Received 10/4/05; revised 4/20/06; accepted 5/12/06. Grant support: Italian Association for Cancer Research (2004). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Amalia Azzariti, Clinical Experimental Oncology Laboratory, National Cancer Institute, Via Amendola 209, 70125 Bari, Italy. Phone: 39-80-5555530; Fax: 39-80-5555561. E-mail: amaliaris@yahoo.com Copyright C 2006 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-05-0402 1807 Mol Cancer Ther 2006;5(7). July 2006 Downloaded from http://aacrjournals.org/mct/article-pdf/5/7/1807/1873468/1807.pdf by guest on 17 June 2022