Cytotoxic Effects of a Novel Pyrazolopyrimidine Derivative Entrapped in Liposomes in Anaplastic Thyroid Cancer Cells in Vitro and in Xenograft Tumors in Vivo
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Endocrine-Related Cancer (2008) 15 499–510 Cytotoxic effects of a novel pyrazolopyrimidine derivative entrapped in liposomes in anaplastic thyroid cancer cells in vitro and in xenograft tumors in vivo M Celano, S Schenone1, D Cosco, M Navarra2, E Puxeddu3, L Racanicchi3, C Brullo1, E Varano, S Alcaro, E Ferretti 4, G Botta5, S Filetti 4, M Fresta, M Botta5 and D Russo Department of Pharmacobiological Sciences, University of Catanzaro ‘Magna Græcia’, Campus Universitario, loc. Germaneto, Viale Europa, 88100 Catanzaro, Italy 1Department of Pharmaceutical Sciences, University of Genova, Genova, Italy 2Pharmaco-Biological Department, University of Messina, Messina, Italy 3Department of Internal Medicine, University of Perugia, Perugia, Italy 4Department of Clinical Sciences, University of Roma ‘La Sapienza’, Roma, Italy 5Pharmaco-Chemical-Technological Department, University of Siena, Siena, Italy (Correspondence should be addressed to D Russo; Email: [email protected]) Abstract In this study, we evaluated the activity of two novel pyrazolopyrimidine derivatives (Si 34 and Si 35) against ARO cells, a human anaplastic thyroid cancer cell line. ARO cells exposed to different concentrations of the drugs showed a reduced growth rate and an increase of mortality. After 72 h incubation, doses of 5 and 10 mM Si 34 determined a decrease of cell counts by w25% and w75% compared with those of control cells respectively. Similar findings were observed using Si 35. Treatment with both Si 34 and Si 35 at 10 mM increased cell mortality also (w29% and w18% respectively). At these concentrations, a decrease in cyclin D1 levels was observed. To improve the biopharmaceutical properties, a liposome formulation was prepared. When entrapped in unilamellar liposomes, Si 34 exerted its cytotoxic effects even at lower doses (maximal inhibition at 5 mM) and after shorter incubation time (48 h) either in ARO or other thyroid cancer cell lines. The effects were associated with weak apoptotic death. Inhibition of epidermal growth factor- stimulated src and ERK phosphorylation, as well as reduction of migration properties of ARO cells was also observed. Moreover, the growth of tumor xenografts induced in severe combined immunodeficiency (SCID) mice was inhibited by i.v. administration of 25–50 mg/kg of the drug liposomal formulation. In conclusion, the liposomal preparation of this novel pyrazolopyrimidine derivative appears to be a promising tool for the treatment of anaplasic thyroid cancer. Endocrine-Related Cancer (2008) 15 499–510 Introduction Targeted molecular therapy is rapidly emerging as a The outcome of anaplastic thyroid carcinoma is far from promising strategy for the treatment of cancer. satisfactory. Most cases are subjected to multimodal Molecular characterization of the development and treatment regimens based on aggressive surgery, external progression of specific types of cancer (Gibbs 2000) beam radiation and chemotherapy, but this approach is has identified various classes of molecules that might rarely effective, and the prognosis for these patients is be productively targeted by novel anticancer agents. still poor (Ain 1998, Pasieka 2003). However, promising One of the most promising is the protein tyrosine results are being obtained in some patients thanks to kinase (PTK) family. These enzymes participate in combined therapies using novel molecular targeting various signaling pathways that control cell proli- agents (Kundra & Burman 2007). feration, secretion, adhesion, and responses to mitogen Endocrine-Related Cancer (2008) 15 499–510 Downloaded from Bioscientifica.comDOI: 10.1677/ERC-07-0243 at 09/24/2021 05:48:02PM 1351–0088/08/015–499 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access M Celano et al.: Novel drugs for thyroid cancer stress (Erpel & Courtneidge 1995, Parsons & Parsons phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1997). PTK malfunction is a hallmark of many types of (Si 35; Fig. 1) were synthesized as described (Carraro human neoplastic disease. Indeed, PTKs are encoded et al. 2004, Schenone et al.2004b). Briefly, ethyl by the vast majority of oncogenes and proto-oncogenes 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazolo-4- that have been implicated in human cancer. Regulation carboxylate was reacted with benzoyl isothionate and of thyrocyte growth also involves activation of PTKs, subsequently cyclized with sodium hydroxide. The including growth factor receptors and elements of resulting pyrazolopyrimidine derivative was then signal transduction pathways whose deregulation is chlorinated and reacted with amines. The pyrazolopyr- thought to lead to their neoplastic transformation imidine structure of Si 34 and Si 35 is identical to that (Fagin 2004). Of the various PTK inhibitors being of PP1 and PP2, but there is a thiomethyl group at C6, tested as potential anticancer agents (Zwick et al. a 2-chloro-2-phenylethyl chain at N1, and different 2001), those that suppress the activity of non-receptor amino groups at C4. Stock solutions of each PTKs of the Src family have attracted particular compound were prepared in 100% DMSO (50 mM/l) interest (Alvarez et al. 2006). Several pyrazolo[2,3-d]- and diluted with culture medium before addition to pyrimidines have been identified as potent and cell cultures. selective inhibitors of c-src activity. Two of these, the pyrazolopyrimidine derivatives PP1 and PP2, have Molecular modeling also been shown to block RET-induced thyroid tumorigenesis (Carlomagno et al. 2002, 2003). The molecular modeling work was carried out In previous studies, a series of novel compounds that coupling two different computational methods (confor- are structurally similar to PP1 and PP2 has shown mational analysis and ClogP estimation) as reported inhibition of epidermal growth factor (EGF)-stimu- lated src activation resulting in arrest of proliferation of A431 epidermoid tumor cells. In particular, two of these compounds, Si 34 and Si 35, exerted a strong antiproliferative effect also against a breast cancer cell line (Carraro et al. 2004, Schenone et al. 2004a,b). The aim of the present study was to investigate the effects of Si 34 and Si 35 against ARO cells, a well- established undifferentiated thyroid cancer cell line widely used to test in vitro the effects of novel potential anticancer drugs (Fagin et al. 1993). Moreover, a liposomal drug delivery system was prepared to improve the biopharmaceutical features of the pyrazo- lopyrimidine derivatives, such as the possibility to achieve a stable aqueous drug dispersion thus avoiding the toxic effect of any organic solvent, e.g., dimethyl- sulfoxide (DMSO), to be used to solubilize lipophilic drugs. The liposomal preparation of Si 34 was also tested in other thyroid cancer cell lines. Moreover, its effects were investigated on cell proliferation signal transduction pathways, apoptosis and invasiveness of ARO cells. Finally, the effect on the growth of ARO cells in non obese diabetic/severe combined immuno- deficiency (NOD-SCID) mice was analyzed. Methods Compounds N-benzyl-1-(2-chloro-2-phenylethyl)-6-(methylthio)- Figure 1 Structure of the two pyrazolopyrimidine derivates used in this study. (A) Chemical structures of compounds Si 34 and 1H-pyrazolo[3,4-d]pyrimidin-4-amine (Si 34) and Si 35. (B) Global minimum energy conformations of compounds 1-(2-chloro-2-phenylethyl)-6-(methylthio)-N-(2- Si 34 and Si 35. Downloaded from Bioscientifica.com at 09/24/2021 05:48:02PM via free access 500 www.endocrinology-journals.org Endocrine-Related Cancer (2008) 15 499–510 in a recent communication (Alcaro et al. 2007). Cell cultures and cell growth analysis The structures of Si 34 and Si 35 were built using the The ARO and FRO cells derived from a human Maestro Graphical interface and modeled using the anaplastic thyroid carcinoma, BCPAP, TPC-1, and Merk Molecular Force Field (MMFF) in Gibbs Born/ BHP 17-10 derived from poorly differentiated papillary Surface Area (GB/SA) water as implemented in thyroid carcinoma, and FTC-133 cells derived from a MacroModel ver 7.2 (Mohamadi et al. 1990). For follicular thyroid cancer were grown as previously each compound, 1000 conformations were generated described (Hoelting et al.1995, Ohta et al.1996, Russo by Monte–Carlo search and de-duplicated using et al.2001). Control cells were exposed to DMSO alone standard conditions. The lipophilicity of both at the same concentration present in cultures exposed to compounds was estimated by the ClogP method (Leo the test compounds (i.e., 1:5000 – 0.02% when final & Hansch 1971) using the SMILE notation format as concentrations of 10 mM/l compounds were used). input of the web interface. For cell growth rate analysis, 104 cells were plated in 60 mm dishes. Twelve hours later, when the cells had Preparation of Si 34-loaded liposomes presumably settled and begun to proliferate, test compounds were added. Cells were harvested after 24, Liposomes were made up of 1,2-dipalmitoyl-sn- 48, and 72 h incubation and counted in a Neubauer glycero-3-phosphocholine monohydrate/cholesterol/ chamber. The growth was also evaluated using the N-(carbonyl-methoxypolyethylene glycol-2000)-1,2- 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium distearoyl-sn-glycero-3-phosphoethanolamine (all pur- bromide (MTT) method and measure of 3H-thymidine chased by Genzyme, Suffolk, UK) in a 6:3:1 molar ratio. incorporation. For MTT assay, 3000 cells were seeded Si 34-loaded liposomes were prepared by dissolving onto 96 well plates in 100 ml medium. Twenty-four lipid components into a pyrex round-bottomed flask hours later, medium was changed, test compounds (1, 5, using a solvent mixture of chloroform/methanol (3:1 and 10 mM/l Si 34 free or entrapped in liposomes or 1, 5, v/v; Sigma–Aldrich S.r.l). Then, a solution (200 ml) of and 10 mM/l Si 35) or equivalent dilutions of DMSO the lipophilic drug (1 mg/ml) in chloroform/dichloro- (control) were added, and plates were incubated for 24, methane was added to the solvent mixture previously 48, and 72 h.