Molecular and Cellular Pharmacology
C(5) modified uracil derivatives showing antiproliferative and erythroid differentiation inducing activities on human chronic myelogenous leukemia K562 cells

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Abstract

The K562 cell line has been proposed as a useful experimental system to identify anti-tumor compounds acting by inducing terminal erythroid differentiation. K562 cells exhibit a low proportion of hemoglobin-synthesizing cells under standard cell growth conditions, but are able to undergo terminal erythroid differentiation when treated with a variety of anti-tumor compounds. In this paper we report a screening study on a set of different modified C(5) uracil derivatives for the evaluation of their antiproliferative effect in connection with erythroid differentiation pathways, and for defining a new class of drug candidates for the treatment of chronic myelogenous leukemia. Activity of the derivatives tested can be classified in two effect: an antiproliferative effect linked to a high level of erythroid differentiation activity and an antiproliferative effect without activation of gamma globin genes The highest antiproliferative effect and erythroid induction was shown by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine did not show any effect, suggesting the importance of the linear alkyl chain in position N(1). To our knowledge this compound should be considered among the most efficient inducers of erythroid differentiation of K562 cells. This work is the starting point for the quest of more effective and specific drugs for the induction of terminal erythroid differentiation, for leading new insights in the treatment of neoplastic diseases with molecules acting by inducing differentiation rather than by simply exerting cytotoxic effects.

Introduction

The pyrimidine system is a very important pharmacophor core of naturally occurring and synthetic bioactive compounds, interacting with the synthesis and function of nucleic acids, interfering with biosynthetic pathways and competing for the same binding sites of naturally occurring pyrimidines (Lagoja, 2005). Modified pyrimidines are currently used as drugs; for example fluorouracil has cytostatic effects and is currently used in cancer therapeutics, azidothymidine (AZT) was the first applied drug for HIV treatment and bacimethrin (4-amino-5-hydroxymethyl-2-methoxypyrimidine) is known as the simplest pyrimidine antibiotics (Reddick et al., 2001).

The K562 cell line (Lozzio and Lozzio, 1975) has been proposed as a very useful experimental system to identify antitumor compounds (Lampronti et al., 2003a, Lampronti et al., 2003b, Lampronti et al., 2006) acting by inducing terminal erythroid differentiation of this as well as other tumor cell lines (Bianchi et al., 1999, Bianchi et al., 2000, Bianchi et al., 2001, Chiarabelli et al., 2003, Gambari and Fibach, 2007, Olivieri, 1996, Osti et al., 1997). K562 cells exhibit a low proportion of hemoglobin-synthesizing cells under standard cell growth conditions, but are able to undergo terminal erythroid differentiation when treated with a variety of compounds, including short fatty acids (Gambari and Fibach, 2007), 5-azacytidine (Gambari and Fibach, 2007), mithramycin and chromomycin (Bianchi et al., 1999, Fibach et al., 2003), cisplatin and cisplatin analogs (Bianchi et al., 2000), tallimustine (Bianchi et al., 2001, Gambari and Fibach, 2007), rapamycin (Fibach et al., 2006), everolimus (Zuccato et al., 2007), psoralens (Lampronti et al., 2003a, Lampronti et al., 2003b) and resveratrol (Bianchi et al., 2009). Following erythroid induction, increase of expression of ε and γ globin genes is observed, leading to accumulation of Hb Portland (ζ2γ2) and Hb Gower 1 (ζ2ε2) (Gambari and Fibach, 2007).

The design and production of antiproliferative molecules targeting the K562 cell system might be of great interest for the development of cocktails exhibiting applications in the treatment of chronic myelogenous leukemia. For instance smenospongine (Aoki et al., 2004a, Aoki et al., 2004b), crambescidin 800 (Aoki et al., 2004a, Aoki et al., 2004b) and doxorubicin derivatives (Szulawska et al., 2007) were reported as molecules of possible interest for inhibiting of chronic myelogenous leukemia cell growth, stimulating terminal differentiation along the erythroid program. Some molecules, such as Pivanex (an HDAC inhibitor) (Rabizadeh et al., 2007) and a morpholine derivative of doxorubicin (Jakubowska et al., 2008), are synergistic with the most common anti-chronic myelogenous leukemia agents, STI571 (Imatinib). In addition to synergistic effects, molecules inducing differentiation might be of great interest for treatment of Imatinib mesylate-resistant human chronic myelogenous leukemia cell lines, as recently demonstrated for the phytoalexin resveratrol (Puissant et al., 2008).

Recently we reported a novel class of C-5 linked N-1 alkylated uracil dimers rationally designed to potentially interact with adenine in biological systems. These pyrimidine derivatives have shown antiproliferative and erythroid differentiation activities toward human chronic myelogenous leukemia K562 cells (Accetta et al., 2009). In this paper we report a screening study on a set of different modified C(5) uracil derivatives (Table 1) for the evaluation of their antiproliferative effect in connection with erythroid differentiation pathways, and for defining a new class of drugs candidates for the treatment of chronic myelogenous leukemia.

Section snippets

Chemical synthesis of uracil compounds

Compounds 1, 2 and 4 were purchased from Sigma-Aldrich and tested without further purification. Synthesis of compounds 5, 8, 9, 10 and 11 was previously reported in Accetta et al. (2009). All syntheses started from uracil, thymine or 5-carboxyuracil (isoorotic acid) as described schematically in the Results section. Full synthetic procedures and characterization of compounds 3, 12, 13, 14, 15, 16, 17 and 18 are reported elsewhere (Accetta et al., 2010).

Cell culture conditions, proliferation assay and cell differentiation

The human K562 cells (Lozzio and Lozzio,

Synthetic scheme of the compounds tested

The C-5 position of pyrimidine nucleosides can be easily modified with a variety of different residues. Substitution at this position is often used for introducing substituents on DNA oligomers with reporter groups. In a general project, aimed at molecular engineering of peptide nucleic acid (PNA) derivatives (A. Accetta, 2010), we prepared a set of N(1) and/or N(3) alkyl-C(5) modified uracil derivatives. Since these compounds are structurally analogs of modified nucleosides, they could be

Discussion

The biological activity of the derivatives tested in this study can lead to the following alternative effects: (a) a strong antiproliferative effect linked to a high level of erythroid differentiation activity and (b) an antiproliferative effect independent from the activation of the erythroid program (Table 2). The highest antiproliferative effect and erythroid induction ability were exhibited by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine

Acknowledgments

This work was partially supported by a grant by MIUR (PRIN2005 grant n. 2005038704). R.G. is granted by Associazione Italiana Ricerca sul Cancro (AIRC), Fondazione Cariparo (Cassa di Risparmio di Padova e Rovigo), Associazione Veneta per la Lotta alla Talassemia, Rovigo, Italy (AVLT), and Telethon (grant GGP10214). We thank the EU Project ITHANET (eInfrasctructure for Thalassemia Research Network) for support.

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