Induction of apoptosis in leukemia cell lines by new copper(II) complexes containing naphthyl groups via interaction with death receptors

https://doi.org/10.1016/j.jinorgbio.2015.09.014Get rights and content

Highlights

  • New copper(II) complexes containing naphthalene rings (alpha and beta)

  • X-ray diffractions studies and several investigations in solution were performed.

  • Complex (2) is almost four times less toxic than cisplatin, based on LD50 values.

  • Complex (2) exhibits higher activity than cisplatin against U937.

  • The apoptosis signal triggered by complex (2) starts from an extrinsic pathway.

Abstract

The synthesis, physico-chemical characterization and cytotoxicity of four new ligands and their respective copper(II) complexes toward two human leukemia cell lines (THP-1 and U937) are reported (i.e. [(HL1)Cu(μ-Cl)2Cu(HL1)]Cl2·H2O (1), [(H2L2)Cu(μ-Cl)2Cu(H2L2)]Cl2·5H2O (2), [(HL3)Cu(μ-Cl)2Cu(HL3)]Cl2·4H2O (3), [(H2L4)Cu(μ-Cl)2Cu(H2L4)]Cl2·6H2O (4)). Ligands HL1 and HL3 contain two pyridines, amine and alcohol moieties with a naphthyl pendant unit yielding a N3O coordination metal environment. Ligands H2L2 and H2L4 have pyridine, phenol, amine and alcohol groups with a naphthyl pendant unit providing a N2O2 coordination metal environment. These compounds are likely to be dinuclear in the solid state but form mononuclear species in solution. The complexes have an antiproliferative effect against both leukemia cell lines; complex (2) exhibits higher activity than cisplatin against U937 (8.20 vs 16.25 μmol dm 3) and a comparable one against THP-1. These human neoplastic cells are also more susceptible than peripheral blood mononuclear cells (PBMCs) toward the tested compounds. Using C57BL/6 mice an LD50 of 55 mg kg 1 was determined for complex (2), suggesting that this compound is almost four times less toxic than cisplatin (LD50 = 14.5 mg kg 1). The mechanism of cell death promoted by ligand H2L2 and by complexes (2) and (4) was investigated by a range of techniques demonstrating that the apoptosis signal triggered at least by complex (2) starts from an extrinsic pathway involving the activation of caspases 4 and 8. This signal is amplified by mitochondria with the concomitant release of cytochrome c and the activation of caspase 9.

Graphical abstract

In this paper we present the synthesis, physico-chemical properties and cytotoxicity toward two human leukemia cell lines of four new copper(II) complexes. Complex (2) showed relevant antiproliferative effect against both cell lines and the mechanism of action starts from an extrinsic pathway and is amplified by the mitochondria.

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Introduction

Leukemia is a broad term covering a spectrum of diseases. It is part of a group of diseases affecting the blood, bone marrow and lymphoid system, and which are all known as hematological neoplasms. An estimated combined total of 156,420 people in the US were diagnosed with leukemia, lymphoma or myeloma in 2014. New cases of leukemia, lymphoma and myeloma accounted for 9% of the estimated 1,665,540 new cancer cases diagnosed in the US in 2014 [1]. About 90% of all leukemia cases are diagnosed in adults. While the treatment of childhood leukemia has improved significantly (with an average 85–95% complete response (CR) rate and 80–90% long–term survival), the prognoses for adult leukemic patients are still unsatisfactory; the average CR rate is 70–80% but long-term survival is only 20–30% [2], [3], [4].

The development of Gleevec® (imatinib mesylate, a tyrosine kinase inhibitor), frequently referred to as the “leukemia pill”, has revolutionized CML (cronic myelogeneous leukemia) treatment. This oral drug was approved by the FDA for use in 2001 [5]. In 2011, Sprycel (dasatinib, another tyrosine kinase inhibitor) was developed to treat CML, a drug that induces hematologic and cytogenic responses in patients who cannot tolerate or are resistant to imatinib mesylate. Its major side effect is reversible myelosuppression, and it was thus suggested that a successful long-term treatment of CML may require a cocktail of kinase inhibitors [6].

Due to its biological relevance and the role copper plays in tumor angiogenesis [7], especially in the early stage, a large number of copper(II) complexes have been synthesized and screened for anticancer activity; many of these derivatives display prominent in vitro cytotoxic activity [8], [9], [10]. However, very few in vitro studies on copper(II) complexes have been translated into preclinical in vivo models [11], [12], [13], [14], [15]. In 2014, Xu and co-workers reported anti-tumor activity of a copper complex containing disulfiram in its structure. This complex targets lymphoid malignant cell lines (Raji and Molt4 cells), in vitro and in vivo [16]. In 2015, Erxleben and co-workers reported results for a dinuclear copper(II) complex, containing the ligand (2,6-bis(1,4,7-triazacyclonon-1-ylmethyl)-4-methylphenol), which displays low toxicity against a human non-tumor cell line and IC50 values comparable or even lower than those of cisplatin against various cancer cell lines. Their complex interacts with DNA and activates the p53-dependent pathway of apoptosis [17]. Zhu and co-workers reported potent antitumoral activity against human cell lines (A549, Eca109 and SGC7901) for three mononuclear complexes containing the ligand H2TBHP (2-(3,5-di-tert-butyl-2-hydroxybenzylamino)-2-benzyl-acetic acid). The associated apoptosis is related to the effect of these copper(II) complexes on the expression of p53, Bax and Bcl-2 [18].

In an earlier study we reported that the complex [Cu(HPClNOL)Cl]Cl·MeOH (HPClNOL = [1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol) was able to promote DNA cleavage in vitro and was toxic to the THP-1 cell line, inducing cell death by apoptosis [19]. In a subsequent study we reported that the cytotoxic activity of the iron(III) complex [Cl(HPClNOL)Fe(III)(μ-O)Fe(III)(HPClNOL)Cl]Cl2·2H2O, employing the same HPClNOL ligand, takes place in the mitochondria, also involving so-called death receptors, which suggested that both pathways (intrinsic and extrinsic) may be involved in the apoptotic stimuli [20]. Attempting to search for more active compounds as well as to expand on our understanding of the events occurring within cells that were exposed to a cytotoxic agent, we have synthesized a family of new ligands (Scheme 1) and their copper complexes (Scheme 2).

HL1–H2L4 (see Scheme 1) are derived from the ligands HPClNOL and H2BPClNOL (= 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol), which have previously led to the synthesis of compounds with interesting biological properties (e.g. antitumoral, antibacterial and antioxidant) [19], [20], [21], [22], [23]. The main difference between those previously reported ligands (HPClNOL and H2BPClNOL) and HL1–H2L4, is the presence of a naphthyl group in the latter. This modification was based on the demonstrated relevance of naphthalene derivatives as valuable pharmacologic compounds possessing various important biological properties such as anti-inflammatory, antibacterial, hypotensive, bradycardiac and antitumoral activities [24], [25]. Ligands HL1 and HL3 are isomers (possessing two pyridinic groups), as are H2L2 and H2L4 (possessing one phenolic and one pyridinic group). The biological activity of the corresponding copper complexes was assessed both in vitro and in vivo through the evaluation of IC50 (on human leukemia cell lines normal cells) and DL50 (mice) values. The interaction of the copper complexes with cancer cells was monitored via fluorescence and electron microscopies, cell cycle analysis, annexin staining, mitochondrial membrane potential (ΔΨm) analysis, release of cytochrome c and the activation of caspases.

Section snippets

Materials

The ligands and their respective copper complexes were synthesized using analytical grade reagents. UV–Vis, electrochemical and MS investigations were carried out employing spectroscopic, HPLC or MS quality solvents. All chemicals and reagents were purchased from Aldrich (Sigma-Aldrich) and used as such.

Spectroscopic measurements

1H and 13C NMR spectra were recorded on a JEOL eclipse 400 + spectrometer. Chemical shifts (δ) are given in ppm, and the spectra were recorded in appropriate, deuterated solvents, as indicated.

Syntheses and structures of copper(II) coordination compounds

A group of similar ligands was developed in order to probe the influence of the chelating groups and structural isomerism introduced by them on their coordination behavior, as well as to gain insight into the influence of these features on the antitumor activity of their copper(II) complexes.

Ligands HL1 and HL3 present two pyridine groups, a tertiary amine and an alcohol function as chelating groups and an α-naphthyl or β-naphthyl group as pendant unit, respectively, providing a N3O

Conclusions

In this study we have introduced four new ligands and their copper complexes. The physico-chemical characterization showed that the compounds have a dinuclear structure in the solid state, which is broken in solution, resulting in mononuclear copper complexes.

The insertion of the naphthyl group into the ligand was an attempt to develop copper complexes with high anticancer activity. In the present family of copper complexes we observed that those which contain the phenol group (complexes (2)

Acknowledgments

The authors are grateful to financial support received from CAPES, (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro) and FINEP (Financiadora de Estudos e Projetos). G. S. also acknowledges the receipt of an ARC Future Fellowship (FT120100694).

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    Dedicated to Prof. Graeme R. Hanson in memoriam.

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