In vitro and in vivo anti-proliferative activity and ultrastructure investigations of a copper(II) complex toward human lung cancer cell NCI-H460

https://doi.org/10.1016/j.jinorgbio.2020.111166Get rights and content

Highlights

  • Cell treatment with complex (2) results in apoptosis cell death by intrinsic pathway.

  • Complex (2) induces mitochondrial damage and endoplasmic reticulum stress.

  • In vivo tumor nodules treatment with complex (2) reduced 48% tumor growth.

Abstract

The aim of our study was to evaluate the in vitro and in vivo anti-proliferative potential of complex (2) [Cu (L1)Cl]Cl.2H2O, where L1 = 1-[2-hydroxybenzyl(2-pyridylmethyl)amino]-3-(1-naphthyloxy)-2-propanol on lung carcinoma cell NCI-H460. Cell viability assay determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay demonstrated that the complex (2) exhibits higher activity against the NCI-H460 cell, with an IC50 value lower than cisplatin (26.5 μM ± 1.1 and 203 ± 1.2 μM respectively). Cell death by apoptosis was investigated by flow cytometer analysis of sub-G1 populations in the cell cycle and Annexin V/Propidium Iodide assay. Changes on the cell surface and ultrastructure were detected by scanning and transmission electron microscopy. Our work revealed that complex (2) induced changes associated with apoptosis, such as plasma membrane blebbing and a lower microvilli amount, fragmentation and condensation of chromatin, alterations in mitochondria, and enlargement of the endoplasmic reticulum. Mitochondrial function of NCI-H460 cells evaluated by 5,5′,6,6′-tetrachloro 1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) probes showed high loss of mitochondrial membrane potential when treated with complex (2). Moreover, caspase-12 measurement showed an expressive activation level, which is related to endoplasmic reticulum stress. In vivo assay using the murine model of human lung cancer cell showed that complex (2) and cisplatin has similar antineoplastic activity.

Introduction

Cisplatin (cis-diamminedichloroplatinum(II)) is a powerful chemotherapeutic agent and was the first platinum containing coordination complex used in oncology therapy. In 1978 its efficacy in treatment of cancer patients was established and approved for use by the FDA [1,2]. Cisplatin is clinically proven to treat numerous types of human cancers and has contributed to increase life expectancy for patients with lung, bladder, head and neck, testicular and ovarian cancer, and is also used to treat other cancers, including carcinomas, germ cell tumors, lymphomas, and sarcomas, making it one of the most clinically successful antineoplastic drugs [1,3]. In general, cisplatin mechanism of action is related to its ability to induce DNA damage by cross-linking inter and intra-filament DNA, interfering with its repair, transcription and replication mechanism, these events in turn, result in apoptosis cell death [1,4].

Apoptosis is considered the main mechanism of cell death induced by most cancer therapeutic agents. This process is accompanied by several morphological changes such as rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), classically little or no ultra-structural modifications of cytoplasmic organelles, plasma membrane blebbing (but maintenance of its integrity until the final stages of the process), and engulfment by resident phagocytes (in vivo) causing minimal damage/inflammation to surrounding tissues [5,6]. Death by apoptosis can be triggered by the extrinsic (death receptor) and intrinsic (mitochondrial) pathways. Activation of death receptor results in binding of the adapter protein tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) and recruitment of the Fas-associated death domain (FADD) and receptor-interacting protein (RIP) proteins followed by the dimerization of the death effector domain, FADD then associates with pro-caspase-8. Death inducing signaling complex is formed and induces activation of pro-caspase-8. Activated caspase 8 subsequently triggers the execution phase of apoptosis by activating the downstream effector caspases 3, 6 and 7 [7,8]. The mitochondrial pathway of cell death can be activated by a variety of stimuli such as radiation, chemotherapeutics, free radicals, viral infections, and serum/growth factor withdrawal. It is well-established that these stimuli induce the loss of mitochondrial transmembrane potential (ΔΨm) followed by the release of pro-apoptotic proteins such as cytochrome c. This process is well controlled by the B-cell lymphoma 2 (BCL-2) protein family. Cytochrome c interacts with apoptotic protease activating factor 1 (APAF-1) in conjunction with dATP, these proteins form apoptosome that recruits and activates caspase 9, leading to the activation of executioner caspases 3, 6 and 7 and the death response [7,8].

Based on high antineoplastic activity of cisplatin associated with its severe side effects such as nephrotoxicity [9], hepatotoxicity, cardiotoxicity [10], ototoxicity, myelosuppression and a high percentage of cancer cell resistance to treatment [1], a considerable amount of research has been devoted to the development of new metallodrug candidates, which are capable of interacting with DNA or other molecular targets such as proteins and enzymes that induce cellular death [11] but with less severe side effects. The discovery of the cytotoxic activity of complexes with other metals besides platinum, including iron [12], gallium [13], copper [14,15], cobalt [16], palladium [4], ruthenium [17] and gold complexes [18], has expanded the arsenal of metallic ions that have been studied for this purpose.

Cytotoxic activity of copper complexes has generated great interest in the search for anticancer agents with lower toxicity compared with platinum drugs [19]. In this context, a considerable number of works have been made by using different tumor cell lines. The cytotoxic mechanism of copper(II) complexes associated with the production of reactive oxygen species (ROS), interaction with DNA and induction of apoptosis have been described in acute lymphoblastic leukemia cell [20,21] histiocytic lymphoma [22], breast carcinoma [14,23], pulmonary carcinoma [24,25] and prostate carcinoma [26].

Our group has synthesized a number of coordination complexes including iron(III) complexes [12], copper(II) [15,22], cobalt(II) [16] and platinum(II) [27] and their biological activity was tested on several neoplastic cell lines. In 2015 we reported on the evaluation of the synthesis, characterization of two complexes having copper(II) as metal nuclei, and cytotoxic activity toward two human leukemia cell lines (THP-1 and U937) [22]. These complexes exhibited a high anti-proliferative effect on both cell lines, whereas complex (2) exhibited higher activity than cisplatin against the U937 cell line (8.20 μM vs 16.2 μM). We also demonstrated that complex (2) induces apoptosis by activating the intrinsic pathway of cell death. The toxicity of complex (2) determined in vivo in C57BL/6 mice (LD50 of 55 mg/kg) [22] when compared to cisplatin (LD50 of 6.6 mg/kg) [28], showed to be 8.3 times less toxic than the standard metallodrug. As the alpha isomer was the most active compound, we decided to investigate the inhibitory effects of this compound on NCI-H460 lung carcinoma cells and the underlying mechanisms of cell death induced by complex (2) on this highly metastatic cancer cell. Additionally, we also investigated the in vivo treatment by complex (2) of BALB/c nude mice bearing cancerous lesions of NCI-H460 cells.

Section snippets

Synthesis of copper(II) complex - complex (2)

The ligand (L1) and its respective copper complex [CuCl] Cl.2H2O, where H2L2 = 1-[2-hydroxybenzyl(2-pyridylmethyl)amino]-3-(1-naphthyloxy)-2-propanol (Fig. 1) were synthesized as described previously [22]. Complex (2) was obtained as green crystals. Aiming to confirm the structure which was reported previously, physico-chemical characterization was carried out employing elemental analysis (CHN), ESI(+)-MS, ESI(+)-MS/MS and conductivimetry measurements. Complex (2): Elemental analysis (CHN):

Complex (2) induces cytotoxicity on neoplastic cells

The screening of the anti-proliferative activity of new biologically active compounds is essential in cancer research regarding the development of new antitumor drugs. Thus, the in vitro cytotoxicity of ligand L1, metallic salt, complex (2), and cisplatin (control) were evaluated against three human cancer cell lines: NCI-H460, COLO 205, and MOLT-4 by MTT assay. Complex (2) showed a significant inhibitory effect on the growth of the neoplastic cell lines NCI-H460, COLO 205, MOLT-4, and HB4a

Discussion

Inorganic chemistry uses in medicine have been described for many centuries [7,37]. However, only after the clinical success of cisplatin in the 1970s was the consolidation of inorganic medicinal chemistry established [7]. At that time the cytotoxic properties of cisplatin were proven and it was considered one of the most potent drugs used against numerous kinds of cancers. Although Pt-based therapies have been largely used in cancer treatment, two major limitations are commonly observed: the

Conclusion

In this study, we showed the anti-proliferative potential of complex (2) by decreasing NCI-H460 cell viability by apoptosis involving the intrinsic pathway. This process is a result of mitochondrial membrane potential depolarization and activation of caspase-12 associated with ER stress. The complex (2) showed a higher in vitro cytotoxicity when compared to cisplatin treatment on NCI-H460 cells. Unfortunately, this result was not prove by in vivo assay with BALB/c nude mice bearing NCI-H460

Funding

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro).

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The studies involving the use of animals were approved by the Animal Ethics Use Committee of Darcy Ribeiro North Fluminense State University (Campos dos Goytacazes, Rio de Janeiro, Brazil) – protocol number CEUA 349.

Declaration of competing interest

Leide L. M. Figueredo declares that she has no conflict of interest. William R. Freitas declares that he has no conflict of interest. Erika S. Bull declares that she has no conflict of interest. Christiane F. Horn declares that she has no conflict of interest. Adolfo Horn Jr. declares that he has no conflict of interest. João C. A. de Almeida declares that he has no conflict of interest. Milton M. Kanshiro declares that he has no conflict of interest.

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    1

    Current address: Departamento de Química, Universidade Federal de Santa Catarina, 88040-900, Florianópolis/SC, Brazil.

    2

    Both authors contributed equally to this manuscript.

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