Elsevier

Toxicology in Vitro

Volume 67, September 2020, 104922
Toxicology in Vitro

Copper transporter 1 affinity as a delivery strategy to improve the cytotoxic profile of rationally designed copper(II) complexes for cancer treatment

https://doi.org/10.1016/j.tiv.2020.104922Get rights and content

Highlights

  • Copper complexes are candidates to efficient and less toxic treatments against cancer.

  • Copper uptake into tumor cells is mostly mediated by copper receptor 1 (CTR1).

  • Complexes (1) and (2) were differently uptake by tumor cells overexpressing CTR1.

  • The less bulky complex (2) was more cytotoxic for tumor cells overexpressing CTR1.

  • The rational design of the complexes is important as a delivery strategy for treatment.

Abstract

Cisplatin is widely used to treat different types of cancer, but its severe side effects are the major disadvantage of this treatment. Therefore, other metals are currently the subject of research in the rational development of anticancer drugs, such as copper, that has been demonstrated to be promising in this scenario. Here, we evaluated the effects of two novel copper complexes against breast cancer cell lines, and also examined the influence of overexpressing copper transporter 1 (CTR1) on the cytotoxicity of these complexes. Complex (1) [Cu(sdmx)2(phen)] showed low IC50 values, induced intense cell morphological changes and arrested the cell cycle at the sub-G1 phase in cancer cells. Complex (1) was tested in transfected cells overexpressing the CTR1 receptor in order to compare its steric effects with a less bulky ligand and more labile complex (2) [CuCl2(impy)]. A significant reduction of IC50 value was observed in CTR1 overexpressing cells for complex (2) (32 μM to 20 μM) as compared to (1) (2.78 μM to 3.41 μM), evidencing a possible uptake through copper reduction (Cu+2 → Cu+1) mediated by CTR1. Thus, considering that CTR1 is a mediator of metallodrugs uptake, the development of strategies that use rational drug design is important in order to improve the therapeutic efficacy through greater specificity and consecutive reduction of side effects. Here we show the example for the case of copper(II) complexes.

Introduction

Breast cancer is the most prevalent cancer type worldwide among women, accounting for about 2.1 million diagnosed cases in 2018, which amounts to almost 1 in 4 cancer cases. The disease is the most frequently diagnosed cancer and is also the leading cause of cancer death in over 100 countries (Bray et al., 2018). The treatment of breast cancer consists mainly of surgery, chemotherapy or radiation therapy. Particularly for chemotherapy, research to find new antitumor drugs still represents a field full of challenges.

The first coordination compound used in the clinic to treat cancer was the metallodrug cisplatin, cis-diamminedichoridoplatinum(II), which interacts with DNA and activates cytoplasmic and nuclear signaling pathways involved in the regulation of the cell cycle, damage repair, and programmed cell death (Roberts et al., 2016). However, due to its several side effects, research on the platinum metallodrugs for anticancer therapy has increased over the years (Roberts et al., 2016).

In addition to platinum, other metals are currently the subject of research in the rational development of anticancer drugs, such as copper. The antitumor properties of copper complexes have gained attention in the literature, since they not only involve damage to DNA and cytoplasmic proteins, but also generate reactive oxygen species (ROS), inducing high toxicity that is lethal to cancer cells (Denoyer et al., 2015; Palanimuthu et al., 2013; Zaidi et al., 2014). Concomitant to the development of metallodrugs, metal transporters are excellent targets since they are useful to researchers wishing to modulate the effects of inorganic complexes, increasing their effectiveness and reducing side effects, by better understanding their mechanisms of transport and action (Mjos and Orvig, 2014; Bergamo et al., 2018). One of these transporters is the copper transporter-1, CTR1, which is responsible for about 70% of the total metal influx to the cell, while 30% is mediated via low-affinity transport mechanisms (Zimnicka et al., 2011). The selectivity of CTR1 is shared with less affinity also by platinum and silver. CTR1 is important for the uptake of cisplatin into tumor cells, and consequently for its therapeutic efficacy (Kilari, 2016), contrarily, silver causes inhibition of the transporter (Bertinato et al., 2010).

In a continuous attempt to find new bioactive copper(II) complexes, we evaluated in the present work the biological effects of two compounds, (1) and (2). Complex (1) [Cu(sdmx)2(phen)], in which sdmx is a deprotonated sulfadimethoxine ligand and phen is 1,10-phenanthroline, showed promising in vitro activity against a panel of cancer cell lines (Nakahata et al., 2018). This complex is sterically hindered and less prone to ligand substitution. Complex (2) [CuCl2(impy)], with impy being 2 = (imidazole-2-yl)pyridine, was chosen in order to evaluate steric effects of a less bulky ligand and more labile complex on its possible uptake through the reduction of copper (Cu+2 → Cu+1) mediated by CTR1. The biological effects of the ligands in such complexes are reported in the literature in different approaches. The copper(II) complex with 1,10-phenanthroline and sulfadimethoxine, [Cu(sdmx)2(phen)], has shown antimycotic (Nakahata et al., 2018), antimicrobial, and antineoplastic activity (Fan et al., 2017). Imidazole derivatives, also have shown to possess antibacterial activities when coordinated with copper (Nakahata et al., 2017; Lewis et al., 2016) as in the case of the complex (2). In this work we present the cytotoxicity of these complexes against tumor and non-tumor breast cells transfected for overexpressing CTR1 and tested in several in vitro assays.

Section snippets

Materials

Copper(II) nitrate trihydrate (99–104%), sulfadimethoxine (sdmxH, ≥ 98.5%), 2-pyridinecarbonitrile (99%) and aminoacetaldehyde, dimethyl acetal (99%) were purchased from Sigma-Aldrich. Copper(II) chloride dihydrate (99%) and 1,10-phenanthroline were purchased from Vetec-Sigma. The copper(II) complexes (Fig. 1) evaluated in this manuscript were synthesized and characterized according to protocols previously published by our research group (Nakahata et al., 2018; Nakahata et al., 2017).

Cell culture

Cellular viability

In order to evaluate the cytotoxicity of the complexes (1) and (2); their precursors [Cu(NO3)2(phen)], CuCl2·2H2O, and ligands sulfadimethoxine (sdmxH) and 2-(imidazol-2-yl)pyridine (impy) on tumor cells MDA-MB-231, SK-BR3, MCF-7 and non-tumor breast MCF-10A cells, MTT was used as described in the methods section.

The complexes presented a broad spectrum of action on the viability of the tested cell lines. The ligands (sdmxH) and (impd) participate in the synthetic route of the copper(II)

Discussion

Copper(II) is a metal ion with considerable potential in therapeutic applications, and the activity of copper complexes has been recently evaluated with promising results. Tardito et al. (Tardito et al., 2011) tested 11 coordinated copper complexes to different pyrazolone-derived ligands and found significant response against HT1080 fibrosarcoma. IC50 values ranged from 3 to 28 μM, with most values at around 13 μM and with three complexes below 7 μM (Tardito et al., 2011). Additional studies on

Conclusion

Complex (1), of molecular formula [Cu(sdmx)2(phen)], was the most toxic to the triple negative MDA-MB-231 breast cancer cell line, being able to inhibit the formation of colonies, alter the morphology, and disrupt cytoskeleton organization at low concentrations. By means of CTR1 overexpression, we demonstrated the importance in designing copper(II) metallodrugs that may have improved affinity for metal transporters for greater cell uptake and activity, particularly for complex (2), of

Funding

The work was supported by the São Paulo Research Foundation, Brazil (FAPESP – scholarships #2015/20882-3, #2017/19504-0 and #2018/17170-0; grants #2013/07600-3 and #2018/12062-4).

Availability of data and material

The dataset supporting the results will be available upon request.

Authors' contributions

RHGT and AEG performed the experimental procedures; AEG, CMRC, MLF and MRC were involved in planning and supervised the work; RHGT, AEG and CMRC processed the experimental data, performed the analysis and designed the figures. DHN, ALBF and PPC synthesized and characterized the copper complexes. All authors discussed the results, commented on the manuscript and were involved in draft the manuscript.

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors would like to thank Dr. Richard Decker and Janelle Weslyn Salameh for their assistance in the cell transfection experiment and Dr. Ana Carolina Baptista Moreno Martin for her assistance in the cell cycle experiment and analysis.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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