Synthesis, characterization, DNA binding, topoisomerase inhibition, and apoptosis induction studies of a novel cobalt(III) complex with a thiosemicarbazone ligand

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Highlights

  • A cobalt(III) complex with a thiosemicarbazone ligand was synthesized and characterized.

  • The complex inhibited human topoisomerases I and IIα.

  • Dissipation of the mitochondrial membrane potential (ΔΨm) by the complex was noted.

  • Intrinsic apoptotic pathway was the major regulator of cell death mechanism.

  • A 59Co NMR spectroscopic study provided new data for the spectrochemical series.

Abstract

In this study, 9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1 and [Co(phen)2(O2CO)]Cl·6H2O 2 (where phen = 1,10-phenanthroline) were synthesized. [Co(phen)2(O2CO)]Cl·6H2O 2 was used to produce anhydrous [Co(phen)2(H2O)2](NO3)3 3. Subsequently, anhydrous [Co(phen)2(H2O)2](NO3)3 3 was reacted with MeATSC 1 to produce [Co(phen)2(MeATSC)](NO3)3·1.5H2O·C2H5OH 4. The ligand, MeATSC 1 and all complexes were characterized by elemental analysis, FT IR, UV–visible, and multinuclear NMR (1H, 13C, and 59Co) spectroscopy, along with HRMS, and conductivity measurements, where appropriate. Interactions of MeATSC 1 and complex 4 with calf thymus DNA (ctDNA) were investigated by carrying out UV–visible spectrophotometric studies. UV–visible spectrophotometric studies revealed weak interactions between ctDNA and the analytes, MeATSC 1 and complex 4 (Kb = 8.1 × 105 and 1.6 × 104 M−1, respectively). Topoisomerase inhibition assays and cleavage studies proved that complex 4 was an efficient catalytic inhibitor of human topoisomerases I and IIα. Based upon the results obtained from the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay on 4T1-luc metastatic mammary breast cancer cells (IC50 = 34.4 ± 5.2 μM when compared to IC50 = 13.75 ± 1.08 μM for the control, cisplatin), further investigations into the molecular events initiated by exposure to complex 4 were investigated. Studies have shown that complex 4 activated both the apoptotic and autophagic signaling pathways in addition to causing dissipation of the mitochondrial membrane potential (ΔΨm). Furthermore, activation of cysteine-aspartic proteases3 (caspase 3) in a time- and concentration-dependent manner coupled with the ΔΨm, studies implicated the intrinsic apoptotic pathway as the major regulator of cell death mechanism.

Graphical abstract

A cobalt(III) complex was utilized in in vitro studies and binding studies with calf thymus DNA. Topoisomerase inhibition assays and cleavage studies proved that the complex was an efficient catalytic inhibitor of human topoisomerases I and IIα. The complex also induces autophagic flux in 4T1-luc cells.

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Introduction

Breast cancer (BCa) is the second leading cause of morbidity and mortality in women [[1], [2], [3], [4], [5]]. Furthermore, triple-negative breast cancer (TNBC) presents considerable therapeutic challenges due to disease heterogeneity, absence of established therapeutic targets, aggressive metastatic potential with higher rate of distant recurrence, and poor prognosis [[6], [7], [8]]. In addition, TNBC is most common in younger patients (TNBC is more likely to occur before age 40 or 50, versus age 60 or older, which is more typical for other breast cancer types [9,10]), especially in African American women [11] often leading to significant disease progression and poor prognosis.

Metastatic breast cancer is the most common disease in western women with approximately 1.7 million new breast cancer diagnoses in 2014 [12,13]. In many patients, it is not the primary tumor but metastases at distant sites that are the main cause of death. While rates of metastasis and mortality have slightly decreased over the last two decades, the disease is still considered incurable even with advances in early detection and systemic adjuvant therapies [14]. Chemotherapy only increases the 15-year survival rate of women under 50 by 10%; in elderly women this increase is only 3% [15,16]. No technology currently exists for the accurate prediction of metastasis; consequently, patients are subject to the toxic side effects of classic chemotherapy, which substantially affect the patients' quality of life [[17], [18], [19]]. Therefore, there is an urgent need to necessitate concerted efforts to identify effective agents against TNBC.

Cisplatin is widely used for the treatment of many cancers [20] despite its high toxicity, undesirable side effects, and problems with drug resistance in primary and metastatic cancers [21,22]. These limitations have spurred a growing interest in novel non-platinum metal complexes that show anti-cancer properties [23]. Several ruthenium- [[24], [25], [26]], gold-, gallium-, titanium-, vanadium, and arsenic-based compounds have been investigated for their anti-cancer potential [[27], [28], [29], [30], [31], [32]]. Although none of these compounds have been approved clinically, significant advancements have been made in their development including clinical studies with some of the most promising candidates. More recently, Jaouen and co-workers reported the use of ferrocenyl tamoxifen derivatives for breast cancer inhibition [[33], [34], [35]]. In one of those studies, Jaouen and co-workers [35] developed stealth FcOHTam, an organometallic ferrocene derivative of hydroxytamoxifen loaded lipid nanocapsules to evaluate this novel drug on a TNBC xenografted model [35]. A significantly lower tumor volume was obtained with a difference of 36% at day 38 when compared to the untreated group [35]. Those results represented the first evidence of an in vivo effect of FcOHTam and ferrocenyl derivatives on xenografted breast tumors [35].

For many years, and more recently, the interaction between a variety of transition metals with polypyridyl ligands and DNA has been extensively studied as part of the efforts to fight cancer [[36], [37], [38], [39], [40], [41], [42], [43], [44], [45]]. Due to unusual binding properties and general photoactivity, these coordination compounds are suitable candidates as DNA secondary structure probes, photocleaving agents, and anti-cancer drugs [46]. Examples of photocleaving activities with ruthenium(II)-containing mixed-metal complexes with rhodium(III) and vanadium(IV) metal centers were reported by Brewer and co-workers [42,43,47] and Holder and co-workers [48], and by other researchers who reported cobalt-, ruthenium-, and vanadium-containing metal centers [[49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80]].

Cobalt is one of the most important trace metals, where in the form of vitamin B12 (cobalamin), it plays a number of critical roles in multiple biological functions, including DNA synthesis, formation of red blood cells, maintenance of the nervous system, and growth and development. Despite their well-known versatility, cobalt-containing compounds have not been studied extensively as inorganic pharmaceuticals in comparison to other metals. To date, the only cobalt-based therapeutic that has reached clinical trials is Doxovir, a Co(III) Schiff base complex effective against drug-resistant herpes simplex virus 1 (HSV1) [81]. While it provides an example for cobalt-based pharmaceuticals, the mechanism of action of Doxovir is not fully understood. A substantial amount of literature on cobalt-containing complexes has been reported while demonstrating their potential for medicinal applications [32,[82], [83], [84], [85], [86], [87], [88], [89]]. However, the rationale behind the design and mechanisms of many of these agents has not been clearly defined.

Cobalt(III) and ruthenium(II) complexes that can intercalate between the stacked base pairs of native DNA have been actively investigated as probes [[90], [91], [92], [93], [94]] of DNA structure in solution and as stereoselective or conformation-specific agents for the photoactivated cleavage of DNA [95,96]. The nature of the interactions between cobalt(III) and ruthenium(II) complexes and others [97,98] and DNA have primarily been studied by cyclic voltammetry [96,[99], [100], [101], [102], [103], [104]], spectroscopic [[105], [106], [107], [108], [109]], and X-ray crystallographic methods [105], to name a few.

Metal-based compounds that contain thiosemicarbazones as ligands possess a wide range of biological activities [[110], [111], [112], [113], [114], [115], [116], [117]]. Thiosemicarbazones and their metal complexes present a wide range of applications that stretch from analytical chemistry, through pharmacology to nuclear medicine [118,119]. The biomedical activity of the thiosemicarbazones is enhanced by coordination to a transition metal ion [[110], [111], [112], [113], [114], [115], [116], [117],120,121]. Approximately 40% of drug candidates produced from combinatorial screening programs have poor aqueous solubility (<10 μM) [122]. Coordination of a drug candidate to a positively-charged metal complex has been shown to greatly improve aqueous solubility in many cases, e.g., the proton pump inhibitor esomeprazole has very low aqueous solubility and is typically administered as esomeprazole magnesium, a water soluble bis(esomeprazole) magnesium complex [123]. Alternatively, coordination of a metal ion by negatively-charged groups such as carboxylates and phosphates can reduce the negative charge of a drug, thus enhancing passive cellular uptake and absorption [124]. In addition, endogenous metal ions and complexes can offer active transport pathways into the cell, allowing greater and more selective cellular accumulation of the drug [125].

Transition metal complexes are particularly suited to controlled drug release, having bonds that are highly responsive to their environment [121]. Such cases are applied when prodrug complexes can be designed that are inert under normal physiological conditions, but become labile with a change in environment such as redox status, pH, or the localized application of light [[126], [127], [128]]. By not activating a drug before it reaches its target, side reactions and premature metabolism that can lead to undesirable side effects to normal cells can be reduced tremendously [[110], [111], [112], [113], [114], [115], [116],127,129]. In addition to overcoming the limitations of an organic drug, the metal complex can itself be biologically active [129]. By generating an active metal complex and active organic molecule from a single prodrug, multiple means of acting upon a target can be achieved [130]. Dual-action drugs may be more potent than the parent organic drug and be able to circumvent drug resistance mechanisms [131].

Cobalt(III) complexes with thiosemicarbazones and methyl-substituted 1,10-phenanthroline [132] as ligands, have been reported as anti-cancer agents in several cancer cell lines [87,133]. However, an understanding of how cobalt-containing complexes interact with biological systems to elicit chemotherapeutic effects is clearly necessary for the development of cobalt-based drugs [83].

Given the antineoplastic activities of thiosemicarbazone metal complexes [87,[134], [135], [136]], it is important to know their sites of action and mechanisms of cell death induction at the molecular level. A Nomenclature Committee on “Cell Death” has defined regulated cell death (RCD) mechanisms that include a number of different signaling scenarios beyond the initial introduction of programmed cell death or apoptosis as an alternative to necrosis [137]. The most common RCD mechanisms include apoptosis, programmed necrosis or necroptosis, and post-autophagic cell death [138,139]. For studies here, we have focused on apoptosis and autophagy. Apoptosis can be induced by extrinsic mechanisms through cell death receptors and/or intrinsic mechanisms that are initiated by heterogeneous intracellular signaling such as DNA damage and oxidative stress or hypoxia, among others. All of these intrinsic mechanisms involve mitochondria. Autophagy serves pro-survival functions as well as RCD signaling, depending on the stimulus, its intensity, and cellular context.

In our efforts to carry out anti-cancer studies with non-platinum-containing complexes, we have decided to focus on a cobalt(III) complex using our previously reported appended thiosemicarbazone with anthracene (9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1) [140]. Anthracene derivatives are the most important class of ligands with high intrinsic fluorescence properties [141]. Anthracene derivatives have been investigated for their binding to DNA, and some are promising chemotherapeutic agents [142,143]. Anthracene itself has been reported to be effective against psoriasis [144]; while other anthracene derivatives tested for their anti-cancer activity include mitoxantrone, ametantrone, and bisantrene [[145], [146], [147]]. Mitoxantrone, ametantrone, and bisantrene have been suggested to elicit their activity by binding to DNA through groove/electrostatic as well as intercalative binding modes [[145], [146], [147]]. The azonafides [148] are a series of anthracene-based DNA intercalators which inhibit tumor cell growth in vitro at low nanomolar concentrations and are not affected by the multidrug resistance phenomenon (MDR), which is a logical choice to explore the use of our cobalt(III) complex with an anthracene-containing thiosemicarbazone ligand, MeATSC 1 [140]. Herein this paper presents mechanistic investigations of the combined effect of thiosemicarbazone pharmacophore with anthracene (MeATCS 1) and complex 4 on a highly aggressive metastatic breast cancer cell line, 4T1-luc [[149], [150], [151]]. Interactions of the novel complex 4 with ctDNA and human topoisomerase, cytotoxicity studies, studies of induction of apoptosis and autophagy signaling pathways, along with the effect on the mitochondrial membrane potential (ΔΨm) are also discussed.

Section snippets

Reagents and instrumentation

All chemicals and reagents were purchased from commercial sources and were used without further purification. Ethidium bromide (EB) and calf thymus DNA (ctDNA) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were obtained from Mediatech, Inc. (Manassas, VA). CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS), and Caspase-3/7 Glo kits were purchased from Promega Corporation (Madison, WI). All buffer

Synthesis and characterization

The MeATSC 1 ligand was synthesized and characterized as by Beckford et al. [140]. Complexes 2 and 3 were synthesized; then characterized by 1H NMR spectroscopy (see Supplementary information for the 1H NMR spectra for complexes 2 and 3) as reported in the literature [[153], [154], [155]]. Scheme 1 shows the synthetic procedure for the formation of complex 4.

Elemental analysis was carried out on complex 4. The percentage for carbon, hydrogen, and nitrogen were found as 53.23%, 3.73%, and

Conclusions

In this study, we investigated the interactions of MeATSC 1, complex 3, and complex 4 with ctDNA. We also analyzed the biochemical effects of complex 4 on a model breast cancer cell line, 4T1-luc. Spectroscopic characterization showed complex 4 to contain the thiosemicarbazone in the thione isomeric form. However, there is tautomerization of the coordinated MeATSC ligand 1 on the cobalt(III) metal center within the mass spectrometer chamber and from conductivity measurements (where there was

Abbreviations

    ΔΨm

    mitochondrial membrane potential

    acetylethTSC

    (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide

    amtp

    3-amino-1,2,4-triazino[5,6-f]1,10-phenanthroline

    ATCC

    American Type Culture Collection

    ATS

    diacetyl bis(thiosemicarbazone)

    ATSC

    9-anthraldehydethiosemicarbazone

    bpy

    2,2′-bipyridine

    BnA

    benzylamine

    C8H4O42−

    terephthalate anion

    caspase 3

    cysteine-aspartic proteases3

    ctDNA

    calf thymus DNA

    DMEM

    Dulbecco's Modified Eagle's Medium

    dmgBF2

    difluoroboryldimethylglyoximato

    dpta

    dipyrido-[3,2-a;2′,3′-c

Acknowledgements

AAH's work to generate compounds 14 used in this study was supported by the Mississippi INBRE (P20RR016476), funded by the National Center for Research Resources, National Institutes of Health (NIH). AAH would like to thank the National Science Foundation (NSF) for the NSF CAREER Award, as this material is based upon work partially supported by the NSF under CHE-1431172 (Formerly CHE-1151832). AAH would also like to thank Old Dominion University's Faculty Proposal Preparation Program (FP3) and

Declaration of competing interest

There is no interest conflict in this paper.

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