Cristina Marzano

Copper Complexes as Anticancer Agents

Metal-based antitumor drugs play a relevant role in antiblastic chemotherapy. Cisplatin is regarded as one of the most effective drugs, even if severe toxicities and drug resistance phenomena limit its clinical use. Therefore, in recent years there has been a rapid expansion in research and development of novel metal-based anticancer drugs to improve clinical effectiveness, to reduce general toxicity and to broaden the spectrum of activity. The variety of metal ion functions in biology has stimulated the development of new metallodrugs other than Pt drugs with the aim to obtain compounds acting via alternative mechanisms of action. Among non-Pt compounds, copper complexes are potentially attractive as anticancer agents. Actually, since many years a lot of researches have actively investigated copper compounds based on the assumption proposal that endogenous metals may be less toxic. It has been established that the properties of copper-coordinated compounds are largely determined by the nature of ligands and donor atoms bound to the metal ion. In this review, the most remarkable achievements in the design and development of copper(I, II) complexes as antitumor agents are discussed. Special emphasis has been focused on the identification of structure-activity relationships for the different classes of copper(I,II) complexes. This work was motivated by the observation that no comprehensive surveys of copper complexes as anticancer agents were available in the literature. Moreover, up to now, despite the enormous efforts in synthesizing different classes of copper complexes, very few data concerning the molecular basis of the mechanisms underlying their antitumor activity are available. This overview, collecting the most significant strategies adopted in the last ten years to design promising anticancer copper(I,II) compounds, would be a help to the researchers working in this field.

Copper in Diseases and Treatments, and Copper-based Anticancer Strategies

Copper is found in all living organisms and is a crucial trace element in redox chemistry, growth and development. It is important for the function of several enzymes and proteins involved in energy metabolism, respiration, and DNA synthesis, notably cytochrome oxidase, superoxide dismutase, ascorbate oxidase, and tyrosinase. The major functions of copper—biological molecules involve oxidation–reduction reactions in which they react directly with molecular oxygen to produce free radicals. Therefore, copper requires tightly regulated homeostatic mechanisms to ensure adequate supplies without any toxic effects. Overload or deficiency of copper is associated, respectively, with Wilson disease (WD) and Menkes disease (MD), which are of genetic origin. Researches on Menkes and Wilson disorders have provided useful insights in the field of copper homeostasis and in particular into the understanding of intracellular trafficking and distribution of copper at molecular levels. Therapies based on metal supplementation with copper histidine or removal of copper excess by means of specific copper chelators are currently effective in treating MD and WD, respectively. Copper chelation therapy is now attracting much attention for the investigation and treatment of various neurodegenerative disorders such as Alzheimer, Parkinson and CreutzfeldtJakob. An excess of copper appears to be an essential co‐factor for angiogenesis. Moreover, elevated levels of copper have been found in many types of human cancers, including prostate, breast, colon, lung, and brain. On these basis, the employment of copper chelators has been reported to be of therapeutic value in the treatment of several types of cancers as anti‐angiogenic molecules. More recently, mixtures of copper chelators with copper salts have been found to act as efficient proteasome inhibitors and apoptosis inducers, specifically in cancer cells. Moreover, following the worldwide success of platinum(II) compounds in cancer chemotherapy, several families of individual copper complexes have been studied as potential antitumor agents. These investigations, revealing the occurrence of mechanisms of action quite different from platinum drugs, head toward the development of new anticancer metallodrugs with improved specificity and decreased toxic side effects.

Platinum(II) and palladium(II) complexes with dithiocarbamates and amines: synthesis, characterization and cell assay

The [M(ESDT)Cl]n (M = Pd or Pt; ESDT = EtO2CCH2(CH3)NCS2, methylamino-acetic acid ethyl ester-dithiocarboxylate) species have been reacted with various amines (py, pyridine; PrNH2, n-propylamine; c-BuNH2, cyclobutylamine; en, ethylenediamine) in dichloromethane or chloroform with the aim to obtain mixed ligand complexes. The neutral complexes [M(ESDT)(L)Cl] (L = py, PrNH2 or c-BuNH2) and the ionic species ([M(ESDT)(L)2]Cl and [M(ESDT)(En)]Cl) have been isolated, and characterized by IR and proton NMR spectroscopies. The crystal structure of [Pd(ESDT)(PrNH2)Cl] has been determined by X-ray crystallography. The behaviour of the complexes in various solvents was described on the basis of the proton NMR spectra. The complexes and the dithiocarbamato intermediates have been tested for in vitro cytostatic activity against human leukemic HL-60 and HeLa cells.

In vitro antitumor activity of the water soluble copper(I) complexes bearing the tris(hydroxymethyl)phosphine ligand.

Monocationic hydrophilic complexes [Cu(thp)4](+) 3 and [Cu(bhpe)2](+) 4 were synthesized by ligand exchange reactions starting from the labile [Cu(CH3CN)4][PF6] precursor in the presence of an excess of the relevant hydrophilic phosphine. Complexes 3 and 4 were tested against a panel of several human tumor cell lines. Complex 3 has been shown to be about 1 order of magnitude more cytotoxic than cisplatin. Chemosensitivity tests performed on cisplatin and multidrug resistance phenotypes suggested that complex 3 acts via a different mechanism of action than the reference drug. Different short-term proliferation assays suggested that lysosomal damage is an early cellular event associated with complex 3 cytotoxicity, probably mediated by an increased production of reactive oxygen species. Cytological stains and flow cytometric analyses indicated that the phosphine copper(I) complex is able to inhibit the growth of tumor cells via G2/M cell cycle arrest and paraptosis accompanied with the loss of mitochondrial transmembrane potential.

In Vitro Antitumour Activity of Water Soluble Cu(I), Ag(I) and Au(I) Complexes Supported by Hydrophilic Alkyl Phosphine Ligands

Hydrophilic, monocationic [M(L)(4)]PF(6) complexes (M = Cu or Ag; L: thp = tris(hydroxymethyl)phosphine, L: PTA = 1,3,5-triaza-7-phosphaadamantane, L: thpp = tris(hydroxypropyl)phosphine) were synthesized by ligand exchange reaction starting from [Cu(CH(3)CN)(4)]PF(6) or AgPF(6) precursors at room temperature in the presence of an excess of the relevant phosphine. The related [Au(L)(4)]PF(6) complexes (L = thp, PTA or thpp) were synthesized by metathesis reactions starting from [Au(L)(4)]Cl at room temperature in the presence of equimolar quantity of TlPF(6). The three series of complexes [M(L)(4)]PF(6) were tested as cytotoxic agents against a panel of several human tumour cell lines also including a defined cisplatin resistant cell line. These investigations have been carried out in comparison with the clinically used metallodrug cisplatin and preliminary structure-activity relationships are presented. The best results in terms of in vitro antitumour activity were achieved with metal-thp species and, among the coinage metal complexes, copper derivatives were found to be the most efficient drugs. Preliminary studies concerning the mechanism of action of these [M(L)(4)]PF(6) species pointed to thioredoxin reductase as one of the putative cellular targets of gold and silver complexes and provided evidence that copper derivatives mediated their cytotoxic effect through proteasome inhibition.

Gold(III)-dithiocarbamato anticancer agents: activity, toxicology and histopathological studies in rodents.

Gold(III)‐dithiocarbamato complexes have recently gained increasing attention as potential anticancer agents because of their strong tumor cell growth–inhibitory effects, generally achieved by exploiting non‐cisplatin‐like mechanisms of action. The rationale of our research work is to combine the antitumor properties of the gold(III) metal center with the potential chemoprotective function of coordinated dithiocarbamates in order to reduce toxic side effects (in particular nephrotoxicity) induced by clinically established platinum‐based drugs. In this context, [AuIIIBr2(ESDT)] (AUL12) was proved to exert promising and outstanding antitumor activity in vitro and to overcome both acquired and intrinsic resistance showed by some types of tumors toward cisplatin. As a subsequent extension of our previous work, we here report on detailed in vivo studies in rodents, including antitumor activity toward three transplantable murine tumor models, toxicity, nephrotoxicity and histopathological investigations. Remarkably, the gold(III) complex AUL12 stands out for higher anticancer activity than cisplatin toward all the murine tumor models examined, inducing up to 80% inhibition of tumor growth. In addition, it shows low acute toxicity levels (lethal dose, LD50 = 30 mg kg−1) and reduced nephrotoxicity. Altogether, these results confirm the reliability of our drug design strategy and support the validation of this gold(III)‐dithiocarbamato derivative as a suitable candidate for clinical trials.

Synthesis and biological activity of ester- and amide-functionalized imidazolium salts and related water-soluble coinage metal N-heterocyclic carbene complexes.

N-Heterocyclic carbene (NHC) ligand precursors, namely, HIm(A)Cl [1,3-bis(2-ethoxy-2-oxoethyl)-1H-imidazol-3-ium chloride] and HIm(B)Cl {1,3-bis[2-(diethylamino)-2-oxoethyl]-1H-imidazol-3-ium chloride}, functionalized with hydrophilic groups on the imidazole rings have been synthesized and were used in the synthesis of corresponding carbene complexes of silver(I) and copper(I), {[Im(A)]AgCl}, {[Im(A)]CuCl}, and {[Im(B)](2)Ag}Cl. Related Au(I)NHC complexes {[Im(A)]AuCl} and {[Im(B)]AuCl} have been obtained by transmetalation using the silver carbene precursor. These compounds were characterized by several spectroscopic techniques including NMR and mass spectroscopy. HIm(B)Cl and the gold(I) complexes {[Im(A)]AuCl} and {[Im(B)]AuCl} were also characterized by X-ray crystallography. The cytotoxic properties of the NHC complexes have been assessed in various human cancer cell lines, including cisplatin-sensitive and -resistant cells. The silver(I) complex {[Im(B)](2)Ag}Cl was found to be the most active, with IC(50) values about 2-fold lower than those achieved with cisplatin in C13*-resistant cells. Growth-inhibitory effects evaluated in human nontransformed cells revealed a preferential cytotoxicity of {[Im(B)](2)Ag}Cl versus neoplastic cells. Gold(I) and silver(I) carbene complexes were also evaluated for their ability to in vitro inhibit the enzyme thioredoxin reductase (TrxR). The results of this investigation showing that TrxR appeared markedly inhibited by both gold(I) and silver(I) derivatives at nanomolar concentrations clearly point out this selenoenzyme as a protein target for silver(I) in addition to gold(I) complexes.

A novel copper complex induces paraptosis in colon cancer cells via the activation of ER stress signalling.

Platinum anticancer drugs have been used for three decades despite their serious side effects and the emerging of resistance phenomena. Recently, a phosphine copper(I) complex, [Cu(thp)4][PF6] (CP), gained special attention because of its strong antiproliferative effects. CP killed human colon cancer cells more efficiently than cisplatin and oxaliplatin and it overcame platinum drug resistance. CP preferentially reduced cancer cell viability whereas non‐tumour cells were poorly affected. Colon cancer cells died via a programmed cell death whose transduction pathways were characterized by the absence of hallmarks of apoptosis. The inhibition of 26S proteasome activities induced by CP caused intracellular accumulation of polyubiquitinated proteins and the functional suppression of the ubiquitin–proteasome pathway thus triggering endoplasmic reticulum stress. These data, providing a mechanistic characterization of CP‐induced cancer cell death, shed light on the signaling pathways involved in paraptosis thus offering a new tool to overcome apoptosis‐resistance in colon cancer cells.

Smart delivery of antitumoral platinum complexes from biomimetic hydroxyapatite nanocrystals

This study widens the role of biomimetic hydroxyapatite (HA) nanocrystals as bone substitutes and describes how they can be used as bone-specific drug delivery devices for in situ treatment of bone tumors upon local implantation. The adsorption and release kinetics of bis-{ethylenediamineplatinum(II)}-2-amino-1-hydroxyethane-1,1-diyl-bisphosphonate and bis-{ethylenediamineplatinum(II)}medronate on two kinds of HA nanocrystals having different morphologies, crystallinity degrees and surface areas have been investigated. The different chemical structures of the two Pt complexes appreciably affect not only the affinity towards the two kinds of HA, but also their release. The Pt complex loading is slightly greater for the HA characterized by lower crystallinity and higher surface area, with respect to the more crystalline one. The cytotoxicity of Pt complexes released from the HA were tested against human cervix carcinoma cells and, interestingly, were found to be more cytotoxic than the unmodified complexes. The released Pt species are therefore the active dichloridoethylenediamineplatinum(II) or related solvato species formed by Pt-bisphosphonate bond breaking.

Selenium induces a multi‐targeted cell death process in addition to ROS formation

Selenium compounds inhibit neoplastic growth. Redox active selenium compounds are evolving as promising chemotherapeutic agents through tumour selectivity and multi‐target response, which are of great benefit in preventing development of drug resistance. Generation of reactive oxygen species is implicated in selenium‐mediated cytotoxic effects on cancer cells. Recent findings indicate that activation of diverse intracellular signalling leading to cell death depends on the chemical form of selenium applied and/or cell line investigated. In the present study, we aimed at deciphering different modes of cell death in a single cell line (HeLa) upon treatment with three redox active selenium compounds (selenite, selenodiglutathione and seleno‐DL‐cystine). Both selenite and selenodiglutathione exhibited equipotent toxicity (IC50 5 μM) in these cells with striking differences in toxicity mechanisms. Morphological and molecular alterations provided evidence of necroptosis‐like cell death in selenite treatment, whereas selenodiglutathione induced apoptosis‐like cell death. We demonstrate that selenodiglutathione efficiently glutathionylated free protein thiols, which might explain the early differences in cytotoxic effects induced by selenite and selenodiglutathione. In contrast, seleno‐DL‐cystine treatment at an IC50 concentration of 100 μM induced morphologically two distinct different types of cell death, one with apoptosis‐like phenotype, while the other was reminiscent of paraptosis‐like cell death, characterized by induction of unfolded protein response, ER‐stress and occurrence of large cytoplasmic vacuoles. Collectively, the current results underline the diverse cytotoxic effects and variable potential of redox active selenium compounds on the survival of HeLa cells and thereby substantiate the potential of chemical species‐specific usage of selenium in the treatment of cancers.