domain: chim sdv

text:
Sílvia A Sousa
email: sousasilvia@tecnico.ulisboa.ptfmarujo@ctn.tecnico.ulisboa.pt

Jorge H Leitão

Rafaela A L Silva

Dulce Belo

Isabel C Santos

Joana F Guerreiro

Marta Martins

Diana Fontinha

Miguel Prudêncio

Manuel Almeida

Dominique Lorcy

Fernanda Marques

On the Path to Gold: Monoanionic Au Bisdithiolate Complexes with Antimicrobial and Antitumor Activities

Keywords: Monoanionic Au complexes, Dithiolates, Antimicrobial, Antitumor, Reactive oxygen species (ROS), DNA interaction

The emergence of resistance to antimicrobial and anticancer drugs poses severe threats to public health worldwide, highlighting the need for more efficient treatments. Here,

) 2 ] -(where cdc = cyanodithioimido carbonate)(3), and [Au(qdt) 2 ] -(where qdt = quinoxaline-2,3-dithiolate)(4) were screened for their antimicrobial and antitumor activities. Complexes 3 and 4 showed antibacterial activity against Staphylococcus aureus (MIC = 15.3 and 14.7 µg/mL, respectively). Complex 3 Journal Pre-proof J o u r n a l P r e -p r o o f 2 also caused significant growth inhibition of Candida glabrata (MIC = 7.0 µg/mL).

Concentrations of complexes 1-4 up to 125 µg/mL had no growth inhibition activity against Escherichia coli. The cytotoxic activity of complexes 1-4 was evaluated against the ovarian cancer cells A2780 and A2780cisR, sensitive and resistant to cisplatin, respectively. All compounds showed high cytotoxic activities against both tumoral cell lines, exhibiting IC 50 values in the low micromolar range (0.9 -5.5 µM) upon 48h incubation. In contrast to complex 1, the complexes 2-4 induced a dose-dependent formation of reactive oxygen species (ROS), similar to the observed for the reference drugs auranofin and cisplatin. Opposite to 4, complexes 1-3 were able to activate caspase 3/7, suggesting the involvement of apoptosis in the mechanism of cell death.

Contrasting with cisplatin, complexes 3, 4 and auranofin did not cause DNA damage.

Combined, these data provide evidence that these monoanionic gold bisdithiolates, particularly complex 3, are potential lead compounds to further explore as therapeutic drugs.

Introduction

The burden of human infectious diseases remains a serious threat to public health worldwide. In addition, the emergence of resistance to the clinically in-use antimicrobials among bacteria, fungi and parasites, lowers the efficacy of available prevention and treatment measures [START_REF]Global Action Plan on Antimicrobial Resistance[END_REF]. The World Health Organization (WHO) released a list of bacteria for which new antibiotics are urgently needed. The top priority species were summarized under the acronym ESKAPE, comprising the Gram-positive Enterococcus faecium and Staphylococcus aureus, and the Gram-negative Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. [START_REF] Rice | Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE[END_REF]. Fungal human infections are also associated with high morbidity and mortality rates, especially among immunocompromised patients, such as HIV-infected patients and cancer patients [START_REF] Cavalheiro | Host-Pathogen Interactions Mediated by MDR Transporters in Fungi: As Pleiotropic as it Gets![END_REF].

Another global health concern is the rise in the incidence of cancer, which remains one of the most common causes of death worldwide [START_REF] Arnold | Recent trends in incidence of five common cancers in 26 European countries since 1988: Analysis of the European Cancer Observatory[END_REF]. With advances in cancer treatment, in particular with the use of chemotherapeutic drugs, the prognosis and survival rate of patients have improved considerably over the past decades for several types of cancers.

However, the toxicity and severe side effects associated with the majority of the anticancer drugs currently available has led to an increase in opportunistic infections [START_REF] Klastersky | Opportunistic infections in patients with cancer[END_REF]. Moreover, chronic infections with pathogenic microorganisms lead to inflammation and decreased immunological defences, contributing to the proliferation of cancer cells [START_REF] Plummer | Global burden of cancers attributable to infections in 2012: a synthetic analysis[END_REF][START_REF] Hattar | Lipoteichoic acids from Staphylococcus aureus stimulate proliferation of human non-small-cell lung cancer cells in vitro[END_REF][START_REF] Ramirez-Garcia | Candida albicans and cancer: Can this yeast induce cancer development or progression?[END_REF]. Therefore, new classes of compounds with both antimicrobial and antitumor properties that act on non-classical targets have been recently developed and studied [START_REF] Felício | Peptides with Dual Antimicrobial and Anticancer Activities[END_REF].

One of those molecules, auranofin, a coordinated gold(I) complex commonly used for the treatment of rheumatoid arthritis, was proposed for drug repositioning as a new antimicrobial and antitumor agent. Auranofin has progressed to FDA approved clinical trials in various types of tumors such as leukemia and ovarian cancer, even though the molecular mechanisms underlying the antitumor activity in ovarian cancer cells are not yet fully understood [START_REF] Landini | Selection and characterization of a human ovarian cancer cell line resistant to auranofin[END_REF][START_REF] Roder | Auranofin: repurposing an old drug for a golden new age[END_REF]. In addition, auranofin is also used as a reference compound when novel gold-based compounds are evaluated as prospective therapeutic drugs [START_REF] Roder | Auranofin: repurposing an old drug for a golden new age[END_REF].

The cytotoxicity of gold complexes usually involves the inhibition of enzymes, especially those containing thiol groups. Therefore, the strong binding affinity of gold to thiol groups makes thioredoxin reductases (TrxR), glutathione reductases (GR) and cysteine proteases, all overexpressed in cancer cells, potential targets for gold complexes [START_REF] Zou | Chemical biology of anticancer gold( iii ) and gold( i ) complexes[END_REF]. Inhibition of these enzymes activity can impair the redox state of the cell and lead to increased production of reactive oxygen species (ROS), causing cellular oxidative stress and leading to intrinsic apoptosis. This mechanism of action is distinct from the mechanism of action of platinum-based metallodrugs currently used in anticancer therapies, such as cisplatin, which mainly rely on direct interaction with the DNA [START_REF] Siddik | Cisplatin: mode of cytotoxic action and molecular basis of resistance[END_REF]. Due to the emergence of drug resistance of cancer cells to these metallodrugs and the undesirable toxic effects that constitute a current barrier to a successful treatment, novel antitumor drugs are currently being pursued [START_REF] Housman | Drug resistance in cancer: an overview[END_REF].

Transition metal bisdithiolates have been extensively studied as building blocks of new molecular conductors and magnets [START_REF] Stiefel | Dithiolene chemistry : synthesis, properties, and applications[END_REF]. Their usual square planar molecular structure, stability, and vivid redox behaviour, make them also suitable candidates to be explored on different areas of research, namely their biological applications. Studies on the biological properties of dithiolate complexes have been limited to a few examples, such as cobalt(II), copper(II), nickel(II) and zinc(II) furanylmethyl-and thienylmethyl dithiolenes, which possess significant antibacterial and antifungal activity [START_REF] Chohan | In-vitro Antibacterial, Antifungal and cytotoxic activity of cobalt (II), copper (II), nickel (II) and zinc (II) complexes with furanylmethyl-and thienylmethyl-dithiolenes: [1, 3-dithiole-2-one and 1,3dithiole-2-thione[END_REF]. More recently, gold(III) 1,2-dithiolene cyclometalated complexes were also found to exhibit antimicrobial and cytotoxic activities [START_REF] Pintus | Au(pyb-H)(mnt)]: A novel gold(III) 1,2dithiolene cyclometalated complex with antimicrobial activity (pyb-H = Cdeprotonated 2-benzylpyridine; mnt = 1,2-dicyanoethene-1,2-dithiolate)[END_REF]. However, the molecular structure of the reported complexes differs significantly from the square planar homoleptic transition metal bisdithiolates studied in this work. We selected four homoleptic monoanionic gold bisdithiolate complexes [START_REF] Belo | Synthesis and characterisation of charge transfer salts based on Au(dcdmp)2 and TTF type donors[END_REF][START_REF] Neves | Cation and ligand roles in the coordination of FeIII bisdithiolene complexes; the crystal structures of (BrBzPy)2[Fe(qdt)2]2 and [Fe(α-tpdt)2]22-salts[END_REF][START_REF] Belo | Synthesis, crystal structure and magnetic properties of bis(3,4;3′,4′-ethylenedithio)2,2′,5,5′tetrathiafulvalene-bis(cyanoimidodithiocarbonate)aurate(III), ([END_REF][START_REF] Silva | α-DT-TTF) 2 [Au(mnt) 2 ]: A Weakly Disordered Molecular Spin-Ladder System[END_REF][START_REF] Silva | Dithiophene-TTF Salts; New Ladder Structures and Spin-Ladder Behavior[END_REF] (Figure 1) to be explored for their antimicrobial and antitumor activities, and to gain clues into their structure-activity relationship.

These complexes were previously used to prepare molecular materials as salts with several organic donors [START_REF] Belo | Synthesis and characterisation of charge transfer salts based on Au(dcdmp)2 and TTF type donors[END_REF][START_REF] Neves | Cation and ligand roles in the coordination of FeIII bisdithiolene complexes; the crystal structures of (BrBzPy)2[Fe(qdt)2]2 and [Fe(α-tpdt)2]22-salts[END_REF][START_REF] Belo | Synthesis, crystal structure and magnetic properties of bis(3,4;3′,4′-ethylenedithio)2,2′,5,5′tetrathiafulvalene-bis(cyanoimidodithiocarbonate)aurate(III), ([END_REF][START_REF] Silva | α-DT-TTF) 2 [Au(mnt) 2 ]: A Weakly Disordered Molecular Spin-Ladder System[END_REF][START_REF] Silva | Dithiophene-TTF Salts; New Ladder Structures and Spin-Ladder Behavior[END_REF], but the structure of 3 and 4 as salts with simple cations was never reported. To our knowledge, the work reported herein constitutes the first systematic study aiming to explore the biological properties of this type of compounds.

Materials and Methods

Compounds

The gold complexes TBA[Au(mnt) 2 ] (1), TBA[Au(i-mnt) 2 ] (2), TBA[Au(cdc) 2 ] (3), and TPP[Au(qdt) 2 ] (4) were prepared as tetrabutyl ammonium (TBA) and tetraphenylphosphonium (TPP) salts by previously described methods [START_REF] Davison | The Preparation and Characterization of Four-Coordinate Complexes Related by Electron-Transfer Reactions[END_REF][START_REF] Werden | Transition Metal Complexes Containing 1,1-Dicyanoethylene-2,2-dithiolate[END_REF][START_REF] Cotton | Transition metal complexes of the N-cyanocarbimate ion[END_REF][START_REF] Ribas | Molecular compounds based on DT-TTF and Au(cdc)2 complex. Structural, magnetic and electrical properties[END_REF][START_REF] Theriot | Metal complexes of 2,3quinoxalinedithiol[END_REF][START_REF] Ganguli | Cobalt and iron complexes of 2,3-quinoxalinedithiol[END_REF][START_REF] Simão | Charge transfer salts based on Cu(qdt)2, Ni(qdt)2 and Au(qdt)2 anions[END_REF].

Auranofin and cisplatin were purchased from Sigma-Aldrich.

Cells and cell culture media

A2780 (cisplatin sensitive) and A2780cisR (cisplatin resistant) ovarian tumour cells were purchased from Sigma-Aldrich. V79 (hamster lung fibroblasts) were purchased from ATCC (American Type Culture Collection). RPMI and DMEM/F12 cell media and medium supplements were purchased from Gibco (Thermo Fisher Scientific).

Bacterial and fungal strains and culture media

The bacterial strains Staphylococcus aureus Newman and Escherichia coli ATCC25922, and the fungal strains Candida glabrata CBS138 and Candida albicans SC5134 were used. The bacterial and fungal strains were isolated from human infections [START_REF] Duthie | Staphylococcal Coagulase: Mode of Action and Antigenicity[END_REF][START_REF] Minogue | Complete Genome Assembly of Escherichia coli ATCC 25922, a Serotype O6 Reference Strain[END_REF][START_REF] Borst | Rapid acquisition of stable azole resistance by Candida glabrata isolates obtained before the clinical introduction of fluconazole[END_REF][START_REF] Gillum | Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations[END_REF]. When in use, bacterial strains were maintained in Lennox Broth (LB) solid medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl and 15 g/L agar), while fungal strains were maintained in Yeast Extract-Peptone-Dextrose (YPD) solid medium (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract and 15 g/L agar), as previously described [START_REF] Costa | Antimicrobial Activity of Silver Camphorimine Complexes against Candida Strains[END_REF].

Cyclic voltammetry

Cyclic voltammetry data were obtained using a BAS C3 Cell Stand. The voltammograms were obtained at room temperature with variable scan rates in the range of 20-500 mV.s -1 , a platinum working wire and counter electrodes and a Ag/AgNO 3 (0.01 M AgNO 3 and 0.1 M TBAPF 6 in acetonitrile) reference electrode, in which the Ag + ion electrode was separated from the bulk solution by a VycorTM frit. The measurements were performed on fresh solutions with a concentration of 110 -3 M, in acetonitrile, that contained TBAPF 6 (110 -1 M) as a supporting electrolyte. Ferrocene was added directly to the solution after analysis of the analyte of interest to allow the potentials normalization in situ, relatively to the ferrocene/ferrocenium couple redox potential.

X-ray crystallography

Suitable crystals for X-ray diffraction were mounted on a loop with protective oil, and X-ray data was collected on a Bruker APEX II CCD detector diffractometer using graphite monochromated MoKα radiation (λ = 0.71073 Å) and operated in a φ and ω scans mode. A semi empirical absorption correction was carried out using SADABS (Sheldrick, G. M. SADABS, Bruker AXS Inc., Madison, Wisconsin, USA, 2004). Data collection, cell refinement and data reduction were done with the SMART and SAINT programs (SMART and SAINT, Bruker AXS Inc., Madison, Wisconsin, USA, 2004).

The structures were solved by direct methods using SIR97 [START_REF] Altomare | SIR 97: a new tool for crystal structure determination and refinement[END_REF] and refined by fullmatrix least-squares methods using the program SHELXL97 [START_REF] Sheldrick | SHELXL-97 program for crystal structure refinement[END_REF] using the winGX software package [START_REF] Farrugia | WinGX suite for small-molecule single-crystal crystallography[END_REF]. Non-hydrogen atoms were refined with anisotropic thermal parameters, except in cases of disorder where these specific atoms were refined as isotropic, whereas H-atoms were placed in idealized positions and allowed to refine riding on the parent C atom. Molecular graphics were prepared using ORTEP [START_REF] Farrugia | IUCr, ORTEP -3 for Windows -a version of ORTEP -III with a Graphical User Interface (GUI)[END_REF].

Intermolecular energy interactions calculations

The interaction energies were calculated with the freeware PrimeColor Software, CAESER [START_REF] Ren | Crystal and electronic structure analysis using CAESAR[END_REF]. The program employs the extended Hückel method [START_REF] Hoffmann | An Extended Hückel Theory. I. Hydrocarbons[END_REF][START_REF] Whangbo | The band structure of the tetracyanoplatinate chain[END_REF][START_REF] Whangbo | Conjugated One and Two Dimensional Polymers[END_REF], where the basis set consists of Slater type orbitals of double- quality. The exponents, contraction coefficients and atomic parameters were taken from previous work [START_REF] Canadell | On the band electronic structure of X [M (dmit)2]2 (X = TTF, (CH3)4N ; M = Ni, Pd) molecular conductors and superconductors[END_REF].

Determination of antibacterial activity

The antibacterial activity of the complexes 1-4 was assessed according to standard methods (NCCLS), and as previously described [START_REF] Cardoso | Antibacterial activity of silver camphorimine coordination polymers[END_REF][START_REF] Alves | Synthesis, antimicrobial activity and toxicity to nematodes of cyclam derivatives[END_REF][START_REF] Carvalho | Ag(I) camphor complexes: antimicrobial activity by design[END_REF], using 96-well polystyrene microtiter plates (Greiner Bio-One). Stock solutions of the tested complexes were prepared in 100% DMSO at final concentrations of 10 mg/mL (complexes 1 and 2), or 5 mg/mL (complexes 3 and 4, and auranofin), depending on the solubility of each complex. Briefly, to each well, 100 l aliquots of serial 1:2 dilutions of stock solutions prepared for each complex in Mueller-Hinton (MH) broth (Fluka Analytical) were added, in order to obtain final concentrations ranging from 125 µg/mL to 0.49 µg/mL. Then, 100 l of adequately diluted bacterial suspensions (S. aureus Newman or E. coli ATCC 25922) were added and mixed with the serially diluted complexes, in order to obtain a final optical density of 0.01, measured at 640 nm (OD 640 ), in a Hitachi U-2000 UV/Vis spectrophotometer. These bacterial suspensions were obtained from 5 h grown bacterial cultures (carried out in MH broth at 37°C and 250 rev.min -1 ), and adequately diluted with fresh MH medium. When testing auranofin towards S. aureus Newman, additional concentrations ranging from 0.49 µg/mL to 0.06 µg/mL were also used. After 24 h of incubation at 37 ºC, the wells were examined for turbidity (growth), resuspended by pipetting, and their optical density was measured in a SPECTROstar Nano microplate reader (BMG Labtech) at 640 nm.

All complexes were tested in three independent experiments and in duplicate wells.

Minimum inhibitory concentration (MIC) values were estimated after data fitting of the OD 640 mean values using a modified Gompertz equation as described by Lambert and Pearson, using the GraphPad Prism software (version 6.07) [START_REF] Lambert | Susceptibility testing: accurate and reproducible minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) values[END_REF]. In each experiment, positive (without complex) and negative controls (no organism inoculum) were performed. The effect of 5 % (V/V) DMSO on bacterial growth was also determined.

Determination of antifungal activity

Antifungal susceptibility testing was carried out according to the standardized microdilution method recommended by EUCAST (European Committee on Antimicrobial Susceptibility Testing) for Candida spp. [START_REF] Arendrup | EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST)[END_REF], and as previously described [START_REF] Costa | Antimicrobial Activity of Silver Camphorimine Complexes against Candida Strains[END_REF][START_REF] Cardoso | Ag(I) camphorimine complexes with antimicrobial activity towards clinically important bacteria and species of the Candida genus[END_REF]. Briefly, 96-well microtiter plates (Greiner Bio-One) were filled with 100 l aliquots of 1:2 serially diluted stock solutions of each complex, prepared as described in 2.7, in order to obtain final concentrations ranging from 125 µg/mL to 0.49 µg/mL. Stock solutions were prepared in RPMI 2% G. RPMI 2% G is composed of RPMI-1640 liquid medium (SIGMA) supplemented with 20 g/L glucose (final concentration), buffered to pH 7.0 with 0.165 M morpholinepropanesulphonic acid (MOPS; SIGMA). To these aliquots, 100 L of diluted fungal suspensions (C. glabrata CBS138 or C. albicans SC5134) were added. Diluted fungal suspensions were prepared from overnight grown cultures (carried out in YPD broth at 30 °C and 250 rev.min -1 ), diluted with RPMI-1640 fresh liquid medium to a final optical density of 0.025, measured at 530 nm (OD 530 ) in a Hitachi U-2000 UV/Vis spectrophotometer. After 24 h of incubation at 35 ºC, the wells were examined for turbidity (growth), resuspended and their optical density was measured in a SPECTROstar Nano microplate reader (BMG Labtech) at 530 nm.

All compounds were tested at least in three independent experiments and in duplicate wells. Minimum inhibitory concentration (MIC) was estimated after data fitting of the OD 530 mean values using a modified Gompertz equation, using the GraphPad Prism software (version 6.07) [START_REF] Lambert | Susceptibility testing: accurate and reproducible minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) values[END_REF]. In each experiment, positive (without compound) and negative controls (no organism inoculum) were carried out. The effect of 1.25 % (V/V) DMSO on fungal growth was also evaluated.

Determination of cytotoxic activity

The cytotoxic activity of the tested compounds was evaluated in ovarian cancer cells A2780 (cisplatin sensitive) and A2780cisR (cisplatin resistant), and in normal fibroblasts V79. All cell lines were grown in RPMI medium supplemented with 10% FBS and maintained in a humidified incubator (Heraeus, Germany) with 5% CO 2 /95% air. Cell viability was measured by the colorimetric MTT assay. The assay measures the conversion of 3-(4,5-dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to an insoluble formazan product by mitochondrial dehydrogenase enzymes active in live cells. For the assay, cells were seeded in 96-well plates at a density of 2 × 10 4 cells in 200 μL medium and allowed to attach for 24 h. Complexes 1-4 were first diluted in DMSO for complete solubility and then in medium to prepare serial dilutions in the range 10 -7 -10 -4 M. The concentration of DMSO in the medium did not exceed 1% and had no cytotoxic effect. The reference compounds cisplatin and auranofin were first diluted in water (cisplatin) and DMSO (auranofin) and then in medium in the same concentration range of the complexes. After careful removal of the medium, 200 μL of each compound's dilution in fresh medium were added to the cells and incubated for 3, 24 and 48 h at 37 ºC. At the end of the treatment, the medium was discarded and 200 μL of MTT solution in PBS (0.5 mg/mL) were applied to each well. After 3 h at 37 °C, the medium was removed and replaced by DMSO (200 μL) to solubilize the formazan crystals formed. The percentage of cellular viability was assessed measuring the absorbance at 570 nm using a plate spectrophotometer (Power Wave Xs, Bio-Tek). The IC 50 values were calculated using the GraphPad Prism software (version 5.0). Results are shown as the mean ± SD of at least two independent experiments done with six replicates each.

Measurement of reactive oxygen species (ROS) 2.10.1 NBT assay

Superoxide anion was detected with nitroblue tetrazolium (NBT) (NBT= 2.2′-bis(4-Nitrophenyl)-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′-diphenylene)ditetrazolium chloride), a yellow water-soluble tetrazolium compound. NBT reacts with cellular superoxide anions to form a dark blue formazan derivative that can be detected by its absorbance at 550 nm. The experimental procedure followed an adaptation of previously described methods [START_REF] Esfandiari | Utility of the nitroblue tetrazolium reduction test for assessment of reactive oxygen species production by seminal leukocytes and spermatozoa[END_REF][START_REF] Fridovich | Superoxide Anion Radical (O•̄ 2 ), Superoxide Dismutases, and Related Matters[END_REF]. For the assay, A2780 cells (2x10 4 /well) in RPMI were seeded in 96-well plates and left to adhere overnight. Then, cells were incubated with 10, 20 or 50 µM of the compounds for 1 h at 37 °C, followed by addition of 20 μL of a NBT solution (10 mg/mL PBS). The incubation was allowed to proceed for an additional 1 h at 37 °C. The medium was then gently discarded and the formazan deposits were solubilized with 200 μL of 90% DMSO:10% 0.01 M NaOH and 0.1% SDS. The absorbance of each well was measured at 550 nm. Each experiment was repeated twice and each concentration tested with at least six replicates per experiment.

Results (mean ± SD) are expressed as % of controls (no treatment).

H 2 DCFDA assay

The production of intracellular ROS, namely hydrogen peroxide, hydroxyl radicals and peroxynitrite, was detected by fluorescence using the cell permeant fluorogenic dye H 2 DCFDA (dihydro-2′7′dichlorofluorescein diacetate) on a microplate-based assay [START_REF] Kalyanaraman | Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations[END_REF]. H 2 DCFDA is deacetylated by intracellular esterases to a non-fluorescent compound, which is later converted into the highly fluorescent 2',7'-dichlorofluorescein (DCF) upon oxidation by ROS. DCF fluorescence was detected using maximum excitation and emission spectra of 492 and 517 nm, respectively.

For the H 2 DCFDA assays, A2780 cells (2x10 4 /well) were seeded in 96-well plates and left to attach overnight. Then, the medium on each well was replaced with a solution of 10 μM H 2 DCFDA in phenol red-free DMEM/F12 and cells were further incubated at 37 °C for 30 min. This solution was then removed and cells were incubated with fresh phenol red-free DMEM/F12 medium containing 10, 20 or 50 μM of each test complex, hydrogen peroxide (positive control) or the reference drugs auranofin and cisplatin at the same concentrations (10, 20 and 50 μM) for 3 h. For the combined assays with the ROS scavengers, the complexes at 50 µM were co-incubated with sodium pyruvate (10 mM), sodium bicarbonate (10 mM) and DMSO (0.5%) for 3 h. Results are shown as the mean±SD of a representative experiment done with four replicates per condition.

Apoptosis (caspase 3/7 assay)

Apoptosis (caspase 3/7 assay) was assessed using a Caspase-Glo®3/7 Assay (Promega).

The assay provides a proluminescent caspase-3/7 substrate that is cleaved in the presence of caspase 3/7 to release aminoluciferin, the luciferase substrate used in the production of light. The luminescent signal is proportional to the caspase activity present in the cells. The assay employed A2780 cells, in phenol red-free DMEM/F12 medium, in 96 wells plates, treated with the compounds for 24 h, at concentrations equivalent to their IC 50 values. Zinc acetate (50 µM) was co-incubated with the complexes. After 24 h of incubation, 100 μL of medium were removed from each well. Caspase 3/7® reagent was added at a 1:1 ratio and the plate was shaken in an orbital shaker for 30 s at 300-500 rpm. The plate was incubated at room temperature for 90 min, protected from light. Luminescence intensity was measured using an Infinite 200 Plate Reader (Tecan). Each experiment was performed in duplicate and each concentration was tested with at least three replicates. Results (mean ± SD) were expressed as relative fluorescence units.

DNA interaction

DNA interaction was assessed by monitoring the electrophoretic mobility of the supercoiled ϕX174 DNA, as previously described [START_REF] Francisco | Pt(ii) complexes with bidentate and tridentate pyrazolyl-containing chelators: synthesis, structural characterization and biological studies[END_REF]. Briefly, each reaction mixture contained, in a final volume of 20 µL, 200 ng of supercoiled ϕX174 DNA (Promega) in 10 mM phosphate buffer (pH 7.2) with different concentrations of cisplatin, auranofin, or the metal complexes. After incubation in the dark for 24 h at 37 ºC, 2 μl of 10× DNA loading buffer (Applichem) was added to each tube and the samples were loaded on an 0.8% agarose gel in TBE buffer (Thermo Fisher Scientific). The electrophoresis was carried out for about 3 h at 90 V. Samples with non-incubated plasmid and plasmid incubated with DMSO were used as controls. The gels were then stained in a 3× GelRed® (Biotium) staining solution in H 2 O, according to the manufacturer's instructions. Bands were visualized using an AlphaImagerEP (Alpha Innotech) under UV light.

Results and Discussion

Crystal Structures

The structure of complexes 1 and 2 were previously reported. In this work we describe the structure of complexes 3 and 4 as tetrabutylammonium and tetraphenylphosphonium salts.

Complex 3

TBA[Au(cdc) 2 ] (3) crystallizes in the monoclinic system, space group P2 1 /c (Table S1).

The unit cell contains an anion and a cation, both molecules at general positions. The anionic molecule presents the usual square coordination geometry and, within the experimental error, it is planar (Figure 2, Table S2).

The crystal structure consists of stair-like chains of anion pairs, running parallel to the a axis (Figure 3). The angle between the molecules' average plane in neighbouring chains is 76 °. There are no interactions between molecules in different chains. Along the chains, the pairs of anions are connected by side-by-side S•••S and S•••N short contacts (Figure 4, Table S3). Between pairs, the overlapping mode between anions places the sulphur atoms on top of the gold atom, inducing a soft molecular slipping both along the longest and minor molecular axis. The cations fill the spaces between anionic chains.

Several C-H•••S and C-H•••N hydrogen bonds connect the anions and the cations.

Complex 4

TPP[Au(qdt) 2 ] (4) crystallizes in the monoclinic system, space group P2/n (Table S1).

The unit cell contains two anions and two cations at an inversion center. The anionic molecules present a square coordination geometry of the Au-S 4 core, with the quinoxaline skeleton of the two ligands pointing up in opposite directions, conferring a chair type distortion to the molecule (Figure 2b-c, Table S4). The crystal structure is composed of a 3D anionic network where each anion is surrounded by other four anions placed almost perpendicularly (86°) to its molecular average plan (Figure 5). The anions in this gridding arrangement are connected by C-H•••S hydrogen bonds, involving the coordinating sulphur atoms and the more external hydrogen atom of the phenyl ring (Table S5). The cations are placed in tunnels shaped by the anionic grid along b, and In the crystal structures of complexes 1-4, the major difference between anions is the size of the electronic cloud (Figure 7). Although the anions in 1 and 2 are isostructural, the i-mnt ligand is slightly larger than the related mnt. Both the ligands in 3 and 1 present a similar electronic cloud; however, in 3 the asymmetric trans configuration potentiates intermolecular interactions less constrained by stereochemical effects.

Although bearing the same four nitrogen atoms as the previous complexes, the ligand in 4 has a quinoxaline moiety, a more extended aromatic system that besides enlarging the molecular size also enables π-π interactions.

One of the most important aspects of transition metal bisdithiolenes is their vivid redox behaviour, making readily accessible several oxidation states, from monoanionic, to dianionic, neutral and even cationic states [START_REF] Coucouvanis | The Chemistry of the Dithioacid and 1,1-Dithiolate Complexes[END_REF]. In the case of gold bisdithiolates, the monoanionic state is the most common and stable. The organic dithiolene ligands play a non-innocent role in the modulation of the redox properties of these complexes by contributing, in different degrees, to the molecular orbitals. This is illustrated in Figure 8, representing the HOMO of the anions in complexes 1-4. These results denote that the oxidation processes are more ligand centered in the case of cdc (3) and qdt [START_REF] Arnold | Recent trends in incidence of five common cancers in 26 European countries since 1988: Analysis of the European Cancer Observatory[END_REF] complexes than in the case of mnt (1) and i-mnt (2).

Cyclic Voltammetry

The redox properties of complexes 1-4, in CH 3 CN solutions, were studied by cyclic voltammetry, at room temperature, using Ag/AgNO 3 as the reference electrode. The redox potential of the ferrocene/ferrocenium couple was used as an internal reference.

The results are summarized in Figure 9, Table 1 and Figure S1.

Complex 1 undergoes a quasi-reversible one electron oxidation process, centered at 793 mV, ascribed to the couple [Au(mnt) 2 ] -1 /[Au(mnt) 2 ] -2 . In the case of compound 2, an irreversible redox process at -829 mV is observed, attributed to the reduction of the monoanionic to the dianionic species ([Au(i-mnt) 2 ] -1  [Au(i-mnt) 2 ] -2 ) [START_REF] Williams | The Toluenedithiolate and Maleonitriledithiolate Square-Matrix Systems[END_REF]. In the range -1/1 V, no redox processes were visible for complex 3, suggesting a higher stability of this [Au(cdc) 2 ] -monoanion. Complex 4 presents a slightly different voltammogram, showing two distant peaks, one cationic and one anionic, at -701 and +901 mV, respectively. The peak at more positive potentials was revealed to be interdependent of the process occurring at more negative potentials.

The irreversible reduction process observed in the case of 4 is likely to reflect the transition from the monoanionic to the dianionic species ([Au(qdt

) 2 ] -1  [Au(qdt) 2 ] -2 ).
With the exception of complex 3, where no processes were observed in the range of the potentials studied, the ligand does not substantially affect the redox potentials concerning the couple ([AuL] -1 /[AuL] -2 ), although only in the case of [Au(mnt) 2 ] -it is a quasi-reversible process.

Studies on complexes stability in solution by UV-vis spectroscopy

In order to evaluate the stability of complexes 1-4 in solution, a series of UV-Vis spectra were collected during 48 h. The spectra of DMSO solutions of complexes 1-4, were recorded in phenol red-free DMEM/F12 medium, in the absence or presence of fetal bovine serum (FBS). As a reference, the spectra of complexes 1-4 in DMSO were also collected. Results from this study support the cyclic voltammetry data, suggesting that complex 3 (Figure 10) is the most stable complex of this series of gold bisdithiolates. Distinctly from the other complexes (Figure S2-S4), the solutions of complex 3 remained translucent throughout the 48 h, with no visible precipitation. Complex 3 also presented a higher solubility in both medium, with and without FBS. In general, all complexes were more soluble in the presence of FBS. In all cases, a new absorption peak appeared around 237 nm, which was ascribed to the interaction of the complexes with the medium. In the medium with FBS, these interactions could be observed thought the peak at around 250 nm.

Antibacterial Activity

The antibacterial properties of complexes 1-4 were assessed based on the determination of the MIC values towards the Gram-positive S. aureus Newman and the Gram-negative E. coli ATCC25922. The MIC values obtained for the four complexes tested and auranofin are summarized in Table 2.

Complexes 3 and 4 inhibited S. aureus Newman growth, with estimated MIC values of 15.3 μg/mL and 14.7 μg/mL, respectively, while complexes 1 and 2 did not inhibit the growth of S. aureus for concentrations ≤ 125 μg/mL. However, complex 2 at 125 μg/mL reduced 65% of the S. aureus Newman growth (data not shown). Contrastingly, no E. coli growth inhibition was detected for the complexes tested at concentrations up to 125 μg/mL. Concentrations higher than 125 μg/mL resulted in the formation of precipitates during the assay and were not considered. Results indicate a higher antibacterial activity of complexes 3 and 4 towards Gram-positive bacteria.

The antibacterial activity of auranofin was also tested as a reference. Auranofin was very active in inhibiting the growth of the Gram-positive S. aureus Newman, with an estimated MIC of 0.2 μg/mL (Table 2). In contrast, the estimated MIC value for the Gram-negative E. coli ATCC 25922 was 35.6 μg/mL (Table 2). These results are in good agreement with previous reports that describe a high potency of auranofin against Gram-positive clinical isolates, with inhibitory activities in the range 0.12-2 μg/mL and higher MIC values for Gram-negative bacteria (> 16 μg/mL) [START_REF] Aguinagalde | Auranofin efficacy against MDR Streptococcus pneumoniae and Staphylococcus aureus infections[END_REF][START_REF] Harbut | Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis[END_REF]. Similar conclusions on relative antibacterial activity of the complexes can be taken if one considers the estimated antibacterial activity of complexes and auranofin in terms of molar concentration instead of mass concentration, as complex 3 and auranofin molecular masses of 671.76 and 678.48 g/mol, respectively. Complexes 1 and 2 are isomers with identical molecular mass (719.80 g/mol) while complex 4 has the highest molecular mass 823.95 g/mol.

Previous studies suggested that auranofin inhibits the bacterial TrxR, a protein essential for the maintenance of the thiol-redox homeostasis in some Gram-positive bacteria, including S. aureus [START_REF] Harbut | Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis[END_REF]. Thiol-based redox metabolism is essential for several cellular processes, such as protection against reactive oxidative species, correct protein folding, and DNA synthesis [START_REF] Lu | The thioredoxin antioxidant system, Free Radic[END_REF]. In many organisms, glutathione (GSH) and GR function in parallel with thioredoxin (Trx) and thioredoxin reductase (TrxR), to provide the cells with a source of reducing equivalents [START_REF] Lu | The thioredoxin antioxidant system, Free Radic[END_REF]. Several Gram-positive bacteria lack the GR-GSH system, making the Trx-TrxR system an interesting new target for antimicrobials [START_REF] Harbut | Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis[END_REF][START_REF] Lu | The thioredoxin antioxidant system, Free Radic[END_REF]. However, recent reports have shown that TrxR is not the primary target of auranofin in bacteria [START_REF] Thangamani | Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens[END_REF]. In their study, Thangamani et al. observed that auranofin inhibited several biosynthetic pathways in bacteria including cell wall, DNA and protein synthesis, but no exact molecular target was found [START_REF] Thangamani | Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens[END_REF]. These authors also observed that the lower activity of auranofin against Gram-negative bacteria was not only due to the presence of GR-GSH system, but also due to the permeability barrier conferred by the outer membrane.

Antifungal activity

The antifungal activities of the complexes 1-4 towards the pathogenic strains C. glabrata CBS138 and C. albicans SC5134 were assessed using the microdilution method. The MIC values for the four compounds tested are presented in Table 3.

Results show that complex 3 was able to inhibit C. glabrata CBS138 growth, with an estimated MIC of 7 μg/mL. The other three complexes tested were unable to inhibit C. glabrata growth. However, complex 2 at 62.5 μg/mL reduced C. glabrata CBS138 growth by 54% (data not shown). C. albicans SC5134 growth was not inhibited by the four complexes tested, although complex 3 at 62.5 μg/mL reduced C. albicans SC5134 growth by 87% (data not shown). These results indicate a higher antifungal activity of complex 3 towards Candida strains, with a higher inhibitory effect against C. glabrata strains.

The antifungal activity of auranofin was also tested against the clinical Candida spp. strains for comparison. Auranofin was very active in inhibiting the growth of the Candida spp. isolates tested, with MIC values of 7.9 and 15.3 μg/mL for C. albicans and C. glabrata, respectively (Table 3). Previous studies have also shown that auranofin inhibits the growth of several clinical Candida spp. isolates [START_REF] Thangamani | Repurposing Approach Identifies Auranofin with Broad Spectrum Antifungal Activity That Targets Mia40-Erv1 Pathway[END_REF]. Chemogenomic profiling of Saccharomyces cerevisiae with auranofin revealed that mitochondrial proteins are the potential targets of this compound [START_REF] Thangamani | Repurposing Approach Identifies Auranofin with Broad Spectrum Antifungal Activity That Targets Mia40-Erv1 Pathway[END_REF].

Cytotoxic activity

The cytotoxic activity of complexes 1-4 was evaluated against the ovarian A2780 and A2780cisR cancer cells, sensitive and resistant to cisplatin, respectively, as well as towards the fibroblast normal cell line V79. Auranofin and cisplatin were included in the assays for comparative purposes.

As depicted in Figure 11 andTable 4, all complexes displayed similar high cytotoxic activity against both the sensitive and the resistant cancer cell lines upon 48 h of exposure to the complexes. At shorter incubation times (3 h), all the complexes presented IC 50 values higher than 100 µM, with the exception of 3 (IC 50 = 64.2 ± 15 µM) (data not shown). These differences in the complexes cytotoxic activity became more evident after 24 h of incubation (Figure 11). In fact, at 24 h, complex 1 was the least active complex of the series for all the cell lines studied, even when compared with its relative complex 2. Auranofin also exhibited potent antitumor activity in both cancer cell lines upon 48 h incubation (Table 4), in agreement with results previously reported for A2780 cells [START_REF] Landini | Selection and characterization of a human ovarian cancer cell line resistant to auranofin[END_REF][START_REF] Marzano | Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatinresistant human ovarian cancer cells[END_REF]. Upon 48 h incubation, the IC 50 values determined for the gold complexes towards the cisplatin sensitive A2780 cells were in the same order of magnitude (0.9 -4.4 µM) as those obtained in this study for cisplatin (IC 50 = 3.6 ± 1.3 µM). However, in the resistant cell line A280cisR, the IC 50 values of the complexes were remarkably lower, about one order of magnitude, when compared to cisplatin (IC 50 = 36 ± 13 µM), which indicates the occurrence of less cross-resistance for the new complexes tested than for the reference drug. The precursor potassium gold(III) chloride (KAuCl 4 ) and the ligand potassium-1,1-dicyano-2,2-ethylenedithiolate (K 2 (i-mnt) were also included in the study but displayed very low activity (IC 50 > 100 µM) (data not shown). These results indicate that the coordination to the gold center resulted in a considerable improvement of the cytotoxic activity and neither the ligands nor the gold precursor seemed to be the active species responsible for the cytotoxicity.

A highly relevant point concerning viable pharmacological applications of anticancer drugs is their relatively high degree of specificity for cancer cells [START_REF] Blagosklonny | Analysis of FDA approved anticancer drugs reveals the future of cancer therapy[END_REF]. Thus, it is important to evaluate the cytotoxic activity of a prospective drug against a normal cell line. In this work, V79 fibroblasts were selected to evaluate the selectivity of the complexes against cancer cells (Table 4). The IC 50 values calculated for the reference drugs cisplatin and auranofin in normal cells revealed auranofin as the more toxic compound towards these cells. These results highlight another potentially advantageous feature of the monoanionic gold bisdithiolate complexes under study in being less toxic to normal cells than the reference drug auranofin, while presenting similar toxicity to cisplatin.

Production of ROS

Several anticancer drugs act by inhibiting and suppressing cancer progression through ROS-mediated cell death [START_REF] Xu | The role of reactive oxygen species in screening anticancer agents[END_REF]. ROS are highly reactive and extremely unstable molecules. Therefore, their detection relies on the measurement of the end products that are formed when these molecules react with particular substances. The potential induction of intracellular ROS by complexes 1-4 and the reference drugs in A2780 cells was analysed using the nitroblue tetrazolium (NBT) colorimetric assay and the H 2 DCFDA fluorescent probe.

The NBT is the most sensitive probe for the detection of superoxide anion and is suitable for the detection of the early steps of oxidative stress by various treatments. As shown in Figure 12, the compounds were able to induce the generation of superoxide anions, detected with NBT. In fact, at concentrations above 20 µM, the formation of superoxide anion was evident for all the complexes. As superoxide is mostly produced within the mitochondria, it is possible that this organelle can mediate the cytotoxic effect of the compounds.

H 2 DCFDA is one of the most widely used methods for directly measuring the redox state of a cell. Therefore, H 2 DCFDA oxidation should be interpreted as an indication of the general cellular oxidative stress due to a variety of factors. However, some challenges for the use of H 2 DCFDA have been discussed, including fluctuations in cellular antioxidants, different cellular permeability to the probe and, very importantly, the esterase activity that may vary among cells [START_REF] Jakubowski | 2,7-dichlorofluorescin oxidation and reactive oxygen species: What does it measure?[END_REF][START_REF] Zhang | Methods for the detection of reactive oxygen species[END_REF]. Hydrogen peroxide, hydroxyl radicals or peroxynitrite can be detected using the H 2 DCFDA probe. The peroxynitrite anion (ONOO -), a product of the reaction between superoxide anion and nitric oxide is a strong oxidant. Once protonated, ONOOH originates a highly reactive product that can induce the oxidation of thiol groups on proteins, thus affecting cellular metabolism. The effect of the complexes and the reference drugs on ROS production by A2780 cells, as well as the results from positive controls, are depicted in Figure 13-A. The formation of ROS was higher for 2 and 3 at concentrations above 20 µM, a trend similar to that obtained for the reference drugs auranofin and cisplatin. These observations are in agreement with previous reports on studies of the mechanism of action of these drugs [START_REF] Hwang-Bo | Auranofin, an inhibitor of thioredoxin reductase, induces apoptosis in hepatocellular carcinoma Hep3B cells by generation of reactive oxygen species[END_REF][START_REF] Choi | Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation[END_REF]. In fact, inhibition of thiol enzymes by auranofin was shown to promote cell death through the activation of apoptosis and ROS accumulation [START_REF] Hwang-Bo | Auranofin, an inhibitor of thioredoxin reductase, induces apoptosis in hepatocellular carcinoma Hep3B cells by generation of reactive oxygen species[END_REF]. Concerning cisplatin, it is well known that its cytotoxicity has been correlated to the generation of mitochondrial ROS that can promote a series of deleterious events and influence multiple metabolic functions which may ultimately lead to cell death [START_REF] Choi | Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation[END_REF].

To determine if the changes in ROS levels reflected a functional role of ROS in the compounds mode of action, the assays mentioned above were repeated using several ROS scavengers, namely sodium pyruvate, DMSO, and sodium bicarbonate. The former protects the cells from H 2 O 2 damage [START_REF] Wang | Pyruvate protects mitochondria from oxidative stress in human neuroblastoma SK-N-SH cells[END_REF], the latter enhances the decomposition of peroxynitrite [START_REF] Khan | The decomposition of peroxynitrite to nitroxyl anion (NO-) and singlet oxygen in aqueous solution[END_REF], while DMSO traps the hydroxide radical OH . [START_REF] Brayton | Dimethyl sulfoxide (DMSO): a review[END_REF][START_REF] Santos | Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects[END_REF]. The results presented in Figure 13-B suggest H 2 O 2 as the ROS species involved in the mechanism of action of both complexes 2 and 3, and also of auranofin, although to a lesser extent.

Despite complexes 2 and 3 exhibited similar cytotoxicity towards the cell lines under study, their ROS production profiles were distinct.

Caspase assays

Apoptotic cell death involves caspases, a family of cysteine proteases that play an essential role in apoptosis. Caspases function in a cascade of cleavage events that culminate in cell death. The initiator caspases, caspases-8 and -9, activate the executioner's caspases-3, -6 and -7, which cleave selected targets, enabling cell death.

We explored the use of the active form of caspase-3 and -7 for the detection of apoptotic events induced by the gold complexes 1-4 [START_REF] Perry | Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis[END_REF][START_REF] Eron | Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8[END_REF].

Zinc is an effective signalling regulator and has emerged as an important caspase regulator by direct inactivation of caspases. Zinc inhibits both the initiator and executioner apoptotic caspases by binding to the caspases zinc-binding sites [START_REF] Perry | Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis[END_REF][START_REF] Eron | Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8[END_REF].

As depicted in Figure 14, the ability of the complexes to activate caspase-3/7 correlated with their cytotoxicity. Complexes 2 and 3 showed the highest caspase activation activity, in contrast to 4, which we hypothesize to induce cell death by other mechanisms. Co-incubation with Zn 2+ (50 µM) prevented the occurrence of apoptosis induced by the complexes.

Complexes -DNA Interactions

As previously mentioned, the monoanionic Au bisdithiolate complexes under study were found to exhibit square planar coordination geometry, which is similar to that of cisplatin. This is also the case of many monoanionic Au complexes of this type, leading to the proposal that they might share similar mechanisms of action through interaction with the DNA, a feature already observed for some gold(III) compounds [START_REF] Mirabelli | Interactions of gold coordination complexes with DNA[END_REF]. In this context, we investigated whether the most promising complexes analysed in this study, complexes 3 and 4, were able to interact with and induce conformational changes in ΦX174 supercoiled DNA, in vitro. As shown in Figure 15, cisplatin was able to alter, in a dose-dependent way, the electrophoretic mobilities of the nicked and supercoiled forms of DNA (top and lower bands in the gel, respectively), a feature characteristic of compounds with DNA intercalation or crosslinking abilities [START_REF] Cohen | Binding of cis-and transdichlorodiammineplatinum(II) to DNA: evidence for unwinding and shortening[END_REF]. In contrast, none of the complexes were able to induce conformational changes in the DNA, following the same pattern observed for auranofin and as previously described [START_REF] Mirabelli | Interactions of gold coordination complexes with DNA[END_REF]. These results demonstrate that the cytotoxic effect of these complexes is not mediated by their interaction with the DNA, suggesting instead a mechanism of action distinct from that of cisplatin.

Conclusions

Four homoleptic monoanionic Au bisdithiolate complexes [START_REF]Global Action Plan on Antimicrobial Resistance[END_REF][START_REF] Rice | Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE[END_REF][START_REF] Cavalheiro | Host-Pathogen Interactions Mediated by MDR Transporters in Fungi: As Pleiotropic as it Gets![END_REF][START_REF] Arnold | Recent trends in incidence of five common cancers in 26 European countries since 1988: Analysis of the European Cancer Observatory[END_REF], two of them structurally analysed for the first time, were characterized and evaluated as prospective therapeutic drugs. Complexes 3 and 4, where the unpaired electrons have density in sp 2 nitrogen atoms, were more reactive than complexes 1 and 2 that possess only nitrogen atoms in a nitrile group. In the case of complex 3, the reactivity of the sp 2 nitrogen is attenuated by the inductive effect of the nitrile group. These differences are also suggested by the cyclic voltammetry studies, that showed a more complex voltamogram in the case of [Au(qdt) 2 ] -, which can be ascribed to the presence of protonated species. The favourable occurrence of protonated species, especially in 3 and 4, would allow the molecules to be positively charged. In the case of complexes 1 and 2, the protonation effects are not present in CV, although the change of the complex to a cationic state through a hydrolyzation (acid or basic medium) of the nitrile group, leading to the formation of primary amines, cannot be entirely excluded. The crystal structure analysis shows that all studied complexes can interact with surrounding molecules through strong Au•••S and S•••S interactions, and charge assisted hydrogen bonds. The present study demonstrates the potential of homoleptic bisdithiolate gold complexes as a novel approach in the design and development of new antibacterial, antifungal and antitumor agents.

Complex 3, the most redox-stable complex, presents very significant antimicrobial activity, both towards the Gram-positive S. aureus and towards the fungal pathogen C. glabrata. Complex 4 only presents antimicrobial activity against the Gram-positive S.

aureus. This result suggests that the biological target(s) of the complex differs in prokaryotic and eukaryotic cells, and/or that the permeability of the two types of cells to the compounds is different. The isomeric complexes 1 and 2 have no antimicrobial activity towards either the bacterial or fungal strains tested. Nevertheless, they present differences in their cytotoxic activity in the ovarian cancer cells and the normal fibroblasts, as well as in their ability to induce ROS production. The dithiolate ligands play a significant role in the redox properties of the complexes, but as demonstrated by specific tests with KAuCl 4 and K 2 (i-mnt) 2 , neither the ligands nor the gold precursor are active species responsible for the cytotoxicity.

In summary, the antimicrobial and antitumor activity of this type of Au bisdithiolate complexes was established against the Gram-positive S aureus, the fungal species C. glabrata, and ovarian cancer cells, with promising results. The exact mechanism of action of these complexes is yet unknown; the targets are unidentified and the exact chemical nature of the active species responsible for their activity are still unclear.

Nevertheless, the properties of these complexes revealed in this study can provide a valuable platform for designing new derivatives with improved biological activities and can contribute to shed more light on the structure-activity relationships. Future studies will also focus on cell membrane permeability by these complexes to enlighten the mechanisms underlying their cytotoxicity. In addition, as superoxide is mostly produced within mitochondria, our results suggest that superoxide mediates the cytotoxic effect of Journal Pre-proof J o u r n a l P r e -p r o o f the complexes. Although speculative at present, the implication of the mitochondria in the mechanism of action of the gold bisdithiolate complexes worth further investigation. of the double helix, Science (80-. ). 203 (1979)

1 - 4 .

 14 interact with the anions throughout C-H•••S and C-H•••N hydrogen bonds and π-π interactions comprising the cation phenyl ring and the extended aromatic ring of the anion ligands. The homoleptic monoanionic gold bisdithiolate complexes are essentially planar molecules with a square planar coordination geometry. This is also the case for the gold complexes Deviations of planarity are usually associated to intermolecular interactions with neighbouring molecules, throughout the ligands, yielding soft chair or boat type molecular configurations or, due to the flexibility of the Au-S bond, inducing different degrees of tetrahedral distortions of the AuS 4 core.Figure 6 represents different intermolecular interactions in complexes 1-4.

Figure 1 -

 1 Figure 1 -Molecular structures of the complexes under study: [Au(mnt) 2 ] -(where mnt = 1,1-dicyanoethylene-2,2´-dithiolate) (1), [Au(i-mnt) 2 ] -(where i-mnt = 2,2dicyanoethylene-1,1-dithiolate) (2), [Au(cdc) 2 ] -(where cdc = cyanodithioimido carbonate) (3) and [Au(qdt)2] -(where qdt = quinoxaline-2,3-dithiolate) (4).

Figure 2 -

 2 Figure 2 -ORTEP top and side views of the monoanionic units in the crystal structure

Figure 3 -

 3 Figure 3 -Crystal structure of TBA[Au(cdc) 2 ] (3) viewed along a.

Figure 4 -

 4 Figure 4 -Detail of an anionic chain in the crystal structure of TBA[Au(cdc) 2 ] (3).

Figure 5 -

 5 Figure 5 -Crystal structure of TPP[Au(qdt) 2 ] (4), viewed along b.

Figure 6 -

 6 Figure 6 -Detail showing the possible intermolecular interactions in complexes 1-4: a)

Figure 7 -

 7 Figure 7 -Details of the relative shape and size of the anions electronic cloud in the crystal structures of complexes 1-4: a) [Au(mnt) 2 ] -, b) [Au(i-mnt) 2 ] -, c) [Au(cdc) 2 ] -and d) [Au(qdt) 2 ] -.

Figure 8 -

 8 Figure 8 -HOMO schematic representation of complexes 1-4: a) [Au(mnt) 2 ] -, b) [Au(i-mnt) 2 ] -, c) [Au(cdc) 2 ] -and d) [Au(qdt) 2 ] -.

Figure 9 -

 9 Figure 9 -Cyclic voltammograms of compounds 1, 2 and 4, in acetonitrile (1×10 -3 M)with n-Bu 4 NPF 6 (1x10 -1 M) as the supporting electrolyte, at room temperature, and at a

Figure 10 -

 10 Figure 10 -UV-Vis spectra of complex 3 in DMSO solution (a), in phenol red-free

Figure 11 -

 11 Figure 11 -Cytotoxic activity, expressed as the IC 50 values for complexes 1-4 towards

Figure 12 -

 12 Figure 12-Superoxide production, using the NBT assay, by A2780 cells upon 3 h

Figure 13 -

 13 Figure 13-ROS production based on 2', 7' -dichlorofluorescein (DCF) formation, by

Figure 14 -

 14 Figure 14-Caspase -3 and -7 activity in A2780 cells, upon 24 h exposure to the

Figure 15 -

 15 Figure 15-Photographs of agarose gels loaded with samples of supercoiled ϕX174

Figure 2 Figure 4 Figure 5 Journal 51 Figure 6 Figure 14 Journal

 24551614 Figure 2

Table 1 -

 1 1014-1016. doi:10.1126/science.370979. Oxidation potentials of complexes 1-2 and 4. § The studies were performed under the same experimental conditions using working and counter Pt electrodes, Ag/AgNO 3 (10 -3 M) as the reference electrode and CH 3 CN as solvent. The scan rate used was 100 mV.s -1 . E 1/2 (vs. Ag/AgNO 3 )= E 1/2 (vs. Ag/AgCl)-300 mV. The redox potentials were normalized relatively to the Fc/Fc + couple, which was used as an internal reference. Half-wave potentials are given by E 1/2 = (Ep a + Ep c )/2.

		E c	E a	E 1/2	E 1/2 Fc
	Complex
		(mV)	(mV)	(mV)	(mV)
	1	-861	-724	-793	143
	2	-829	-	-	155
	4	-701	901	-	135
	§

Table 2 -

 2 Estimated minimum inhibitory concentration (MIC) of complexes 1-4, and auranofin, towards the Gram-negative E. coli ATCC 25922 and the Gram-positive S. aureus Newman. Results are the mean ± SD of three independent experiments done with two replicates.

		MIC (µg/mL)
	Complex
		E. coli ATCC25922	S. aureus Newman
	1	> 125	> 125
	2	> 125	> 125
	3	> 125	15.3 ± 1.3
	4	> 125	14.7 ± 0.9
	Auranofin	35.6 ± 0.5	0.2

Table 3 -

 3 Estimated minimum inhibitory concentration (MIC) of complexes 1-4, and auranofin, towards C. albicans SC5134 and C glabrata CBS138 strains. Results are the mean ± SD of three independent experiments done with two replicates.

		C. albicans SC5134	C. glabrata CBS138
	Complex
		MIC (µg/mL)	MIC (µg/mL)
	1	> 125	> 125
	2	> 125	> 125
	3	> 62.5	7.0 ± 0.8
	4	> 62.5	> 62.5
	Auranofin	7.9 ± 0.6	15.3 ± 0.4

Table 4 -

 4 IC 50 values (µM) determined after 48 h incubation for the complexes and reference drugs in the ovarian cancer cells A2780 and A2780cisR and the normal V79 fibroblasts. Results are shown as the mean ± SD of two independent experiments done with six replicates.

	Compounds	A2780	A2780cisR	V79	SI*
	1	4.4 ± 1.3	5.5 ± 1.5	26 ± 4.8	6.0
	2	1.5 ± 0.5	2.3 ± 1.0	2.4 ± 0.7	1.6
	3	0.9 ± 0.2	1.7 ± 0.5	3.4 ± 1.0	3.8
	4	1.2 ± 0.3	1.3 ± 0.4	5.3 ± 1.1	4.4
	Auranofin	0.4 ± 0.3	0.5 ± 0.17	0.7 ± 0.3	1.5
	Cisplatin	3.6 ± 1.3	36 ± 13	6.7 ± 2.7	1.9

*SI= the selectivity index calculated as IC 50 (V79)/IC 50 (A2780).

Acknowledgments

The kind gift of C. albicans SC5134 and C. glabrata CBS138 by Professor Nuno Mira from iBB is gratefully acknowledge. This work was supported by Fundação para a Ciência e Tecnologia (FCT) through contracts UID/MULTI/04349/2013, PTDC/BIA-MIC/1615/2014, UID/BIO/04565/2019, PTDC/SAU/INF/29550/2017 and PTDC/QUI-QIN/29834/2017.

Competing interests

The authors declare no conflict of interest. The funding source had no role on the study design, collection, analysis and data interpretation, writing and decision to submit the manuscript for publication.

A

Graphical abstract

Highlights

 Four homeleptic monoanionic gold bisdithiolate complexes were characterized.  Complexes redox properties were characterized by cyclic voltammetry.  The complexes were tested for their antimicrobial and antitumor activities.  The most redox stable complex showed higher antimicrobial and antitumoral activities.  The mechanism of action may involve the induction of ROS, mainly superoxide.


Xavier Mignard

Anne-Marie Ruppert

Armelle Lavolé

Thibault Vieira

Nathalie Rozensztajn

Jacques Cadranel

Marie Wislez
email: marie.wislez@aphp.fr

cours du traitement du cancer pulmonaire non à petites cellules de stade avancé?

Keywords: NSCLC, chemotherapy, rechallenge Résumé CBNPC, chimiothérapie, ré-introduction

Is chemotherapy rechallenge feasible in advanced-stage non-small-cell lung cancer?

La ré-introduction de chimiothérapie est-elle faisable au

Introduction

Despite recent progress, mortality from non-small-cell lung cancer (NSCLC) remains high.

During the last decade, therapeutic advances have included targeted therapies, mainly smallmolecule tyrosine kinase receptor inhibitors (TKIs) [START_REF] Lin | Five-Year Survival in EGFR-Mutant Metastatic Lung Adenocarcinoma Treated with EGFR-TKIs[END_REF]. However, these advances involve nonsquamous NSCLC only, and targetable anomalies are harbored by a minority of NSCLC patients, for instance by 10 to 15% for EGFR [START_REF] Shigematsu | Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers[END_REF], by around 5% for ALK [START_REF] Sequist | Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice[END_REF], and by less than 2% for ROS1.

More recently, immunotherapy in the form of checkpoint inhibitors, such as anti-PD-1 (Programmed Death 1) and anti-PD-L1 (Programmed Death Ligand 1) antibodies, have resulted in improved overall survival in second-line [START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF] for all comers and in first-line therapy, but only in patients with strong PD-L1 expression (>50%) [START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF].

In patients who are ineligible for TKIs or immunotherapy, which remains the majority of patients, standard first-line treatment consists of a platinum-based chemotherapy doublet combined with pemetrexed, paclitaxel, gemcitabine, or vinorelbine [START_REF] Bluthgen | Second-line combination therapies in nonsmall cell lung cancer without known driver mutations[END_REF]. This standard is also recommended for progressive patients after first-line immunotherapy (more than 40% at six months of treatment). The use of either cisplatin or carboplatin depends on age, performance status, and comorbidities like chronic kidney or heart failure [START_REF] Smit | Cisplatin and carboplatin-based chemotherapy in the first-line treatment of non-small cell lung cancer: Analysis from the European FRAME study[END_REF]. The benefit of a second-line treatment has been proven for pemetrexed [START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF], docetaxel [START_REF] Shepherd | Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy[END_REF], erlotinib [START_REF] Shepherd | Erlotinib in previously treated non-small-cell lung cancer[END_REF], and immunotherapy [START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF]. Third-line treatment proves also effective for eligible patients [START_REF] Song | Third-line therapy for advanced non-small-cell lung cancer patients: feasible drugs for feasible patients[END_REF][START_REF] Girard | Third-line chemotherapy in advanced non-small cell lung cancer: identifying the candidates for routine practice[END_REF], without any guidelines for treatment beyond this point.

Rechallenge with a conventional cytotoxic chemotherapy may be attempted if the disease has initially responded. Chemotherapy rechallenge is recommended in other cancer treatments.

Pemetrexed rechallenge is indicated in malignant pleural mesothelioma when initial progressionfree survival exceeds 12 months [START_REF] Ceresoli | Retreatment with pemetrexed-based chemotherapy in patients with malignant pleural mesothelioma[END_REF]. In disseminated relapsing small-cell lung cancer, rechallenge with a platinum doublet plus etoposide is considered an option in platinum-sensitive disease [START_REF] Genestreti | Outcomes of Platinum-Sensitive Small-Cell Lung Cancer Patients Treated With Platinum/Etoposide Rechallenge: A Multi-Institutional Retrospective Analysis[END_REF]. In the same manner, carboplatin rechallenge is common practice in platinum-sensitive relapsing ovarian cancer [START_REF] Spriggs | Optimal sequencing in the treatment of recurrent ovarian cancer[END_REF]. In metastatic castration-resistant prostate cancer, taxanes like docetaxel or cabazitaxel can likewise be reintroduced [START_REF] Oudard | Docetaxel rechallenge after an initial good response in patients with metastatic castration-resistant prostate cancer[END_REF]. Following its introduction, immunotherapy became an additional treatment option; prior to its advent, chemotherapy rechallenge was practiced whenever other alternatives proved scarce. Chemotherapy rechallenge may still be worth considering, especially after progression on immunotherapy. Rechallenge with the various immunotherapy agents is presently being discussed. Little data currently exists on chemotherapy rechallenge in NSCLC. This work sought to better describe the characteristics of patients who underwent chemotherapy rechallenge in a retrospective study, while assessing the efficacy and safety of this practice.

Patients and Methods

Inclusion criteria

All consecutive adult patients with advanced-stage NSCLC receiving first-line treatment from January 1, 2011, to December 30, 2011, in a French university hospital were included. Advancedstage NSCLC was defined as Stage IIIB (ineligible for curative therapy) and Stage IV NSCLC according to the 7 th edition of the International Association for the Study of Lung Cancer (IASLC) classification. First-line treatment consisted of either chemotherapy or TKI for patients with oncogenic addiction. Patients with metastatic relapse after previous curative surgery could be included. Neo-adjuvant or adjuvant chemotherapy as well as radiotherapy were accepted and were not considered as a first-line treatment. Patients list was generated from the chemotherapy prescription software.

Data collection

The following data were collected: age at diagnosis; gender; smoking history, namely number of pack-years, years of smoking, and current smoking status defined as never-smoker (<100 cigarettes in life), former smoker (>100 cigarettes in life but stopped >1 year) or current smoker (>100 cigarettes in life and either still smoking or stopped <1 year); performance status at diagnosis according to the ECOG scale; disease stage according to the 7 th edition of the IASLC clinical TNM classification; occurrence of central nervous system (CNS) or sub-diaphragmatic metastases.

Comorbidities like cardiovascular diseases (high blood pressure, stroke, myocardial ischemia, or arteriosclerosis obliterans), diabetes, neoplasms of other organs, chronic respiratory failure (CRF) or chronic obstructive pulmonary disease (COPD), chronic renal failure, HIV infection, psychiatric disorder, or alcoholism were recorded. Histological types were defined according to the 2004 WHO classification [START_REF] Travis | Pathology and genetics of tumours of the lung, pleura, thymus and heart[END_REF] as adenocarcinoma, squamous-cell carcinoma, large cell carcinoma, and sarcomatoid carcinoma. Adenocarcinoma subtypes were defined as acinar, mucinous, lepidic, papillary or undifferentiated. Oncogenic addiction was defined as EGFR mutation or ALK or ROS1 rearrangement. ALK or ROS1 rearrangements were detected by immunohistochemistry and confirmed by fluorescence in situ hybridization. EGFR, KRAS, BRAF, HER2, and PI3KCA mutations were screened by previously described techniques [START_REF] Beau-Faller | Crossvalidation study for epidermal growth factor receptor and KRAS mutation detection in 74 blinded non-small cell lung carcinoma samples: a total of 5550 exons sequenced by 15 molecular French laboratories (evaluation of the EGFR mutation status for the administration of EGFR-TKIs in nonsmall cell lung carcinoma [ERMETIC] project--part 1)[END_REF]. The number of treatment lines received by each patient, including targeted therapies, and for each line of treatment were recorded, as well as its duration, how many cycles were administered (including maintenance if relevant), and whether the dose was reduced. Patients were treated in accordance with routine clinical care.

Chemotherapy rechallenge was defined as the re-use of a drug that had been administered earlier in the course of treatment and stopped due to disease progression or toxicity. At least one line of treatment had to have been received between first use and re-use. Resuming treatment after a break was not considered rechallenge, even if treatment was halted for a long period. The same agent had to be used, except for platinum-derived products like cisplatin or carboplatin. Re-using an agent as monotherapy when it was first used in a platinum-based doublet was considered as a rechallenge (Fig. 1). Rechallenge with targeted therapy was not taken into account for this study.

The following data were collected for each chemotherapy rechallenge: treatment duration, performance status at rechallenge, number of previous lines, use as maintenance or not, progression-free survival at first-use and at rechallenge, line of treatment after rechallenge, and toxicity (hematological, gastrointestinal, or other) graded according to the WHO classification.

Rechallenged patients were divided into five groups: platinum-based doublet or triplet (cisplatin/carboplatin + other agent ± bevacizumab), or pemetrexed, gemcitabine, paclitaxel and vinorelbine monotherapy.

Statistical analyses

Patients were analyzed according to whether they received chemotherapy rechallenge or not.

Results

Patients characteristics

In 2011, 149 patients underwent first-line treatment for advanced-stage NSCLC, in our French university hospital. Median age was 60 years . Overall, 66% of the patients were men, 47.5% were smokers, 39% were former smokers, and 8.5% were never-smokers. Histology showed 66.5% had adenocarcinoma, 21% squamous-cell carcinoma, 10.5% large cell carcinoma, and 2% sarcomatoid carcinoma. Among non-squamous NSCLCs, 6.5% were EGFR mutated, 16% KRAS mutated, 0.5% BRAF mutated, and 5% harbored ALK rearrangement. No ROS1 rearrangement was observed. The mutation status of 11 patients (7.5%) was unknown. Regarding comorbidities, 39% of the patients had at least one cardiovascular comorbidity, 11.5% had diabetes, 14.5% had a current neoplasm or had had a prior neoplasm in another organ, and 11.5% had either CRF or COPD. Most patients were in good general condition (77% had a performance status of 0-1) at first-line treatment. Patients received a mean of 2.8, or a median of 2.0 [START_REF] Lin | Five-Year Survival in EGFR-Mutant Metastatic Lung Adenocarcinoma Treated with EGFR-TKIs[END_REF][START_REF] Shigematsu | Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers[END_REF][START_REF] Sequist | Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice[END_REF][START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF][START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Bluthgen | Second-line combination therapies in nonsmall cell lung cancer without known driver mutations[END_REF][START_REF] Smit | Cisplatin and carboplatin-based chemotherapy in the first-line treatment of non-small cell lung cancer: Analysis from the European FRAME study[END_REF][START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF][START_REF] Shepherd | Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy[END_REF][START_REF] Shepherd | Erlotinib in previously treated non-small-cell lung cancer[END_REF], treatment lines, and almost half of them received TKI (regardless of mutation status) in accordance with current guidelines.

Characteristics of chemotherapy rechallenge

Altogether, 18 of 149 patients (12%) underwent chemotherapy rechallenge: 13 had asingle rechallenge and five two rechallenges with different agents. Hence, 23 distinct rechallenges were noted across our five groups, namely eight rechallenges with a platinum-based doublet (carboplatin, n=5; cisplatin, n=3), seven with pemetrexed, four with gemcitabine, three with paclitaxel and one with vinorelbine. No rechallenge with docetaxel was observed.

The median number of cycles was 4 [START_REF] Lin | Five-Year Survival in EGFR-Mutant Metastatic Lung Adenocarcinoma Treated with EGFR-TKIs[END_REF][START_REF] Shigematsu | Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers[END_REF][START_REF] Sequist | Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice[END_REF][START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF][START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Bluthgen | Second-line combination therapies in nonsmall cell lung cancer without known driver mutations[END_REF][START_REF] Smit | Cisplatin and carboplatin-based chemotherapy in the first-line treatment of non-small cell lung cancer: Analysis from the European FRAME study[END_REF][START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF][START_REF] Shepherd | Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy[END_REF][START_REF] Shepherd | Erlotinib in previously treated non-small-cell lung cancer[END_REF][START_REF] Song | Third-line therapy for advanced non-small-cell lung cancer patients: feasible drugs for feasible patients[END_REF][START_REF] Girard | Third-line chemotherapy in advanced non-small cell lung cancer: identifying the candidates for routine practice[END_REF][START_REF] Ceresoli | Retreatment with pemetrexed-based chemotherapy in patients with malignant pleural mesothelioma[END_REF][START_REF] Genestreti | Outcomes of Platinum-Sensitive Small-Cell Lung Cancer Patients Treated With Platinum/Etoposide Rechallenge: A Multi-Institutional Retrospective Analysis[END_REF][START_REF] Spriggs | Optimal sequencing in the treatment of recurrent ovarian cancer[END_REF] at both first use and rechallenge. For platinum-based doublets, the median cycle number at rechallenge was 4 [2-5] versus 4 [START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF] at first use. The median cycle number was lower at rechallenge than at first use for pemetrexed (3 [2-8] vs. 7 [START_REF] Lin | Five-Year Survival in EGFR-Mutant Metastatic Lung Adenocarcinoma Treated with EGFR-TKIs[END_REF][START_REF] Shigematsu | Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers[END_REF][START_REF] Sequist | Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice[END_REF][START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF][START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Bluthgen | Second-line combination therapies in nonsmall cell lung cancer without known driver mutations[END_REF][START_REF] Smit | Cisplatin and carboplatin-based chemotherapy in the first-line treatment of non-small cell lung cancer: Analysis from the European FRAME study[END_REF][START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF][START_REF] Shepherd | Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy[END_REF][START_REF] Shepherd | Erlotinib in previously treated non-small-cell lung cancer[END_REF][START_REF] Song | Third-line therapy for advanced non-small-cell lung cancer patients: feasible drugs for feasible patients[END_REF][START_REF] Girard | Third-line chemotherapy in advanced non-small cell lung cancer: identifying the candidates for routine practice[END_REF][START_REF] Ceresoli | Retreatment with pemetrexed-based chemotherapy in patients with malignant pleural mesothelioma[END_REF][START_REF] Genestreti | Outcomes of Platinum-Sensitive Small-Cell Lung Cancer Patients Treated With Platinum/Etoposide Rechallenge: A Multi-Institutional Retrospective Analysis[END_REF][START_REF] Spriggs | Optimal sequencing in the treatment of recurrent ovarian cancer[END_REF]) and for gemcitabine (2 [1-16] vs. 5.5 [START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF][START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Bluthgen | Second-line combination therapies in nonsmall cell lung cancer without known driver mutations[END_REF][START_REF] Smit | Cisplatin and carboplatin-based chemotherapy in the first-line treatment of non-small cell lung cancer: Analysis from the European FRAME study[END_REF]). At rechallenge, patients were mostly at their fourth line of treatment (4 [START_REF] Sequist | Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice[END_REF][START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF][START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF]) and mostly (78%) with a favorable PS (0-1). A subsequent line of treatment was feasible in 61% of patients. Pemetrexed and gemcitabine were initially used as maintenance therapy in respectively 43 and 50% of the patients (Table 1).

Characteristics of patients according to chemotherapy rechallenge

In univariate analyses, rechallenged patients were younger (median age 56 vs. 61 years, p = 0.04), were more often women (61% vs. 30.5%, p = 0.021), had fewer cardiovascular comorbidities (16.5% vs. 42%, p = 0.039), and had a better PS (100% 0-1 performance status at diagnosis vs. 74%, p = 0.043). Rechallenged patients tended to smoke less (median smoking duration of 30.5 vs. 40 years, p = 0.086). They received a higher number of treatment lines (median 5.0 vs. 2.0, p <0.0001) and received a targeted therapy (TKI) more often (89% vs. 43.5%, p = 0.0003), regardless of the presence or absence of oncogenic addiction. A higher proportion of adenocarcinomas was noted in rechallenged patients (72% vs. 65% other histology, p = 0.57) particularly of the lepidic subtype (23% vs. 3.5%, p = 0.037). No difference was found according to EGFR or ALK status.

KRAS-mutated patients tended to be less rechallenged than others (1/24 rechallenged vs. 23/24 not rechallenged, p = 0.18) (tables 2 and 3). Regarding overall survival, patients with chemotherapy rechallenge had a longer overall survival (median 32.7 months vs. 13.4 months, p = 0.0002).

Progression-free survival at first use and rechallenge

Progression-free survival was shorter at rechallenge than at first use, namely 2.7 months [0.2-22.5] versus 9.2 months [2.7-31.6] (p = 0.0021). Progression-free survival by drug is given in Table 4.

Toxicity at first use and rechallenge

During the 23 rechallenges, at least one Grade-3 or -4 toxicity was observed in seven cases (30%) at first use and in five cases (24%) at rechallenge (p = 0.62). No toxicity gain was found at rechallenge compared to first use, except one case of Grade-4 neutropenia during paclitaxel rechallenge.

Toxicity was mostly hematological. There was no Grade-3 or -4 neurological or renal toxicity (data not shown) nor iatrogenic death.

Discussion

Of 149 patients with advanced NSCLC, 18 underwent at least one chemotherapy rechallenge during treatment, with five being rechallenged twice. Hence, we recorded a total of 23 distinct rechallenges. The most commonly used drugs for rechallenge were pemetrexed and platinum-based doublets, which were given in, respectively, 7 and 8 cases. Rechallenge was carried out with gemcitabine in four cases, with paclitaxel in three cases, and with vinorelbine in one case.

Those who received chemotherapy rechallenge were characterized as patients exhibiting favorable prognostic factors. Younger age, female gender, better general condition, fewer comorbidities, and lepidic histological subtype have previously been shown to be associated with longer overall survival [START_REF] Sculier | The impact of additional prognostic factors on survival and their relationship with the anatomical extent of disease expressed by the 6th Edition of the TNM Classification of Malignant Tumors and the proposals for the 7th Edition[END_REF], [START_REF] Grose | The impact of comorbidity upon determinants of outcome in patients with lung cancer[END_REF], [START_REF] Hung | Predictive value of the international association for the study of lung cancer/American Thoracic Society/European Respiratory Society classification of lung adenocarcinoma in tumor recurrence and patient survival[END_REF]. Our results are consistent with the study of Girard et al., in which patients who benefited the most from third-line therapy were those with good prognostic factors [START_REF] Girard | Third-line chemotherapy in advanced non-small cell lung cancer: identifying the candidates for routine practice[END_REF].

KRAS-mutated patients received chemotherapy rechallenge less than others (1 patient rechallenged vs. 23 non-rechallenged, p = 0.18). KRAS mutation seems to be associated with worse survival for patients with advanced NSCLC [START_REF] Mascaux | The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis[END_REF], but its involvement in chemotherapy resistance is still debatable [START_REF] Roberts | KRAS mutation: should we test for it, and does it matter?[END_REF].

Progression-free survival at rechallenge was 2.7 months for all treatments, which was significantly shorter than progression-free survival at first use (9.2 months). For platinum-based doublets, progression-free survival at rechallenge was non-significantly shorter (3.0 months) than at first use (7.4 months), which might be due to lack of statistical power. For pemetrexed, progressionfree survival was significantly shorter at rechallenge (2.5 months) than at first use (7.7 months). The longest progression-free survival at rechallenge was obtained with paclitaxel (5.4 months) and the shortest with gemcitabine (1.8 months).

Petrelli et al. reported a progression-free survival of 3.9 months with platinum/pemetrexed rechallenge in a pooled analysis of 11 studies [START_REF] Petrelli | Platinum rechallenge in patients with advanced NSCLC: a pooled analysis[END_REF]. In a retrospective study, when pemetrexed rechallenge was given as monotherapy or as combination therapy with a platinum salt, it showed a progression-free survival of 3.8 months in a second-line setting only [START_REF] Zhuo | Rechallenge with pemetrexed-based chemotherapy improves the survival of patients with advanced non-squamous non-small-cell lung cancer[END_REF]. Median age was similar and patients were in good general condition (97% had a performance status of 1). In both studies, patients were rechallenged earlier than in ours (second line vs. fourth), which may explain why they observed longer progression-free survival. In another retrospective study, 25 patients had pemetrexed rechallenge [START_REF] Song | Retreatment with pemetrexed chemotherapy in advanced non-small cell lung cancer patient[END_REF]. Progression-free survival was shorter (1.5 month). The patients were mostly men (64%) and mostly had a performance status of 2 (60%). The discrepancies between all these studies may be due to their retrospective design and small sample sizes.

In our study, Grade-3 or -4 toxicities did not increase at rechallenge except for one patient with paclitaxel (Grade-4 neutropenia). A rise in toxicity could have been expected, since chemotherapy rechallenge is practiced on heavily pre-treated patients. For example, docetaxel rechallenge in metastatic castration-resistant prostate cancer is risky, owing to cumulative neurotoxicity [START_REF] Oudard | Docetaxel rechallenge after an initial good response in patients with metastatic castration-resistant prostate cancer[END_REF]. No cumulative toxicity was found in our study, especially no renal toxicity.

A further line of treatment was administered after rechallenge in 61% of our patients.

The rechallenge was not, therefore, their last line of treatment, which suggests that their general condition permitted them to receive the additional treatment line. PFS was shorter at rechallenge than at first-use in our study. These results are coherent with other studies describing a decreased disease control between first-line and subsequent lines [START_REF] Massarelli | A retrospective analysis of the outcome of patients who have received two prior chemotherapy regimens including platinum and docetaxel for recurrent non-small-cell lung cancer[END_REF]. This retrospective study does not answer the question of whether rechallenge proves cost-effective or of benefit in terms of quality of life. Our study reflects the realities of routine practice in this patient population. Immune checkpoint inhibitors are considered a new option and may delay the need for chemotherapy rechallenge in the course of the disease. Our study's limitations were its retrospective design, single-center data, small sample size, and heterogeneity of administered treatments (including those administered twice). However, our findings may further improve our understanding of chemotherapy rechallenge.

Conclusion

Of 149 patients with advanced NSCLC, 18 (12%) underwent chemotherapy rechallenge. These patients had good prognostic factors. Progression-free survival was shorter at rechallenge than at first use. No toxicity gain was noted at rechallenge. Thus chemotherapy rechallenge should be considered a treatment option in suitable patients.

  Continuous variables were expressed as median and associated range [min-max]. Categorical variables were expressed as percentages. Comparisons between non-parametric continuous variables were conducted using the Mann-Whitney test. Comparisons between categorical variables were conducted by means of the the Chi-squared test or Fisher's exact test when the number of observations was less than 5. Progression-free survival rates at first use and at rechallenge were compared with the Wilcoxon test. Toxicity at first use and at rechallenge were compared using McNemar's Chi-squared test. Results were considered statistically significant when p <0.05. Analyses were performed with SPSS 20.0 (IBM France) software. Data were reviewed by a statistician.

Figure 1 :Figure 1 :

 11 Figure 1: Illustration of chemotherapy rechallenge in the course of treatment

  Comparisons between categorical variables were conducted with the Chi-squared test or Fisher's exact test when n < 5.

Table 2 : Histological subtypes according to rechallenge of treatment

 2

		Overall	Rechallenged	Not rechallenged	Univariate
	Variable	n=99	n=13	n=86	analysis
		n (%)	n (%)	n (%)	(p)
	Adenocarcinoma
	histologic subtype
	Acinar	26 (26.5)
	Mucinous	14 (14)
	Lepidic	6 (6)
	Papillary	3 (3)
	Undifferentiated	34 (34.5)
	Unknown	16 (16)

Table 3 : Characteristics of rechallenged and non-rechallenged patients

 3

Acknowledgments

The authors wish to thank Dr. Pierre Levy for his review of the statistics, Mr. J. Rose for his help generating the list of patients, Ms. V. Poulot and Ms. L. Pinon for their help with molecular and histological data, and Ms. N. Mathiot and Ms. G. Pontdeme for their overall contribution to this work.

Ethics and regulations

The local database was declared, and it obtained authorization from the bodies that monitor the application of data privacy laws according to French guidelines.

6 (75) 2 ( 25) 0 (0) 0 (0) 6 (86) 0 (0) 0 (0) 1 ( 14)

3 (100) 0 (0) 0 (0) 0 (0) 0 (0) 1 (100) 0 (0) 0 (0)

Number of lines at rechallenge

Number of cycles at rechallenge Median