Blockage of aquaporin‐3 peroxiporin activity by organogold compounds affects melanoma cell adhesion, proliferation and migration

Aquaporin‐3 (AQP3) is a membrane channel with dual aquaglyceroporin/peroxiporin activity, facilitating the diffusion of water, glycerol and H2O2 across cell membranes. AQP3 shows aberrant expression in melanoma and its role in cell adhesion, migration and proliferation is well described. Gold compounds were shown to modulate AQP3 activity with reduced associated toxicity, making them promising molecules for cancer therapy. In this study, we validated the phenotype resulting from AQP3‐silencing of two melanoma cell lines, MNT‐1 and A375, which resulted in decreased H2O2 permeability. Subsequently, the AQP3 inhibitory effect of a new series of organogold compounds derived from Auphen, a potent AQP3 inhibitor, was first evaluated in red blood cells (RBCs) that highly express AQP3, and then in HEK‐293T cells with AQP3 overexpression to ascertain the compounds’ specificity. The first screening in RBCs unveiled two organogold compounds as promising blockers of AQP3 permeability. Moderate reduction of glycerol permeability but drastic inhibition of H2O2 permeability was detected for some of the gold derivatives in both AQP3‐overexpressing cells and human melanoma cell lines. Additionally, all compounds were effective in impairing cell adhesion, proliferation and migration, although in a cell type‐dependent manner. In conclusion, our data show that AQP3 peroxiporin activity is crucial for melanoma progression and highlight organogold compounds as promising AQP3 inhibitors with implications in melanoma cell adhesion, proliferation and migration, unveiling their potential as anticancer drugs against AQP3‐overexpressing tumours.


Introduction
Skin cancer accounts for over 1.5 million cases diagnosed globally in 2020, and although melanoma accounts only for 1% of the total, it is one of the most invasive human cancers with a high potential for metastasis and a fatal outcome.Among all human cancers, melanoma represents the highest mutation burden, allowing cancer cells to resist and avoid treatments.Therefore, despite the considerable advances in clinical management of cancer, therapies against metastatic melanoma often fail (Siegel et al., 2017).
Amongst the possible anticancer drug targets, the family of transmembrane water channels, commonly named aquaporins (AQPs), are particularly attractive.
Interestingly, amongst the various isoforms, the dual-acting aquaglyceroporin/peroxiporin AQP3 has been recently proposed as a new player in the biology of skin cancer due to its pivotal role in its progression and metastasis (da Silva et al., 2021;Marlar et al., 2017).The fact that AQP3-knockout mice do not develop tumours even when exposed to a chemical tumour initiator demonstrates that AQP3 is crucial for skin cancer development (Hara-Chikuma & Verkman, 2008).The peroxiporin activity of AQP3 makes it a key player in redox balance maintenance modulating the intracellular levels of H 2 O 2 (Prata et al., 2019).When the intracellular concentration of reactive oxygen species (ROS), such as H 2 O 2 , increases above the physiological range, the redox signalling is disrupted promoting oxidative stress (Pizzino et al., 2017) and favouring molecular pathways that trigger inflammation, tumour growth, angiogenesis and metastasis, thus prompting tumour malignancy (Taniyama & Griendling, 2003).As AQP3 is also an aquaglyceroporin, its overexpression confers cancer cells a higher glycerol content for ATP synthesis, promoting cell proliferation (Galan-Cobo et al., 2015).Additionally, the implication of AQP3 in epithelial-mesenchymal transition (EMT) has been exhaustively investigated.AQP3 is correlated with EMT-related proteins, migration and invasion (Chen et al., 2014;Login et al., 2019;Marlar et al., 2017).In this context, the development of small-molecule selective inhibitors of AQP3 is of interest as a new therapeutic strategy against skin cancer.
The discovery of inhibitors with proven AQP3 selectivity has been challenging due to the structural similarity between AQP isoforms.In the last years a few small organic molecules (Sonntag et al., 2019), metal-containing scaffolds (Pimpao et al., 2020) and natural compounds (Paccetti-Alves et al., 2023) were unveiled with activity against AQP3 (for a review see Pimpão et al., 2022).However, among the AQP3 inhibitors reported so far, metallodrugs exhibited concomitant anticancer properties in vitro and in vivo (Nave et al., 2016;Pinho et al., 2019Pinho et al., , 2021) ) underlining the crucial role of AQP3 in cancer pathophysiology.
The investigation of metal complexes as selective inhibitors for AQP3 began in 2010 with the identification of the gold(III) coordination complex [Au(phen)Cl 2 ]Cl (Auphen, phen = 1,10-phenanthroline, Fig. 1A) as a potent and selective inhibitor of AQP3 (Aikman et al., 2018;Martins et al., 2012).The mechanism of inhibition was postulated to be due to the formation of a gold-sulfur bond between gold(III) and the Cys-40 residue within the AQP3 channel (Serna et al., 2014), which was evidenced by various complementary studies (de Almeida et al., 2017;Martins et al., 2013).Computational studies conducted on Au(III) complexes-AQP3 adducts evidenced that protein conformational changes, resulting from gold(III) binding to Cys-40, are responsible for the inhibition of glycerol permeation (de Almeida et al., 2017;
In 2021, we expanded the library of cyclometalated complexes as inhibitors for human aquaglyceroporin 1A) (Pimpao et al., 2021).Interestingly, complexes 1 and 2 showed a stronger inhibitory effect (70% and 74%, respectively) compared to complex 3 (47%); however, none of the complexes outperformed Auphen (Pimpao et al., 2021).It should be noted that the three investigated cyclometalated Au(III) compounds (1-3) have also shown interesting reactivity with cysteine (Cys) residues, forming Au(CˆN)-Cys adducts (CˆN = bidentate ligand) with peptides, resulting in cysteine arylation with the CˆN ligand (Thomas et al., 2020;Wenzel et al., 2019).Mechanistic studies suggest that the formation of the CˆN-peptide adduct is templated by the Au(III) centre facilitating the C-S cross-coupling reaction via reductive elimination (Fig. 1B) (Wenzel et al., 2019).The rate at which cysteine arylation takes place for each organogold compound follows the same trend as the inhibition of AQP10 (Pimpao et al., 2021).Functional assays demonstrated the irreversible inhibition of AQP10 solute permeation with complex 2, due to the covalent modification of cysteine residues in the AQP10 pore, leading to its shrinkage as evidenced via atomistic simulations (Pimpao et al., 2021).Instead, the benchmark compound Auphen, following ligand exchange reactions, can only form coordination bonds with the thiol group of cysteine residues which are reversible per se in nature.
In this study, the permeability features of AQP3 in two melanoma cell lines (MNT-1 and A375) were first validated using an AQP3 knock-down strategy.Then, taking advantage of the high expression of AQP3 in human red blood cells (RBCs), the effect of AQP3 blockage by organogold compounds 1−3 on melanoma cell biology in comparison to the benchmark AQP3 inhibitor Auphen was investigated and further confirmed using AQP3-overexpressing HEK-293T cells.AQP3 blockage by the selected compounds was then validated by measuring the water, glycerol and H 2 O 2 permeability of the two melanoma cell lines.Subsequently, the impact of AQP3 modulation was evaluated on cell adhesion, proliferation and migration of melanoma cells.

Ethical approval
Venous blood samples were obtained from healthy human volunteers following a protocol approved by the Ethics Committee of the Faculty of Pharmacy of the University of Lisbon (Instituto Português de Sangue Protocol SN-22/05/2007).Informed consent was obtained from all participants.The study conformed to the standards set by the Declaration of Helsinki, except for registration in a database.

Organogold compounds
The organogold complexes used in this study, and Auphen, were synthesized according to published procedures (Pimpao et al., 2021).Stock solutions of gold compounds were freshly prepared by dissolving the solid compound in water (Auphen) or dimethyl sulfoxide (DMSO) (compounds 1−3).The stock solutions' concentrations were 10 mM for Auphen (in water) and 5 mM for the other compounds (in DMSO).Dilutions of the compounds were freshly prepared in aqueous solution before each experiment.

Erythrocyte sampling and preparation
Venous blood samples were collected from anonymous human donors in citrate anticoagulant (2.7% citric acid, 4.5% trisodium citrate and 2% glucose) to prevent coagulation.The blood was centrifuged at 750 g for 10 min at room temperature to isolate RBCs.After washing three times with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4), RBCs were diluted to a 0.5% suspension and kept on ice to be used immediately in the experiments.

Transfection of cell lines
For AQP3 knock-down in MNT1 and A375 cells, small interfering RNA (siRNA)-containing expression vectors targeting human AQP3 (ID: s1521, Ambion, Thermo Fisher Scientific, Waltham, MA, USA) combined with Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) were used according to the manufacturer's protocol.A control of silencing (Silencer Negative Control siRNA #1, Ambion, Thermo Fisher Scientific) was performed in parallel.For cell transfection, cells were seeded with an inoculum of 40,000 cells cm −2 in 12-well plates or 15 mm Ø coverslips, for qPCR and H 2 O 2 permeability assays, respectively.Cells were used for experiments 24 h post-transfection.Experimental groups were established as follows: cells transfected with negative control (siControl), MNT1 cells transfected with AQP3 siRNA (MNT1-siAQP3) and A375 cells transfected with AQP3 siRNA (A375-siAQP3).For AQP3 overexpression in HEK-293T cells, human AQP3 ORF-encoding expression vectors (Genecopeia, Rockville, MD, USA) were used according to the manufacturer's recommendations combined with Endofectin MAX.Control of overexpression was produced in parallel.For cell transfection, cells were seeded with an inoculum of 70,000 cells cm −2 in six-well plates and the instructions recommended by Genecopeia were followed.Five days post-transfection cells were trypsinized and when cells reached a confluence of 70-80% selection with 1.7 μg/ml puromycin (Sigma, Kawasaki, Kanagawa, Japan) was initiated.Transfections were validated by qPCR and functional assays.

Immunofluorescence
Cells were inoculated in 15 mm Ø coverslips at a density of 40,000 cells cm −2 .After 24 h, cells were fixed with 4% paraformaldehyde solution for 20 min.Fixed cells were permeabilized with 0.1% Triton X-100 for 10 min, followed by blocking solution of 3% BSA in PBS for 30 min at room temperature.Cells were then incubated overnight with the primary antibody (anti-AQP3 rabbit polyclonal antibody ab125219, 1:50, Abcam) diluted in blocking solution, at 4°C.The following day, cells were incubated with secondary antibody consisting of Alexa 594 anti-rabbit (1:500, Thermo Fisher Scientific), in blocking solution, for 1 h, at room temperature.Nuclei were stained with Hoechst 33258 dye (1:1000, Sigma-Aldrich, St. Louis, MO, USA), 2 min, at room temperature.After staining, coverslips were mounted J Physiol 602.13 in microscope slides using ProLong Glass Antifade Mountant (Thermo Fisher Scientific) and fluorescence images were acquired using a Zeiss LSM 710 META confocal point scanning microscope (Zeiss Microscopy, Jena, Germany) with a ×63 oil immersion objective.

Permeability assays
Before assays, RBCs, HEK-293T and melanoma cells were centrifuged at 150 g to obtain a cellular pellet and were resuspended in isotonic saline buffer.The cell suspension was homogeneous with cells showing a spherical shape, as observed under light microscopy.The diameter of cells was measured for all the preparations using ImageJ software.
Water and glycerol permeability assays of RBCs, HEK-293T and melanoma cells were performed by stopped-flow light scattering (Martins et al., 2012) on a Hi-Tech Scientific (Jaipur, Rajasthan, India) PQ/SF-53 stopped-flow apparatus, with a 2 ms dead time, temperature-controlled.After challenging cell suspensions with an equal volume of shock solution at 23°C, the time course of volume change was measured by following the 90°scattered light intensity at 400 nm.Four-to-eight runs were stored and analysed in each experimental condition.In each run, 0.1 ml cellular suspension was mixed with an equal amount of hyperosmotic solution to reach inwardly directed gradients of solute.For osmotic water permeability, a hyperosmotic solution containing a non-permeant solute was used (450 mM mannitol in PBS, pH 7.4) producing an inwardly directed gradient of solute and water outflux until an osmotic equilibrium was reached.To measure glycerol permeability, a hyperosmotic solution containing glycerol (300 mM glycerol in PBS pH 7.4 for RBCs and 450 mM glycerol in PBS pH 7.4 for HEK-293T and melanoma cells) was used, creating an inwardly directed glycerol gradient.After the first fast cell shrinkage due to water outflow, glycerol influx in response to its chemical gradient was followed by water influx with subsequent cell reswelling.In all the permeability assays, the tonicity of the osmotic shocks (given by the ratio of the initial-to-final medium osmolarity after the applied osmotic challenges) was similar (tonicity of 1.5).Baselines were acquired using the respective incubation buffers as isotonic shock solutions.The kinetics of cell shrinkage and reswelling were measured from the time course of 90°scattered light intensity at 530 nm until a stable light scatter signal was attained.P f was calculated as , where V w is the molar volume of water, V 0 /A is the initial cell volume-to-area ratio, (osm out ) ∞ is the final medium osmolarity after the applied osmotic gradient, and k (s −1 ) is the single exponential time constant fitted to the light scatter signal of cell shrinkage; P gly was calculated by P gly = k(V 0 /A), where V 0 /A is the initial cell volume-to-area ratio and k (s −1 ) is the single exponential time constant fitted to the light scatter signal of glycerol influx.For inhibition assays, cells were incubated with gold compounds (5 μM) 30 min prior to permeability experiments.
The inhibitor concentration that corresponds to 50% inhibition (IC 50 ) was calculated by nonlinear regression of dose-response curves (GraphPad Prism software, GraphPad Software, Inc., San Diego, CA, USA) using the following equation: y = 100/(1 + 10((logIC 50x) × Hill slope)), where Hill slope describes the steepness of the family of curves.

H 2 O 2 influx assay
For H 2 O 2 permeability assays, adherent cells on 15 mm-diameter coverslips in a density of 5500, 7000 or 10,000 cells cm −2 (for MNT-1, A375 and HEK-293T, respectively) were loaded with the reactive oxygen species (ROS) sensitive probe 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 -DCFDA, Thermo Fisher Scientific) at 10 μM for 30 min at 37°C in 5% CO 2 .After incubation with the fluorescent probe and treatment with gold compounds, coverslips were mounted in a closed chamber (Warner Instruments, Hamden, CT, USA) on the stage of a Zeiss Axiovert 200 inverted microscope, using a ×40 epifluorescence objective.The oxidation kinetics of H 2 -DCFDA was measured after challenging cells with H 2 O 2 , according to previously described methods (Silva et al., 2022).Fluorescence was recorded with excitation at 495 nm, with 10 nm bandwidth, and emission collected with a 515/10 nm bandpass filter.Data were recorded and analysed using Metafluor software (Molecular Devices, San Jose, CA, USA), connected to a CCD camera (Cool Snap EZ, Photometrics, Tucson, AZ, USA).Fluorescence signal was acquired for cells in HEPES buffer for 40 s, followed by 100 μM H 2 O 2 , freshly prepared in HEPES buffer and directly added to the cells.The H 2 O 2 influx was measured by following the time course of intracellular ROS accumulation upon H 2 O 2 challenge, reported as a first order rate constant obtained from the slope of a semi-logarithmic plot of fluorescence intensity vs. time (Rodrigues et al., 2019).

Cytotoxicity assays
Gold compound-associated toxicity was evaluated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazium bromide (MTT) and lactate dehydrogenase (LDH) leakage assays.In the MTT assay, cells were inoculated at a density of 11,000, 14,000 or 100,000 cells cm −2 (for MNT-1, A375 and HEK-293T, respectively) in 96-well plates and grown at 37°C, 5% CO 2 until reaching 80% confluence.Cells were then incubated for 24 h with compounds 1−3 and Auphen at different concentrations (0, 2.5, 5 and 10 μM).After incubation, cells were washed with PBS and incubated with 0.5 mg ml −1 MTT reagent in PBS for 1 h at 37°C, 5% CO 2 .Finally, purple formazan crystals were solubilized with DMSO for 10 min, and the absorbance was measured at 570 nm (with correction at 690 nm).In the LDH leakage assay, cells were inoculated at a density of 30,000 or 40,000 cells cm −2 (for MNT-1 and A375, respectively) in 12-well plates and grown at 37°C, 5% CO 2 until reaching 80% confluence.The presence of LDH in cell-free supernatants was measured using the cytotoxicity detection kit (Roche, Barcelona, Spain) following the protocol previously described (da Silva, Cardoso, Martinez-Banaclocha et al., 2020) to evaluate gold compound-associated cell death.Cell death was defined as the percentage of released LDH compared with maximal LDH levels in cell lysates obtained using lysis buffer on non-treated cells (positive control).The percentage of the LDH release was calculated using saline buffer as negative control.These procedures were performed in triplicate.

Adhesion and proliferation assays
Adhesion and proliferation assays were conducted using an MTT assay as previously described (da Silva, Cardoso, Mendez-Gimenez et al., 2020).For the adhesion assay, cells were inoculated at a density of 11,000 or 14,000 cells cm −2 (for MNT-1 and A375, respectively) in 96-well plates and grown at 37°C, 5% CO 2 until adherence.Cells were resuspended in complete medium and treated with the gold compounds at the moment of inoculation.Adhesion was evaluated at 3, 4, 5 and 7 h after inoculation.For the proliferation assay, cells were inoculated at a density of 5500 or 7000 cells cm −2 (for MNT-1 and A375, respectively) in 96-well plates and grown at 37°C, 5% CO 2 .After adherence, cells were treated with 5 μM gold compounds.Proliferation was evaluated at 0, 8, 23 and 30 h after treatment.These procedures were performed in triplicate.

Migration assay
Cells were inoculated at a density of 6000 or 8000 cells cm −2 (for MNT-1 and A375, respectively) in 12-well plates and grown at 37°C, 5% CO 2 until reaching 80% confluence.The cell monolayer was wounded with an even trace using a sterile 200 μl tip and washed with PBS to remove debris.Cells were incubated with 5 μM gold compounds at 37°C, 5% CO 2 .Images of the wound closure were captured at 0, 9 and 24 h under a light microscope and analysed using the software ImageJ.Wound closure was normalized to initial wound area at time 0. This procedure was performed in triplicate.

Statistical analysis
All the experiments were performed in at least three biological and three technical triplicates.Statistical analysis between groups was performed by Student's t test or one-way ANOVA followed by Tukey's multiple comparisons test.All values are expressed as means (±SD).A P-value <0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism software.

AQPs expression in melanoma cells
AQP3 upregulation has been described in melanoma (da Silva et al., 2021;Gao et al., 2012), suggesting that its aberrant expression could be used for targeted therapeutics.Thus, we first screened the level of AQPs gene expression, validated the high level of AQP3 mRNA in the two melanoma cell lines, MNT-1 and A375, and confirmed the expression of the AQP3 protein.AQP3 and AQP11 were shown to be the most expressed in both melanoma cell lines (Fig. 2), with MNT-1 cells presenting higher transcript levels than A375 cells.In addition, AQP1 and AQP5 were also detected in lower amounts, and transcripts for AQP4, AQP6, AQP7 and AQP8 were found in the A375 cell line.Western blot and immunofluorescence analysis validated the expression of AQP3 in both cell lines; however, at variance with what was observed at the transcript level, A375 cells present higher AQP3 protein levels than MNT-1 cells.

AQP3 role in melanoma cellular oxidative stress
AQP3 has been shown to facilitate H 2 O 2 movement across membranes with implications for cell redox balance and, consequently, for oxidative stress.Thus, we investigated the impact of silencing AQP3 on the intracellular oxidative status of melanoma cells (Fig. 3).For the assessment of H 2 O 2 permeability, the rate of intracellular ROS accumulation was evaluated following the change of intensity of the ROS-sensitive fluorescent probe (H 2 -DCFDA) after an H 2 O 2 challenge.In both cell lines, knocking down AQP3 expression resulted in reduced H 2 O 2 permeability and consequently in a lower ROS accumulation when cells are challenged by H 2 O 2 , demonstrating the involvement of AQP3 on the physiological response to oxidative challenges.

Effect of organogold compounds on the glycerol permeability of RBCs
The inhibitory effect of gold compounds (compounds 1−3 and Auphen) on AQP3 function was assessed by  measuring the glycerol permeability coefficient (P gly ) of RBCs, which highly express AQP1 (strict water channel) and AQP3 (water/glycerol channel) (Preston et al., 1992;Roudier et al., 1998), by stopped-flow light scattering.To assess glycerol permeability, cells were confronted with a hyperosmotic solution of glycerol resulting in water efflux and cell shrinkage, followed by cell reswelling due to glycerol and water influx via AQP3 (Fig. 4A).P gly was calculated using the cell volume changes rate before and after cell treatment with 20 μM of gold compounds for 30 min.Our results show that compound 3 at 20 μM has a strong inhibitory effect (ca. 93%) of AQP3-mediated glycerol transport, comparable to Auphen, followed by compound 2 with an inhibitory effect of ca.85%, while compound 1 reveals a lower potency with inhibition around 50% (Fig. 4B).
Next, we obtained dose-response curves for RBC glycerol permeability with compound concentrations ranging from 0.1 to 40 μM (Fig. 4C-E), to calculate the IC 50 value for each compound.Compound 3 showed the highest potency (IC 50 8.91 ± 1.10 μM) followed by compound 2 (9.76 ± 0.98 μM).Compound 1 exhibited the highest IC 50 value (22.32 ± 0.83 μM) of all the tested gold complexes.Considering the previously reported IC 50 for Auphen in RBCs (0.8 ± 0.08 μM) (Martins et al., 2012), a weaker potency can be inferred for the new gold series herein tested.However, among the three organogold compounds, compound 3 showed the strongest AQP3 inhibition ability.

Effect of organogold compounds on the permeability of AQP3-overexpressing cells
To ascertain the specificity of this series of gold compounds towards AQP3 glyceroporin and peroxiporin activity, the inhibitory effect of compounds 1−3 and Auphen was assessed by measuring glycerol permeability and ROS accumulation in HEK-293T cells with AQP3 overexpression (Fig. 5A).Toxicity assays ensured that cell viability was not significantly reduced when cells were confronted with a range of 0-10 μM of gold compounds for 30 min (Fig. 5B).
The effect of 5 μM gold compounds in the activity of AQP3 as a glycerol channel was assessed by evaluating glycerol permeability (Fig. 5C).As shown in Fig. 5C, compounds 2, 3 and Auphen were partially effective in inhibiting glycerol permeation (36% and 45% inhibition, respectively).Auphen resulted in 50% inhibition of glycerol permeability compared to non-treated cells.
The effect of gold compounds on AQP3 H 2 O 2 channelling was evaluated through the rate of ROS accumulation after H 2 O 2 challenge.As shown in Fig. 5D, the peroxiporin activity of AQP3 was drastically reduced by compounds 2, 3 and Auphen.Compound 2 reduced H 2 O 2 permeability by 96%, compound 3 by 99% and Auphen by 97% (Fig. 5D).

Cytotoxic effect of organogold compounds on melanoma cells
Before the characterization of the gold compounds' effect on melanoma AQP3 activity and cellular assays (adhesion, proliferation and migration), we first performed toxicity assays to evaluate the compounds' non-toxic concentration to ensure that cell viability would not be compromised during the assays.Cytotoxicity was assessed by two complementary experiments: evaluation of cell viability by MTT assay and membrane integrity by lactate dehydrogenase (LDH) leakage assay.In the MTT assay, the gold compounds (at concentrations ranging from 2.5 to 10 μM) were tested in both MNT-1 and A375 cell lines, and cell viability was measured after 24 h of incubation.
In this assay, compounds 1, 2 and 3, as well as Auphen, did not show cytotoxic effects in MNT-1 cells up to 5 μM over 24 h.Above this concentration, compound 2 reduced cell viability (ca.70%) (Fig. 6A).In A375 cells, the organogold compounds did not affect cell viability even at the highest concentration of 10 μM.Instead, above 5 μM, Auphen induced moderate cell viability loss (around 30%) (Fig. 6B).Based on these results, the concentration chosen for subsequent experiments was 5 μM for all the gold compounds, assuring that cell viability would not be reduced.
The LDH leakage assay gives information on plasma membrane integrity, usually associated with cell death by apoptosis or pyroptosis (Bertheloot et al., 2021).This experiment was performed to understand if the gold complexes would compromise melanoma cells' membrane integrity by inducing apoptosis.Our data demonstrate that none of the gold complexes affected melanoma cell membrane integrity.Additionally, the results show that the interaction of compounds 1, 2 and 3 with AQP3 does not activate mechanisms of cell death in either MNT-1 or A375 melanoma cells (Fig. 6C and D), which is reflected by the residual LDH leakage detected during the experiment.

Effect of organogold compounds on AQP3 activity in melanoma cells
To evaluate the inhibitory effects of gold-compounds on AQP3 channel activity in melanoma cells, we measured membrane water, glycerol and H 2 O 2 permeability in MNT-1 and A375 cells treated with 5 μM of gold compounds for 30 min, by stopped flow spectroscopy (for water and glycerol permeability) or single cell fluorescence microscopy (for H 2 O 2 permeation).Our results demonstrate that none of the gold complexes significantly impaired water permeability of MNT-1 (Fig. 7A) and A375 (Fig. 7B) cells, in line with previous results showing that Auphen poorly affects cells' water permeability (Martins et al., 2012).
The effect of the gold compounds on the glyceroporin activity of AQP3, the most expressed glycerol channel in both melanoma cell lines, was evaluated by assessing glycerol permeability.Our results show that compounds 2, 3 and Auphen were moderately active inhibitors, although with compound-specific potency.In MNT-1, compound 3 Aquaporin-3 modulation by gold compounds impairs melanoma cell progression 3121 inhibited 40% of the glycerol transport, while compound 2 inhibited around 25%. Auphen resulted in 10% inhibition of glycerol permeability compared to control cells (Fig. 7C).The gold compounds showed inferior AQP3 inhibition in A375 cells, with only compound 2 and Auphen showing an inhibitory effect on glycerol transport of 25% and 23% respectively, when compared to control (Fig. 7D).
For H 2 O 2 permeability assays, the rate of intracellular ROS accumulation after H 2 O 2 challenge was evaluated for each cell line.Remarkably, in MNT-1 cells, peroxiporin activity was reduced by 86% for Auphen, 73% by compound 2, and 68% by compound 3 (Fig. 7E).Additionally, in A375 cells, the H 2 O 2 influx rate was reduced by 88% for compound 2, 86% by compound 3 and 92% by Auphen.Compound 1 did not affect H 2 O 2 influx in both MNT-1 and A375 cells (Fig. 7F).

Effect of organogold compounds on melanoma cell adhesion, proliferation and migration
To further investigate whether AQP3 inhibition by gold compounds underlies signal transduction mechanisms that impair melanoma progression, we evaluated melanoma cell adhesion, proliferation and migration before and after treatment with compounds 1−3 and Auphen.
Cell adhesion and proliferation were evaluated in MNT-1 and A375 cells by MTT assay.For adhesion, cells were incubated with 5 μM compounds 1−3 and Auphen during seeding, while for proliferation, cells were inoculated, allowed to adhere, and then incubated with the gold compounds.
The organogold complexes were also shown to have a cytostatic effect on melanoma cells.Compounds 2, 3 and Auphen reduced proliferation by around 35% in MNT-1 cells.Compound 1 was not efficient in slowing cell proliferation (Fig. 8C).In A375 cells, Auphen was the only gold compound hampering cell proliferation (90% impairment) (Fig. 8D).
Cell migration was assessed in melanoma cells by the wound closure assay.Thus, a wound was opened in the cell monolayer by scratch and the wound closure was followed at 0, 9 and 24 h after scratch.Cell migration rate was measured as the first derivative resulting from the area of the wound vs. time plot.
In MNT-1 cells, the gold compounds 2, 3 and Auphen delayed melanoma cell migration when compared to the control cells (Fig. 9A and C).The potency of the gold complexes in inhibiting cell migration was slightly higher for compound 2 (61%), followed by compound 3 (52%) and Auphen (34%) (Fig. 9B).In A375 cells, despite the delay in wound closure, the gold compounds seem to have lower potency than observed in MNT-1 cells, with significant differences only observed 24 h after the scratch (Fig. 9D and F).Similar to MNT-1 cells, non-treated A375 cells closed the wound before the 24 h time point, while all of the treated monolayers remained wounded.Auphen exhibited the strongest inhibitory effect on cell migration (30%), followed by compound 3 (22%) and 2 (18%) (Fig. 9E).

Discussion
Human AQP3 has a very well-characterized role in cancer biology.As a glycerol facilitator, AQP3 plays a part in energy balance and lipid metabolism, skin wound healing, cell proliferation and migration, and consequently cancer progression (da Silva et al., 2021;Verkman et al., 2014).On the other hand, the H 2 O 2 externally produced by superoxide dismutase (SOD) resulting from the − generated by NADPH oxidase 2 (NOX2) is transported into the cell by AQP3 (Miller et al., 2010), mediating downstream intracellular signalling and promoting cancer progression via tumour-related signalling pathways (Moon et al., 2022;Prata et al., 2019;Satooka & Hara-Chikuma, 2016).Thus, AQP3 influences cell redox status and oxidative stress, activating signalling pathways to promote inflammation, tumour growth and metastasis, and thus triggering the development of malignant tumours (Taniyama & Griendling, 2003).The development of new gold-based molecules that impact AQP3 function could result in a promising strategy for the treatment of AQP3-overexpressing cancers.In the present study, we investigated the potential anticancer properties of a new series of AQP3-inhibiting organogold compounds in vitro.Gold compounds are promising anticancer drug candidates since they represent a class of molecules with broad anticancer activity and different modes of action with respect to cisplatin (Marmol et al., 2019;Ott & Gust, 2007;Yue et al., 2020).Initial studies with RBCs enabled the detection of a strong AQP3 inhibitory effect of organogold compounds in the order 3 > 2 > 1, with reduction of glycerol permeability of ca.95%, 85% and 60%, respectively.The benchmark Au(III) coordination complex Auphen showed a similar effect to compound 3, with 95% inhibition of glycerol transport.When comparing the potency of this new gold series with previously described gold-based AQP3-inhibitors and other metallic compounds, Auphen is still the most potent (Martins et al., 2012(Martins et al., , 2013;;Pimpao et al., 2020), although not ideal as drug lead due to the lability of the N-Au coordination bonds between the phenanthroline ligand and Au(III) centre.Additionally, the specificity of these organogold compounds for AQP3 was assessed by measuring glycerol and H 2 O 2 permeability in AQP3-overexpressing cells treated with these complexes.Compounds 2, 3 and Auphen decreased glycerol trans-port in these cells by 36%, 45% and 50% respectively.Moreover, compound 2 impaired H 2 O 2 permeation by 96%, compound 3 by 99%, and Auphen by 97%, showing a strong blockage of AQP3 peroxiporin activity.
The AQP3 inhibitory effects of these gold compounds hold promise as drug leads for AQP3-overexpressing cancers.Thus, after evaluating their cytotoxic effect in two melanoma cell lines, their impact in cell adhesion, proliferation and migration was evaluated.The aberrant expression of AQP3 in melanoma biopsies has been previously reported (Gao et al., 2012).Therefore, here we selected two melanoma cell lines, MNT-1 and A375.AQP3 gene and protein expression were validated in both cell lines.The AQP expression level and isoform type can vary based on cell type, cancer stage and even the patient from which cells are derived.In fact, MNT-1 cells showed higher AQP3 gene expression than A375 cells, while A375 showed higher AQP3 protein expression than MNT-1, suggesting an increased RNA stability in MNT-1 cells.
Since AQP3 is the most expressed plasma membrane AQP in MNT-1 and A375 melanoma cells, we evaluated MNT-1 (B) and A375 (E) cells, non-treated and treated with 5 μM of gold compounds.C and F, representative images of wound closure progression in MNT-1 (C) and A375 (F) cells, non-treated and treated with 5 μM of gold compounds during the experiment.Data are represented as the means (±SD) of n = 3 independent experiments.( * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001, n.s., time point after wound opening vs. initial time point (0 h), gold compounds inhibited vs. control non-inhibited cells.)[Colour figure can be viewed at wileyonlinelibrary.com] the effect of the gold compounds on water, glycerol and H 2 O 2 permeability, which represent AQP3 channel activity.We previously reported that gold compounds exhibit a modest effect on AQP3 water permeability, while clearly decreasing glycerol transport (Martins et al., 2013), which may be explained by their unique inhibition mechanism in which, rather than a direct steric blockage, gold binding to Cys residues in each one of the four protein monomers induces conformational changes and constriction of the channel pores in a monomer cooperative manner, and this might not be sufficient to hinder the water permeability (de Almeida et al., 2014).In MNT-1 and A375 melanoma cells, none of the gold compounds showed an inhibitory effect on water permeability.However, glycerol permeability was impaired by compounds 2 and 3 in MNT-1 cells, while in A375 only compound 3 and Auphen had an inhibitory effect.Importantly, except for compound 1, the H 2 O 2 influx rate was considerably reduced by all the compounds in both melanoma cell lines, and this was even more pronounced in A375 cells.Notably, when both cell lines were silenced for AQP3, H 2 O 2 influx and intracellular ROS accumulation were decreased, demonstrating the importance of AQP3 for melanoma cells' oxidative stress.
AQP3-facilitated glycerol transport, a major source of ATP, participates in epidermal proliferation and tumour formation (Hara-Chikuma & Verkman, 2008).Reduced glycerol transport and lipid synthesis due to AQP3 knockdown impact energy-demanding cancer cells impairing cancer development and tumour growth (Wang et al., 2015).Additionally, AQP3-mediated H 2 O 2 transport may promote cancer progression via tumour-related signalling pathways (Moon et al., 2022;Prata et al., 2019).H 2 O 2 concentration can modulate the cellular oxidation status and play a role as a signalling molecule in cancer cell survival (Taniyama & Griendling, 2003).Impairment of H 2 O 2 transport deeply impacts cancer biology.
In MNT-1 and A375 cells, a high level of AQP11 transcript was also detected.An efficient transport of H 2 O 2 via AQP11 has been reported (Bestetti et al., 2020).However, unlike AQP3, AQP11 is localized intracellularly mainly in the endoplasmic reticulum membranes (Ishibashi et al., 2014) and thus cannot account for extracellular H 2 O 2 influxes nor their inhibition by the gold complexes herein detected.
Therefore, we subsequently investigated if the organogold compounds could affect melanoma cell adhesion, proliferation and migration, as these processes are strongly dependent on cellular energy and activation of signalling cascades in which AQP3 has an established role (Edamana et al., 2021;Marlar et al., 2017;Moon et al., 2022;Wang et al., 2022).
Interestingly, although all the tested compounds impaired the cell adhesion of MNT-1, only compound 2 affected the adhesion of A375 cells.Of note, our previous study showed that cells silenced for AQP3 and AQP5 had a lower cell-cell adhesion (Silva et al., 2022).A high cell adhesion can result in high cohesion within a tumour mass, although, on the other hand, decreased adhesion is associated with metastasis.In addition, compounds 2, 3 and Auphen also compromised cell proliferation in MNT-1 cells, showing cytostatic properties; however, only Auphen slowed down cell division in A375 cells.Interestingly, all compounds except compound 1 significantly reduced cell migration in both MNT-1 and A375 cells.
Previous studies showed that cell migration can be reduced by silencing expression of AQP3 (De Ieso & Yool, 2018;Pimpao et al., 2020;Rodrigues et al., 2019;Silva et al., 2022), evidencing that AQP3 has a pivotal role in cancer metastasis.Moreover, overexpression of AQP3 in cancer cells was shown to stimulate several intracellular signalling pathways, promoting cell proliferation, migration and invasion as well as aggravation of epithelial-to-mesenchymal transition (Smith et al., 2023).The role of AQP3 in signalling mechanisms in which H 2 O 2 acts as an important second messenger may explain, at least in part, the effect of gold compounds on cell migration of both MNT-1 and A375 cells.
Overall, a relationship between blockage of AQP3 peroxiporin activity by organogold compounds and impairment of cell adhesion, proliferation and migration of melanoma cells can be established, although dependent on melanoma cell type and tumour stage.The use of selective gold-based AQP3 inhibitors may contribute to further elucidating the role of AQP3 in melanoma and pave the way to the design of new gold-based therapeutics for the treatment of skin cancers.

Conclusions
Our data show that AQP3-mediated glycerol and H 2 O 2 permeability correlate to cell adhesion, proliferation and migration of melanoma cells.The present study highlights the role of AQP3 in melanoma progression and unveils organogold compounds as anticancer drug leads in AQP3-overexpressing cancers.

J
Physiol 602.13

Figure 3 .
Figure 3. AQP3 silencing correlates with reduced ROS accumulation in melanoma cells A and B, validation of AQP3 knockdown by qPCR in MNT1 (A) and A375 (B) melanoma cells.Data are represented as the means (±SD) of three independent experiments.C and D, reactive oxygen species (ROS) accumulation after adding 100 μM H 2 O 2 to MNT1 (C) and A375 (D) melanoma cells loaded with ROS-sensitive probe H 2 -DCFDA.Data are represented as the means (±SD) of n = 8 from a total of three independent experiments.( * P < 0.05, * * P < 0.01, * * * * P < 0.0001, n.s.non-significant vs. siCTL.)siCTL, control of transfection; siAQP3, AQP3-silenced cells.[Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4 .
Figure 4. Inhibitory effect of the gold compounds 1−3 and Auphen on RBCs glycerol permeability A, representative stopped-flow signal for glycerol transport after imposing a hyperosmotic glycerol solution to RBCs non-treated (control) and treated with 20 μM of compound 3 (30 min incubation at room temperature).B, glycerol permeability of RBCs control and treated with 20 μM gold compounds 1−3 and Auphen.C-E, concentration-dependent inhibition of RBCs glycerol permeability by compound 1 (C), 2 (D) and 3 (E) (incubated for 30 min at room temperature).Data are represented as the means (±SD) of n = 3-5 independent experiments.( * * P < 0.01, * * * * P < 0.0001 vs. Control.)[Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6 .
Figure 6.Effect of gold compounds 1−3 and Auphen on cell viability and membrane integrity of human melanoma cells A and B, cell viability determined by MTT assay after cell exposure to a range of concentrations of five gold compounds (0, 2.5, 5, 7.5, 10 μM) for 24 h in MNT-1 (A) and A375 (B) cells.C and D, membrane integrity assessed by LDH leakage assay after cell exposure to 5 μM of compounds 1−3 and Auphen for 24 h in MNT-1 (C) and) A375 (D cells.Results are expressed as means (±SD) of n = 3 independent experiments.(n.s. vs. Control.)[Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7 .
Figure 7. Effect of gold compounds 1−3 and Auphen on water, glycerol and hydrogen peroxide permeability of human melanoma cells A-D, water (A and B) and glycerol (C and D) permeabilities after cell exposure to 5 μM of gold compounds for 30 min in MNT-1 (A and C) and A375 (B and D) cells.E and F, ROS accumulation rate after a 100 μM H 2 O 2 challenge, in MNT-1 (E) and A375 (F) cells untreated and treated with 5 μM of gold compounds for 30 min before the experiment.Data are represented as the means (±SD) of n = 10-12 from a total of three experiments.( * P < 0.05, * * P < 0.01, * * * * P < 0.0001, n.s. vs. Control.)[Colour figure can be viewed at wileyonlinelibrary.com]

Figure 8 .
Figure 8.Effect of gold compounds 1−3 and Auphen on cell adhesion and proliferation of human melanoma cells Cell adhesion (A and B) and proliferation (C and D) were obtained using MTT assay while exposing MNT-1 (A and C) and A375 (B and D) to 5 μM of gold compounds.Data are represented as the means (±SD) of n = 3 of a total of three independent experiments.( * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001, n.s. vs. Control.)[Colour figure can be viewed at wileyonlinelibrary.com]