antioxidants Article Impact of the APE1 Redox Function Inhibitor E3330 in Non-Small Cell Lung Cancer Cells Exposed to Cisplatin: Increased Cytotoxicity and Impairment of Cell Migration and Invasion Rita Manguinhas 1 , Ana S. Fernandes 2 , João G. Costa 2 , Nuno Saraiva 2 , Sérgio P. Camões 1 , Nuno Gil 3 , Rafael Rosell 4,5 , Matilde Castro 1 , Joana P. Miranda 1 and Nuno G. Oliveira 1, * 1 Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003 Lisboa, Portugal; rmanguinhas@campus.ul.pt (R.M.); sergiocamoes@campus.ul.pt (S.P.C.); mcastro@ff.ulisboa.pt (M.C.); jmiranda@ff.ulisboa.pt (J.P.M.) 2 Research Center for Biosciences & Health Technologies (CBIOS), Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-024 Lisboa, Portugal; ana.fernandes@ulusofona.pt (A.S.F.); jgcosta@ulusofona.pt (J.G.C.); nuno.saraiva@ulusofona.pt (N.S.) 3 Lung Cancer Unit, Champalimaud Centre for the Unknown, Av. Brasília, 1400-038 Lisboa, Portugal; nuno.gil@fundacaochampalimaud.pt 4 Laboratory of Cellular and Molecular Biology, Institute for Health Science Research Germans Trias i Pujol (IGTP), Campus Can Ruti, Ctra de Can Ruti, Camí de les Escoles, s/n, 08916 Badalona, Barcelona, Spain; rrosell@iconcologia.net 5 Internal Medicine Department, Universitat Autónoma de Barcelona, Campus de la UAB, Plaça Cívica, 08193 Bellaterra, Barcelona, Spain * Correspondence: ngoliveira@ff.ulisboa.pt  Received: 12 May 2020; Accepted: 22 June 2020; Published: 24 June 2020  Abstract: Elevated expression levels of the apurinic/apyrimidinic endonuclease 1 (APE1) have been correlated with the more aggressive phenotypes and poor prognosis of non-small cell lung cancer (NSCLC). This study aimed to assess the impact of the inhibition of the redox function of APE1 with E3330 either alone or in combination with cisplatin in NSCLC cells. For this purpose, complementary endpoints focusing on cell viability, apoptosis, cell cycle distribution, and migration/invasion were studied. Cisplatin decreased the viability of H1975 cells in a time- and concentration-dependent manner, with IC50 values of 9.6 µM for crystal violet assay and 15.9 µM for 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. E3330 was clearly cytotoxic for concentrations above 30 µM. The co-incubation of E3330 and cisplatin significantly decreased cell viability compared to cisplatin alone. Regarding cell cycle distribution, cisplatin led to an increase in sub-G1, whereas the co-treatment with E3330 did not change this profile, which was then confirmed in terms of % apoptotic cells. In addition, the combination of E3330 and cisplatin at low concentrations decreased collective and chemotactic migration, and also chemoinvasion, by reducing these capabilities up to 20%. Overall, these results point to E3330 as a promising compound to boost cisplatin therapy that warrants further investigation in NSCLC. Keywords: non-small cell lung cancer; cisplatin; apurinic/apyrimidinic endonuclease 1; E3330; cytotoxicity; apoptosis; migration; invasion Antioxidants 2020, 9, 550; doi:10.3390/antiox9060550 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 550 2 of 18 1. Introduction Worldwide, lung cancer (LC) is the first cause of cancer-related deaths and the most diagnosed type of cancer for men and women combined. In the US, LC is by far the main cause of death by cancer [1,2]. Non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases and has a low survival rate due to metastasis progression [3]. The most common chemotherapy regimens used in this type of cancer comprise the platinum-based drug cisplatin (Figure 1A), which exerts its effect by cross-linking DNA, inhibiting its replication and transcription, resulting in cell death [4,5]. Even though cisplatin is associated with slightly better survival rates, it is still associated with inherited and acquired resistance to therapy, making it inactive against some tumor types [6]. Indeed, cisplatin resistance is one of the major limitations to its clinical use [4]. Nevertheless, the mechanisms responsible for the resistance of tumor cells are not yet completely understood. For this reason, a number of possible mechanisms for cisplatin resistance were proposed, including reduced intracellular accumulation of cisplatin; Antioxidants enhanced 2020, drug 9, x FOR PEER inactivation by metallothionein and glutathione; altered expression REVIEW of 3 of 18 oncogenes and regulatory proteins; and increased repair activity of DNA damage [5]. The mechanistic findings on nicotine-induced administration by enabling a cisplatin chemoresistance synergistic [7,8] should effect. This evaluation wasalso be mentioned performed for theinfirst the time contextin of lung the cancer context ofinNSCLC tobacco by smokers. Moreover, integrating both nicotine multiple endpoints[9] and cigarette related smoking to cell [10] cell viability, havecycle been associated with progression, oxidative apoptosis, stress. and cell migration and invasion. Figure 1. Figure Chemical structure 1. Chemical structure of of cisplatin cisplatin (A) (A) and and (2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4- (2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4- cyclohexadien-1-yl)methylene] undecanoic acid (E3330) cyclohexadien-1-yl)methylene] undecanoic acid (E3330) (B).(B). The human apurinic/apyrimidinic endonuclease 1 (APE1) is an essential enzyme with two key 2. Materials and Methods functions, i.e., repairing DNA damage through the base excision repair (BER) pathway and also a redox signaling protein, modulating the activation of several transcription factors related to cell survival, 2.1. Chemicals proliferation, migration/invasion, inflammation, angiogenesis and metastases formation [11–13]. RPMI-1640 Several with L-glutamine transcription factors havewas beenpurchased related tofrom APE1 Biowest 0 s redox(Nuaillé, activity,France). Cisplatin, particularly E3330, the nuclear penicillin-streptomycin solution (10,000 units/mL of penicillin; 10 mg/mL of streptomycin), factor-κB (NF-κB), activator protein 1 (AP-1), early growth response protein-1 (Egr-1), hypoxia-inducible crystal violet factor (CV), sodiump53, 1α (HIF-1α), bicarbonate, extracellular signal transducer andmatrix (ECM) activator gel and dimethylsulfoxide of transcription 3 (STAT3), and (DMSO) were Nuclear factor purchased from Sigma-Aldrich (Madrid, Spain). Sodium pyruvate was purchased (erythroid-derived 2)-like 2 (Nrf-2) [12,14–20]. In addition, upon oxidative stress, APE1 is known to from Lonza (Basel, Switzerland) and trypsinredox control the intracellular (0.25%), stateand via fetal bovine serum the inhibition (FBS) oxygen of reactive from Gibcospecies(Eugene, OR, USA). (ROS) production Ethanol absolute, or by binding withpropidium transcription iodide (PI),(such factors ethylenediaminetetraacetic as p53, HIF-1α, and Nrf-2), acidpromoting (EDTA), and acetic acid an antioxidant were obtained from Merck (Darmstadt, Germany). HEPES and D-Glucose response [21–23]. Furthermore, multiple studies have demonstrated that APE1 is overexpressed were purchased from AppliChem in numerous(Darmstadt, Germany). types of cancer, such as CellTiter NSCLC. 96Increased ® Aqueousexpression MTS (3-(4,5-Dimethylthiazol-2-yl)-5-(3- is also associated with more carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) aggressive tumor phenotypes and poor prognosis [13,18,24]. wasFor acquired from Promega these reasons, APE1 has(Madison, gained WI, USA) and the Alexa Fluor 488 ® Annexin V/PI Kit was acquired from Molecular Probes (Eugene, increasing attention as an emerging druggable target in cancer therapy. A study by Wang et al. [24] OR, USA). that APE1 could be a promising target for the combination of cisplatin-based chemotherapy suggested A 25 mM in NCSLC stocksince patients, solution of inhibition its total E3330 wasusing prepared siRNAinenhanced DMSO, aliquoted, sensitivityand stored at to cisplatin −20 °C. activity in Working solutions were freshly diluted in complete cell culture medium A549 cells by enabling a synergistic relationship. Other complementary studies also demonstrated so that, in each final solution, the DMSO that the increased concentration was chemoresistance kept at treatment to cisplatin 0.2% (v/v).could Cisplatin was dissolved be related in saline (0.9% to APE1 overexpression NaCl) at a concentration of 2 mM, aliquoted, and stored at −20 °C. In all cell-based up-regulating transcription factors related to cell survival, such as NF-κB and AP-1, and adaptive assays, vehicle- treated response controls were also to apoptotic included, stimulation in which [25–27]. H1975 All this cells were evidence exposed reinforces thattoAPE1 the respective solvents, plays a vital role as i.e., DMSO (final an upstream concentration effector of 0.2% (v/v)) in cancer progression andorreducing saline. its expression levels could help with cisplatin therapy efficiency. 2.2. Cell Culture The human NSCLC cell line H1975 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). This cell line was established in July 1988, originated from lung adenocarcinoma (NSCLC) and the tissue donor was a non-smoker female. H1975 cells were cultured in monolayer in RPMI-1640 medium with L-glutamine supplemented with 10 mM HEPES, 2.5 g/L D- Antioxidants 2020, 9, 550 3 of 18 The quinone derivative (2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl) methylene] undecanoic acid (E3330 or APX3330, Figure 1B) has been described as a direct and highly specific inhibitor of APE1 redox function, being the first to be identified [28]. For this reason, it has been tested to reduce growth-promoting, inflammatory and anti-apoptotic activities in cells, as well as tumor invasion and metastatic disease in different types of cancer [12,13,28,29]. Its therapeutic potential has been addressed in several cancer-cell-based studies, with interesting results including those from a previous study from our group with human breast cancer cells [13]. A study with prostate cancer also demonstrated important results by reducing cancer cell proliferation and inducing cell cycle arrest upon selective inhibition of the reduction-oxidation function with E3330 [30]. In pancreatic cancer, E3330 was able to impair tumor growth and blocked the activity of NF-κB, AP-1, and HIF-1α [31]. Furthermore, E3330 therapeutic efficacy has been shown to reduce the activity of the transcriptional activators, described previously, regulated by the APE1 redox activity [14–17]. As a result, E3330 has been evaluated in phase I clinical trials in patients with advanced solid tumors [29]. In this context, the present work aimed to assess the impact of APE1’s redox function inhibition by E3330 in NSCLC cells in vitro and if this active compound could improve the efficacy of cisplatin administration by enabling a synergistic effect. This evaluation was performed for the first time in the context of NSCLC by integrating multiple endpoints related to cell viability, cell cycle progression, apoptosis, and cell migration and invasion. 2. Materials and Methods 2.1. Chemicals RPMI-1640 with l-glutamine was purchased from Biowest (Nuaillé, France). Cisplatin, E3330, penicillin-streptomycin solution (10,000 units/mL of penicillin; 10 mg/mL of streptomycin), crystal violet (CV), sodium bicarbonate, extracellular matrix (ECM) gel and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (Madrid, Spain). Sodium pyruvate was purchased from Lonza (Basel, Switzerland) and trypsin (0.25%), and fetal bovine serum (FBS) from Gibco (Eugene, OR, USA). Ethanol absolute, propidium iodide (PI), ethylenediaminetetraacetic acid (EDTA), and acetic acid were obtained from Merck (Darmstadt, Germany). HEPES and d-Glucose were purchased from AppliChem (Darmstadt, Germany). CellTiter 96® Aqueous MTS (3-(4,5-Dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was acquired from Promega (Madison, WI, USA) and the Alexa Fluor 488® Annexin V/PI Kit was acquired from Molecular Probes (Eugene, OR, USA). A 25 mM stock solution of E3330 was prepared in DMSO, aliquoted, and stored at −20 ◦ C. Working solutions were freshly diluted in complete cell culture medium so that, in each final solution, the DMSO concentration was kept at 0.2% (v/v). Cisplatin was dissolved in saline (0.9% NaCl) at a concentration of 2 mM, aliquoted, and stored at −20 ◦ C. In all cell-based assays, vehicle-treated controls were also included, in which H1975 cells were exposed to the respective solvents, i.e., DMSO (final concentration of 0.2% (v/v)) or saline. 2.2. Cell Culture The human NSCLC cell line H1975 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). This cell line was established in July 1988, originated from lung adenocarcinoma (NSCLC) and the tissue donor was a non-smoker female. H1975 cells were cultured in monolayer in RPMI-1640 medium with l-glutamine supplemented with 10 mM HEPES, 2.5 g/L d-glucose, 1 mM Sodium pyruvate, 1.5 g/L Sodium bicarbonate, 10% FBS and 1% Pen/Strep (complete cell culture medium) and were maintained at 37 ◦ C, under a humidified atmosphere containing 5% CO2 in air. Antioxidants 2020, 9, 550 4 of 18 2.3. Crystal Violet (CV) Staining Assay The cytotoxicity of cisplatin/E3330 alone or in combination, in H1975 cells, was evaluated according to a previously described CV staining protocol [13]. Cells were seeded for 24 h at a density of approximately 3 × 103 cells/well in 200 µL of complete culture medium in 96-well plates. After the 24 h period, culture medium was changed and the cells were incubated with a range of cisplatin (1–50 µM) or E3330 (5–50 µM) concentrations, for another 72 h (and also 48 h for cisplatin), in order to assess concentration-response profiles. For combinatory assays, cells were pre-incubated with E3330 (30 µM) for 3 h and then simultaneously exposed to E3330 and different concentrations of cisplatin (5–20 µM) for 72 h. After the incubation periods, H1975 cells were washed with phosphate-buffered saline (PBS) to remove non-adherent cells (non-viable cells). The adherent cells were then fixed with ice-cold 96% ethanol for 10 min and stained with 0.1% crystal violet in 10% ethanol for 5 min. After rinsing with tap water, the stained cells were dissolved in 200 µL of 96% ethanol with 1% acetic acid. Absorbance was measured at 595 nm (OD595) using a SPECTROstar OMEGA microplate reader (BMG Labtech, Offenburg, Germany). Absorbance values presented by vehicle-treated control cells (untreated cells) corresponded to 100% of cell viability. Three to five independent experiments were carried out and six replicates were used for each condition in each independent experiment. The IC50 was calculated based on the concentration–response curve using GraphPad Prism® 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). 2.4. MTS Reduction Assay The MTS reduction assay was carried out as a confirmatory assay of cell viability by applying the same experimental conditions as in the CV assay and following an already described protocol [13]. Briefly, after treatment with the compounds and removal of the incubation medium, cells were washed with PBS, followed by the addition of 100 µL of fresh complete growth medium plus 20 µL of MTS substrate prepared from the CellTiter 96® Aqueous MTS, according to the manufacturer’s instructions. Cells were incubated for 2 h with the MTS reagent and the results were measured in terms of absorbance at 490 nm and 690 nm (reference wavelength) using a SPECTROstar OMEGA microplate reader (BMG Labtech, Offenburg, Germany). Absorbance values presented by vehicle-treated control cells corresponded to 100% of cell viability. Three to four independent experiments were carried out and three replicates were used for each condition in each independent experiment. The IC50 was also calculated based on the concentration–response curve using GraphPad Prism® 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). 2.5. Cell DNA Content Analysis Cell DNA content analysis by flow cytometry was performed in order to evaluate the effect of the combination of both compounds in cell cycle distribution. This procedure was carried out according to previously described protocols [32,33]. Briefly, 6 × 104 cells/well were seeded in 6-well plates and cultured for 21 h. Afterwards, cells were incubated with E3330 (30 µM) for 3 h. The culture medium was then changed and both cisplatin (20 µM) and E3330 (30 µM) were added to the cells and incubated for an additional 72-h period. Cells were harvested using 5 mM EDTA in PBS at 37 ◦ C, washed with cold PBS and fixed with chilled 80% ethanol. Cells were stained with PI (10 µg/mL) and simultaneously treated with RNase A (20 µg/mL) for 15−20 min. PI staining was analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Data acquisition and analysis were performed using CellQuest® software (Becton Dickinson, San Jose, CA, USA ) and FlowJo® (Tree Star Inc., San Carlos, CA, USA), respectively. Three independent experiments were performed. 2.6. Apoptosis Assay The percentage of apoptotic cells was measured using the flow cytometry dead cell apoptosis kit with Alexa® Fluor 488 Annexin V and PI according to the manufacturers’ instructions (Molecular Antioxidants 2020, 9, 550 5 of 18 Probes, Eugene, OR, USA). Roughly, 9 × 104 cells were seeded in 6-well plates and grown for 21 h at 37 ◦ C in complete growth medium. After the 21 h period, E3330 (30 µM) was added to the cells for 3 h (pre-incubation). The culture medium was then renewed and both cisplatin (20 µM) and E3330 (30 µM) were added to the cells and incubated for further 72 h. Afterwards, the cells were detached with 5 mM EDTA in PBS at 37 ◦ C and washed with cold PBS. Cells were then stained with PI and Alexa Fluor® 488 annexin V according to the manufacturer’s instructions and analysed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Three independent experiments were performed. Data acquisition and analysis were performed using CellQuest® software (Becton Dickinson, San Jose, CA, USA) and FlowJo® (Tree Star Inc., San Carlos, CA, USA), respectively. 2.7. Selection of Cisplatin and E3330 Concentrations for the Migration/Invasion Assays To select non-toxic concentrations of both compounds for the migration and invasion assays, CV and MTS assays were performed as described previously, but using medium with a low serum content instead. This step is of utmost importance to avoid misleading conclusions in migration assays due to cytotoxic effects. Taking this into account, 8 × 103 cells/well were seeded in 96-well plates in complete culture medium containing 10% FBS for 24 h. After the 24 h period, the complete cell culture medium was replaced by culture medium containing 2% FBS and cells were then incubated with a range of low concentrations of cisplatin (0.1−5 µM) or E3330 (10−30 µM) for another 24 h. The CV and MTS assays were subsequently performed and non-toxic concentrations of both E3330 and cisplatin were selected for the following assays. Three to seven independent experiments were performed, each one comprising three (MTS) or six (CV) replicates. 2.8. In Vitro Wound-Healing Assay For the evaluation of collective cell migration, an in vitro wound-healing assay was performed according to a previously described method [34,35]. H1975 cells were seeded in 24-well plates at a density of approximately 5.5 × 104 cells/well and incubated for 21 h in complete cell culture medium. E3330 was then added to the well at a concentration of 10 µM and incubated for 3 h. Afterwards, the cell culture medium was removed, and a scratch was performed using a 200 µL sterile pipette tip on the cell monolayer. Cells were then washed twice with warm PBS, in order to remove cellular debris, and were left to migrate in cell culture medium containing 2% FBS in the presence of cisplatin (1 µM) and E3330 (10 µM) for further 20 h. Wound closure was evaluated using a Motic AE2000 Inverted Phase Contrast Microscope (Motic, Barcelona, Spain) and pictures of the same areas were captured using a magnification of 40× with a camera Moticam 2500 (Motic, Barcelona, Spain). The scratch width was measured with Motic Images plus v2.0 software (Motic, Barcelona, Spain) at 0, 8, and 20 h after the scratch was performed. The percentage of cell migration was measured in relation to the initial distance between the wound edges. At each time-point, two pictures of the scratch were taken for each condition. Three independent experiments were performed. 2.9. Chemotaxis and Chemoinvasion Assays As a single-cell migration evaluation, a chemotactic migration assay was performed by adopting a protocol already described by Flórido et al. [36] and Fernandes et al. [34]. Briefly, 3 × 104 cells/well were seeded in cell culture medium containing 2% FBS on the top of a transwell insert with transparent polyethylene terephthalate (PET) membranes containing 8 µm pores (BD Falcon, Bedford, MA, USA) inside 24-well plates. Complete cell culture medium was added to the lower chamber, containing 10% FBS as the chemoattractant. Right after seeding, E3330 (10 µM) alone was added to both chambers and incubated for 3 h. After this 3 h period, cisplatin (1 µM) was also added to both chambers and cells were allowed to migrate through the membrane for another 16 h. Subsequently, non-migrating cells were carefully removed from the upper chamber with a cotton swab and migrating cells (bottom of each membrane) were fixed with cold 96% ethanol for 10 min and then stained with 0.1% crystal violet in 10% ethanol for 15 min. The inserts were thoroughly rinsed using tap water and were allowed to Antioxidants 2020, 9, 550 6 of 18 dry for at least 24 h. Five randomly selected fields were photographed for each condition, using a Moticam 2500 (Motic, Barcelona, Spain) placed on a Motic AE2000 Inverted Phase Contrast Microscope (Motic, Barcelona, Spain) with an amplification of 100×. For each picture, migrated cells were manually counted using the software Motic Images plus v3.0 (Motic, Barcelona, Spain). The counted cells were expressed as percentages of vehicle-treated control cells and three independent experiments were performed. A procedure similar to the abovementioned chemotactic migration assay was carried out for the evaluation of chemoinvasion. The difference between the two setups was the addition of 75 µL of ECM gel (1:25 dilution in serum-free medium) in order to coat the porous membranes of the transwell inserts. The 2020, Antioxidants initial 9, xseeding REVIEW was also adapted to 1.5 × 104 cells/well. The analysis of the6 results FOR PEERdensity of 18 was performed similarly to the abovementioned chemotactic migration assay, and five independent was performed experiments weresimilarly to the abovementioned chemotactic migration assay, and five independent performed. experiments were performed. 3. Results 3. Results 3.1. Cytotoxicity Profile of Cisplatin in H1975 Cells 3.1. Cytotoxicity Profile of Cisplatin in H1975 Cells In order to determine the impact of cisplatin treatment in H1975 cells viability, a concentration- response In profile order towas determine the impact established, of cisplatin resorting treatment to CV staining andinMTS H1975 cells viability, reduction assays. aInconcentration- the CV staining response profile was established, resorting to CV staining and MTS reduction assays. In the CV assay, after 48 and 72 h of cisplatin exposure (Figure 2A), the viability of H1975 cells decreased in a time- staining assay, after 48 and 72 h of cisplatin exposure (Figure 2A), the viability of H1975 cells and concentration-dependent manner (1–50 µM). Additionally, The MTS reduction assay (Figure 2B) decreased in a time- and concentration-dependent manner (1–50 µ M). Additionally, The MTS was performed as a mechanistically complementary method and the concentration–response curves reduction assay (Figure 2B) was performed as a mechanistically complementary method and the showed similar cytotoxicity profiles. The IC50 values for the CV assay were 27.5 and 9.6 µM for 48 and concentration–response curves showed similar cytotoxicity profiles. The IC50 values for the CV assay 72 h, respectively. In addition, the IC50 value calculated for the MTS assay at 72 h was 15.9 µM, in the were 27.5 and 9.6 µ M for 48 and 72 h, respectively. In addition, the IC50 value calculated for the MTS same assayrange, at 72although slightly h was 15.9 µ M, inhigher. the same Cisplatin was demonstrated range, although to be Cisplatin slightly higher. toxic at low wasconcentrations demonstratedfor a 72 to hbeincubation toxic at lowperiod, starting for concentrations to compromise cell viability a 72 h incubation at 1 µMtoand period, starting dramatically compromise decreasing cell viability at it at 150µ M µM,and dramatically decreasing it at 50 µ M, for both MTS and CV assays. Considering these results, 10, for both MTS and CV assays. Considering these results, the cisplatin concentrations of 5, and 20 µM the cisplatinwere selected for of concentrations the5,subsequent 10, and 20 µ combinatory M were selected assays, since for the they represent subsequent different combinatory levels assays, of since cytotoxicity, comprising they represent the range different levels of cytotoxicity, IC50 values calculated comprisingasthe well as concentrations range slightly above of IC50 values calculated as andwellbelow this parameter. as concentrations slightly above and below this parameter. Figure 2. Cytotoxic effects of cisplatin (1–50 µM) in H1975 cells. The viability of cells treated with Figure for cisplatin 2. Cytotoxic 48 h and effects 72 h wasof assessed cisplatin (1–50 µ M) violet by crystal in H1975 cells. (CV) The viability staining assay (A)ofand cellsfor treated with 72 h by MTS reduction assay (B). Values represent mean ± SD (n = 3–4) and are expressed as percentages of the cisplatin for 48 h and 72 h was assessed by crystal violet (CV) staining assay (A) and for 72 h by MTS reduction assay vehicle-treated (B). Values control cells. represent mean ± SD (n = 3–4) and are expressed as percentages of the vehicle-treated control cells. 3.2. Impact of E3330 in the Viability of H1975 Cells 3.2. Impact of E3330 in the Viability of H1975 Cells The effect of E3330 was evaluated by exposing H1975 cells during 72 h to a range of concentrations from 5The to 50effect µM. of E3330 Both was MTS CV and evaluated assaysby exposing revealed H1975 that E3330cells wasduring 72 h to a toxic not considerably rangeatoflow concentrations from 5 to 50 µ M. Both CV and MTS assays revealed that E3330 was not considerably toxic at low concentrations (Figure 3A and 3B, respectively). Both assays demonstrated a similar concentration–response curve for E3330. Nevertheless, E3330 at 50 µ M showed decreased cell viability in about 45% with the CV assay whereas, with the MTS assay, the decrease was lower, approximately 30%. A similar trend in the differences between these two methods was also observed in the previous cisplatin assays, reflecting the inherent sensitivities of these two mechanistically Antioxidants 2020, 9, 550 7 of 18 concentrations (Figure 3A,B, respectively). Both assays demonstrated a similar concentration–response curve for E3330. Nevertheless, E3330 at 50 µM showed decreased cell viability in about 45% with the CV assay whereas, with the MTS assay, the decrease was lower, approximately 30%. A similar trend in the differences between these two methods was also observed in the previous cisplatin assays, reflecting the inherent sensitivities of these two mechanistically distinct endpoints. Since the range of E3330 concentrations applied for these experimental conditions did not lead to a 50% loss in cell viability, it was not possible to calculate the IC50 values for H1975 cells. The concentration of 30 µM was chosen for the combinatory assays since it was the higher concentration of E3330 tested that displayed aAntioxidants relatively2020, low9,impact x FOR PEER REVIEW on cell viability. 7 of 18 Figure 3. Evaluation of E3330 (5–50 µM) cytotoxicity in H1975 cells. The cell viability of E3330-exposed Figure 3. Evaluation of E3330 (5–50 µ M) cytotoxicity in H1975 cells. The cell viability of E3330- cells (72 h) was evaluated by CV staining (A) and MTS reduction (B) assays. Values represent mean ± SD exposed cells (72 h) was evaluated by CV staining (A) and MTS reduction (B) assays. Values represent (n = 3) and are expressed as percentages of the vehicle-treated control cells. mean ± SD (n = 3) and are expressed as percentages of the vehicle-treated control cells. 3.3. The Combination of E3330 and Cisplatin Displays a Synergistic Effect in Cell Viability 3.3. The Combination of E3330 and Cisplatin Displays a Synergistic Effect in Cell Viability With the purpose of evaluating if E3330 enhanced cisplatin treatment in NSCLC, H1975 cells were With thewith co-incubated purpose theseof twoevaluating compounds if E3330 and theenhanced cisplatin effects were treatment evaluated usinginthe NSCLC, H1975assay CV staining cells werevalidated and co-incubated withwith the MTSthesereduction two compounds assay. Inand thethe CVeffects assay, were E3330evaluated using the CV staining (30 µM) demonstrated a slight assay and validated with the MTS reduction assay. In the CV assay, decrease in cell viability of around 11% (p < 0.01) when compared to the vehicle-treated E3330 (30 µ M) demonstrated control cellsa slight decrease (Figure 4A). In inthecell MTSviability assay,ofthis around decrease11% was(p < 0.01) lowerwhen and compared to thesignificant not statistically vehicle-treated control (Figure 4B). cells (Figure 4A). In the MTS assay, this decrease was lower and not statistically All the concentrations of cisplatin (5, 10, and 20 µM) tested in the CV assay revealed an impairment significant (Figure 4B). in cellAll the concentrations viability that was clearly of intensified cisplatin (5,when 10, and 20 µ M) the APE1 tested redox in theE3330 inhibitor CV assay revealed an was co-incubated. impairment This in cell significant viabilityeffect combined that was wasclearly intensifiedinwhen also confirmed the APE1 the MTS assay.redox inhibitor In this E3330 case, the was cells co- were incubated. This significant combined effect was also confirmed in the MTS treated with 20 µM of cisplatin and 30 µM of E3330. In absolute percentage values, the decreases in assay. In this case, the cellsviability cell were treated observedwithfor205,µ10Mand of cisplatin 20 µM ofand 30 µ Minofthe cisplatin, E3330. In absolute presence of E3330,percentage were 18.5%values, the (p < 0.05), decreases 22.8% (p < in celland 0.05) viability 12.4%observed (p < 0.01),for 5, 10 and 20 respectively, forµthe M ofCVcisplatin, assay, and in 17.1% the presence of E3330, (p < 0.05) for the were MTS assay. Considering the relative decreases in cell viability observed, the concentration of17.1% 18.5% (p < 0.05), 22.8% (p < 0.05) and 12.4% (p < 0.01), respectively, for the CV assay, and E3330(pat< 0.05) 30 µMfor the MTS reduced in 36%assay. and 78% Considering the relative the cell viability of 20 µMdecreases in cell cells cisplatin-treated viability for theobserved, CV and MTS the concentration of E3330 at 30 µ M reduced in 36% and 78% the cell viability of 20 assays, respectively. As such, this combination was selected for further cell cycle distribution studies. µ M cisplatin-treated cells for thethese Altogether, CV and MTS results assays,that suggest respectively. As such, this combination for all the concentrations and endpoints was selected tested, for further a synergistic cell effect cyclepresent. was distribution studies. Altogether, these results suggest that for all the concentrations and endpoints tested, a synergistic effect was present. 18.5% (p < 0.05), 22.8% (p < 0.05) and 12.4% (p < 0.01), respectively, for the CV assay, and 17.1% (p < 0.05) for the MTS assay. Considering the relative decreases in cell viability observed, the concentration of E3330 at 30 µ M reduced in 36% and 78% the cell viability of 20 µ M cisplatin-treated cells for the CV and MTS assays, respectively. As such, this combination was selected for further cell cycle distribution Antioxidants 2020, 9, 550 studies. Altogether, these results suggest that for all the concentrations and 8 of 18 endpoints tested, a synergistic effect was present. Figure 4. Impact of E3330 on the viability of H1975 cells treated with cisplatin. Cells were pre-incubated with E3330 Figure 4. (30 µM)offor Impact 3 h and E3330 thenviability on the simultaneously of H1975 exposed to E3330 cells treated with and cisplatin cisplatin. Cells(5–20 pre-for wereµM) 72incubated h. The effects in terms of cell viability were evaluated using the CV staining assay (A) with E3330 (30 µ M) for 3 h and then simultaneously exposed to E3330 and cisplatin (5–20 and MTS reduction assay (B). Values represent mean ± SD (n = 3–5) and are expressed as percentages relative µ M) for 72 h. The effects in terms of cell viability were evaluated using the CV staining assay (A) and to vehicle-treated control cells. * p < 0.05 and ** p < 0.01 relative to respective cisplatin-treated cells (Student’s t-test). 3.4. Effect of the Combination of E3330 and Cisplatin in Cell Cycle Distribution and Cell Death Since cytotoxicity is frequently accompanied by cell cycle arrest and/or cell death, the impact of E3330 and cisplatin combination in H1975 cells was evaluated by cell DNA content using PI staining for flow cytometry (Figure 5A,B). As expected, the exposure to cisplatin (20 µM, 72 h) alone substantially increased (around five-fold) the sub-G1 population and lead to a decrease in the G0/G1 population when compared to vehicle-treated control cells. E3330 (30 µM, 72 h) did not significantly modify the cell cycle distribution of vehicle-treated or cisplatin-treated cells. In fact, the cell cycle distribution of cisplatin alone Antioxidants 2020,or 9, xcisplatin FOR PEERwith E3330 remained similar. G2/M population maintained unaltered for REVIEW 9 ofall 18 the conditions tested. Figure 5. Cont. Antioxidants 2020, 9, 550 9 of 18 Figure 5. Cell cycle progression and apoptosis of H1975 cells treated with E3330 and/or cisplatin. Cells Figure were 5. Cell cyclewith pre-incubated progression E3330 (30andµM)apoptosis for 3 h of andH1975 then cells treated cisplatin (20 with µM) E3330 and/or was added forcisplatin. Cells co-incubation were pre-incubated with E3330 (30 µ M) for 3 h and then cisplatin (20 µ M) was added for for further 72 h. After this exposure period, cell DNA content analysis with PI staining was performed co-incubation byforflow further 72 h. After cytometry. (A)this exposure period, Representative flowcell DNA content cytometry analysis(B) histograms. with PI staining Sub-G1, wasS,performed G0/G1, and G2/M by flow cytometry. populations’ summary (A)results. Representative flow cytometry The percentage histograms. of apoptotic cells was(B) Sub-G1, G0/G1, determined by PI S, and and G2/M Annexin Vpopulations’ staining after summary the sameresults. The percentage incubation profile as inof the apoptotic cell DNAcellscontent was determined analysis. by (C)PIRepresentative and Annexin V staining flow afterdot-plots. cytometry the same incubation profile (D) Percentage of as in thecells, viable cell cells DNAundergoing content analysis. early (C) andRepresentative late apoptosis, and necrotic cells summary results. (E) Summary results demonstrate the percentage oflate flow cytometry dot-plots. (D) Percentage of viable cells, cells undergoing early and apoptosis, apoptotic cells (Annexin V positive cells). Values represent mean ± SD (n = 3), * p < 0.05 (one-way ANOVA cells and necrotic cells summary results. (E) Summary results demonstrate the percentage of apoptotic with (Annexin Tukey’s V positive cells). Values represent mean ± SD (n = 3), *p < 0.05 (one-way ANOVA with test). Tukey’s test). The induction of apoptosis was analyzed by flow cytometry after staining with Annexin V-FITC and PI (Figure 5C–E). Representative graphs obtained by flow cytometry are displayed in Figure 5C. Incubation with cisplatin (20 µM, 72 h) alone led to a ~3-fold increase in the % of apoptotic cells when compared to vehicle-treated cells (Figure 5E; ~26% vs 9%, respectively), which is in line with the observed increase in the sub-G1 population. Moreover, although the exposure to E3330 did not significantly alter the % of apoptotic cells in cisplatin-treated cultures, a small trend towards a synergistic effect was observed (Figure 5E). 3.5. E3330 in Combination with Cisplatin Reduces Both Collective and Chemotactic Cell Migration Considering an impairment in cell viability would interfere with possible results in cell migration and invasion processes, it was necessary to ascertain that non-toxic concentrations of E3330 and cisplatin were used. As such, the H1975 cells were exposed to a range of low concentrations of either cisplatin (0.1–5 µM) or E3330 (10–30 µM) for 24 h in order to select the conditions of migration/invasion assays. Accordingly, in these experiments, a complete culture medium with 2% FBS was used. The effect of cisplatin was assessed using the CV assay (Figure 6A). Cell viability was not markedly affected up to 2.5 µM (~10% reduction). At a concentration of 5 µM, this decrease reached around 17%. As for E3330, Considering an impairment in cell viability would interfere with possible results in cell migration and invasion processes, it was necessary to ascertain that non-toxic concentrations of E3330 and cisplatin were used. As such, the H1975 cells were exposed to a range of low concentrations of either cisplatin (0.1–5 μM) or E3330 (10–30 μM) for 24 h in order to select the conditions of migration/invasion Antioxidants 2020, 9, 550 assays. Accordingly, in these experiments, a complete culture medium with 10 2%of 18 FBS was used. The effect of cisplatin was assessed using the CV assay (Figure 6A). Cell viability was not markedly affected up to 2.5 µ M (~10% reduction). At a concentration of 5 μM, this decrease cell viability reached started around 17%.to As be for affected E3330,atcell theviability concentration of be started to 20 affected µM with atathe reduction of 12% concentration ofin 20the μMCV assay with a reduction of 12% in the CV assay (Figure 6B), although in the MTS assay this concentration6C). (Figure 6B), although in the MTS assay this concentration level was not cytotoxic (Figure Overall, considering level was both(Figure not cytotoxic assays 6C). and Overall, compounds, we decided considering both to selectand assays thecompounds, representative wenon-cytotoxic decided to concentrations of 1 µM fornon-cytotoxic select the representative cisplatin andconcentrations 10 µM for E3330of 1for μMtheformigration and10 cisplatin and invasion μM for assays. E3330 for the migration and invasion assays. Figure 6. Viability of H1975 cells exposed to low concentrations of cisplatin or E3330 in culture medium Figure with 6. Viability 2% FBS. ofofH1975 (A) Effect cells cisplatin exposed (0.1–5 µM; 24to h) low onconcentrations cell viability, inofthe cisplatin presence orofE3330 in culture 2% FBS, evaluated medium with 2% FBS. (A) Effect of cisplatin (0.1–5 μM; 24 h) on cell viability, in the presence by the CV assay. Effect of E3330 (10–30 µM; 24 h) on cell viability in the presence of 2% FBS evaluated of 2% FBS, evaluated by the CV assay. Effect of E3330 (10–30 μM; 24 h) on cell viability in the presence of by both CV (B) and MTS assays (C). Values for cell viability represent mean ± SD (n = 4–7) and are 2% FBS evaluated by both CV (B) and MTS assays (C). Values for cell viability represent mean ± SD expressed as percentages relative to vehicle-treated control cells. (n = 4–7) and are expressed as percentages relative to vehicle-treated control cells. Metastases development comprises multiple biological mechanisms, including an increase in Metastases development comprises multiple biological mechanisms, including an increase in cell motility. For this reason, after the selection of non-cytotoxic concentrations of both E3330 and cell motility. For this reason, after the selection of non-cytotoxic concentrations of both E3330 and cisplatin, the migration capacity of H1975 cells was evaluated by resorting to two mechanistically cisplatin, the migration capacity of H1975 cells was evaluated by resorting to two mechanistically different methods. Firstly, collective cell migration was assessed with the wound-healing assay as different methods. Firstly, collective cell migration was assessed with the wound-healing assay as an an evaluation of the cells’ movement across a horizontal surface with the conservation of functional evaluation of the cells’ movement across a horizontal surface with the conservation of functional cell– cell–cell junctions cell junctions (Figure (Figure 7A). 7A). Both Both E3330 E3330 andand cisplatin cisplatin alone alone diddid notnot demonstrate demonstrate an an effect effect on on wound wound closure. closure.Importantly, Importantly, their their combination significantlyreduced combination significantly reducedthisthisclosure closureinin about about 20%20%(p (p < 0.05) < 0.05) when compared to vehicle-treated control cells. This decrease was also statistically when compared to vehicle-treated control cells. This decrease was also statistically significant whensignificant when comparing the combination of both compounds with cisplatin-treated cells comparing the combination of both compounds with cisplatin-treated cells (p < 0.01). (p < 0.01). Microphotographs were also taken at a timepoint Microphotographs of 8 hatofaco-incubation, were also taken timepoint of 8 halready revealing aalready of co-incubation, slight revealing impairment for the a slight Antioxidants 2020, 9, x FOR PEER REVIEW 11 of 18 combinatory impairmentcondition (data not shown). for the combinatory condition (data not shown). For the determination of chemotactic individual cell migration, the transwell assay was performed (Figure 7C) as a measurement of the ability of single cells to directionally respond to a chemoattractant gradient. In this assay, E3330 and cisplatin alone also did not influence chemotactic migration, but again their combination reduced this type of migration in approximately 12% (p < 0.05) when compared to vehicle-treated control cells. The decrease observed was also statistically significant when compared to cisplatin-treated H1975 cells (p < 0.05). Representative images of the wound-healing assay and the chemotaxis migration assay are presented in Figure 7B,D, respectively. Figure 7. Cont. Figure 7. E3330 effect on collective and chemotactic migration of H1975 cells exposed to cisplatin. Collective cell migration was evaluated by the wound-healing assay (A) and chemotaxis was Antioxidants 2020, 9, 550 11 of 18 Figure 7. E3330 effect on collective and chemotactic migration of H1975 cells exposed to cisplatin. Figure Collective 7. migration cell E3330 effect wason collective evaluated byand chemotactic migration the wound-healing assay (A)ofand H1975 cells exposed chemotaxis to cisplatin. was measured usingCollective a transwell cell migration assay was evaluated (C). Representative by the wound-healing microscopy assay (A) andassay images of the wound-healing chemotaxis (40×, B) was measured and the usingassay chemotaxis a transwell assaycells (migrating (C).stained Representative microscopy with crystal imagesD). violet—100×, of Scale bars = 200 µm.assay the wound-healing Values for the wound-healing assay represent mean ± SD (n = 3) and are expressed as percentage (40×, B) and the chemotaxis assay (migrating cells stained with crystal violet – 100×, D). Scaleofbars = wound200closure, μm. Values for the calculated wound-healing relative to the initial width; * p < 0.05 and assay represent mean** p±