Synergistic Growth Inhibition of HT-29 Colon and MCF-7 Breast Cancer Cells with Simultaneous and Sequential Combinations of Antineoplastics and CNS Drugs

Home , Benzatropine, Fluphenazine, Imipramine, Latrepirdine, Mibefradil, Nepicastat

International Journal of Molecular Sciences

Article Synergistic Growth Inhibition of HT-29 Colon and MCF-7 Breast Cancer Cells with Simultaneous and Sequential Combinations of Antineoplastics and CNS Drugs

Diana Duarte 1,2 , Armando Cardoso 3,4 and Nuno Vale 1,5,*

1 OncoPharma Research Group, Center for Health Technology and Services Research (CINTESIS), Rua Doutor Plácido da Costa, 4200-450 Porto, Portugal; [email protected] 2 Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal 3 NeuroGen Research Group, Center for Health Technology and Services Research (CINTESIS), Rua Doutor Plácido da Costa, 4200-450 Porto, Portugal; [email protected] 4 Unit of Anatomy, Department of Biomedicine, Faculty of Medicine, University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal 5 Department of Community Medicine, Health Information and Decision (MEDCIDS), Faculty of Medicine, University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal * Correspondence: [email protected]

Abstract: Several central nervous system (CNS) drugs exhibit potent anti-cancer activities. This study aimed to design a novel model of combination that combines different CNS agents and antineoplastic drugs (5-fluorouracil (5-FU) and paclitaxel (PTX)) for colorectal and breast cancer therapy, respectively. Cytotoxic effects of 5-FU and PTX alone and in combination with different CNS agents were evaluated on HT-29 colon and MCF-7 breast cancer cells, respectively. Three antimalarials alone and in combination with 5-FU were also evaluated in HT-29 cells. Different Citation: Duarte, D.; Cardoso, A.; schedules and concentrations in a fixed ratio were added to the cultured cells and incubated for Vale, N. Synergistic Growth Inhibition 48 h. Cell viability was evaluated using MTT and SRB assays. Synergism was evaluated using the of HT-29 Colon and MCF-7 Breast Chou-Talalay, Bliss Independence and HSA methods. Our results demonstrate that fluphenazine, Cancer Cells with Simultaneous and fluoxetine and benztropine have enhanced anticancer activity when used alone as compared to being Sequential Combinations of used in combination, making them ideal candidates for drug repurposing in colorectal cancer (CRC). Antineoplastics and CNS Drugs. Int. J. Mol. Sci. 2021, 22, 7408. https:// Regarding MCF-7 cells, sertraline was the most promising candidate alone for drug repurposing, with doi.org/10.3390/ijms22147408 the lowest IC50 value. For HT-29 cells, the CNS drugs sertraline and thioridazine in simultaneous combination with 5-FU demonstrated the strongest synergism among all combinations. In MCF-7 Academic Editor: Maria E. Mycielska breast cancer cells, the combination of fluoxetine, fluphenazine and benztropine with PTX resulted

in synergism for all concentrations below IC50. We also found that the antimalarial artesunate Received: 2 July 2021 administration prior to 5-FU produces better results in reducing HT-29 cell viability than the inverse Accepted: 8 July 2021 drug schedule or the simultaneous combination. These results demonstrate that CNS drugs activity Published: 10 July 2021 differs between the two selected cell lines, both alone and in combination, and support that some CNS agents may be promising candidates for drug repurposing in these types of cancers. Additionally, Publisher’s Note: MDPI stays neutral these results demonstrate that 5-FU or a combination of PTX with CNS drugs should be further with regard to jurisdictional claims in evaluated. These results also demonstrate that antimalarial drugs may also be used as antitumor published maps and institutional afﬁl- agents in colorectal cancer, besides breast cancer. iations.

Keywords: colorectal cancer; breast cancer; drug synergism; antineoplastic drugs; drug repurposing; CNS drugs; combination therapy

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and conditions of the Creative Commons Cancer represents a major health problem worldwide and is the second leading cause Attribution (CC BY) license (https:// of death in the United States of America (USA). In 2021, there were an estimated 1,898,160 creativecommons.org/licenses/by/ new cancer cases and 608,570 cancer deaths in the USA. Colorectal cancer (CRC) represents 4.0/). the second leading cause of death by cancer in the USA, and in 2021, there were an

Int. J. Mol. Sci. 2021, 22, 7408. https://doi.org/10.3390/ijms22147408 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 7408 2 of 39

estimated 149,500 newly diagnosed cases and 52,980 deaths caused by this type of cancer. Of these, 17,930 new cases and 3640 deaths occurred in people under the age of 50. Breast cancer represents the second leading cause of death by cancer among women, with an estimated 281,550 new cases and 43,600 deaths in 2021 in the USA [1]. Although surgery and chemotherapy play a major role in the treatment of CRC, the efficacy rate remains very low. The development of new drugs for cancer therapy is, therefore, urgent, but this process is time-consuming, costly and has low approval rates [2]. Additionally, the majority of the new chemotherapeutics have problems related to toxicity, leading to side effects [3]. Thus, it is important to develop and explore novel pharmaceutical strategies to overcome the obstacles associated with the development of new drugs for cancer therapy. Drug repurposing (or repositioning) and drug combination are two strategies that have gained the attention of many research groups in recent years. Drug repurposing is a strategy that uses drugs that are already approved by the Food and Drug Administration (FDA) in new therapeutic indications besides the original. This strategy presents advantages concerning the development of new drugs, since repurposed drugs are already approved by the FDA and have known safety and toxicity profiles. This allows saving time and money, increasing the likelihood of these drugs entering clinical trials [4]. The drug combination is a strategy that consists of the administration of a cocktail of two or more drugs [5]. This methodology allows overcoming the intratumoral and intertumoral heterogeneity. Intratumoral heterogeneity results from the differential drug response between the different cells of the same tumour, contributing to the progression of the disease and the appearance of drug resistance. The intertumoral heterogeneity corresponds to the heterogeneity between patients with the same type of cancer and makes it difficult to predict the response of different patients to the same therapy [6]. Combination therapies help to overcome these problems and several studies have demonstrated that they are indeed more effective than monotherapy [7–12]. The efficacy of the drug combination depends on the schedule of administration (e.g., simultaneous or sequential) and on the design of the combination models [13,14], to make the most of the interaction between the drugs. Pharmacologically, a combination of two or more drugs will be more effective the greater the synergism between the drugs, i.e., the greater the potentiation of its effectiveness compared to the two drugs alone [15]. The repurposing of central nervous system (CNS) drugs has been explored, and several studies demonstrate the effectiveness of this class of drugs in reducing the viability of tumour cells [16–20]. CNS drugs can be divided into three main classes: antipsychotics, antidepressants and anticonvulsants. Antipsychotics and antidepressants can be subdivided according to their mechanism of action in tricyclic antidepressants (TCA), monoamine oxidase inhibitors (MAOI), selective serotonin reuptake inhibitors (SSRI), serotonin and norepinephrine reuptake inhibitors (SNRI), norepinephrine and dopamine reuptake inhibitors (NDRI) and atypical antidepressants [20]. Several CNS drugs have demonstrated potential for drug repurposing, such as imipramine, phenothiazines, trifluoperazine, pimozide and valproic acid. Imipramine, for example, has been studied in different types of cancer, such as glioma [19], breast [21], head and neck carcinoma [22], acute/chronic myeloid leukaemia [23,24], etc. Phenothiazines, a conventional antipsychotic drug family, the members of which work mainly as dopamine D2 antagonists, have also been studied in breast cancer [25], small cell lung carcinoma [18] and oral cancer [26]. Trifluoperazine, an FDA-approved phenothiazine and a D2 receptor antagonist, was already studied in glioblastoma [27] and lung cancer [28], among others. Pimozide, another D2 blocking agent used for Tourette’s Disorder, can fight cancer cells, including the apoptotic effects in cancer cells and the decreased expression of Bcl-2 [29]. Valproate (Valproic acid) is an anti-epileptic drug that blocks Na+ channels, GABA transaminase and Ca2+ channels. This drug is used in epilepsy, migraine seizures and acute manic episodes. Several studies suggest its beneficial role in fighting lymphoma [30], prostate [31] and breast cancer [32] and bladder [33] and hepatocellular carcinoma [34], among others. Int. J. Mol. Sci. 2021, 22, 7408 3 of 39

In this work, we hypothesised that different CNS agents (Scheme1A and Table1 ) could synergistically act with 5-fluorouracil (5-FU) and paclitaxel (PTX) in the CRC and breast cancer treatments, respectively. 5-FU is an antineoplastic drug commonly used in CRC therapy, but its use has several limitations, including its short half-life, high cytotoxicity, and low bioavailability [35]. PTX is a chemotherapeutic agent that is a mitotic inhibitor, used for the treatment of advanced carcinoma of the ovary, and other various cancers including breast and lung cancer. PTX use is limited by the appearance of drug resistance and its side effects [36]. This combination model consists of the combination of an antineoplastic drug and different repurposed drugs, and aims to improve the activity of the reference drug and simultaneously reduce its therapeutic dose, by using drugs with acceptable toxicological profiles. Recently, our group also developed a new combination model using different antimalarials and antineoplastic drugs in MCF-7 breast cancer cells [37]. Several antimalarials have been combined with doxorubicin and paclitaxel, two antineoplastic agents commonly used in breast cancer therapy. The results were very promising, and it was found that the best combinations corresponded to the antimalarials mefloquine, chloroquine and artesunate [37]. Although the relationship between (familial) breast cancer and colorectal cancer is a controversial subject, recently, it was discovered that rare mutations in the NTHL1 gene, which was originally associated with CRC, also cause breast cancer [38]. For this reason, we decided to also include these antimalarials (Scheme1B) in this study to confirm the anti-tumoral activity of this class of drugs in the HT-29 colon cancer cells. We have demonstrated that the combination of 5-FU and some antimalarials also induces anti-tumour effects in HT-29 cells. Interestingly, for artesunate, we discovered that the drug schedule influences the anticancer effect of this combination, being greater when artesunate is given before 5-FU to HT-29 cells. We demonstrate that some CNS drugs such as fluphenazine, fluoxetine and benztropine work better alone than in combination with 5-FU to reduce the viability of HT-29 colon cancer cells. In MCF-7 cells, sertraline was the most promising repurposed drug when used alone, with the lowest IC50 value. Compared to HT-29 cells, the IC50 obtained for the tested CNS drugs was higher in breast cancer cells, demonstrating a better efficacy of these drugs in CRC. We also found that the combination of 5-FU with sertraline and thioridazine induces a greater anti-tumour effect compared to each drug alone in these cells. In combination, results for MCF-7 were more promising than in HT-29 cells, with the combinations of PTX with fluoxetine, benztropine and fluphenazine resulting in a higher number of synergistic pairs. Int. J. Mol. Sci. 2021, 22, 7408 4 of 39 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 39

Scheme 1. Chemical structur structureses of of the the drugs drugs used used in in combination combination.. (A (A) CNS) CNS drugs: drugs: (1 ()1 selegiline,) selegiline, (2) ( 2safinamide,) safinamide, (3) ( 3entaca-) enta- pone,capone, (4) ( 4tolcapone,) tolcapone, (5 ()5 latrepirdine,) latrepirdine, (6 ()6 fluphenazine,) fluphenazine, (7 ()7 )thioridazine, thioridazine, (8 (8) )fluoxetine, fluoxetine, ( (99)) benztropine, benztropine, ( (1010)) carbidopa, carbidopa, ( (11)) bromocriptine, ((1212)) nepicastat,nepicastat, ((1313)) scopolamine,scopolamine, ( 14(14)) carbamazepine, carbamazepine, ( 15(15)) sertraline sertraline and and (16 (16) rivastigmine.) rivastigmine (.B (B) Antimalarial) Antimalar- ial drugs: (18) mefloquine, (19) chloroquine and (20) artesunate. drugs: (18) mefloquine, (19) chloroquine and (20) artesunate. Table 1. CNS drugs used in this work and their mechanism of action.

Drug Mechanism of Action Ref. Irreversible inhibitor of monoamine oxidase type B (MAO-B). Binds to MAO-B and blocks the microsomal metabolism of dopamine, enhancing Selegiline [39] the dopaminergic activity in the substantial nigra. It can also inhibit monoamine oxidase type A (MAO-A). Reversible inhibitor of MAO-B; blocks voltage-dependent Na+ and Ca2+ Safinamide [40] channels and inhibits the glutamate release. Entacapone Int. J. Mol. Sci. 2021, 22, 7408 5 of 39

Table 1. CNS drugs used in this work and their mechanism of action.

Drug Mechanism of Action Ref. Irreversible inhibitor of monoamine oxidase type B (MAO-B). Binds to MAO-B and blocks the microsomal metabolism of dopamine, Selegiline [39] enhancing the dopaminergic activity in the substantial nigra. It can also inhibit monoamine oxidase type A (MAO-A). Reversible inhibitor of MAO-B; blocks voltage-dependent Na+ and Safinamide [40] Ca2+ channels and inhibits the glutamate release. Adjunct to levodopa/carbidopa therapy and reversible inhibitor of Entacapone catechol-O-methyltransferase (COMT) in peripheral tissues, altering the plasma pharmacokinetics of levodopa. Tolcapone [41,42] Blocks H1 histamine receptor activity and interact with calcium Latrepirdine [43] channels and a wide range of other neurotransmitter receptors. Blocks postsynaptic mesolimbic dopaminergic D1 and D2 receptors Fluphenazine in the brain and depresses the release of hypothalamic and hypophyseal hormones Thioridazine [44,45] Selective serotonin reuptake inhibitor (SSRI); inhibits the presynaptic Fluoxetine reuptake of the neurotransmitter serotonin, increasing the levels of [46] 5-hydroxytryptamine (5-HT) in the brain. Selective inhibitor of dopamine transporter; presents affinity for Benztropine [47] histamine and M1 muscarine receptors. Inhibitor of the aromatic amino acid decarboxylase (DDC) and the Carbidopa [48] peripheral metabolism of levodopa. Bromocriptine Agonist of dopaminergic D2 and D3 receptors in the brain. [49] Nepicastat Inhibitor of Dopamine β-hydroxylase (DβH) [50] Non-selective competitive inhibitor of G-protein-coupled muscarinic Scopolamine [51] receptors (mAChRs), with anticholinergic action. Inhibits sodium channel firing, treating seizure activity. In bipolar Carbamazepine disorder, carbamazepine is thought to increase dopamine turnover [52] and increase GABA transmission. Selective inhibitor of serotonin reuptake at the presynaptic neuronal Sertraline [53] membrane, thereby increasing serotonergic activity. Binds reversibly with and inactivates cholinesterase (e.g., acetylcholinesterase and butyrylcholinesterase), preventing the Rivastigmine [54] hydrolysis of acetylcholine, and thus, leading to an increased concentration of acetylcholine at cholinergic synapses. Allosteric agonist and modulator of the 5-HT receptor and an m-chlorophenilbiguanide 3 [55,56] antagonist of the α2A-adrenergic receptor.

2. Results 2.1. HT-29 Colorectal Cancer Cells 2.1.1. The Effect of 5-FU as the Single Agent on Cellular Viability We analysed the anti-tumour potential of the antineoplastic drug 5-FU in the HT-29 colorectal cancer cell line, to confirm its efficacy in this type of cancer. Cells were treated with 5-FU in concentrations ranging 0.1–100 µM for 48 h and cell survival was evaluated by MTT, a viability assay that measures mitochondrial activity. The results of the MTT assay for 5-FU are given in Figure1A. Based on these results, a dose-response curve was obtained and the IC50 value for 5-FU was calculated (Figure1B). This value was further used in the combinations. Our results revealed a significant activity of 5-FU at concentrations above 10 µM, with little differences in cell viability among the higher concentrations. The cells displayed a mild response to the cytotoxic effect of 5-FU, with less than 4 µM killing almost 50% of cells. These results support the anti-cancer activity of 5-FU in the treatment of CRC and justify its use in the combinations proposed in this study. Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, 7408x FOR PEER REVIEW 66 ofof 3939

FigureFigure 1.1. The effect ofof 5-FU5-FU onon HT-29HT-29 cells.cells. ((AA)) CellCell viabilityviability andand ((BB)) dose-response.dose-response. CellsCells werewere culturedcultured inin thethe presencepresence ofof increasingincreasing concentrationsconcentrations ofof 5-FU,5-FU, andand afterafter 4848 h,h, thethe MTTMTT assayassay waswas performedperformed to measure thethe cellularcellular viability.viability. ValuesValues areare expressedexpressed inin percentagepercentage ofof controlcontrol andand representrepresent means means± ± SEM. Each experiment was done three times independently ((nn == 3) ****** statisticallystatistically signiﬁcantsignificant vs.vs. controlcontrol atat pp << 0.001. **** statistically signiﬁcantsignificant vs. control at p < 0.0001.

2.1.2. The Effect of CNS Drugs and Antimalarial DrugsDrugs asas SingleSingle AgentsAgents onon Cellular Via- Cellularbility Viability We nextnext evaluatedevaluated thethe probableprobable antitumorantitumor effecteffect ofof differentdifferent CNSCNS drugsdrugs asas singlesingle agents, namely selegiline, entacapone, tolcapone, latrepirdine, fluphenazine,fluphenazine, safinamide,safinamide, carbidopa, scopolamine, benztropine, thioridazine, fluoxetine, fluoxetine, nepicastat and bromocrip- tinetine inin HT-29HT-29 coloncolon cancercancer cells.cells. InIn thisthis study,study, wewe havehave alsoalso includedincluded threethree antimalarialantimalarial drugs (mefloquine, (mefloquine, chloroquine chloroquine and and artesunate) artesunate) based based on onour our previous previous results results [37], [ 37to ],con- to confirmfirm if these if these drugs drugs would would maintain maintain their their anti anti-cancer-cancer a activityctivity in in another another cell cell line besidesbesides thethe MCF-7MCF-7 breastbreast cancercancer cells.cells. HT-29HT-29 cellscells werewere treatedtreated withwith increasingincreasing concentrationsconcentrations ofof each drug, starting from 11 µµMM toto 100100 µµMM toto evaluateevaluate cellcell viabilityviability afterafter 4848 hh ofof treatment.treatment. Based on on the the MTT MTT results, results, we we found found that that latrepirdine, latrepirdine, fluphenazine, fluphenazine, fluoxetine, fluoxetine, benztropine,benztropine, thioridazine, thioridazine, sertraline, sertraline, mefloquine mefloquine and artesunateand artesunate displayed displayed significant significant anti- tumouranti-tumour activity activity in HT-29 in HT cells.-29 Cytotoxiccells. Cytotoxic effects effects of latrepirdine of latrepirdine (Figure (Figure2A) were 2A) significant were sig- evennificant in concentrationseven in concentrations of 1 µM, withof 1 µM 7.75, µwithM causing 7.75 µM a reductioncausing a ofreduction more than of 50%more of than the cells50% (Figureof the cells2B). (Figure Fluphenazine 2B). Fluphenazine anti-tumour anti effect-tumo wasu ther effect strongest was the among strongest all drugs among tested all alonedrugs andtested concentrations alone and concentrations above 10 µM killed above almost 10 µM all killed cells (Figurealmost2 allC). cells Indeed, (Figure the IC 2C).50 obtained for fluphenazine was the lowest and it was less than 2 µM (Figure2D). The MTT Indeed, the IC50 obtained for fluphenazine was the lowest and it was less than 2 µM (Fig- assayure 2D). for The fluoxetine MTT assay treatment for fluoxetine demonstrated treatment a strongdemonstrated cytotoxic a strong effect ofcytotoxic this CNS effect drug of µ inthis HT-29 CNS cellsdrug for in allHT concentrations-29 cells for all testedconcentrations above 10 testedM (Figure above2 10E). µM The (Figure dose-response 2E). The curve for fluoxetine revealed an IC value of 6.12 µM (Figure2F). Benztropine showed dose-response curve for fluoxetine revealed50 an IC50 value of 6.12 µM (Figure 2F). Benztro- µ significantpine showed anti-tumour significant effectsanti-tumo in concentrationsur effects in concentrations above 10 M above (Figure 102G) µM and (Figure the IC 2G)50 value obtained was 18.23 µM. Thioridazine treatment also significantly decreased HT-29 and the IC50 value obtained was 18.23 µM. Thioridazine treatment also significantly de- cell viability, from 1 µM to 100 µM, with strong effects for all concentrations above 5 µM creased HT-29 cell viability, from 1 µM to 100 µM, with strong effects for all concentra- (Figure3A) and an IC 50 value of 4.26 µM (Figure3B). Treatment with sertraline at doses tions above 5 µM (Figure 3A) and an IC50 value of 4.26 µM (Figure 3B). Treatment with above 1 µM for 48 h had a strong effect on the cell viability (Figure3C), resulting in an IC sertraline at doses above 1 µM for 48 h had a strong effect on the cell viability (Figure 3C),50 value of less than 3 µM (Figure3D). All concentrations of the antimalarial drug mefloquine resulting in an IC50 value of less than 3 µM (Figure 3D). All concentrations of the antima- above 10 µM showed a strong effect on the cell viability of HT-29 cells, with more than 50% larial drug mefloquine above 10 µM showed a strong effect on the cell viability of HT-29 of the cells being not viable (Figure3E). The dose-response curve resulted in a value of cells, with more than 50% of the cells being not viable (Figure 3E). The dose-response 11.49 µM for the IC50 (Figure3F). Artesunate, another antimalarial drug, also demonstrates curve resulted in a value of 11.49 µM for the IC50 (Figure 3F). Artesunate, another antima- good efficacy against these cells in all concentrations above 10 µM (Figure3G) and an IC larial drug, also demonstrates good efficacy against these cells in all concentrations above50 value under 20 µM (Figure3H). MTT assays for the others CNS drugs and chloroquine 10 µM (Figure 3G) and an IC50 value under 20 µM (Figure 3H). MTT assays for the others demonstrate a lack of efficacy of these drugs on the reduction of HT-29 cell viability or IC50 aboveCNS drugs 20 µM and and chloroquine were discarded demonstrate from the a druglack of combinations. efficacy of these These drugs results on the demonstrate reduction thatof HT both-29 CNScell viability agents and or IC antimalarial50 above 20 drugsµM and are were good discarded candidates from for usethe indrug combination combinations. These results demonstrate that both CNS agents and antimalarial drugs are good with 5-FU. Table2 summarises the IC 50 obtained for all drugs tested alone in this work. candidates for use in combination with 5-FU. Table 2 summarises the IC50 obtained for all drugs tested alone in this work. Int. J. Mol. Sci. 2021, 22, 7408 7 of 39 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 39

FigureFigure 2. The 2. effects The effects of some of some CNS CNS drugs drugs on on HT-29 HT-29 cells. cells. ((AA)) TheThe e effectffect of of latrepirdine latrepirdine onon cell cell viability viability and and(B) the (B )dose the- dose- responseresponse curve. curve. (C) The (C) effectThe effect of fluphenazine of fluphenazine on on cell cell viability viability andand (D) the the dose dose-response-response curve. curve. (E) (TheE) The effect effect of fluoxetine of fluoxetine on cell viability and (F) the dose-response curve. (G) The effect of benztropine on cell viability and (H) the dose-response on cell viability and (F) the dose-response curve. (G) The effect of benztropine on cell viability and (H) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, the MTT assay was curve.performed Cells were to cultured measure inthe the cellular presence viability. of increasing Values are concentrationsexpressed in percentage of each of drug, control and and after represent 48 h, the means MTT ± assaySEM. was performed to measure the cellular viability. Values are expressed in percentage of control and represent means ± SEM. Each experiment was done three times independently (n = 3); ** statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 39

Int. J. Mol. Sci. 2021, 22, 7408 8 of 39 Each experiment was done three times independently (n = 3); statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.

FigureFigure 3. The 3.effects The effects of some of some CNS CNS drugs drugs and and antimalarial antimalarial drugsdrugs on HT HT-29-29 cells. cells. (A ()A The) The effect effect of thioridazine of thioridazine on cell on cell viabilityviability and (B and) the (B dose-response) the dose-response curve. curve. (C ()C The) The effect effect of of sertraline sertraline on cell cell viability viability and and (D ()D the)the dose dose-response-response curve. curve. (E) (E) The effect of mefloquine on cell viability and (F) the dose-response curve. (G) The effect of artesunate on cell viability and The effect of mefloquine on cell viability and (F) the dose-response curve. (G) The effect of artesunate on cell viability and (H) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, (H) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, the MTT assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means ± SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001. Int. J. Mol. Sci. 2021, 22, 7408 9 of 39

Table 2. Cytotoxicity of 5-FU, several CNS drugs and some antimalarials agents in HT-29 colon

cancer cells. IC50 values are given as mean.

Drug IC50 (µM) 5-Fluorouracil 3.79 ‡ Selegiline >100 Entacapone 40.89 Tolcapone 35.47 Latrepirdine 7.75 ‡ Fluphenazine 1.86 ‡ Saﬁnamide >100 Fluoxetine 6.12 ‡ Benztropine 18.23 ‡ Thioridazine 4.26 ‡ Carbidopa >100 Bromocriptine >100 Nepicastat 61.24 Scopolamine >100 Carbamazepine >100 Sertraline 2.45 ‡ Chloroquine 32.13 Meﬂoquine 11.49 ‡ Artesunate 17.88 ‡ Rivastigmine >100 m-Chlorophenilbiguanide >100 ‡ Drugs selected for combinations with 5-FU.

2.1.3. The Effect of Various Combinations of 5-FU and Different CNS Agents and Antimalarial Drugs After finding the best candidates for drug repurposing in CRC therapy and their IC50 value, we evaluated the combination of 5-FU with each drug using the model of combination developed in our previous work [37]. Specifically, HT-29 cells were treated with the two drugs alone or combined in a fixed ratio, in the concentrations of 0.25 × IC50, 0.5 × IC50, IC50, 2 × IC50 and 4 × IC50, and two cell-based assays were performed: MTT and SRB. Morphological evaluation of cells treated with each drug alone and in combination was also done. The most promising drugs for drug combination were selected according to their IC50 value. To do so, the combination of 5-FU and each drug of Table2 with an IC50 value under 20 µM was evaluated: latrepirdine, fluphenazine, fluoxetine, benztropine, thioridazine, sertraline, mefloquine and artesunate. When combined with 5-FU, latrepirdine did not have any significant anti-cancer effects, at any concentration, both by MTT and SRB assay (Figure4A,B, respectively). The combination with thioridazine resulted in a similar reduction of cell viability and cell protein synthesis as thioridazine alone, at concentrations higher than IC50. At a concentration of 4 × IC50, the combination of 5-FU plus thioridazine demonstrated enhanced but not significant anticancer effects than thioridazine (Figure4C,D). The combination with 5-FU and sertraline also demonstrated significant anticancer effects compared to 5-FU alone, at a higher concentration, for both assays (Figure4E,F). A small difference between sertraline and sertraline+5-FU is seen at the concentration of 4 × IC50, but this is not significant. The combination with mefloquine resulted in all concentrations showing a greater anticancer effect than 5-FU alone (Figure4G,H). The activity seen on these combinations can be the result of the strong anticancer activity of mefloquine alone. Morphologically, the results are in agreement with the MTT and SRB assays. At concentrations of 4 × IC50, all combinations resulted in a decrease of cell number and smaller and rounder cells, comparing with control cells and 5-FU, which is indicative of cell death (Figure5). In the combinations 5-FU plus fluphenazine, fluoxetine, benztropine and artesunate, we found out that for a concentration of 2 × IC50, the results of the combined drugs were worse than the repurposed drugs alone, demonstrating a kind of competition mechanism between the two drugs when Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 39

effect than 5-FU alone (Figure 4G,H). The activity seen on these combinations can be the result of the strong anticancer activity of mefloquine alone. Morphologically, the results are in agreement with the MTT and SRB assays. At concentrations of 4 × IC50, all combinations resulted in a decrease of cell number and smaller and rounder cells, comparing with control cells and 5-FU, which is indicative of cell death (Figure 5). In the combina- Int. J. Mol. Sci. 2021, 22, 7408 10 of 39 tions 5-FU plus fluphenazine, fluoxetine, benztropine and artesunate, we found out that for a concentration of 2 × IC50, the results of the combined drugs were worse than the repurposed drugs alone, demonstrating a kind of competition mechanism between the administeredtwo drugs when together, administered mainly intogether, the MTT mainly assays. in Additionally, the MTT assays. forthe Additionally higher concentra-, for the tionshigher (4 concentrations× IC50), the results (4 × IC obtained50), the results for the obtained combined for drugs the combined did not show drugs improvements did not show concerningimprovements the concerning repurposed the drugs repurposed alone (Figure drugs6 alone). Microscopically, (Figure 6). Micr atoscopically, concentrations at con- of 4centrations× IC50, differences of 4 × IC between50, differences cells were between only cells found were between only 5-FU,found control between cells 5- andFU, treatedcontrol cells;cells and differences treated betweencells; differences single drugs between and single drug combinationsdrugs and drug were combinations very subtle were and bothvery treatmentssubtle and resultedboth treatments in the decreasing resulted ofin cellthe number,decreasing less of aggregate cell number, formation less aggregate and rounded for- cellsmation (Figure and 7rounded). cells (Figure 7).

Figure 4. Cont. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 39 Int.Int. J.J. Mol.Mol. Sci. 2021,, 22,, 7408x FOR PEER REVIEW 1111 ofof 3939

FigureFigure 4. 4.Growth Growth inhibition inhibition ofof HT-29HT-29 after after 48 48 h h of of combination combinationtherapy, therapy, by by MTT MTT (left) (left) and and SRB SRB assays assays (right). (right). CellsCells werewere exposedFigureexposed 4. to toGrowth concentrations concentrations inhibition of of of each each HT drug -drug29 after of of 0.25, 0.25,48 h 0.5, 0.5,of combination 1, 1, 2 2 and and 4 4 times times therapy, their their ICby IC50 50MTTand and the(left)the cellcell and viabilityviability SRB assays waswas evaluated (right).evaluated Cells by by MTTwere MTT andexposedand SRB SRB assays.to assays. concentrations TheThe drugsdrugs of inin each combinationcombination drug of 0.25, werewere 0.5, co-administered co -1,administered 2 and 4 times at at the theirthe same same IC50 time. andtime. the ((AA )cell) The The viability effecteffect ofof was 5-FU5-FU evaluated plusplus latrepirdinelatrepirdine by MTT and SRB assays. The drugs in combination were co-administered at the same time. (A) The effect of 5-FU plus latrepirdine onon cell cell viability viability and and ( B(B)) cell cell protein protein synthesis. synthesis. ( C(C)) The The effect effect of of 5-FU5-FU plusplus thioridazinethioridazine on on cell cell viability viability and and ( D(D)) cell cell protein protein on cell viability and (B) cell protein synthesis. (C) The effect of 5-FU plus thioridazine on cell viability and (D) cell protein synthesis.synthesis. ((EE)) TheThe effecteffect ofof 5-FU5-FU plusplus sertralinesertraline on on cellcell viability viability and and ( F(F)) cell cell protein protein synthesis. synthesis. ( G(G)) The The effect effect of of 5-FU 5-FU plus plus synthesismefloquine. (E )on The cell effect viability of 5- FUand plus (H) sertralinecell protein on synthesis cell viability. Value ands are(F) cellexpressed protein in synthesis percentage. (G )of The control effect and of 5 represent-FU plus mefloquine on cell viability and (H) cell protein synthesis. Values are expressed in percentage of control and represent mefloquinemeans ± SEM. on Eachcell viability experiment and was (H) donecell protein three times synthesis independently. Values are (n expressed= 3); * statistically in percentage significant of control vs. control and atrepresent p < 0.05. means ± SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. means** statistically ± SEM. Eachsignificant experiment vs. control was doneat p < three0.01. ***times statistically independently significant (n = 3);vs. *control statistically at p < significant0.001. **** statisticallyvs. control atsignificant p < 0.05. ****vs. statisticallystatistically control at significantsignificantp < 0.0001. vs. vs. controlcontrol atatp p< < 0.01.0.01. *** statistically significantsignificant vs.vs. controlcontrol atat pp << 0.001. **** statistically significantsignificant vs.vs. control atat pp << 0.0001. A B A B

Control 5-FU Control 5-FU C D C D

Latrepirdine Latrepirdine + 5-FU Latrepirdine Latrepirdine + 5-FU E F E F

Thioridazine Thioridazine + 5-FU Thioridazine Thioridazine + 5-FU G H G H Figure 5. Cont.

Sertraline Sertraline + 5-FU Sertraline Sertraline + 5-FU I J I J

Mefloquine Mefloquine + 5-FU Mefloquine Mefloquine + 5-FU A B

A B

Control 5-FU

C D

Control 5-FU

C D

Latrepirdine Latrepirdine + 5-FU E F

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 12 of 39

Int. J. Mol. Sci. 2021, 22, 7408 12 of 39 Int. J. Mol. Sci. 2021, 22, x FOR PEERThioridazine REVIEW Thioridazine + 5-FU 12 of 39

G H Thioridazine Thioridazine + 5-FU

G H

Sertraline Sertraline + 5-FU

I Sertraline SertralineJ + 5-FU I J

MefloquineMefloquine MefloquineMefloquine + 5+-FU 5-FU

FigureFigureFigure 5. 5.Microscopic5. MicroscopicMicroscopic cellular cellular visuali visualisationssationation of of HT-29HT HT-29-29 cells cells afterafter after 4848 hh48 ofof h incubationincubation of incubation with with vehicle vehicle (A),), (A), 5-FU5-FU5-FU (B (),(BB latrepirdine),), latrepirdinelatrepirdine ( (C(CC),),), latrepirdine latrepirdinelatrepirdine + + + 5-FU5 5-FU-FU ((D D(D),), thioridazine),thioridazine thioridazine ((EE),), ( thioridazineEthioridazine), thioridazine + + 5-FU5-FU + 5 ((F-FFU),), sertralinesertraline (F), sertraline (G),(G sertraline), sertraline + +5 -5FU-FU ( H(H),), mefloquinemefloquine ((II)) and and mefloquine mefloquine + 5 +-FU 5-FU (J) at(J )concentrations at concentrations of 4 × of IC 450 ×of IC 50 of (G), sertraline + 5-FU (H), meﬂoquine (I) and meﬂoquine + 5-FU (J) at concentrations of 4 × IC50 of each drug. eacheach drug drug..

Figure 6. Cont. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 13 of 39 Int. J. Mol. Sci. 2021, 22, 7408 13 of 39

Figure 6. Growth inhibition of HT-29 after 48 h of combination therapy, by MTT (left) and SRB assays (right). Cells were Figure 6. Growth inhibition of HT-29 after 48 h of combination therapy, by MTT (left) and SRB assays (right). Cells were exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their IC50 and cell viability were evaluated by MTT and exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their IC50 and cell viability were evaluated by MTT SRB assays. The drugs in combination were co-administered at the same time. (A) The effect of 5-FU plus fluphenazine and SRB assays. The drugs in combination were co-administered at the same time. (A) The effect of 5-FU plus fluphenazine B C D on cellon viability cell viability and and (B) ( cell) cell protein protein synthesis. synthesis. ( (C) The effecteffect of of 5-FU 5-FU plus plus fluoxetine fluoxetine on cellon cell viability viability and (and) cell (D protein) cell protein synthesis.synthesis. (E) The (E) Theeffect effect of 5 of-FU 5-FU plus plus benztropine benztropine on on cell cell viability viability and and ( (FF)) cell cell protein protein synthesis.synthesis. ((GG)) The The effect effect of of 5-FU 5-FU plus artesunateplus artesunate on cell onviability cell viability and ( andH) cell (H) cellprotein protein synthesis. synthesis. Values Values are are expressedexpressed in in percentage percentage of control of control and representand represent ± meansmeans ± SEM.SEM. Each Each experiment experiment was was done done three three times times independently (n (=n 3);= 3); * statistically * statistically significant signific vs.ant control vs. control at p < 0.05.at p < 0.05. ** statistically** statistically significant significant vs. vs. control control at at pp << 0.01. 0.01. *** *** statistically significant significant vs. vs. control control at p at< 0.001.p < 0.001. **** statistically**** statistically significant significant vs. controlvs. control at pat < p0.0001.< 0.0001. 2.1.4. Synergistic Combinations of 5-FU and CNS Agents/Antimalarial Drugs 2.1.4. Synergistic Combinations of 5-FU and CNS Agents/Antimalarial Drugs To investigate the effects of the combinations of 5-FU with the previous drugs, and afterTo finding investigate the most the promisingeffects of onesthe combinations based on MTT of and 5-FU SRB with assays, the the previous combination drugs, and afterindex finding (CI) was the calculated most promising using the ones Chou-Talalay based on method, MTT usingand SRB the CompuSynassays, the software. combination indexCI was (CI) plotted was calculated on the y-axis using as a the function Chou of-Talalay effect level method, (Fa) on using the x the-axis CompuSyn to assess drug software. CIsynergism. was plotted The on fractional the y-axis effect as is a a function value between of effect 0 and level 1, where (Fa) 0 on means the thatx-axis the to drug assess did drug synergism.not affect cellThe viability fractional and effect 1 means is thata value the drugbetween produced 0 and a full1, where effect on0 means decreasing thatcell the drug didviability. not affect A combination cell viability of 5-FUand 1 plus means latrepirdine that the demonstrated drug produced little a synergism full effect with on decreasing only one synergic pair (Figure8A), with an Fa value of 0.44 (Table3). Both combinations of 5-FU cell viability. A combination of 5-FU plus latrepirdine demonstrated little synergism with plus fluoxetine and benztropine demonstrated synergism just for one pair (Figure8B,C, onlyrespectively), one synergic with pair Fa values (Figure of 0.738A), and with 0.87, an respectively Fa value of (Table 0.443 (Table). The combination 3). Both combinations with ofthioridazine 5-FU plus fluoxetine was one of theand most benztropi promisingne demonstrated ones, with three synergism synergic pairs just (Figure for one8D) pair and (Figure 8Ba and Fa value C, respectively), reaching 0.75 (Table with3 Fa). For values sertraline, of 0.73 all and combinations 0.87, respectively were synergic (Table (Figure 3). 8TheE) com- binationand produced with thioridazine a Fa value of was 0.85 one (Table of3 the). The most combination promising of ones, 5-FU andwith mefloquine three synergic also pairs (Figureresulted 8D) in oneand synergic a Fa value pair (Figurereaching8F), 0.75 with (Table an Fa value 3). For of 0.848sertraline, (Table3 ).all A combinations combination were synergicof artesunate (Figure and 8E) fluphenazine and produced with a 5-FU Fa value did not of result 0.85 (Table in any synergism3). The combination (Figure8G,H, of 5-FU andrespectively), mefloquine with also CI resulted > 1 for all in pairs one ofsynergic concentrations pair (Figure (Table 38F),). Together, with an these Fa value results of 0.848 demonstrate that some CNS agents, such as sertraline and thioridazine, may be promising (Table 3). A combination of artesunate and fluphenazine with 5-FU did not result in any to evaluate future combinations. synergism (Figure 8G and H, respectively), with CI > 1 for all pairs of concentrations (Ta- ble 3). Together, these results demonstrate that some CNS agents, such as sertraline and thioridazine, may be promising to evaluate future combinations. Int. J. Mol. Sci. 2021, 22, 7408 14 of 39 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 14 of 39

A B

Control 5-FU

C D

Fluphenazine Fluphenazine + 5-FU

E F

Fluoxetine Fluoxetine + 5-FU

G H

Benztropine Benztropine + 5-FU

I J

Artesunate Artesunate + 5-FU

FigureFigure 7. 7.Microscopic Microscopic cellular visuali visualisationsation of of HT HT-29-29 cells cells after after 48 48h of h ofincubation incubation with with vehicle vehicle (A), (A), 5-FU (B), fluphenazine (C), fluphenazine + 5-FU (D), fluoxetine (E), fluoxetine + 5-FU (F), benztro- 5-FU (B), fluphenazine (C), fluphenazine + 5-FU (D), fluoxetine (E), fluoxetine + 5-FU (F), benztropine pine (G), benztropine + 5-FU (H), artesunate (I) and artesunate + 5-FU (J) at concentrations of 4 × (G), benztropine + 5-FU (H), artesunate (I) and artesunate + 5-FU (J) at concentrations of 4 × IC of IC50 of each drug. 50 each drug. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 15 of 39

Besides the Chou-Talalay method, drug interactions were also evaluated by the Bliss Independence and Highest Single Agent (HSA) methods, using the SynergyFinder 2.0 software. This software is a web application for interactive analysis and visualisation of multi-drug combination profiling data by different synergism evaluation methods. The Bliss independence model assumes a stochastic process in which two drugs produce their effects independently, and the expected combination effect can be calculated based on the probability of independent events [57]. The HSA model is one of the simplest reference models for synergism evaluation and states that the expected combination effect is the maximum of the single drug responses at corresponding concentrations. In this software, the synergy score for a drug combination is averaged over all the dose combination measurements, giving a positive or negative value, corresponding to synergism or antagonism, Int. J. Mol. Sci. 2021, 22, 7408 15 of 39 respectively. The 2D and 3D synergy maps highlight synergistic and antagonistic dose regions in red and green colours, respectively [57].

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 16 of 39

FigureFigure 8. 8. ChouChou-Talalay-Talalay method method Fa Fa-CI-CI plot plot of of5-FU 5-FU plus plus latrepirdine latrepirdine (A), ( Afluoxetine), fluoxetine (B), (benztropineB), benztropine (C), thioridazine (C), thioridazine (D), sertraline(D), sertraline (E), mefloquine (E), mefloquine (F), artesunate (F), artesunate (G) and (G fluphenazine) and fluphenazine (H). CI was (H). plotted CI was on plotted the y-axis on theas a yfunction-axis as of a functioneffect level of (Fa)effect on level the x (Fa)-axis on to the evaluatex-axis drug to evaluate synergism. drug CI synergism. < 1, CI = 1 CI and < 1,CI CI > =1 1refers and CIto synergism, > 1 refers to additivity synergism, and additivity antagonism, and respectively.antagonism, respectively.

LatrepirdineBesides the Chou-Talalay in combination method, with 5 drug-FU, interactionsboth by Bliss were and also HSA evaluated models by(Figure the Bliss 9A andIndependence B, respectively) and, Highestdemonstrated Single a Agentnegative (HSA) synergy methods, score, in using line thewith SynergyFinder the Chou-Talalay 2.0 results,software. indicating This software antagonism is a web forapplication all pairs. Thioridazine for interactive demonstrated analysis and synergism visualisation by the of multi-drug combination proﬁling data by different synergism evaluation methods. The Bliss model, with a positive synergy score of 5.178 (Figure 9C). The results for the HSA Bliss independence model assumes a stochastic process in which two drugs produce their model demonstrated antagonism, but some regions of synergy, as represented in red in effects independently, and the expected combination effect can be calculated based on the Figure 9D. In line with the previous results, the combination of 5-FU with sertraline resulted in strong synergism, both in the Bliss (Figure 9E) and HSA models (Figure 9F), with synergy scores of 22.203 and 3.042, respectively. For mefloquine, no synergism was observed using the Bliss and HSA models (Figure 9G and H, respectively). By the Bliss model, fluphenazine combined with 5-FU resulted in a negative synergy score, demonstrating antagonism (Figure 10A). By the HSA models, the general synergy score was also negative but with a region in the 2D/3D plot demonstrating a pair of concentrations with synergic behaviour (Figure 10B). Fluoxetine and benztropine did not show any synergism in Bliss and HSA models, demonstrating an antagonistic behaviour between these drugs and 5-FU (Figure 10C–F). Contrary to the previous results obtained by the Chou-Talalay method, the combination of 5-FU plus artesunate as evaluated by the Bliss Method resulted in a positive synergy score of 0.411, with a red region on the 2D/3D plot in the lowest concentrations (Figure 10G). Using the HSA model, the synergy score was negative, demonstrating antagonism (Figure 10H). These results demonstrate that the choice of synergy evaluation model can give slightly different results regarding the synergy evaluation of drug combinations, although these reference models produce similar results most of the time.

Table 3. CI values and the respective fractional effect of different combinations of 5-FU plus CNS agents and antimalarial drugs. CI in red indicates concentrations of drug pairs that are synergic. Cells were treated with 0.25, 0.5, 1, 2 and 4 times the IC50 of each drug (total dose).

Combination Total Dose Fractional CI (Drug A + Drug B) (Dose A + Dose B) Effect (Fa) Value 2.75 0.14129 1.65359 5.5 0.16103 2.00449 5-FU + latrepirdine 11.0 0.17389 2.97604 22.0 0.21248 2.69396 44.0 0.44126 0.21700 2.25 0.1497 1.83049 4.5 0.1688 2.64547 5-FU + fluoxetine 9.0 0.364 1.36014 18.0 0.73678 0.98008 Int. J. Mol. Sci. 2021, 22, 7408 16 of 39

probability of independent events [57]. The HSA model is one of the simplest reference models for synergism evaluation and states that the expected combination effect is the maximum of the single drug responses at corresponding concentrations. In this software, the synergy score for a drug combination is averaged over all the dose combination measurements, giving a positive or negative value, corresponding to synergism or antagonism, respectively. The 2D and 3D synergy maps highlight synergistic and antagonistic dose regions in red and green colours, respectively [57]. Latrepirdine in combination with 5-FU, both by Bliss and HSA models (Figure9A,B, respectively), demonstrated a negative synergy score, in line with the Chou-Talalay results, indicating antagonism for all pairs. Thioridazine demonstrated synergism by the Bliss model, with a positive synergy score of 5.178 (Figure9C). The results for the HSA model demonstrated antagonism, but some regions of synergy, as represented in red in Figure9D. In line with the previous results, the combination of 5-FU with sertraline resulted in strong synergism, both in the Bliss (Figure9E) and HSA models (Figure9F), with synergy scores of 22.203 and 3.042, respectively. For meﬂoquine, no synergism was observed using the Bliss and HSA models (Figure9G,H, respectively). By the Bliss model, ﬂuphenazine combined with 5-FU resulted in a negative synergy score, demonstrating antagonism (Figure 10A). By the HSA models, the general synergy score was also negative but with a region in the 2D/3D plot demonstrating a pair of concentrations with synergic behaviour (Figure 10B). Fluoxetine and benztropine did not show any synergism in Bliss and HSA models, demonstrating an antagonistic behaviour between these drugs and 5-FU (Figure 10C–F). Contrary to the previous results obtained by the Chou-Talalay method, the combination of 5-FU plus artesunate as evaluated by the Bliss Method resulted in a positive synergy score of 0.411, with a red region on the 2D/3D plot in the lowest concentrations (Figure 10G). Using the HSA model, the synergy score was negative, demonstrating antagonism (Figure 10H). These results demonstrate that the choice of synergy evaluation model can give slightly different results regarding the synergy evaluation of drug combinations, although these reference models produce similar results most of the time.

Table 3. CI values and the respective fractional effect of different combinations of 5-FU plus CNS agents and antimalarial drugs. CI in red indicates concentrations of drug pairs that are synergic.

Cells were treated with 0.25, 0.5, 1, 2 and 4 times the IC50 of each drug (total dose).

Combination Total Dose Fractional CI (Drug A + Drug B) (Dose A + Dose B) Effect (Fa) Value 2.75 0.14129 1.65359 5.5 0.16103 2.00449 5-FU + latrepirdine 11.0 0.17389 2.97604 22.0 0.21248 2.69396 44.0 0.44126 0.21700 2.25 0.1497 1.83049 4.5 0.1688 2.64547 5-FU + ﬂuoxetine 9.0 0.364 1.36014 18.0 0.73678 0.98008 36.0 0.8799 1.09726 5.25 0.1984 1.94601 10.5 0.3913 1.17167 5-FU + benztropine 21.0 0.5293 1.27070 42.0 0.6237 1.67649 84.0 0.8716 0.74841 Int. J. Mol. Sci. 2021, 22, 7408 17 of 39

Table 3. Cont.

Combination Total Dose Fractional CI (Drug A + Drug B) (Dose A + Dose B) Effect (Fa) Value 1.75 0.00001 3.28032 3.5 0.0882 0.57237 5-FU + thioridazine 7.0 0.5245 0.63129 14.0 0.7502 0.99126 28.0 0.8692 1.63800 1.375 0.0001 0.91640 2.75 0.001 0.95924 5-FU + sertraline 5.5 0.1469 0.54137 11.0 0.4412 0.76907 22.0 0.8507 0.99970 3.625 0.2138 29.9325 7.25 0.5659 4.07663 5-FU + meﬂoquine 14.5 0.8481 0.68334 29.0 0.861 1.14685 58.0 0.873 1.92463 5.25 0.00001 1461.26 10.5 0.0782 1.27561 5-FU + artesunate 21.0 0.1521 1.35742 42.0 0.2489 1.62381 84.0 0.5575 1.07643 5.25 0.11608 11.6103 10.5 0.17321 13.0845 5-FU + ﬂuphenazine 21.0 0.4477 8.40182 42.0 0.5522 12.1699 84.0 0.8399 8.04387

2.1.5. The Effect of Different Combination Schedules of 5-FU and Different CNS Agents and Antimalarial Drugs Based on the MTT assay results (Figure6), the combination of 5-FU with fluphenazine, fluoxetine, benztropine and artesunate seem to demonstrate some kind of competition between the two drugs, with the results for the combination being worse than for the repurposed drugs alone. We design a new model of combination for these pairs of drugs and evaluated the influence of the drug schedule on HT-29. We hypothesise that if we administered the drugs at different times (sequential), the results would be better, due to non-competition between the two drugs. To do so, we tested three schedules (Figure 11): simultaneous administration (Schedule A), drug A prior drug B (Schedule B) and drug B prior drug A (Schedule C). We found out that for all CNS drugs, simultaneous administration produced better results in reducing cell viability than other schedules (Figure 12A–F). Interestingly, we found that all CNS drugs alone demonstrated better antitumor activity than in combination, indicating these drugs are ideal candidates for drug repurposing. For artesunate, we found out that the administration of artesunate prior to 5-FU produced better results than other drug schedules (Figure 12G,H). Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, 7408x FOR PEER REVIEW 1818 ofof 3939

Figure 9. Bliss (left) and HAS (right) synergy plots of 5-FU plus latrepirdine (A,B), thioridazine (C,D), sertraline (E,F) and Figure 9. Bliss (left) and HAS (right) synergy plots of 5-FU plus latrepirdine (A,B), thioridazine (C,D), sertraline (E,F) and meﬂoquine (G,H). mefloquine (G,H). Int.Int. J. J. Mol. Mol. Sci. Sci.2021 2021, 22, 22, 7408, x FOR PEER REVIEW 1919 of of 39 39

Figure 10. Bliss (left) and HAS (right) synergy plots of 5-FU plus ﬂuphenazine (A,B), ﬂuoxetine (C,D), benztropine (E,F) Figure 10. Bliss (left) and HAS (right) synergy plots of 5-FU plus fluphenazine (A,B), fluoxetine (C,D), benztropine (E,F) and artesunate (G,H). and artesunate (G,H). Int.Int. J. J. Mol. Mol. Sci. Sci. 20212021, ,2222, ,x 7408 FOR PEER REVIEW 2020 of of 39 39

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 20 of 39

FigureFigure 11. 11. TheThe three three combination combination schedules schedules designed designed for for evaluating evaluating 5 5-FU-FU combination combination with with fluphenazine,ﬂuphenazine,Figure 11. fluoxetine,The ﬂuoxetine, three combination benztropine benztropine schedules and and artesunate. artesunate. designed Schedule for Schedule evaluating A represents A 5represents-FU combination cells treated cells treated with concomi- con- fluphenazine, fluoxetine, benztropine and artesunate. Schedule A represents cells treated concomi- tantlycomitantly with 5 with-FU and 5-FU each and repurposed each repurposed drug for drug 48 h. for Schedule 48 h. Schedule B represents B represents cells pre cells-treated pre-treated with 5- FU tantlyfor 24 with h followed 5-FU and by each each repurposed repurposed drug drugfor 48 h.for Schedule another B 24represents h. For schedulecells pre-treated C, cells with were 5- pre- withFU 5-FU for 24 for h 24followed h followed by each by repurposed each repurposed drug for drug another for 24 another h. For 24schedule h. For C, schedule cells were C, pre cells- were treated with each repurposed drug for 24 h, followed by 5-FU for another 24 h. pre-treatedtreated with with each each repurposed repurposed drug drug for 24 for h, 24followed h, followed by 5-FU by for 5-FU another for another24 h. 24 h.

Figure 12. Cont.

Int. J. Mol.Int. J. Sci. Mol.2021 Sci., 202122, 7408, 22, x FOR PEER REVIEW 21 of 39 21 of 39

FigureFigure 12. HT-29 12. HT cells-29 cells were were exposed exposed to sequential to sequential 5-FU 5-FU and and (A ,(BA),B fluphenazine) fluphenazine (C (,CD,D) fluoxetine) fluoxetine( E(E,F,F)) benztropinebenztropine and (G,H) (G,H) artesunate using constant ratios of the IC50 dose. Values are expressed in percentage of control and represent means artesunate using constant ratios of the IC50 dose. Values are expressed in percentage of control and represent means ± SEM. ± SEM. Each experiment was done three times independently (n = 3). Each experiment was done three times independently (n = 3). 2.1.6. Synergism Evaluation of Different Combination Schedules of 5-FU and CNS 2.1.6.Agents Synergism/Antimalarial Evaluation Drugs of Different Combination Schedules of 5-FU and CNS Agents/Antimalarial Drugs Based on the previous results, we also analysed the drug interactions in these com- binationsBased to on evaluate the previous if thereresults, were differences we also in analysed the CI values the drug between interactions the three schedules in thesecombi- nationsof administration. to evaluate For if there fluphenazine, were differences there were in no the differences CI values between between the thesimultaneous three schedules ofadministration administration. and For the fluphenazine, sequential administration there were of no the differences drugs, and betweenall pairs were the simultaneousantago- administrationnists (Figure 13 andA). For the fluoxetine, sequential only administration one pair in the of simultaneous the drugs, and administration all pairs were was antago- nistssynergic (Figure, and 13 inA). sequential For fluoxetine, administration, only one no pairsynergism in the could simultaneous be seen, so administrationthe simultane- was synergic,ous combination and in sequential seems to be administration, advantageous over no synergism the sequential could (Figure be seen, 13B) so. The the same simultane- ouswas combination observed for seemsbenztropine, to be advantageousdemonstrating aover lack of the efficacy sequential in sequential (Figure administra- 13B). The same tion (Figure 13C). Contrary to these drugs, a combination with artesunate in sequential was observed for benztropine, demonstrating a lack of efficacy in sequential administra- form, with artesunate being given prior to 5-FU, seems to have better results compared to tion5-FU(Figure prior 13 artesunateC). Contrary and simultaneous to these drugs, administration, a combination resulting with artesunatein three synergistic in sequential form,pairs with (CI < artesunate 1) (Figure 1 being3D). Table given 4 s priorhows tothe 5-FU, CI values seems obtained to have for better each combination, results compared todepending 5-FU prior on artesunate the drug schedule. and simultaneous administration, resulting in three synergistic pairs (CI < 1) (Figure 13D). Table4 shows the CI values obtained for each combination, depending on the drug schedule. Int. J. J. Mol. Mol. Sci. Sci. 20212021,, 2222,, x 7408 FOR PEER REVIEW 22 of 39

Figure 13. ChouChou-Talalay-Talalay method Fa Fa-CI-CI plot of three schedule schedule-dependent-dependent combinations of of 5 5-FU-FU plus fluphenazine ﬂuphenazine ( (AA),), fluoxetineﬂuoxetine (BB),), benztropinebenztropine (C(C)) and and artesunate artesunate ( D(D).). CI CI was was plotted plotted on on the they-axis y-axis as as a functiona function of effectof effect level level (Fa) (Fa) on theon thex-axis x- axisto evaluate to evaluate drug drug synergism. synergism. CI < CI 1, CI< 1, = CI 1 and= 1 and CI > CI 1 refers> 1 refers to synergism, to synergism, additivity additivity and and antagonism, antagonism, respectively. respectively.

Table 4. CI values and respective fractional effect for three different combination schedules of 5 5-FU-FU plus fluphenazine, fluoxetine, benztropine and artesunate. CI in red indicates concentrations of plus ﬂuphenazine, ﬂuoxetine, benztropine and artesunate. CI in red indicates concentrations of drug drug pairs that are synergic. pairs that are synergic. Combination Indexes for the Different Drug Combinations Combination Indexes for the Different Drug Combinations Drug CombinationDrug Combination ScheduleSchedule A A ScheduleSchedule B B ScheduleSchedule C C (µM) (CI) (CI) (CI) 5-FU(µM) Fluphenazine (CI) (CI) (CI) 5-FU 0.75Fluphenazine 0.5 11.61 >100 4.09 1.5 1 13.08 >100 24.81 0.75 30.5 211.61 8.40>100 >100 4.09 1.77 6 4 12.17 4.36 1.96 1.5 121 813.08 8.04>1004.68 24.81 1.76 3 5-FU 2 Fluoxetine 8.40 >100 1.77 0.75 1.5 1.83 >100 7.30 6 1.5 4 3 12.17 2.65 4.36>100 1.963.53 3 6 1.36 >100 2.32 12 6 8 12 8.04 0.98 4.68 2.56 1.761.48 12 24 1.10 1.74 1.11 5-FU 5-FUFluoxetine Benztropine 0.75 0.751.5 4.51.83 1.95>100 34.97 7.30 4.01 1.5 9 1.17 2.86 2.54 1.5 33 182.65 1.27>1003.42 3.53 2.12 6 36 1.68 4.33 3.58 3 126 72 1.36 0.75 >1004.31 2.32 3.14 6 5-FU 12 Artesunate 0.98 2.56 1.48 0.75 4.5 >100 7.88 0.40 12 1.5 24 9 1.10 1.28 1.74 4.85 1.110.49 3 18 1.36 4.22 0.81 5-FU 6 Benztropine 36 1.62 2.56 1.16 0.75 12 4.5 72 1.95 1.08 34.971.64 4.011.26 1.5 9 1.17 2.86 2.54 3 18 1.27 3.42 2.12 6 36 1.68 4.33 3.58 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 23 of 39

12 72 0.75 4.31 3.14 5-FU Artesunate Int. J. Mol. Sci. 2021, 22, 7408 0.75 4.5 >100 7.88 0.40 23 of 39 1.5 9 1.28 4.85 0.49 3 18 1.36 4.22 0.81 6 36 1.62 2.56 1.16 2.2. MCF-712 Breast Cancer72 Cells 1.08 1.64 1.26 2.2.1. The Effect of CNS Drugs as Single Agents on Cellular Viability 2.2.Finally, MCF-7 weBreast evaluated Cancer Cells the cytotoxic effect of the most promising CNS drugs in MCF-7 breast2.2.1. cancer The Effect cells, of bothCNS Drugs alone as and Single in combination. Agents on Cellular This Viability time, we combined these drugs with paclitaxelFinally, we (PTX), evaluated an antineoplastic the cytotoxic effect drug of that the is most used promising for the treatment CNS drugs of in breast MCF- cancer instead7 breast of cancer 5-FU, cells, as previous both alone results and in from combination. our group This revealed time, we combined that this drugthese drugs is not very effectivewith paclitaxel against MCF-7(PTX), an breast antineoplastic cancer cells. drug Based that is on used HT-29 for the results, treatment we selected of breast thioridazine,cancer benztropine,instead of 5 fluoxetine,-FU, as previous fluphenazine, results from sertraline our group and revealed latrepirdine that this and drug evaluated is not theirvery effect oneffective MCF-7 viability.against MCF As- previous,7 breast cancer MCF-7 cells. cells Based were on treated HT-29 withresults, increasing we selected concentrations thiori- of eachdazine, repurposed benztropine, drug, fluoxetine, starting fluphenazine, from 1 µM sertraline to 100 µandM latr to evaluateepirdine and cell evaluate viabilityd after their effect on MCF-7 viability. As previous, MCF-7 cells were treated with increasing 48 h treatment. concentrations of each repurposed drug, starting from 1 µM to 100 µM to evaluate cell Based on the MTT results, we found that all tested CNS drugs displayed significant viability after 48 h treatment. anti-tumourBased on activity the MTT in results, MCF-7 we cells. found Cytotoxic that all tested effects CNS of drugs fluoxetine displayed (Figure significant 14A) were significantanti-tumour in concentrations activity in MCF- above7 cells. 10CytotoxicµM, with effects 7.78 ofµ fluoxetineM causing (Figure a reduction 14A) were of more sig- than 50%nificant of the in cells concentrations (Figure 14B). above The 1 anti-tumour0 µM, with 7.7 effect8 µM ofcausing sertraline a reduction was the of strongest more than among all50% drugs of the tested cells alone, (Figure and 14B). concentrations The anti-tumour above effect 10 ofµ sMertraline killed was almost the strongest all cells (Figureamong 14C). Theall IC drugs50 value tested obtained alone, and for concentrations sertraline was above the 10 lowest,µM killed i.e., almost about all 2.22cells (FigureµM (Figure 14C). 14D). MTTThe results IC50 value for thioridazineobtained for sertraline demonstrated was the a lowest strong, i.e., cytotoxic about 2 effect.22 µM of (Figure this CNS 14D). drug in HT-29MTT cells results for for all thioridazine concentrations demonstrated tested above a strong 10 cytotoxicµM (Figure effect 14 ofE). this The CNS dose-response drug in HT-29 cells for all concentrations tested above 10 µM (Figure 14E). The dose-response curve for thioridazine resulted in an IC50 value of 5.72 µM (Figure 14F). Fluphenazine showedcurve significantfor thioridazine anti-tumour resulted effectsin an IC similar50 value to of sertraline 5.72 µM (Figure in concentrations 14F). Fluphenazine above 10 µM showed significant anti-tumour effects similar to sertraline in concentrations above 10 µM (Figure 14G) and the IC50 value obtained was 2.68 µM (Figure 14H). Benztropine and (Figure 14G) and the IC50 value obtained was 2.68 µM (Figure 14H). Benztropine and latrepirdine effects on MCF-7 viability were the worst among all drugs tested. Benztropine latrepirdine effects on MCF-7 viability were the worst among all drugs tested. Benztropine treatmenttreatment significantly significantly decreaseddecreased MCF MCF-7-7 breast breast cancer cancer cell cell viability viability for all for concentrations all concentrations µ µ aboveabove 15 15 µMM (Figure (Figure 14I14I)) and and an an IC50 IC value50 value of 21.71 of 21.71µM (FigureM (Figure14J). Only 14 tJ).reatment Onlys treatments with withlatrepirdine latrepirdine at doses at doses above above 25 µM 25 µforM 48 for h 48had h hada significant a significant effect effect on the on cell the viability cell viability (Figure(Figure 14 14KK),), resulting resulting in in anan IC50 valuevalue of of more more than than 70 70µMµ (FigureM (Figure 14L ).14 L).

Figure 14. Cont. Int. J. Mol. Sci. 2021, 22, 7408 24 of 39 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 24 of 39

FigureFigure 14. The 14. effectsThe effects of some of some CNS CNS drugs drugs on on MCF-7 MCF-7 cells.cells. ( A)) The The effect effect of of fluoxetine fluoxetine on oncell cellviability viability and ( andB) the (B dose) the- dose- responseresponse curve. curve. (C) The(C) The effect effect of sertralineof sertraline on on cell cell viability viability andand (DD)) dose dose-response-response curve. curve. (E) (TheE) Theeffect effect of thioridazine of thioridazine on on cell viabilitycell viability and (andF) the (F) dose-responsethe dose-response curve. curve. ( G(G)) The The effecteffect of fluphenazine fluphenazine on on cell cell viability viability and and (H) (theH) dose the- dose-responseresponse curve. (I) The effect of benztropine on cell viability and (J) the dose-response curve. (K) The effect of latrepirdine on cell curve. (I) The effect of benztropine on cell viability and (J) the dose-response curve. (K) The effect of latrepirdine on cell viability and (L) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, the MTT assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means ± SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.01. **** statistically significant vs. control at p < 0.0001. Int. J. Mol. Sci. 2021, 22, 7408 25 of 39

These results demonstrate that CNS agents, such as ﬂuoxetine, sertraline, benztropine, ﬂuphenazine and thioridazine, are good candidates to be used in combination with PTX. Table5 shows a comparison between the IC 50 obtained for these drugs in the two cell lines (MCF-7 and HT-29). Compared to the previous results, it is possible to verify that all IC50 values obtained for MCF-7 breast cancer cells were higher than the ones obtained for HT-29 colon cancer cells, except for sertraline, demonstrating that these drugs alone have less potency in breast cancer cells.

Table 5. Comparison between the IC50 of several CNS drugs in HT-29 and MCF-7 cancer cells. IC50 values are given as mean.

HT-29 MCF-7 Drug IC50 (µM) IC50 (µM) Fluphenazine 1.86 2.68 ‡ Fluoxetine 6.12 7.78 ‡ Benztropine 18.23 21.71 ‡ Thioridazine 4.26 5.72 ‡ Sertraline 2.45 2.22 ‡ Latrepirdine 7.75 75.37 ‡ Drugs selected for combinations with PTX.

2.2.2. The Effect of Various Combinations of PTX and Different CNS Agents We next evaluated the combination of PTX with each drug using the model of combination developed in our previous work [37]. The IC50 value for PTX adopted in these drug combinations was obtained in our previous work [37]. MCF-7 cells were treated with the two drugs alone or combined in a fixed ratio, in the concentrations of 0.25 × IC50, 0.5 × IC50, IC50, 2 × IC50 and 4 × IC50, and two cell-based assays were performed: MTT and SRB. Morphological evaluation of cells treated with each drug alone and in combination was also done. The combination of PTX plus fluphenazine, fluoxetine, benztropine, thioridazine and sertraline was evaluated. When combined with PTX, fluoxetine demonstrates significant anti-cancer effects both by MTT and SRB assays (Figure 15A,B, respectively), mainly at the concentration of 2 × IC50, where the combined effect was statistically significant compared to each drug alone. The combination with sertraline resulted in a similar reduction of cell viability and cell protein synthesis as PTX alone, at all concentrations. At the concentrations of IC50 and 2 × IC50, the combination of PTX plus sertraline demonstrated significative anticancer effects compared to sertraline alone (Figure 15C,D). The combination with PTX and thioridazine demonstrated significant anticancer effects compared to both drugs alone, at concentrations of IC50 and 2 × IC50 (Figure 15E,F). The combination with fluphenazine resulted in all intermediate concentrations showing a greater anticancer effect than fluphenazine alone (Figure 15G,H). The activity seen for these combinations can be the result of the strong anticancer activity of PTX alone. The combination with benztropine resulted in a statistically significant reduction of cell viability at concentrations of IC50 and 2 × IC50 compared to PTX alone (Figure 15I,J), demonstrating that the activity of this combination can be the result of the repurposed drug alone, contrary to the previous combinations. Together, these results demonstrate that both CNS drugs and PTX can have different pharmacological actions in the combined effects. Morphologically, the results are in agreement with the MTT and SRB assays. At concentrations of 4 × IC50, all combinations resulted in a decrease of cell number and smaller and rounder cells, compared to control cells and PTX, which is indicative of cell death (Figure 16). Int. J. Mol. Sci. 2021, 22, 7408 26 of 39 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 26 of 39

Figure 15.FigureGrowth 15. Growth inhibition inhibition of MCF-7 of MCF after-7 after 48 48 h h of of combination combination therapy therapy with with PTX PTX,, by MTT by MTT(left) and (left SRB) and assays SRB (right assays). (right). Cells were exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their IC50 and the cell viability was evaluated Cells were exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their IC50 and the cell viability was evaluated by MTT and SRB assays. The drugs in combination were co-administered at the same time. (A) The effect of PTX plus fluoxetine on cell viability and (B) cell protein synthesis. (C) The effect of PTX plus sertraline on cell viability and (D) cell protein synthesis. (E) The effect of PTX plus thioridazine on cell viability and (F) cell protein synthesis. (G) The effect of PTX plus fluphenazine on cell viability and (H) cell protein synthesis. (I) The effect of PTX plus benztropine on cell viability and (J) cell protein synthesis. Values are expressed in percentage of control and represent means ± SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 27 of 39

by MTT and SRB assays. The drugs in combination were co-administered at the same time. (A) The effect of PTX plus fluoxetine on cell viability and (B) cell protein synthesis. (C) The effect of PTX plus sertraline on cell viability and (D) cell protein synthesis. (E) The effect of PTX plus thioridazine on cell viability and (F) cell protein synthesis. (G) The effect of PTX plus fluphenazine on cell viability and (H) cell protein synthesis. (I) The effect of PTX plus benztropine on cell viability and (J)Int. cell J. protein Mol. Sci. synthesis.2021, 22, 7408 Values are expressed in percentage of control and represent means ± SEM. Each experiment 27 of 39 was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.

Figure 16. Cont. Int. J. Mol. Sci. 2021, 22, 7408 28 of 39 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 28 of 39

Figure 16. Microscopic cellular visualisation of MCF-7 cells after 48 h of incubation with vehicle (A), Figure 16. Microscopic cellular visualisation of MCF-7 cells after 48 h of incubation with vehicle (A), PTX (B), fluoxetine PTX (B), fluoxetine (C), fluoxetine + PTX (D), sertraline (E), sertraline + PTX (F), thioridazine (G), C D E F G H I ( ), fluoxetine +thioridazine PTX ( ), sertraline + PTX (H ( ),), fluphenazine sertraline + PTX(I) fluphenazine ( ), thioridazine + PTX ( (J),), thioridazinebenztropine +(K PTX) and ( benztropine), fluphenazine + ( ) J K L × fluphenazine + PTXPTX ((L),) benztropineat concentrations ( ) and of 4 benztropine × IC50 of each + PTXdrug. ( Scale) at concentrations bar: 50 µm. of 4 IC50 of each drug. Scale bar: 50 µm. 2.2.3. Synergistic Combinations of PTX and CNS Agents Next, 2.2.3.we calculated Synergistic the Combinations combination index of PTX (CI) and using CNS the Agents Chou-Talalay method, using the CompuSynNext, software. we calculated A combination the combination of PTX index plus (CI)fluoxetine using thedemonstrated Chou-Talalay syner- method, using gism for thethe lowest CompuSyn concentrations software. with A combination three synergic of PTX pair pluss (Figure fluoxetine 17A), demonstratedwith Fa values synergism of 0.1184, 0.2472for the and lowest 0.3621 concentrations (Table 6). The withcombination three synergic with sertraline pairs (Figure resulted 17A), in only with one Fa values of synergic pair,0.1184, for 0.2472the lowest and concen 0.3621tration (Table 6(Figure). The 17B). combination The combinations with sertraline of PTX resulted plus in only thioridazineone demonstrated synergic pair, synergism for the lowest for two concentration pairs (Figure (Figure17C), with 17B). Fa Thevalues combinations of 0.1895 of PTX and 0.5027plus (Table thioridazine 6). Combination demonstrated with fluphenazine synergism forand two benztropine pairs (Figure resulted 17C), in with three Fa values of synergic pairs0.1895 (Figure and 0.5027 17D and (Table E, respectively6). Combination), with with Fa values fluphenazine lower than and 0. benztropine60 (Table 6). resulted in Together, thesethree results synergic demonstrate pairs (Figure that 17 D,E,these respectively), CNS agents may with be Fa promising values lower candidates than 0.60 (Table6). to evaluateTogether, in future these combinations. results demonstrate that these CNS agents may be promising candidates to Theseevaluate drug interactions in future combinations.were also evaluated by the Bliss Independence method, using the SynergyFinderThese drug 2.0 software. interactions Fluoxetine were also combination evaluated by with the BlissPTX Independenceanalysed with method, the using Bliss modelthe (Figure SynergyFinder 18A) demonstrated 2.0 software. the highest Fluoxetine synergy combination score, in line with with PTX the analysed Chou- with the Talalay results,Bliss indicating model (Figure synergism 18A) demonstrated for three pairs. the Sertraline highest combination synergy score, demonstrated in line with the Chou- Talalay results, indicating synergism for three pairs. Sertraline combination demonstrated synergism using the Bliss model, with a positive synergy score of 2.127 (Figure 18B). The synergism using the Bliss model, with a positive synergy score of 2.127 (Figure 18B). The combination of PTX with thioridazine also resulted in synergism using the Bliss method combination of PTX with thioridazine also resulted in synergism using the Bliss method (Figure 18C) with a synergy score of 2.938. The combinations of PTX plus fluphenazine (Figure 18C) with a synergy score of 2.938. The combinations of PTX plus fluphenazine and benztropine resulted in the lowest synergy scores using the Bliss method, with scores and benztropine resulted in the lowest synergy scores using the Bliss method, with scores of 0.569 and -8.262 (Figure 18 D and E, respectively). of 0.569 and -8.262 (Figure 18D,E, respectively). The Bliss method results demonstrate slightly different results regarding the synergy The Bliss method results demonstrate slightly different results regarding the synergy evaluation of drug combinations compared to the Chou-Talalay results, especially regard- evaluation of drug combinations compared to the Chou-Talalay results, especially regarding ing fluphenazine and benztropine combinations. Despite this, these reference models pro- fluphenazine and benztropine combinations. Despite this, these reference models produce duce similar results most of the time. similar results most of the time. Int.Int. J. Mol. J. Mol. Sci. Sci.2021 2021, 22, ,22 7408, x FOR PEER REVIEW 29 of 39

FigureFigure 17. 17.Chou-Talalay Chou-Talalay method method Fa-CI Fa-CI plot plot of of PTX PTX plus plus ﬂuoxetine fluoxetine ( A(A),), sertraline sertraline( (BB),), thioridazinethioridazine ((CC),), ﬂuphenazinefluphenazine ( D) andand benztropine benztropine (E ).(E CI). CI was was plotted plotted on on the they -axisy-axis as as a a function function of of effect effect level level (Fa)(Fa) onon thethe xx-axis-axis to evaluate drug synergism. CICI < 1,< 1, CI CI = 1= and1 and CI CI > > 1 1 refers refers to to synergism, synergism, additivity additivity and and antagonism,antagonism, respectively.respectively.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 30 of 39

Table 6. CI values and respective fractional effect of different combinations of PTX plus CNS agents. CI in red indicates concentrations of drug pairs that are synergic. Cells were treated with 0.25, 0.5, 1, 2 and 4 times the IC50 of each drug (total dose).

Int. J. Mol. Sci. 2021, 22, 7408 Combination Total Dose Fractional 30 ofCI 39 (Drug A + Drug B) (Dose A + Dose B) Effect (Fa) Value 2.75 0.1184 0.33562 Table 6. CI values and respective fractional effect5.5 of different combinations0.2472 of PTX plus CNS agents.0.48322 CI inPTX red indicates + fluoxetine concentrations of drug pairs11.0 that are synergic. Cells were0.3621 treated with 0.25,0.79198 0.5, 1, 2 and 4 times the IC50 of each drug (total dose).22.0 0.5227 1.24993 Combination Total Dose 44.0 Fractional 0.7167 CI 1.85505 (Drug A + Drug B) (Dose A + Dose B)1.375 Effect (Fa) 1.0 × 10−4Value 5.01852 2.75 2.75 0.1184 0.0768 0.33562 0.73865 5.5 0.2472 0.48322 PTXPTX + ﬂuoxetine+ sertraline 11.0 5.5 0.3621 0.1987 0.79198 0.99737 22.0 11.0 0.5227 0.4012 1.24993 1.40860 44.0 22.0 0.7167 0.6326 1.85505 2.04261 1.375 1.0 × 10−4 5.01852 2.752.25 0.0768 0.0109 0.73865 0.98541 PTX + sertraline 5.54.5 0.19870.1895 0.99737 0.69489 PTX + thioridazine 11.09.0 0.40120.5027 1.40860 0.86789 22.0 0.6326 2.04261 2.25 18.0 0.0109 0.7077 0.98541 1.32210 4.5 36.0 0.1895 0.7128 0.69489 2.62409 PTX + thioridazine 9.0 0.5027 0.86789 18.0 1.5 0.7077 0.01908 1.32210 0.63552 36.0 3.0 0.7128 0.057 2.62409 0.83495 PTX + fluphenazine 1.56.0 0.01908 0.33804 0.63552 0.78096 3.0 0.057 0.83495 PTX + ﬂuphenazine 6.012.0 0.33804 0.44769 0.78096 1.33256 12.024.0 0.447690.67095 1.33256 1.95345 24.06.25 0.670950.09897 1.95345 0.45763 6.25 0.09897 0.45763 12.5 12.5 0.2321 0.2321 0.65346 0.65346 PTXPTX + + benztropine benztropine 25.0 25.0 0.51629 0.51629 0.86840 0.86840 50.0 50.0 0.66485 0.66485 1.42716 1.42716 100.0 0.71074 2.66897 100.0 0.71074 2.66897

Figure 18. Cont. Int.Int. J. J. Mol. Mol. Sci. Sci.2021 2021,,22 22,, 7408 x FOR PEER REVIEW 3131 of of 39 39

FigureFigure 18. 18.Bliss Bliss synergy synergy plots plots of of PTX PTX plus plus fluoxetine fluoxetine (A (),A sertraline), sertraline (B ),(B thioridazine), thioridazine (C), (C fluphenazine), fluphenazine (D) and(D) and benztropine benztropine (E). (E). 3. Discussion 3. DiscussionDrug repurposing and drug combination are strategies that have become more pop- ular overDrug the repurposing years, representing and drug acombination faster and cheaper are strategies strategy that to have identify become new more potential pop- candidatesular over the for years, cancer representing therapy. Repurposed a faster and drugs cheaper are already strategy available to identify on the new market potential for othercandidates diseases for andcancer have therapy. pharmacokinetics, Repurposed pharmacodynamics drugs are already available and toxicological on the market profiles for thatother are diseases well established, and have facilitatingpharmacokinetics, their approval pharmacodynamics for novel indications. and toxicological The combination profiles ofthat drugs are well allows established, decreasing facilitating the therapeutical their approval dose, reducingfor novel indications. the side effects The ofcombination the drugs. Theof drugs combination allows decreasing of antineoplastic the therapeutical drugs with dose, other reducing drug classes the side has effects been of explored the drugs. in severalThe combination studies, but of antineoplastic few studies report drugs the with CNS other drugs drug benefits classes forhas cancerbeen explor therapy,ed in both sev- aloneeral studies, and in combination.but few studies 5-FU report is an the essential CNS drugs agent benefits in the treatmentfor cancer oftherapy, CRC, butboth its alone use isand limited in combination. by its short 5 half-life,-FU is an highessential cytotoxicity agent in andthe treatment low bioavailability, of CRC, but which its use limit is lim- its benefits.ited by its PTX short is anhalf antineoplastic-life, high cytotoxicity drug commonly and lowused bioav forailability, the treatment which oflimit breast its benefits. cancer, butPTX its is maximum an antineoplastic therapeutic drug dosage commonly is limited used by for the the appearance treatment of of drug breast resistance cancer, andbut itsits sidemaximum effects. therapeutic To overcome dosage these problems,is limited by higher the appearance doses and long-term of drug resistance use of these and antineo- its side plasticeffects. drugs To overcome is necessary, these which problems, increases highe its sider doses effects. and Current long-term research use of aims these todecrease antineo- the chemotherapeutic drugs doses and exposure time. Recent studies have investigated plastic drugs is necessary, which increases its side effects. Current research aims to de- new drugs that can synergise with 5-FU or PTX, but, to our knowledge, none have explored crease the chemotherapeutic drugs doses and exposure time. Recent studies have investi- CNS drugs in combination with 5-FU or PTX for CRC or breast cancer therapy. gated new drugs that can synergise with 5-FU or PTX, but, to our knowledge, none have We studied the potential anticancer activity of different CNS drugs in HT-29 colon explored CNS drugs in combination with 5-FU or PTX for CRC or breast cancer therapy. and MCF-7 breast cancer cells and evaluated the potential synergistic effects of this class of We studied the potential anticancer activity of different CNS drugs in HT-29 colon drugs with 5-FU or PTX, antineoplastic drugs used for CRC and breast cancer treatment, and MCF-7 breast cancer cells and evaluated the potential synergistic effects of this class respectively. First, several CNS drugs were screened by MTT assay to treat HT-29 and of drugs with 5-FU or PTX, antineoplastic drugs used for CRC and breast cancer treat- MCF-7 cells to evaluate their potential as repurposed drugs. Besides CNS drugs, we also ment, respectively. First, several CNS drugs were screened by MTT assay to treat HT-29 evaluated three antimalarial drugs (chloroquine, artesunate and mefloquine) in this study and MCF-7 cells to evaluate their potential as repurposed drugs. Besides CNS drugs, we based on our previous results in MCF-7 cells, to evaluate if their anticancer behaviour was also evaluated three antimalarial drugs (chloroquine, artesunate and mefloquine) in this Int. J. Mol. Sci. 2021, 22, 7408 32 of 39

maintained in a different cell line (HT-29). After an evaluation using MTT, the IC50 for each drug was determined, and those with an IC50 under 20 µM were selected for combination with 5-FU or PTX, depending on the cell type. We employed our previously described combination model in which cells were treated with the concentrations of 0.25, 0.5, 1, 2 and 4 times the IC50 of each drug, alone and in combination, using MTT and SRB assays. We next evaluated synergism by three different methods: Chou-Talalay, Bliss (for HT-29 and MCF-7 cells) and HSA (only for HT-29 cells). The Chou-Talalay method is based on the median-effect equation, derived from the mass-action law principle. This unified theory encompasses the Michaelis–Menten, Hill, Henderson–Hasselbalch and Scatchard equations in biochemistry and biophysics and provides a quantitative definition for additive effect (CI = 1), synergism (CI < 1) and antagonism (CI > 1) in drug combinations [58]. The Bliss independence model adopts a stochastic process in which two drugs produce their effects independently, and the expected combination effect can be calculated based on the probability of independent events [57]. The HSA model is one of the simplest reference models for synergism evaluation and states that the expected combination effect is the maximum of the single drug responses at corresponding concentrations. The synergy score for a drug combination is averaged over all the dose combination measurements, giving a positive or negative value, corresponding to synergism or antagonism, respectively. The 2D and 3D synergy maps highlight synergistic and antagonistic dose regions in red and green colours, respectively. Our results demonstrated that CNS drugs as single agents have the ability to decrease cell viability in a concentration-dependent manner in both cell lines. In HT-29 colon cancer cells, the most promising drugs were latrepirdine, fluphenazine, fluoxetine, benztropine, thioridazine, sertraline, mefloquine and artesunate, all with IC50 values under 20 µM, with fluphenazine being the most potent with an IC50 of 1.86 µM. For MCF-7 breast cancer cells, we found out that these drugs were less potent, with IC50 values higher than the ones obtained for colon cancer cells, except for sertraline, whose IC50 was 2.22 µM. In simultaneous combination, we found that sertraline and thioridazine were the most promising candidates for the improvement of the anti-cancer activity of 5-FU in HT-29 colon cancer cells. For MCF-7 cells, almost all tested combinations resulted in synergic pairs, for the lowest concentrations. Drugs such as fluoxetine and thioridazine combined with PTX resulted in an enhanced reduction in the viability of MCF-7 cells, compared to a single treatment with repurposed drugs or PTX. Compared to HT-29 cells, the combination of CNS drugs with PTX in MCF-7 revealed more synergistic interactions than with 5-FU, except for sertraline. Curiously, when tested alone in MCF-7, sertraline was the most potent repurposed drug, but its combination with PTX resulted in only one synergistic pair. Altogether, these results suggest that the dominant behaviour of drug combinations comes from the antineoplastic drug and that the mechanism of these drug combinations may be related to this class of drugs. Specifically for fluphenazine, fluoxetine, benztropine and artesunate in HT-29 cells, we found out that the combination of these drugs with 5-FU resulted in worse results than the repurposed drug alone, probably due to competition between the two drugs, so we designed a model of drug combination based on the sequential addition of the two drugs, with an interval of 24 h. For most drugs, we did not find significant differences between the drug schedules, except for artesunate, in which we found that artesunate prior to 5-FU administration resulted in enhanced anticancer effects. For fluphenazine, fluoxetine and benztropine, we found that these drugs act better alone than in combination, being ideal candidates for drug repurposing. These results demonstrate for the first time that CNS agents may be potential candidates for drug repurposing in colon and breast cancer therapy. We have found that all tested CNS drugs can synergistically decrease MCF-7 cell viability when combined with PTX, with fluoxetine, benztropine and fluphenazine being the most promising drugs at lower concentrations. We also concluded that sertraline and thioridazine combination with 5-FU can synergistically decrease cancer viability in HT-29 colon cancer cells. We prove that artesunate, an antimalarial drug, has anticancer potential Int. J. Mol. Sci. 2021, 22, 7408 33 of 39

in these cells and that the combination with 5-FU is beneficial if given in a sequential schedule and prior to 5-FU. Mechanistically, several studies suggest that combination treatment with 5-FU synergistically induces apoptosis in colon cancer cells [59–64]. Despite inducing apoptosis, the observed synergistic effect can also be the result of the combined impact on autophagy, a catabolic process exerted in cells in response to stressful conditions, such as nutrient deprivation or damage to proteins/DNA, which can ultimately trigger cell death. Indeed, in human colon cancer cell lines and colorectal cancer-xenografted mice, sertraline demonstrated proapoptotic activity by mitogen-activated protein kinase cascade activation and Bcl-2 inhibition [65]. Regarding thioridazine, recently, it was found that this drug significantly suppresses the proliferation and invasion of colon cancer stem cells and induced cell apoptosis in a concentration-dependent manner. It was found that apoptosis genes such as Bax and caspase-3 are overexpressed after treatment, and anti-apoptosis gene Bcl-2 was downregulated. Accordingly, the mitochondrial potential of these cells was downregulated [66]. Based on these literature findings, we propose that simultaneous apoptosis and autophagic cell death can be occurring in our combinations. We believe that 5-FU combined with sertraline and thioridazine mainly increases the concentration of caspase-3 enzyme and other apoptotic proteins in HT-29 cells causing apoptosis-dependent cell death. This inhibition of autophagy and induction of apoptosis can be proposed to be the basis of synergy in the case of the combined treatment of 5-FU and these CNS drugs in colon cells. Regarding the MCF-7 results, fluoxetine, fluphenazine and benztropine combined with PTX revealed to be the most promising combinations. PTX belongs to the taxanes drug class and acts by blocking cell mitosis through the stabilisation of microtubules, leading to cell cycle arrest preferentially in the G2/M phase and apoptosis [67]. Some studies showed that drugs acting on serotonin (5-HT) signalling, including selective serotonin reuptake inhibitors (SSRIs), inhibit tumour sphere formation in human breast tumour cells in in vitro and in vivo models [68]. Particularly, fluoxetine was found to significantly decrease the proliferation of several breast cancer cell lines by inducing apoptosis and autophagy- mediated cell death or endoplasmic reticulum stress and autophagy, respectively [68–71]. In triple-negative breast cancer cells, fluphenazine inhibited breast cancer cell growth and induced G0/G1 cell cycle arrest and induced mitochondria-mediated apoptosis in breast cancer cells [72]. In the case of MCF-7 cells, as they do not express caspase-3, they do not undergo normal apoptosis and autophagy can represent the main alternative cell death pathway [73]. Recent studies suggest that benztropine reduces the activity of oncogenic signalling transducers and trans-activators for MMP9, including STAT3, NF- κB and β-catenin [74]. We believe that the PTX mechanism of synergy in combination with CNS drugs can be related to enhanced cell cycle arrest, interference with important oncogenic signalling and increased autophagy-mediated cell death. We also believe that CNS drugs can act as chemosensitizers by slowing down drug efflux, increasing drug accumulation. As several CNS drugs are substrates and modulators of the P-glycoprotein (P-gp) protein [75–77], we also believe they can inhibit P-gp to stop effusing drugs from intracellular, to increase the intracellular concentration of anticancer drugs such as PTX. These results imply that CNS drugs may be promising chemosensitizers compounds and enhance the cytotoxic effects of 5-FU and PTX in HT-29 and MCF-7 cancer cells, respectively. Since these drugs are already accessible in the market, their use for cancer therapy is achievable. Since different colon cancer cells lines are metabolically different and have specific characteristics, more research should be made on other colon cancer cells, such as HCT116, SW480, LoVo, etc. The same is true for breast cancer cells, and these combinations can be further explored in other cell lines such as tumoral MDA-MB-231 cells or normal MCF-10A cells. Deeper mechanistic studies are strongly recommended to evaluate the anticancer mechanisms underlying these drugs and these combinations. This class of drugs should also be investigated both alone and in combination for other types of cancer, such as pancreatic, prostate, lung, etc. These are promising results and should be further confirmed in animal models and clinical trials. Our results demonstrate that Int. J. Mol. Sci. 2021, 22, 7408 34 of 39

the use of CNS and antimalarial drugs, both alone and in combination, may lead to new therapeutic strategies for colon and breast cancer therapy.

4. Materials and Methods 4.1. Materials McCoy’s 5A Modified Medium, Dulbecco’s Modified Eagle Medium (DMEM), foetal bovine serum (FBS) and a penicillin-streptomycin solution were purchased from Millipore Sigma (Merck KGaA, Darmstadt, Germany). Other cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). 5-FU (cat. no. F6627), selegiline (cat. no. M003), entacapone (cat. no. SML0654), tolcapone (cat. no. SML0150), latrepirdine (cat. no. D6196), fluphenazine (cat. no. F4765), safinamide (cat. no. SML0025), carbidopa (cat. no. PHR1655), scopolamine (cat. no. S1013), Thiazolyl Blue Tetrazolium Bromide (MTT, cat. no. M5655) and sulforhodamine B (SRB, cat. no. S1402) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Benztropine (cat. no. 16214), thioridazine (cat. no. 14400), fluoxetine (cat. no. 14418) and artesunate (cat. no. 11817) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Nepicastat (cat. no. 5037) and paclitaxel (cat. no. 1097) were obtained from Tocris Bioscience (Bristol, UK). Mefloquine (cat. no. sc-211784) and chloroquine (cat. no. C6628) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Bromocriptine was used in tablets and diluted in water before stock preparation.

4.2. Cell Line and Cell Culture Human colorectal cancer HT-29 and breast cancer MCF-7 cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained ◦ according to ATCC’s recommendations at 37 C and 5% CO2 in appropriate medium supplemented with 10% foetal bovine serum, 100 U/mL penicillin G and 100 µg/mL streptomycin. Cells were maintained in the logarithmic growth phase at all times. The media was changed every 2 days and trypsinised with 0.25% trypsin-EDTA. A total of 200 µL of HT-29 cells (7500 cells/well) or MCF-7 cells (5000 cells/well) were seeded in 96-well plates and allowed to adhere overnight before drug exposure. After 24 h, the cell culture media were replaced with 200 µL of drug-containing media. Cells were exposed to drugs for 48 h, followed by MTT and SRB assays to evaluate single and combination drug treatments in the cell viability and protein synthesis rate of these cells.

4.3. Drug Treatment

The half-maximal inhibitory concentration (IC50) value was first determined for each drug alone in HT-29 and MCF-7cells. Drug concentrations ranged from 0.1 to 100 µM for the single-drug treatment. Combination studies were performed by combining 5-FU or PTX (Drug A) according to each cell line, with different repurposed drugs (Drug B). Drug A was 5-FU for HT-29 cells and PTX for MCF-7 cells. Only drugs that present the most promising pharmacological profile (IC50 < 20 µM) were tested in simultaneous combination with 5-FU or PTX, following schedule A (Figure 11). Both Drug A and Drug B concentrations were variable, and the combined effects of equipotent concentrations (fixed ratio) of the IC50 values for each drug were evaluated. The combinations of fluoxetine, fluphenazine, benztropine and artesunate with 5-FU were also tested in sequential schedules of administration (Schedule B and C, Figure 11). For schedule A, cells were treated concomitantly with 5-FU or PTX and each repurposed drug for 48 h. For schedule B, cells were pre-treated with 5-FU 24 h followed by each repurposed drug for 24 h. For schedule C, cells were pre-treated with each repurposed drug for 24 h followed by 5-FU for another 24 h.

4.4. Cell Viability Assay To determine the effects of 5-FU or PTX and the repurposed drugs on the viability of HT-29 and MCF-7 cells, respectively, MTT and SRB assays were used. For the MTT protocol, after drug treatment, the cell medium was removed and 100 µL/well of MTT solution Int. J. Mol. Sci. 2021, 22, 7408 35 of 39

(0.5 mg/mL in PBS) was added. Cells were incubated for 3 h, protected from light. After this period, the MTT solution was removed, and DMSO (100 µL/well) was added to solubilise the formazan crystals. Absorbance was measured at 570 nm in an automated microplate reader (Tecan Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). For SRB assay, after treatments, the cultured cells were fixed with ice-cold 10% trichloroacetic acid for 30 min and stained with 0.4% SRB for 1 h at room temperature. Excess dye was removed by rinsing several times with tap water. Protein-bound dye was dissolved with 200 µL 10 mM Tris base solution for the determination of absorbance with a microplate reader with a filter wavelength of 540 nm (Tecan Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). The IC50 of the therapeutic drug was determined as each drug concentration showing 50% cell growth inhibition as compared with control. All conditions were performed three times independently, in triplicate.

4.5. Cell Morphology Visualisation After each treatment, cell morphology was assessed on a Leica DMI 6000B microscope equipped with a Leica DFC350 FX camera and then analysed with the Leica LAS X imaging software (v3.7.4).

4.6. Data Analysis GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) was used to produce concentration-response curves by nonlinear regression analysis. The viability of cells treated with each drug was normalised to the viability of control cells and cell viability fractions were plotted vs. drug concentrations in the logarithmic scale.

4.7. Analysis of Drug Interactions To quantify drug interaction between 5-FU and CNS drugs, we first estimated the Combination Index (CI) by the unified theory, introduced by Chou and Talalay [15] using the CompuSyn software (ComboSyn, Inc., New York, NY, USA). We used the mutually exclusive model, based on the assumption that drugs act through entirely different mechanisms [78]. The two drugs were combined in a fixed ratio of doses that correspond to 0.25, 0.5, 1, 2 and 4 times that of the individual IC50 values. CI was plotted on the y-axis as a function of effect level (Fa) on the x-axis to assess drug synergism between drug combinations. The CI is a quantitative representation of pharmacological interactions. CI < 1 indicates synergism, CI = 1 indicates additive interaction and CI > 1 indicates antagonism. We also estimated the expected drug combination responses based on the highest single agent (HSA) and Bliss reference model using SynergyFinder [57]. Deviations between observed and expected responses with positive and negative values denote synergy and antagonism, respectively.

4.8. Statistical Analysis The results are presented as mean ± SEM for n experiments performed. All data were assayed in three independent experiences, in triplicate. Statistical comparisons between control and treatment groups, at the same time point, were performed with Student’s t-test and one-way ANOVA test. Statistical signiﬁcance was accepted at p values < 0.05.

Author Contributions: Conceptualisation, N.V.; methodology, D.D., A.C. and N.V.; software, D.D.; validation, A.C. and N.V.; formal analysis, D.D., A.C. and N.V.; investigation, D.D., A.C. and N.V.; resources, A.C. and N.V.; data curation, D.D.; writing—original draft preparation, D.D.; writing— review and editing, A.C. and N.V.; visualisation, A.C. and N.V.; supervision, N.V.; project administration, N.V.; funding acquisition, A.C. and N.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was ﬁnanced by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e a Tecnologia (FCT) in the framework of the project IF/00092/2014/CP1255/CT0004. Int. J. Mol. Sci. 2021, 22, 7408 36 of 39

Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: This article was supported by National Funds through FCT-Fundação para a Ciência e a Tecnologia, I.P., within CINTESIS, R & D Unit (reference UIDB/4255/2020). D.D. acknowledges FCT for funding her PhD grant (SFRH/BD/140734/2018). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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