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Article The Up-Regulation of Oxidative Stress as a Potential Mechanism of Novel MAO-B Inhibitors for Glioblastoma Treatment

Guya Diletta Marconi 1, Marialucia Gallorini 1 , Simone Carradori 1,* , Paolo Guglielmi 2, Amelia Cataldi 1 and Susi Zara 1

1 Department of Pharmacy, University “G. d’Annunzio” of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy; [email protected] (G.D.M.); [email protected] (M.G.); [email protected] (A.C.); [email protected] (S.Z.) 2 Department of Drug Chemistry and Technologies, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy; [email protected] * Correspondence: [email protected], Tel.: +39-0871-355-4583

 Academic Editor: Simona Collina  Received: 24 April 2019; Accepted: 23 May 2019; Published: 25 May 2019

Abstract: Gliomas are malignant brain tumors characterized by rapid spread and growth into neighboring tissues and graded I–IV by the World Health Organization. Glioblastoma is the fastest growing and most devastating IV glioma. The aim of this paper is to evaluate the biological effects of two potent and selective B (MAO-B) inhibitors, Cmp3 and Cmp5, in C6 glioma cells and in CTX/TNA2 astrocytes in terms of cell proliferation, apoptosis occurrence, inflammatory events and cell migration. These compounds decrease C6 glioma cells viability sparing normal astrocytes. Cell cycle analysis, the Mitochondrial Membrane Potential (MMP) and (ROS) production were detected, revealing that Cmp3 and Cmp5 induce a G1 or G2/M cell cycle arrest, as well as a MMP depolarization and an overproduction of ROS; moreover, they inhibit the expression level of inducible 2, thus contributing to fatal drug-induced oxidative stress. Cmp5 notably reduces glioma cell migration via down-regulating Matrix Metalloproteinases 2 and 9. This study demonstrated that our novel MAO-B inhibitors increase the oxidative stress level resulting in a cell cycle arrest and markedly reduces glioma cells migration thus reinforcing the hypothesis of a critical role-played by MAO-B in mediating oncogenesis in high-grade gliomas.

Keywords: Glioblastoma; MAO-B inhibitors; oxidative stress; migration

1. Introduction Gliomas are the first brain tumors assumed to originate from the neuroglial stem or from progenitor cells, characterized by inappropriate proliferation, infiltration into neighboring normal brain tissue and the disruption of the normal functioning of the brain. Based on their histological appearance, gliomas are conventionally classified as astrocytic, oligodendroglial or ependymal tumors and attributed by the World Health Organization grades I–IV, which denote disparate levels of malignancy [1]. Glioblastoma (GBM), a grade IV astrocytoma notoriously treatment–resistant brain cancer, is the greatest destructive brain tumor, largely due to the limited effects of conventional post-surgical chemotherapeutic agents and to irradiation [2]. A better understanding of the mechanism underlying the destructive nature of GBM could improve radio-, chemo- and therapy. More effective GBM therapeutic strategies are urgently needed. Unfortunately, the present therapies for high-grade gliomas are not an effective approach; after preliminary diagnosis, patients receive a maximal surgical resection, concomitant radiotherapy

Molecules 2019, 24, 2005; doi:10.3390/molecules24102005 www.mdpi.com/journal/molecules Molecules 2019, 24, 2005 2 of 16 and chemotherapy with Temozolomide (TMZ), the currently used chemoterapeutic agent for GBM treatment.Molecules The 2019 TMZ, 24, x FOR raised PEER the REVIEW prognosis of GBM patients because it can cross the blood-brain2 of barrier17 and promote glioma cell death by alkylating DNA, even if TMZ resistance could be one of the major reasonsand why chemotherapy treatments with are Temozolomide not effective [3 (TMZ),]. the currently used chemoterapeutic agent for GBM Monoaminetreatment. The Oxidase TMZ raised (MAO) the prognosis catalyzes of GBM the oxidative patients because deamination it can cross of the a range blood-brain of monoamines, barrier and promote glioma cell death by alkylating DNA, even if TMZ resistance could be one of the major including 5-hydroxytryptamine (5-HT, ), and the , reasons why treatments are not effective [3]. noradrenaline and adrenaline. The two isoenzymes of MAO (MAO-A and MAO-B) are present in Monoamine Oxidase (MAO) catalyzes the oxidative deamination of a range of monoamines, mostincluding mammalian 5-hydroxytryptamine tissues. They are (5-HT, not uniformly serotonin), distributed histamine inand the the human catecholamines brain, and dopamine, the principal form innoradrenaline the basal ganglia and adrenaline. is MAO-B. The Over two theisoenzymes past several of MAO decades, (MAO-A MAO-B and MAO-B) inhibitors are showed presentto inhave potentialmost use mammalian in the treatment tissues. They of several are not neurodegenerative uniformly distributed disorders, in the human including brain, and Parkinson’s the principal disease and Alzheimer’sform in the basal disease ganglia [4]. is MAO-B. Over the past several decades, MAO-B inhibitors showed to have However,potential use in in the the paper treatment of Gabilondo of several neurodeg et al. [5],enerative it was reporteddisorders, thatincluding a remarkable Parkinson’s and disease selective increaseand in Alzheimer’s MAO-B activity disease is [4]. also detected in human gliomas. This could be explained by an augmented number ofHowever, astrocytes in the as paper well asof aGabilondo higher content et al. [5], of it MAO-Bwas reported in tumoral that a remarkable astrocytes and with selective respect to increase in MAO-B activity is also detected in human gliomas. This could be explained by an normal ones [5], highlighting that the overexpression of MAO-B could be related with high-grade augmented number of astrocytes as well as a higher content of MAO-B in tumoral astrocytes with glioma, and thus defining MAO-B as a promising target for the development of novel therapies respect to normal ones [5], highlighting that the overexpression of MAO-B could be related with high- for cancer.grade glioma, To better and understandthus defining theMAO-B role as played a promising by MAO-B target for inhibitors the development in the treatmentof novel therapies of glioma, the aimfor iscancer. to evaluate To better two understand newly synthesized the role played compounds, by MAO-B inhibitors namely in Cmp the 3treatmentand Cmp of5 glioma,(Figure the1)[ 6], alongaim with is to two evaluate approved two newly selective synthesized MAO-B compounds, inhibitors (deprenyl namely Cmp and3 and safinamide). Cmp5 (Figure These 1) [6], compounds along werewith the best-in-class two approved derivatives, selective MAO-B within inhibitors the hydrazothiazole (deprenyl and ). scaffold, endowed These compounds with a potent were and selectivethe best-in-class hMAO-B inhibition derivatives, in thewithin low the nanomolar hydrazothiazole range comparablescaffold, endowed to the with clinically a potent used and drugs. selective hMAO-B inhibition in the low nanomolar range comparable to the clinically used drugs. The Selectivity Index (SI) was expressed as IC50 hMAO-A/IC50 hMAO-B. They were shown to act as reversibleThe Selectivity and competitive Index (SI) inhibitors. was expressed We also as IC assessed50 hMAO-A/IC the in50 vitro hMAO-B.stability They of were pH andshown temperature to act as in reversible and competitive inhibitors. We also assessed the in vitro stability of pH and temperature forced conditions [7]. in forced conditions [7]. The aimThe ofaim this of this work work is tois investigateto investigate the the biological biological response response of of rat rat C6 C6 glioma glioma cell cellline lineand andCTX CTX TNA2TNA2 astrocytes astrocytes after after treatment treatment with with these these two two novelnovel MAO-B inhibitors inhibitors in interms terms of cell of cellproliferation, proliferation, apoptosisapoptosis occurrence, occurrence, inflammatory inflammatory events events and and cell cell migration.migration.

Figure 1. Structures and monoamine oxidase (MAO) inhibitory activity of the tested compounds. Figure 1. Structures and monoamine oxidase (MAO) inhibitory activity of the tested compounds. 2. Results 2. Results 2.1. Effects of MAO-B Inhibitors on Rat C6 Glioma and on CTX TNA2 Astrocytes Cell Viability 2.1. Effects of MAO-B Inhibitors on Rat C6 Glioma and on CTX TNA2 Astrocytes Cell Viability Two novel, selective and potent MAO-B inhibitors, Compound 3 (Cmp3) and Compound 5 (Cmp5), wereTwo selectednovel, selective and kept and for potent further MAO-B investigations inhibitors, (FigureCompound1). The3 (Cmp e ffects3) and of CmpCompound3 and Cmp5 5 were(Cmp evaluated5), were by selected means and of MTT kept assayfor further on rat inve C6stigations glioma (Figure cell line 1). compared The effects withof Cmp non-tumoral3 and Cmp5 CTX were evaluated by means of MTT assay on rat C6 glioma cell line compared with non-tumoral CTX TNA2 astrocytes. Cmp3 and Cmp5 were tested individually and compared to TMZ, the widely used TNA2 astrocytes. Cmp3 and Cmp5 were tested individually and compared to TMZ, the widely used drugdrug in GBM in GBM treatment, treatment, to evaluateto evaluate the the eff effectect on on cell cell viability.viability. CmpCmp3 and3 and Cmp Cmp5 were5 were administered administered at at doses doses rangingranging from from 3.125 3.125 up up to to100 100 μM,µ M,whereas whereas TMZ TMZ was administeredwas administered at doses at doses ranging ranging from from 15.625 15.625 upup toto 500 μµM,M, based based on on the the results results already already reported reported

MoleculesMolecules 20192019, ,2424, ,x 2005 FOR PEER REVIEW 33 of of 17 16 in literature [8,9]. In CTX TNA2 astrocytes, Cmp5 at 100 μM evidences cell viability percentages of in literature [8,9]. In CTX TNA2 astrocytes, Cmp5 at 100 µM evidences cell viability percentages ~60% and ~50% after 24 h (Figure 2A) and 72 h of treatment (Figure 2B), respectively, whereas Cmp3 of ~60% and ~50% after 24 h (Figure2A) and 72 h of treatment (Figure2B), respectively, whereas at 100 μM does not affect the cell viability after 24 h (Figure 2A) and displays a cell viability Cmp3 at 100 µM does not affect the cell viability after 24 h (Figure2A) and displays a cell viability percentage of ~60% after 72 h (Figure 2B) of treatment. Conversely, TMZ at 500 μM is shown to percentage of ~60% after 72 h (Figure2B) of treatment. Conversely, TMZ at 500 µM is shown to present present cell viability percentages of ~70% and ~60% after 24 h (Figure 2C) and 72 h of treatment cell viability percentages of ~70% and ~60% after 24 h (Figure2C) and 72 h of treatment (Figure2D), (Figure 2D), respectively. Cmp3 and Cmp5 drastically reduce the viable C6 cells at doses between 50 respectively. Cmp3 and Cmp5 drastically reduce the viable C6 cells at doses between 50 and 100 µM and 100 μM after 24 h and 72 h of treatment (Figure 3A and 3B, respectively), while TMZ at 500 μM after 24 h and 72 h of treatment (Figure3A,B, respectively), while TMZ at 500 µM shows similar C6 shows similar C6 cell viability percentages reported for CTX TNA2 at the same time points. (Figure cell viability percentages reported for CTX TNA2 at the same time points. (Figure3C,D, respectively). 3C and 3D, respectively). These results lead us to keep 50 μM for the Cmp5 and 100 μM for the Cmp3 These results lead us to keep 50 µM for the Cmp5 and 100 µM for the Cmp3 as suitable doses for as suitable doses for further investigations. further investigations. For the sake of comparison, we also tested for the first time two well-recognized, potent and For the sake of comparison, we also tested for the first time two well-recognized, potent selective hMAO-B inhibitors (deprenyl and safinamide) in order to better corroborate our proof-of- and selective hMAO-B inhibitors (deprenyl and safinamide) in order to better corroborate our concept. Our newly synthesized compounds (Cmp3 and Cmp5) were nanomolar inhibitors of proof-of-concept. Our newly synthesized compounds (Cmp3 and Cmp5) were nanomolar inhibitors hMAO-B working in the same range of these two approved drugs. Preliminary MTT experiments of hMAO-B working in the same range of these two approved drugs. Preliminary MTT experiments regarding these two approved hMAO-B inhibitors against C6 and CTX TNA2 cells at 24 h and 72 h regarding these two approved hMAO-B inhibitors against C6 and CTX TNA2 cells at 24 h and 72 h (Figure S1) assessed that, at lower concentrations (0.625–10 µM), safinamide and deprenyl display no (Figure S1) assessed that, at lower concentrations (0.625–10 µM), safinamide and deprenyl display statistically significant differences between tumoral and non-tumoral cell lines (a biological effect no statistically significant differences between tumoral and non-tumoral cell lines (a biological effect which can be further stressed at higher concentrations), differently from what is shown by our novel which can be further stressed at higher concentrations), differently from what is shown by our novel compounds (Cmp3 and Cmp5). compounds (Cmp3 and Cmp5).

Figure 2. MTT cell viability assay on CTX. (A,B) Histograms represent the viability dose-response of Figure 2. MTT cell viability assay on CTX. (A,B) Histograms represent the viability dose-response of CTX cells exposed to different concentrations of Cmp3 and Cmp5 (from 3.125 to 100 µM) for 24 h and CTX cells exposed to different concentrations of Cmp3 and Cmp5 (from 3.125 to 100 μM) for 24 h and 72 h, respectively. (C,D) Histograms represent the viability dose-response of CTX cells exposed to 72 h, respectively. (C,D) Histograms represent the viability dose-response of CTX cells exposed to different concentrations of TMZ (from 15.636 to 500 µM) for 24 h and 72 h. Proliferation was assessed different concentrations of TMZ (from 15.636 to 500 μM) for 24 h and 72 h. Proliferation was assessed using MTT assay and normalized to control cells treated with DMSO (0.2% as final concentration). using MTT assay and normalized to control cells treated with DMSO (0.2% as final concentration).

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FigureFigure 3. 3.MTTMTT cell cell viability viability assay assay on on C6 C6.. ( (A,BA,B)) Histograms Histograms represent thethe viabilityviability dose-response dose-response of of C6 C6cells cells exposed exposed to to di differentfferent concentrations concentrations of of Cmp Cmp3 and3 and Cmp Cmp5 (from5 (from 3.125 3.125 to 100to 100µM) μM) for for 24 h24 and h and 72 h. 72( C,D h. ) (C,D Histograms) Histograms represent represent the viability the viability dose-response dose-response of C6 cells of exposed C6 cells to di exposedfferent concentrations to different µ concentratiof TMZ (fromons of 15.636 TMZ to(from 500 15.636M) for to 24 500 h andμM) 72 for h. 24 Proliferation h and 72 h. was Proliferation assessed usingwas assessed MTT assay using and MTTnormalized assay and to normalized control cells to treated control with cells DMSO treated (0.2% with as DMSO final concentration).(0.2% as final concentration). 2.2. Influence of Cmp3 and Cmp5 on Programmed Cell Death and Necrosis 2.2. Influence of Cmp3 and Cmp5 on Programmed Cell Death and Necrosis The influence of MAO-B inhibitors on the induction of apoptosis in the C6 rat glioma cell cultures The influence of MAO-B inhibitors on the induction of apoptosis in the C6 rat glioma cell after 24 h and 48 h of treatment is shown in Figure4. DMSO-treated cells remain viable over the time of cultures after 24 h and 48 h of treatment is shown in Figure 4. DMSO-treated cells remain viable over the culture, being the percentage of cells stained negative for both Annexin-V and PI assessed at 92.94% the time of the culture, being the percentage of cells stained negative for both Annexin-V and PI and 92.28%, after 24 h and 48 h, respectively. As for cells exposed to Cmp3, the percentage of viable assessed at 92.94% and 92.28%, after 24 h and 48 h, respectively. As for cells exposed to Cmp3, the cells is comparable to that detected for DMSO both after 24 h (94.30%) and 48 h (92.47%). Apoptosis percentage of viable cells is comparable to that detected for DMSO both after 24 h (94.30%) and 48 h and/or necrosis are not observed. A slight, but not significant decrease in the viability of cells exposed (92.47%). Apoptosis and/or necrosis are not observed. A slight, but not significant decrease in the to the Cmp5 can be established after 48 h of treatment (90.07%) as well as a rise in the percentage of the viability of cells exposed to the Cmp5 can be established after 48 h of treatment (90.07%) as well as a PI-stained necrotic population (8.05%) compared to that of DMSO-treated cells (6.47%). rise in the percentage of the PI-stained necrotic population (8.05%) compared to that of DMSO-treated cells (6.47%).

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Figure 4. Induction of apoptosis and necrosis in C6 cells in the presence of Cmp5 and Cmp3. Percentages of cellsFigure found 4. inInduction the various ofphases apoptosis of cell and death, necrosis analyzed in byC6 flowcells cytometry in the presence after staining of Cmp with5 Annexinand Cmp3. V-FITCPercentages and propidium of cells iodide found (PI), in arethe shown various in thephases upper of panel. cell death, Bars represent analyzed the by percentage flow cytometry of viable after cellsstaining (unstained), with cellsAnnexin in apoptosis V-FITC (Annexin and propidium V-positive), iodide cells (PI), in lateare apoptosisshown in (Annexin the upper V+ PI),panel. and Bars cellsrepresent in necrosis the (PI) percentage as medians of viableSD calculated cells (unstained), from three cells independent in apoptosis experiments. (Annexin V-positive), Representative cells in ± dual-parameterlate apoptosis fluorescence (Annexin densityV+PI), and dot plotscells atin 48necros h areis displayed (PI) as medians in the lower ± SD panel. calculated from three independent experiments. Representative dual-parameter fluorescence density dot plots at 48 h are 2.3. Regulationdisplayed of thein the Cell lower Cycle panel. in the C6 Cells Exposed to Cmp3 and Cmp5 In order to establish whether the two MAO-B inhibitors could induce cell cycle arrest checkpoints and2.3. decrease Regulation the C6of the cells Cell proliferation, Cycle in the C6 the Cells DNA Exposed content to Cmp profiles3 and of Cmp cultures5 exposed to the Cmp3 and CmpIn5 afterorder 24 to h (Figureestablish5, upperwhether panel) the obtainingtwo MAO-B percentages inhibitors of cellscould found induce in G1,cell Scycle and G2arrest phasecheckpoints (Figure5, and lower decrease panel) the were C6 analyzed. cells proliferation, The DNA the content DNA content fluorescence profiles emission of cultures peaks exposed and to barsthe related Cmp3 to and the Cmp DMSO5 after sample 24 h (Figure display 5, a upper typical panel) cell cycle obtaining profile, percentages with proliferative of cells found and active in G1, S cellsand (G1 G2 phase phase= 48.18%;(Figure 5, S phaselower =panel)33.46%; wereG2 analyz/M phaseed. The= 18.37%). DNA content The Cmp fluorescence3 causes a delayemission of thepeaks celland cycle bars in therelated G1 phase,to the DMSO being thesample percentage display of a typical cells increased cell cycle in profile, respect with to DMSO-exposed proliferative and cells active (58.46%).cells (G1 Likewise, phase = the 48.18%; percentage S phase of cells = 33.46%; found G2/M in the G1phase phase = 18.37%). after the The Cmp Cmp5 administration3 causes a delay is even of the higher,cell beingcycle in assessed the G1 atphase, 61.51%. being In parallel,the percentage a consistent of cells and increased remarkable in respect change to in DMSO-exposed the percentage of cells cells(58.46%). found in Likewise, the S phase the is percentage registered inof thecells presence found in of the the G1 Cmp phase3 (26.27%) after the and Cmp is further5 administration enhanced is witheven the Cmphigher,5 (18.34%). being assessed at 61.51%. In parallel, a consistent and remarkable change in the percentage of cells found in the S phase is registered in the presence of the Cmp3 (26.27%) and is further enhanced with the Cmp5 (18.34%).

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Figure 5. Cell cycle analysis in C6 rat glioma cells in the presence of Cmp5 and Cmp3. The upper Figure 5. Cell cycle analysis in C6 rat glioma cells in the presence of Cmp5 and Cmp3. The upper panel displays the different DNA profile of cells after a 24 h treatment. Peaks are generated by the panel displays the different DNA profile of cells after a 24 h treatment. Peaks are generated by the emission of PI in the FL3 fluorescence channel. The percentage of cells in the G1, S and G2 phases emission of PI in the FL3 fluorescence channel. The percentage of cells in the G1, S and G2 phases are are shown in the bar graph in the lower panel. Bars represent medians SD from three independent shown in the bar graph in the lower panel. Bars represent medians ±± SD from three independent experiments. *** p < 0.002 between G1 phase of DMSO and Cmp5; *** p < 0.002 between S phase of DMSOexperiments. and Cmp ***5 ;p ** < p0.002< 0.02 between between G1 G1 phase phase of of DMSO DMSO and and Cmp Cmp53; .*** p < 0.002 between S phase of DMSO and Cmp5; ** p < 0.02 between G1 phase of DMSO and Cmp3. 2.4. Generation of Reactive Oxygen Species (ROS) and Depolarization of the Mitochondrial Membrane Potential2.4. Generation (MMP) of in Reactive Cells Exposed Oxygen to Species Cmp3 and (ROS Cmp) and5 Depolarization of the Mitochondrial Membrane PotentialOxidative (MMP) stress, in Cells as detected Exposed byto Cmp the oxidation3 and Cmp of5 CM-H2DCF-DA, significantly increases when the C6 cellsOxidative are exposed stress, to as Cmp detected3 and by Cmp the5 afteroxidation 6 h (Figure of CM-H2DCF-DA,6). Both Cmp significantly3 and Cmp5 increasesgenerate ROS,when registeringthe C6 cells a are 2.4- exposed (Cmp3) to and Cmp a 4-fold3 and (Cmp Cmp5) after increase 6 h (Figure in the DCF 6). Both fluorescence Cmp3 and intensity Cmp5 generate compared ROS, to DMSO-treatedregistering a 2.4- culture. (Cmp After3) and a 24a 4-fold h exposure, (Cmp the5) increase Cmp3 dramatically in the DCF risesfluorescence the ROS intensity production, compared with a 6.2-foldto DMSO-treated increase in culture. respect toAfter cells a exposed24 h exposure, to DMSO the while Cmp the3 dramatically DCF levels relatedrises the to ROS Cmp production,5-exposed culturewith a are6.2-fold comparable increase within respect the one to cells exposed expo tosed DMSO. to DMSO According while the to DCF the inductionlevels related of oxidative to Cmp5- stress,exposed MMP culture is found are depolarizedcomparable inwith the the presence one ex ofposed the twoto DMSO. MAO inhibitors According as to shown the induction in Figure 6of. Inoxidative more detail, stress, after MMP 6 h treatmentis found depolarized Cmp3 halves in thethe MMPpresence as compared of the two to MAO exposure inhibitors to DMSO as shown control. in TheFigure depolarization 6. In more detail, of the after MMP 6 h caused treatment by theCmp Cmp3 halves3 exposure the MMP is remarkableas compared as to compared exposure to DMSO MMP depolarizationcontrol. The depolarization upon Cmp5 treatment of the MMP after caused the same by exposurethe Cmp3 period, exposure being is remarkable the MMP level as compared comparable to toMMP the DMSO depolarization sample. upon Cmp5 treatment after the same exposure period, being the MMP level comparableAfter longer to the experimental DMSO sample. times (24 h), Cmp3 retains a consistent and significant disturbance of the MMP,After in longer respect experimental to the DMSO times sample, (24 h), being Cmp3 Mean retains Fluorescence a consistent Intensities and significant (MFIs) disturbance assessed at of 2.23the MMP,105 (Cmp in respect3) and to 3.13 the DMSO105 (DMSO). sample, In parallel,being Mean Cmp Fluorescence5 considerably Intensities lowers MMP (MFIs) if compared assessed to at × × 62.23 h exposure, × 105 (Cmp revealing3) and values3.13 × 10 comparable5 (DMSO). with In parallel, those registered Cmp5 considerably for Cmp3 (MFI lowers of Cmp MMP5 = if2.18 compared105) × (Figureto 6 h 6exposure,). revealing values comparable with those registered for Cmp3 (MFI of Cmp5 = 2.18 × 105) (Figure 6).

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FigureFigure 6. Generation6. Generation of of intracellular intracellular reactive oxyge oxygenn species species (ROS) (ROS) and and mitochondrial mitochondrial membrane membrane potentialpotential (MMP) (MMP) modulation modulation in in C6 C6 cellscells in the presence presence of of Cmp Cmp5 and5 and Cmp Cmp3. Bars3. Bars in the in lower the lower panel panel representrepresent median median values values ±SD SD of of thethe meanmean fluoresce fluorescencence intensity intensity (MFI) (MFI) generated generated by the by oxidation the oxidation of of ± CM-H2DCF-DACM-H2DCF-DA (generation (generation of of intracellular intracellular ROS) ROS) an andd by by the the emission emission of TMRE of TMRE (MMP) (MMP) measured measured by by flowflow cytometry cytometry in cellsin cells exposed exposed to toMAO-B MAO-B inhibito inhibitors.rs. Representative Representative fluorescence fluorescence emission emission peakspeaks are are shownshown in the in the upper upper panel panel and and are are provided provided to to display display the theshift shift inin thethe fluorescencefluorescence emissions emissions in in the the FL1 FL1 (FITC) and FL2 (PE) channels. **** p < 0.0005, *** p < 0.005, ** p < 0.02. (FITC) and FL2 (PE) channels. **** p < 0.0005, *** p < 0.005, ** p < 0.02.

2.5. Nitric2.5. Nitric Oxide Oxide Synthase Synthase 1 1 (NOS-1), (NOS-1), NitricNitric Oxide Synthase Synthase 2 2(NOS-2) (NOS-2) and and Vascular Vascular Endothelial Endothelial Growth Growth FactorFactor (VEGF) (VEGF) Expression Expression in in Response Response toto MAO-BMAO-B Inhibitors Inhibitors in in Rat Rat C6 C6 cells cells To identifyTo identify the etheffects effects of Cmp of Cmp3 at 1003 at µ100M andμM Cmpand Cmp5 at 505 atµ M50 on μM the on inflammatory the inflammatory event event induction, induction, a Western Blot Analysis of neuronal NOS-1 and inducible NOS-2 was performed after 6 a Western Blot Analysis of neuronal NOS-1 and inducible NOS-2 was performed after 6 and 24 h of and 24 h of treatment. After 6 h of treatment, no significant difference in NOS-1 expression level is treatment.recorded After in samples 6 h of treated treatment, with noboth significant Cmp3 and diCmpfference5 within respect NOS-1 to the expression DMSO sample. levelis After recorded 24 in samplesh of treatment, treated with the bothNOS-1 Cmp expression3 and Cmp level5 iswith significantly respect tolower the in DMSO cells treated sample. with After Cmp 245 in h ofrespect treatment, the NOS-1to cells treated expression with levelCmp3 is. Moreover, significantly from lower 6 h to 24 in h cells of treatment, treated with a statistically Cmp5 in significant respect to decrease cells treated withof Cmp the NOS-13. Moreover, expression from is detectable 6 h to 24 hfor of Cmp treatment,3 and Cmp a statistically5 (Figure 7A, significant B). decrease of the NOS-1 expressionAfter is detectable6 h of treatment for Cmp a 3statisticallyand Cmp 5significant(Figure7A,B). increase in the NOS-2 expression level is appreciableAfter 6 h of in treatment samples treated a statistically with both significant Cmp3 and increase Cmp5 with in the respect NOS-2 to expressionDMSO sample. level After is appreciable 24 h in samplesof treatment treated the withNOS-2 both expression Cmp3 and is markedly Cmp5 with reduced respect in cells to DMSOtreated sample.with both After Cmp3 24 and h of Cmp treatment5 the NOS-2in respect expression to the DMSO is markedly sample. A reduced notable indecrease cells treated of the protein with both expression Cmp3 andin samples, Cmp5 inexposed respect to to the both Cmp3 and Cmp5, is reported from 6 h to 24 h of exposure (Figure 7A, C). DMSO sample. A notable decrease of the protein expression in samples, exposed to both Cmp3 and Furthermore, a western blot of VEGF, a major contributor of tumorigenesis and angiogenesis, Cmpwas5, is performed. reported fromAfter 66 hh, tothe 24 protein h of exposure is significantly (Figure higher7A,C). in cells treated with Cmp3 in respect to theFurthermore, DMSO sample; a western conversely, blot no of significant VEGF, a diff majorerences contributor are found ofin tumorigenesisC6 cells treated with and Cmp angiogenesis,5 as wascompared performed. to DMSO. After 6In h, addition, the protein a significant is significantly reduction higher of the VEGF in cells expression treated is with evidenced Cmp3 forin respectcells to the DMSOtreated with sample; Cmp conversely,3 from 6 to 24 no h significant(Figure 7A, D). diff erences are found in C6 cells treated with Cmp5 as compared to DMSO. In addition, a significant reduction of the VEGF expression is evidenced for cells treated with Cmp3 from 6 to 24 h (Figure7A,D). MoleculesMolecules2019, 201924, 2005, 24, x FOR PEER REVIEW 8 of 178 of 16

Figure 7. Western blotting analysis of NOS-1, NOS-2 and VEGF expression in rat C6 glioma cell lines Figure 7. Western blotting analysis of NOS-1, NOS-2 and VEGF expression in rat C6 glioma cell lines treated with Cmp5 and Cmp3.(A) Cells treated with DMSO (0.2%) were loaded as the negative control. treated with Cmp5 and Cmp3. (A) Cells treated with DMSO (0.2%) were loaded as the negative β Eachcontrol. membrane Each has membrane been probed has been with probed–actin with antibody β–actin to antibody verify loading to verify consistency. loading consistency. Western blot is theWestern most representative blot is the most of representative three different of experiments.three different (experiments.B–D) Histograms (B–D) Histograms represent densitometricrepresent measurementsdensitometric of proteinsmeasurements bands of expressed proteins bands as integrated expressed optical as integrated intensity optical (IOI) intensity mean of (IOI) three mean separate experiments.of three separate The error experiments. bars on these The graphserror bars show on standardthese graphs deviation show standard ( SD). Densitometricdeviation (± SD). values ± analysedDensitometric by ANOVA values (post analysed hoc applicationby ANOVA (post of Tukey’s hoc application multiple of comparisonTukey’s multiple test) comparison return significant test) differences.return significant **** p < 0.0001, differences. *** p <****0.0002, p < 0.0001, ** p ***< 0.0005, p < 0.0002, * p <** 0.005.p < 0.0005, * p < 0.005.

2.6. Prostaglandin2.6. Prostaglandin E2 (PGE-2)E2 (PGE-2) Secretion Secretion Level Level in inCells Cells Exposed to to Cmp Cmp3 3andand to toCmp Cmp5 5 The ELISAThe ELISA assay assay for for PGE-2 PGE-2 secretion secretion was was performedperformed on on the the C6 C6 cell cell line line treated treated with with Cmp Cmp5 and5 and Cmp3Cmpat 503 atµ M50 μ andM and 100 100µM, μM, respectively, respectively, for for 24 24 andand 72 h (Figure (Figure 8).8). As As evidenced evidenced in Figure in Figure 8, both8, both compoundscompounds notably notably enhanced enhanced the the secretion secretion ofof PGE-2 on on rat rat C6 C6 glioma glioma cell cell lines lines at 24 at h 24 and h and72 h 72 h Moleculescompared 2019, to24 , cellsx FOR treated PEER REVIEW with DMSO. Furthermore, Cmp5 at 50 μM is responsible for a higher9 ofPGE- 17 compared to cells treated with DMSO. Furthermore, Cmp5 at 50 µM is responsible for a higher PGE-2 2 secretion level with respect to cells treated with Cmp3 at 100 μM after 24 and 72 h. secretion level with respect to cells treated with Cmp3 at 100 µM after 24 and 72 h.

Figure 8. ELISA assay in C6 rat glioma cells in the presence of Cmp3 and Cmp5. PGE2 secretion levels, measured after 24 and 72 h, are reported as pg/mL/OD MTT values. Data shown are the mean ( SD) Figure 8. ELISA assay in C6 rat glioma cells in the presence of Cmp3 and Cmp5. PGE2 secretion levels, ± of threemeasured different after experiments.24 and 72 h, are Cmp reported5 50 µasM pg/m vs.L/OD ctrl, *Cmp MTT 3values.100 µ DataM vs. shown Cmp 5are50 theµM meanp < (±0.05. SD) of three different experiments. Cmp5 50 μM vs ctrl, *Cmp3 100 μM vs Cmp5 50 μM p < 0.05.

2.7. Effects of MAO-B Inhibitors on Cell Migration and Metalloproteinases Expression A transwell migration assay was executed using an 8 μm pore size polycarbonate membrane in C6 cells in the absence or presence of 100 μM Cmp3 and 50 μM Cmp5. Cells were exposed for 24 h to medium with or without chemoattractant (Foetal Bovine Serum) in the presence or absence of compounds where migrated cells were then stained with crystal violet. Both compounds provoke a remarkable reduction of cell migration with respect to the DMSO sample (Figure 9). To evaluate the degradation of extracellular matrix, Matrix Metalloproteinases 2 and 9 (MMP-2 and MMP-9) expression levels were investigated with Western Blot Analysis. The MMP-2 expression is significantly reduced when cells are treated with Cmp5 with respect to the DMSO sample after both time points. After 24 h of treatment, the MMP-2 expression appears significantly increased in cells treated with Cmp3 in respect to DMSO and Cmp5 samples (Figure 10A, B). In parallel, the MMP-9 protein level is markedly augmented in cells treated for 6 h with Cmp3 compared to Cmp5 and DMSO samples. After 24 h of treatment, the MMP-9 expression was markedly reduced in cells treated with both compounds in respect to the DMSO sample (Figure 10 A,C). In addition, Cmp3 provokes a marked reduction of MMP-9 from 6 to 24 h treatment, whereas Cmp5 downregulates both MMPs (Figure 10 A–C).

Molecules 2019, 24, 2005 9 of 16

2.7. Effects of MAO-B Inhibitors on Cell Migration and Metalloproteinases Expression A transwell migration assay was executed using an 8 µm pore size polycarbonate membrane in C6 cells in the absence or presence of 100 µM Cmp3 and 50 µM Cmp5. Cells were exposed for 24 h to medium with or without chemoattractant (Foetal Bovine Serum) in the presence or absence of compounds where migrated cells were then stained with crystal violet. Both compounds provoke a remarkable reduction of cell migration with respect to the DMSO sample (Figure9). To evaluate the degradation of extracellular matrix, Matrix Metalloproteinases 2 and 9 (MMP-2 and MMP-9) expression levels were investigated with Western Blot Analysis. The MMP-2 expression is significantly reduced when cells are treated with Cmp5 with respect to the DMSO sample after both time points. After 24 h of treatment, the MMP-2 expression appears significantly increased in cells treated with Cmp3 in respect to DMSO and Cmp5 samples (Figure 10A,B). In parallel, the MMP-9 protein level is markedly augmentedMolecules 2019, in 24, cells x FOR treated PEER REVIEW for 6 h with Cmp3 compared to Cmp5 and DMSO samples. After10 24 of h17 of treatment, the MMP-9 expression was markedly reduced in cells treated with both compounds in respect to the DMSO sample (Figure 10 A,C). In addition, Cmp3 provokes a marked reduction of MMP-9 from 6 to 24 h treatment, whereas Cmp5 downregulates both MMPs (Figure 10A–C).

Figure 9. Transwell migration assay in C6 rat glioma cell line in the presence of Cmp5 and Cmp3. Dose treatmentsFigure 9. Transwell are 50 and migration 100 µM for assay Cmp in5 and C6 rat Cmp glio3, respectivelyma cell line (upperin the presence panel). Histogram of Cmp5 and represents Cmp3. densitometricDose treatments analysis are 50 determined and 100 μM by for quantifying Cmp5 and thresholded Cmp3, respectively area for violet (upper color panel). in ten Histogram fields for eachrepresents of three densitometric slides per sample analysis (lower determined panel). Data by quantifying are presented threshol as meanded standardarea for violet deviation. color Cmp in ten5 ± 50fieldsµM versusfor each ctrl; of *Cmpthree3 slides100 µ Mper versus sample ctrl: (lowerp < 0.0001. panel). Data are presented as mean ± standard deviation. Cmp5 50 μM versus ctrl; *Cmp3 100 μM versus ctrl: p < 0.0001.

Molecules 2019, 24, x FOR PEER REVIEW 11 of 17 Molecules 2019, 24, 2005 10 of 16

Figure 10. Western blotting analysis of MMP-2, MMP-9 expression in rat C6 glioma cell lines treated withFigure Cmp 10.5 Westernand Cmp blotting3.(A) Cellsanalys treatedis of MMP-2, with DMSO MMP-9 (0.2%) expression were loaded in rat C6 as negativeglioma cell control. lines treated Each membranewith Cmp5 has and been Cmp probed3. (A) Cells with treatedβ–actin with antibody DMSO to (0.2%) verify were loading loaded consistency. as negative Western control. blot Each is themembrane most representative has been probed of three with di βff–actinerent antibody experiments. to verify (B,C loading) Histograms consistency. represent Western densitometric blot is the measurementsmost representative of proteins of bandsthree expresseddifferent asexperiments. integrated optical (B) Histograms intensity (IOI) represent mean of threedensitometric separate experiments.measurements The of proteins error bars bands on expressed these graphs as integrat showed standard optical intensity deviation (IOI) ( meanSD). ****of threep < separate0.0001, ± ***experiments.p < 0.0002, **Thep < error0.0005, bars * pon< 0.005.these graphs show standard deviation (± SD). **** p < 0.0001, *** p < 0.0002, ** p < 0.0005, * p < 0.005. 3. Discussion 3. DiscussionIt is widely reported in scientific literature that the MAO-B activity appears to increase with agingIt as is well widely as in reported patients in aff ectedscientific by neurodegenerativeliterature that the conditionsMAO-B activity including appears Parkinson’s to increase disease with andaging Alzheimer’s as well as in disease, patients thus affected explaining by neurod theegenerative administration conditions of MAO-B including inhibitors Parkinson’s to treat disease the abovementionedand Alzheimer’s diseases disease, [10 thus]. Nevertheless, explaining Gabilondothe administration and colleagues of MAO-B demonstrated inhibitors a significant to treat and the selectiveabovementioned increase indiseases the MAO-B [10]. activityNevertheless, in human Gab gliomasilondo and compared colleagues with demonstrated non-tumoral tissues a significant [5,11]. and Therefore,selective increase we evaluated in the MAO-B for the firstactivity time in the human biological gliomas effects compared of two novelwith non-tumoral MAO-B inhibitors, tissues which[5,11]. were synthesized in our medicinal chemistry laboratory, on the C6 glioma cell line and on CTX TNA2Therefore, non-tumoral we astrocytes, evaluated infor terms the first of cell time proliferation, the biological apoptosis effects occurrence,of two novel inflammatory MAO-B inhibitors, events andwhich cell were migration synthesized in order in toour improve medicinal the chemistry design and laboratory, the assessment on the of C6 anti-glioma glioma cell therapies. line and on CTX TNA2The non-tumoral two novel MAO-B astrocytes, inhibitors, in terms Cmp of3 andcell Cmpproliferation,5, appeared apoptosis as promising occurrence, molecules inflammatory targeting MAO-Bevents and and cell reporting migration in glioma in order cells to aimprove percentage the ofdesign cell viability and the notablyassessment lower of thananti-glioma that recorded therapies. for TMZ,The which two is novel the most MAO-B used inhibitors, drug in GBM. Cmp Surprisingly,3 and Cmp5, appeared we also found as promising that Cmp molecules3 and Cmp targeting5 were capableMAO-B of and blocking reporting tumor in glioma cell proliferation cells a percentage while sparing of cell normal viability astrocytes. notably lower We further than that investigated recorded thisfor aspectTMZ, which by flow is cytometricthe most used Annexin-V drug in assay GBM. discovering Surprisingly, that we the also block found of tumor that Cmp cell proliferation3 and Cmp5 waswere unrelated capable toof apoptosis blocking ortumor to necrosis. cell proliferation while sparing normal astrocytes. We further investigatedAs commonly this aspect reported by flow in scientific cytometric literature Annexin-V in diff erentassay experimentaldiscovering that models the block of glioma, of tumor several cell cytotoxicproliferation drugs was provoked unrelated an to anti-proliferativeapoptosis or to necrosis. effect inducing the G1 or G2/M cell cycle phase accumulationAs commonly leading reported to a final in cellscientific cycle arrestliterature [12, 13in]. different Based on experimental this knowledge, models the of cell glioma, cycle phasesseveral cytotoxic drugs provoked an anti-proliferative effect inducing the G1 or G2/M cell cycle phase

Molecules 2019, 24, 2005 11 of 16 were measured by flow cytometry to further elucidate the biological mechanism underlying MAO-B inhibitory activity in tumor cells. We found that our novel MAO-B inhibitors, especially Cmp5, were responsible for an accumulation of C6 cells in the G1 phase and in parallel to a notable reduction of cells in the S phase. Our data are in accordance to previous studies demonstrating that MAO inhibitors, both MAO-A and MAO-B inhibitors, as well as and , are able to inhibit the proliferation of prostate and breast cancer cells [14,15]. Furthermore, we measured the ROS production leading us to assume that the ROS increase after treatment with both MAO-B inhibitors could be largely responsible for the reduction of the S phase of C6 glioma cells, as already hypothesized [16]. It is possible to assume that our data on cell cycle report a clear temporal distinction between the ROS induction by Cmp3 and Cmp5. Indeed, Cmp5 is an early ROS inducer, whereas Cmp3 is a stronger, but late inducer. This could represent a valuable and promising result considering that the increase of intracellular ROS can be therapeutically exploited. In fact, several chemotherapeutic agents may exert a selective toxic activity against cancer cells because they raise oxidative stress forcing the already stressed cells behind their limit [17]. Given that a notable reduction of the MMP predicts a marked oxidative stress, our data are further supported by the MMP, which appears markedly depolarized when the two MAO inhibitors are administrated. This trend could be related to the chemical-physical differences between the two compounds. Cmp3 has a CLogP value of 4.30, whereas Cmp5 has a CLogP value of 4.09, thus reinforcing our data on the early mitochondrial membrane depolarization possibly due to the Cmp3 higher lipophilicity (associated to a better membrane permeability). The ability of our tested compounds to increase the base level of oxidative stress is also confirmed by the augmented PGE-2 secretion level measured after 24 h and 72 h of treatment, thus underlining the critical role played by oxidative stress in the mechanism of action of chemotherapeutic agents, as also hypothesized by Sun and colleagues [18]. Since it is well known that NOS activation is regulated by ROS and Reactive Nitrogen Species and that NOS-2 is implicated in human malignant tumors [19,20], we investigated the NOS expression. At an early stage, both our compounds did not modify the expression level of constitutive subtype of NOS-1, whereas they evidenced a remarkable ability to upregulate the expression level of inducible isoform NOS-2, thus contributing to the dramatic induction of a fatal drug-induced oxidative stress. However, the tested compounds did not reveal an up-regulation of NOS-2 at long time exposure, this could be due to a possible cell mechanism of resistance triggered after a longer drug administration. Malignant gliomas are also well known for high vascularization suggesting that the recruitment of VEGF, the most important contributor of angiogenesis, represents a critical step for the progression of this malignancy [21]. Given that the VEGF expression is Nitric Oxide dependent [22], we evaluated the protein level, finding as expected, that the signaling pathway triggered by MAO-B inhibitors, involving ROS and NOS-2, is also supported by VEGF recruitment. It appears significantly augmented only at an early stage with Cmp3, thus supporting the hypothesis of the oxidative stress-promoted mechanism of action. Moreover, the acquisition of a migratory phenotype is the prerequisite for its metastatic spreading and invasive potential [23]. Based on this evidence, we evaluated the effects of Cmp3 and Cmp5 on C6 cell migration. Surprisingly, both novel MAO-B inhibitors markedly reduced glioma cell migration, thus significantly preventing the invasiveness of brain tumors and their ability to infiltrate the neighbouring tissues as already demonstrated for several anti-glioma agents [24,25]. To clarify the molecular mechanism underlying this result, we evaluated the expression levels of MMP-2 and MMP-9, two proteases of the Matrix Metalloproteinase family involved in the remodeling and turnover of local extracellular matrix components. The MMP-2 and MMP-9 received particular attention given that their increased expression appeared related to the malignant potential of several types of cancer, including human gliomas [26]. We evidenced that Cmp5 notably reduces glioma cell migration via the down-regulation of MMP-2 and MMP-9. These results are in agreement with data reported in scientific literature such as those reported by Webb [27] who explained that an up-regulation of MMP-2 and MMP-9 may result in cellular invasion, migration and in cancer metastasis. On the other hand, Cmp3, Molecules 2019, 24, 2005 12 of 16 downregulating MMP-9 only at long time exposure, led us to assume that an alternative pathway could be involved in the reduction of cell migration. In conclusion, this study demonstrates that our novel MAO-B inhibitors are able to contrast glioma proliferation by arresting the cell cycle and drastically increasing oxidative stress conditions, as well as the invasiveness by markedly reducing the migration of malignant cells. These findings reinforce the critical role played by MAO-B in mediating oncogenesis in high-grade glioma. Therefore, targeting the MAO-B protein could be a new approach to achieve improved therapeutic efficacy for glioblastoma.

4. Materials and Methods

4.1. Drugs and Reference Inhibitors The two compounds were synthesized, purified and characterized as recently reported [6], whereas the two approved hMAO-B inhibitors (deprenyl and safinamide) were provided by Sigma-Aldrich (Milan, Italy). CLogP values for the newly-synthesized compounds were generated by ChemBioDraw Ultra 12.0.

4.2. Cell Lines C6 rat glioma cell line (ECACC 92090409, Sigma Aldrich, Milan, Italy) and CTX/TNA2 rat astrocytes (ATCC CRL-2006TM) were cultured in Ham’s F12 and in DMEM high Glucose medium, respectively, supplemented with 10% of foetal bovine serum (FBS), 1% of penicillin/streptomycin and 1% of l-glutamine (all purchased by EuroClone, Milan, Italy). Cell cultures were maintained in an incubator in a humidified atmosphere with 5% CO2 at 37 ◦C.

4.3. MTT Assay The C6 and CTX/TNA2 cells were seeded at cell density of 8000/well into a 96-well tissue culture plate. The metabolic activity of C6 and CTX/TNA2 cell lines was evaluated after 24 h and 72 h of treatment with Cmp3 and Cmp5 at 3.125, 6.25, 12.5, 25, 50, 100 µM and with TMZ at doses ranging from 15.625 up to 500 µM, on a 96-well polystyrene plate through MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Sigma Aldrich, Milan, Italy). The assay is based on the capability of viable cells to reduce MTT into a colored formazan product. Compounds were dissolved in DMSO with a final concentration of 0.2%. At the established time points, the medium was replaced by a new one containing 0.5 mg/mL MTT and probed with cells for 4 h at 37 ◦C. The plate was incubated in DMSO solution for 30 min at 37 ◦C to solubilize salts and then read at 540 nm by means of a microplate reader (Multiskan GO, Thermo Scientific, MA, USA). Values obtained in the absence of cells were considered as background. Viability was normalized to control cells treated with the vehicle DMSO.

4.4. Detection of Apoptosis by Flow Cytometry C6 rat glioma cells (2.375 105/well) were cultured in 6-well plates for 24 h at 37 C in a humidified × ◦ atmosphere. The growth medium was then removed and the cells were subsequently exposed to treatments. Apoptosis detection was performed after 24 and 48 h collecting supernatants in tubes, cells were washed once with PBS at room temperature. Next, cells were trypsinized and collected by centrifugation together with supernatants. Apoptotic and necrotic cells were detected after staining cells with an Annexin-V and Propidium Iodide (PI) kit (eBioscience, Thermo Fisher Scientific, MA, USA), following the manufacturer’s instructions. Briefly, samples were incubated in 197 µL of binding buffer and 3 µL of Annexin-V FITC for 15 min at room temperature in the dark. Volumes were afterwards doubled, cells were washed once by centrifugation and re-suspended into 300 µL of binding buffer with PI. The fluorescence was determined by a CytoFlex flow cytometer (Beckman Coulter, CA, USA). FITC fluorescence (FL-1) was analyzed by a 530/30 band pass filter, and PI fluorescence (FL-3) by a 650 nm long pass filter. Data acquisition (2 104 events/sample) and data analysis were performed × Molecules 2019, 24, 2005 13 of 16 with the CytExpert Software (Beckman Coulter, CA, USA). The percentages of viable cells (Annexin V ; PI ) were detected in the lower left quadrant (unstained) of density plots, as well as the cells − − in apoptosis (Annexin V+/PI , lower right quadrant), late apoptosis (Annexin V+/PI+, upper right − quadrant) and necrosis (Annexin V /PI+, upper left quadrant). − 4.5. Cell Cycle Analysis The C6 rat glioma cells were seeded and exposed as previously described in this section. After 24 h exposure, the medium was removed and cells were washed once with PBS, trypsinized and collected by centrifugation. Next, the cells were counted by means of Trypan blue dye exclusion test and 3 105 cells/sample were fixed in cold ethanol 70% v/v and kept at 4 C overnight. After that, the × ◦ cells were gently washed with cold PBS and centrifuged at 2700 g for 10 min at 4 ◦C. After having discarded supernatants, each sample was incubated in 300 µL of the staining solution containing PBS without calcium and magnesium, RNase 100 µg/mL (stock solution 10 mg/mL in 10 mM sodium acetate buffer, pH 7.4) and PI 10 µg/mL (stock solution 1 mg/mL in water) (all purchased by Sigma Aldrich, MI, USA) and kept at 4 ◦C overnight in the dark. PI fluorescence was detected by a flow cytometer equipped with a 488 nm laser (CytoFlex flow cytometer, Beckman Coulter, CA, USA) in the FL-3 channel (620 nm of wavelength emission). 2 104 events/sample were collected and analyzed × with CytExpert Software (Beckman Coulter, CA, USA). The percentage of cells in G1, S, or G2 phase of the cell cycle were calculated after mathematical modeling of histograms using the FCS Express Flow Cytometry Data Analysis (De Novo Software, CA, USA).

4.6. Flow Cytometry Analysis of ROS and MMP The C6 rat glioma cells were cultured and stimulated as described for the apoptosis assay. The intracellular generation of ROS was determined using the chloromethyl derivative of 20,70-Dichlorofluorescein Diacetate (DCF-DA) (CM-H2DCF-DA, Molecular Probes, Invitrogen, CA, USA), an oxidation-sensitive probe, while the fluctuations of the mitochondrial membrane potential was monitored by means of the TMRE (Tetramethylrhodamine, Ethyl Ester) Assay Kit (Abcam, Cambridge, UK). After 6 and 24 h, exposure medium was removed and cells were incubated with PBS/FBS 10% and CM-H2DCFDA 2.5 µM at 37 ◦C in a humidified atmosphere in the dark. After 30 min, 100 nM TMRE was added and cells were incubated for an additional 30 min. Next, cells were washed once with PBS, trypsinized and collected by centrifugation. After having resuspended cells in 30 µL of PBS, DCF and TMRE fluorescence emissions were measured by flow cytometry (CytoFlex flow cytometer, Beckman Coulter, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 527 nm (DCF-FITC) and 575 nm (TMRE-PE). Mean Fluorescence Intensity (MFI) was obtained by histogram statistics using the CytExpert Software (Beckman Coulter, CA, USA). Individual values of MFI were normalized on negative controls (unstained samples) and are provided as mean values of MFI ratio SD. ± 4.7. Western Blot Analyses The C6 cell lysates (20 µg) were electrophoresed and transferred to the nitrocellulose membrane. Nitrocellulose membranes, blocked in 5% of non-fat milk in PBS 0.1% Tween-20, were probed with mouse monoclonal anti-β actin antibody (antibody dilution 1:5000) (A5316 Sigma, MO, USA), rabbit polyclonal anti-VEGF anti-NOS-2 antibodies (antibodies dilution 1:200) (sc-152, sc-651, respectively, both purchased by Santa Cruz biotechnology, CA, USA), mouse monoclonal anti-HIF-1α, anti-MMP-9, anti-MMP-2, anti-NOS-1 antibodies (antibodies dilution 1:200) (sc-5354, sc-393859, sc-13595, sc-5302, respectively, all purchased by Santa Cruz biotechnology, CA, USA), then incubated in the presence of specific conjugated IgG horseradish peroxidase. Immunoreactive bands were detected by ECL system (Amersham Int., Buckunghamshire, UK) and analyzed by densitometry. Densitometric values, expressed as Integrated Optical Intensity (IOI), were estimated in a CHEMIDOC XRS system by the Molecules 2019, 24, 2005 14 of 16

QuantiOne 1-D analysis software (BIORAD, Richmond, CA, USA). Values obtained were normalized based on densitometric values of loading control β-actin.

4.8. ELISA Test for PGE-2 The PGE-2 secretion in the culture medium was detected by following the instructions provided by the manufacturer’s protocol. EIA kit (Enzo Life Sciences, Farmingdale, NY, USA) was used to measure PGE-2 concentrations. The absorption values were obtained by the spectrophotometric reading of plates at 450 and 405 nm, respectively, by means of a microplate reader (Multiskan GO, Thermo Scientific, MA, USA). Secretion levels of PGE-2 were analyzed in different wells after treatment with Cmp5 at 50 µM and Cmp3 at 100 µM, normalized for relative optical density (pg/mL/OD) as previously determined by the MTT assay.

4.9. Transwell Migration Assay The C6 cell migration was evaluated by means of a 24-well Transwell Boyden chamber containing 8 µm pore size membranes (Corning, Lowell, MA, USA). For this purpose, suspended C6 rat glioma cells were separately treated with 50 µM Cmp5 and 100 µM Cmp3 in serum-free Ham’s F12 at cell density of 50,000/150 µL, and then added to the upper chamber of a 8 µm pore size insert. Ham’s F12 supplemented with 10% FBS was added to the lower chamber as a chemoattractant and allowed to migrate towards a 10% FBS containing medium present in the lower chamber. Cells were incubated for 24 h at 37 ◦C, the non-migrating cells on the upper chamber were then removed with the aid of a cotton swab, whereas the cells migrated to the lower surface of the membrane were stained with crystal violet for 10 min at room temperature. Images were captured at 20x magnification with a light microscope (Leica DM 4000, Leica Cambridge Ltd., Cambridge, UK) equipped with a Leica DFC 320 camera (Leica Cambridge Ltd.). Images were analyzed by means of Leica Application Suite–X (LAS-X) analysis software. The migration rate was calculated as the area covered by stained cells, given that they represent the migrated cells.

4.10. Statistics Statistical analysis was executed with GraphPad 7 software using t-test and Ordinary One-Way ANOVA followed by post-hoc Tukey’s multiple comparisons tests. Values of p < 0.05 were considered statistically significant.

Supplementary Materials: The following are available online. Figure S1: MTT cell viability assay on C6 and CTX TNA2. (A, C) Histograms represent the viability dose-response of C6 cells exposed to different concentrations of deprenyl and safinamide (0.625 up to 10 µM) for 24 h and 72 h. (B, D) Histograms represent the viability dose-response of CTX cells exposed to different concentrations (0.625 up to 10 µM) of deprenyl and safinamide for 24 h and 72 h. Proliferation was assessed using MTT assay and normalized to control cells treated with DMSO (0.2% as final concentration). Author Contributions: G.D.M. performed the experiments and wrote the manuscript, M.G. executed and analyzed flow cytometry experiments, S.C. and P.G. designed, synthesized and purified the novel drugs, A.C. coordinated the research project and S.Z. designed and funded the experimental project. Funding: This research has been financed by Cataldi RES, FABBR 2018 Zara Susi and FAR2017 Zara Susi. Acknowledgments: The authors would like to thank Filomena Natarelli for the English language editing. Conflicts of Interest: All the authors state that there is no conflict of interest.

References

1. Weller, M.; Wick, W.; Aldape, K.; Brada, M.; Berger, M.; Pfister, S.M.; Nishikawa, R.; Rosenthal, M.; Wen, P.Y.; Stupp, R.; et al. Glioma. Nat. Rev. Dis. Primers 2015, 1, 15017. 2. Ulasov, I.; Yi, R.; Guo, D.; Sarvaiya, P.; Cobbs, C. The emerging role of MMP14 in brain tumorigenesis and future therapeutics. Biochim. Biophys. Acta 2014, 1846, 113–120. [CrossRef][PubMed] Molecules 2019, 24, 2005 15 of 16

3. Perazzoli, G.; Prados, J.; Ortiz, R.; Caba, O.; Cabeza, L.; Berdasco, M.; Gónzalez, B.; Melguizo, C. Temozolomide Resistance in glioblastoma cell lines: Implication of MGMT, MMR, P-glycoprotein and CD133 expression. PLoS ONE 2015, 10, e0140131. [CrossRef][PubMed] 4. Finberg, J.P.; Rabey, J.M. Inhibitors of MAO-A and MAO-B in psychiatry and neurology. Front. Pharmacol. 2016, 7, 340. [CrossRef][PubMed] 5. Gabilondo, A.M.; Hostalot, C.; Garibi, J.M.; Meana, J.J.; Callado, L.F. Monoamine oxidase B activity is increased in human gliomas. Neurochem. Int. 2008, 52, 230–234. [CrossRef] 6. Secci, D.; Carradori, S.; Petzer, A.; Guglielmi, V.; D’Ascenzio, M.; Chimenti, P.; Bagetta, V.; Alcaro, S.; Zengin, G.; Petzer, J.P.; et al. 4-(3-Nitrophenyl)thiazol-2-ylhydrazone derivatives as antioxidants and selective hMAO-B inhibitors: Synthesis, biological activity and computational analysis. J. Enzym. Inhib. Med. Chem. 2019, 34, 597–612. [CrossRef] 7. Secci, D.; Locatelli, M.; Kabir, A.; Salvatorelli, E.; Macedonio, G.; Mollica, A.; Carradori, S. Investigation on the stability of new biologically active thiosemicarbazone-derived compounds by a validated HPLC-PDA method. Curr. Anal. Chem. 2019, 15, 313–320. [CrossRef] 8. Azzalin, A.; Nato, G.; Parmigiani, E.; Garello, F.; Buffo, A.; Magrassi, L. Inhibitors of GLUT/SLC2A enhance the action of BCNU and Temozolomide against high-grade gliomas. Neoplasia 2017, 19, 364–373. 9. Lan, F.; Yang, Y.; Han, J.; Wu, Q.; Yu, H.; Yue, X. Sulforaphane reverses chemo-resistance to temozolomide in glioblastoma cells by NF-κB-dependent pathway downregulating MGMT expression. Int. J. Oncol. 2016, 48, 559–568. [CrossRef][PubMed] 10. Tipton, K.F.; Boyce, S.; O’Sullivan, J.; Davey, G.P.; Healy, J. Monoamine oxidases: Certainties and uncertainties. Curr. Med. Chem. 2004, 11, 1965–1982. [CrossRef] 11. Callado, L.F.; Jesús, M.; Garibi, J.; Meana, J. Gliomas: Role of Monoamine Oxidase B in Diagnosis. In Tumors of the Central Nervous System (Part 1); Hayat, M., Ed.; Springer: Dordrecht, The Netherlands, 2011; Volume 1, pp. 53–59. 12. He, X.; Maimaiti, M.; Jiao, Y.; Meng, X.; Li, H. Sinomenine induces G1-phase cell cycle arrest and apoptosis in malignant glioma cells via downregulation of Sirtuin 1 and induction of p53 acetylation. Technol. Cancer Res. Treat. 2018, 17, 1533034618770305. [CrossRef] 13. Niu, H.; Li, X.; Yang, A.; Jin, Z.; Wang, X.; Wang, Q.; Yu, C.; Wei, Z.; Dou, C. Cycloartenol exerts anti-proliferative effects on Glioma U87 cells via induction of cell cycle arrest and p38 MAPK-mediated apoptosis. J. BU ON 2018, 23, 1840–1845. [PubMed] 14. Lee, H.T.; Choi, M.R.; Doh, M.S.; Jung, K.H.; Chai, Y.G. Effects of the monoamine oxidase inhibitors pargyline and tranylcypromine on cellular proliferation in human prostate cancer cells. Oncol. Rep. 2013, 30, 1587–1592. [CrossRef][PubMed] 15. Lee, H.T.; Jung, K.H.; Kim, S.K.; Choi, M.R.; Chai, Y.G. Effects of pargyline on cellular proliferation in human breast cancer cells. Mol. Cell Toxicol. 2012, 8, 393–399. [CrossRef] 16. Liu, H.; Zhang, W.; Wang, K.; Wang, X.; Yin, F.; Li, C.; Wang, C.; Zhao, B.; Zhong, C.; Zhang, J.; et al. Methionine and cystine double deprivation stress suppresses glioma proliferation via inducing ROS/autophagy. Toxicol. Lett. 2015, 232, 349–355. [CrossRef] 17. Schumacker, P.T. Reactive oxygen species in cancer cells: Live by the sword, die by the sword. Cancer Cell 2006, 10, 175–176. [CrossRef] 18. Sun, Y.; Chen, J.; Rigas, B. Chemopreventive agents induce oxidative stress in cancer cells leading to COX-2 overexpression and COX-2-independent cell death. Carcinogenesis 2009, 30, 93–100. [CrossRef] 19. Gào, X.; Schöttker, B. Reduction-oxidation pathways involved in cancer development: A systematic review of literature reviews. Oncotarget 2017, 8, 51888–51906. [CrossRef] 20. Vannini, F.; Kashfi, K.; Nath, N. The dual role of iNOS in cancer. Redox Biol. 2015, 6, 334–343. [CrossRef] 21. Tuettenberg, J.; Friedel, J.C.; Vajkoczy, P. Angiogenesis in malignant glioma-a target for antitumor therapy? Crit. Rev. Oncol. Hematol. 2006, 59, 181–193. [CrossRef] 22. Kimura, H.; Weisz, A.; Kurashima, Y.; Hashimoto, K.; Ogura, T.; D’Acquisto, F.; Addeo, R.; Makuuchi, M.; Esumi, H. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: Control of hypoxia-inducible factor-1 activity by nitric oxide. Blood 2000, 95, 189–197. [PubMed] 23. Kramer, N.; Walzl, A.; Unger, C.; Rosner, M.; Krupitza, G.; Hengstschläger, M.; Dolznig, H. In vitro cell migration and invasion assays. Mutat. Res. 2013, 752, 10–24. [CrossRef][PubMed] Molecules 2019, 24, 2005 16 of 16

24. Meskelevicius, D.; Sidlauskas, K.; Bagdonaviciute, R.; Liobikas, J.; Majiene, D. Juglone exerts cytotoxic, anti-proliferative and anti-invasive effects on Glioblastoma Multiforme in a cell culture model. Anticancer Agents Med. Chem. 2016, 16, 1190–1197. [CrossRef] 25. Chandrika, G.; Natesh, K.; Ranade, D.; Chugh, A.; Shastry, P. Suppression of the invasive potential of Glioblastoma cells by mTOR inhibitors involves modulation of NFκB and PKC-α signaling. Sci. Rep. 2016, 6, 22455. [CrossRef][PubMed] 26. Zhang, J.F.; Wang, P.; Yan, Y.J.; Li, Y.; Guan, M.W.; Yu, J.J.; Wang, X.D. IL-33 enhances glioma cell migration and invasion by upregulation of MMP2 and MMP9 via the ST2-NF-κB pathway. Oncol. Rep. 2017, 38, 2033–2042. [CrossRef][PubMed] 27. Webb, A.H.; Gao, B.T.; Goldsmith, Z.K.; Irvine, A.S.; Saleh, N.; Lee, R.P.; Lendermon, J.B.; Bheemreddy, R.; Zhang, Q.; Brennan, R.C.; et al. Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma. BMC Cancer 2017, 17, 434. [CrossRef]

Sample Availability: Samples of the compounds Cmp3 and Cmp5 are available from the authors.

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