Genotoxicity and Epigenotoxicity of Carbazole-Derived Molecules on MCF-7 Breast Cancer Cells

International Journal of Molecular Sciences

Article Genotoxicity and Epigenotoxicity of Carbazole-Derived Molecules on MCF-7 Breast Cancer Cells

Claudio Luparello 1,* , Ilenia Cruciata 1 , Andreas C. Joerger 2,3, Cory A. Ocasio 4,† , Rhiannon Jones 4, Raysa Khan Tareque 4, Mark C. Bagley 4 , John Spencer 4,* , Martin Walker 5, Carol Austin 5, Tiziana Ferrara 1, Pietro D0Oca 1, Rossella Bellina 1, Rossella Branni 1 and Fabio Caradonna 1,*

1 Department of Biological Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Viale delle Scienze, Edificio 16, 90128 Palermo, Italy; [email protected] (I.C.); [email protected] (T.F.); [email protected] (P.D.); [email protected] (R.B.); [email protected] (R.B.) 2 Institute of Pharmaceutical Chemistry, Goethe University, 60438 Frankfurt am Main, Germany; [email protected] 3 Buchmann Institute for Molecular Life Sciences, Structural Genomics Consortium (SGC), 60438 Frankfurt am Main, Germany 4 Department of Chemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QJ, UK; [email protected] (C.A.O.); [email protected] (R.J.); [email protected] (R.K.T.); [email protected] (M.C.B.) 5 Eurofins Integrated Discovery UK Ltd., Fyfield Business & Research Park, Fyfield Road, Ongar, Essex CM5 0GS, UK; [email protected] (M.W.); [email protected] (C.A.) * Correspondence: [email protected] (C.L.); [email protected] (J.S.); [email protected] (F.C.) † Current address: The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

Citation: Luparello, C.; Cruciata, I.; Abstract: The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N- methyl- Joerger, A.C.; Ocasio, C.A.; Jones, R.; methanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), Tareque, R.K.; Bagley, M.C.; Spencer, second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore J.; Walker, M.; Austin, C.; et al. p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice Genotoxicity and Epigenotoxicity of Carbazole-Derived Molecules on and acting as molecular chaperones. In the present paper, these three molecules have been tested MCF-7 Breast Cancer Cells. Int. J. Mol. for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast ade- Sci. 2021, 22, 3410. https://doi.org/ nocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet 10.3390/ijms22073410 assay and methylation-sensitive arbitrarily primed PCR to analyze their intrinsic DNA damage- inducing and DNA methylation-changing abilities. We demonstrate that small modiﬁcations in Academic Editor: the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and Daniela Trisciuoglio epigenetic properties, with PK9320 and PK9323 being eligible candidates as “anticancer compounds” and “anticancer epi-compounds” and PK083 a “damage-corrective” compound on human breast Received: 1 March 2021 adenocarcinoma cells. Such different properties may be exploited for their use as anticancer agents Accepted: 23 March 2021 and chemical probes. Published: 26 March 2021

Keywords: genomic instability; carbazole derivatives; epigenetics; DNA methylation; breast cancer Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Genotoxicity, as defined by toxicological genetics and mutagenesis, describes the ability of chemical agents to induce modifications to the DNA nucleotide sequence or double helix structure of an organism, damaging its genetic information, thereby causing Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. mutations that can lead to the onset of cancer [1]. DNA damage is one of the most This article is an open access article dangerous threats to cellular homeostasis, and, for this reason, the cellular response to distributed under the terms and this event is immediate and vigorous. This DNA-damage response (DDR) encompasses conditions of the Creative Commons numerous metabolic pathways, and its defects leave cells susceptible to genomic instability, Attribution (CC BY) license (https:// predispose to further genomic errors and, lastly, transformation. creativecommons.org/licenses/by/ Since the early discovery of the antibiotic properties of the carbazole alkaloids ellip- 4.0/). ticine and murrayanine extracted from Ochrosia elliptica Labill and Murraya koenigii Spreng,

Int. J. Mol. Sci. 2021, 22, 3410. https://doi.org/10.3390/ijms22073410 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 15

Since the early discovery of the antibiotic properties of the carbazole alkaloids ellip- Int. J. Mol. Sci. 2021, 22, 3410 ticine and murrayanine extracted from Ochrosia elliptica Labill and Murraya koenigii2 of 14 Spreng, respectively [2,3], natural carbazoles and their synthetic derivatives have been extensively studied and incorporated as key structural motifs in a number of approved respectively [2,3], natural carbazoles and their synthetic derivatives have been extensively drugs acting against a wide spectrum of diseases, including cancer, microbial infections studied and incorporated as key structural motifs in a number of approved drugs acting and neurological disorders [4–6]. However, carbazoles have also been shown to induce against a wide spectrum of diseases, including cancer, microbial infections and neurological epigenetic anomalies, such as changes in DNA methylation status, and also to exert gen- disorders [4–6]. However, carbazoles have also been shown to induce epigenetic anomalies, otoxic effects. To cite some examples, exposure to mahanine, a carbazole alkaloid from such as changes in DNA methylation status, and also to exert genotoxic effects. To cite Micromelum minutum, was found to be responsible for the negative modulation of the cel- some examples, exposure to mahanine, a carbazole alkaloid from Micromelum minutum, lular levels of selected DNA methyltransferases in prostate tumor cells, thereby restoring was found to be responsible for the negative modulation of the cellular levels of selected the expression of the epigenetically silenced tumor suppressor gene RASSF1A (Ras Asso- DNA methyltransferases in prostate tumor cells, thereby restoring the expression of the ciation Domain Family Member 1). Moreover, SH-I-14 was shown to determine the pro- epigenetically silenced tumor suppressor gene RASSF1A (Ras Association Domain Family moter demethylation-induced reactivation of tumor-suppressor gene expression in triple- Member 1). Moreover, SH-I-14 was shown to determine the promoter demethylation- inducednegative reactivationbreast cancer of tumor-suppressor cells. The epigenetic gene expressionimpact of in2,7-dibromocarbazole triple-negative breast and cancer 6- cells.formylindolo The epigenetic [3 ,2-b]carbazole impact of 2,7-dibromocarbazolehas been also acknowledged. and 6-formylindolo In particular, [3,2-b]carbazole the former mol- hasecule been was also proven acknowledged. to demethylate In particular, Ang2 (angiopoietin the former molecule 2) gene promoter was proven and to potentiate demethylate its Ang2expression,(angiopoietin resulting 2) in gene deficient promoter angiogenesis and potentiate by endothelial its expression, cells. The resulting latter one, in deficient a ligand angiogenesisof the aryl hydrocarbon by endothelial receptor, cells. Thewas latterfound one, to repress a ligand the of expression the aryl hydrocarbon of the CYP1A1 receptor, (aryl washydrocarbon found to represshydroxylase) the expression gene in ofhepatoma the CYP1A1 cells(aryl by hydrocarbontargeting DNA hydroxylase) methyltransferase gene in hepatoma3A, which cellsled to by impairment targeting DNA of the methyltransferase cellular detoxification 3A, which system led to[7–10]. impairment Furthermore, of the cellularValovič detoxificationová et al. [11] systemdemonstrated [7–10]. Furthermore,the occurrence Valoviˇcov of significantá et al. [levels11] demonstrated of DNA strand the occurrencebreaks, micronuclei, of significant and levels DNA of adducts DNA strand after breaks, exposure micronuclei, of human and keratinocytes DNA adducts to after 7H- exposuredibenzo[c,g of]carbazole, human keratinocytes and Zhanataev to 7H-dibenzo[ et al. [12] reportedc,g]carbazole, genotoxic and Zhanataeveffects of two et al. carba- [12] reportedzole-derived genotoxic xenomycins, effects ofPLX01107 two carbazole-derived and PLX01008, xenomycins, initially developed PLX01107 as andpotential PLX01008, anti- initiallyfungal agents. developed Although as potential the genotoxic antifungal and agents. epigenotoxic Although side the genotoxiceffects of carbazoles and epigenotoxic can be sidean issue effects when of carbazoles administered can be to anhealthy issue whentissues, administered thus limiting to healthythe use of tissues, the compounds thus limiting in theclinical use ofpractice, the compounds these properties in clinical make practice, them these promising properties “anticancer make them (epi)drugs” promising effica- “anti- cancercious as (epi)drugs” preventative efficacious and/or as treatment preventative agents and/or in a treatment combinatorial agents molecular in a combinatorial therapy molecularagainst metastatic therapy cancer against histotypes. metastatic cancer histotypes. CarbazoleCarbazole derivativesderivatives have alsoalso beenbeen usedused asas chemicalchemical chaperoneschaperones toto reactivatereactivate aa thermolabilethermolabile p53p53 cancercancer mutant.mutant. The p53 cancer hotspot mutation Y220C greatly destabi- lizeslizes thethe p53p53 DNA-bindingDNA-binding domain,domain, whichwhich unfoldsunfolds andand aggregatesaggregates atat bodybody temperature,temperature, resultingresulting inin thethe lossloss ofof p53p53 tumortumor suppressorsuppressor functionfunction [[13].13]. StructuralStructural studiesstudies havehave furtherfurther shownshown thatthat thethe mutationmutation createscreates aa druggabledruggable cavitycavity onon thethe surfacesurface thatthat cancan bebe targetedtargeted byby smallsmall molecules.molecules.PK083 PK083(Figure (Figure1 )1) is is a carbazole-baseda carbazole-based small small molecule molecule selected selected as aas starting a start- scaffolding scaffold to stabilize to stabilize and reactivate and reactivate the Y220C the mutantY220C mutant [14]. Structure-guided [14]. Structure-guided design then design led tothen second-generation led to second-generation analogues, analogues, including includingPK9320 and PK9320PK9323 and, PK9323 which bound, which the bound mutant the withmutant higher with affinity higher andaffinity were and more were potent more in potent stabilizing in stabilizing and reactivating and reactivating the mutant the than mu- thetant parent than the compound parent compound (Figure1). Importantly, (Figure 1). Importantly, those second-generation those second-generation carbazoles restored carba- p53zoles signaling restored in p53 the signaling liver cancer in cellthe lineliver HUH-7 cancer bearingcell line aHUH-7 homozygous bearing Y220C a homozygous mutation, withY220C potential mutation, future with application potential future in personalized application cancer in personalized therapy [15 cancer,16]. therapy [15,16].

FigureFigure 1.1. Chemical structuresstructures ofof thethe carbazolecarbazole derivativesderivatives PK083PK083,, PK9320PK9320 andand PK9323PK9323..

Here,Here, wewe werewere interestedinterested inin gaininggaining insightsinsights intointo potentialpotential mutantmutant p53-independentp53-independent genotoxicgenotoxic oror epigeneticepigenetic effectseffects ofof thisthis carbazolecarbazole seriesseries atat highhigh compoundcompound concentrations,concentrations, usingusing compoundscompounds PK083PK083,, PK9320PK9320 andand PK9323PK9323 asas examples.examples. We havehave thereforetherefore employedemployed a combination of Western blot, Comet assay and methylation-sensitive arbitrarily primed PCR techniques to analyze their DNA damage-inducing and DNA methylation-changing abilities in wild-type p53 MCF-7 breast adenocarcinoma cells.

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a combination of Western blot, Comet assay and methylation-sensitive arbitrarily primed Int. J. Mol. Sci. 2021, 22, 3410 PCR techniques to analyze their DNA damage-inducing and DNA methylation-changing3 of 14 abilities in wild-type p53 MCF-7 breast adenocarcinoma cells.

2. Results In order to elucidate the potential genotoxic mechanismmechanism exhibitedexhibited byby PK083PK083,, PK9320PK9320 and PK9323,, histone histone H2AX H2AX phosphorylation phosphorylation and and Co Cometmet assays assays were were conducted conducted after after ex- exposureposure of MCF-7 of MCF-7 breast breast cancer cancer cells cellsto each to eachof the of molecules. the molecules. It is known It is that, known in response that, in responseto ionizing to ionizingradiation radiation or DNA-damaging or DNA-damaging chemotherapeutic chemotherapeutic agents, agents,DNA double-strand DNA double- strandbreaks breaks(DSB) are (DSB) generated are generated that rapidly that rapidly result resultin the inphosphorylation the phosphorylation of the of histone the histone H2A H2Avariant variant H2AX H2AX [17]. Because [17]. Because the phosphorylation the phosphorylation of H2AX of H2AXat Ser139 at Ser139 (γ-H2AX) (γ-H2AX) is abun- is abundant,dant, fast and fast correlates and correlates with DSB, with it DSB, represents it represents a very sensitive a very sensitive marker markerof DNA ofdamage DNA damageand the subsequent and the subsequent repair, and repair, is therefore and is thereforea commonly a commonly used test usedto identify test to genotoxic identify genotoxiccompounds compounds [18]. [18]. As shown in Figure2 2,, PK9323, at 10 μµMM concentration, concentration, led to enhanced enhanced γH2AXH2AX phosphorylation compared with the untreated control,control, correlating with more extensiveextensive DNADNA damage. In contrast, PK083 and PK9320 were not found to affect the level of histone phosphorylation and were genotoxicallygenotoxically inactive in that aspect.aspect. However, PK9320 showedshowed aa cytotoxic effect,effect, as as evidenced evidenced by by the the drastic drastic decrease decrease in tubulin in tubulin levels, levels, suggesting suggesting cell death cell ordeath inhibition or inhibition of cell of growth. cell growth. The exposure–response The exposure–response relationship relationship was further was further investigated, inves- µ identifyingtigated, identifyingPK9323 asPK9323 relatively as relatively highly genotoxic, highly genotoxic, with an ECwith50 ofan 2.2 EC50M, of which2.2 μM, exceeds which µ thatexceeds of cisplatin that of cisplatin (EC50 = 4(ECM),50 = as 4 shownμM), as in shown Figure in3. Figure The optical 3. The densities optical ofdensities the Western of the blotWestern bands blot shown bands in shown both ﬁgures in both can figures be found can be in Figurefound in S1. Figure S1.

Figure 2. MCF-7 cells were treated with DMSO control or an indicated concentration ofof compound.com- Afterpound. 20 After h, cells 20 were h, cells harvested, were harvested, lysed and lysed probed and for probed DNA for damage DNA by damage Western by blot Western analysis blot using anal- a ysis using a phosphorylated γH2AX antibody. Western blot analysis of tubulin was used as a phosphorylated γH2AX antibody. Western blot analysis of tubulin was used as a loading and cell loading and cell viability control. viability control.

To gain more insight into this latter aspect, MCF-7 cells were exposed to PK9323, and its effect on cell morphology and growth rate was analyzed by microscopic observations and direct cell counting also comparing it to those exerted by PK083 and PK9320. No sig- niﬁcant morphological change indicative of cell suffering or death could be observed with all treatments, even when exposing cells to the maximum concentration of the compounds (Figure S2). The numbers of control and treated cells were then evaluated by counting in a haemo- cytometer, and the results conﬁrm that cell exposure to 10 µM PK9320 appeared to drastically decrease the cell count to circa 20% vs. control. Interestingly, also in the presence of PK083 and PK9323 at 10 µM concentrations, the amount of cells decreased, although to aFigure lesser 3.degree MCF-7 cells than were with treatedPK9320 with(ca. 2-fold 39% serial and 40%dilutions vs. control,of PK9323 respectively). from 10 down Atto 0 a μ lowerM. concentrationAfter 20 h, cells ofwere 2.2 harvested,µM, PK9320 lysedand andPK9323 probedexerted for DNA only damage a negligible by Western effect blot on analysis cell growth (A) (ca.using 119% a phosphorylated and 112% vs.γH2AX control, antibody. respectively), Western blot whereas, analysis conversely,of tubulin wasPK083 used asinduced a loading an average 76% increase in the cell number with respect to the untreated samples (Figure4).

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a combination of Western blot, Comet assay and methylation-sensitive arbitrarily primed PCR techniques to analyze their DNA damage-inducing and DNA methylation-changing abilities in wild-type p53 MCF-7 breast adenocarcinoma cells.

2. Results In order to elucidate the potential genotoxic mechanism exhibited by PK083, PK9320 and PK9323, histone H2AX phosphorylation and Comet assays were conducted after exposure of MCF-7 breast cancer cells to each of the molecules. It is known that, in response to ionizing radiation or DNA-damaging chemotherapeutic agents, DNA double-strand breaks (DSB) are generated that rapidly result in the phosphorylation of the histone H2A variant H2AX [17]. Because the phosphorylation of H2AX at Ser139 (γ-H2AX) is abundant, fast and correlates with DSB, it represents a very sensitive marker of DNA damage and the subsequent repair, and is therefore a commonly used test to identify genotoxic compounds [18]. As shown in Figure 2, PK9323, at 10 μM concentration, led to enhanced γH2AX phosphorylation compared with the untreated control, correlating with more extensive DNA damage. In contrast, PK083 and PK9320 were not found to affect the level of histone phosphorylation and were genotoxically inactive in that aspect. However, PK9320 showed a cytotoxic effect, as evidenced by the drastic decrease in tubulin levels, suggesting cell death or inhibition of cell growth. The exposure–response relationship was further investigated, identifying PK9323 as relatively highly genotoxic, with an EC50 of 2.2 μM, which exceeds that of cisplatin (EC50 = 4 μM), as shown in Figure 3. The optical densities of the Western blot bands shown in both figures can be found in Figure S1.

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Figure 2. MCF-7 cells were treated with DMSO control or an indicated concentration of com- andpound. cell viability After 20 control. h, cells B) were Dose-response harvested, lysedcurve andfor γprobedH2AX phosphorylationfor DNA damage after by Western treatment blot of anal- Int. J. Mol. Sci. 2021, 22, 3410 4 of 14 MCF-7ysis usingcells with a phosphorylated PK9323 for 20 γh;H2AX DMSO antibody. was used Western as a no blotcompound analysis control. of tubulin The was EC50 used of PK9323 as a is 2.2loading ± 0.3 μandM and cell cisplatinviability wascontrol. tested in parallel at 4 μM as a reference DNA lesion-inducing drug. The error bars correspond to the SD of three independent measurements.

To gain more insight into this latter aspect, MCF-7 cells were exposed to PK9323, and its effect on cell morphology and growth rate was analyzed by microscopic observations and direct cell counting also comparing it to those exerted by PK083 and PK9320. No significant morphological change indicative of cell suffering or death could be observed with all treatments, even when exposing cells to the maximum concentration of the compounds (Figure S2). The numbers of control and treated cells were then evaluated by counting in a hae- mocytometer, and the results confirm that cell exposure to 10 μM PK9320 appeared to drastically decrease the cell count to circa 20% vs. control. Interestingly, also in the pres- enceFigureFigure of PK083 3.3. MCF-7 and cells cells PK9323 were were treated at treated 10 μwithM with concentrations, 2-fold 2-fold serial serial dilutions dilutionsthe amount of PK9323 of PK9323 of from cells 10 from decreased, down 10 downto 0 μalt-M. to 0 µM. houghAfterAfter to 2020 a h, lesser cells were weredegree harvested, harvested, than with lysed lysed PK9320 and and probed (ca. probed 39% for forDNA and DNA 40%damage damage vs. bycontrol, Western by Western respectively). blot analysis blot analysis At(A) (A) a lowerusingusing aaconcentration phosphorylatedphosphorylated of γ 2.2H2AX μM, antibody. PK9320 Western and PK9323 blot analysis analysis exerted of of onlytubulin tubulin a negligiblewas was used used as aseffect a aloading loading on and cellcell growth viability (ca. control. 119% B)and Dose-response 112% vs. control, curve respectively), for γH2AX phosphorylation whereas, conversely, after treatment PK083 in- of MCF-7 ducedcells withan averagePK9323 76%for 20 increase h; DMSO in wasthe cell used number as a no compoundwith respect control. to the The untreated EC of PK9323samplesis 2.2 ± 50 (Figure0.3 µM 4). and cisplatin was tested in parallel at 4 µM as a reference DNA lesion-inducing drug. The error bars correspond to the SD of three independent measurements.

FigureFigure 4. Effect 4. Effect of the of thecarbazole carbazole derivatives derivatives on the on number the number of MCF-7 of MCF-7cells after cells 20 h after of exposure. 20 h of exposure. CellCell numbers numbers were were measured measured by bydirect direct counting counting in a inBürker a Bürker chamber chamber afterafter trypsinization. trypsinization. Error Error bars barscorrespond correspond to to the the SEM SEM of of three three independent independent measurements. measurements. The The black black line line representsrepresents thethe IQRIQR values. values.* = p <* = 0.05 p < vs.0.05 control; vs. control; ** = **p <<= p 0.001<< 0.001 vs. vs. control. control.

TheThe possible possible occurrence occurrence of of genomic genomic DNA DNA lesions lesions induced by thethe carbazolecarbazole compoundscom- poundswas quantitatively was quantitatively evaluated evaluated by Cometby Comet assay assay in in terms terms ofof averageaverage Olive Olive Tail Tail Mo- Moment ment(OTM), (OTM), indicative indicative of persistent of persistent single- single- and and double-strand double-strand breaks. breaks. Preliminary Preliminary data data showed showedthat the that Student the Student0s t test′s t didtest notdid revealnot reveal significant significant differences differences between between the the OTM OTM values valuesobtained obtained from from the untreatedthe untreated and and the the control control DMSO-vehicle-only DMSO-vehicle-only exposed exposed culturescultures (data (datanot shown),not shown), thereby thereby ensuring ensuring that that the vehiclethe vehicle did notdid influencenot influence the experimentalthe experimental data. The data.data The shown data inshown Figure in 5Figure indicate 5 indicate that the that OTM the ofOTM the of DNA the DNA methyltransferase methyltransferase inhibitor inhibitor5-azacitidine 5-azacitidine (5-azaC)-treated (5-azaC)-treated cells was cells not was significantly not significantly different different from from that ofthat the of DMSO thecontrol, DMSO confirming control, confirming that no toxic that effectno toxic was effect exerted was byexerted the methylation by the methylation inhibitor inhibi- on the cells tor on the cells under the conditions used. This suggested a very low probability of the under the conditions used. This suggested a very low probability of the onset of clonal onset of clonal selection, an important premise for the validation of the subsequent meth- selection, an important premise for the validation of the subsequent methylation analyses. ylation analyses. Moreover, PK9320 and PK083, at both concentrations tested, appeared Moreover, PK9320 and PK083, at both concentrations tested, appeared unable to cause unable to cause damage to the genomic DNA, with PK083 rather exerting a protective damage to the genomic DNA, with PK083 rather exerting a protective action, as shown by the OTM value significantly lower than that of untreated cells. Conversely, as expected from the results of the histone γH2AX phosphorylation assay, PK9323 induced a considerable degree of genomic DNA damage at 2.2 µM concentration, whereas at 10 µM concentration the difference with the DMSO control was not statistically significant, probably due to a clonal selection phenomenon leading to the preferential survival of one or more clones better equipped to repair DNA breakages. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 15

action, as shown by the OTM value significantly lower than that of untreated cells. Con- versely, as expected from the results of the histone γH2AX phosphorylation assay, PK9323 induced a considerable degree of genomic DNA damage at 2.2 μM concentration, whereas at 10 μM concentration the difference with the DMSO control was not statistically Int. J. Mol. Sci. 2021, 22, 3410 significant, probably due to a clonal selection phenomenon leading to the preferential sur- 5 of 14 vival of one or more clones better equipped to repair DNA breakages.

Figure 5. Effect of the carbazole derivatives on the occurrence of genomic DNA lesions in MCF-7 Figure 5. Effect of the carbazole derivatives on the occurrence of genomic DNA lesions in MCF-7 cellscells after after 20 20 h of h ofexposure. exposure. (A) Representative (A) Representative micrographs micrographs of comet ofimages comet obtained images from obtained MCF-7 from MCF-7 cellscells exposed exposed to DMSO to DMSO vehicle vehicle as control as control (a), 10 μ (a),M 5-azaC 10 µM (b) 5-azaC and PK083 (b) and(c,d), PK083PK9320 (c,d),(e,f), orPK9320 (e,f), or PK9323PK9323 (g,h)(g,h) at at2.2 2.2 μMµ (c,e,g)M (c,e,g) or 10 or μM 10 concentrationsµM concentrations (d,f,h), stained (d,f,h), with stained Gel Red with DNA Gel Dye Red and DNA Dye and observed with a Nikon Microphot SA fluorescence microscope. Each spot corresponds to the DNA observed with a Nikon Microphot SA fluorescence microscope. Each spot corresponds to the DNA of an individual cell: when round it represents the intact DNA which is a large macromolecule thatof an poorly individual migrates cell: in the when electrophoretic round it represents field, when the “comet intact shaped” DNA it which is symptomatic is a large of macromolecule DNA that breakspoorly rendering migrates the in molecule the electrophoretic small enough field, to mo whenve in the “comet gel. Microscopic shaped” it magnification is symptomatic = 400×. of DNA breaks (renderingB) Histogram the showing molecule the induced small enough genomic to DNA move damage in the evaluated gel. Microscopic as OTM by magnification Comet assay. = 400× g.(B) OTM is the product of the percentage of total DNA in the tail and the distance between the bulk of headHistogram and tail showingregions. The the numb induceder of scored genomic cells DNA was 50 damage for each evaluatedexperimental as condition. OTM by The Comet assay. OTM blackis the line product represents of the the percentageIQR values. * of = totalp < 0.05 DNA vs. control; in the ** tail = p and << 0.001 the distancevs. control between. the bulk of head and tail regions. The number of scored cells was 50 for each experimental condition. The black line representsIn a last the set IQR of assays, values. we * = investigatedp < 0.05 vs. control;in more **detail = p << the 0.001 epigenetic vs. control. modulation carried out by the three compounds through the analysis of the global methylation status of the genomicIn a last DNA. set ofFor assays, this purpose, we investigated MeSAP-PCR in experiments more detail were the epigeneticcarried out on modulation single- carried (SDD)out by and the double-digested three compounds DNA through (DDD) the samples analysis obtained of the from global control methylation and PK083 status-, of the ge- PK9320nomic- DNA. or PK9323 For this-treated purpose, MCF-7 MeSAP-PCR cells at 2.2 and experiments 10 μM concentrations. were carried The outDNA on fin- single- (SDD) gerprintings were analyzed by densitometric scanning, and the numbers of variations, in and double-digested DNA (DDD) samples obtained from control and PK083-, PK9320- or terms of appearing/disappearing (A/D) and attenuation/intensification (A/I) of the band µ patternsPK9323 in-treated the SDD MCF-7 vs. DDD cells preparations, at 2.2 and were 10 determinedM concentrations. (Figure 6). The The obtained DNA fingerprintings data arewere summarized analyzed in by Table densitometric S1 and shown scanning, in the histogram and thein Figure numbers 7 and ofindicated variations, that 20 in terms of happearing/disappearing treatments with both concentrations (A/D) and of PK9320 attenuation/intensification and PK9323 were effective (A/I) in modifying of the band patterns thein theglobal SDD methylation vs. DDD pattern preparations, of cancer werecell DNA, determined as shown (Figure by the 6different). The obtainednumber, data are intensitysummarized and size in of Table the bands S1 and in the shown matched in the control histogram and exposed in Figure samples;7 and in particular, indicated that 20 h thetreatments most numerous with both changes concentrations were observed of afterPK9320 cell exposureand PK9323 to 2.2were μM PK9323 effective. Con- in modifying versely,the global the difference methylation between pattern the modulatory of cancer activities cell DNA, of PK083 as shown and the by vehicle the different alone number, wasintensity not significant. and size of the bands in the matched control and exposed samples; in particular, As expected, the results obtained with 5-azaC-treated cells were significantly differ- the most numerous changes were observed after cell exposure to 2.2 µM PK9323. Con- ent from those of the control. This finding, in the absence of clonal selection towards an epigeneticallyversely, the differenceunbalancedbetween cell population, the modulatory demonstrates activities that MCF-7 of PK083cells areand sensitive the vehicle to alone thewas modulation not significant. of DNA methylation mediated by the “DNA methyltransferase-S adeno- sylmethionineAs expected, (DNMT-SAM)” the results obtainedsystem, the with main 5-azaC-treated pathway by which cells weregenomic significantly DNA meth- different from ylationthose ofis theensured. control. This finding, in the absence of clonal selection towards an epigenetically unbalanced cell population, demonstrates that MCF-7 cells are sensitive to the modulation of DNA methylation mediated by the “DNA methyltransferase-S adenosylmethionine (DNMT-SAM)” system, the main pathway by which genomic DNA methylation is ensured. All three carbazoles in this study have been investigated in pharmacokinetic (PK) studies, notably looking at cytotoxicity in HepG2 liver cancer cells. Further studies looked at aque- ous solubility and all compounds had acceptable values at pH 7 of >100 mM. Measurement of clearance (compound metabolic stability), in mouse microsomes, showed PK9323 to have a reasonable half-life (>1 h), coupled with a relatively low clearance with around 3% unbound drug in plasma protein binding studies (PPB). PK9320 had the shortest half-life and highest clearance of the three compounds studied; moreover, it had a very low % PPB. Concerning compound cytotoxicity, the following results were obtained: PK083 (23 µM); PK9320 (4.8 µM); PK9323 (1.1 µM), with PK083 showing mild and PK9323 high cytotoxicity in this assay (Table S2). These data appear to align well with the other studies outlined above. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 15

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Figure 6. AnalysisFigure of 6. genome-wideAnalysis of genome-wide DNA methylation DNA methylationstatus. Repr status.esentative Representative MeSAP fingerprintings MeSAP ﬁngerprintings and corresponding densitometryand profiles corresponding of single-digested densitometry DNA proﬁles(SDD) and of single-digested double-digested DNA DNA (SDD) (DDD) and samples double-digested from DMSO DNA vehicle (A) and 5-azaC- (DDD)(B), PK083- samples (C,D), from PK9320 DMSO (E,F vehicle) or PK9323-treated (A) and 5-azaC- MCF-7 (B cells), PK083- (G,H) (atC, D2.2), ( PK9320C,E,G) and (E, F10) orμM PK9323- concentrations (D,F,H). Changestreated of MCF-7the DNA cells band (G ,patternH) at 2.2s are (C indicated,E,G) and with 10 µ Mblack concentrations arrows (A/D) ( Dor,F grey,H). arrowheads Changes of (A/I). the DNA band patterns are indicated with black arrows (A/D) or grey arrowheads (A/I).

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FigureFigure 7.7. Effect of the carbazole derivatives on the gl globalobal methylation methylation pattern pattern of of MCF-7 MCF-7 cells cells after after 2020 hh ofof exposure.exposure. TheThe histogramhistogram showsshows thethe percentagepercentage of of genomic genomic demethylation demethylation of of exposed exposed MCF-7 cellsMCF-7 vs. cells controls, vs. controls, as calculated as calculated by the numberby the nu ofmber band of pattern band pattern variations. variations. The black The line black represents line represents the IQR values. * = p < 0.05; ** = p < 0.001. the IQR values. * = p < 0.05; ** = p < 0.001.

3.3. Discussion ItIt isis widelywidely acknowledgedacknowledged thatthat membersmembers ofof thethe familyfamily of carbazole-based molecules, eithereither natural or or synthetic, synthetic, represent represent promis promisinging compounds compounds with with the ability the ability to act to as act DNA as DNAintercalating intercalating agents agentsand to interfere and to interfere with the withactivity the of activity crucial ofenzymes, crucial such enzymes, as topoiso- such asmerases topoisomerases and telomerases and telomerases [19]. The topology [19]. The of topology DNA and of the DNA maintenance and the maintenance of telomere oflength telomere are two length crucial are endpoints two crucial whereby endpoints cells, whereby physiologically, cells, physiologically, especially transformed especially transformedones, make nicks ones, in make the DNA nicks and in the manage DNA andthem, manage thus exposing them, thus themselves exposing to themselves the risk of tocreating the risk free of creatingDNA ends free prone DNA to ends produce prone chromosomal to produce chromosomal aberrations that aberrations can initiate/pro- that can initiate/promotemote tumorigenesis tumorigenesis or induce cell or death induce [20,21]. cell death [20,21]. Moreover,Moreover, somesome carbazole-derived carbazole-derived molecules molecules affect affect DNA DNA methylation methylation at speciﬁc at specific CpG islandsCpG islands [9]. Because [9]. Because of this of property,this property, they currentlythey currently represent represent the mainstay the mainstay of treatment of treat- decisionsment decisions for leukemia for leukemia patients patients who are who not suitableare not forsuitable intensive for intensive chemotherapy chemotherapy [22], also because[22], alsothey because can they be safely can be metabolized safely metaboli byzed CYP by enzymes CYP enzymes [23]. [23]. It is It also is also important important to rememberto remember that that the the expression expression of someof some members members of theof the cytochrome cytochrome P450 P450 family, family, which which is composedis composed of of several several genes genes [24 [24],], is regulatedis regulated by by DNA DNA methylation methylation [25 [25].]. Here,Here, wewe havehave analyzedanalyzed thethe DNADNA damage-inducingdamage-inducing potentialpotential andand methylation-methylation- changingchanging capability of of three three carbazoles, carbazoles, PK083, PK083, PK9320, PK9320, and and PK9323, PK9323, in human in human breast breast ad- adenocarcinomaenocarcinoma cells cells (MCF-7) (MCF-7) to evaluate to evaluate their their potential potential use useas “anticancer as “anticancer (epi)drugs” (epi)drugs” in a incombinatorial a combinatorial molecular molecular therapy therapy [26], [in26 ],additi in additionon to their to their reported reported action action as Y220C as Y220C mu- mutanttant p53 p53 reactivators reactivators [15,16]. [15,16 ]. WeWe havehave chosenchosen toto investigateinvestigate thethe genotoxicgenotoxic potencypotency of the three molecules, quanti- tativelytatively andand qualitatively,qualitatively, byby performingperforming thethe histonehistone γγH2AXH2AX phosphorylationphosphorylation assayassay andand thethe CometComet assay.assay. UsingUsing thesethese twotwo complementarycomplementary techniquestechniques enabledenabled usus toto looklook atat twotwo facetsfacets ofof thethe samesame kindkind ofof DNADNA damage:damage: fromfrom thethe molecularmolecular pointpoint ofof viewview andand fromfrom thethe cellularcellular one.one. In fact,fact, thethe extentextent ofof H2AXH2AX phosphorylationphosphorylation correlatescorrelates withwith thethe numbernumber ofof regionsregions withwith DNA DNA DSB, DSB, which which are are involved involved in in the the recruitment recruitment of DNAof DNA repair repair factors; factors; at the at chromatinthe chromatin level, level, a DNA a DNA damage damage site, evensite, ineven a transformed in a transformed cell and cell within and within certain certain limits, canlimits, still can be repairedstill be repaired and its effectand its be effect silenced be silenced or underestimated. or underestimated. Also for Also this reason,for this thisrea- assayson, this was assay called was “a called surrogate “a surrogate marker” marker of DSB” study of DSB [27 study]. In contrast,[27]. In contrast, the alkaline the alkaline Comet assay,Comet a assay, useful a technique useful technique for detecting for detectin the cyto-electrophoreticg the cyto-electrophoretic migration migration of nuclear of DNA nu- withclear single-DNA with and single- double-strand and double-strand breaks, visually breaks, and visually quantitatively and quantitatively shows DNA shows damage DNA atdamage the cellular/nuclear at the cellular/nuclear level originating level originat froming several from sources, several evensources, in the even long in term the [long28], providingterm [28], anproviding overall picturean overall of anpicture entire of population an entire population of cells “sensitively of cells “sensitively and precisely”, and

Int. J. Mol. Sci. 2021, 22, 3410 8 of 14

as was more recently reported [29]. Hence, both points of view were considered to better characterize the genotoxic potency and to hypothesize more precisely on the mechanism of action of the inducing molecules. An innovative feature of this study is its epigenomic aspect. The in vitro/in vivo study of the epigenetic effects of carbazole derivatives is a relatively new field, and only a few publications have addressed this topic to date (A PubMed search in February 2021, with “carbazole derivative epigenetic” as key words retrieved only 14 hits). Given that the modulation of genomic DNA methylation is one of the crucial epigenetic modifications in the oncogenic process and a biomarker implicated in various human pathologies, including cancer [30], we also wanted to evaluate the capacity of the three molecules to act as epigenomic modulators. Taking the obtained results and the literature data into consideration, the findings reported here allow us to make the following comments: (1) First of all, it must be highlighted that the Comet assay performed on 5-azaC- treated cells showed that there was no direct link between 5-azaC-induced genomic DNA demethylation and DNA strand breaks. Therefore, these two toxicological endpoints can be considered to be independent of one another. (2) Our data showed significantly different genotoxic and epigenotoxic effects of the tested molecules, with PK9323 being the most active in inducing molecular modifications. In fact, all the biological and epigenetic endpoints, at both the molecular and nuclear/cellular level, clearly showed that this specific carbazole derivative possesses a high (epi)genotoxic potency, as demonstrated by the highly significant differences in the γH2AX phosphorylation, cell viability, Comet and MeSAP-PCR assays relative to the control and the other tested carbazoles. This is the first description of the genotoxicity of this newly synthesized molecule and, more generally, the first description of combined effects, both genomic and epigenomic, of a carbazole derivative. (3) Compound PK9320 might be labeled as genotoxically inactive, given that it was not found to affect the level of histone phosphorylation; however, it is noteworthy that it caused a drastic decrease in tubulin levels, indicative of cell death or inhibition of cell growth. This result prompted us to examine the toxicity of this specific molecule from different angles by probing its endocellular/endonuclear and epigenetic action, because carbazole derivatives have been reported as non-toxic mainly when tested at the molecular level, but highly genotoxic when submitted to other tests, including the Comet assay [12]. Consistent with these observations, we found that PK9320, at 10 µM concentration, was able to reduce cell numbers in a highly significant way. Moreover, it was unable to cause significant damage to nuclear DNA, as revealed by the Comet assay, at both 2.2 and 10 µM concentrations, but induced a statistically significant reduction of genomic DNA methylation in both experimental conditions. PK9320, in summary, at the concentration used in this study, appears to be a carbazole derivative endowed with a certain capability to exert DNA damage, but mainly coming from its epigenomic modifying action at least with regard to DNA methylation: an “epidrug” as defined by Ghasemi [31], which could be better renamed as “pure epidrug”, given its specificity of action. (4) Finally, PK083, a molecule structurally similar to PK9320 and PK9323, has shown surprisingly attenuated, and in some cases even opposite, effects in almost all the analyses performed in this study. In fact, this compound exerted a non-genotoxic effect when assayed by phospho-γH2AX test and a protective effect on basal DNA damage, which was highly significant at both administered concentrations with respect to the control. Moreover, MeSAP-PCR data revealed that PK083 displayed a non-significant demethy- lating potency on the CpG sites of genomic DNA, lower than that of 5-azaC. It may be concluded that PK083 is a non-(epi)genotoxic molecule with a similar behavior to that reported for ellipticine [32], a molecule with a tumor cell growth inhibitory ability, judged as non-genotoxic by γH2AX assay. In our experimental system, the sole activity performed by PK083 that can be considered toxic was the reduction of MCF-7 cell number when administered at 10 µM concentration. Int. J. Mol. Sci. 2021, 22, 3410 9 of 14

Although there are still several unanswered questions that our study did not address, such as the effect of carbazoles on histone acetylation/deacetylation balances, miRNA variations and DNA methylation at the gene level, overall our data show that small modiﬁcations in the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and epigenetic properties, which may be exploited for their use as anticancer agents and probe molecules.

4. Materials and Methods 4.1. Cell Culture and Treatments The MCF-7 breast tumor cell line was cultured in high glucose–DMEM medium plus 10% foetal calf serum (FCS; ThermoFisher, Waltham, MA, USA), 100 U/mL peni- cillin, 100 µg/mL streptomycin, and 2.5 mg/L amphotericin B (Invitrogen, Carlsbad, CA, ◦ USA), at 37 C in a 5% CO2 atmosphere. The cells were detached from flasks with 0.05% trypsin-EDTA (Invitrogen), counted, and plated at the necessary density for treatment after achieving 60−80% confluency. PK083, PK9320 and PK9323 were synthesized as reported by Bauer et al. [15], dissolved in dimethyl sulfoxide (DMSO) firstly at 330 and 1500 mM stock concentrations and then in 0.33 and 0.15 mM work concentrations, aliquoted in several tubes to avoid freezing–thawing cycles and stored at −20 ◦C. The biological assays were carried out for 20 h in the presence of the respective compound at the concentrations indicated in the specific paragraphs; the working concentrations were obtained by dilution of either stock solution with a constant volume of DMSO. Negative controls were prepared by exposing cells to the same volume of DMSO vehicle only for 20 h. No significant difference between untreated cells and DMSO-treated ones was found in relation to each biological endpoint investigated. As a positive control for the epigenomic studies, cells were treated with a 10 µM concentration of the DNA methyltransferase inhibitor 5-azaC for 48 h [25].

4.2. Morphological Observations and Cell Number Evaluation MCF-7 cells were plated at a density of 1.0 × 106 cells/mL in 60-mm Petri dishes, cultured in control conditions or in the presence of either PK083, PK9320 or PK9323 and observed under an inverted OPTIKA phase-contrast microscope (Ponteranica, Italy) at the end of the treatment. At least 50 microscopic ﬁelds were examined in three independent experiments. Cell numbers were measured by direct counting in a Bürker chamber after trypsinization [33].

4.3. DNA Damage Assays 4.3.1. Western Blot for Phospho-γH2AX Histone MCF-7 breast cancer cells were seeded at a density of 1.0 × 105 cells/well in a 12-well plate and allowed to settle for 48 h before exposure to PK083, PK9320, or DMSO. At the end of the treatments, the cells were washed with phosphate-buffered saline and lysed using 1x SDS-loading buffer containing β-mercaptoethanol (5x buffer: 0.25% w/v bromophenol blue, 0.5 M DTT (dithiothreitol), 50% v/v glycerol, 10% v/v SDS (sodium dodecyl sulphate) and 0.25M Tris-HCl, pH 6.8). The lysates were sonicated and clariﬁed by centrifugation at 13,000 r.p.m. before being loaded into 12% SDS-polyacrylamide gels. Proteins were separated by SDS-PAGE at 200 V for 35 min. Western blot analysis of phospho-γH2AX histone and the loading control α-tubulin was accomplished through transfer of protein onto PVDF membranes using the Trans-Blot Turbo System (Bio-Rad, Hercules, CA, USA). Mouse mon- oclonal anti-α-tubulin (DM1A) was purchased from Abcam (Cambridge, UK) and mouse anti-phospho-histone H2AX (Ser139) (JBW301) was purchased from Millipore (Darmstadt, Germany). Secondary antibodies were HRP-conjugated, polyclonal goat-derived antibodies against mouse and rabbit (Dako, Agilent Technologies, Santa Clara, CA, USA). After blotting and immunoreactions, protein bands were detected with a chemiluminescent Western blotting substrate (SuperSignal West Femto, ThermoFisher) and the ImageQuant LAS 4000 system (GE Healthcare, Chicago, IL, USA). Protein bands were quantiﬁed by densitometry using ImageJ 1.50C and phospho-histone H2AX bands were normalized Int. J. Mol. Sci. 2021, 22, 3410 10 of 14

to the α-tubulin control band. DNA damage was reported as the percent of normalized phospho-histone H2AX. DNA damage assays were repeated in triplicate and the half maximal effective concentration (EC50) value for PK9323 compound compared with that of cisplatin as a reference DNA lesion-inducing drug using the non-linear regression module of Prism 6.0 (GraphPad, San Diego, CA, USA). In light of the results obtained from the dose–response assays with PK9323, the following experiments were carried out for 20 h in the presence of either 2.2 (PK9323 EC50) or 10 µM (maximum concentration tested) of either of the three compounds; both working concentrations were obtained by dilution of either stock solution with a constant volume of DMSO. Negative controls were prepared by exposing cells to the same volume of DMSO vehicle only for 20 h.

4.3.2. Alkaline Comet Assay The extent of the DNA damage in MCF-7 cells by PK083, PK9320 and PK9323 was examined via single cell gel electrophoresis with the OxiSelect Comet assay kit (Cell Biolabs, San Diego, CA, USA) as previously described [34,35]. Essentially, control and treated cells were mechanically detached, resuspended at a density of 1.0 × 105/mL in Ca++/Mg++-free phosphate-buffered saline and mixed with Comet agarose. The samples were then loaded onto the OxiSelect Comet slides, allowed to gel, and treated with the lysis buffer followed by an alkaline solution allowing DNA unwinding and denaturation. The damaged DNA exhibiting both single- and double-strand breaks was separated from the intact fraction through alkaline gel electrophoresis and, at the end of the run, the slides were ﬁxed, air- dried, and stained with Vista Green DNA or Gel red (Biotium, Fremont, CA, USA) Dye. At least 50 nuclei were selected randomly per experiment, observed and photographed under a Nikon Microphot SA ﬂuorescence microscope and the olive tail moment (OTM) was evaluated with the CASP Version 1.2.3b1 software (Sourceforge, Diceholdings Inc., New York, NY, USA).

4.4. DNA Extraction and Methylation-Sensitive Arbitrarily Primed Polymerase Chain Reaction (MeSAP PCR) Extraction of the genomic DNA of control and treated MCF-7 cells was performed using the PureLink Genomic DNA Kit (Invitrogen) according to the manufacturer’s in- structions, and the extracted DNA was quantitated using a NanoDrop® ND-1000 spec- trophotometer (ThermoFisher), as previously described [36]. In order to evaluate the modulatory effects of PK083, PK9320 and PK9323 on the genome-wide DNA methylation level, MeSAP-PCR experiments were carried out as previously reported [37,38]. Essentially, single-digested DNA (SDD) samples were obtained by treating the genomic DNA with AfaI restriction endonuclease (ThermoFisher) whose cleav- age site is (GT*AC). Subsequently, in order to obtain double-digested DNA (DDD), samples aliquots of SDD were further treated with the methylation-sensitive HpaII restriction endonuclease (ThermoFisher), ineffective in cleaving the DNA when its recognition site (CG*GG) contains a methylated cytosine. An arbitrarily primed two-step PCR experiment was performed to amplify both SDD and DDD samples. The first PCR step (cycle profile: 94 ◦C for 5 min, four cycles at 94 ◦C for 30 s, 40 ◦C for 60 s and 72 ◦C for 90 s) was carried out in the presence of a 21-mer arbitrary primer (50-AACTGAAGCAGTGGCCTCGCG-30) in low stringency conditions, i.e., a permissive annealing temperature and a high salt and primer concentration. This allows for the best matches between the arbitrary primer and the genomic CpG sites, due to the complementary 30 end of the former one. The second highly stringent PCR step (cycle profile: 94 ◦C for 1 min, four cycles at 60 ◦C for 1 min and 72 ◦C for 2 min) was carried out in continuity, and, at the end, the amplified DNA was separated through a non-denaturing 6% polyacrylamide gel electrophoresis, stained with Gel Red nucleic acid gel stain (Biotium, Hayward, CA, USA), and analyzed with SigmaGel v.1.0 image analysis software (SPSS, Chicago, IL, USA). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 12 of 15

amplified DNA was separated through a non-denaturing 6% polyacrylamide gel electrophoresis, stained with Gel Red nucleic acid gel stain (Biotium, Hayward, CA, USA), and analyzed with SigmaGel v.1.0 image analysis software (SPSS, Chicago, IL, USA). Int. J. Mol. Sci. 2021, 22, 3410 11 of 14 4.5. Pharmacokinetic (PK) Studies PK studies were carried out as outlined previously [39,40]. A detailed description of 4.5. Pharmacokinetic (PK) Studies the protocols can be found in the supporting information file. PK studies were carried out as outlined previously [39,40]. A detailed description of 4.6. Statistics the protocols can be found in the supporting information ﬁle.

Data are presented4.6. as Statistics mean ± SEM (SD in Figure 3) from three independent experiments. A Student’s t test was used and p < 0.05 was taken as the minimum level of statis- Data are presented as mean ± SEM (SD in Figure3) from three independent experi- tical significance between treated and control samples. The IQR values were calculated ments. A Student’s t test was used and p < 0.05 was taken as the minimum level of statistical and represented as a black line in the histograms. signiﬁcance between treated and control samples. The IQR values were calculated and represented as a black line in the histograms. 5. Conclusions In conclusion, we5. Conclusionshave studied the genotoxic and epigenotoxic properties of three carbazole-derived moleculesIn conclusion, and demonstrated we have that, studied despite the their genotoxic similar and structure, epigenotoxic they properties of three have very different biologicalcarbazole-derived activities, moleculesin particular and towards demonstrated the DNA that, molecule despite as their a tar- similar structure, they get. have very different biological activities, in particular towards the DNA molecule as a target. The physicochemicalThe properties physicochemical of this carbazole properties series of thiswarrant carbazole some seriescomments. warrant In some comments. In the mass spectra of thesethe mass analogues, spectra we of sometimes these analogues, observed we sometimesspecies with observed a reduced species mass with a reduced mass that would be consistentthat wouldwith the be loss consistent of the withamine the side loss group of the and amine the sideformation group of and a the formation of a reactive iminium-methidereactive species iminium-methide (Scheme 1; [15,41]). species Given (Scheme that1;[ the15 ,NHMe41]). Given group that is an the NHMe group is an important interactingimportant moiety for interacting carbazoles moiety in the forp53-Y220C carbazoles cavity in the (it p53-Y220Cforms an H-bond cavity (it forms an H-bond with a D228 backbonewith carbonyl a D228 group backbone as reported carbonyl by group [41]), assuch reported a reduction by [41 ]),of binding such a reduction of binding affinity would reduceafﬁnity their wouldmutant reduce p53 reacti theirvation mutant potential p53 reactivation and possibly potential lead and to possiblyoff- lead to off-target target effects. This aspecteffects. may This be aspectconsidered may bewhen considered designing when the designingnext generation the next of car- generation of carbazole- bazole-based Y220C basedmutant Y220C stabilizers mutant to stabilizersincorporate to an incorporate interacting an moiety interacting that cannot moiety that cannot result result in an eliminationin an reaction, elimination for exampl reaction,e, by for introducing example, by an introducing additional anmethylene additional methylene group group between the carbazolebetween thering carbazole and the amin ring ande group. the amine However, group. so However, far there sois no far evi- there is no evidence that dence that this eliminationthis elimination occurs in occurscells. in cells.

Scheme 1. Potential amineScheme loss 1.in carbazolesPotential amine observed loss in in high-resolution carbazoles observed electrospray in high-resolution ionization electrospray ionization mass spectrometry (ESI-HRMS).mass spectrometry (ESI-HRMS).

Apart from expandingApart the frombiological expanding knowle thedge biological of the effects knowledge of specific of the carbazoles effects of speciﬁc carbazoles on cellular genome status,on cellular the molecular genome status,aspects the put molecular in evidence aspects in our put study in evidence may provide in our study may provide promising targets forpromising anti-cancer targets therapeutic for anti-cancer interventions. therapeutic In perspective, interventions. we can In define perspective, we can deﬁne these molecules, andthese PK9320 molecules, and PK9323 and PK9320to a greaterand extent,PK9323 asto pharmacologically a greater extent, as active pharmacologically active substances and, althoughsubstances further and, investigation although furtherwill be investigationnecessary, as eligible will be necessary,candidates as for eligible candidates for “anticancer drugs” and“anticancer “anticancer drugs” epi-drugs” and “anticancer on human epi-drugs” breast adenocarcinoma on human breast cells. adenocarcinoma In cells. In particular, the lower particular,dose of both the molecules lower dose used of both in our molecules experimental used in conditions our experimental appears conditions appears to to be the most promising,be the because most promising, the higher because dose could the higherbe so genotoxic dose could to beoperate so genotoxic a dan- to operate a danger- gerous clonal selectionous toward clonal the selection developmen towardt theof clones development more able of clonesto repair more DNA able break- to repair DNA breakages ages and thus more andaggressive. thus more In addition, aggressive. the In property addition, of the the property PK083 ofmolecule, the PK083 whichmolecule, which according to our data is even corrective of the basal DNA damage, will be worth investigating, thereby opening new potential scenarios for its future biomedical applications.

Supplementary Materials: The following is available online at https://www.mdpi.com/article/10

.3390/ijms22073410/s1, Figure S1: Optical densities of the Western blot bands; Figure S2: Phase- contrast micrographs of control and treated cells; Table S1: Variation of DNA band pattern as index of genomic demethylation; Protocols for PK studies; Table S2: Results from PK studies. Int. J. Mol. Sci. 2021, 22, 3410 12 of 14

Author Contributions: Conceptualization, C.L., I.C., J.S. and F.C.; Data curation, C.L., I.C., A.C.J., C.A.O., J.S. and F.C.; Investigation, C.A.O., R.J., R.K.T., M.C.B., M.W., C.A., T.F., P.D., R.B. (Rossella Bellina) and R.B. (Rossella Branni); Writing—original draft, C.L. and F.C.; Writing—review and editing, C.L., I.C., A.C.J., C.A.O., J.S. and F.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the University of Palermo (FFR 2018 and FFR 2020 funds for FC and CL), Worldwide Cancer Research grants 18-0043, 14-1002, the German Research Foundation (DFG grants JO 1473/1-1 and JO 1473/1-3) and the ERDF (LabFact: InterReg V project 121). We would also like to thank the School of Life Sciences, University of Sussex, and the Ewart Bequest fund for extra support. A.C.J. is also grateful for support by the SGC, a registered charity (no: 1097737) that receives funds from; AbbVie, Bayer AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Diamond, Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Merck & Co (aka MSD outside Canada and US), Pfizer, São Paulo Research Foundation- FAPESP, Takeda and Wellcome [106169/ZZ14/Z]. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest: The authors declare no conflict of interest.

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