Gene 524 (2013) 232–240

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Exploiting RNA as a new biomolecular target for synthetic polyamines

Anna Minarini a, Andrea Milelli b, Vincenzo Tumiatti b, Michela Rosini a, Monia Lenzi a, Lorenzo Ferruzzi a,Eleonora Turrini b, Patrizia Hrelia a, Piero Sestili c, Cinzia Calcabrini c, Carmela Fimognari b,⁎a Department of Pharmacy and BioTechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126 Bologna, Italyb Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Corso d’Augusto 237, 47921 Rimini, Italyc Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino “Carlo Bo”, Via Maggetti 21, 61029 Urbino, Italy

Abbreviations: DL5, 4-(((3-((4-((3-aminopropylamino)methyl)-2-methoxy phenol tetrahydrochloritetraazahexadecane-1,16-diyl)bis(2-methoxyphenol) tetrN1′-(butane-1,4-diyl)bis(N3-(2-methoxybenzyl)propane-1salt; MEB4, N1-(3-aminopropyl)-N4-(3-((2-methoxyb1,4-diamine tetrahydrochloride salt; PA, polyamine; Pinterfering RNA; RIN, RNA integrity number.⁎ Corresponding author. Tel.: +39 0512095636; fax:

E-mail address: carmela.fimognari@unibo.it (C. Fimo

0378-1119/$ – see front matter © 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.gene.2013.04.016

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 1 April 2013Available online 19 April 2013

Keywords:Synthetic polyaminesRNARNA integrity numberMethoctramineSpermine

Anticancer chemotherapy is strongly hampered by the low therapeutic index of most anticancer drugs andthe development of chemoresistance. Therefore, there is a continued need for the identification of new mo-lecular targets in order to selectively hit cancer cells. RNA has been recently validated as a cancer target bythe use of different specific ligands and/or by different agents able to destroy its diverse forms. The abilityof synthetic polyamines to interact and to alter the RNA structure has been already reported. In the presentpaper the interaction and the ability to damage RNA structure by several synthetic polyamines were evaluatedand quantified by microfluid capillary electrophoresis. This technique allowed us to visualize both the RNAimpairment through different electropherograms and to assess the RNA integrity number. Finally, the abilityto discriminate between RNA and DNA by these synthetic polyamines was also evaluated.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Antitumor chemotherapy is strongly hampered by the low ther-apeutic index of most anticancer drugs and the development ofchemoresistance, although advances in treatment of neoplastic diseaseshave been developed. In this research field there is a continued need forthe identification of newpharmacological strategies based on the charac-terization of molecular targets potentially exploitable for drugs withhighly selective mode of action.

The ever growing realization of the variety of biochemical roles ofRNA in all living organisms is leading to an increasing appreciation thatcellular and viral RNAs provide inviting targets to treat both infectiousand chronic diseases. In the entire pharmacopeia there are few, if any,drugs developedwith the intent to target RNA. However, some clinicallyuseful antibiotics such as tetracyclines, macrolides, and aminoglycosidesbind to conserved ribosomal RNA structural motifs as their mode ofaction. Certainly, RNA-binding drugs have precedence (Gallego andVarani, 2001). Furthermore, RNA is the genetic material of pathogenicviruses such as HIV or hepatitis C virus (HCV), and thus it provides

)amino)butyl)amino)propyl)de salt; DL6, 4,4′-(2,6,11,15-ahydrochloride salt; MEB1, N1,,3-diamine) tetrahydrochlorideenzyl)amino)propyl)butane-As, polyamines; siRNA, small

+39 0512095624.gnari).

l rights reserved.

numerous opportunities for the discovery of new drugs to treat the dev-astating illnesses caused by these agents. Finally, the complex functionsof RNA molecules in the control of gene expression in humans providenumerous opportunities to target specific RNA structures for treating avariety of chronic and degenerative conditions. However, the identifica-tion of RNA-binding drugs is in its infancy because, with the exception ofthe bacterial ribosome, RNA has only recently been considered as valu-able drug targets, for instance for the discovery of new antitumor agents(Leland and Raines, 2001).

Past investigations demonstrated the ability of some syntheticpolyamines (PAs) to interact with RNA (Hermann, 2003; Lawton andAppella, 2004). Although simple in structure, the polycationic PAsspermidine and spermine and their diamine precursor putrescine are es-sential factors for growth in eukaryotic cells (Wallace, 2009). Importantly,synthetic PAs are able to interact with awide number of biological targetsif suitably chemically modified (Bolognesi et al., 1998; Bonaiuto et al.,2012; Melchiorre et al., 2010; Minarini et al., 2010; Simoni et al., 2010).Regarding more specific cellular targets, it has been observed thatcaspase-dependent and -independent apoptotic cell death has been trig-gered after treatment with several PA analogs (Chen et al., 2003; Hegardtet al., 2002). Moreover, specific PA analogs have demonstrated effectson cell cycle. A selected group of asymmetrically substituted analogsproduces a profound G2/M block in the cell cycle that results from alteredtubulin polymerization in treated cells (Ha et al., 1997;Webb et al., 1999).Experimental andmodelingdata demonstrated that PAs and their analogsundoubtedly interact with naked DNA and affect DNA conformation andaggregation (Casero and Marton, 2007; Valasinas et al., 2003; Zini et al.,2009). Recent results also showed that PAs were able to interact with

Table 1Chemical structure of synthetic PAs.

Compound Chemical structure

Sperminea

Methoctraminea

DL5a

DL6a

MEB4a

MEB1a

a Tetrahydrochloride salt.

233A. Minarini et al. / Gene 524 (2013) 232–240

RNA and altered its integrity (Fimognari et al., 2008, 2009, 2012;Ouameur et al., 2010) and stabilize the tertiary structure of RNAmolecules (Imai et al., 2009).

Current available technologies used for the destruction of RNA in-clude approaches that utilize the activity of protein ribonucleases suchas antisense oligonucleotide, small interfering RNA (siRNA), RNase P-associated external guide sequence, and sequence-specific approachesthat do not utilize activity of protein ribonucleases, such as ribozyme(Tafech et al., 2006). Endogenous protein RNases can be utilized to targetspecific RNA for degradation by employing antisense oligonucleotidesand double-stranded siRNA molecules. Alfacell Corporation hasdeveloped an entirely novel approach to induce cytotoxicity in can-cer cells, based on the ability of the amphibian endoribonuclease(RNase) Onconase® (P-30 protein) to kill rapidly proliferating cells(Iordanov et al., 2000). Addition of exogenous protein RNases andbovine seminal-RNase (BS-RNase) to cells is known to degrade RNAs. Insuch cases, degradation of specific RNA cannot be achieved, but rathertheir effect on gene inactivation and cell cytotoxicity is believed tooccur through the destruction of various forms of RNAs leading toprogrammed cell death (Tafech et al., 2006). Onconase® is currently instage 3 clinical trials and was recently licensed by Par Pharmaceutical,following earlier studies demonstrating inhibition of multiple forms ofcancer cell growth both in vitro and in vivo (Halicka et al., 2007; Leeet al., 2007; Ramos-Nino et al., 2005). These studies of Onconase® car-ried out byAlfacell clearly validated RNAas a target for anticancer agents.

Even if several interesting effects were described, there are somechallenges that need to be considered before molecules that target RNAfor degradation warrant clinical testing. The major challenges associatedwith the approaches previously described are the methods of adminis-tration and delivery. Although the oral route is a preferred method ofdrug delivery, due to its non-invasive nature, adequate nucleic acid,peptide or protein drug delivery has not yet proven successful via thisroute (Tafech et al., 2006). This is largely due to the susceptibility ofthese large macromolecules to elimination by the digestive system.Moreover, regardless ofwhether themolecule to be delivered is a protein(RNase and onconase) or a nucleic acid (antisense oligonucleotide,siRNA, ribozyme), it encounters possible degradation by enzymes, bothextracellularly and intracellularly, before finding means to cross themembrane of target cells and destroy its target molecule.

Moreover, there are specific problems for some RNA-damagingmolecules. Once inside the cell, nucleic acid-based molecule is expectedto destroy the targeted nucleic acids without affecting other molecules.Unfortunately, this is often not the case, as the so-called off-target effectusually arises, which is a worrisome issue to many (Tafech et al., 2006).Even the siRNA technology, which was earlier hailed for its laser-likespecificity, has recently been shown to exhibit some off-target effects(Lim et al., 2005). In a clinical setting, the presence of off-target genesilencing could result in toxic side-effects of the RNA interferencebased drug.

In the case of the RNase therapeutic approaches where the goal is tokill targeted cells, the off-target effects within target cells do not appearto be an issue. It is also worth of mention that antisense oligonucleotidescan induce immune system (Tafech et al., 2006).

In this context, we tried to have insight into the effect of PAs on RNAdamage. To fulfill this aim, several synthetic spermine analogs, MEB1,MEB4, DL5 and DL6, were investigated about their ability to interactwith RNA and to quantify the damage caused through microfluidiccapillary electrophoresis (Mueller et al., 2000). These spermine analogswere selected to verify the effect on PA–RNA interaction of the insertionof one or two substituted aromatic rings on the spermine scaffold.Methoctramine, a PA acting as muscarinic receptor antagonist, used asa pharmacological tool for receptor characterization, was included inthis investigation in order to better define its pharmacological profile.The amount of the RNA damage was visualized by different electro-pherograms obtained for each polyamine (PA) synthesized, and asoftware algorithm allowed us to calculate the RNA integrity number

(RIN) (Schroeder et al., 2006). To better understand the globalimpact of PAs on nucleic acids, and to evaluate the selectivity ofsuch PAs, the effects on RNA were compared with those on DNA.

2. Results and discussion

PA analogs of spermine were selected for this first approach takinginto account our previous results in literature (Fimognari et al., 2008)(Table 1). It is worth noting that MEB4 and DL5 are monosubstitutedspermine derivatives, while MEB1 and DL6 are disubstituted sperminederivatives. These differences in chemical features allowed us to prelim-inarily determine the chemical requests on the PA scaffold to provideRNA damage. Methoctramine, a muscarinic receptor antagonist usedas pharmacological tool for receptor characterization, was included inthis investigation in order to better define its cytotoxic profile and to in-vestigate the effect of different polymethylene chains between nitrogenatoms of PA scaffold (Zini et al., 2009).

Jurkat cells were treated with different concentrations of PAs. Cellviability is shown in Fig. 1. For all the compounds, a gradual dose-dependent decrease in the number of viable cells was recorded;however, their IC50 values, which represent the concentration ofpolyamine required to decrease the cell viability activity by 50%(and calculated by interpolation from the dose–response curve),indicated a different inhibitory potency. DL6, MEB4 and spermineslightly modified the viability up to the highest concentrations testedand within the considered time interval, and the IC50 value did not fallwithin the range of concentrations tested. The cytotoxic activity of DL5,MEB1 and methoctramine was more pronounced, with IC50 values of98.33, 142.79 and 225.28 μM, respectively, and higher in comparisonwith that of spermine (IC50 = 1145 μM).

A cytotoxic lesion is typically characterized by the disruption ofcellular material, plasma membrane and organelles. In this case, itis possible to detect DNA or RNA damage as a consequence of a cyto-toxic insult. We therefore excluded the testing of doses that decreaseviability, compared to the concurrent control cultures, by more than50%.

In the second part of the study, we analyzed the RNA-damagingactivity of PAs. To this aim, cells were treated with PAs for 3, 6 or 24 h.The RNA-damaging effect of the different compounds was assessed byRIN measurement.

0 100 200 300 400 5000

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0 100 200 300 400 5000

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% o

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cel

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IC50: 5440.51 µMIC50: 98.33 µM

IC50: 142.79 µM IC50: 1860.08 µM

IC50: 225.28 µM IC50: 1145.88 µM

Fig. 1. Effect of PAs on viability of Jurkat cells. The viability was determined immediately after treatments, as detailed in the Materials and methods section. The data presented areaveraged from three independent experiments with error bars denoting S.D. of the mean.

234 A. Minarini et al. / Gene 524 (2013) 232–240

As reported in Fig. 2, a prototype electropherogram of total RNAcontaining a marker peak at about 24S (I) as well as 3 prominent peakscorresponding to small RNAs (peak II), 18S (peak III), and 28S (peak IV)rRNA was obtained for untreated cells. In cells treated with RNA-damaging compounds, aside from three prominent peaks (small RNAs,18S and 28S rRNA), an electropherogram of the size distribution of cellu-lar RNAs shows a broad range of molecular weights with much weakersignals. With increasing RNA damage, heights of 18S and 28S peaksgradually decrease, additional peak signals appear in a molecular weightrange between small RNAs and the 18S peak, and the baseline signalincreases.

For all the PAs, no effect on RNA integrity was recorded after 1 and3 h of treatment (data not shown). A significant decrease in RNA integ-rity was evident after 24 h of treatment. A decrease of the heights of18S and 28S peaks was well evident in cells treated with MEB1 andmethoctramine (Figs. 3D and F). An increase in the baseline signalwas particularly marked in DL6- and spermine-treated cells, alongwith signals from cellular RNAs with a broad range of molecularweights especially in spermine-treated cells (Figs. 4B and F).

Although all the tested agents were able to damage RNA, theirRNA-damaging potency was different. This can be easily illustrated by

comparing the RNA-lesive potency of equimolar concentrations ofPAs. The lowest concentration tested common to all PAs correspondsto 15.6 μM. At this concentration, the lowest RIN value (about 0.1)was recorded for DL5 (Fig. 3A), followed by methoctramine, with aRIN value of about 4.9 (Fig. 3E) and MEB1, with a RIN value of 7.9(Fig. 3C). A nonsignificant decrease of RIN value with respect to theuntreated cultures was observed for DL6 (RIN = 9.5) (Fig. 4A), MEB4(RIN = 9.6) (Fig. 4C) and spermine (RIN = 9.2) (Fig. 4E) at 15.6 μM.

PAs were tested for their DNA damaging activity towards culturedJurkat cells with the FHA under conditions allowing the detection ofDNA single and double strand breaks. Using this technique, DNAfragments resulting from the cleavage of DNA diffuse out of the nuclearcage as an inverse function of their size, a process that can be easilyobserved and quantified with a fluorescence microscope coupled to animage analysis software (Sestili, 2009; Sestili et al., 2006). Representa-tive micrographs of damaged (treated with H2O2 included as a refer-ence DNA damaging agent), DL6-treated or control cells are shown inFig. 5. Damaged cells show bright and large halos surrounding a smallnuclear remnant, while control or undamaged cells show no or negligi-ble halos (see micrographs in Fig. 5). 1, 3, and 24 h exposure of Jurkatcells to concentrations comparable to those used in RNA damage

I IIIIV

II

Fig. 2. Electropherograms of RNA size distribution of untreated cultures.

235A. Minarini et al. / Gene 524 (2013) 232–240

experiments did not cause any appreciable DNA cleavage: as an exam-ple, Fig. 5 (main graph) compares the results of DL6-treated cells andof H2O2-treated cells (included as a reference DNA damaging agent).Accordingly, the representative micrograph of DL6-treated cells indi-cates no sign of damaged DNA. It is worth noting that cells treatedwith a scarcely cytotoxic dose of H2O2 (75 μM) show signs of extensiveDNA damage at 1 and 3 h exposure, but none at 24. This phenomenon istypical of agentswhich rapidly cleave DNA producing rapidly repairablelesions and that are progressively removed by metabolizing cellularsystems (antioxidants for H2O2): since no damage was found at anyof these exposure times, it can be inferred that none of the tested PAsfalls into this category, at least at concentrations causing b50% cellkilling. We also tested the effect of the most active RNA damaging PAs(DL5, MEB1 and methoctramine) at concentrations exceeding theirIC50 values: in this case DNA was damaged to some extent, as in thecase of DL5 used at 600 μM for 1 and 3 h (Fig. 5). However a direct com-parison with the corresponding RNA damaging potential at 24 h expo-sure was impossible: indeed, we could only score cells treated for 6 hbecause of the extensive cell demise observed at longer exposuretimes with these high PA concentrations, a circumstance incompatiblewith the execution of FHA. Taken collectively these data indicate thatDL5, methoctramine and MEB1 display a low DNA damaging activitywhich can be appreciated only at exceedingly toxic concentrations, afinding which would also suggest that DNA damage caused by thesethree agents has a scarce, if any, cytotoxic relevance.

3. Conclusion and perspectives

DNA and RNA have a different sensitivity to PAs. In particular, in ourexperimental settings, RNA appears to bemore susceptible to damagingagents than DNA. This is not surprising; RNA is indeed mostly single-stranded, its bases are neither protected by hydrogen binding nor locat-ed inside the double helix, and ismainly localized in the cytoplasm. RNAis thus more accessible than DNA. Moreover, almost all of the cellularRNA has functional capacity for protein synthesis whereas only 28% ofthe human genomic DNA is transcribed into RNA and only 5% of thesetranscribed sequences actually encode proteins. Finally, RNA is moreabundant than DNA (Fimognari et al., 2008). Taken together, thoseobservations underline that it is highly probable that significant damageto RNA occurswhen cells are exposed to nucleic acids damaging agents.On the other hand, those observations do not allow predicting theability of a xenobiotic to damage DNA and/or RNA. Indeed, ethylmethanesulfonate, a well-known DNA-damaging agent (Fimognariet al., 2005), was devoid of RNA-damaging properties, whilespermine and S-nitroso-N-acetylpenicillamine, although lacking DNA-damaging properties (Guidarelli et al., 1998; Stopper et al., 1999), are

able to damage RNA (Fimognari et al., 2009). On the whole, these obser-vations along with the data presented herein, would suggest that theDNA and RNA damaging capacities of a given genotoxic agent areindependent and not necessarily interdependent. That a given agentpreferentially attacks RNA, DNA or both targets may then depend onintrinsic featureswhichmight probably relate to themechanism respon-sible for genomic damage, the affinity for double or single strandednucleic acids and its intracellular compartmentalization.

This investigation represents a first attempt to quantify the damage toRNA by synthetic PAs. The different RNA-damaging properties of the PAstestedmaybe due to a different cellular uptake.We are aware that amoreextensive study should be undertaken to analyze cellular contents ofsynthetic PAs which might be relevant to define their structure–activityrelationships. It is known that PA transport is carrier mediated, time-,temperature- and concentration-dependent, energy requiring, andsaturable (Palmer and Wallace, 2010). This last aspect deserves in-depth in vitro study aimed at clarifying the role of PA transport systemin the uptake of our synthetic PAs and their RNA-damaging activitiesin PA transport system-deficient cell lines. However, data derivingfrom this study might contribute to assemble the rather complicatepuzzle related to the role(s) of the PAs inside the cells. In fact, it isknown that the biological functions of the natural PAs have, at themoment, not been completely clarified and all information whichshed light on their functions should be taken into account.

4. Materials and methods

4.1. Materials

All chemicals were of analytical grade and purchased from Sigma(St. Louis, MO, USA).

4.2. Chemistry

1H and 13C NMR spectra were recorded on Varian VXR 400 and200 spectrometers in CDCl3, CD3OD, and D2O as solvents. Chemicalshifts are given in ppm, J values are given in Hz, and spin multiplici-ties are given as s (singlet), brs (broad singlet), d (doublet), t (triplet),q (quartet), or m (multiplet). Electron spray ionization (ESI) massspectra were recorded on a VG 7070E instrument. Chromatographicseparations were performed on silica gel columns by flash (Kieselgel40, 0.040–0.063 mm; Merck), or gravity column (Kieselgel 60,0.063–0.200 mm; Merck) chromatography. Reactions were followedby thin-layer chromatography (TLC) on Merck (0.25 mm) glass-packed precoated silica gel plates (60 F254) that were visualizedin an iodine chamber, UV lamp and bromocresol green. The term“dried” refers to the use of anhydrous Na2SO4. Methoctramine wassynthesized as previously reported (Minarini et al., 1989).

Although monosubstituted spermine MEB4 was already known(Rosini et al., 1999), it was synthesized following themore advantageousprocedure reported in Scheme 1. In particular, the tri-Boc-spermine(Wellendorph et al., 2003) was coupled with the suitable substitutedbenzaldehyde via Schiff base, followed by deprotection of the aminoprotecting groups by acidic hydrolysis. The disubstituted spermine de-rivatives were easily synthesized by direct condensation of the suitablesubstituted benzaldehyde via Schiff base.

Tert-butyl (4-((tert-butoxycarbonyl)(3-((2-methoxybenzyl)amino)propyl)amino) butyl)(3-((tert-butoxycarbonyl) amino)propyl)carba-mate (1, Scheme 1). A solution of tri-Boc-spermine (0.502 g, 1 mmol)and 2-methoxybenzaldehyde (0.150 g, 1.1 mmol) in toluene (50 mL)was refluxed in a Dean–Stark apparatus for 5 h and on cooling solventwas removed in vacuum. NaBH4 (0.151 g, 4 mmol) was added inportions to a cooled (0 °C) solution of the residue in EtOH (30 mL) andstirring was continued at room temperature for 4 h. The solvent wasthen removed and the residue was dissolved in CH2Cl2 (30 mL) andwashed with H2O (3 × 20 mL). Removal of the dried solvent in vacuum

236 A. Minarini et al. / Gene 524 (2013) 232–240

gave a residue thatwas purified by flash chromatographywith amixtureof CH2Cl2/MeOH/33% aq. NH4OH (9:1:0.05) as eluent, yielding thedesired product 1 (0.448 g, 72%) as yellow oil; 1H NMR (200 MHz,D2O) δ 1.42–1.65 (m, 29H), 1.63–1.78 (m, 4H), 2.16 (brs, 1H exchange-able with D2O), 2.68 (t, 2H, J = 6.8), 3.12–3.23 (m, 12H), 3.85 (s, 2H),3.79 (s, 3H), 5.18 (brs, 1H exchangeable with D2O), 6.65–6.81 (m, 1H),6.81–6.84 (m, 2H), 7.30–7.37 (m, 1H).

N1-(3-aminopropyl)-N4-(3-((2-methoxybenzyl)amino)propyl)butane-1,4-diamine tetrahydrochloride salt (MEB4, Scheme 1). Asolution of 1 (0.400 g, 0.69 mmol) in 30 mL of MeOH and 20 mLof aq. HCl 3 M (10 mL) was stirred overnight at room temperature.Following solvent removal, the residue was washed with Et2O(5 × 20 mL). The resulting solid was filtered and dried to affordthe desired product MEB4 tetrahydrochloride as a white solid

0.0 0.9 1.9 3.9 7.8 15.6

31.2

62.5

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101112

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***

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Concentration (µM)

RIN

A

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***

***

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C

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0123456789

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***

*** *** *** ***

RIN

E

Fig. 3. RIN values and electropherograms of RNA size distribution calculated after cell treatmaveraged from three measures with error bars denoting S.D. of the mean. Electropherogram

(0.281 g, 87%); mp 123–125 °C; 1H NMR (400 MHz, D2O) δ 1.62–1.66(m, 4H), 1.93–2.01 (m, 4H), 2.94–3.05 (m, 12H), 3.78 (s, 2H), 4.13(s 3H), 6.91–6.94 (m, 3H), 7.26–7.31 (m, 1H); 13C NMR (400 MHz,D2O) δ 22.6, 22.7, 23.7, 36.5, 43.9, 44.4, 46.9, 51.0, 55.4, 115.0, 115.3,122.3, 130.5, 132.1, 159.2; MS (ESI+) m/z = 162 (M + 2H)2+.

Tert-butyl (4-((tert-butoxycarbonyl)(3-((4-hydroxy-3-methoxy-benzyl) amino) propyl) amino)butyl)(3-((tert-butoxycarbonyl)amino) propyl) carbamate (Leland and Raines, 2001). A solution oftri-Boc-spermine (Wellendorph et al., 2003) (0.502 g, 1 mmol)and 4-hydroxy-3-methoxybenzaldehyde (0.167 g, 1.1 mmol) in toluene(50 mL) was refluxed in a Dean–Stark apparatus for 5 h and on coolingsolvent was removed in vacuum. NaBH4 (0.151 g, 4 mmol) was addedin portions to a cooled (0 °C) solution of the residue in EtOH (30 mL)and stirring was continued at room temperature for 4 h. The solvent

B

D

F

ent for 24 h with DL5 (A, B), MEB1 (C, D), methoctramine (E, F). The data presented ares are representative of three different experiments with similar results.

0.0 15.6 31.2 62.5 125.0 250.0 500.00123456789

101112

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Concentration (µM)

Concentration (µM)

Concentration (µM)

RIN

***

***

*** *** ***

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101112

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***RIN

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101112

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***

RIN

***

***

***

***

B

C D

E F

A

Fig. 4. RIN values and electropherograms of RNA size distribution calculated after cell treatment for 24 h with DL6 (A, B), MEB4 (C, D), spermine (E, F). The data presented areaveraged from three measures with error bars denoting S.D. of the mean. Electropherograms are representative of three different experiments with similar results.

237A. Minarini et al. / Gene 524 (2013) 232–240

was then removed and the residue was dissolved in CH2Cl2 (30 mL) andwashed with H2O (3 × 20 mL). Removal of the dried solvent in vacuumgave a residue thatwas purified by flash chromatographywith amixtureof CH2Cl2/MeOH/33% aq. NH4OH (9:1:0.05) as eluent, yielding the de-sired product 2 (0.543 g, 85%) as yellow oil; 1H NMR (200 MHz, CDCl3)δ 1.43–1.47 (m, 29H), 1.63–1.68 (m, 4H), 2.23 (brs, 1H exchangeablewith D2O), 2.64 (t, 2H, J = 6.8), 3.09–3.25 (m, 12H), 3.90 (s, 2H), 5.27(brs, 1H exchangeable with D2O), 7.16–7.38 (m, 4H).

4-(((3-((4-((3-aminopropyl)amino)butyl)amino)propyl)amino)methyl)-2-methoxy phenol tetrahydrochloride salt (DL5, Scheme 1).A solution of 2 (0.400 g, 0.69 mmol) in 30 mL ofMeOH and 20 mL of aq.HCl 3 M (10 mL) was stirred overnight at room temperature. Followingsolvent removal, the residue was washed with Et2O (5 × 20 mL). Theresulting solid was filtered and dried to afford the desired product DL5tetrahydrochloride as a brown solid (0.278 g, 97%); mp 123–125 °C; 1HNMR (400 MHz, D2O) δ 1.63–1.67 (m, 4H), 1.93–2.03 (m, 4H),

2.96–3.05 (m, 12H), 3.75 (s, 3H), 4.06 (s, 2H), 6.82–6.87 (m, 2H),6.99 (s, 1H); 13C NMR (400 MHz, D2O) δ 22.5, 22.6, 22.7, 36.5, 43.5,44.4, 44.5, 46.9, 51.0, 55.9, 113.9, 115.7, 122.7, 123.3, 145.9, 147.6;MS (ESI+) m/z = 170 (M + H)+.

N1,N1′-(butane-1,4-diyl)bis(N3-(2-methoxybenzyl)propane-1,3-diamine) tetrahydrochloride salt (MEB1, Scheme 1). A solution ofspermine (0.500 g, 2.47 mmol) and 2-methoxybenzaldehyde (0.841 g,6.18 mmol) in MeOH (30 mL) was refluxed for 6 h and after coolingthe solvent was removed in vacuum. NaBH4 (0.38 g, 10 mmol) wasadded in portions to a solution of the residue in EtOH (30 mL), stir-ring was continued at room temperature for 6 h, aq. HCl (10 mL,6 M) was added to pH = 3 and the solvents were evaporated to dry-ness in vacuum. The residue was washed with Et2O (5 × 20 mL) andthe resulting solid was filtered and dried to afford the desiredproduct MEB1 tetrahydrochloride (1.23 g, 85%) as a white solid;mp = 118°–120 °C. 1H-NMR (200 MHz, CD3OD) δ 1.41–1.53 (m, 8H),

1 3 24

0

1

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6

Exposure time (h)

Nu

clea

r d

iffu

sio

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acto

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DL5 1h DL5 3h

** **

**

*

Fig. 5. Effect of DL6 on cellular DNA. Cells were treated for 1, 3 or 24 h with 300 μMDL6(striped bars), and immediately assayed for DNA damage with the fast halo assay. Solidbars refer to the effect of 75 μM H2O2 given to cells for the same lengths of time; openbars refer to control cells. Also shown (insets) representative micrographs of FHAprocessed-cells treated with 300 μM DL6 (24 h), 75 μM H2O2 (3 h and 24 h), 600 μMDL5 (1 and 3 h), or of control cells.

238 A. Minarini et al. / Gene 524 (2013) 232–240

2.0 (m, 4H), 2.55 (m, 12H), 3.73 (s, 6H), 3.81 (s, 4H), 6.65–6.96 (s, 8H).(ESI+) m/z = 443 (M + H)+.

4,4′- (2,6,11,15-tetraazahexadecane-1,16-diyl)bis(2-methoxy-phenol) tetrahydrochloride salt (DL6, Scheme 1). A solution of spermine(0.500 g, 2.471 mmol) and 4-hydroxy-3-methoxybenzaldehyde(0.841 g, 6.18 mmoles) in MeOH (30 mL) was refluxed for 6 h andafter cooling the solvent was removed in vacuum. NaBH4 (0.38 g,10 mmol) was added in portions to a solution of the residue in EtOH(30 mL), stirring was continued at room temperature for 6 h, aq. HCl(10 mL, 3 M) was added to pH = 3 and the solvents were evaporatedto dryness in vacuum. The residue was washed with Et2O (5 × 20 mL)and the resulting solid was filtered and dried to afford, after drying invacuum, the desired product DL6 tetrahydrochloride (1.27 g, 83%) asa white solid; mp = 250°–252 °C. 1H NMR (200 MHz, CD3OD) δ1.40–1.60 (m, 4H), 1.65–1.80 (m, 4H), 2.50–2.70 (m, 12), 3.66 (s, 4H),3.83 (s, 6H), 6.65–6.80 (m, 4H), 6.93 (s, 2H). (ESI+) m/z = 474(M + H)+.

5. Cell cultures and treatments

Jurkat T-leukemia cells were purchased from Istituto ZooprofilatticoSperimentale della Lombardia e dell'Emilia-Romagna (Brescia, Italy)andwere free frommycoplasma infections. Cells were grown in suspen-sion and propagated in RPMI 1640 supplemented with 10% heat-inactivated bovine serum, 1% antibiotics (all obtained from Sigma,St. Louis, MO, USA). To maintain exponential growth, the cultureswere divided every third day by dilution to a concentration of1 × 105 cells/mL.

Cells were treated with different concentrations of PAs for 1, 3or 24 h at 37 °C. The range of concentrations of the potentialRNA-damaging agents was selected considering the quantity oftotal RNA extracted per cell, as recently suggested (Hofer et al.,2005; Martinet et al., 2004). In our experimental conditions, thehighest concentration tested corresponded to 0.5 mM.

6. Cell viability

Viability was determined immediately after treatments by usingGuava EasyCyte Mini flow cytometry (Guava Technologies, Hayward,CA, USA), according to the manufacturer's recommendations. Briefly,cells were mixed with an adequate volume of Guava ViaCount Reagent(Guava Technologies) and allowed to stain for at least 5 min at roomtemperature. The Guava ViaCount Reagent provides absolute cellcount and viability data based on the differential permeability of DNA-binding dyes and the analysis of forward scatter. The fluorescence ofeach dye is resolved operationally to allow the quantitative assessmentof both viable and non-viable cells present in a suspension.

7. Extraction of RNA

After cell treatment, RNA was isolated with an Agilent Total RNAisolation Mini Kit (Agilent Technologies, Palo Alto, CA, USA), accordingto themanufacturer's recommendations. Briefly, 350–400 μL of lysis solu-tion were added to cell pellet and the cell homogenate was centrifugedthrough a mini-prefiltration column. The flow-through was mixed withan equal volumeof 70%ethanol, incubated for 5 minat room temperatureand centrifuged through a mini-isolation column. The flow-through wasdiscarded and the RNA-loaded column was transferred into an RNase-free final collection tube. Then, the purified RNA was eluted by additionof 10–15 μL of nuclease-free water.

8. Analysis of RNA damage

RNA analysiswas performedbymicrofluidic capillary electrophoresiswith the Agilent 2100 bioanalyzer. The bioanalyzer is an automatedbio-analytical device using microfluidics technology that provides elec-trophoretic separations in an automated and reproducible manner(Mueller et al., 2000). Tiny amounts of RNA samples are separated inthe channels of the microfabricated chips according to their molecularweight and subsequently detected via laser-induced fluorescencedetection. The result is visualized as an electropherogram wherethe amount of measured fluorescence correlates with the amountof RNA of a given size. A software algorithm then allows the calcula-tion of an RNA integrity number (RIN). The RIN algorithm is based ona selection of informative features from the electropherograms. Forthis purpose, each electropherogram is divided into the following nineadjacent segments covering the entire electropherogram: a pre-region,a marker-region, a 5S-region, a fast-region, an 18S-region, an inter-region, a 28S-region, a precursor-region and a post-region. In addition,several global features are extracted, i.e. features that span severalsegments. Among these, the average and maximum height, areas andtheir ratios, total RNA ratio and the 28S area ratio are themost importantfeatures. The gradual degradation of rRNA is reflected by a continuousshift towards shorter fragment sizes. For classification of RNA integrity,ten categories are defined from ≤1 (totally degraded RNA) to 10 (fullyintact RNA) (Schroeder et al., 2006).

9. Analysis of DNA damage by fast halo assay (FHA)

The assay has been carried out as previously described (Sestili, 2009;Sestili et al., 2006). After the treatments, the cellswerewashed two timeswith PBS and then resuspended at 4.0 × 104/mL in ice-cold PBScontaining 5 mM EDTA: 25 μL of this cell suspension was diluted withan equal volume of 2% low melting agarose in PBS and immediately

H2N NN

HN

R

R

Ri

NH

NN

HN

R

R

RH

O

1: R = Boc, R' = 2-OCH32: R = Boc, R' = 3-OCH3, 4-OH

MEB4: R = H, R' = 2-OCH3DL5: R = H, R' = 3-OCH3, 4-OH

ii

R'

R' = 2-OCH3R' = 3-OCH3, 4-OH

R'

H2N NH

HN NH2H

OR'

R' = 2-OCH3R' = 3-OCH3, 4-OH

iiiNH

NH

HN

HN

MEB1: R' = 2-OCH3DL6: R' = 3-OCH3, 4-OH

R'R'

R = Boc

(a, b)

(a, b)

Scheme 1. (i) (a) Toluene/Δ/3 h; (b) NaBH4/EtOH/rt/4 h; (ii) HCl 3 M/rt/overnight; (iii) (a) MeOH/Δ/6 h; (b) NaBH4/EtOH/rt/6 h. Boc: (CH3)3OCO\.

239A. Minarini et al. / Gene 524 (2013) 232–240

sandwiched between an agarose-coated slide and a coverslip. Aftercomplete gelling on ice, the coverslips were removed and the slideswere immersed in NaOH 300 mM for 15 min at room temperature.Ethidium bromide (10 μg/mL) was directly added to NaOH during thelast 5 min of incubation. The slides were then washed and destainedfor 5 min in distilled water. The ethidium bromide-labeled DNA wasvisualized using a Leica DMLB/DFC300F fluorescence microscope (LeicaMicrosystems,Wetzlar, Germany) equippedwith anOlympus ColorviewIIIU CCD camera (Olympus Italia Srl, Segrate, Italy) and the resultingimages were digitally recorded on a PC and processed with an imageanalysis software (Scion Image, Scion Corporation, Frederick, MD, USA).The amount of fragmented DNA diffusing out of the nuclear cage,i.e. the extent of strand scission,was quantified by calculating the nucleardiffusion factor, which represents the ratio between the total area of thehalo and nucleus and that of the nucleus.

10. Statistical analysis

All results are expressed as the mean ± S.D. of at least three ex-periments. Differences among treatments were evaluated by ANOVA,followed by Dunnett or Bonferroni t-test, using GraphPad InStat version3.00 forWindows 95 (GraphPad Software, San Diego, CA, USA). P b 0.05was considered significant.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This research was supported by grants from Ministero dell'Istruzionedell'Università e della Ricerca, Programmi di Ricerca Scientifica diRilevante Interesse Nazionale (PRIN), grant numbers: 200974K3JCand 2009ESXPT2_001; University of Bologna (RFO) and PoloScientifico-Didattico di Rimini.

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