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Induction of erythroid differentiation and increased globin mRNA productionwith furocoumarins and their photoproducts

Alessia Salvador a,⇑, Eleonora Brognara c, Daniela Vedaldi a, Ignazio Castagliuolo b, Paola Brun b,Cristina Zuccato c, Ilaria Lampronti c, Roberto Gambari c,d,⇑a Department of Drug Sciences, University of Padova, Italyb Department of Molecular Medicine, University of Padova, Italyc Department of Life Sciences and Biotechnology, University of Ferrara, Italyd Center of Biotechnology, University of Ferrara, Italy

a r t i c l e i n f o

Article history:Received 14 November 2012Received in revised form 14 February 2013Accepted 18 February 2013Available online 27 February 2013

Keywords:PsoralenAngelicinPhotoproductsK562 cellsErythroid differentiationFetal hemoglobin

a b s t r a c t

Differentiation-therapy is an important approach in the treatment of cancer, as in the case of erythroidinduction in chronic myelogenous leukemia. Moreover, an important therapeutic strategy for treatingbeta-thalassemia and sickle-cell anemia could be the use of drugs able to induce erythroid differentiationand fetal hemoglobin (HbF) accumulation: in fact, the increased production of this type of hemoglobincan reduce the clinical symptoms and the frequency of transfusions. An important class of erythroid dif-ferentiating compounds and HbF inducers is composed by DNA-binding chemotherapeutics: however,they are not used in most instances considering their possible devastating side effects. In this contest,we approached the study of erythrodifferentiating properties of furocoumarins. In fact, upon UV-A irra-diation, they are able to covalently bind DNA. Thus, the erythrodifferentiation activity of some linear andangular furocoumarins was evaluated in the experimental K562 cellular model system. Quantitative real-time reverse transcription polymerase-chain reaction assay was employed to evaluate the accumulationof different globin mRNAs. The results demonstrated that both linear and angular furocoumarins arestrong inducers of erythroid differentiation of K562 cells. From a preliminary screening, we selectedthe most active compounds and investigated the role of DNA photodamage in their erythroid inducingactivity and mechanism of action. Moreover, some cytofluorimetric experiments were carried out to bet-ter study cell cycle modifications and the mitochondrial involvement. A further development of the workwas carried out studying the erythroid differentiation of photolysis products of these molecules. 5,50-Dimethylpsoralen photoproducts induced an important increase in c-globin gene transcription in K562cells.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Furocoumarins are well known natural or synthetic compounds,which derive from a linear (psoralens) or angular (angelicins) con-densation of a coumarin with a furan ring. Some of them are em-ployed in PUVA (Psoralen + UVA) therapy for the treatment ofautoimmune or hyper-proliferative skin diseases, including psoria-sis and vitiligo. PUVA therapy efficacy is due to a combination ofpsoralen administration and UV-A irradiation. In fact, when acti-vated by UV-A light, furocoumarins induce many biological effects,

such as photocycloadditions to DNA, immune system modulation,reactions with proteins, RNA and lipids [1]. Thanks to the develop-ment of the photopheresis, the PUVA therapy has amplified itsapplication to some specific tumor forms such as cutaneous T-celllymphoma [2].

Although the first furocoumarin was introduced in clinical prac-tice as early as 1974 [3], these molecules still draw the attention ofthe scientific community. In fact, many new potential therapeuticapplications for furocoumarins are found. For instance, some psor-alen derivatives, such as 8-methoxypsoralen, showed anticonvul-sant properties [4]; 4,6,40-trimethylangelicin demonstrated to bepotentially useful in the treatment of cystic fibrosis thanks to itsanti-inflammatory activity and its potentiating action on the CFTRmembrane channel whose dysfunction causes that disease [5].Moreover, furocoumarins were found to induce various processesof differentiation. Psoralen is able to stimulate osteoblast differen-tiation without irradiation as demonstrated by Tang et al. [6],

1011-1344/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.jphotobiol.2013.02.011

⇑ Corresponding authors. Addresses: Department of Sciences of Drug, Universityof Padova, Via Marzolo 5, 35131 Padova, Italy. Tel.: +39 049 8275034; fax: +39 0498275366 (A. Salvador), Department of Life Sciences and Biotechnology, Universityof Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy. Tel.: +39 532 974443; fax:+39 532 974500 (R. Gambari).

E-mail addresses: [email protected] (A. Salvador), [email protected](R. Gambari).

Journal of Photochemistry and Photobiology B: Biology 121 (2013) 57–66

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while with or without light activation, many furocoumarins induceerythroid differentiation in different cellular models [7–9]. Thislatter property can be useful for the treatment of hematologic dis-eases, such as b-thalassemia: at present, an important therapeuticstrategy is the administration of fetal hemoglobin (Hb) inducers toreduce clinical symptoms and blood transfusion requirement [10].

The aim of our study was to evaluate the activity of six linearand five angular furocoumarins on the induction of erythroid dif-ferentiation expression of globin genes in the human leukemia cellline K562. These molecules were not fully checked for their poten-tial erythro-differentiation so far. The K562 cell line, isolated froma patient with chronic myelogenous leukemia in blast crisis, is of-ten used as in vitro experimental system for the first screening ofnew fetal Hb inducers [11]. The K562 cell line presents a lowamount of Hb-synthesizing cells under standard cell-growth con-ditions. After the treatment with suitable inducing compounds,massive erythroid induction occurs, with a clear increase in theexpression of human a and c globin genes and a cytoplasmic accu-mulation of Hb Portland (f2c2) and Hb Gower 1 (f2e2) [10,12,13].

We then focused our attention on the most active compounds inorder to obtain preliminary data about their mechanism of actionand about the mode of cellular death induced by them.

A second part of our study was related to the well establishedobservation that after UV-A irradiation, psoralens undergo photol-ysis with the formation of new species in solution, the so calledphotooxidation photoproducts (POPs). POPs also present some bio-logical activity: in fact, some papers showed their antileukemicand immunosuppressive effects, which led us to hypothesize theirpossible biological contribution in PUVA therapy [14,15]. Recently,we also isolated and reported the erythroid differentiation induc-tion by a specific 5-methoxypsoralen photoproduct [16].

Thus, the effect of POPs was also evaluated on the expression ofembryo-fetal globin genes in K562 cells by quantititative real-timereverse transcription polymerase-chain reaction assay (RT-qPCR).

2. Materials and methods

2.1. Chemicals and cellular buffers and media

Psoralens and angelicins belong to the collection of the Sciencesof Drug Department in Padova University [17–19]. If not specifiedelsewhere, all chemicals, biological buffers and cellular media werepurchased from Sigma–Aldrich.

2.2. Irradiation procedure

Two HPW 125 Philips lamps, mainly emitting at 365 nm, wereused for irradiation experiments. The spectral irradiance of thesource was 4.0 mW cm�2 as measured at the sample level by aCole-Parmer Instrument Company radiometer (Niles, IL, USA)equipped with a 365-CX sensor.

2.3. Cell culture

The human leukemia K562 cells were cultured in a humidifiedatmosphere of 5% CO2/air in RPMI 1640 medium, supplementedwith 10% fetal bovine serum (FBS; Invitrogen), 100 units/mL peni-cillin and 100 mg/mL streptomycin.

2.3.1. Phototoxicity and benzidine testSuspensions of 30,000 K562 cells/mL in complete medium were

seeded in individual wells of a 24-well tissue culture microtiterplate. The plates were incubated at 37 �C for 24 h prior to theexperiments. Stock solutions of furocoumarin derivatives wereprepared in methanol and then diluted with Hank’s balanced salt

solution (HBSS pH 7.2; the concentration of methanol was alwayslower than 0.5%) for irradiation experiments. After medium re-moval, 1 mL of the drug solution was added to each well, incubatedat 37 �C for 30 min and then irradiated (1 and 2 J/cm2, which cor-respond to 4 and 8 min of irradiation at 0.25 J/cm2). After irradia-tion, the solution was replaced with complete medium and theplates were incubated for 5–7 days. The medium was never chan-ged during this period. Erythroid differentiation was determinedby counting blue benzidine-positive cells after suspending the cellsin a solution containing 0.2% benzidine in 10% H2O2 and 0.5 Mglacial acetic acid [7]. Cell phototoxicity was assessed by the MTT[(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)]test 5 days after irradiation [20]. The irreversibility of erythroid dif-ferentiation was also assessed: 6 days after irradiation, 10,000 cellswere plated in a new plate with fresh medium and after further4 days, they were counted after benzidine-staining as elsewheredescribed [21].

2.3.2. Cell cycle analysis1 � 106 K562 cells were incubated for 24 h and then irradiated

(1 J/cm2) in HBSS with or without the test compounds. After 24 hfrom irradiation, cells were fixed with ice-cooled ethanol (70% v/v), treated overnight with RNAse A (0.1 mg/mL) in phosphate sal-ine buffer and finally stained with propidium iodide (PI, 0.1 mg/mL). Samples were analyzed on a BD FACS Calibur flow cytometercollecting 10,000 events. Results of cell-cycle analysis were exam-ined using WinMDI 2.9 (Windows Multiple Document Interface forFlow Cytometry) [20].

2.3.3. Mitochondrial dysfunctionK562 cells (300,000 cells/mL) were seeded in 24-well micro-

plate and incubated for 24 h prior irradiation. After medium re-moval, 1 mL of the drug solution was added to each well,incubated at 37 �C for 30 min and then irradiated (1 J/cm2). Afterirradiation, the solution was replaced with complete mediumand the plates were incubated for 24 h. Cells were collected by cen-trifugation and re-suspended in 1 lM JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazol-carbocyanine) solution in HBSSor in 100 nM NAO (10-N-nonyl acridine orange) solution in RPMImedium. The cytofluorimetric analysis (BD FACS Calibur flowcytometer) was performed collecting green (FL1) and orange(FL2) fluorescence for JC-1 staining and only the green one (FL1)for NAO staining in at least 10,000 events for each sample [22,23].

2.4. Preparation of photoproducts

Solutions of derivatives in methanol were irradiated in a quartzcuvette with different UV-A doses (0, 8, 16 and 32 J/cm2). After theirradiation, the solution was lyophilized, suspended in a knownvolume of methanol and stored at �20 �C. Concentrations of un-known photoproduct mixtures in this paper were expressed as ifthe initial psoralen was not photodegraded.

2.5. mRNA measurements

RNA was isolated from K562 cells and measured by reversetranscription quantitative real-time polymerase chain reaction(RT-qPCR) as described [24] using gene-specific double fluores-cence labeled probes in an ABI Prism 7700 Sequence Detection Sys-tem version 1.7.3 (Applied Biosystems). The following primer andprobe sequences were used: a-globin forward primer, 50-CACGCG CAC AAG CTT CG-30; a-globin reverse primer, 50-AGG GTCACC AGC AGG CAG T-30; a-globin probe, 50-FAM-TGG ACC CGGTCA ACT TCA AGC TCC T-TAMRA-30; c-globin forward primer,50-TGG CAA GAA GGT GCT GAC TTC-30; c-globin reverse primer,50-TCA CTC AGC TGG GCA AAG G-30; c-globin probe, 50-FAM-TGG

58 A. Salvador et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 57–66


GAG ATG CCA TAA AGC ACC TGG-TAMRA-30. The kit for quantita-tive RT-PCR for f-globin mRNA and e-globin mRNA were fromApplied Biosystems (f-globin mRNA: Hs00923579_m1; e-globinmRNA: Hs00362216_m1). The fluorescent reporter and thequencher were 6-carboxyfluorescein (FAM) and 6-carboxy-N,N,N0,N0-tetramethylrhodamine (TAMRA), respectively. For real-timePCR, the reference gene was 18S; this probe was fluorescent-labeled with VIC (Applied Biosystems) [24,25].

2.6. Statistical analysis

Unless indicated otherwise, results are presented as mean ±SEM. The differences between different treatments were analyzedusing the two-sided Student’s t test; p values lower than 0.05 wereconsidered significant (� = p < 0.05).

3. Results

3.1. Furocoumarins induce erythroid differentiation and phototoxicity

The erythroid differentiation of K562 cells was investigatedafter the treatment with six psoralens and five angelicins, whosestructures are described in Fig. 1. K652 cells were irradiated withtwo UV-A doses (1 and 2 J/cm2) and then erythroid differentiationwas measured by benzidine test after 5, 6 and 7 days from irradi-ation. At the same time, cellular viability was evaluated by MTT,obtaining evidences suggesting antiproliferative effects and photo-toxicity. Different concentrations of compounds were employedbecause of their different phototoxic effects; accordingly, concen-trations were chosen to maximize the erythroid effect withoutextensive reduction of cell viability. Moreover, considering the factthat some angelicins were powerful c-globin inducers withoutirradiation [26], the same tests were performed with higher con-centration of furocumarins in the absence of irradiation. With theexception of 4,6,40-trimethylangelicin (4,6,40-TMA) [26], withoutirradiation all furocoumarins, when administered at 50 lM,

showed a very low capability of causing an increase of benzidinepositive cells (lower than 10%) with respect to control (data notshown). On the contrary, after irradiation all tested molecules wereable to induce a clear increase of benzidine positive cells, as dis-played in Table 1. Table 1 reports the percentages of benzidine po-sitive cells and cellular viability after 6 days after irradiation at thehighest concentration for each compound. In general, psoralens in-duced a higher proportion of erythroid differentiating cells (38.4–78.1%) in comparison to angelicins (24.3–58.7%), and these dataconfirmed the ones obtained with other furocoumarins [7]. Fur-thermore, the induction of erythroid differentiation was dependenton the UV-A dose with the exception of some cases in which theantiproliferative effect was a major effect (see for example 5,50-dimethylpsoralen or 4,6,40-TMA or 4,40,50-trimethylangelicin). Inthe panel A of Fig. 2, the concentration-dependent increase of theratio of benzidine positive cells was illustrated for some com-pounds as examples. Moreover, the panel B of Fig. 2 shows repre-sentative pictures of treated cells after benzidine staining: cellsirradiated in the presence of 40,50-dimethylpsoralen (40,50-DMP)clearly were blue-colored1 and became larger with respect to con-trol (this effect is not unusual within already reported inducers oferythroid differentiation, such as cytosine arabinoside [10]). Fur-ther experiments were carried out to determine whether the in-duced erythroid differentiation was reversible. To this aim,6 days after irradiation (1 J/cm2), K562 cells were incubated foradditional 4 days with fresh medium, and the benzidine test wasperformed at this point. After 10 days, the percentage of erythroiddifferentiation did not present any significant variation in compar-ison to the ones of the sixth day, with the exception of 5,50-DMP,which exhibited a significant increase in the proportion of benzi-dine-positive cells (from 50.6 ± 5.0 to 66.8 ± 2.0). Considering thefact that in erythroid-induced K562 cells the growth efficiency islower (see Tables 1 and 2), these evidences support the concept

Fig. 1. Furocoumarin structures.

1 For interpretation of color in Fig. 2, the reader is referred to the web version ofthis article.

A. Salvador et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 57–66 59


Table 1Differentiation and cellular viability after 6 days from irradiation with furocoumarins.

% Benzidine positive cellsa % Cellular viabilityb

1 J/cm2 2 J/cm2 1 J/cm2 2 J/cm2

Control 1.5 ± 0.5 1.8 ± 0.4 98.0 ± 2.5 97.5 ± 1.4

Psoralens50-MP 1 lM 58.7 ± 1.9 78.1 ± 5.1 65.5 ± 2.5 45.7 ± 3.640 ,50-DMP 5 lM 60.7 ± 1.1 72.2 ± 5.0 73.7 ± 3.7 54.0 ± 2.55,50-DMP 1 lM 50.6 ± 5.0 44.5 ± 2.7 48.8 ± 6.7 19.5 ± 2.68,40-DMP 1 lM 38.4 ± 1.9 42.7 ± 1.7 60.9 ± 3.7 35.8 ± 3.93,4,8,40-TMP 1 lM 43.5 ± 7.1 38.7 ± 1.7 39.4 ± 5.7 22.5 ± 1.950-Br-4,40-DMP 1 lM 40.2 ± 4.3 46.7 ± 4.5 88.7 ± 11.2 42.6 ± 3.7

Angelicins50-MA 5 lM 24.3 ± 3.2 28.9 ± 4.8 97.7 ± 3.2 99.9 ± 4.53,4,40-TMA 1 lM 30.2 ± 2.2 39.4 ± 2.0 102.2 ± 11.2 57.8 ± 5.44,6,40-TMA 0.5 lM 58.7 ± 4.0 40.7 ± 4.3 60.9 ± 4.6 15.9 ± 3.44,40 ,50-TMA 5 lM 47.8 ± 6.9 18.9 ± 2.5 31.1 ± 2.5 9.2 ± 2.26,40 ,50-TMA 1 lM 33.5 ± 4.0 42.4 ± 1.9 74.3 ± 8.2 44.4 ± 5.5

a Data are expressed as media ± S.E.M. of at least four different independent experiments.b Data are obtained by MTT and are expressed as media ± S.E.M. of at least four different independent experiments.

Fig. 2. Erythroid differentiation photoinduced by furocoumarins. Panel A. Percentage of benzidine positive cells after 6 days from irradiation (1 J/cm2) of K562 cells in thepresence of the indicated concentrations of 50-MP, 40 ,50-DMP, 5,50-DMP and 4,6,40-TMA. Results are expressed as media ± S.E.M. of at least 4 different experiments. Panel B.Representative pictures (20�) of benzidine stained K562 cells 6 days after irradiation (1 J/cm2) by light microscope: control in the left picture, 5 lM 40 ,50-DMP in the right one.



that benzidine-negative cells at day 6 still can differentiate even inthe absence of irradiated compounds in the medium (this ‘‘com-mitment-like’’ effect is present in several inducers of K562 cell dif-ferentiation). In any case, the data suggest that the induceddifferentiation observed at day 6 is irreversible.

Since 50-methylpsoralen (50-MP), 40,50-DMP and 5,50-dimethylp-soralen (5,50-DMP) for psoralens and 4,6,40-TMA for angelicinswere the most active compounds, further experimental activitywas carried out with these molecules. Moreover, the lower UV-A(1 J/cm2) dose was chosen to minimize the phototoxic effect.

3.2. Erythroid differentiation is linked to DNA damage

The mechanism by which erythroid differentiation induced byfurocoumarin takes place is still unknown. However, the DNAphotobinding is considered the main effect for the photoantiprolif-erative activity of the PUVA therapy. Thus, some preliminaryexperiments were carried out to verify whether furocoumarinDNA photodamage could be involved also in the erythroid differen-tiation process. K562 cells were irradiated in the presence of thetested compounds and of the inhibitors of some phosphoinositidekinase-related kinases, such as DNA-dependent protein kinase(DNA-PK), ataxia telangiectasia mutated (ATM) and the ataxia-and Rad3-related protein (ATR), which can be activated after differ-ent kinds of DNA damage [27]. In particular, wortmannin was used

as inhibitor of the catalytic subunit of the PI3-kinase family of en-zymes [28], and caffeine as inhibitor of ATM and ATR but not ofDNA-PK [29]. Cell viability was not affected by the presence ofthese two inhibitors (data not shown). As it can be observed inFig. 3, the amount of benzidine positive cells was significantly re-duced, even if not completely abolished, for all tested compounds,when the irradiation was carried out in the presence of thoseinhibitors. Thus, the processes activated by DNA damage couldbe involved, at least in part, in the erythroid differentiationprocess.

3.3. Furocoumarin induce increased expression of globin genes

The effects of furocoumarins on the expression of human globingenes were determined by RT-qPCR analysis using probes amplify-ing the a-like a-globin and f-globin and the b-like e-globin andc-globin mRNA sequences. Effects on production of b-globin mRNAwere not analyzed, since it is well known that K562 cells do notefficiently transcribe the b-globin genes [10,30]. In Fig. 4, globinmRNA expression for 40,50-DMP and 4,6,60-TMA is presented; thesetwo molecules were selected as an example for linear (40,50-DMP)and angular (4,6,40-TMA) most active furocoumarins in inducingerythroid differentiation (Table 1). The results were reproducibleand demonstrate that: (a) when K562 cells were irradiated(1 J/cm2) in the absence of the compounds, only minor effects wereobserved on accumulation of globin mRNAs; (b) the induction ofglobin gene expression is not enhanced by low concentrations offurocoumarins without irradiation; however, (c) when irradiation

Fig. 3. Involvement of DNA photodamage in erythroid differentiation byfurocoumarins. Percentage of benzidine positive cells after 6 days from irradiationfrom irradiation (1 J/cm2) of K562 cells in the presence of 1 lM 50-MP, 5 lM 40 ,50-DMP, 1 lM 5,50-DMP and 0.5 lM 4,6,40-TMA. The irradiation was carried out in thepresence or absence of 10 lM Wortmannin (upper panel) or of 1 mM caffeine(lower panel). Results are expressed as media ± S.E.M. of at least four differentexperiments. C = irradiated control.

Fig. 4. Increase in a-, c-, e- and f-globin mRNA content in K562 cells irradiated(1 J/cm2) or incubated (dark) in the presence of 40 ,50-DMP (upper panel) and 4,6,40-TMA (lower panel) at the indicated concentrations. Expression of globin genes wasassayed by quantitative reverse transcriptase polymerase-chain reaction (qRT-PCR)after 6 days from the irradiation/incubation. CI = irradiated control.



was carried out in the presence of increasing concentrations of thetwo furocoumarins, a dose-dependent increase in globin mRNAexpression was observed. The level of induction was found to bedose-dependent, all the analyzed globin mRNAs were clearly in-duced, the level of induction was dramatic for a-globin, f-globinand c-globin mRNA sequences, but clearly evident also for e-globin

mRNA. When the experiment was repeated (n = 3) using the high-est furocoumarin concentration reproducible results were ob-served, and if the results were compared to reference K562 cellstreated with a control HbF inducer, this induction level was higherthan the most effective K562 erythroid inducer available, 1-octyl-thymine [30]. In fact the induction of f-globin mRNA was48.5-fold ± 8.5 for 40,50-DMP, 64.6-fold ± 8.2 for 4,6,40-TMA and37-fold ± 6.8 for 1-octylthymine (data not shown and Ref. [30]).

3.4. Furocoumarins induce a block in G2-M phase and cause cell deathby apoptosis

To further study the effects of furocoumarins on cell prolifera-tion, a cell cycle analysis was carried out after 24 h from the irra-diation of K562 in the presence of two different concentrations ofthe compounds (Fig. 5). This test is based on the fact that each cellcycle phase presents a different DNA content, which was quanti-fied by propidium iodide (PI) staining. The irradiation of K562 withall tested furocoumarins caused a reduction of G1 phase togetherwith a clear accumulation of cells in G2-M phase (see Table 2). ThisG2-M block was consistent with the effect of other furocoumarinsin the same cell line [7]. Moreover, indications of cell death byapoptosis were detected as DNA fragments in sub-G1 phase.

3.5. Furocoumarins induce cell death and erythroid differentiationwith the involvement of mitochondria

As furocoumarins are known to photoinduce apoptosis with theinvolvement of mitochondria, the role of these organelles was eval-uated with two different flow cytometry tests [31]. Impairment inmitochondrial function is an early event in the executive phase ofprogrammed cell death in different cell types and appears as theconsequence of a preliminary reduction of the mitochondrialtransmembrane potential (DWM). The lipophilic cation JC-1 wasused to monitor the changes in DWM induced by the tested com-pounds in combination with UV-A irradiation. Another conse-quence of mitochondrial dysfunction is the production of reactiveoxygen species which oxidized the mitochondrial phospholipidcardiolipin (CL). CL oxidation was monitored by staining irradiatedcells with N-nonyl acridine orange (NAO) as described in Section2.3.3.

A concentration-dependent increase of the percentage of cellswith a collapsed DWM can be observed after JC-1 staining (Fig. 6,upper panel): this may be an indication of the opening of the mito-chondrial mega-channels also called the permeability transitionpores (PTPs).

NAO interacts stoichiometrically with intact non-oxidized car-diolipin. In good agreement with the collapsed mitochondrialpotentials, treated cells showed an increase in the oxidized CL(Fig. 6, lower panel).

Fig. 5. Representative histograms of cell cycles after irradiation of K562 cells in thepresence of furocoumarins. Cells were irradiated (1 J/cm2) at the indicatedconcentrations of 50-MP, 40 ,50-DMP, 5,50-DMP and 4,6,40-TMA and after 24 h ofincubation were labeled with propidium iodide and analyzed.

Table 2Percentage of K562 cells in each cell cycle phase after 24 h from irradiation (1 J/cm2)in the presence of the indicated concentration of furocoumarins.

G1a S G2-M subG1

Control 43.9 25.1 23.0 8.050-MP 1b 14.0 12.0 63.2 10.850-MP 0.5 22.8 14.6 50.5 12.040 ,50-DMP 5 7.3 15.1 59.5 18.440 ,50-DMP 2.5 8.9 9.3 70.8 11.05,50-DMP 1 9.2 24.0 35.2 31.65,50-DMP 0.5 7.1 14.3 63.6 15.04,6,40-TMA 0.5 9.4 12.6 64.0 14.04,6,4’-TMA 0.25 17.2 17.6 44.2 21.0

a Percentage of cells.b lM.



Moreover, we decided to evaluate a correlation between ery-throid differentiation and mitochondrial impairment. In particular,the involvement of the mitochondrial pathway via activation ofcaspase-3 and caspase-9 was evaluated; to this aim, K562 cellswere irradiated in the presence of the pancaspase inhibitor z-VAD.fmk and then benzidine test was performed. As shown inFig. 7, z-VAD.fmk suppressed erythroid differentiation induced byall furocumarins.

3.6. 5,50-DMP photoproducts induce erythroid differentiation

We also studied the possible erythroid differentiation activity ofirradiated mixtures of some tested furocoumarins. These com-pounds were 50-MP, 40,50-DMP and 5,50-DMP and were chosen onthe basis of their higher sensitivity to UV-A photodegradation (fol-lowed by UV–vis spectroscopy- data not shown).

After their irradiation in methanol solution with different UV-Adoses (0, 8, 16 and 32 J/cm2), psoralens were concentrated by sol-vent evaporation and then resuspended in methanol. The erythroiddifferentiation of photoproducts was investigated by benzidinetest incubating K562 with psoralen irradiated mixtures at two dif-ferent concentrations (50 and 200 lM) for 5–7 days. Cell growthwas also evaluated using the MTT assay after 6 days of treatment(Table 3). After 6 days of incubation, cells treated with 50 lMpre-irradiated mixtures did not show a clear increase of benzidinepositive cells (Fig. 8, upper panel) nor a decrease in cellular viabil-ity in comparison to control (Table 3); on the contrary, using thehigher concentration, an induction of erythroid differentiation(26–36% benzidine positive cells) (Fig. 8, lower panel) togetherwith a reduction of cellular viability was observed only with 5,50-DMP (Table 3); the other POP mixtures exhibited low activity orwere inactive. The irreversibility of the erythroid differentiationinduction by 5,50-DMP photoproduct mixtures was also assessed.The first 6 days of treatment were sufficient for K562 cells to differ-entiate irreversibly since during additional 4 days of culturing inthe absence of the inducer of washed cells, the population of ben-zidine-positive cells still increased (from 26.3 ± 3.1 to 44.3 ± 2.2 inthe case of 8 J and from 35.1 ± 2.0 to 40.5 ± 1.1 in the case of 16 J).

3.7. Furocoumarin photoproducts induce increased expression ofglobin genes

RT-qPCR was also employed to quantify the expression of globinmRNA following treatment of K562 cells with 5,50-DMP photo-products. There is a clear positive relationship between UV-A dosesused to obtain the photoproducts and the extent of increased glo-bin mRNAs in respect to control K562 cells (Fig. 9). As far as a pos-sible differential activity of furocoumarin photoproducts on globingene expression is concerned, the data clearly indicate that accu-mulation of both the a-like a-globin mRNA and f-globin mRNAare strongly induced. On the contrary, among the b-like e-globinand c-globin mRNAs, the c-globin mRNA is preferentially induced.This conclusion is well in agreement with the data shown in Fig. 4

Fig. 6. Mitochondrial dysfunction photoinduced by furocoumarins. Percentage ofcells with loss of mitochondrial membrane potential (upper panel) and withoxidized cardiolipin (lower panel) after 24 h from the irradiation (1 J/cm2) in thepresence of the indicated concentrations of 50-MP, 40 ,50-DMP, 5,50-DMP and 4,6,40-TMA. C = irradiated control. Data are expressed as mean ± S.E.M. of three indepen-dent experiments.

Fig. 7. Involvement of mitochondrial pathway in erythroid differentiation byfurocoumarins. Percentage of benzidine positive cells after 6 days from irradiationfrom irradiation (1 J/cm2) of K562 cells in the presence of 1 lM 50-MP, 5 lM 40 ,50-DMP, 1 lM 5,50-DMP and 0.5 lM 4,6,40-TMA. The irradiation was carried out in thepresence or absence of 50 lM z-VAD.fmk. Results are expressed as media ± S.E.M. ofat least three different experiments. C = irradiated control.

Table 3Percentage of cellular viability after incubation of 6 days of K562 with pre-irradiatedmixtures of 50-MP, 40 ,50-DMP and 5,50-DMP.

0 J/cm2 8 J/cm2 16 J/cm2 32 J/cm2

50-MP 50a 95.0 ± 4.5 93.0 ± 3.5 92.6 ± 4.8 89.8 ± 3.540 ,50-DMP 50 93.8 ± 2.3 94.5 ± 5.0 94.0 ± 4.6 92.9 ± 0.45,50-DMP 50 98.7 ± 5.8 94.8 ± 6.5 93.4 ± 3.5 94.3 ± 4.750-MP 200 88.9 ± 7.8 80.2 ± 3.6 84.5 ± 3.9 81.9 ± 4.940 ,50-DMP 200 86.0 ± 2.4 86.9 ± 6.2 96.8 ± 6.3 83.3 ± 2.85,50-DMP 200 99.8 ± 2.9 49.5 ± 2.3 22.4 ± 2.2 22.1 ± 1.9

a lM.



and concerning the effects of other furocoumarins on globin geneexpression in irradiated K562 cells.

4. Discussion

In this study, we reported the antiproliferative effects and theinducing activity on erythroid differentiation of some psoralenand angelicin analogs in the human chronic myelogenous leukemiaK562 cell line. Some of us previously demonstrated that fur-ocoumarins, in combination with UV-A, present the capability ofinducing erythroid differentiation like other DNA binders. Thus,we decided to continue our research evaluating new derivatives,some of them chosen on the basis of some considerations aboutthe structure–activity relationship. For instance, we focused ourattention on angelicin with trimethylation as this substitutionseemed to be successful for erythroid differentiation [26]. In fact,trimethylangelicins resulted to induce higher percentages of benzi-dine positive cells with respect to 50-MA (see Table 1). In the case ofpsoralens, our aim was also to verify the role of the substitution offuran ring, considering preliminary data demonstrating thatmonomethylation on furan leads to a very active compound andconfirmed the higher inducible power of methylpsoralens [7].The dimethylation involving one or both furan positions led to veryinteresting compounds, especially when the substitution on posi-tion 8 is avoided. We also decided to evaluate new substitutions,as tetramethylation or the introduction of an halogen, but theydo not seem to increase erythroid induction activity (see Table 1).

Interestingly, the most active compounds were able to induce aclear and important increase of globin mRNA expression which wasmuch higher than that reported elsewhere for 5-methoxypsoralen[8] (Fig. 4). It should be underlined that the level of inductionreached in these experimental conditions is even higher than thatexhibited by the most powerful inducer described [30].

Moreover, since the mechanism of erythroid differentiationmediated by furocoumarins (in the presence or absence of UV-Aexposure) is not well understood, first of all, some preliminaryanalyses were performed to investigate the role of DNA damage.Central to the DNA damage response are the ATM (ataxia-telangi-ectasia mutated), ATR (ataxia-telangiectasia and Rad3-related) andDNA-dependent protein kinases that modulate cell cycle progres-sion, DNA repair, and sometimes, apoptosis. We observed a signif-icant reduction of the levels of erythroid differentiation induced byfurocoumarins when irradiation was performed in the presence ofinhibitors of these kinases (see Fig. 3): this suggests that furocoum-arin-mediated erythroid differentiation is at least partially medi-ated by the DNA damage activated proteins. A role of DNAdamage can be also found by the analysis of cell cycle of irradiatedcells in the presence of furocoumarins since a clear block in G2-Mphase was observed (see Fig. 5 and Table 2). Furthermore, cell cyclestudies demonstrated that furocoumarins plus UV-A induced a cer-tain degree of cell death (see Fig. 5) by apoptosis thanks to thepresence of a percentage of cells with a lower DNA content thanG1 phase. The role of mitochondria in cell death was also demon-strated (Fig. 6). We also evaluated a possible role of mitochondrialdysfunction and of apoptosis in erythroid differentiation and weobserved a clear suppression of the proportion of benzidine posi-tive cells after mitochondrial pathway inhibition. These data indi-cate that erythroid differentiation may be a consequence of a stressresponse in which mitochondrial and DNA damage signaling areinvolved.

In this report, we also aimed at studying a possible role ofphotodegradation products in furocoumarin activity. The mostinteresting photoproducts mixtures were those obtained with5,50-DMP: in fact, the efficiency of these photoproducts in inducingincrease of globin mRNA content is dramatic and much higher thanthose exhibited by other inducers of K562 erythroid differentia-tion, such as cytosine arabinoside, butyric acid, mithramycin. Thissupports the concept that this strategy might be of some interest in

Fig. 8. Erythroid differentiation induced by furocoumarin photoproducts. Percent-age of benzidine positive cells after 6 days of K562 cell incubation in the presence ofirradiated (0, 8, 16 and 32 J/cm2) mixtures of 50-MP, 40 ,50-DMP and 5,50-DMP: in theupper panel, the concentration is 50 lM while in the lower panel it is 200 lM.Results are expressed as media ± S.E.M. of at least three different experiments.

Fig. 9. Increase in a-, c-, e- and f-globin mRNA content in K562 cells incubated inthe presence of pre-irradiated mixtures of 5,50-DMP (0, 8 and 16 J/cm2) for 6 days.The final concentration was 200 lM. Expression of globin genes was assayed byquantitative reverse transcriptase polymerase-chain reaction (qRT-PCR).



the design of novel agents against chronic myelogenous leukemiato be used in differentiation therapy.

The design and production of antiproliferative molecules target-ing the K562 cell system might be of great interest for the develop-ment of cocktails exhibiting applications in the treatment of chronicmyelogenous leukemia. For instance smenospongine [32], crambe-scidin 800 [33] and doxorubicin derivatives [21] were reported asmolecules of possible interest for inhibiting of CML cell growth,stimulating terminal differentiation along the erythroid program.Some molecules, such as Pivanex (an HDAC inhibitor) [34] and amorpholine derivative of doxorubicin [35], are synergistic withthe most common anti-CML agents, STI571 (Imatinib). In additionto synergistic effects, molecules inducing differentiation might beof great interest for treatment of Imatinib mesylate-resistanthuman CML cell lines, as recently demonstrated for the phytoalexinresveratrol [36].

As far as a possible differential activity of furocoumarin photo-products on globin gene expression is concerned, the preferentialeffects on c-globin mRNA might be also of interest for the develop-ment of novel HbF inducers in thalassemia. At present, one of themost promising novel approaches for the clinical management ofb-thalassaemia is the treatment of patients with chemical inducersof endogenous HbF. On the basis of recent achievements obtainedin this research field, several studies focusing on the mechanismsregulating reactivation of HbF production in humans have been re-ported. Relevant to these issues are studies showing that there is astrong negative correlation between HbF levels and morbidities.

In conclusion, the results presented in this paper demonstratethat the effects on erythroid differentiation of UV-A exposure ofK562 cells in the presence of linear and angular psoralens are partof a general phenomenon. The differentiating activity of these com-pounds in the presence of UV-A irradiation was associated with adramatic induction of accumulation of the a-like a-globin andf-globin mRNA and the b-like e-globin and c-globin mRNA se-quences. Of particular interest is our finding that erythroid induc-tion and accumulation of c-globin mRNA can be also obtained withpsoralen plus UVA induced photolysis products. It will be of inter-est to identify and characterize the active products involved.

Acknowledgements

This work was supported by the Associazione Veneta per laLotta alla Talassemia (AVLT) of Rovigo, by Fondazione Telethon(Contract GGP010214) and by Fondazione CARIPARO. R.G. isfunded by FP7 THALAMOSS Project.

References

[1] F. Dall’Acqua, G. Viola, D. Vedaldi, in: W.F. Hoorspool, F. Lenci (Eds.), Molecularbasis of psoralen photochemotherapy, CRC Handbook of OrganicPhotochemistry and Photobiology, CRC Press, Boca Raton, 2004. pp. 142/1–17.

[2] R. Edelson, C. Berger, F. Gasparro, B. Jegasothy, P. Heald, B. Wintroub, E.Vonderheid, R. Knobler, K. Wolff, G. Plewig, G. McKiernan, I. Christiansen, M.Oster, H. Höningsmann, H. Wilford, E. Kokoschka, T. Rehle, M. Perez, G. Stingl,L. Laroche, Treatment of cutaneous T-cell lymphoma by extracorporealphotochemotherapy, New Engl. J. Med. 316 (1987) 297–303.

[3] J.A. Parrish, T.B. Fitzpatrick, L. Tanenbaum, M.A. Pathak, Photochemotherapy ofpsoriasis with oral methoxsalen and long-wave ultraviolet light, New Engl. J.Med. 291 (1974) 1207–1212.

[4] J.J. Luszczki, M. Andres-March, M. Glensk, K.S. Skalicka-Wozniak,Anticonvulsant effects of four linear furanocoumarins, bergapten,imperatorin, oxypeucedanin, and xanthotoxin, in the mouse maximalelectroshock-induces seizure model: a comparative study, Pharmacol. Rep.62 (2010) 1231–1236.

[5] A. Tamanini, M. Borgatti, A. Finotti, L. Piccagli, V. Bezzerri, M. Favia, L. Guerra, I.Lampronti, N. Bianchi, F. Dall’Acqua, D. Vedaldi, A. Salvador, E. Fabbri, I.Mancini, E. Nicolis, V. Casavola, G. Cabrini, R. Gambari, Trimethylangelicinreduces IL-8 transcription and potentiates CFTR function, Am. J. Physiol. LungCell. Mol. Physiol. 300 (2011) L380–L390.

[6] D.Z. Tang, F. Yang, Z. Yang, J. Huang, Q. Shi, D. Chen, Y.L. Wang, Psoralenstimulates osteoblast differentiation through activation of BMP signalling,Biochem. Biophys. Res. Commun. 405 (2011) 256–261.

[7] G. Viola, D. Vedaldi, F. Dall’Acqua, E. Fortunato, G. Basso, N. Bianchi, C. Zuccato,M. Borgatti, I. Lampronti, R. Gambari, Induction of c-globin mRNA, erythroiddifferentiation and apoptosis in UVA-irradiated human erythroid cells in thepresence of furocoumarin derivatives, Biochem. Pharmacol. 75 (2008) 810–825.

[8] G. Viola, A. Salvador, D. Vedaldi, F. Dall’Acqua, N. Bianchi, C. Zuccato, M.Borgatti, I. Lampronti, R. Gambari, Differentiation and apoptosis in UVA-irradiated cells treated with furocoumarin derivatives, Ann. NY Acad. Sci. 1171(2009) 334–344.

[9] I. Lampronti, N. Bianchi, M. Borgatti, E. Fibach, E. Prus, R. Gambari,Accumulation of c-globin mRNA in human erythroid cells treated withangelicin, Eur. J. Haematol. 71 (2003) 189–195.

[10] R. Gambari, E. Fibach, Medicinal chemistry of fetal hemoglobin inducers fortreatment of b-thalassemia, Curr. Med. Chem. 14 (2007) 199–212.

[11] C.B. Lozzio, B.B. Lozzio, Human chronic leukemia cell-line with positivephiladelphia chromosome, Blood 45 (1975) 321–333.

[12] N. Bianchi, C. Zuccato, A. Finotti, I. Lampronti, M. Borgatti, R. Gambari,Involvement of miRNA in erythroid differentiation, Epigenomics 4 (2012) 51–65.

[13] R. Gambari, Alternative options for DNA-based experimental therapy of b-thalassemia, Expert Opin. Biol. Ther. 12 (2012) 443–462.

[14] S. Caffieri, F. Di Lisa, F. Bolesani, M. Facco, G. Semenzato, F. Dall’Acqua, M.Canton, The mitochondrial effects of novel apoptogenic molecules generatedby psoralen photolysis as a crucial mechanism in PUVA therapy, Blood 109(2007) 4988–4994.

[15] A.Y. Potapenko, A.A. Kyagova, L.N. Bezdetnaya, E.P. Lysenko, I.Y.Chernyakhovskaya, V.A. Bekhalo, E.V. Nagurskaya, V.A. Nesterenko, N.G.Korotky, S.N. Akhtyamov, T.M. Lanshchikova, Products of psoralenphotooxidation possess immunomodulative and antileukemic effects,Photochem. Photobiol. 60 (1994) 171–174.

[16] A. Salvador, S. Dall’Acqua, M. Sutera Sardo, S. Caffieri, D. Vedaldi, F. Dall’Acqua,M. Borgatti, C. Zuccato, N. Bianchi, R. Gambari, Erythroid induction of chronicmyelogenous leukemia K562 cells following treatment with photoproductderived from the UV-A irradiation of 5-methoxypsoralen, ChemMedChem 5(2010) 1506–1512.

[17] S. Chimichi, M. Boccalini, B. Cosimelli, G. Viola, D. Vedaldi, F. Dall’Acqua, Aconvenient synthesis of psoralens, Tetrahedron 58 (2002) 4859–4863.

[18] F. Bordin, F. Dall’Acqua, A. Guiotto, Angelicins, angular analogs of psoralens:chemistry, photochemical, photobiological and phototherapeutic properties,Pharmacol. Ther. 52 (1991) 331–363.

[19] G. Viola, M. Boccalini, E. Fortunato, F. Dall’Acqua, S. Chimichi, Synthesis andphotobiological properties of bromo- and alkoxymethylfurocoumarins, Lett.Drug Des. Discov. 5 (2008) 93–103.

[20] P. Barraja, L. Caracausi, P. Diana, A. Carbone, A. Montalbano, G. Cirrincione, P.Brun, G. Palù, I. Castagliuolo, F. Dall’Acqua, D. Vedaldi, A. Salvador, Synthesis ofpyrrolo[3,2-h]quinolinones with good photochemotherapeutic activity and noDNA damage, Bioorg. Med. Chem. 18 (2010) 4830–4843.

[21] A. Szulawska, J. Arkusinska, M. Czyz, Accumulation of c-globin mRNA andinduction of irreversible erythroid differentiation after treatment of CML cellline K562 with new doxorubicin derivatives, Biochem. Pharmacol. 73 (2007)175–184.

[22] S. Salvioli, A. Ardizzoni, C. Franceschi, A. Cossarizza, JC-1, but not DiOC6(3) orrhodamine 123, is a reliable fluorescent probe to assess delta psi changes inintact cells: implications for studies on mitochondrial functionality duringapoptosis, FEBS Lett. 411 (1997) 77–82.

[23] P. Kaewsuya, N.D. Danielson, D. Ekhterae, Fluorescent determination ofcardiolipin using 10-N-nonyl acridine orange, Anal. Bioanal. Chem. 387(2007) 2775–2782.

[24] C. Chiarabelli, N. Bianchi, M. Borgatti, E. Prus, E. Fibach, R. Gambari, Inductionof gamma-globin gene expression by tallimustine analogs in human erythroidcells, Haematologica 88 (2003) 826–827.

[25] N. Bianchi, F. Osti, C. Rutigliano, F.G. Corradini, E. Borsetti, M. Tomassetti, C.Mischiati, G. Feriotto, R. Gambari, The DNA-binding drugs mithramycin andchromomycin are powerful inducers of erythroid differentiation of humanK562 cells, Brit. J. Haematol. 104 (1999) 258–265.

[26] I. Lampronti, N. Bianchi, C. Zuccato, F. Dall’Acqua, D. Vedaldi, G. Viola, R.Potenza, F. Chiavilli, G. Breveglieri, M. Borgatti, A. Finotti, G. Feriotto, F.Salvatori, R. Gambari, Increase in globin mRNA content in human erythroidcells treated with angelicin analogs, Int. J. Hematol. 90 (2009) 318–327.

[27] C.J. Bakkenist, M.B. Kastan, Initiating cellular stress response, Cell 118 (2004)9–17.

[28] J.N. Sarkaria, R.S. Tibbetts, E.C. Busby, A.P. Kennedy, D.E. Hill, R. Tt Abraham,Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizingagent wortmannin, Cancer Res. 57 (1998) 4375–4382.

[29] J.N. Sarkaria, E.C. Busby, R.S. Tibbetts, P. Roos, Y. Taya, L.M. Karnitz, R.T.Abraham, Inhibition of ATM and ATR kinase activity by the radiosensitizingagent, caffeine, Cancer Res. 59 (1999) 4375–4382.

[30] E. Brognara, I. Lampronti, G. Breveglieri, A. Accetta, R. Corradini, A. Manicardi,M. Borgatti, A. Canella, C. Multineddu, R. Marchelli, R. Gambari, C(5) modifieduracil derivatives showing antiproliferative and erythroid differentiationinducing activities on human chronic myelogenous leukemia K562 cells, Eur.J. Pharmacol. 672 (2011) 30–37.



[31] M. Canton, S. Caffieri, F. Dall’Acqua, F. Di Lisa, PUVA-induced apoptosisinvolves mitochondrial dysfunction caused by the opening of the permeabilitytransition pore, FEBS Lett. 522 (2002) 168–172.

[32] S. Aoki, D. Kong, K. Matsui, M. Kobayashi, Smenospongine, a spongeansesquiterpene aminoquinone, induces erythroid differentiation in K562 cells,Anticancer Drugs 15 (2004) 363–369.

[33] S. Aoki, D. Kong, K. Matsui, M. Kobayashi, Erythroid differentiation in K562chronic myelogenous cells induced by crambescidin 800, a pentacyclicguanidine alkaloid, Anticancer Res. 24 (2004) 2325–2330.

[34] E. Rabizadeh, V. Merkin, I. Belyaeva, M. Shaklai, Y. Zimra, Pivanex, a histonedeacetylase inhibitor, induces changes in BCR–ABL expression and when

combined with STI571, acts synergistically in a chronic myelocytic leukemiacell line, Leukemia Res. 31 (2007) 1115–1123.

[35] J. Jakubowska, M. Wasowska-Lukawska, M. Czyz, STI571 and morpholinederivative of doxorubicin collaborate in inhibition of K562 cell proliferation byinducing differentiation and mitochondrial pathway of apoptosis, Eur. J.Pharmacol. 596 (2008) 41–49.

[36] A. Puissant, S. Grosso, A. Jacquel, N. Belhacene, P. Colosetti, J.P. Cassuto, P.Auberger, Imatinib mesylate-resistant human chronic myelogenous leukemiacell lines exhibit high sensitivity to the phytoalexin resveratrol, FASEB J. 22(2008) 1894–1904.


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