Efficacy of Different Sequencesof Radio- and Chemotherapy inExperimental Models of HumanMelanomaCHIARA ARIENTI,1 WAINER ZOLI,1 SARA PIGNATTA,1 SILVIA CARLONI,1

GIULIA PAGANELLI,1 PAOLA ULIVI,1 ANTONINO ROMEO,2 ENRICO MENGHI,3

ANNA SARNELLI,3 LAURA MEDRI,4 ROLANDO POLICO,2 ROSELLA SILVESTRINI,1

AND ANNA TESEI1*1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy2 Radiotherapy Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy3Medical Physics Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy4Pathology Unit, Morgagni-Pierantoni Hospital, Forlì, Italy

Although combination chemotherapy and radiotherapy havebecome the standardof care in numerous tumors, themechanismsof interactionare often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigatetheir mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation-induced cytotoxicity before and aftercisplatin treatment. Expression levels of a panel of genes were determined by real-time RT-PCR. Cytotoxic effect was evaluated by flowcytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the twotreatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells.Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from16 to>100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet-shapednucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin onlyinducedweak cytotoxicity.Our findings highlight the efficacyof the sequence radiation! cisplatin in reducing cell proliferation and in inducingapoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair.

J. Cell. Physiol. 229: 1548–1556, 2014. © 2014 Wiley Periodicals, Inc.

The worldwide incidence of melanoma, considered until fairlyrecently a rare malignancy, is increasing faster than any othercancer histotype, with the exception of lung malignancies inwomen. Over the last three decades the incidence ofmelanoma has doubled in the United States. TheWorld HealthOrganization reports that about 132,000 new cases of this typeof cancer are diagnosed each year worldwide.When diagnosedearly, melanoma can be cured by surgical resection and about80% of cases are dealt with in this way. However, metastaticmelanoma is largely refractory to existing therapies and has avery poor prognosis, with a median survival of 6 months and a5-year survival of less than 5%. Hence new treatment strategiesare urgently needed (Gray-Schopfer et al., 2007).

Although complete surgical excision of stage I/II disease hasover a 95% cure rate, the success of systemic therapy formetastatic melanoma is minimal (Soengas and Lowe, 2003).Despite progress made in recent years with the introduction ofnew drugs and new combinations of chemoimmunotherapy,prognosis remains poor for advanced disease. Although it isconsidered a radioresistant tumor, radiation treatment issometimes used in patients with lentigo maligna melanoma asadjuvant therapy in selected patients with regional metastaticdisease and also with palliative intent, especially in those withbone and brain metastases (Testori et al., 2009).

In the treatment of melanoma, there is no consensus on theeffects of total radiation dose, fraction size, or time intervalbetween fractions. It was recently suggested that a higherresponse to radiotherapy can be achieved by delivering singlehigh-dose fractions of 5Gy over a short time period(Olivier et al., 2007; Khan et al., 2011). Clinical trials showed

that the use of cisplatin in combination with temozolomide asadjuvant therapy was an effective and safe choice for patientswith melanoma (Daponte et al., 2005; Lian et al., 2013).Furthermore, cisplatin is often used for the treatment of otherneoplastic diseases, e.g. head and neck cancer, in combinationwith low-dose radiotherapy, because of its radiosensitivity andlow toxicity. Such positive properties of cisplatin are due to itsmechanism of action that induces DNA adduct formation and,consequently, apoptosis (Kelland, 2007).

In the last decade, chemotherapy and radiotherapycombinations have become the standard of care for locallyadvanced tumors because combined treatment leads totoxicity independence, spatial cooperation, and additive or

There are no conflicts of interest to declare.

Contract grant sponsor: Italian Ministry of Health;Contract grant number: RO Strategici 11/07.

*Correspondence to: Anna Tesei, Biosciences Laboratory, IstitutoScientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST)IRCCS, Via P. Maroncelli 40, Meldola 47014, Italy.E-mail: anna.tesei@irst.emr.it

Manuscript Received: 28 November 2013Manuscript Accepted: 27 February 2014

Accepted manuscript online in Wiley Online Library(wileyonlinelibrary.com): 4 March 2014.DOI: 10.1002/jcp.24598

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synergic cell killing. However, the main mechanisms ofinteraction of numerous agents remain unclear (Riestereret al., 2007).

The purpose of this study was to identify the most effectivetreatment among schedules of cisplatin or radiation treatmentused alone or in combination and to explore and clarify themechanisms of interaction at the DNA level.

Materials and MethodsCell lines

The study was performed on three melanoma cell lines, M66, M79and M14. M14, a commercially available cell line derived from aprimary tumor, was obtained from the American Type CultureCollection (Rockville, MD), while M66 and M79 cell lines wereisolated in our laboratory from a primary tumor and a metastaticbrain lesion, respectively. The cytotoxic effect of the differentradio-chemotherapy schedules was also tested on HUDE, anestablished normal human dermal fibroblast cell line purchasedfrom Istituto Zooprofilattico Sperimentale della Lombardia edell’Emilia Romagna (Brescia, Italy). Cell lines were maintained as amonolayer at 37 °C and passaged weekly. The culture mediumwascomposed of DMEM/Ham’s F12 (1:1) supplemented with fetal calfserum (10%), glutamine (2mM), non-essential amino acids (1%)(Mascia Brunelli S.p.A., Milan, Italy), and insulin (10mg/ml) (Sigma-Aldrich, St. Louis, MO). Cells were used in the exponential growthphase in all experiments. For growth analysis, cells were plated in12-well plates in triplicate at a concentration of 2� 104 cells/well.Cells were collected and counted after 1, 2, 3, 4, 5, 6, and 7 days.

Treatments

Drug. Cisplatin was tested at scalar concentrations of 0.1, 1,and 10mM. An exposure time of 6 h followed by a 24-h washout(w.o.) was chosen on the basis of peak plasma levels defined inrecent pharmacokinetic studies (Souid et al., 2003; Urien andLokiec, 2004).Radiation. Cells were irradiated in 25-cm2

flasks with 2.4 and5Gy using the linear acceleration Elekta Synergy Platform system.Combination of chemo- and radiation treatment. M66,

M79, and M14 cell lines were treated with a combination of 10mMcisplatin (peak plasma level, PPL) and 2.4 or 5Gy as follows: cellswere exposed for 6 h to cisplatin followed by a 24-hwashout beforeor after 2.4 or 5Gy followed by 72-h washout.

Clonogenic assay

Following treatment, 500 cells were seeded in 10 cm2 dishes in500ml of medium. After 14 days, the resulting colonies were fixedand stained using 0.5% crystal violet in 25%methanol; colonies withmore than 50 cells were quantified under inverted microscope(I500X, Olympus) by two independent observers. Five seriesof samples were prepared for each treatment dose (Leonardet al., 1996; Latz et al., 1998).

Data analysis

Of the different methods available to evaluate the interactionbetween drugs or between drug and irradiation treatment, wechose the interaction analysis method of Kern et al. (1988),subsequently modified by Romanelli et al. (1998)—because of thelow dose effect and no dose-response curves induced by cisplatin.In brief, the expected cell survival (Sexp, defined as the product ofthe survival observed with treatment A alone and the survivalobserved with treatment B alone) and the observed cell survival(Sobs) for the combination of A and B were used to construct anindex (RI): RI¼ Sexp/Sobs. An RI of �0.5 indicated the absence ofsynergism or antagonism. Synergism was defined as any value ofRI> 1.5. In all experiments, the SD did not exceed 10%.

Flow cytometry

Flow cytometric analysis was performed using a FACS Canto flowcytometer (Becton Dickinson, San Diego, CA) equipped with anargon laser (488 nm). Data acquisition and analysis wereperformed using FACSDiva (Becton Dickinson) and ModFit 2.0(DNA Modelling System, Verity Software House, Inc., Topsham,ME). Samples were run in triplicate and 10,000 events werecollected for each replica. Data were the average of threeexperiments, with errors under 5%.Annexin V assay. After exposure to 2.4 or 5Gy, or to 10mM

of cisplatin alone or in sequence, cells were washed once in PBSand incubated with 25ml/ml of Annexin V-FITC in binding buffer(Bender MedSystems, Vienna, Austria) for 15min at 37 °C in ahumidified atmosphere in the dark. Cells were then washed againin PBS and re-suspended in binding buffer. Immediately before flowcytometric analysis, propidium iodide was added to a finalconcentration of 5mg/ml to discriminate between apoptotic(Ann-Vþ and PIþ or �) and necrotic cells (Ann-V� and PIþ).15,000 events were recorded for each sample.Cell cycle distribution. After exposure to cisplatin, cells

were fixed in 70% ethanol, stained with propidium iodide(10mg/ml, MP Biomedicals, Verona, Italy), RNAse (10 kunits/ml,Sigma Aldrich) and NP40 (0.01%, Sigma Aldrich) overnight at37 °C in the dark and analyzed by flow cytometry. Data wereexpressed as fractions of cells in the different cycle phases.

Western blot

Cell proteins were isolated by lysing cells in 50mM of Tris-HCl(pH 8.0), 150mM of NaCl, 1% Triton X-100, and 0.1% SDSsupplemented with 1mM of phenylmethylsulfonyl fluoride and aprotease inhibitor mixture (Sigma Aldrich). Identical amounts oftotal proteins were denatured and separated on 12% SDS–polyacrylamide gel and then electroblotted onto Immobilon-PTransfer Membrane (Millipore Corporation, Billerica, MA). Themembrane was stained with Ponceau S (Sigma Aldrich) to verifyequal amounts of sample loading and then incubated for 2 h atroom temperature with 5% non-fat dry milk in PBS-T. Themembrane was probed overnight at 4 °C with the specific primaryantibody and then with a horseradish peroxidase-conjugatedsecondary antibody diluted 1:5000 (Santa Cruz Biotechnology,Santa Cruz, CA). The bound antibodies were detected byImmobilon Western chemiluminescent HRP substrate(Millipore Corporation) using Chemidoc XRS Molecular Imager(Bio-Rad, Laboratories, Hercules, CA). The following primaryantibodies were used: anti-p21WAF1 mouse monoclonalantibody, 1:100 (NeoMarkers, Fremont, CA); anti-cleavedcaspase-3 rabbit polyclonal antibody 1:500 (Cell SignalingTechnology, Beverly, MA); anti-PUMA polyclonal rabbit antibody1:500 (Upstate Biotechnology, Charlottesville, VA); and anti-actinrabbit polyclonal antibody 1:5000 (Sigma Aldrich). Quantity OneSoftware was used for analysis.

Comet assay

The assay was performed according to the manufacturer’sprotocol (Comet assay, Trevigen, Gaithersburg, MD). Briefly,at the end of the treatments, 5� 105 cells were suspended inLMAgarose (at 37 °C) at a ratio of 1:10 (v/v), and 75ml wereimmediately transferred onto the comet slide. The slides wereimmerged for 1 h at 4 °C in a lysis solution, washed in the dark for1 h at room temperature in an alkaline solution, then washed twicein TBE electrophoresis solution and electrophoresed for 10min at20 V. Slides were then dipped in 70% ethanol and stained with theSilver Staining Kit (Trevigen). The extent of DNA damage wasevaluated quantitatively by Axiovert microscope (Zeiss, Arese,Milan, Italy) (40x) by scoring at least 1,000 nucleoids and wasexpressed, as previously described (Zoli et al., 2004), as the

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percentage of comet-shaped nucleoids with respect to the totalnumber of nucleoids scored. All samples were evaluated blindly bytwo independent observers.

Real time RT-PCR

Total RNA was extracted from cell lines using TRIzol1 reagentaccording to the manufacturer’s instructions (Invitrogen, LifeTechnologies, Carlsbad, CA). Reverse transcription (RT)reactions were performed in a 20-ml volume containing 800 ng oftotal RNA using an iScript TM cDNA Synthesis kit (Bio-Rad) andanalyzed by Real Time RT-PCR (MyiQ System, Bio-Rad) to detectexpression levels of BRCA1, ERCC1, DNA-PK, PARP, MGMT, ATM,and TGM2 genes. Primers for mRNA amplification were designedusing Beacon Designer Software (version 4, BioRad). The standardreaction volume was 25ml containing 2ml of cDNA template, 1 xSYBR Green Mix and 5mM of forward and reverse primers. Themixture was subjected to the following cycling conditions: 95 °Cfor 1min and 30 sec followed by 40 cycles of amplification for15 sec at 95 °C and 30 sec at 54.5 °C (for PARP), 60 °C (for BRCA1,ERCC1, DNA-PK, MGMT, TGM2 and b2-microglobulin) or 62 °C (forATM). The expression of each gene marker was normalized to theendogenous reference (b2-microglobulin) using Gene ExpressionMacro Software (Version 1.1) (BioRad).

Gene-specific amplification efficiency was used to calculate therelative expression of target genes using Gene Expression MacroSoftware. Intra-experiment variability did not exceed 5%. Thereproducibility of Real-Time PCR results was verified in triplicatesamples and the coefficient of variation (CV), calculated from thethree Ct values, never exceeded 1.5%.

Immunofluorescence staining

Cells were harvested by trypsinization, washed with serum andPBS and sedimented onto glass coverslips for 20min at 500 r.p.m.in a Shandon Cytospin 3 tabletop centrifuge. The nuclei wereimmediately fixed with acetone for 10min and then withchloroform for 5min at room temperature. The coverslips wereincubated overnight at room temperature in primary antibody(anti Rad51 diluted 1:100, Cell Signaling, Danvers, MA), diluted inblocking buffer, and then washed again and incubated withfluorochrome-conjugated secondary antibodies (FITC green,

Alexafluor 488 goat, diluted 1: 1, Life Technologies) for 2 h at roomtemperature. Cells were analyzed with Zeiss Imager M1microscope (Zeiss, Milan, Italy) and the imageswere recordedwithZeiss AxioVision camera.

A minimum of 100 cells were analyzed for each experiment andresults are presented as the average of at least three independentcell preparations. Cells containing a minimum of five RAD51 fociper nucleus were scored as positive.

Fig. 1. Cytotoxic effect of cisplatin or radiation treatment inmelanoma cell lines assessed by clonogenic assay. M66 (solid line),M79 (dashed line), and M14 (dotted line) were exposed to cisplatinor radiation treatment. A) Cisplatin treatment for 6 h followed by24-h washout (w.o.). B) Radiation therapy at doses of 2.4Gy and5Gy followed by a 72-h w.o.

Fig. 2. Activity of radiation treatment and cisplatin combination in different sequences evaluated by clonogenic assay. Before (dotted line) orafter (solid line) 2.4 or 5Gy radiation doses followed by a 72-h w.o., cells were exposed to cisplatin 10mM for 6h followed by a 24-h w.o.Standard deviation (SD) never exceeded 5%.

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Data analysis

Dose response curves were created byMicrosoft Excel1 softwareand 50% inhibiting concentration (IC50) values were determinedgraphically from the plots. Paired proportions were comparedusingMcNemar’s test and statistical analyses were carried out withSAS Statistical Software (SAS Institute Inc, 1989). P< 0.05 wasconsidered significant.

ResultsEffect of single or combination agents

All the cell lines investigated showed poor sensitivity tocisplatin treatment. In particular, after a 6-h exposure tocisplatin followed by a 24-h w.o., the concentration inhibitingcell survival by 50% (IC50) was never reached in any cell line orat any concentration (Fig. 1A). Seventy-two h after the end of

TABLE I. Interaction between radiation and cisplatin

TreatmentRatio Index (mean value)*

M66 M79 M14

6-h Cis !24-h w.o.!2.4 Gy!72-h w.o. 0.71 0.6 1.46-h Cis !24-h w.o.!5Gy !72-h w.o. 0.55 0.52 1.12.4 Gy!72-h w.o.!6-h Cis!24-h w.o. >100 16 >1005Gy !72-h w.o.!6-h Cis!24-h w.o. >100 >100 >100

Cis [10mM]RI �1.5, synergistic; RI< 1.5-> 0.5, additive; RI� 0.5, antagonisticw.o., washout*Ratio Index (RI)

TABLE II. Distribution of cells in different cycle phases (%)

G0-G1 S G2-M

M6624-h w.o.Control 74.3 16.9 8.8Cis 6 h 4.1 82.0 13.9

48-h w.o.Control 78.2 15.1 6.7Cis 6 h 2.7 97.1 0.2

72-h w.o.Control 73.4 14.7 11.9Cis 6 h 3.1 49.8 47.1

M7924-h w.o.Control 52.1 39.0 8.9Cis 6 h 16.3 82.2 1.5

48-h w.o.Control 53.8 44.3 1.9Cis 6 h 5.8 70.2 24.0

72-h w.o.Control 56.6 33.9 9.5Cis 6 h 10.0 67.9 22.1

M1424-h w.o.Control 42.6 42.8 14.6Cis 6 h 11.9 88.1 0.0

48-h w.o.control 60.9 30.4 8.7Cis 6 h 1.0 73.1 25.9

72-h w.o.Control 55.9 32.2 11.9Cis 6 h 8.0 87.9 4.1

Bold: P< 0.05w.o., washout

Fig. 3. Proteins involved in apoptotic processes. A) Median percentage of apoptotic cells after radiation treatment, cisplatin, or combinationtreatment (radiation at 2.4 or 5Gy followed by cisplatin). Values are the mean�SD of three independent experiments. B) Western blotanalysis was performed for all cell lines at basal level, 72 h after irradiation (2.4Gy or 5Gy), after a 6-h cisplatin treatment followed by a 24-hw.o. and after the inverse sequence (radiation at 2.4 or 5Gy followed by cisplatin). Values were the mean�SD of three independentexperiments.

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radiation treatment, which corresponds to three and sixdoubling times, cells were seeded for the clonogenic assay orfor treatment with cisplatin. The panel of cells exhibited adifferent degree of sensitivity when subjected to irradiationtreatment with 2.4Gy or 5Gy (Fig. 1B). At the highest dosetested, M66 and M14 cells reached IC50, whereas the M79 linewas resistant to the irradiation treatment. In particular, M66and M14 showed similar cell survival percentages. The sameresults were obtained after a 144-h wash out followingradiation treatment (data not shown).

Interaction between cisplatin and radiation was investigatedusing different treatment schedules (Fig. 2). In the firstschedule, cells were treated for 6 h with cisplatin at the PPL(10mM) followed by a 24-h w.o., and then irradiated with 2.4 or5Gy.

A modest dose-dependent cytotoxic effect was observed inM79 cells in which IC50 was not reached at the highest dosetested. In the other two cell lines, a cytotoxic irradiation dose-related effect was observed, especially in M14 where the IC50was reached at the lowest dose used (Fig. 2). Furthermore,interaction analysis showed that pretreatment with cisplatinenhanced the effect of radiation in all cell lines but producedonly an additive interaction with RI values ranging from 0.52 to1.472 h after the end of radiation treatment (Table I).

Conversely, either radiation dose followed by cisplatinproduced a significant decrease (P< 0.05) in cell survival withrespect to the inverse sequence in all cell lines. In particular, asynergistic interaction was observed with RI values rangingfrom 16 in M79 cell line to >100 in M14 and M66 cell lines(Table I).

Effect of cisplatin on cell cycle

Cell cycle distribution was analyzed after a 6-h exposure tocisplatin followed by a 24-, 48- and 72-h w.o. A significantlyhigher S phase cell fraction (P< 0.05) was observed in all celllines. This remained constant up to the 72-h wash out in M14cell line but decreased slightly in M66 and M79 (Table II).

Apoptosis

Apoptosis was present in a small fraction of cells (around 5%) inall cell lines at baseline and increased up to 20% when theradiation dose was increased or after exposure to cisplatin,especially in M79 cell lines (Fig. 3A). A synergistic interactionproducing about 40% apoptosis in M66 and M79 cells and up to48% in M14 was observed in all cell lines after the sequenceradiation treatment ! cisplatin, regardless of the radiationdose used. p21, caspase 3, and PUMA expression was analyzedin all cell lines after cisplatin or radiation exposure, singly or insequence (Fig. 3B). Treatment with cisplatin or either of theradiation doses did not induce p21 expression. Conversely, weobserved a weak induction of p21 expression in all cell lines,especially in M14, after irradiation with 2.4Gy or 5Gy followedby cisplatin 10mM. Conversely, strong PUMA and caspase 3induction was observed after either radiation dose followed bycisplatin.

DNA damage

In the Comet assay analysis, cells exposed to cisplatin followedby irradiation showed a significant percentage of non-damagednuclei (about 50% of nuclei in all cell lines). Conversely, thegreatest DNA damage was observed in all 3 lines when cellswere exposed to either radiation dose followed by cisplatin,and consisted mainly of an increase in the percentage of comet-shaped nucleoids (from 54% to 96–98%) (p< 0.05 by t-test)(Fig. 4). In particular, about 50% of the nuclei showed high ortotal cellular DNA damage. Furthermore, after exposure to

cisplatin followed by radiation, the evaluation of RAD51 fociformation positivity in all cell lines investigated, in particular,83% positivity in M66, 40% in M79, and 38% in M14 cell lines(Fig. 5). Conversely, the inverse sequence produced a muchlower percentage of positive nuclei (25% in M66, 10% in M79,and 15% in M14) (data not shown). When the two types oftreatment were used singly, only 15% of cells exhibited RAD51foci (data not shown).

Modulation of genes involved in DNA damage repair andsensing processes

Genes involved in DNA damage repair (BRCA1, ERCC1,MGMT), DNA damage sensing (TGM2, ATM, PARP), or bothprocesses (DNA-PK) were analyzed. Gene expression profileswere defined before and after different treatment schedules inall three cell lines (Fig. 6). Although DNA-PK was notinfluenced by radiation or cisplatin alone, significantly lowerexpression levels were observed in M66 and M14 cell lines onlyafter the sequence using the highest radiation dose (5Gy).

PARP expression levels were not influenced by radiationtreatment, slightly increased after cisplatin treatment in M66and M14 cell lines, and generally decreased following theradiation treatment ! cisplatin sequence.

Fig. 4. Single Cell Gel Electrophoresis assay to detect the DNAdamage. Cells with damaged DNA (comets) evaluated afterradiation treatment, cisplatin, or combination treatment (before orafter 2.4 or 5Gy radiation doses at 2.4 or 5Gy followed by cisplatin).*significance at P< 0.05 by t-test.

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BRCA-1 significantly increased after irradiation or cisplatinalone and after the sequence irradiation-cisplatin in only M79cell line, whereas a significant decrease was observed in M66cell line after the same sequence.

Similarly, MGMT expression substantially increased under alltreatment conditions with single agents or sequence schedulesin M79 cell line and decreased after cisplatin only and radiation-cisplatin sequences in M14. A significant increase in MGMTwasonly seen in M66 following radiation treatments alone. ATM,ERCC1, and TGM2 showed a similar expression profile afterthe different treatments, with a general increase in expressionafter radiation treatment, especially in M66 and M79, a slight ornon significant decrease after cisplatin, and a significantreduction after the radiation-cisplatin sequence, mainly forTGM2, in M79 and M14.

Toxic effect in normal cells

The effect of the two different radio-chemotherapy scheduleswas tested on HUDE fibroblasts. Both treatment schedulesinduced a dose-related cytotoxic effect, as highlighted by thereduced clonogenic kill of HUDE cells evaluated 14 days afterthe end of treatment (Fig. 7). However, a significantly lowertoxicity was observed after radiation treatment ! cisplatincompared to the inverse sequence (P< 0.05). These data werealso confirmed by the 50% reduction in the number of Rad51

foci-positive cells with respect to that observed after theinverse sequence (15% vs 30%) (Fig. 7B).

Discussion

Melanoma is considered extremely resistant to chemotherapyand relatively resistant to radiotherapy, especially in theadvanced stages of the disease. Recent studies have shown thatmelanoma cells have a low apoptotic index and generally showmodest levels of spontaneous or drug-induced apoptosiscompared to other tumor histotypes (Staunton andGaffney, 1995; Glinsky et al., 1997). For this reason, strategiesaimed at exploiting melanoma cell killing by bypassing orovercoming upstream death defects could be used to improveclinical response.

The combination of radio- and chemotherapy has becomethe standard care for the majority of patients with solid tumorsand aims to improve locoregional disease control and patientsurvival (Wilson et al., 2006). The classic framework definingthe possible interactions between combined radio- andchemotherapy, first defined by Steel (1979), involves thefunctions of spatial cooperation, toxicity independence, normaltissue protection, and varying degrees of additivity. However,the exact mechanisms underlying the potentiation of radio-therapy by chemotherapeutic agents are still not fullyunderstood.

Fig. 5. RAD51 focus formation in melanoma cells. Treated and untreated cultures were stained with an anti-RAD51 pAb (FBE1) and fociwere visualized by microscopy.

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In the present study, we evaluated the antitumor activity ofradiation therapy in combination with cisplatin treatment inmelanoma cell lines. Our results indicate that cisplatin andradiation used singly are not particularly effective in reducingproliferation. The only positive interaction observed whencisplatin was followed by radiation was an additive effect in allcell lines. The absence of a positive interaction may beattributable to the S-phase cell cycle block induced by cisplatin,which prevents the cytotoxic activity of radiation exertedthrough the induction of DNA damage (Bartek andLukas, 2001). Conversely, radiation treatment followed bycisplatin showed a strong synergistic interaction in all cell linesthat was associated with a marked increase in apoptosis andDNA damage, suggesting that this schedule may sensitizemelanoma cells by activating the apoptotic program. Inparticular, our data indicate that radiation treatment followed

by cisplatin caused such severeDNAdamage that the apoptoticpathway was triggered, as shown by the significant increase inpro-apoptotic PUMA expression and by weak p21 activation.The p21 expression was probably mediated by p53-indepen-dent mechanisms (Fig. 3) (Gartel and Tyner, 1999; Agrawalet al., 2002). Like M66 and M79 lines, M14 also harborsmutated, non-functioning p53, as previously reported byVannini et al. (2007).

The RAD51 protein forms subnuclear complexes thatcontain many of the enzymatic activities required for theefficient repair of DNA double-strand breaks. RAD51foci havebeen observed in mitotic S-phase cells and are thought toidentify sites where stalled or broken replication forks undergorepair (Tashiro et al., 1996; Raderschall et al., 1999). In ourstudy, an increase in the number of cells containing RAD51 foci(up to 70%) was observed in response to radiation treatmentfollowed by cisplatin with respect to untreated cells.

Several mechanisms may lie at the basis of the radiation–cisplatin interaction (Hennequin and Favaudon, 2002), amongwhich are homologous recombination and nonhomologousend-joining (Frit et al., 1999; Chistiakov et al., 2008). For thisreason, we selected a number of DNA repair genes and DNAdamage sensors on the basis of their known function (Jeggoet al., 2011; Lord and Ashworth, 2012) to evaluate themodulation induced by the synergistic radio-chemo sequence.As already observed by our group (Arienti et al., 2013),expression levels of most of the genes evaluated increasedsignificantly after radiation, in particular ATM, ERCC1, andTGM2. After cisplatin treatment, a slight but significant increasein only PARP and DNA-PK expression levels was seen in M14.This increase, indicating DNA repair activity, was completelynullified by irradiation pretreatment. In the other two cell lines agenerally increase in expression was observed in most of genesanalyzed after chemo- or radiation treatment administeredsingly. Once again, this increase disappeared when cells wereexposed to radiation followed by cisplatin. Notably, TGM2expression levels significantly decreased in all cell lines. Althoughthe physiological function of TGM2 is not yet fully understood(Lorand and Graham, 2003), we hypothesized its direct role inthe regulation of DNA damage response, in agreement with Aiet al. (2012). In particular, our data highlight that radiationfollowed by cisplatin consistently affected DNA break-sensingmolecules such as DNA-PK, PARP, ATM, and TGM2, as shownby their decreased expression in all the cell lines used. Such aneffect was not as evident in the molecules involved in DNAdamage repair such as BRCA1, MGMT, and ERCC1, whoseexpression was reduced simultaneously only in M14 cells.ERCC1, BRCA1, and MGMT were also highly expressed in theM79 cell line after exposure to the radio-chemotherapycombination, albeit to a lesser degree than the cisplatin-onlytreatment, potentially indicating a residual defense mechanism.

We also highlighted the lower toxicity of the radiationtreatment followed by cisplatin compared to the inverseschedule (cisplatin followed by radiation) in a cell line of healthytissue. This was also confirmed by Rad51 foci analysis, whichrevealed a reduced number of positive cells after exposure toradiation followed by cisplatin.

Our results show the effectiveness of radiation and cisplatincombination in reducing proliferation via the induction ofapoptosis in melanoma cell lines. We also identified the mosteffective schedule in vitro of the two treatments, observing thatit modulated a network of proteins involved in DNA damagerepair. In conclusion, our data highlight the potentialsuperiority of radiation treatment followed by cisplatin overthe inverse sequence, routinely used in clinical practice.Further studies in animal models are needed to validate thesefindings which, if confirmed, could provide a scientific rationalefor a clinical trial aimed at defining the best treatment schedulefor patients with melanoma.

Fig. 6. Modulation of gene expression after treatment with agentsused singly or in sequence. Real Time PCR analysis was performedfor M66, M79, and M14 cell lines 72h after radiation doses of 2.4 and5Gy followed by a 72-h w.o., cisplatin treatment for 6 h followed by a24-h w.o. and sequence (radiation at 2.4 or 5Gy followed bycisplatin) (normalized to b2-microglobulin; � SD, standarddeviation of three replicates).

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Acknowledgments

This work was supported by a grant from the Italian Ministry ofHealth (RO Strategici 11/07) “p53 family interaction networkas a target of antitumor peptide therapy”. The funding body didnot have a role in the study design, in the collection, analysis,and interpretation of data; in the writing of the manuscript; orin the decision to submit the manuscript for publication.

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