review article radiation oncology in vitro : trends...

Review ArticleRadiation Oncology In Vitro Trends to Improve Radiotherapythrough Molecular Targets

Nataacutelia Feofanova1 Jony Marques Geraldo23 and Liacutedia Maria de Andrade4

1 Research Institute of Internal and Preventive Medicine FSBI Boris Bogatkov Street 1751 Novosibirsk 630089 Russia2 School of Medicine Federal University of Minas Gerais Avenida Alfredo Balena 190 Santa Efigenia30130-100 Belo Horizonte MG State Brazil

3 Alberto Cavalcanti Hospital Minas Gerais State Hospital Foundation-FHEMIG Rua Camilo de Brito 636 Padre Eustaquio30730-540 Belo Horizonte MG Brazil

4Department of Physics Institute of Mathematical Sciences Federal University of Minas Gerais Avenida Antonio Carlos 662731270-901 Belo Horizonte MG Brazil

Correspondence should be addressed to Lıdia Maria de Andrade lidiapesquisagmailcom

Received 5 June 2014 Accepted 16 July 2014 Published 15 September 2014

Academic Editor Jack Yang

Copyright copy 2014 Natalia Feofanova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Much has been investigated to improve the beneficial effects of radiotherapy especially in that case where radioresistant behavioris observed Beyond simple identification of resistant phenotype the discovery and development of specific molecular targets havedemonstrated therapeutic potential in cancer treatment including radiotherapy Alterations on transduction signaling pathwayrelated with MAPK cascade are the main axis in cancer cellular proliferation even as cell migration and invasiveness in irradiatedtumor cell lines then for that reason more studies are in course focusing on among others DNA damage enhancement apoptosisstimulation and growth factors receptor blockages showing promising in vitro results highlighting molecular targets associatedwith ionizing radiation as a new radiotherapy strategy to improve clinical outcome In this review we discuss some of the mainmolecular targets related with tumor cell proliferation and migration as well as their potential contributions to radiation oncologyimprovements

1 Introduction

To achieve a better understanding of the different targetedcancer responsiveness a wide range of experimental tumorsof various histologic types have been developed for radiobio-logical studies [1] whose effects induced by ionizing radiation(IR) can be investigated through many approaches allowingthe identification of radioresistance or radiosensitivity ofhuman cancer cell lines [2] There are for instance similarbehaviors between clonogenic repopulation in vitro applyingfractioned schedule on human squamous cell carcinoma andpatients that were treatedwith radiotherapy [3] Neverthelessseveral target molecules into different subcellular compart-ments related with radioresistance were already identified asan attempt to improve cellular radiation responses and in

another aspect a number of agents targeting components ofcell signaling pathways and processes critical to neoplastictransformation and progression are ongoing clinical devel-opment [4] Amongst other signaling cascades targets thereare some related with cell dynamics in an integrin dependentfashion that increases cancer cell migration induced by IRlike beta-galactoside alpha-(26)-sialyltransferase (ST6Gal I)that was found overexpressed in ovarian and other cancerswhose expression has been correlated to metastasis and poorprognosis [5 6] In colon cancer cells IR increases the expres-sion of ST6Gal I which in turn is involved in radioresistanceand radiation-induced migration via sialylation of integrin1205731that may be a novel target for overcoming radiation-

induced survival especially adhesion and migration of thiskind of tumor [7 8] Another well-known type of molecules

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 461687 13 pageshttpdxdoiorg1011552014461687

2 BioMed Research International

involved in cancer progression after IR is the members of adisintegrin and metalloproteinase (ADAM) family that arethought to mediate the shedding of epidermal growth factorreceptor (EGFR) ligands an important signaling pathwayto cell proliferation and migration and this event is criticalfor a more soluble functional EGFR ligands yield ADAM isactivated by IR leading to an increased triggering cell prolif-eration cascade in irradiated melanoma cells [9] specifyingADAMrsquos blockage as an attractive target to radiosensitizethis tumor type Another important subject is radio-inducedDNA damage and its repair ability that have been largelyinvestigated including the role of tyrosine kinase pathwayssuch as the ataxia-telangiectasia mutated (ATM) a proteinkinase that is best known for its role in the DNA damageresponse [10] and one recent work describes a new functionfor 51015840-adenosine monophosphate- (AMP-) activated proteinkinase (AMPK) an established metabolic stress sensor thathas the ability to control cellular growth and mediate cellcycle checkpoints in cancer cells in response to low energylevels as a sensor of genomic stress and a participant ofthe DNA damage response (DDR) pathway highlighting theimportance of targeting AMPK as novel cancer therapeuticsfor a radiosensitization of human cancer cells mediated bya simultaneous inhibition of the Akt and activation of theAMPK signaling pathways [11ndash13]

The knowledge in tumor biology has been increased inthe last years due to cellular signaling pathways discoveringmechanistically identified by molecular tools that allowedinvestigation of an enormous range of possibilities evenas new mathematical models have also been subsidizingradiotherapy enhancement The understanding and identi-fication of specific molecular targets with significant ther-apeutic implications in order to develop new strategies forradiotherapy are crucial to improve patient survival withoutsubstantially increasing toxicity

2 Radiobiological Models

Delivery of advanced radiotherapy techniques has taken newapproaches to treatment including stereotactic irradiationand intensity-modulated X-rays beam in order to improveoutcomes of cancer treatment and reduce damage to normalsurrounding tissues [14] The classical well-known radiobi-ological models are linear-quadratic (LQ) and biologicallyeffective dose (BED) that are widely used to estimate theeffects of various radiation schedules but it has been sug-gested that LQ is not applicable to high doses per fraction [15]due to the fact that LQoverestimates effects of high daily radi-ation doses proving that better models should be proposed[16] Thus different treatment schedules applying hypofrac-tionated radiotherapy (hRT) and other radiation modalitiessuch as light ions have been used with the same proposaleven though the pattern of received dose is different from thatin conventional radiation and therefore the radiobiologicalaspects of cell death are shown to be modified Prolongedor short radiation delivery makes sublethal damage repairrepopulation and reoxygenation be better evaluated speciallywhen amathematical model is used for dose conversion fromconventional treatment to high daily hypofractionated doses

[17] For instance dose conversion models as repairable-conditionally repairable (RCR) model [18] and multitarget(MT) model are currently recommended When those mod-els were compared LQ seemed to fit relatively well at dosesof 5Gy or less at 6Gy or higher doses RCR and MT modelsseemed to be more reliable than LQ [19] In hypofractionatedstereotactic radiotherapy LQ model should not be used andconversion models incorporating the concept of RCR or MTmodels such as generalized linear-quadratic (gLQ) modelsappear to be more suitable [20 21]

Recent investigations have highlighted differential cellu-lar responses when submitted to intensity-modulated radi-ation fields [22] particularly in areas outside the primarytreatment fields [23] Differential DNA damage responsesfollowingmodulated radiation field delivery were found pro-viding an evidence for a role of intercellular communicationin mediating cellular radiobiological response to modulatedradiation fields suggesting that advanced radiotherapy treat-ment plans require a refinement of existing radiobiologicalmodeling [24] Concerning radiobiology DNAdouble strandbreaks (DSB) are considered to be the kind of DNA damageresponsible for most end points such as chromosome aberra-tions and cell killing However due to high number of DSBsinduced by radiation at sublethal doses it is immediatelyobvious that DSB is not lethal in general indicating thatmost of induced DSBs can be rejoined or repaired correctlydisplaying the spatial distribution of DSBs as major factorin determining lethality [25ndash27] Nevertheless a mechanisticdose-response model has been proposed based on the con-cept of giant loops which constitute a level of chromatinorganization on a megabase pair length scale [28ndash30] thatsuggests DSBs are induced within different loop domainsof DNA assumed to be processed independently by cellularrepair mechanism Given giant loop chromatin organizationand assumption of two damage classes representing themain point Giant LOop Binary LEsion (GLOBLE) approacharises as promising model [31] This model is able to revealimportant features of dose-response curves describing cellsurvival especially transitions from low to high doses in adose-response correlation

The effects of combined modality treatments are investi-gated by using mathematical models to predict cell death asan attempt to fit LQ MT and gLQ models to experimentaldata based on in vitro assays demonstrating that gLQequation is superior to LQ and MT models in predictingcellular death at high doses of radiotherapy [32] A significantincreasing in biologically equivalent dose may be achievedafter addition of radiosensitizing agents to hRT as wellas linear accelerators containing new technologies such asflattening filter free (FFF) increases instantaneous dose-rateof X-ray pulses by a factor of 2ndash6 compared to conventionalflattened output [33]

New models for radiobiological cell responses have beenproposed and one of those is a simple two-parameteralgorithmic model which captures the essential biologicalfeatures of irradiation-induced cell death and associated cellcycle delays This approach estimates directly the underlyingirradiation-induced cell survival and was investigated inmammary carcinoma cell line EMT6Rowhere a comparison

BioMed Research International 3

of estimated underlying cell survival probability with invitro survival probability data confirms an optimal timingof mixed irradiationchemotherapy treatments leading toa development of an accurate spatial and temporal modelof tumor progression and cell cycle dynamics [34] It wasalready proposed that concurrent chemotherapy with hRTcould be beneficial for a number ofmalignancies takingmorevariables for survival cell curves and BED calculations [35ndash37] Apparently mathematical models concepts associatedwith in vitro assays and also applying chemotherapy ormonoclonal antibodies might be contributed to radiotherapyenhancement

3 Radiation Sensitivity Targets of CellularProliferation Signaling Pathways

A multiplicity of approaches has been investigated in theefforts to enhance ionizing radiation (IR) effects particularlysignaling cascade involved in cell proliferationThemitogen-activated protein kinase (MAPK) pathway transduces signalsfrom the cell membrane to the nucleus in response toa variety of different stimuli and participates in variousintracellular signaling pathways that control a wide spectrumof cellular processes including growth differentiation andstress responses and it is known to have a key role in cancerprogression [38] It was already demonstrated in breast cancercell lines MDAMB-231 that association of SphK1 antagonistFTY720 with IR significantly increased antiproliferative andproapoptotic effects through promoting alterations inMAPKsignaling [39] As is already knownRaf-MEK-ERKpathway isa key downstream effector of the Ras small GTPase the mostfrequentlymutated oncogene in human cancers thus this sig-naling network has been subject of intense research and phar-maceutical scrutiny to identify novel target-based approachesfor cancer treatment [40] then small molecule inhibitorsof MEK (PD0325901) and Akt (API-2) were subsequentlyevaluated for their radiosensitizing potential alone and incombination with pancreatic tumor cell lines demonstratingthat MEK inhibition results in growth arrest apoptosis andradiosensitization of multiple preclinical pancreatic tumormodels and these effects can be enhanced by associationwithan Akt inhibitor [41]

Since PI3KAKTmTOR signaling axis controls cell pro-liferation and survival this pathway has achieved majorimportance as a target for cancer therapy [42] It is alreadyknown that activation of these signals is a contributing factorto decreased radiation sensitivity [43] indicating target ofmTOR a downstream kinase of the phosphatidylinositol 3-kinase (PI3K)AKT survival pathway may be a target forradiation sensitizing several human cancer cell lines Itsinhibition currently being proved increased radiosensitivityof some human cell lines including SQ20B head and neckcarcinoma cells and U251 glioblastoma cells [44] Radia-tion sensitivity effect of NVP-BEZ235 a dual PI3KmTORinhibitor reveals enhancement of apoptosis in human gliomacells as well as cell cycle arrest resulting in striking tumorradiosensitization which extended the survival of braintumor-bearing mice [45ndash47] Likewise NVP-BEZ235 promi-nently improved the radiosensitivity of PC-3 prostate cancer

cells through inducing a G2M arrest and enhanced proapop-

totic effect after combined IR [48] Another study appliedthe inhibitor RAD001 associated with ionizing radiation insix bladder tumor cell lines UM-UC3 UM-UC5 UM-UC6KU7 253J-BV and 253-JP showing arrest in both G

1and G

2

phases of cell cycle when treatments are carried out togetherprimarily regulated by changes in the levels of cyclin D1 p27and p21 suggesting that alterations of cell cycle by inhibitingthe mTOR signaling pathway in combination with radiationhave favorable outcomes and it is a promising therapeuticmodality for bladder cancer [49]

The epidermal growth factor receptor (EGFR) is fre-quently overexpressed in malignant tumors and its level iscorrelated with increased cellular radioresistance [50] Oneof the defined mechanisms is that EGFR amplification orRas activation by mutations results in increased clonogeniccell survival and decreased tumor growth delay followingirradiation [51] There is for instance a current opinionabout therapeutics that target EGFR might enhance thecytotoxic effects of IR One of these approaches are thehumanized monoclonal antibodies used as anticancer ther-apy and expected to improve the effectiveness of currenttherapy to stimulate radiation sensitization amongst othertargets EGFR blockage (cetuximab) EGFR tyrosine kinaseinhibitors (gefitinib) and vascular endothelial growth factor(VEGF) inhibitors such as bevacizumab are still under inves-tigation [52ndash56] Cetuximab and IR have shown promisingresults when performed concomitantly Previous data haveshown that use of monoclonal antibody cetuximab (C225)improves local tumor control after irradiation in FaDuhuman squamous cell carcinoma (hSCC) due to decreasingrepopulation and improving reoxygenation effects as well[57] Recently cetuximab was approved for the treatment ofpatients with recurrent metastatic head and neck squamouscell carcinoma (HNSCC) [58] and phase III clinical trialscombining bevacizumab with conventional treatments havebeen performed in advancedrecurrent HNSCC patientsEGFR tyrosine kinase inhibitor (E-TKI) promoted radiosen-sitization of non-small cell lung cancer (NSCLC) A549 andH3255 cells with low nitric oxide levels due to suppressionof cell viability when associated with IR [59] A positivecorrelation between the presence of a KRAS mutation andradiosensitization after treatment with the EGFR inhibitorserlotinib and cetuximab in several non-small cell lunglineages was lately demonstrated [60] In addition it wasnoticed for instance that radiation-induced upregulationof hypoxia-inducible factor-1 alpha (HIF-1120572) was completelyabolished by simultaneous treatment of HNSCC cells withcetuximab [61] Despite the promising results of applyingcetuximab radiosensitization effect was lost in head and necktumor cells overexpressing Ras family members such as K-Ras N-Ras and H-Ras proteins even as EGFR-independentactivation of the RASRAFMEKMAPK pathway [62ndash64]

The humanized anti-VEGF monoclonal antibody beva-cizumab has single agent activity in previously treated andrecurrent cervical ovarian and colorectal cancer diseases[65ndash68] even as tumor sensitivity to adjuvant radiother-apy improvement [69 70] The mechanisms of interac-tion between antiangiogenic agents and IR are complex


and involve interactions between tumor cells and tumormicroenvironment including tumor oxygenation stromaand vasculature Radiation resistance of solid tumors towardphoton irradiation is caused by attenuation of DNA damagein less oxygenated tumor areas and by increased hypoxia-inducible factor- (HIF-) 1 signaling [71] When the antian-giogenesis drug Endostar combined with radiotherapy wasapplied on A549 cells increased radiation sensitivity bytranscriptional factors expression reduction of TGF-120573

1and

HIF-1120572was noticed [72] and it was observed in human colonadenocarcinoma cell line WiDr surviving after radiationtherapy by acquiringHIF-1 activity and translocation towardstumor blood vessels in a dependent cellular dynamics afterirradiation recurrence what might suggest basis for targetingHIF-1 after radiation therapy [73 74] especially in hypoxictumors

Interestingly another promising target to radiosensitizetumors resistant to irradiation is nucleoplasmic calciumThe role of nuclear calcium in tumor cell proliferation waspreviously determined in HepG2 cells showing decreasedproliferation rate under low nuclear Ca2+ concentrationsdue to a mitotic blocking induced by buffering of nuclearCa2+ [75] Even though the mechanism by which nuclearCa2+ regulates cell proliferation is not completely understoodthere are reports demonstrating that activation of tyrosinekinase receptors (RTKs) leads to translocation of RTKs to thenucleus to generate localized nuclear Ca2+ signaling whichare believed to modulate cell proliferation [76] We werethe first research group who established that nuclear Ca2+buffering decreases EGFR expression and also the radiosen-sitization effect of association between nucleoplasmic Ca2+buffering and X-rays in human squamous cell carcinomaA431 preventing ADAM-17 overexpression induced by IRFurthermore this association promoted less tumor cellsproliferation and reduced their survival fractions [77] sug-gesting nucleoplasmic Ca2+ as a new target to radiosensitizesquamous cell carcinoma

4 DNA Damage Improvements via PARP andDNA-PKcs Inhibitors

The concept of DNA repair centers and the meaning ofradiation-induced foci in human cells have remained con-troversial in spite of evidences for formation of these repaircenters in a dose-response nonlinearity manner [78] WhileIR induces a variety of DNA lesions including base damageand single strand breaks DNA double strand break (DSB)is widely considered as the lesion responsible not only forthe aimed cell killing of tumor cells but also for the generalgenomic instability [79] As part of an intricate repair com-plex poly(ADP-ribose)polymerase 1 (PARP1) functioningas DNA nick-sensor interacting with base excision repairDNA intermediates containing single strand breaks [80 81]Some researchers have shown that PARP inhibitors (PARPi)enhance the cytotoxicity effects of gamma and X-irradiationand alkylating agents at least when tumor sensitizationexceeds effects on normal tissues which could improveclinical outcomes [82 83]These inhibitors have gained recent

attention due to their unique selectivity in killing tumorswith defective DNA repair therefore achieving interestingresults by improved radiation sensitivity in UM-SCC1 UM-SCC6 and FaDu cancer cells that used with cetuximabdecreased nonhomologous end joining (NHEJ) and homol-ogous recombination (HR) mediated DNA double strandbreak [84] When olaparib a potent PARP-1 inhibitor wasinvestigated in Calu-6 and A549 cells a human NSCLCpersistentDNAdouble strand breaks for at least 24 hours aftertreatment in combinationwith IRwere found demonstratingradiosensitization to lung cancer cells [85] Moreover PARPiwas also proposed as a radiosensitizer to Glioblastoma-initiating cells [86 87] even as another PARP inhibitor ABT-888 (veliparib) enhanced the radiation response of prostatecancer cell lines DU-145 and PC-3 that efficiently promotedabundant senescent cells displaying persistent DNA damagefoci and in human head and neck cancer cells improvedcytotoxicity with ABT-888 and IR was found compared toeither agent alone [88 89] Inhibition of histone deacetylases(HDACs) also increases DNA damage as was noted in A549lungDLD-1 colorectalMiaPaCa2 pancreatic andUT-SCC15head and neck squamous cell carcinoma cells treated withNDACI054 histone deacetylase inhibitor that showed a sig-nificant intensification of residual 120574-H2AXp53BP1-positivefoci leading to radiosensitization of these cell lines [90] Inthe same way decay of 120574-H2AX foci correlates with p53functionality and potentially lethal damage repair in humancolorectal carcinoma RKO and prostate cancer DU-145 cells[24 91] An alternative treatment strategy to interferewith theproliferative pathways is to apply nimotuzumab a humanizedIgG1monoclonal antibody that specifically targets EGFR in

combinationwith IR [92] Because of the inhibition of nucleartranslocation of EGFR nimotuzumab and also cetuximabboth antibodies induce radiosensitization increasing thepercentage of deaddying cells and the yield of 120574-H2AXfoci being able to promote intensification of radiosensitivityof malignant cells expressing EGFR and offer potentialimproving of therapeutic index of radiotherapy [93] Asignificant inhibition of radio-induced DNA damage repairdue to inhibited activation of DNA-dependent kinase cat-alytic subunits (DNA-PKcs) through blocking the PI3KAKTpathway in A549 cells and MCF-7 breast cancer cells wasobserved [92] In another work transfected HeLa cells withthe anti-DPK3-scFv gene resulted in more sensitivity to IRand diminished DNA repair which could indicate blockageof DPK3-scFv via targeting DNA-PKcs as a novel biologicalradiosensitizer for cancer treatment [94] Table 1 summarizessomemolecular targets investigated in their respective tumorcell lines

5 Apoptosis Signaling Pathway Target

Apoptosis is a programmed cell death that is currently ofintense research interest in cancer biology and it was alreadyestablished that expression levels of Bcl-2 family proteins intumors can modulate apoptosis influencing tumor behaviorand treatment [95] and also in another way apoptosis canbe triggered intrinsically or extrinsically by DNA damage orother types of severe cellular injures such as reactive oxygen


Table 1 Molecular targets related with intracellular signaling pathways in different tumor cell lines

Tumor type Cell line Proliferationtargets Migration targets Angiogenesis

targets DNA targets

Breast MDAMB-231MCF-7

SphK1Akt

PGE2MMP-2 DNA-PKcs

Head and neck

SQ20BFaDuA431

UM-SCC1UM-SCC6UT-SCC15

PI3KmTOREGFR

ADAM-17nuclear Ca2+

HIF-1120572PARPHDACp53

Glioblastoma U251 PI3KmTOR 120572v1205733

Bladder

UM-UC3UM-UC5UM-UC6KU7

253J-BV253-JP

mTORcyclin D1

p27p21

LungA549H3255Calu-6

EGFRE-TKIPI3KAkt

Akt2TGF120573

12057221205731Src

COX-2MMP-9

VEGFRTGF-1205731

PARP-1HDAC

ColorectalWiDrDLD-1RKOKM20

EGFRPI3KmTOR

Akt2TGF120573

ST6Gal IHIF-1120572 HDAC

Prostate PC-3DU-145 PI3KmTOR PARP-1

p53

Cervix HeLaFIR PI3K p65 NF-kB DNA-PKcs

LiverHepG2

McA-RH77KM20

nuclear Ca2+PI3KAkt

MMP-2MMP-9 VEGF

species [96] For example Bcl-2 gene has been revealedto be overexpressed in oral cancers predicting outcome inpatients treated with definitive radiotherapy [97] One ofthe investigated mechanisms of cell death is related withradiation-induced resistance of tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL) receptor an importantprotein related with failure of recurrent laryngeal cancer Itwas suggested that hypermethylation of DR4 CpG islandcan promote TRAIL radioresistance [98] Changes in geneexpression levels have largely been studied in several cancertypes even as their regulatory mechanisms and additionallymicroRNAs a class of endogenous small noncoding RNAsthat negatively regulates gene expression are considered anew subject of cancer therapy investigation Recent studiesshowed that miR-193a-3p was able to radiosensitize both U-251 and HeLa cells by accumulation of intracellular reactiveoxygen species increasing DNA damage and also apop-tosis directly targeting the antiapoptotic Mcl-1 gene [99]while silencing miR-21 in radioresistant NSCLC A549 cellssensitized them to IR by inhibiting cell proliferation and

enhancing cell apoptosis through inhibition of PI3KAktsignaling pathway [100] The synergistic effect of resvera-trol and IR has been shown in different cancer cell lineseffectively acting by enhancing expression of antiproliferativeand proapoptotic molecules and inhibiting proproliferativeand antiapoptotic molecules leading to induction of apop-tosis through various pathways suggesting resveratrol plusradiotherapy as a therapeutic promise in the near future[101]

Recent investigations have been suggesting autophagy asa cell death pathway that may mediate cancer cells sensitivityto IR even though it could originate a protective mecha-nism against the treatment itself by removing proteins andorganelles that are damaged or alternatively produce aneffective cell death process [102] Inhibition of autophagycould sensitize tumor cells tomany cytotoxic drugs or reverseresistance to chemotherapeutic drugs representing a promis-ing strategy to improve the efficacy of cancer treatment[103] However the autophagic responses of cancer cells to


antineoplastic therapy including IR remain a controversialissue

6 Migration and Invasion Pathways Target

Irradiation of primary tumor might promote invasion andfavor metastasis by upregulating the expression of genes andactivating signaling pathways that are involved in migrationand motility One of the principal pathways involved in alter-ation of migratory activities is PI3KAkt signaling pathwaythat has been implicated in driving metastatic phenotypein thyroid [104] breast [105] and other cancers [106] andPI3K activity is further increased by radiotherapy in cer-tain tumors [107 108] Activation of this signaling pathwaypromotes metastatic transition via stimulation of epithelial-mesenchymal transition (EMT) even as enhancement ofmigration and invasion [106]

Besides being related to proliferation cascade responsesAkt2 one of the specific isoforms of Aktwhich is downstreamof PI3K plays important role in promotion of cell migrationand invasion In a xenograft model of colorectal cancerknockdown of Akt2 in KM20 cell line inhibits liver metasta-sis the converse is observed when constitutively active Myr-Akt2 is expressed [109] It has been noticed that activationof Akt2 increases cell invasion and metastasis of breast andovarian cancer cells through upregulated integrin signaling[110] its inactivation also inhibits glioma cell invasion [111]and knockdown of Akt2 rather than Akt1 in the cell lineA549 dramatically abolishes its invasive potential [112]

A growing number of studies demonstrate that IR mayenhance the migratory and invasive properties of cancer cellsvia induction of epithelial-mesenchymal transition (EMT)EMT is an embryonic program important for organogenesisin normal development but its dysfunction can help the sur-vival and dissemination of cancer cells that is characterizedby loss of cell-cell contacts decreased expression of epithelialmarkers E-cadherin beta-catenin and ZO-1 remodeling ofthe actin cytoskeleton and increased expression of mes-enchymal markers N-cadherin fibronectin and vimentinSeveral transcription factors have been discovered that caninitiate and maintain this process including Snail Twist andZeb [113] Furthermore transforming growth factor beta1(TGF120573) is a tumor promoter and potent inducer of EMT

though it can be a tumor suppressor during the initial stage oftumorigenesis Ionizing radiation is able to enhance expres-sion of TGF

120573in various cell lines This enhancement occurs

along with increase of mesenchymal markers and decreaseof epithelial markers as well as alterations in migratory andinvasive capabilities of the cells [114 115] However IR isshown to induce TGF

120573activation in vivo and sensitize even

nonmalignant mammary epithelial cells to undergo TGF120573-

mediated EMT [116] In colorectal cancer IR induces analteration to a malignant phenotype consistent with EMT invitro [117]Moreover TGF

120573is known to bemaster regulator of

EMT and IR-induced EMT can be reversed by its inhibitionnonetheless other pathways of EMT induction exist as wellEvents associated with EMT induced by IR could be reversedthrough inhibition of TGF

120573signaling with TGF

120573R inhibitor

SB431542 as was already found in A549 cell line [115]

Another mechanism observed in cervical cancer cells (FIRcells) showed EMT induced by irradiation is dependent onactivation of p65 subunit of NF-120581B [118] Pharmacologicalinhibition of Akt with GSK690693 blocks the expressionof ZEB1 and vimentin and restores the expression of E-cadherin following IR thus preventing the migration andEMT of nasopharyngeal carcinoma cell lines [119] even asknockdown of Akt2 induces reversion of the EMT process inmammary epithelial cell lines [120] Cell motility on varioussubstrates as well as penetration of membranes is mediatedby integrins expressed on the cell surface Expression of120572v1205733in glioma cells [108] and 120572

51205731in pancreatic cancer

[121] is upregulated after IR facilitating cell migration andinvasion Likewise integrin 120572

31205731is overexpressed after IR

promoting the migration of meningioma cells via focaladhesion kinase and extracellular signal-regulated kinase[122] Moreover integrin 120572

21205731is selectively upregulated in

irradiated lung cancer cells and is required for aggressivephenotype and invasion in 3D collagen gels and such inva-siveness ismediated via PI3KAkt signaling pathway and theninvasion speed in vitro can be reduced significantly by PI3Kinhibitor LY294002 [123] Additionally integrin expressionplays role in activation of MMP-2 interaction of MMP-2with 120572v120573

3integrin is required during its maturation and

activation demonstrating localization of active MMP-2 and120572v1205733integrin at the migration front accelerates cancer cell

migration [124 125]Pharmacological inhibition of integrin dependent sig-

naling pathways can be assumed as one of the promisingapproaches for combined therapy with IR The function-blocking anti-120572v120573

3monoclonal antibody 17E6 and 120572v120573

3

120572v1205735specific antagonist EMD121974 (cilengitide) inhibit in

vitro matrigel invasion and lung metastasis formation oftumor cells growing in a preirradiated microenvironment[126] and Cilengitide demonstrates strong antimigratoryproperties inmeningioma cells in vitro through combinationwith IR that allows achieving significant decrease of tumorvolumes using intracranial model of human meningioma[127]

Nonreceptor tyrosine kinase Src is often activated invarious types of cancer via mutations or growth factor signal-ing pathways including insulin-like growth factor-1 receptor(IGFR-1) EGFR and platelet-derived growth factor receptor(PDGFR)Then Src plays an important role in focal adhesiondisassembly since its expression results in disruption of focaladhesions and stress fibers leading to the loss of adhesion tothe extracellular matrix [128] This Src-mediated disruptionof focal adhesions leads to a decrease in cell-cell and cell-ECMadhesion and is an important process central to cellmigrationand invasion In addition to its effects on motility Src mayenhance cellular invasion by regulating the expression ofMMPs and tissue inhibitors of metalloproteinases [129] Inlung cancer cells EGFR signaling appears to be the dominantmechanism of Src activation It was already noted that inhibi-tion of Src with submicromolar concentrations of AZD0530blocks Src and focal adhesion kinase resulting in significantinhibition of cell migration and matrigel invasion in NSCLCcells [130] suggesting key tyrosine kinase target moleculescombiningwith IR as a promising radiotherapy enhancement


NR

Cytosol

RTK

ADAM-17EGFRligands

Soluble EGFR

Plasma membrane

PI3k

Akt

Ras

Raf

Mek

Erk

VEGFR

MAPK

MMP

Akt

p

p

p p

PI3k

p p

p

p

p

mTOR

mTOR

HIF-1120572

Cell proliferation

Cell survival

Angiogenesis

PARP

Cyclins

DNA-PKc

Integrins

Src

Migration

Apoptosis

Bcl-2

Caspases

VEGF

Transcription factors

DNA damage

MAPK

IR

Nucleus

IP3RCa2+

Figure 1 Network of signaling cascade involved in tumor progression and radioresistance The scheme shows tyrosine kinase axis andits signaling cascade leading to cell proliferation migration angiogenesis apoptosis and DNA damage some of them activated byphosphorylation (p) These signaling pathways are involved in radiation resistance and are promising targets to improve radiotherapy (IP

3R

= receptor of inositol 145-trisphosphate and NR = nucleoplasmic reticulum)

to impair proliferation and invasivenessTherefore inhibitorsof these signaling pathways as part of combination therapywith IR can significantly ameliorate side effect of irradiationFigure 1 shows a scheme of cellular signaling pathways relatedwith tumor cell progression

7 Ionizing Radiation andMicroenvironment Interface

Enhancement of invasiveness in response to IR can becaused not only by alterations in cancer cell gene expressionprofile which increase cell motility and migratory capacitiesbut also due to modulation of tumor microenvironmentIrradiated tumor microenvironment may exert potentialtumor-promoting effects and tumors growing within a pre-viously irradiated bed tend to be more metastatic [126]Extracellular cellmatrixmodifications that favor invasivenessare dependent on expression of such enzymes as matrixmetalloproteinases (MMPs) MMP-2 MMP-9 and urokinaseplasminogen activator (uPA) as well It is known thatexpression of these molecules as well as cytokines which

promote invasion in cancer cells can be induced by IR bothin cancer and in stromal cells Increased levels of MMP-9 and uPA are found in conditioned medium of irradi-ated neuroblastoma cells [131] Furthermore conditionedmedium from irradiated nonparenchymal liver cells containselevated amounts of MMP-2 MMP-9 EGF and VEGF andpromotes invasiveness of sublethally irradiated cultures inhepatoma cell line McA-RH77 [132] Notwithstanding pro-teolytic enzyme urokinase plasminogen activator is upreg-ulated after irradiation in the IOMM-Lee meningioma cellsvia activation of EGFRMEK12 and p38 signaling pathwayswhich results in increased tumor invasion and migrationin vitro Additionally inhibitors of these signaling pathwayswith specific inhibitors AG1478 U0126 and SB203580 showdecrease of uPA levels in both basal and irradiated-IOMM-Lee cells [133] Some proteinases which are expressed byadvancing cells of metastatic tumor MMPs are believed toplay major role in tumor invasion as they can destroy almostall of basement membrane macromolecules Injury to thebasement membrane can result in the release of proinvasivegrowth factors which can further stimulate the expression of


MMPs [134 135] Lewis lung carcinoma cells irradiated indose of 75 Gy demonstrate enhanced expression of MMP-9and increased invasiveness in vitro When transplanted sub-cutaneously after subsequent irradiation in xenograft modelthese cells also have greater lung metastatic potential whichis MMP-9 dependent and can be reduced by prototypicalMMP-9 inhibitor zoledronic acid [136] Proteasome inhibitorMG132 potentiates the effect of radiation against the growthandmetastasis inNSCLC cells in nontoxic dose Pretreatmentwith MG132 followed by irradiation in dose of 4Gy in vitrois shown to suppress cell migration and invasion abilities inA549 and H1299 cancer cell lines which is accompanied bydecreased expression of MMP-2 and MMP-9 in NSCLC celllines [137] Irradiation in dose of 5Gy is shown to induceCOX-2 activation in fibroblasts which leads to increasedinvasiveness of MDAMB-231 cells cocultivated with theseirradiated fibroblasts This effect is due to PGE2-dependentinduction ofMMP-2 expression inMDAMB-231 cells and canbe completely reversed by COX-2 inhibitor NS-398 [138]

Pharmacological inhibition of PI3KAkt signaling path-way has a great therapeutic potential when combined withIR In thyroid carcinoma cells PI3K inhibitor GDC0941 sig-nificantly inhibits lung metastasis in mice bearing irradiatedfollicular thyroid carcinoma cells It is of interest that PI3Kis not activated in these cells by IR in vitro which meansthat tumor microenvironment is involved in antimetastaticactivities of GDC0941 [139]

Many protocols of irradiation therapy involve irradiationof not only the tumor itself but also nonmalignant cellswhich surround tumor As a stress stimulus irradiationchanges significantly gene expression profile of these cellsand therefore causes modulation of microenvironment [140]Enhanced expression of genes associated with proinflamma-tory response like COX2 andMMPs leads to reorganizationof extracellular matrix facilitating invasiveness of tumor cellsInhibition of these signaling pathways in cells of tumormicroenvironment can become a promising approach forenhancement of positive effect of radiotherapy

8 Conclusions and Future Perspectives

The increased understanding of the molecular processesunderlying cellular sensitivity to IR has led to the identi-fication of novel targets for intervention [87] combiningmolecular targeted therapies and radiation may allow forreducing radiation toxicities and improving treatment out-comes [141] Not only traditional photon therapy but alsoother modalities applying charged particles can be improvedand investigations are being developed for instance hadron-therapy a form of external radiation therapy which usesbeams of charged particles such as carbon ions [71] Inter-esting changes at gene level response were achieved inPC-3 prostate cancer cells applying carbon ion irradiation[142] and the hypoxia-induced radioresistance to X-rayscan be overcome by carbon-ion beams in SCCVII cell line[143] Gene therapy also rises as new approach to improveradiosensitivity combining two or more targeting genesFor example the coexpression of doublecortin (DCX) withsecreted protein and rich in cysteine (SPARC) collaboratively

diminished radioresistance of glioma cells interfering withcell cycle turnover and increased irradiation-induced apop-tosis [144] Interestingly anticancer effects of metforminthe most widely used drug for type II diabetes alone or incombination with IR were found radiosensitizing MCF-7human breast cancer cells and FSaIImouse fibrosarcoma cellsby inactivation of mTOR and suppression of its downstreameffectors S6K1 and 4EBP1 [145]

The trend of radiation therapy improvements has beenfocused on tyrosine kinase cascade the principal signalingpathway to development of new target molecules whosemembers play a pivotal role on cellular proliferation asmigra-tion and tumor invasivenessmediators Predictable responsesmight be achieved based on combined IR with specifictarget molecules able to inhibit overexpressed proteins afterradiation exposure In addition molecular targeted therapybased on signal transduction pathway alterations detectedin cancer offers a tailored treatment possibility includingimprovements on radiation therapy Thereby uses of radio-therapy according to predictive markers would potentiallyreduce costly over treatment improve the treatment risk-benefit ratio and cancer outcomes [146] providing furtherevidence for the importance of intercellular signaling inmodulated exposures where dose gradients are present andmay inform the refinement of established radiobiologicalmodels to facilitate the optimization of advanced radio-therapy treatment plans [147] Furthermore new modelsreproducing clinical conditions as closely as possible areneeded for radiobiological studies to provide informationthat can be translated from bench to bedside [148]

Some questions arise concerning how to maximize IReffects combinedwith these newmolecular targets as possiblestrategies including optimization of dosage and radiationschedule leading tomanagement of toxicities Further in vitrostudies are necessary even as systematic reviews focusing onradiobiology and broad molecular targets especially againstPI3KAKTmTOR pathway that is involved on cellular prolif-eration as cell migration signaling for the purpose of achiev-ing the best outcome associated with IR to the radiotherapyof the future

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Brazilian funding agencies FAPEMIGCAPES and CNPq for financial support of their researches

References

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[2] L M Andrade T P R de Campos M F Leite and A MGoes ldquoIn vitro response of the human breast cancer cell lineMDAMB-231 and human peripheral blood mononuclear cells


exposed to 60Co at single fractionrdquoBrazilian Archives of Biologyand Technology vol 48 no 2 pp 205ndash213 2005

[3] C Petersen D Zips M Krause et al ldquoRepopulation of FaDuhuman squamous cell carcinoma during fractionated radio-therapy correlateswith reoxygenationrdquo International Journal ofRadiation Oncology Biology Physics vol 51 no 2 pp 483ndash4932001

[4] J E Dancey ldquoRecent advances of molecular targeted agentsopportunities for imagingrdquo Cancer Biology and Therapy vol 2no 6 pp 601ndash609 2003

[5] M Lee H-J Lee W D Seo K H Park and Y-S LeeldquoSialylation of integrin beta1 is involved in radiation-inducedadhesion and migration in human colon cancer cellsrdquo Interna-tional Journal of Radiation Oncology Biology and Physics vol76 no 5 pp 1528ndash1536 2010

[6] M J Schultz A F Swindall J W Wright E S Sztul C NLanden and S L Bellis ldquoST6Gal-I sialyltransferase conferscisplatin resistance in ovarian tumor cellsrdquo Journal of OvarianResearch vol 6 no 1 article 25 2013

[7] M Lee J-J Park Y-G Ko and Y-S Lee ldquoleavage of ST6GalI by radiation-induced BACE1 inhibits golgi-anchored ST6GalI-mediated sialylation of integrin 1205731 and migration in coloncancer cellsrdquo Radiation Oncology vol 7 no 1 article 47 2012

[8] P-C Lee Y-C Chiou J-MWong C-L Peng andM-J ShiehldquoTargeting colorectal cancer cells with single-walled carbonnanotubes conjugated to anticancer agent SN-38 and EGFRantibodyrdquo Biomaterials vol 34 no 34 pp 8756ndash8765 2013

[9] H Kataoka ldquoEGFR ligands and their signaling scissorsADAMs as new molecular targets for anticancer treatmentsrdquoJournal of Dermatological Science vol 56 no 3 pp 148ndash1532009

[10] S Ditch and T T Paull ldquoThe ATM protein kinase and cellularredox signaling beyond the DNA damage responserdquo Trends inBiochemical Sciences vol 37 no 1 pp 15ndash22 2012

[11] T Sanli A Rashid C Liu et al ldquoIonizing radiation activatesAMP-activated kinase (AMPK) a target for radiosensitizationof human cancer cellsrdquo International Journal of RadiationOncology Biology Physics vol 78 no 1 pp 221ndash229 2010

[12] T Sanli G R Steinberg G Singh and T Tsakiridis ldquoAMP-activated protein kinase (AMPK) beyond metabolism a novelgenomic stress sensor participating in the DNA damageresponse pathwayrdquo Cancer Biology ampTherapy vol 15 no 2 pp156ndash169 2014

[13] T Sanli C Liu A Rashid et al ldquoLovastatin sensitizes lungcancer cells to ionizing radiation modulation of molecularpathways of radioresistance and tumor suppressionrdquo Journal ofThoracic Oncology vol 6 no 3 pp 439ndash450 2011

[14] Y Shibamoto C Sugie and H Iwata ldquoRadiotherapy for meta-static brain tumorsrdquo International Journal of Clinical Oncologyvol 14 no 4 pp 281ndash288 2009

[15] H Iwata Y Shibamoto R Murata et al ldquoEstimation of errorsassociatedwith use of linear-quadratic formalism for evaluationof biologic equivalence between single and hypofractionatedradiation doses an in vitro studyrdquo International Journal ofRadiation Oncology Biology Physics vol 75 no 2 pp 482ndash4882009

[16] Y Shibamoto S Otsuka H Iwata C Sugie H Ogino and NTomita ldquoRadiobiological evaluation of the radiation dose asused inhigh-precision radiotherapy effect of prolonged deliv-erytime and applicability of the linear-quadraticmodelrdquo Journalof Radiation Research vol 53 no 1 pp 1ndash9 2012

[17] A Brahme ldquoAccurate description of the cell survival andbiological effectat low and high doses and LETrsquosrdquo Journal ofRadiation Research vol 52 no 4 pp 389ndash407 2011

[18] B K Lind L M Persson M R Edgren I Hedlof and ABrahme ldquoRepairable-conditionally repairable damage modelbased on dual poisson processesrdquo Radiation Research vol 160no 3 pp 366ndash375 2003

[19] H Iwata N Matsufuji T Toshito T Akagi S Otsuka andY Shibamoto ldquoCompatibility of the repairable-conditionallyrepairablemulti-target and linear-quadraticmodels in convert-ing hypofractionated radiation doses to single dosesrdquo Journal ofRadiation Research vol 54 no 2 pp 367ndash373 2013

[20] Z Huang N A Mayr S S Lo et al ldquoA generalized linear-quadratic model incorporating reciprocal time pattern of radia-tion damage repairrdquoMedical Physics vol 39 no 1 pp 224ndash2302012

[21] J Z Wang Z Huang S S Lo W T C Yuh and N A Mayr ldquoAgeneralized linear-quadratic model for radiosurgery stereotac-tic body radiation therapy and high-dose rate brachytherapyrdquoScience Translational Medicine vol 2 no 39 Article ID 39ra482010

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radiosensitization using in vitro datardquo International Journal ofRadiation Oncology Biology and Physics vol 83 no 1 pp 385ndash393 2012

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[34] S D Angus and M J Piotrowska ldquoA numerical model ofEMT6Ro spheroid dynamics under irradiation calibration andestimation of the underlying irradiation-induced cell survivalprobabilityrdquo Journal of Theoretical Biology vol 320 pp 23ndash322013

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[36] J-P Pignon A L Maıtre E Maillard and J Bourhis ldquoMeta-analysis of chemotherapy in head and neck cancer (MACH-NC) an update on 93 randomised trials and 17346 patientsrdquoRadiotherapy and Oncology vol 92 no 1 pp 4ndash14 2009

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[38] A Munshi and R Ramesh ldquoMitogen-activated protein kinasesand their role in radiation responserdquo Genes Cancer vol 4 no9-10 pp 401ndash408 2013

[39] G Marvaso A Barone N Amodio et al ldquoSphingosine analogfingolimod (FTY720) increases radiation sensitivity of humanbreast cancer cells in vitrordquo Cancer Biology amp Therapy vol 15no 6 pp 797ndash805 2014

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[45] C R Gil del Alcazar M C Hardebeck B Mukherjee etal ldquoInhibition of DNA double-strand break repair by thedual PI3KmTOR inhibitor NVP-BEZ235 as a strategy forradiosensitization of glioblastomardquo Clinical Cancer Researchvol 20 no 5 pp 1235ndash1248 2014

[46] W J Wang L M Long N Yang et al ldquoNVP-BEZ235 a noveldual PI3KmTOR inhibitor enhances the radiosensitivity ofhuman glioma stem cells in vitrordquo Acta Pharmacologica Sinicavol 34 no 5 pp 681ndash690 2013

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rapamycin inhibitor elicits multifaceted antitumor activities inhuman gliomasrdquo Molecular Cancer Therapeutics vol 8 no 8pp 2204ndash2210 2009

[48] W Zhu W Fu and L Hu ldquoNVP-BEZ235 dual phosphatidyli-nositol 3-kinasemammalian target of rapamycin inhibitorprominently enhances radiosensitivity of prostate cancer cellline PC-3rdquo Cancer Biotherapy amp Radiopharmaceuticals vol 28no 9 pp 665ndash673 2013

[49] R Nassim J J Mansure S Chevalier F Cury and W KassoufldquoCombining mTOR inhibition with radiation improves anti-tumor activity in bladder cancer cells in vitro and in vivo anovel strategy for treatmentrdquo PLoS ONE vol 8 no 6 ArticleID e65257 2013

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[54] S Y Park Y M Kim and H Pyo ldquoGefitinib radiosensitizesnon-small cell lung cancer cells through inhibition of ataxiatelangiectasia mutatedrdquo Molecular Cancer vol 9 article 2222010

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[82] T Hirai H Shirai H Fujimori R Okayasu K Sasai andM Masutani ldquoRadiosensitization effect of poly(ADP-ribose)polymerase inhibition in cells exposed to low and high linerenergy transfer radiationrdquo Cancer Science vol 103 no 6 pp1045ndash1050 2012

[83] D A Loser A Shibata A K Shibata L J Woodbine PA Jeggo and A J Chalmers ldquoSensitization to radiation andalkylating agents by inhibitors of poly(ADP-ribose) polymeraseis enhanced in cells deficient in DNA double-strand breakrepairrdquo Molecular Cancer Therapeutics vol 9 no 6 pp 1775ndash1787 2010

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[86] M Venere P Hamerlik Q Wu et al ldquoTherapeutic targeting ofconstitutive PARP activation compromises stem cell phenotypeand survival of glioblastoma-initiating cellsrdquo Cell Death ampDifferentiation vol 21 no 2 pp 258ndash269 2014

[87] M Verheij C Vens and B van Triest ldquoNovel therapeutics incombination with radiotherapy to improve cancer treatmentrationale mechanisms of action and clinical perspectiverdquo DrugResistance Updates vol 13 no 1-2 pp 29ndash43 2010

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[89] S Nowsheen J A Bonner and E S Yang ldquoThe poly(ADP-ribose) polymerase inhibitor ABT-888 reduces radiation-induced nuclear EGFR and augments head and neck tumorresponse to radiotherapyrdquo Radiotherapy and Oncology vol 99no 3 pp 331ndash338 2011

[90] S Hehlgans K Storch I Lange and N Cordes ldquoThe novelHDAC inhibitor NDACI054 sensitizes human cancer cells toradiotherapyrdquo Radiotherapy and Oncology vol 109 no 1 pp126ndash132 2013

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[92] Y Y Qu S L Hu X Y Xu et al ldquoNimotuzumab enhances theradiosensitivity of cancer cells in vitro by inhibiting radiation-induced DNA damage repairrdquo PLoS ONE vol 8 no 8 ArticleID e70727 2013

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[99] J-E Kwon B-Y Kim S-Y Kwak I-H Bae and Y-H HanldquoIonizing radiation-inducible microRNA miR-193a-3p inducesapoptosis by directly targeting Mcl-1rdquo Apoptosis vol 18 no 7pp 896ndash909 2013

[100] Y Ma H Xia Y Liu and M Li ldquoSilencing miR-21 sensitizesnon-small cell lung cancer A549 cells to ionizing radiationthrough inhibition of PI3KAktrdquo Int vol 2014 Article ID617868 6 pages 2014

[101] L Kma ldquoSynergistic effect of resveratrol and radiotherapy incontrol of cancersrdquo Asian Pacific Journal of Cancer Preventionvol 14 no 11 pp 6197ndash6208 2013

[102] S Palumbo and S Comincini ldquoAutophagy and ionizing radia-tion in tumors the ldquosurvive or not surviverdquo dilemmardquo Journalof Cellular Physiology vol 228 no 1 pp 1ndash8 2013

[103] H Rikiishi ldquoAutophagic action of new targeting agents in headand neck oncologyrdquo Cancer Biology and Therapy vol 13 no 11pp 978ndash991 2012

[104] J E Paes andMD Ringel ldquoDysregulation of the phosphatidyli-nositol 3-kinase pathway in thyroid neoplasiardquo EndocrinologyandMetabolism Clinics of North America vol 37 no 2 pp 375ndash387 2008

[105] MQiao J D Iglehart andA B Pardee ldquoMetastatic potential of21T human breast cancer cells depends on Aktprotein kinase Bactivationrdquo Cancer Research vol 67 no 11 pp 5293ndash5299 2007

[106] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

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[109] P G Rychahou J Kang P Gulhati et al ldquoAkt2 overexpressionplays a critical role in the establishment of colorectal cancermetastasisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 51 pp 20315ndash203202008

[110] M J Arboleda J F Lyons F F Kabbinavar et al ldquoOverexpres-sion of AKT2protein kinase B120573 leads to up-regulation of 1205731integrins increased invasion and metastasis of human breastand ovarian cancer cellsrdquoCancer Research vol 63 no 1 pp 196ndash206 2003

[111] P Pu C Kang J Li and H Jiang ldquoAntisense and dominant-negative AKT2 cDNA inhibits glioma cell invasionrdquo TumorBiology vol 25 no 4 pp 172ndash178 2004

[112] G Sithanandam LW Fornwald J Fields and L M AndersonldquoInactivation of ErbB3 by siRNA promotes apoptosis and atten-uates growth and invasiveness of human lung adenocarcinomacell line A549rdquo Oncogene vol 24 no 11 pp 1847ndash1859 2005

[113] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[114] X Zhang X Li N Zhang Q Yang and M S Moran ldquoLowdoses ionizing radiation enhances the invasiveness of breastcancer cells by inducing epithelial-mesenchymal transitionrdquoBiochemical and Biophysical Research Communications vol 412no 1 pp 188ndash192 2011

[115] Y-C Zhou J-Y Liu J Li et al ldquoIonizing radiation promotesmigration and invasion of cancer cells through transforminggrowth factor-120573-mediated epithelial-mesenchymal transitionrdquoInternational Journal of Radiation Oncology Biology Physics vol81 no 5 pp 1530ndash1537 2011

[116] K L Andarawewa A C Erickson W S Chou et al ldquoIonizingradiation predisposes nonmalignant human mammary epithe-lial cells to undergo transforming growth factor 120573-inducedepithelial to mesenchymal transitionrdquo Cancer Research vol 67no 18 pp 8662ndash8670 2007

[117] A Kawamoto T Yokoe K Tanaka et al ldquoRadiation inducesepithelial-mesenchymal transition in colorectal cancer cellsrdquoOncology Reports vol 27 no 1 pp 51ndash57 2012

[118] S Yan Y Wang Q Yang et al ldquoLow-dose radiation-inducedepithelial-mesenchymal transition through NF-120581B in cervicalcancer cellsrdquo International Journal of Oncology vol 42 no 5pp 1801ndash1806 2013

[119] W Chen S Wu G Zhang W Wang and Y Shi ldquoEffectof AKT inhibition on epithelial-mesenchymal transition andZEB1-potentiated radiotherapy in nasopharyngeal carcinomardquoOncology Letters vol 6 no 5 pp 1234ndash1240 2013

[120] H Y Irie R V Pearline D Grueneberg et al ldquoDistinct rolesof Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transitionrdquo Journal of Cell Biology vol 171 no 6pp 1023ndash1034 2005

[121] H Yao Z-Z Zeng K S Fay et al ldquoRole of 12057251205731integrin up-

regulation in radiation-induced invasion by human pancreaticcancer cellsrdquo Translational Oncology vol 4 no 5 pp 282ndash2922011


[122] V R Gogineni A K Nalla R Gupta et al ldquo12057231205731 integrinpromotes radiation-induced migration of meningioma cellsrdquoInternational Journal of Oncology vol 38 no 6 pp 1615ndash16242011

[123] B Xue W Wu K Huang et al ldquoStromal cell-derived factor-1(SDF-1) enhances cells invasion by 120572v1205736 integrin-mediated sig-naling in ovarian cancerrdquo Molecular and Cellular Biochemistryvol 380 no 1-2 pp 177ndash184 2013

[124] P C Brooks S Stromblad L C Sanders et al ldquoLocalization ofmatrixmetalloproteinaseMMP-2 to the surface of invasive cellsby interaction with integrin 120572v1205733rdquo Cell vol 85 no 5 pp 683ndash693 1996

[125] E I Deryugina B Ratnikov E Monosov et al ldquoMT1-MMPinitiates activation of pro-MMP-2 and integrin 120572v1205733 promotesmaturation of MMP-2 in breast carcinoma cellsrdquo ExperimentalCell Research vol 263 no 2 pp 209ndash223 2001

[126] C Ruegg YMonnier F Kuonen andN Imaizumi ldquoRadiation-induced modifications of the tumor microenvironment pro-mote metastasisrdquo Bulletin du Cancer vol 98 no 6 pp E47ndashE572011

[127] AWilisch-Neumann N Kliese D Pachow et al ldquoThe integrininhibitor cilengitide affects meningioma cell motility and inva-sionrdquo Clinical Cancer Research vol 19 no 19 pp 5402ndash54122013

[128] T J Yeatman ldquoA renaissance for SRCrdquo Nature Reviews Cancervol 4 no 6 pp 470ndash480 2004

[129] D A Hsia S K Mitra C R Hauck et al ldquoDifferential reg-ulation of cell motility and invasion by FAKrdquo Journal of CellBiology vol 160 no 5 pp 753ndash767 2003

[130] P R Purnell P C Mack C G Tepper et al ldquoThe src inhibitorAZD0530 blocks invasion and may act as a radiosensitizer inlung cancer cellsrdquo Journal ofThoracic Oncology vol 4 no 4 pp448ndash454 2009

[131] U Jadhav and S Mohanam ldquoResponse of neuroblastomacells to ionizing radiation modulation of in vitro invasivenessand angiogenesis of human microvascular endothelial cellsrdquoInternational Journal of Oncology vol 29 no 6 pp 1525ndash15312006

[132] L-Y Zhou Z-M Wang Y-B Gao L-Y Wang and Z-CZeng ldquoStimulation of hepatoma cell invasiveness andmetastaticpotential by proteins secreted from irradiated nonparenchy-mal cellsrdquo International Journal of Radiation Oncology BiologyPhysics vol 84 no 3 pp 822ndash828 2012

[133] O Kargiotis C Chetty V Gogineni et al ldquouPAuPAR down-regulation inhibits radiation-induced migration invasion andangiogenesis in IOMM-Lee Meningioma cells and decreasestumor growth in vivordquo International Journal of Oncology vol33 no 5 pp 937ndash947 2008

[134] H L Fillmore T E VanMeter and W C Broaddus ldquoMem-brane-type matrix metalloproteinases (MT-MMPs) expres-sion and function during glioma invasionrdquo Journal of Neuro-Oncology vol 53 no 2 pp 187ndash202 2001

[135] H K Rooprai and D McCormick ldquoProteases and theirinhibitors in human brain tumours a reviewrdquo AnticancerResearch vol 17 no 6 pp 4151ndash4162 1997

[136] C H Chou C M Teng K Y Tzen Y C Chang J H HChen and J C H Cheng ldquoMMP-9 from sublethally irradiatedtumor promotes Lewis lung carcinoma cell invasiveness andpulmonary metastasisrdquo Oncogene vol 31 no 4 pp 458ndash4682012

[137] J Liu W Shen Y Tang et al ldquoProteasome inhibitor MG132enhances the antigrowth and antimetastasis effects of radiationin human nonsmall cell lung cancer cellsrdquo Tumor Biology 2014

[138] B Paquette H Therriault G Desmarais R Wagner RRoyer and R Bujold ldquoRadiation-enhancement of MDA-MB-231 breast cancer cell invasion prevented by a cyclooxygenase-2inhibitorrdquo British Journal of Cancer vol 105 no 4 pp 534ndash5412011

[139] N Burrows M Babur J Resch et al ldquoGDC-0941 inhibits met-astatic characteristics of thyroid carcinomas by targeting boththe phosphoinositide-3 kinase (PI3K) and hypoxia-induciblefactor-1120572 (HIF-1120572) pathwaysrdquo Journal of Clinical Endocrinologyand Metabolism vol 96 no 12 pp E1934ndashE1943 2011

[140] C Chargari C Clemenson I Martins J-L Perfettini and EDeutsch ldquoUnderstanding the functions of tumor stroma inresistance to ionizing radiation emerging targets for pharma-cological modulationrdquo Drug Resistance Updates vol 16 no 1-2pp 10ndash21 2013

[141] Y Du N D Peyser and J R Grandis ldquoIntegration of moleculartargeted therapy with radiation in head and neck cancerrdquoPharmacology andTherapeutics vol 142 no 1 pp 88ndash98 2014

[142] A Suetens M Moreels R Quintens et al ldquoCarbon ionirradiation of the human prostate cancer cell line PC3 a wholegenomemicroarray studyrdquo International Journal of Oncolog vol44 no 4 pp 1056ndash1072 2014

[143] R Hirayama A Uzawa N Takase et al ldquoEvaluation ofSCCVII tumor cell survival in clamped and non-clamped solidtumors exposed to carbon-ion beams in comparison to X-raysrdquoMutation Research vol 756 no 1-2 pp 146ndash151 2013

[144] Y Xu L Yang X Jiang et al ldquoAdenovirus-mediated coex-pression of DCX and SPARC radiosensitizes human malignantglioma cellsrdquo Cellular and Molecular Neurobiology vol 33 no7 pp 965ndash971 2013

[145] C W Song H Lee R P M Dings et al ldquoMetformin kills andradiosensitizes cancer cells and preferentially kills cancer stemcellsrdquo Scientific Reports vol 2 article 362 2012

[146] F E Langlands D Dodwell A M Hanby et al ldquoPSMD9expression predicts radiotherapy response in breast cancerrdquoMolecular Cancer vol 13 no 1 article 73 2014

[147] C Trainor K T Butterworth C K McGarry et al ldquoCellsurvival responses after exposure tomodulated radiation fieldsrdquoRadiation Research vol 177 no 1 pp 44ndash51 2012

[148] A Tesei A Sarnelli C Arienti et al ldquoIn vitro irradiation systemfor radiobiological experimentsrdquoRadiation Oncology vol 8 no1 pp 1ndash11 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


involved in cancer progression after IR is the members of adisintegrin and metalloproteinase (ADAM) family that arethought to mediate the shedding of epidermal growth factorreceptor (EGFR) ligands an important signaling pathwayto cell proliferation and migration and this event is criticalfor a more soluble functional EGFR ligands yield ADAM isactivated by IR leading to an increased triggering cell prolif-eration cascade in irradiated melanoma cells [9] specifyingADAMrsquos blockage as an attractive target to radiosensitizethis tumor type Another important subject is radio-inducedDNA damage and its repair ability that have been largelyinvestigated including the role of tyrosine kinase pathwayssuch as the ataxia-telangiectasia mutated (ATM) a proteinkinase that is best known for its role in the DNA damageresponse [10] and one recent work describes a new functionfor 51015840-adenosine monophosphate- (AMP-) activated proteinkinase (AMPK) an established metabolic stress sensor thathas the ability to control cellular growth and mediate cellcycle checkpoints in cancer cells in response to low energylevels as a sensor of genomic stress and a participant ofthe DNA damage response (DDR) pathway highlighting theimportance of targeting AMPK as novel cancer therapeuticsfor a radiosensitization of human cancer cells mediated bya simultaneous inhibition of the Akt and activation of theAMPK signaling pathways [11ndash13]

The knowledge in tumor biology has been increased inthe last years due to cellular signaling pathways discoveringmechanistically identified by molecular tools that allowedinvestigation of an enormous range of possibilities evenas new mathematical models have also been subsidizingradiotherapy enhancement The understanding and identi-fication of specific molecular targets with significant ther-apeutic implications in order to develop new strategies forradiotherapy are crucial to improve patient survival withoutsubstantially increasing toxicity

2 Radiobiological Models

Delivery of advanced radiotherapy techniques has taken newapproaches to treatment including stereotactic irradiationand intensity-modulated X-rays beam in order to improveoutcomes of cancer treatment and reduce damage to normalsurrounding tissues [14] The classical well-known radiobi-ological models are linear-quadratic (LQ) and biologicallyeffective dose (BED) that are widely used to estimate theeffects of various radiation schedules but it has been sug-gested that LQ is not applicable to high doses per fraction [15]due to the fact that LQoverestimates effects of high daily radi-ation doses proving that better models should be proposed[16] Thus different treatment schedules applying hypofrac-tionated radiotherapy (hRT) and other radiation modalitiessuch as light ions have been used with the same proposaleven though the pattern of received dose is different from thatin conventional radiation and therefore the radiobiologicalaspects of cell death are shown to be modified Prolongedor short radiation delivery makes sublethal damage repairrepopulation and reoxygenation be better evaluated speciallywhen amathematical model is used for dose conversion fromconventional treatment to high daily hypofractionated doses

[17] For instance dose conversion models as repairable-conditionally repairable (RCR) model [18] and multitarget(MT) model are currently recommended When those mod-els were compared LQ seemed to fit relatively well at dosesof 5Gy or less at 6Gy or higher doses RCR and MT modelsseemed to be more reliable than LQ [19] In hypofractionatedstereotactic radiotherapy LQ model should not be used andconversion models incorporating the concept of RCR or MTmodels such as generalized linear-quadratic (gLQ) modelsappear to be more suitable [20 21]

Recent investigations have highlighted differential cellu-lar responses when submitted to intensity-modulated radi-ation fields [22] particularly in areas outside the primarytreatment fields [23] Differential DNA damage responsesfollowingmodulated radiation field delivery were found pro-viding an evidence for a role of intercellular communicationin mediating cellular radiobiological response to modulatedradiation fields suggesting that advanced radiotherapy treat-ment plans require a refinement of existing radiobiologicalmodeling [24] Concerning radiobiology DNAdouble strandbreaks (DSB) are considered to be the kind of DNA damageresponsible for most end points such as chromosome aberra-tions and cell killing However due to high number of DSBsinduced by radiation at sublethal doses it is immediatelyobvious that DSB is not lethal in general indicating thatmost of induced DSBs can be rejoined or repaired correctlydisplaying the spatial distribution of DSBs as major factorin determining lethality [25ndash27] Nevertheless a mechanisticdose-response model has been proposed based on the con-cept of giant loops which constitute a level of chromatinorganization on a megabase pair length scale [28ndash30] thatsuggests DSBs are induced within different loop domainsof DNA assumed to be processed independently by cellularrepair mechanism Given giant loop chromatin organizationand assumption of two damage classes representing themain point Giant LOop Binary LEsion (GLOBLE) approacharises as promising model [31] This model is able to revealimportant features of dose-response curves describing cellsurvival especially transitions from low to high doses in adose-response correlation

The effects of combined modality treatments are investi-gated by using mathematical models to predict cell death asan attempt to fit LQ MT and gLQ models to experimentaldata based on in vitro assays demonstrating that gLQequation is superior to LQ and MT models in predictingcellular death at high doses of radiotherapy [32] A significantincreasing in biologically equivalent dose may be achievedafter addition of radiosensitizing agents to hRT as wellas linear accelerators containing new technologies such asflattening filter free (FFF) increases instantaneous dose-rateof X-ray pulses by a factor of 2ndash6 compared to conventionalflattened output [33]

New models for radiobiological cell responses have beenproposed and one of those is a simple two-parameteralgorithmic model which captures the essential biologicalfeatures of irradiation-induced cell death and associated cellcycle delays This approach estimates directly the underlyingirradiation-induced cell survival and was investigated inmammary carcinoma cell line EMT6Rowhere a comparison








1and G

2






1and












targets DNA targets


SphK1Akt

PGE2MMP-2 DNA-PKcs

Head and neck

SQ20BFaDuA431


PI3KmTOREGFR

ADAM-17nuclear Ca2+



Bladder


253J-BV253-JP

mTORcyclin D1

p27p21

LungA549H3255Calu-6

EGFRE-TKIPI3KAkt

Akt2TGF120573

12057221205731Src

COX-2MMP-9

VEGFRTGF-1205731

PARP-1HDAC


EGFRPI3KmTOR

Akt2TGF120573



p53


LiverHepG2

McA-RH77KM20

nuclear Ca2+PI3KAkt

MMP-2MMP-9 VEGF



















120573R inhibitor














3





NR

Cytosol

RTK

ADAM-17EGFRligands

Soluble EGFR

Plasma membrane

PI3k

Akt

Ras

Raf

Mek

Erk

VEGFR

MAPK

MMP

Akt

p

p

p p

PI3k

p p

p

p

p

mTOR

mTOR

HIF-1120572

Cell proliferation

Cell survival

Angiogenesis

PARP

Cyclins

DNA-PKc

Integrins

Src

Migration

Apoptosis

Bcl-2

Caspases

VEGF


DNA damage

MAPK

IR

Nucleus

IP3RCa2+


3R

















Acknowledgments


References




































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























1and G

2






1and












targets DNA targets


SphK1Akt

PGE2MMP-2 DNA-PKcs

Head and neck

SQ20BFaDuA431


PI3KmTOREGFR

ADAM-17nuclear Ca2+



Bladder


253J-BV253-JP

mTORcyclin D1

p27p21

LungA549H3255Calu-6

EGFRE-TKIPI3KAkt

Akt2TGF120573

12057221205731Src

COX-2MMP-9

VEGFRTGF-1205731

PARP-1HDAC


EGFRPI3KmTOR

Akt2TGF120573



p53


LiverHepG2

McA-RH77KM20

nuclear Ca2+PI3KAkt

MMP-2MMP-9 VEGF



















120573R inhibitor














3





NR

Cytosol

RTK

ADAM-17EGFRligands

Soluble EGFR

Plasma membrane

PI3k

Akt

Ras

Raf

Mek

Erk

VEGFR

MAPK

MMP

Akt

p

p

p p

PI3k

p p

p

p

p

mTOR

mTOR

HIF-1120572

Cell proliferation

Cell survival

Angiogenesis

PARP

Cyclins

DNA-PKc

Integrins

Src

Migration

Apoptosis

Bcl-2

Caspases

VEGF


DNA damage

MAPK

IR

Nucleus

IP3RCa2+


3R

















Acknowledgments


References




































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















1and












targets DNA targets


SphK1Akt

PGE2MMP-2 DNA-PKcs

Head and neck

SQ20BFaDuA431


PI3KmTOREGFR

ADAM-17nuclear Ca2+



Bladder


253J-BV253-JP

mTORcyclin D1

p27p21

LungA549H3255Calu-6

EGFRE-TKIPI3KAkt

Akt2TGF120573

12057221205731Src

COX-2MMP-9

VEGFRTGF-1205731

PARP-1HDAC


EGFRPI3KmTOR

Akt2TGF120573



p53


LiverHepG2

McA-RH77KM20

nuclear Ca2+PI3KAkt

MMP-2MMP-9 VEGF



















120573R inhibitor














3





NR

Cytosol

RTK

ADAM-17EGFRligands

Soluble EGFR

Plasma membrane

PI3k

Akt

Ras

Raf

Mek

Erk

VEGFR

MAPK

MMP

Akt

p

p

p p

PI3k

p p

p

p

p

mTOR

mTOR

HIF-1120572

Cell proliferation

Cell survival

Angiogenesis

PARP

Cyclins

DNA-PKc

Integrins

Src

Migration

Apoptosis

Bcl-2

Caspases

VEGF


DNA damage

MAPK

IR

Nucleus

IP3RCa2+


3R

















Acknowledgments


References




































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















targets DNA targets


SphK1Akt

PGE2MMP-2 DNA-PKcs

Head and neck

SQ20BFaDuA431


PI3KmTOREGFR

ADAM-17nuclear Ca2+



Bladder


253J-BV253-JP

mTORcyclin D1

p27p21

LungA549H3255Calu-6

EGFRE-TKIPI3KAkt

Akt2TGF120573

12057221205731Src

COX-2MMP-9

VEGFRTGF-1205731

PARP-1HDAC


EGFRPI3KmTOR

Akt2TGF120573



p53


LiverHepG2

McA-RH77KM20

nuclear Ca2+PI3KAkt

MMP-2MMP-9 VEGF



















120573R inhibitor














3





NR

Cytosol

RTK

ADAM-17EGFRligands

Soluble EGFR

Plasma membrane

PI3k

Akt

Ras

Raf

Mek

Erk

VEGFR

MAPK

MMP

Akt

p

p

p p

PI3k

p p

p

p

p

mTOR

mTOR

HIF-1120572

Cell proliferation

Cell survival

Angiogenesis

PARP

Cyclins

DNA-PKc

Integrins

Src

Migration

Apoptosis

Bcl-2

Caspases

VEGF


DNA damage

MAPK

IR

Nucleus

IP3RCa2+


3R

















Acknowledgments


References




































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































120573R inhibitor














3





NR

Cytosol

RTK

ADAM-17EGFRligands

Soluble EGFR

Plasma membrane

PI3k

Akt

Ras

Raf

Mek

Erk

VEGFR

MAPK

MMP

Akt

p

p

p p

PI3k

p p

p

p

p

mTOR

mTOR

HIF-1120572

Cell proliferation

Cell survival

Angiogenesis

PARP

Cyclins

DNA-PKc

Integrins

Src

Migration

Apoptosis

Bcl-2

Caspases

VEGF


DNA damage

MAPK

IR

Nucleus

IP3RCa2+


3R

















Acknowledgments


References




































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NR

Cytosol

RTK

ADAM-17EGFRligands

Soluble EGFR

Plasma membrane

PI3k

Akt

Ras

Raf

Mek

Erk

VEGFR

MAPK

MMP

Akt

p

p

p p

PI3k

p p

p

p

p

mTOR

mTOR

HIF-1120572

Cell proliferation

Cell survival

Angiogenesis

PARP

Cyclins

DNA-PKc

Integrins

Src

Migration

Apoptosis

Bcl-2

Caspases

VEGF


DNA damage

MAPK

IR

Nucleus

IP3RCa2+


3R

















Acknowledgments


References




































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Acknowledgments


References




































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article radiation oncology in vitro : trends...

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