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DNA damage, protein expression and migration of melanoma cells irradiated with proton beam M. Elas1, S. Kdracka-Krok1, U. Jankowska1, . Skalniak1, J. Jura1, E.Zuba-Surma1, K. Jasiska1, A. Pawlak1, U. Sowa2, P. Olko2, B. Romanowska-Dixon3, K. Urbaska1 1Faculty of Biochemistry, Biophysics and Biotechnology, JU2Institute of Nuclear Physics, PAS 3Department of Ophthalmology and Ophthalmic Oncology, Jagiellonian University Medical CollegeBLM cells irradiated with 1-7 Gy of proton beam

58 MeV proton beam AIC-144 cyclotron at Institute of Nuclear Physics, Polish Academy of Sciences, Krakw

LET 2n presentCell cycle redistribution: Increase in G2/M with dose, day 6DNA content of melanoma cells following proton irradiation. Panel A shows the flow cytometric protocol for analysis of DNA content in melanoma cells following staining with propidium iodide. Each histogram represents cells from indicated experimental group (control cell as well as cells irradiated with proton beams in doses of 1, 2 and 3Gy at day .???). The gates marked on each histogram enclose cells in three major phases of cell cycle (G0/G1, S and G2/M) as well as polyploids (>2n) with multiplied number of chromosomes. Panel B represents quantitative analysis of the histograms and shows the percentages of cell with different DNA content corresponding to distinct phases of cell cycle. The values represent average data from three experiments (N=3) plotted as Mean SE. Panel C shows average Q/P index of indicated experimental groups which was computed as a ratio of percent content of quiescence cells (G0/G1 phase) and cells with increased number of DNA resembling proliferating cells (G2/M phase). The values represent average data from three experiments (N=3) plotted as Mean SE.4

dose-dependent DNA damage

2 days after irradiation: no difference 5 days after: 11% more for 3 Gy, 14% for 5 Gy, 17% for 7 Gy=> delayed DNA damage

DNA damageNot all lethality was due to direct effect of proton irradiation, some damage was incurred later5

increase in caspases 3 and 7 activity with timeCaspases activity increases with time

day 5Caspase 3 and 7 activity in blm cells treated with indicated proton dose. Two days following irradiation whole cell lysates from cells were prepared and subjected to chemiluminescence-based caspase activity assay. Graph presents mean SD from 3 independent experiments 6

Apoptotic CellsHealthy CellsS91S91 day 9-10S91 day 53 Gy3 GyProteomics2DE and mass spectroscopy to identify proteins influenced by 3 Gy proton beam irradiation

ctrl3 GyKdracka-Krok et al., Plos One, 2014> 1.5 xVCP MVP STRAPFAB-2 Lamine A/CGAPDH

P-bodyPDCD6

StressgranuleCaprin-1

STRAPMCM7Annexin 7 MVP Caprin-1 PDCD6 VCP HSP70TIM, GAPDH VCPMoesinActinin 4FAB-2VimentinAnnexin 7Lamine A/CLamine B NucleusMitochodriumCytoplasmupregulateddownregulated13 up, 4 downKdracka-Krok et al., Plos One, 20149VCP MVP STRAPFAB-2 Lamine A/CGAPDH

P-bodyPDCD6

StressgranuleCaprin-1

STRAPMCM7Annexin 7 MVP Caprin-1 PDCD6 VCP TIM, GAPDH VCPMoesinActinin 4FAB-2VimentinAnnexin 7Lamine A/CLamine B NucleusMitochodriumCytoplasmMVP (1.9 ), Lamine A/C (1.8 ), GAPDH (2.4 ) DNA repairVCP (1.9 ) chromatin associated degradationMVP PTEN translocation regulationMVP, VCP transcription regulationSTRAP (4.1 ) , FAB-2 (1.9 ) mRNA regulationCaprin-1 (1.9), PDCD6 (1.5 ) mRNA regulationHSP70 & G3BP1 (1.8 ) stress & mRNA stability and stress granule formationDNA repair, RNA regulation Kdracka-Krok et al., Plos One, 2014VCP MVP STRAPFAB-2 Lamine A/CGAPDH

P-bodyPDCD6

StressgranuleCaprin-1

STRAPMCM7Annexin 7 MVP Caprin-1 PDCD6 VCP TIM, GAPDH VCPMoesinActinin 4FAB-2VimentinAnnexin 7Lamine A/CLamine B NucleusMitochodriumCytoplasmSTRAP (4.1 ) survival - apoptosis balanceMCM7 (1.6 ) proliferation balanceAnnexin 7 (2.5 ) proliferation inhibitionMVP survival enhancementCaprin-1 (1.9) proliferationPDCD6 (1.5 ) apoptosis, ubiquitination regulationVCP (1.9 ) aggregates management, lysosomal degradationCell survival Kdracka-Krok et al., Plos One, 2014VCP MVP STRAPFAB-2 Lamine A/CGAPDH

P-bodyPDCD6

StressgranuleCaprin-1

STRAPMCM7Annexin 7 MVP Caprin-1 PDCD6 VCP TIM, GAPDH VCPMoesinActinin 4FAB-2VimentinAnnexin 7Lamine A/CLamine B NucleusMitochodriumCytoplasmTIM (2.4 ), GAPDH (2.4 ) glycolysisVCP (1.9 ) mitochondria associated degradationCell metabolism Kdracka-Krok et al., Plos One, 2014VCP MVP STRAPFAB-2 Lamine A/CGAPDH

P-bodyPDCD6

StressgranuleCaprin-1

STRAPMCM7Annexin 7 MVP Caprin-1 PDCD6 VCP TIM, GAPDH VCPMoesinActinin 4Fab-2VimentinAnnexin 7Lamine A/CLamine B NucleusMitochodriumCytoplasmCytoskeleton and motility Moesin (2.4 ) actin remodelling, motilityActinin 4 (1.9 ) migration and metastasisFAB-2 (1.9 ) migration, microtubule destabilizerVimentin (2.0, 2.1, 3.4, 1.6 ) metastasis, EMT markerAnnexin 7 (2.5 ) motilityLamine A/C (1.6, 2,4, 1,5 ) PI3K/AKT/PTEN, adhesion, motilityLamine B (1.4 ) - motility

Kdracka-Krok et al., Plos One, 2014Proteomics4 groups: DNA repair & mRNA regulationSurvival and apoptosisGlycolytic metabolismCytoskeleton and migrationSignaling pathways:p53TGFPTENAKTBAX, Bcl-2Kdracka-Krok et al., Plos One, 2014Interaction with tumor microenvironment Heavy ions shown to inhibit metastasis (Takahashi, 2003; Tsuboi, 2005)

After proton beam irradiation:

strongly inhibited matrix metaloproteinase-2 activity in highly aggressive HT1080 human fibrosarcoma cells in vitro, and significantly decreased the number of pulmonary metastasis in mouse osteosarcoma in vivo (Ogata et al. 2005).

the expression level or activity of molecules related to metastasis such as V3, 1 integrin, and MMP-2 (Takahashi, 2003)

Real-time PCR analysis using human angiogenesis TaqMan Array Plates

Blm cells, 3 Gy , 48 hr culture

Genes with 1.5 change vs control shown

Angiogenesis gene activation Real-time PCR analysis result with the use of human angiogenesis TaqMan Array Plates (LifeTechnologies). Blm cells were irradiated with 3 Gy of proton beam and cultured for two days. Total RNA was isolated and subjected to cDNA synthesis with the oligo-dT primer. Results shown are means from two independent experiments. Listed are only the genes, for which at least 1.5 change versus control (untreated cells) was observed.

16Blm, speed Migration properties of melanoma cells after protonotherapy

Omm1.3, speed

Blm, distance

Omm 1.3, distance Inhibition of metastases of eye-implanted BHM melanoma

10 GyRomanowska et al., ABP, 2013Inhibition of metastases of eye-implanted BHM melanoma

Time [days]Rel mean diameter [mm]

WYNIKIInhibition of metastases of eye-implanted BHM melanoma

x 4.4

p=0.0052

-irradiationRomanowska et al., ABP, 20131-3 Gy sublethal, 5-7 Gy lethal damage of BLM cellsDelayed DNA damage, leading to apoptosis, resulting from endogenous ROS generation due to cell signallingUpregulation of proteins involved in DNA repair and stress, apoptosis and survival, glycolytic metabolism and migration and cytoskeletonIn the latter group, vimentin was heavily suppressed, together with Annexin 7, whereas expression of Moesin 7, Lamins A/C and B, Actinin 4 and FAB-2 were increased Interaction with tumor microenvironment: Upregulation of angiogenic genes, in vivo inhibition of metastases in BHM eye model Conclusions

Obrazowanie mysich guzw: mechanizm odpowiedzi na radio- i fototerapi

MRI, angiografia ToF

MRI, perfuzja ASL

Dept of Ophthalmology and Ophthalmic Oncology, Jagiellonian University Medical College

Boena Romanowska-DixonInstitute of Nuclear Physics, PASPawe Olko Jan SwakoUrszula Sowa Marta PtaszkiewiczDept of General BiochemistryJolanta Juraukasz SkalniakAgnieszka CierniakDept Cell BiologyEwa Zuba-SurmaMarta MichalikDept BiophysicsKrystyna UrbaskaKatarzyna JasiskaMagorzata SzczygieMartyna Krzykawska-SerdaMicha GonetAgnieszka DrzaDept Physical BiochemistrySylwia Kdracka-Krok Urszula Jankowska23ODLEGE PRZERZUTY CZERNIAKAPrzerzuty do puc s inicjowane u czci zwierzt (37%) gdy guz pierwotny zajmuje zaledwie 0,10 powierzchni PK (ju po 2 dniach wzrostu guza)

WCZESNE I PNE EFEKTY RADIOTERAPIICakowitej regresji guza po 10 Gy 125I (4,2,2,2) towarzyszy brak przerzutw w pucach u 50% leczonych chomikw nawet po 70 dniach od enukleacji

rednica guza (mm)Czas (dni)

29 34 dni po ENU100% zwierzt70 dni po ENU50 % zwierzt (Sawow Aneta, 2001)Gene categoryGroupGene symbolGene nameMean change3 Gy vs. control

Extracellular matrixExtracellular matrix glycoproteinTHBS2thrombospondin 2 2.50

HSPG2heparan sulfate proteoglycan 2 1.88

Extracellular matrix linker proteinFN1fibronectin 1 1.83

ReceptorOther receptorNRP1neuropilin 1 1.50

Protein kinase receptorKDRkinase insert domain receptor (a type III receptor tyrosine kinase) 5.68

TIE1tyrosine kinase with immunoglobulin-like and EGF-like domains 1 2.97

PDGFRAplatelet-derived growth factor receptor, alpha polypeptide 1.85

ReceptorCD44CD44 molecule (Indian blood group) 1.69

Select regulatory moleculeProtease inhibitorSERPINF1serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1 2.49

SERPINC1serpin peptidase inhibitor, clade C (antithrombin), member 1 1.93

Signaling moleculeCytokineIL12Ainterleukin 12A (natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1, p35) 1.56

Growth factorVEGFBvascular endothelial growth factor B 1.64

Other signaling moleculeANGPTL2angiopoietin-like 2 2.89

Cell adhesion moleculeOther cell adhesion moleculeEDIL3EGF-like repeats and discoidin I-like domains 3 0.48