M. Elas 1, S. Kdracka-Krok 1, U. Jankowska 1, . Skalniak 1, J.
Jura 1, E.Zuba-Surma 1, K. Jasiska 1, A. Pawlak 1, U. Sowa 2, P.
Olko 2, B. Romanowska-Dixon 3, K. Urbaska 1 1 Faculty of
Biochemistry, Biophysics and Biotechnology, JU 2 Institute of
Nuclear Physics, PAS 3 Department of Ophthalmology and Ophthalmic
Oncology, Jagiellonian University Medical College Slide 2 BLM 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 LETSlide 3 Dose-dependent slowing down of the
proliferation rate 5 & 7 Gy lethal damage 1-3 Gy repair Slide 4
G0&G1/G2&M Increase in G2/M >2n present Cell cycle
redistribution: Increase in G2/M with dose, day 6 Slide 5
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 damage Slide 6 increase in caspases 3 and 7
activity with time Caspases activity increases with time day 5
Slide 7 Apoptotic Cells Healthy Cells S91 S91 day 9-10 S91 day 5 3
Gy Slide 8 Proteomics 2DE and mass spectroscopy to identify
proteins influenced by 3 Gy proton beam irradiation ctrl3 Gy
Kdracka-Krok et al., Plos One, 2014 Slide 9 > 1.5 x VCP MVP
STRAP FAB-2 Lamine A/C GAPDH P-body PDCD6 Stress granule Caprin-1
STRAP MCM7 Annexin 7 MVP Caprin-1 PDCD6 VCP HSP70 TIM, GAPDH VCP
Moesin Actinin 4 FAB-2 Vimentin Annexin 7 Lamine A/C Lamine B
Nucleus Mitochodrium Cytoplasm upregulated downregulated 13 up, 4
down Kdracka-Krok et al., Plos One, 2014 Slide 10 VCP MVP STRAP
FAB-2 Lamine A/C GAPDH P-body PDCD6 Stress granule Caprin-1 STRAP
MCM7 Annexin 7 MVP Caprin-1 PDCD6 VCP TIM, GAPDH VCP Moesin Actinin
4 FAB-2 Vimentin Annexin 7 Lamine A/C Lamine B Nucleus Mitochodrium
Cytoplasm MVP (1.9 ), Lamine A/C (1.8 ), GAPDH (2.4 ) DNA repair
VCP (1.9 ) chromatin associated degradation MVP PTEN translocation
regulation MVP, VCP transcription regulation STRAP (4.1 ), FAB-2
(1.9 ) mRNA regulation Caprin-1 (1.9 ), PDCD6 (1.5 ) mRNA
regulation HSP70 & G3BP1 (1.8 ) stress & mRNA stability and
stress granule formation DNA repair, RNA regulation Kdracka-Krok et
al., Plos One, 2014 Slide 11 VCP MVP STRAP FAB-2 Lamine A/C GAPDH
P-body PDCD6 Stress granule Caprin-1 STRAP MCM7 Annexin 7 MVP
Caprin-1 PDCD6 VCP TIM, GAPDH VCP Moesin Actinin 4 FAB-2 Vimentin
Annexin 7 Lamine A/C Lamine B Nucleus Mitochodrium Cytoplasm STRAP
(4.1 ) survival - apoptosis balance MCM7 (1.6 ) proliferation
balance Annexin 7 (2.5 ) proliferation inhibition MVP survival
enhancement Caprin-1 (1.9 ) proliferation PDCD6 (1.5 ) apoptosis,
ubiquitination regulation VCP (1.9 ) aggregates management,
lysosomal degradation Cell survival Kdracka-Krok et al., Plos One,
2014 Slide 12 VCP MVP STRAP FAB-2 Lamine A/C GAPDH P-body PDCD6
Stress granule Caprin-1 STRAP MCM7 Annexin 7 MVP Caprin-1 PDCD6 VCP
TIM, GAPDH VCP Moesin Actinin 4 FAB-2 Vimentin Annexin 7 Lamine A/C
Lamine B Nucleus Mitochodrium Cytoplasm TIM (2.4 ), GAPDH (2.4 )
glycolysis VCP (1.9 ) mitochondria associated degradation Cell
metabolism Kdracka-Krok et al., Plos One, 2014 Slide 13 VCP MVP
STRAP FAB-2 Lamine A/C GAPDH P-body PDCD6 Stress granule Caprin-1
STRAP MCM7 Annexin 7 MVP Caprin-1 PDCD6 VCP TIM, GAPDH VCP Moesin
Actinin 4 Fab-2 Vimentin Annexin 7 Lamine A/C Lamine B Nucleus
Mitochodrium Cytoplasm Cytoskeleton and motility Moesin (2.4 )
actin remodelling, motility Actinin 4 (1.9 ) migration and
metastasis FAB-2 (1.9 ) migration, microtubule destabilizer
Vimentin (2.0, 2.1, 3.4, 1.6 ) metastasis, EMT marker Annexin 7
(2.5 ) motility Lamine A/C (1.6, 2,4, 1,5 ) PI3K/AKT/PTEN,
adhesion, motility Lamine B (1.4 ) - motility Kdracka-Krok et al.,
Plos One, 2014 Slide 14 Proteomics 4 groups: DNA repair & mRNA
regulation Survival and apoptosis Glycolytic metabolism
Cytoskeleton and migration Signaling pathways: p53 TGF PTEN AKT
BAX, Bcl-2 Kdracka-Krok et al., Plos One, 2014 Slide 15 Interaction
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) Slide 16 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 Slide 17 Blm, speed Migration properties of melanoma
cells after protonotherapy Omm1.3, speed Blm, distance Omm 1.3,
distance Slide 18 Inhibition of metastases of eye-implanted BHM
melanoma 10 Gy Romanowska et al., ABP, 2013 Slide 19 Inhibition of
metastases of eye-implanted BHM melanoma Time [days] Rel mean
diameter [mm] Slide 20 WYNIKI Inhibition of metastases of
eye-implanted BHM melanoma x 4.4 p=0.0052 -irradiation Romanowska
et al., ABP, 2013 Slide 21 1-3 Gy sublethal, 5-7 Gy lethal damage
of BLM cells Delayed DNA damage, leading to apoptosis, resulting
from endogenous ROS generation due to cell signalling Upregulation
of proteins involved in DNA repair and stress, apoptosis and
survival, glycolytic metabolism and migration and cytoskeleton In
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 Slide 22
Obrazowanie mysich guzw: mechanizm odpowiedzi na radio- i
fototerapi MRI, angiografia ToFMRI, perfuzja ASL Slide 23 Dept of
Ophthalmology and Ophthalmic Oncology, Jagiellonian University
Medical College Boena Romanowska-Dixon Institute of Nuclear
Physics, PAS Pawe Olko Jan Swako Urszula Sowa Marta Ptaszkiewicz
Dept of General Biochemistry Jolanta Jura ukasz Skalniak Agnieszka
Cierniak Dept Cell Biology Ewa Zuba-Surma Marta Michalik Dept
Biophysics Krystyna Urbaska Katarzyna Jasiska Magorzata Szczygie
Martyna Krzykawska-Serda Micha Gonet Agnieszka Drza Dept Physical
Biochemistry Sylwia Kdracka-Krok Urszula Jankowska Slide 24 ODLEGE
PRZERZUTY CZERNIAKA Przerzuty do puc s inicjowane u czci zwierzt
(37%) gdy guz pierwotny zajmuje zaledwie 0,10 powierzchni PK (ju po
2 dniach wzrostu guza) Slide 25 WCZESNE I PNE EFEKTY RADIOTERAPII
Cakowitej regresji guza po 10 Gy 125 I (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 ENU 100%
zwierzt 70 dni po ENU 50 % zwierzt (Sawow Aneta, 2001)
