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M. Elas 1, S. Kędracka-Krok 1, U. Jankowska 1, Ł. Skalniak 1, J. Jura 1, E.Zuba-Surma 1, K. Jasińska 1, A. Pawlak 1, U. Sowa 2, P. Olko 2, B. Romanowska-Dixon.

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Presentation on theme: "M. Elas 1, S. Kędracka-Krok 1, U. Jankowska 1, Ł. Skalniak 1, J. Jura 1, E.Zuba-Surma 1, K. Jasińska 1, A. Pawlak 1, U. Sowa 2, P. Olko 2, B. Romanowska-Dixon."— Presentation transcript:

1 M. Elas 1, S. Kędracka-Krok 1, U. Jankowska 1, Ł. Skalniak 1, J. Jura 1, E.Zuba-Surma 1, K. Jasińska 1, A. Pawlak 1, U. Sowa 2, P. Olko 2, B. Romanowska-Dixon 3, K. Urbańska 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

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, Kraków LET <20 keV/µm dose rate 0.15 Gy/s Methods

3 Dose-dependent slowing down of the proliferation rate 5 & 7 Gy lethal damage 1-3 Gy repair

4 G0&G1/G2&M Increase in G2/M >2n present Cell cycle redistribution: Increase in G2/M with dose, day 6

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

6 increase in caspases 3 and 7 activity with time Caspases activity increases with time day 5

7 Apoptotic Cells Healthy Cells S91 S91 – day 9-10 S91 – day 5 3 Gy

8 Proteomics 2DE and mass spectroscopy to identify proteins influenced by 3 Gy proton beam irradiation ctrl3 Gy Kędracka-Krok et al., Plos One, 2014

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 Kędracka-Krok et al., Plos One, 2014

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 Kędracka-Krok et al., Plos One, 2014

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 Kędracka-Krok et al., Plos One, 2014

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 Kędracka-Krok et al., Plos One, 2014

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 Kędracka-Krok et al., Plos One, 2014

14 Proteomics 4 groups: » DNA repair & mRNA regulation » Survival and apoptosis » Glycolytic metabolism » Cytoskeleton and migration Signaling pathways: p53 TGFβ PTEN AKT BAX, Bcl-2 Kędracka-Krok et al., Plos One, 2014

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 αVβ3, β1 integrin, and MMP-2 (Takahashi, 2003)

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

17 Blm, speed Migration properties of melanoma cells after protonotherapy Omm1.3, speed Blm, distance Omm 1.3, distance

18 Inhibition of metastases of eye-implanted BHM melanoma 10 Gy Romanowska et al., ABP, 2013

19 Inhibition of metastases of eye-implanted BHM melanoma Time [days] Rel mean diameter [mm]

20 WYNIKI Inhibition of metastases of eye-implanted BHM melanoma x 4.4 p= β-irradiation Romanowska et al., ABP, 2013

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

22 Obrazowanie mysich guzów: mechanizm odpowiedzi na radio- i fototerapię MRI, angiografia ToFMRI, perfuzja ASL

23 Dept of Ophthalmology and Ophthalmic Oncology, Jagiellonian University Medical College Bożena Romanowska-Dixon Institute of Nuclear Physics, PAS Paweł Olko Jan Swakoń Urszula Sowa Marta Ptaszkiewicz Dept of General Biochemistry Jolanta Jura Łukasz Skalniak Agnieszka Cierniak Dept Cell Biology Ewa Zuba-Surma Marta Michalik Dept Biophysics Krystyna Urbańska Katarzyna Jasińska Małgorzata Szczygieł Martyna Krzykawska-Serda Michał Gonet Agnieszka Drzał Dept Physical Biochemistry Sylwia Kędracka-Krok Urszula Jankowska

24 ODLEGŁE PRZERZUTY CZERNIAKA Przerzuty do płuc są inicjowane u części zwierząt (37%) gdy guz pierwotny zajmuje zaledwie 0,10 powierzchni PK (już po 2 dniach wzrostu guza)

25 WCZESNE I PÓŹNE EFEKTY RADIOTERAPII Całkowitej regresji guza po 10 Gy 125 I (4,2,2,2) towarzyszy brak przerzutów w płucach u 50% leczonych chomików nawet po 70 dniach od enukleacji Średnica guza (mm) Czas (dni) 29 –34 dni po ENU 100% zwierząt 70 dni po ENU 50 % zwierząt (Sawow Aneta, 2001)


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