Dna damage protein expression and migration of melanoma cells irradiated with proton beam
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DNA damage, protein expression and migration of melanoma cells irradiated with proton beam. 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

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DNA damage, protein expression and migration of melanoma cells irradiated with proton beam

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Dna damage protein expression and migration of melanoma cells irradiated with proton beam

DNA damage, protein expression and migration of melanoma cells irradiated with proton beam

M. Elas1, S. Kędracka-Krok1, U. Jankowska1, Ł. Skalniak1, J. Jura1, E.Zuba-Surma1, K. Jasińska1, A. Pawlak1, U. Sowa2,

P. Olko2, B. Romanowska-Dixon3, K. Urbańska1

1Faculty of Biochemistry, Biophysics and Biotechnology, JU

2Institute of Nuclear Physics, PAS

3Department of Ophthalmology and Ophthalmic Oncology,

Jagiellonian University Medical College


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

Methods

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


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

Dose-dependent slowing down of the proliferationrate

5 & 7 Gy lethal damage

1-3 Gy repair


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

Cell cycleredistribution: Increasein G2/M with dose, day 6

G0&G1/G2&M

Increase in G2/M

>2n present


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

DNA damage

dose-dependent DNA damage

2 daysafterirradiation: no difference

5 daysafter: 11% more for 3 Gy, 14% for 5 Gy, 17% for 7 Gy

=>delayedDNA damage


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

Caspases activity increases with time

day 5

increase in caspases 3 and 7 activity with time


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

S91

S91 – day 9-10

S91 – day 5

3 Gy

3 Gy

Apoptotic Cells

Healthy Cells


Proteomics

Proteomics

  • 2DE and mass spectroscopy to identifyproteinsinfluenced by 3 Gy proton beamirradiation

ctrl

3 Gy

Kędracka-Krok et al., Plos One, 2014


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

upregulated

downregulated

13 up, 4 down

> 1.5 x

Nucleus

VCP

MVP

STRAP

FAB-2

Lamine A/C

GAPDH

Moesin

Actinin 4

FAB-2

Vimentin

Annexin 7

Lamine A/C

Lamine B

Cytoplasm

STRAP

MCM7

Annexin 7

MVP

Caprin-1

PDCD6

VCP

HSP70

P-body

PDCD6

Caprin-1

TIM, GAPDH

VCP

Stress

granule

Mitochodrium

Kędracka-Krok et al., Plos One, 2014


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

DNA repair, RNA regulation

VCP

MVP

STRAP

FAB-2

Lamine A/C

GAPDH

Nucleus

Moesin

Actinin 4

FAB-2

Vimentin

Annexin 7

Lamine A/C

Lamine B

Cytoplasm

STRAP

MCM7

Annexin 7

MVP

Caprin-1

PDCD6

VCP

MVP (1.9 ), LamineA/C (1.8 ), GAPDH(2.4 ) – DNA repair

VCP (1.9 ) – chromatinassociateddegradation

MVP – PTEN translocationregulation

MVP, VCP – transcriptionregulation

STRAP (4.1 ) , FAB-2(1.9 ) – mRNAregulation

Caprin-1 (1.9), PDCD6 (1.5 )– mRNAregulation

HSP70 & G3BP1 (1.8 ) – stress & mRNA stability and stress granule formation

PDCD6

P-body

Caprin-1

TIM, GAPDH

VCP

Stress

granule

Mitochodrium

Kędracka-Krok et al., Plos One, 2014


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

Cell survival

VCP

MVP

STRAP

FAB-2

Lamine A/C

GAPDH

Nucleus

Moesin

Actinin 4

FAB-2

Vimentin

Annexin 7

Lamine A/C

Lamine B

Cytoplasm

STRAP

MCM7

Annexin 7

MVP

Caprin-1

PDCD6

VCP

STRAP (4.1 ) – survival - apoptosisbalance

MCM7 (1.6 )– proliferationbalance

Annexin 7(2.5 ) – proliferationinhibition

MVP – survivalenhancement

Caprin-1 (1.9) – proliferation

PDCD6 (1.5 )– apoptosis, ubiquitinationregulation

VCP (1.9 ) – aggregatesmanagement, lysosomaldegradation

PDCD6

P-body

Caprin-1

TIM, GAPDH

VCP

Stress

granule

Mitochodrium

Kędracka-Krok et al., Plos One, 2014


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

Cell metabolism

VCP

MVP

STRAP

FAB-2

Lamine A/C

GAPDH

Nucleus

Moesin

Actinin 4

FAB-2

Vimentin

Annexin 7

Lamine A/C

Lamine B

Cytoplasm

STRAP

MCM7

Annexin 7

MVP

Caprin-1

PDCD6

VCP

TIM (2.4 ), GAPDH(2.4 ) – glycolysis

VCP(1.9 ) – mitochondria associateddegradation

PDCD6

P-body

Caprin-1

TIM, GAPDH

VCP

Stress

granule

Mitochodrium

Kędracka-Krok et al., Plos One, 2014


Dna damage protein expression and migration of melanoma cells irradiated with proton beam

Cytoskeleton and motility

VCP

MVP

STRAP

FAB-2

Lamine A/C

GAPDH

Nucleus

Moesin

Actinin 4

Fab-2

Vimentin

Annexin 7

Lamine A/C

Lamine B

Cytoplasm

STRAP

MCM7

Annexin 7

MVP

Caprin-1

PDCD6

VCP

Moesin(2.4 ) – actinremodelling, motility

Actinin 4 (1.9 ) – migration and metastasis

FAB-2 (1.9 ) – migration, microtubuledestabilizer

Vimentin (2.0, 2.1, 3.4, 1.6 ) –metastasis, EMT marker

Annexin 7(2.5 ) – motility

LamineA/C (1.6, 2,4, 1,5 ) – PI3K/AKT/PTEN, adhesion, motility

Lamine B (1.4 ) - motility

PDCD6

P-body

Caprin-1

TIM, GAPDH

VCP

Stress

granule

Mitochodrium

Kędracka-Krok et al., Plos One, 2014


Proteomics1

Proteomics

  • 4 groups:

    • DNA repair & mRNAregulation

    • Survival and apoptosis

    • Glycolyticmetabolism

    • Cytoskeleton and migration

  • Signalingpathways:

    • p53

    • TGFβ

    • PTEN

    • AKT

    • BAX, Bcl-2

  • Kędracka-Krok et al., Plos One, 2014


    Dna damage protein expression and migration of melanoma cells irradiated with proton beam

    Interaction with tumor microenvironment

    • Heavy ionsshown to inhibitmetastasis (Takahashi, 2003; Tsuboi, 2005)

    • After proton beamirradiation:

    • strongly inhibited matrix metaloproteinase-2 activity in highly aggressive HT1080 human fibrosarcomacells 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)


    Dna damage protein expression and migration of melanoma cells irradiated with proton beam

    Angiogenesisgeneactivation

    Real-time PCR analysis usinghuman angiogenesis TaqMan® Array Plates

    Blm cells, 3 Gy, 48 hrculture

    Genes with 1.5 change vs controlshown


    Dna damage protein expression and migration of melanoma cells irradiated with proton beam

    Migration properties of melanoma cellsafterprotonotherapy

    Blm, speed

    Omm1.3, speed

    Omm 1.3, distance

    Blm, distance


    Inhibition of metastases of eye implanted bhm melanoma

    Inhibition of metastases of eye-implanted BHM melanoma

    10 Gy

    Romanowska et al., ABP, 2013


    Inhibition of metastases of eye implanted bhm melanoma1

    Inhibition of metastases of eye-implanted BHM melanoma

    Rel mean diameter [mm]

    Time [days]


    Dna damage protein expression and migration of melanoma cells irradiated with proton beam

    WYNIKI

    Inhibition of metastases of eye-implanted BHM melanoma

    x 4.4

    p=0.0052

    β-irradiation

    Romanowska et al., ABP, 2013


    Dna damage protein expression and migration of melanoma cells irradiated with proton beam

    Conclusions

    • 1-3Gysublethal, 5-7 Gylethaldamage of BLM cells

    • Delayed DNA damage, leading to apoptosis, resulting from endogenous ROS generationdue to cellsignalling

    • Upregulation of proteinsinvolved in DNA repair and stress, apoptosis and survival, glycolyticmetabolism and migration and cytoskeleton

    • In the lattergroup, vimentin was heavilysuppressed, together with Annexin 7, whereasexpression of Moesin 7, Lamins A/C and B, Actinin 4 and FAB-2 wereincreased

    • Interaction with tumor microenvironment: Upregulation of angiogenicgenes, in vivo inhibition of metastases in BHM eye model


    Dna damage protein expression and migration of melanoma cells irradiated with proton beam

    Obrazowanie mysich guzów: mechanizm odpowiedzi na radio- i fototerapię

    MRI, angiografia ToF

    MRI, perfuzja ASL


    Dna damage protein expression and migration of melanoma cells irradiated with proton beam

    Dept of General Biochemistry

    Jolanta Jura

    Łukasz Skalniak

    Agnieszka Cierniak

    Institute of Nuclear Physics, PAS

    Paweł Olko

    Jan Swakoń

    Urszula Sowa

    Marta Ptaszkiewicz

    Dept PhysicalBiochemistry

    Sylwia Kędracka-Krok

    Urszula Jankowska

    Dept Cell Biology

    Ewa Zuba-Surma

    Marta Michalik

    Dept of Ophthalmology and Ophthalmic Oncology, Jagiellonian University Medical College

    Bożena Romanowska-Dixon

    Dept Biophysics

    Krystyna Urbańska

    Katarzyna Jasińska

    Małgorzata Szczygieł

    Martyna Krzykawska-Serda

    Michał Gonet

    Agnieszka Drzał


    Dna damage protein expression and migration of melanoma cells irradiated with proton beam

    ODLEGŁE PRZERZUTY CZERNIAKA

    Przerzuty do płucsą inicjowane u części zwierząt (37%)gdy guz pierwotny zajmuje zaledwie 0,10 powierzchni PK(już po 2 dniach wzrostu guza)


    Wczesne i p ne efekty radioterapii

    WCZESNE I PÓŹNE EFEKTY RADIOTERAPII

    Całkowitej regresji guza po 10 Gy 125I (4,2,2,2) towarzyszy brak przerzutów w płucach u 50% leczonych chomików nawet po 70 dniach od enukleacji

    29 –34 dni po ENU

    Średnica guza (mm)

    100% zwierząt

    70 dni po ENU

    Czas (dni)

    50 % zwierząt

    (Sawow Aneta, 2001)


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