The application of 1.6 MeV proton microbeam to investigate radiation-induced bystander effect in MIA PaCa-2 pancreatic cancer cell line

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Artūras PLUKIS, Vita PAŠUKONIENĖ, Kęstutis SUŽIEDĖLIS,

1 The application of 1.6 MeV proton microbeam to investigate radiation-induced bystander effect in MIA PaCa-2 pancreatic cancer cell line Elena LAGZDINA, Mindaugas GASPARIŪNAS, Rita PLUKIENĖ, Artūras PLUKIS, Vita PAŠUKONIENĖ, Kęstutis SUŽIEDĖLIS, Vitalij KOVALEVSKIJ 11th International Conference Medical Physics in the Baltic States 2013 Kaunas, 10-12th of October

2 Radiation-Induced Bystander Effect RIBE Potential signaling pathways: Via intercellular gap junctions; Via cell culture medium. Potential signaling molecules: Reactive oxygen species (ROS); Reactive nitrogen species (RNS); Cytochrome c; Ca 2+ ; etc. JAEA R&D review, 2009, Quantum beam science research

Experimental equipment Ion accelerator Tandetron 4110A

6 MeV Beam current in vacuum chamber 5 na Microdosimetry

3 Experimental equipment Ion accelerator Tandetron 4110A (General Ionex Corp.) at the State Research Institute Center for Physical Sciences and Technology Initial energy of protons 1.6 MeV Beam current in vacuum chamber 5 na Microdosimetry problem: DOSE EVALUATION Simplified schematic diagram of the experimental arrangement used to irradiate cell samples. 3

R, mm Dose rate calibration (1/2) Experimental measurements using CR-39 nuclear track detector TASTRAK (Track analysis systems Ltd., UK); C 12 H 18 O 7 polymer with density of 1.

4 R, mm Dose rate calibration (1/2) Experimental measurements using CR-39 nuclear track detector TASTRAK (Track analysis systems Ltd., UK); C 12 H 18 O 7 polymer with density of 1.3 g/cm 3 Etching in 6M NaOH solution at 70 C for 45 min MCNPX code simulation. MCNPX (Monte Carlo N-Particle Code) : applied for the particle transport calculation proton flux to dose conversion E+02 H, MeV/cm 3 0.0E E E E+04 0E z, m 1.6E+04 Proton tracks on the surface of CR-39 nuclear track detector TASTRAK. 1.6 MeV proton beam energy deposition density along the 30 µm penetration path in the cell layer. 4

5 Proton flux, tracs/(100 m) 2 (a) Dose rate calibration (2/2) proton tracs on CR39 Fit Voigt 100pA exp 1 exp 1 100pA exp 2 exp 2 500pA exp 3 exp 3 model 100pA Proton beam parameters and calculation results of dose deposition in cell layer : I p, na D, Gy D TASTRAK, Gy D*, Gy Proton flux, tracs/(100 m) 2 (b) x, mm Experimental points at 5nA Voigt fit D* corrected modeling dose, taking into account the medium layer (of 20 µm) above cells (a) Proton tracks profile simulated using MCNPX 10 3 and experimentally obtained tracks on the CR-39 detector at different current intensity and 10 2 approximation using Voigt function. (b) Proton tracks profile and it's Voigt fit in the case of 5 na. x, mm 5

6 Bystander experiment CELL LINE: Human pancreatic cancer cell line MIA PaCa-2 Purchased from European Collection of Cell Cultures (ECACC) WORKFLOW: CELLS Irradiation IRRADIATED CELLS Medium transfer experiment DSBs measurement; Superoxide measurement BYSTANDER CELLS DNA DSBs and superoxide measurements were performed using FACSort flow cytometer (BD Biosciences). 6

DNA double strand breaks (DSBs) detection DNA DSB γ phosphorylation γ phosphorylation immunostaining

participates in signaling pathways; leads to the formation of other ROS; a common marker for oxidative stress.

2-Hydroethidium (EOH)??? Dihydroethidium (DHE) Chen et al.

7 DNA double strand breaks (DSBs) detection DNA DSB γ phosphorylation γ phosphorylation immunostaining one-electron reduction product of molecular oxygen; one of the major reactive oxygen species (ROS); participates in signaling pathways; leads to the formation of other ROS; a common marker for oxidative stress. DHE + O 2 - Superoxide (O 2 - ) : Superoxide detection F: Fluorescent product (F) Ethidium (E + ) 2-Hydroethidium (EOH)??? Dihydroethidium (DHE) Chen et al., 2013, Annals of Biomedical Engineering, 41: Muslimovic et al., 2012, Current Topics in Ionizing Radiation visualization Research, Dr. Mitsuru Nenoi (Ed.), ISBN: , InTech 7

8 Superoxide production and DSBs formation (a) (b) (a) Bystander cells show greater superoxide production than control cells, but lower compared with irradiated ones. (b) The same results were got in the case of DSBs measurement. 8

9 Conclusions CR-39 nuclear track detectors TASTRAK are suitable for obtaining quantitatively confident result of the proton dose deposited in the cell sample; Medium transfer from directly irradiated cells leads to increase of superoxide level and DSBs formation in the cells that are not directly affected by radiation, so this pilot study allowed to perceive the evidence of bystander effect; Finally, additional bystander experiments are required for obtaining statistically significant results. 9

10 Thank you for attention

Venue: IEEE NSS/MIC/RTSD Conference, Seoul, South Korea, 27 th October 2013 Workshop: NWK3/RD1 Radiation Protection and Dosimetry

Venue: IEEE NSS/MIC/RTSD Conference, Seoul, South Korea, 27 th October 2013 Workshop: NWK3/RD1 Radiation Protection and Dosimetry M. Caresana a, A. Sashala Naik a,c, S. Rollet b, M. Ferrarini a,d a Polytechnic