In vitro studies of radiobiological effects of boron neutron capture therapy (BNCT) on differentiated thyroid cancer.

PONTIGGIA, O; CARPANO, M; PERONA, M; THOMASZ, L; THORP, S; POZZI, E; SIMIAN, M; KAHL, S; JUVENAL, G; PISAREV , M; DAGROSA, MA

Paris

Congreso; 14th International Thyroid Congress; 2010

American Thyroid Association (ATA)

Introduction:  In previous studies we demonstrated that relapsed differentiated thyroid cancer can be treated by boron neutron capture therapy (BNCT). BNCT is based on the selective uptake by the tumor of boron compounds enriched with 10B, like boronophenylalanine (BPA) or BOPP 2,4-bis (a,b-dihydroxyethyl)-deutero-porphyrin IX). The irradiation with an appropriate neutron beam activates 10B to 11B which decays releasing alpha particles and 7Li with a high ionizing activity, and therefore will kill tumoral cells. Little is known about the mechanisms of cellular response to double-strand DNA damage induced by BNCT. Cell cycle regulation and repair mechanisms such as homologous recombination (HR) and non homologous end-joining (NHE) are important determinants of ionizing radiation sensitivity. The p53 protein plays a role in the activation of multiple genes that can induce: temporary G1-S cell cycle, DNA repair, irreversible growth arrest and apoptosis. On the other hand, Ku70 protein is involved in the repair of DNA double strand breaks via NHE pathway and can also play an important role in the apoptosis. In the present work we analyzed the expression of p53 and Ku70 involved in cell cycle regulation and DNA repair. We also evaluated the mechanisms of death induced by BNCT and compared them with the conventional gamma radiation. Materials and Methods: Exponentially growing cells of follicular thyroid carcinoma (WRO) were distributed into the following groups: 1) BPA (10 ppm 10B) + neutrons; 2) BOPP (10 ppm 10B) + neutrons; 3) neutrons alone; 4) gamma-rays. A control group without irradiation for each treatment was added. The cells were irradiated in the thermal neutron beam of the RA-3 (flux= 7.5 109 n/cm2 sec) or with 60Co (1Gy/min) in order to obtain a total physical dose of 3 Gy (±10%). After 24 and 48 h post irradiation, the protein expression of p53 and ku70 was analyzed by Western blotting. The frequency of cell death (apoptosis and necrosis) was measured by Hoechst 33258, 4´-6 diamino 2-phenylindole (DAPI) and propidium iodide (PI) staining using fluorescence microscope. Cell cycle analysis was performed by flow cytometry. Results and Discussion: An up regulation of Ku 70 was observed in the BNCT (neutrons + BOPP) group at 48 h. While the p53 protein showed an increase at 24 h for the neutron alone and gamma groups. Accumulation of cells in G1/S was observed at both studied times for all the irradiated groups (1, 2, 3 and 4). The study of mechanisms of death showed that at 24 h the frequency of apoptosis and necrosis increased in all the groups irradiated with neutrons (Groups 1, 2 and 3) respect to gamma rays and control groups. On the other hand at 48 h an increase in the death cell was observed in the BNCT group irradiated with BOPP (around 5% apoptosis and 20% necrosis). These preliminary results suggest different cellular response for all the irradiated groups. Only the treatment with neutron plus 10BOPP seems to induce the repair system NHE by increase of Ku 70 at 48 h.