Radiosensitizing Effect of 5-Aminolevulinic Acid and Protoporphyrin IX on Carbon-ion Beam Irradiation

Materials and Methods

Materials. 5-ALA hydrochloride was purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan), PpIX from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), MitoSOX Red and Mitotracker Green FM from Thermo Fisher Scientific Inc. (Waltham, MA, USA), Hoechst 33342 from PromoCell (Heidelberg, Germany).

Cell culture. Mouse mammary EMT6 tumor cells (supplied by Dr. Shin-ichiro Masunaga, Kyoto University, Kyoto, Japan) were maintained in Eagle's minimum essential medium (Sigma-Aldrich, Tokyo, Japan) supplemented with 10% fetal bovine serum (Biosera Ltd., Kansas City, MO, USA). Cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C.

Carbon-ion beam irradiation and evaluation of sensitizing activity of ALA and PpIX. EMT6 cells were seeded in flasks at 1×10⁵ cells and cultured overnight. PpIX (1 μM) and ALA (1 mM) were added 24 and 5 hours, respectively, prior to carbon-ion beam irradiation. The cells were transported to the Hyogo Ion Beam Medical Center within 3 hours at 37°C. They were then irradiated with 1.0 and 1.5 Gy carbon-ion beam (320 MeV/nucleon, 83.3 keV/μm). Twenty-four hours after the irradiation, the cells were reseeded in a Petri dish and cultured for 8 days in 5% CO₂ at 37°C. The medium was then removed and the cell surface was washed with 2 ml of 1× phosphate-buffered saline (PBS). Two milliliters of methanol were added and allowed to stand for about 10 min to fix the cells. Next, 2 ml of 5% Giemsa solution (Merck & Co., Inc., Kenilworth, NJ, USA) was added and colony staining was carried out for 1 h. After staining, the dish was washed with water and then dried. Thereafter, the number of colonies was counted to calculate the colony plating efficiency (PE) and the survival rate (SF).

Mitochondrial ROS detection. Mitochondrial ROS content was determined by measuring the fluorescence of MitoSOX Red. The EMT6 cells were seeded in a slide chamber (1×10³ cells/well) and PpIX (1 μM) and ALA (1 mM) were added 24 and 5 h, respectively, prior to carbon-ion beam irradiation. The cells were transported to the Hyogo Ion Beam Medical Center within 3 hours at 37°C, and then irradiated with 1.0-Gy carbon-ion beam (320 MeV/nucleon, 83.3 keV/μm). Three hours after irradiation, the cells were washed with 1 × PBS. MitoSOX Red (5 μM) was added and incubated for 30 min at 37°C. After incubation, cells were washed twice with 1 × PBS and 500 μl of E-MEM was added. The fluorescence intensity of MitoSOX Red was measured at 640 nm excitation and 660 nm emission wavelengths using a fluorescence microscope BZ-X700 (KEYENCE Co. Ltd., Osaka, Japan).

DNA double-strand break detection. EMT6 cells were seeded in slide chamber (WATSON Co. Ltd., Tokyo, Japan) at 1×10⁴ cells/well and cultured overnight. PpIX (1 μM) and ALA (1 mM) were added 24 and 5 h, respectively, prior to the carbon-ion beam irradiation. The cells were transported to the Hyogo Ion Beam Medical Center within 3 h at 37°C and irradiated with 1.0 and 1.5 Gy carbon-ion beam (320 MeV/nucleon, 83.3 keV/μm). Three hours after the carbon-ion beam irradiation, observation slides were prepared as follows: First, the cells were fixed for 15 min at 25°C using 500 μl of 4% paraformaldehyde/phosphate buffer solution. They were then washed twice with 1 × PBS and treated with 500 μl of 0.5% Triton solution at 4°C for 5 min. The cells were again washed twice with 1 × PBS and blocked with 500 μl of blocking solution at room temperature for 30 min. The fixed and blocked cells were then incubated in 300 μl of the primary antibody (Anti-phospho-Histone H2A.X (Ser139), clone JBW301, Merck KGaA, Darmstadt, Germany) solution at room temperature for 1 h. After incubation, washing was carried out three times with 300 μl of 0.5% Triton solution for 3 min each with shaking. This was followed by incubation for 30 min with 200 μl of the diluted secondary antibody (donkey anti mouse IgG affinity purified; fluorescein conjugated absorbed for dual labeling secondary antibody, Merck KGaA, Darmstadt, Germany) solution in a dark room. After incubation, washing was again carried out three times with 200 μl of 0.5% Triton solution for 3 min each with shaking. After washing, the slide was incubated for 15 minutes with 300 μl of DAPI (WAKO, Tokyo, Japan) solution, and then washed again three times with 1 × PBS. The slide was then removed from the treated chamber and dried. A cover glass was made using the Marinol (MUTO Pure Chemicals Co. Ltd., Tokyo, Japan), and the prepared slide was kept refrigerated in a dark room until observation. The fluorescence intensity of γ-H2AX focus was measured at 490 nm excitation and 525 nm emission wavelengths using a fluorescence microscope BZ-X700. Wells were divided into six sections, and the number of γ-H2AX focus of 50 cells per division was counted, and the average of six sections was calculated.

Localization of intracellular PpIX. EMT6 cells (5×10³ cells) were inoculated on a glass dish and cultured overnight. Cultured cells were incubated with PpIX (1 μM) and ALA (1 mM) for 24 h and 5 h, respectively, washed once with 1 × PBS, and 100 nM of Mitotracker Green FM was added. Furthermore, 2 ng/ml of Hoechst 33342 was added. After incubation for 30 min, the cells were washed twice with 1 × PBS and observed under a serum-free E-MEM using a fluorescence microscope.

Statistical analysis. Data are expressed as the mean and standard deviations of at least three independent experiments. The statistical significance of the differences between the results was analyzed using Student's t-test. A p<0.05 was considered statistically significant.