Abstract
Background/Aim: Carbon-ion beam is one of the most advanced radiations used for cancer treatment. However, there are tumors that are difficult to suppress with carbon-ion beam alone, thus necessitating development of drugs that can enhance its therapeutic effect. In this regard, the radiosensitizing effect of 5-aminolevulinic acid (ALA) and protoporphyrin IX (PpIX), that is a metabolic intermediate of ALA, on carbon-ion beam was investigated. Materials and Methods: Radiosensitizing activity, mitochondrial ROS and DNA double-strand break production of ALA and PpIX were evaluated by irradiation with 1.0 or 1.5-Gy carbon-ion beam to mouse mammary EMT6 tumor cells. Results: Combination of carbon-ion beam and ALA or PpIX showed a significant enhancement of its cytotoxic activity through a significant increase in ROS production in mitochondria. Furthermore, the combined activity of carbon-ion beam and ALA resulted in a significant increase in DNA double-strand breakage. Conclusion: ALA selectively accumulates in the mitochondria and PpIX synthesized from ALA reacts with carbon-ion beam to produce ROS that exert antitumor activity.
Carbon-ion beam is one of the most advanced radiations used for cancer treatment (1-3). They have higher LET (linear energy transfer) than conventional x-rays and induce cell death mainly by initiating DNA double-strand breaks (4, 5). In addition, since it has a Bragg peak where energy deposition reaches a maximum at a certain depth of the body, it can be irradiated with high accuracy in the cancer tissue, facilitating minimum side effects and shortened treatment period (6). Although it is said to be effective for the treatment of refractory cancer, there are tumors that are difficult to suppress with carbon-ion beam irradiation alone. Hence, the development of a drug that can further enhance its therapeutic effect is necessary. However, carbon-ion beams have strong DNA-damaging properties. Since the antitumor effect of reactive oxygen species (ROS) is relatively small, drugs that enhance the antitumor effect of carbon-ion beam have rarely been reported (7-9).
Therefore, this study, is focused on 5-aminolevulinic acid (ALA), a drug already in clinical use in photodynamic diagnosis/therapy (10-12). In cancer cells, protoporphyrin IX (PpIX), a metabolic intermediate of ALA, selectively accumulates in the mitochondria (13). It has been reported in recent years that irradiation of X-ray results in local enhancement of ROS production, thus improving its therapeutic effect (14-16). Therefore, we postulated that ALA can also increase ROS production in response to carbon-ion beam and may exhibit carbon-ion beam sensitizing action. In this study, the effect and the mechanism of ALA and PpIX on carbon-ion beam sensitization was examined, by monitoring their effects on ROS production in mitochondria and DNA double strand breakage.
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.
Results
Radiosensitizing activity of ALA and PpIX on carbon-ion beam. According to previous studies, ALA and PpIX showed no cytotoxicity up to 1 mM and 1 μM as maximum intracellular PpIX concentrations were reached at 5 and 24 h, respectively (17, 18). We, therefore, chose the same condition in this experiment. Combined use of carbon-ion beam and ALA has an enhancement ratio (ER) of 1.16 (1 Gy) and 1.29 (1.5 Gy), showing a significant radiosensitizing effect as compared to the control (Figure 1). In addition, the combination of carbon-ion beam and PpIX showed a significant radiosensitizing effect compared to the control with ER=1.14 (1 Gy) and 1.18 (1.5 Gy) (Figure 1). There was no significant difference in the radiosensitizing activity to carbon-ion beam between ALA and PpIX, but ALA displayed a slightly higher radiosensitizing activity than PpIX.
Production of mitochondrial ROS in reaction to combination of carbon-ion beam with ALA or PpIX. The radiosensitizing effect of ALA and PpIX to carbon-ion beam may be related to the production of ROS in mitochondria, as previously shown in the case of X-rays (14-16). Therefore, mitochondrial ROS production was measured when carbon-ion beam was combined with ALA or PpIX. At 1 Gy radiation, the combined use of carbon-ion beam and ALA was about 1.46 times that of control, and the combined use of carbon-ion beam and PpIX was about 1.26 times (Figure 2), indicating significant increase in ROS production in mitochondria. Similar to results shown in Figure 1, addition of ALA showed higher mitochondrial ROS production than the addition of PpIX.
Radiosensitizing activity of ALA and PpIX on carbon-ion beam. C: Control; A: ALA; P: PpIX. Each experiment was performed three times; data are the mean±SD (*p<0.05).
DNA double-strand breaks by combination of carbon-ion beam and ALA or PpIX. DNA double-strand breakage is pivotal in cancer cell death by radiation. DNA double strand breakage activity was measured 3 h after irradiation with 1 Gy carbon-ion beam. Combined use of ALA with carbon-ion beam showed a significant increase of 1.16 times in γ-H2AX focus number when compared to the control (Figure 3). On the contrary, combination of carbon-ion beam with PpIX showed no significant increase in γ-H2AX focus number compared to the control (Figure 3).
Localization of intracellular PpIX. In order to clarify whether the intracellular localization of PpIX is related to its effects on the radiosensitization of carbon-ion beam, ROS productivity in mitochondria, and DNA double strand breakage activity, intracellular PpIX localization at the time of carbon irradiation was evaluated. PpIX synthesized from ALA was mainly localized in mitochondria and cytoplasm and confined slightly in the nucleus (Figure 4). On the contrary, when PpIX was added, it was mainly localized in the nucleus, and almost no localization of PpIX in mitochondria and cytoplasm was observed (Figure 4).
Production of mitochondrial ROS by combination of carbon beam with ALA or PpIX. Each experiment was performed three times; data are the mean±SD (*p<0.05).
DNA double-strand breaks by combination of carbon beam with ALA or PpIX. Each experiment was performed three times; data are the mean±SD (*p<0.05).
Localization of intracellular PpIX. Mitotracker: Mitochondria; Hoechst33342: nucleus.
Discussion
In this study, the radiosensitizing activity of ALA and PpIX on carbon-ion beam and its mode of action were analyzed. Since ALA is converted into PpIX in mitochondria and PpIX, which is a porphyrin compound, has antitumor activity in combination with visible light (photodynamic therapy) and ultrasound (sonodynamic therapy) (12), PpIX was predicted to be the active component. However, since ALA displayed a higher carbon-ion beam sensitizing effect than PpIX, we concluded that the localization of PpIX to mitochondria, where PpIX is biosynthesized from ALA, is crucial. Indeed, the examination of the intracellular localization of PpIX supported our hypothesis: PpIX derived from ALA was mainly localized in the mitochondria, but extraneous PpIX was predominantly present in the nucleus.
In recent years, it has been thought that not only nuclear DNA damage due to irradiation induced by ROS, but also delayed ROS production centered on mitochondria, plays an important role as a biological response to tumor irradiation. It has also been reported that ALA increases delayed ROS production occurring after irradiation, centered on mitochondria (19, 20). Our results support the conclusion that there is delayed ROS production in the mitochondria after ALA treatment.
In conclusion, ALA is converted to PpIX in the mitochondria and interacts with carbon-ion beam to produce ROS and exert its cytotoxic activity.
- Received April 11, 2018.
- Revision received May 14, 2018.
- Accepted May 15, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved