2016 年 39 巻 7 号 p. 1224-1230
Radiosensitizers are used in cancer therapy to increase the γ-irradiation susceptibility of cancer cells, including radioresistant hypoxic cancer cells within solid tumors, so that radiotherapy can be applied at doses sufficiently low to minimize damage to adjacent normal tissues. Radiation-induced DNA damage is repaired by multiple repair systems, and therefore these systems are potential targets for radiosensitizers. We recently reported that the transient receptor potential vanilloid type 1 (TRPV1) channel is involved in early responses to DNA damage after γ-irradiation of human lung adenocarcinoma A549 cells. Therefore, we hypothesized that TRPV1 channel inhibitors would have a radiosensitizing effect by blocking repair of radiation-induced cell damage. Here, we show that pretreatment of A549 cells with the TRPV1 channel inhibitors capsazepine, AMG9810, SB366791 and BCTC suppressed the γ-ray-induced activation of early DNA damage responses, i.e., activation of the protein kinase ataxia-telangiectasia mutated (ATM) and accumulation of p53-binding protein 1 (53BP1). Further, the decrease of survival fraction at one week after γ-irradiation (2.0 Gy) was enhanced by pretreatment of cells with these inhibitors. On the other hand, inhibitor pretreatment did not affect cell viability, the number of apoptotic or necrotic cells, or DNA synthesis at 24 h after irradiation. These results suggest that inhibition of DNA repair by TRPV1 channel inhibitors in irradiated A549 cells caused gradual loss of proliferative ability, rather than acute facilitation of apoptosis or necrosis. TRPV1 channel inhibitors could be novel candidates for radiosensitizers to improve the efficacy of radiation therapy, either alone or in combination with other types of radiosensitizers.
Radiation therapy plays an important role in treatment of numerous cancers,1) because ionizing radiation induces potentially lethal DNA damage, such as DNA double-strand breaks (DSBs). Although DSBs cause cell death or genomic instability,2,3) they can be repaired by mechanisms such as non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ rejoins the DNA ends without requiring sequence homologies, and is mediated by a DNA-dependent protein kinase holoenzyme. On the other hand, HR processes use undamaged homologous DNA sequences (sister chromatid or the homologous chromosome) to ensure the fidelity of the repair process.4–6) The DNA damage response pathways protect genomic integrity and act as a barrier against cancer,7) and are important for recovery of irradiated normal cells. However, overexpression of DNA repair proteins is involved in radioresistance of cancer cells,8–10) and therefore these mechanisms also decrease the efficiency of radiation therapy.
Since ionizing radiation does not discriminate between cancer cells and normal cells, damage to normal tissues is usually a dose-limiting factor in radiation therapy. In addition, many solid cancers develop a hypoxic central domain,11,12) and the hypoxic cells in this region are less susceptible to radiation.13) It is possible to use fractionated-dose irradiation, but this is less effective than a single dose because DNA repair (sublethal-damage repair) occurs during the inter-fraction intervals.14,15) Due to these issues, there is great interest in the use of radiosensitizers, such as gold nanoparticles or heat-shock-protein (Hsp) inhibitors, to improve the efficacy of cancer radiotherapy.16,17)
After irradiation of cells, early DNA repair responses, such as activation of ataxia telangiectasia mutated (ATM; a serine/threonine protein kinase), formation of phosphorylated histone variant H2AX (γH2AX), and accumulation of tumor suppressor p53-binding protein 1 (53BP1), are induced within 1 h.7,18) Activation of ATM induces phosphorylation of H2AX at DNA damage sites and triggers formation of a DNA damage response complex of RAD80, BRCA1 and 53BP1.19) Recently, we showed that transient receptor potential vanilloid type 1 (TRPV1) channel is involved in the early responses to radiation-induced DNA damage in A549 cells.20) TRPV1 channel is a non-selective cation channel, which serves as a pain receptor, and is activated by capsaicin, increased temperature (>43°C), oxidative stress, or hypertonicity.21,22) However, the role of TRPV1 channel in radiation-induced cellular responses has not been clarified.
Therefore, in this study, we investigated the radio-sensitizing effect of TRPV1 channel inhibitors in cancer cells. We found that TRPV1 channel inhibitors suppressed DNA damage response and enhanced radiation-induced reproductive death. They appear to be promising candidates as radiosensitizers utilizing a previously unexploited mechanism.
A549 human lung adenocarcinoma cells and B16 mouse melanoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Biowest, France), penicillin (100 U/mL) and streptomycin (100 µg/mL) in a humidified atmosphere of 5% CO2 in air at 37°C. The cells were irradiated with γ-rays from a Gammacell 40 (137Cs source) (Nordin International, Inc., Canada; 0.865 Gy/min) at room temperature for a suitable time. For example, the cells were irradiated with 2.0 Gy of γ-rays from a 137Cs source (0.865 Gy/min) for 139 s. After irradiation, the cells were further incubated in a humidified atmosphere of 5% CO2 in air at 37°C.
Cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature and permeabilized in 0.1% Triton X-100 for 5 min on ice. After incubation in blocking buffer (10% FBS in PBS) for 1 h, the fixed cells were incubated with primary antibody (phospho-histone H2AX (ser139) rabbit monoclonal antibody (Cell Signaling Technology, U.S.A.) or mouse anti-H2AX phosphorylated (Ser139) antibody (Bio Legend, U.S.A.) 1 : 200, 53BP1 antibody rabbit polyclonal (Novus, U.S.A.) 1 : 200, ATM (phosphoS1981) antibody (Abcam, U.K.) 1 : 1000) for 24 h at 4°C, and then with secondary antibody (Alexa Fluor 594 goat anti-mouse immunoglobulin G (IgG) (Invitrogen, U.S.A.) 1 : 200, goat anti-rabbit IgG-fluorescein isothiocyanate (FITC) (Sigma-Aldrich, U.S.A.) 1 : 200) for 1 h. Counterstaining with Hoechst 33258 (1 mg/mL) was used to verify the location and integrity of nuclei. Fluorescence images were obtained with a laser-scanning confocal microscope (FV1000 IX81; Olympus, Japan). In some experiments, a TRPV1 channel antagonist, capsazepine (CPZ) (Cayman Chemical Co., U.S.A.), AMG9810 (Wako Pure Chemical Industries, Ltd.), SB366791 (Wako Pure Chemical Industries, Ltd.), or BCTC (Wako Pure Chemical Industries, Ltd.), was added to the medium at 30 min before irradiation.
Cells (1.0×105 cells/mL) were incubated for 24 h, then exposed to γ-rays at a dose of 1.0–16.0 Gy, incubated for another 24 h, and seeded at 1.0×103 cells per 100 mm dish. After incubation for 1 week, the cells were stained with 0.5% crystal violet. Colonies containing more than 50 cells were counted. The results were normalized with respect to corresponding non-irradiated cells, and the survival rate was calculated. When required, TRPV1 agonist capsaisin (CPS) (Wako Pure Chemical Industries, Ltd.) was added to the medium at 5 min after irradiation, or TRPV1 antagonist was added to the medium at 30 min before irradiation.
Cells were seeded in 35 mm dishes at 1.0×105 cells/mL were incubated for 24 h, then exposed to γ-rays at a dose of 2.0 Gy and incubated for another 24 h. Next, all cells of each samples were re-suspended in Binding buffer (85 µL) were incubated with Annexin V-FITC (10 µL) and propidium iodide (5 µL) (MEBCYTO Apoptosis kit, Medical & Biological Laboratories, Japan) for 15 min at room temperature, and then 400 µL of Binding buffer was added. Further, 1 mL of ice cold PBS was added into the samples. The cells (10000 cells) were analyzed by flow cytometry (BD-LSR flow cytometer, Becton Dickinson, U.S.A.) with laser excitation at 488 nm. Fluorescence of FITC was examined using an FL-1 filter and fluorescence of propidium iodide was examined using an FL-3 filter. When required, TRPV1 antagonist was added to the medium at 30 min before irradiation.
Cells were lysed in cold lysis buffer containing 1% Triton X-100 and protease inhibitor cocktail (Sigma-Aldrich) at 4°C for 30 min. Lysates were centrifuged at 10000×g for 15 min. Cell lysate were dissolved in 2× sample buffer (50% glycerin, 2% sodium dodecyl sulfate (SDS), 125 mM Tris–Cl, 10 mM dithiothreitol (DTT)) and boiled for 10 min at 95°C. Protein (3 µg/lane) was analyzed by means of 7.5% SDS-polyacrylamide gel electrophoresis (PAGE), and bands were transferred onto a polyvinylidene difluoride (PVDF) membrane. The blots were blocked overnight in 1% bovine serum albumin (BSA) at 4°C. For detection of TRPV1 channel, the blots were incubated with rabbit anti-TRPV1 antibody (Novus) (1 : 1000) overnight at 4°C, and further incubated with goat horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (Cell Signaling Technology) (1 : 20000) for 1.5 h at room temperature. On the other hand, blots were incubated with anti β-actin, monoclonal antibody peroxidase conjugated (Wako Pure Chemical Industries, Ltd.) (1 : 10000) for 1 h at room temperature. Specific proteins were visualized by using ImunoStar® LD (Wako Pure Chemical Industries, Ltd.) according to the manufacturer’s instructions.
Results are expressed as the mean±standard error (S.E.). The statistical significance of differences between control and other groups was calculated using Dunnett’s t-test. Calculations were done with the Instat version 3.0 statistical software package (Graph Pad Software, U.S.A.). The criterion of significance was p<0.05.
We have reported that the TRPV1 channel inhibitor CPZ suppresses formation of DNA repair foci at DNA damage sites after γ-irradiation.20) First, to investigate whether other TRPV1 inhibitors also suppress DNA damage response, we examined the effect of AMG9810, SB366791 and BCTC on activation of ATM and accumulation of 53BP1 at DNA damage sites. The number of co-stained activated ATM and γH2AX foci was decreased by treatment with CPZ, AMG9810, SB366791 or BCTC before irradiation (Figs. 1A, 2A). The number of co-stained accumulations of 53BP1 and γH2AX foci was also decreased by pretreatment with these inhibitors (Figs. 1B, 2B), confirming that all of these TRPV1 channel inhibitors suppress the early DNA damage response after irradiation.
A549 cells were irradiated with 2.0 Gy of γ-rays, and further incubated for 30 min. At the end of incubation, co-stained foci activated ATM and γH2AX were detected by immunostaining (A). At the end of incubation, co-stained accumulations of 53BP1 and γH2AX were detected by immunostaining (B). The immunostaining without primary antibody was shown in (C).
(A) A549 cells were pretreated for 30 min with CPZ (10 µM), AMG9810 (10 µM), SB366791 (1 µM), or BCTC (10 µM) before irradiation, then irradiated with 2.0 Gy of γ-rays, and further incubated for 30 min. At the end of incubation, co-stained foci activated ATM and γH2AX were detected by immunostaining (n=19–34). (B) At the end of incubation, co-stained accumulations of 53BP1 and γH2AX were detected by immunostaining (n=19–34). (C) A549 cells were pretreated for 30 min with ATM kinase inhibitor (10 µM), and then irradiated with 2.0 Gy of γ-rays. Survival fraction was examined by colony formation assay (n=5). Each value represents the mean±S.E. Significant differences between irradiated control cells and indicated groups are shown by *** p<0.001.
Second, we confirmed that the ATM-mediated DNA repair mechanism is essential to rescue γ-irradiation-induced cell death. As shown in Fig. 2C, pretreatment with ATM kinase inhibitor significantly increased γ-irradiation-induced cell death, suggesting that γ-irradiation-induced cell death is dependent on DNA repair via activation of ATM and subsequent accumulation of DNA repair proteins such as 53BP1.
Next, we investigated whether the TRPV1 channel contributes to cell survival after γ-irradiation. We measured the survival rate at 1 week after exposure to 1–16 Gy of γ-rays. As shown in Fig. 3A, the survival fraction in the control decreased in a dose-dependent-manner, indicating radiation-induced loss of cell proliferation and reproductive death. Treatment with TRPV1 inhibitor CPZ before irradiation caused a larger decrease of the survival fraction (Fig. 3A). On the other hand, treatment with TRPV1 agonist CPS attenuated the decrease of survival fraction (Fig. 3B). Further, we investigated the effect of other TRPV1 channel inhibitors on γ-irradiation-induced reproductive cell death. Pretreatment with CPZ, AMG9810, SB366791 or BCTC significantly increased the decrease of survival fraction caused by 2.0 Gy of γ-irradiation (Fig. 3C). These results suggest that inhibition of the TRPV1 channel blocked rescue of cells from γ-irradiation-induced potentially lethal damage, resulting in enhancement of cell death. These data confirm an important role of TRPV1 channel in cellular recovery from irradiation-induced damage.
(A) A549 cells were pretreated for 30 min with the TRPV1 antagonist CPZ (10 µM) and then irradiated (1.0–16 Gy) (n=9). Significant differences between control cells and CPZ-treated cells are shown by * p<0.05. (B) A549 cells were treated with CPS (10 µM) at 5 min after irradiation (1.0–16 Gy) (n=3). Significant differences between control cells and CPS-treated cells are shown by * p<0.05 and **p<0.01. (C) A549 cells were pretreated for 30 min with CPZ (10 µM), AMG9810 (10 µM), SB366791 (1 µM), or BCTC (10 µM) before irradiation (2.0 Gy) (n=12). Significant differences between irradiated control cells and indicated groups are shown by ***p<0.001. (D) A549 cells were pretreated for 30 min with BCTC (10 µM) before irradiation. Cells were irradiated with 2.0 Gy γ-rays and incubated for 0.5–24 h. At the end of incubation, γH2AX was detected by immunostaining (n=15–28). Significant differences between irradiated cells treated with vehicle and irradiated cells treated with BCTC are shown by *** p<0.001. Each value represents the mean±S.E.
To determine whether pretreatment with TRPV1 inhibitors suppressed or delayed the DNA damage response, we examined the formation of DNA repair foci at DNA damage sites at 0.5–24 h after irradiation in the presence or absence of TRPV1 inhibitor BCTC. The number of γH2AX foci decreased depending on elapsed time after irradiation in both cases, and γH2AX focus formation was suppressed for up to 24 h in BCTC-treated cells (Fig. 3D). The results indicate that the damage response was suppressed rather than merely delayed. It is implicated that TRPV1-mediated Ca2+ influx would indirectly facilitate DNA repair, probably at upstream of ATM, because intracellular Ca2+ elevation could not be required for activation of ATM directoly.
Next, we examined the profile of TRPV1 inhibitor-enhanced cell death of irradiated cells. It is known that irradiation of cells causes reproductive death after mitosis, rather than apoptosis or necrosis.23–26) Therefore, reproductive death was analyzed by measuring survival fraction. As shown in Fig. 3C, TRPV1 inhibitors enhanced reproductive death. To examine the mechanism involved, we investigated the changes of apoptosis and necrosis at 24 h after irradiation. We measured the rate of apoptosis or necrosis at 24 h after 2.0 Gy of irradiation. The percentages of apoptotic cells and necrotic cells were not significantly increased by irradiation (Figs. 4A, B). Among the inhibitors, only AMG9810 showed substantial cytotoxicity. Therefore, it is suggested that blockade of the TRPV1 channel does not induce apoptosis or necrosis in irradiated cells.
(A, B) A549 cells were pre-incubated for 30 min with CPZ (10 µM), AMG9810 (10 µM), SB366791 (1 µM), or BCTC (10 µM), then irradiated with γ-rays (2 Gy), and further incubated for 24 h. Percentages of apoptotic cells (Annexin V-positive, PI-negative cells) and necrotic cells (Annexin V-positive, PI-positive cells) were evaluated by flow cytometry (n=4). Each value represents the mean±S.E. Significant differences between irradiated control cells and indicated groups are shown by ** p<0.01.
Furthermore, we confirmed the radiosensitizing effect of TRPV1 inhibitor on another cancer cell line, B16 melanoma cells. The protein expression of TRPV1 channel both in A549 cells and in B16 cells were confirmed by Western blotting (Fig. 5A). The numbers of co-stained activated ATM and γH2AX foci and the numbers of co-stained accumulations of 53BP1 and γH2AX foci were decreased by pretreatment with CPZ (Figs. 5B, C). Pretreatment with CPZ significantly enhanced the decrease of survival fraction caused by 2.0 Gy of γ-irradiation (Fig. 5D). These results indicate that the TRPV1 inhibitor has a radiosensitizing effect in melanoma cells.
(A) The expressions of TRPV1 channel in A549 cells and B16 cells were detected by Western blotting. (B) B16 cells were pretreated for 30 min with CPZ (10 µM), before irradiation. Cells were irradiated with 2.0 Gy of γ-rays and incubated for 30 min. At the end of incubation, co-stained foci of activated ATM and γH2AX were detected by immunostaining (n=29–42). (C) At the end of incubation, co-stained accumulations of 53BP1 and γH2AX were detected by immunostaining (n=25–31). (D) B16 cells were pre-incubated for 30 min with CPZ (10 µM). Cells were irradiated with γ-rays (2.0 Gy), and survival fraction was examined by colony formation assay (n=9). Each value represents the mean±S.E. Significant differences between irradiated control cells and indicated groups are shown by *** p<0.001.
Various radiosensitizers have been reported. Recently, loading of gold nanoparticles into tumors has been introduced for radiosensitization,27,28) but issues regarding photon energy and gold nanoparticle size remain.16) Another approach is inhibition of Hsp,17) which is abundantly overexpressed in many human tumor cells and blocks formation of apoptosome (ApaS-1/caspase-9/cytochrome c), leading to activation of caspase-3 and induction of apoptosis. But, although Hsp90 inhibitors enhance the efficacy of radiation therapy, they also activate heat shock factor 1, which induces the synthesis of cytoprotective Hsp70 in cancer cells.17,29) Gadolinium, hydrogen peroxide, and tirapazamine have also been investigated as radiosensitizers, but have various disadvantages.30–32) Based on our finding that activation of the TRPV1 channel promotes the repair response to DNA damage,20) we speculated that inhibitors of this channel might be available as novel radiosensitizers. Although the mechanism of TRPV1 channel activation after irradiation is still unknown, possible mediators include radiation-induced reactive oxygen species and secondary generation of nitric oxide in irradiated cells,33) or radiation-induced extracellular release of ATP from cells, we have reported.34–37)
On the other hand, it is well known that TRPV1 channel is involved in inflammation and pain sensation,38–41) and TRPV1 channel inhibitors are candidates as analgesic drugs to relieve cancer pain.42,43) Therefore, TRPV1 inhibitors might offer dual benefits in cancer therapy, having both anti-inflammatory and radiosensitizing actions.
In conclusion, our results indicate that TRPV1 channel inhibitors show radiosensitizing action through inhibition of early DNA damage response. Blockade of the TRPV1 channel causes attenuation of ATM activation and accumulation of 53BP1, resulting in enhancement of reproductive death in irradiated cells, without induction of acute apoptosis or necrosis. Our results imply that activation of TRPV1 channel contributes to recovery of cells from potentially lethal radiation-induced damage, providing a new insight into the mechanisms of radiation-induced cellular responses. TRPV1 channel inhibitors are a novel class of candidate radiosensitizers for cancer therapy. Further, since their mechanism of action is different from those of other radiosensitizers previously reported, it may also be possible to use TRPV1 inhibitors in combination with other radiosensitizers.
Parts of this work were supported by Grants-in-Aid for Pharmaceutical Scientific Research (to MT) from Takeda Science Foundation.
The authors declare no conflict of interest.