Radiotherapy and Oncology xxx (2014) xxx–xxx
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Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
Original article
C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe Takahiro Oike a,b, Mayumi Komachi a, Hideaki Ogiwara b, Napapat Amornwichet a, Yuka Saitoh a, Kohta Torikai c, Nobuteru Kubo a, Takashi Nakano a, Takashi Kohno b,⇑ a c
Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi; b Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku; Gunma University Heavy Ion Medical Center, Maebashi, Japan
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 3 March 2014 Accepted 18 March 2014 Available online xxxx Keywords: Radiosensitizer p300 Histone acetyltransferase Mitotic catastrophe G2 checkpoint
a b s t r a c t Background and purpose: Chromatin remodeling through histone modifications, including acetylation, plays an important role in the appropriate response to DNA damage induced by ionizing radiation (IR). Here we investigated the radiosensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase, and explored the underlying mechanisms. Materials and methods: A549, H157 and H460 human non-small cell lung carcinoma (NSCLC) cells, and HFL-III human lung fibroblasts were assessed by clonogenic survival assay. Apoptosis and necrosis were assessed by annexin V staining. Senescence was assessed by Senescence-associated b-galactosidase staining. Mitotic catastrophe was assessed by evaluating nuclear morphology with DAPI staining. Cell cycle profiles were analyzed by flow cytometry. Protein expression was analyzed by immunoblotting. Results: C646 sensitized A549, H460 and H157 cells to IR with a dose enhancement ratio at 10% surviving fraction of 1.4, 1.2 and 1.2, respectively. C646 did not radiosensitize HFL-III cells. In A549 cells, but not in HFL-III cells, C646 (i) enhanced mitotic catastrophe but not apoptosis, necrosis, or senescence after IR; (ii) increased the hyperploid cell population after IR; and (iii) suppressed the phosphorylation of CHK1 after IR. Conclusions: C646 radiosensitizes NSCLC cells by enhancing mitotic catastrophe through the abrogation of G2 checkpoint maintenance. Ó 2014 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2014) xxx–xxx
Ionizing radiation (IR)-induced DNA damage leads to the generation of DNA double-strand breaks (DSBs), resulting in cell death. A critical component of the DNA damage response (DDR) after IR is the induction of dynamic changes in chromatin structure called chromatin remodeling [1]. Post-translational histone modifications play critical roles in chromatin remodeling after IR by mediating the recognition and signaling of DNA damage, repair of DNA damage sites, and the regulation of cell cycle checkpoints. Therefore, compounds that specifically target the enzymes responsible for these histone modifications have high potential as radiosensitizing agents. Histone acetyltransferases (HATs) catalyze the acetylation of histone proteins, which is a process required for chromatin remodeling at DSB sites. We and others have previously demonstrated that natural compounds with multi-HAT-inhibitory activity such as garcinol, curcumin and anacardic acid have a radiosensitizing effect in vitro [2–4]. Moreover, we have shown that siRNA-mediated ⇑ Corresponding author. Address: Division of Genome Biology, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. E-mail address: [email protected] (T. Kohno).
ablation of the HATs p300 and CBP sensitizes cancer cells to IR [5]. These data indicate that the inhibition of HATs is a promising strategy for radiosensitization. However, radiosensitization by selective small molecule inhibitors targeting a specific HAT, which could have a significant clinical potential, has not been well investigated. Recently, Bowers et al. showed that the small molecule compound C646 has selective inhibitory activity against p300 [6]. In that study, an in silico virtual ligand screening based on the Xray structure of the HAT active site of p300 that included approximately 500,000 commercially available small molecules identified C646 as a competitive inhibitor of p300 that antagonizes acetylCoA, a substrate of p300 for acetylation, and C646 was shown to suppress histone acetylation in human cells in vitro. These findings prompted us to investigate the effect of C646 on radiosensitization of human cancer cells. Here, we show that C646 radiosensitizes non-small cell lung carcinoma (NSCLC) cells but not non-cancerous cells, and that C646 suppresses the phosphorylation of the checkpoint kinase CHK1 after IR in cancer cells, leading to the enhancement of mitotic catastrophe.
http://dx.doi.org/10.1016/j.radonc.2014.03.015 0167-8140/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Oike T et al. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.015
2
Radiosensitization by a p300 inhibitor
Materials and methods Materials The human NSCLC cell lines A549 (adenocarcinoma), H157 (squamous cell carcinoma) and H460 (large cell carcinoma), and human lung fibroblast lines HFL-III and MRC-5, were cultured in RPMI-1640 supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. C646 was purchased from R&D (Minneapolis, MN, USA). Dimethyl sulfoxide (DMSO) purchased from Sigma (St Louis, MO, USA) was used as a solvent for the drugs. Antibodies were purchased from Epitomics (Burlingame, CA, USA) (Chk1, 1740-1), Cell Signaling Technology (Danvers, MA, USA) (Phospho-Chk1 Ser345, 133D3; Phospho-Chk2 Thr68, C13C1; b-actin, 3700P), and BD (Franklin Lakes, NJ, USA) (Chk2, 611571). Irradiation Cells received IR using a 60Co c-ray source (Atomic Energy of Canada, Ltd., Ontario, Canada) with a dose rate of 9.1 Gy/min.
using Prolong Gold (Life Technologies, Carlsbad, CA, USA). Confocal images were taken using an inverted microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 100 oil lens. Images were acquired using ZEN Software (2008, Carl Zeiss). Two or more distinct nuclear lobes within a single cell were scored as positive for mitotic catastrophe [7]. The percentage of cells undergoing mitotic catastrophe was quantified by counting at least 300 cells in each experimental condition. Cell cycle analysis Cells were exposed (or not) to C646 at 37 °C for 2 h, and subjected (or not) to IR. After an additional incubation for 72 h, the cells were trypsinized, centrifuged, washed in PBS, and fixed in ice-cold 70% ethanol. The cells were then re-centrifuged and incubated with PBS containing 200 lg/mL RNaseA and 5 lg/mL propidium iodide (PI), and cell cycle distribution was analyzed by Guava easyCyte HT. Immunoblot analysis
Clonogenic survival assay
Immunoblot analysis was performed as described previously [5].
Cells were seeded in 6-well plates, incubated at 37 °C for 4–10 h for attachment, and exposed (or not) to C646. After incubation for 2 h, the cells were subjected (or not) to IR and incubated for 10 days for colony formation. The cells were fixed with methanol and stained with crystal violet. Colonies of at least 50 cells were counted. The surviving fraction was normalized to the corresponding controls. The dose required to reduce the surviving fraction to 10% (D10) of the irradiated cells was calculated by using the linearquadratic model as described previously [2].
Experiments were performed at least in triplicate. The significance of differences was determined by the unpaired Student’s t-test and Welch’s test in the cases where the standard deviations (SDs) of two populations were equal or not equal, respectively, using StatMateIII ver. 3.17 software (ATMS, Tokyo, Japan). A P value 4 N DNA) cell population, consistent with the induction of mitotic catastrophe [10]. Meanwhile, combined treatment did not significantly increase the hyperploid cell population in HFL-III cells. Taken together, these data indicate that the phenotype of cancer cells (but not that of non-cancerous cells) increases their susceptibility to C646-induced mitotic catastrophe after IR. C646 suppresses phosphorylation of CHK1 after IR in A549 cells To explore the mechanisms underlying the enhancement of mitotic catastrophe by C646 in irradiated cells, we examined the effect of C646 on the phosphorylation of the checkpoint kinases CHK1 and CHK2, which is critical for the activation and maintenance of DNA damage checkpoints in response to IR (Fig. 4) [11–15]. The kinetics of phosphorylated CHK1 pSer345 (p-CHK1) expression in A549 and HFL-III cells after IR (D10) were comparable; p-CHK1 expression increased at 30 min after IR and was sustained for 120 min. Interestingly, in the presence of C646 (IC50), p-CHK1 levels in A549 cells (but not those in HFL-III cells) were significantly reduced at 60 min and 120 min after IR. The kinetics of phosphorylated CHK2 pThr68 (p-CHK2) expression after IR were also comparable between the two cell lines; however, in these cases the kinetics were not significantly affected by C646 treat-
Fig. 2. Radiosensitizing effect of C646 assessed by clonogenic survival assay. (a) A549, (b) H157, (c) H460, and (d) HFL-III cells. Average ± SD is shown.
Please cite this article in press as: Oike T et al. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.015
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Radiosensitization by a p300 inhibitor
Fig. 3. Effect of C646 on cell death in irradiated A549 (a) and HFL-III (b) cells. Treatment with IR and C646 was performed at D10 and IC50 in each cell, respectively. Induction of apoptosis and necrosis was assessed by annexin V staining 72 h after IR. Induction of senescence was assessed by SA-b-Gal staining 6 days after IR. Induction of mitotic catastrophe (MC) was assessed based on the morphology of DAPI-stained nuclei 72 h after IR. Average ± SD is shown. ⁄, P < 0.05; , P < 0.01. Note that the percentages of senescent cells are shown in different scales from the others.
Fig. 4. Effect of C646 on the phosphorylation of CHK1 and CHK2 in irradiated A549 (a) and HFL-III (b) cells. Treatment with IR and C646 was performed at D10 and IC50 in each cell, respectively. The cells harvested at the indicated time points were analyzed by immunoblot with antibodies against CHK1, CHK2, p-CHK1, p-CHK2, and b-actin (loading control). Representative immunoblot images are shown (upper panels). The lower panels show the relative band intensities for p-CHK1 and CHK1 (left hand panel in A and B) and for p-CHK2 and CHK2 (right hand panel in A and B). The intensities of the p-CHK1 bands relative to the CHK1 bands at the same time points are shown after normalization to samples treated with IR alone and harvested at 60 min (the time at which the greatest band intensity was observed). The intensities of the p-CHK2 bands relative to the CHK2 bands at the same time points are shown after normalization to samples treated with IR alone and harvested at 30 min post-RT (the time at which the greatest band intensity was observed). Data are expressed as the mean ± SD. ⁄P < 0.05; P < 0.01.
ment. Taken together, these results suggest that C646 suppresses CHK1 phosphorylation in irradiated cancer cells, which may then alter the cell cycle checkpoint and lead to premature entry into mitosis and the enhancement of mitotic catastrophe.
Discussion In the present study, we showed that C646, a selective small molecule inhibitor of p300, radiosensitized NSCLC cell lines of
Please cite this article in press as: Oike T et al. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.015
T. Oike et al. / Radiotherapy and Oncology xxx (2014) xxx–xxx
three different histological types, but not a non-cancerous cell line. C646 enhanced IR-induced mitotic catastrophe and suppressed the phosphorylation of CHK1 after IR in A549 cells but not in HFL-III cells. Targeting HATs critical for chromatin remodeling in the DDR after IR is a potential radiosensitizing strategy [1]. Therefore, we and others have investigated the radiosensitizing effect of several natural compounds with multiple HAT-inhibitory activities such as garcinol, curcumin and anacardic acid, and showed that they sensitized cancer cells to IR in vitro [2–4]. Garcinol inhibits p300, CBP and PCAF [16]; curcumin inhibits p300 and CBP [17]; and anacardic acid inhibits p300 and TIP60 [4,17]. In the present study, C646 was shown to sensitize NSCLC cells to IR (Fig. 2a–c). Together, these findings demonstrate that p300 is a candidate target for radiosensitization in cancer cells. However, the effects of C646 on proteins other than p300 have not been fully elucidated [6]. Thus, the possibility that C646 may target other proteins in addition to p300 for radiosensitization should be further pursued. The radiosensitizing effect of C646 was not evident in noncancerous HFL-III cells (Fig. 2d), which was consistent with our previous data showing that garcinol, a specific inhibitor of CBP, p300 and PCAF HATs, had no radiosensitizing activity in non-cancerous cells [2]. It is considered that, in the process of malignant transformation from normal cells, cancer cells acquire aberrations in DDR-related factors leading to genomic instability which contributes to the higher vulnerability of cancer cells to IR than normal cells [18]. This may in part explain the cancer-specific radiosensitizing effect by HAT inhibition since the present and previous studies suggested the involvement of HAT in DDR including non-homologous end joining, homologous recombination and cell cycle checkpoints [2,19]. The present study suggests that the type of IR-mediated cell death in cancer and normal cells is different, i.e., IR mainly induced mitotic catastrophe in A549 cells, but induced senescence in the majority of HFL-III cells (Fig. 3). This may also reflect the differences in HAT-mediated radiosensitization in these two cell lines. However, the mechanisms underlying the stronger radiosensitizing effect of HAT inhibition in cancer cells than in normal cells have not been fully elucidated. Since sufficient anti-tumor effect with limited toxicity to normal tissues is an essential factor for the clinical application of radiosensitizers, the tumor-specific radiosensitizing effect of C646 should be further investigated in animal models. Our results suggest that the radiosensitizing effect of C646 is mainly mediated by the enhancement of mitotic catastrophe after IR. G2 checkpoints activated after IR allow the repair of DNA damage before it is replicated and passed on to daughter cells, and abrogation of the G2 checkpoint forces the cells with prematurely repaired DNA damage into M phase, leading to mitotic catastrophe [20]. CHK1 and CHK2 are key signal transducers in the DNA damage checkpoint signaling pathway. Especially, the phosphorylation of CHK1 at Ser345 by ATR (ataxia telangiectasia mutated and Rad3 related) has a pivotal role in the activation and maintenance of the G2 checkpoint [12–15]. In the present study, C646-treated A549 cells (but not C646-treated HFL-III cells) showed a significant reduction in p-CHK1 expression at 60 min and 120 min after IR (Fig. 4). These results indicate that the C646-induced enhancement of mitotic catastrophe in irradiated cancer cells can be attributed, at least in part, to the suppression of the phosphorylation of CHK1 after IR, which may abrogate G2 checkpoint. However, the mechanisms by which C646 affects the phosphorylation of CHK1 after IR remain unknown. Since p300 has been shown to interact with more than 400 proteins [21], it is difficult to identify the proteins responsible for these mechanisms. 53BP1 and MDC1, which are involved in the maintenance of the G2 checkpoint [13] and interact with p300 [22], can be possible candidates. Besides, p300-mediated down-regulation of BRCA1 in cancer cells may, in
5
part, be involved in these mechanisms, since BRCA1 acts upstream of the ATR-CHK1 signaling pathway [19]. Further investigation on this issue is underway. In conclusion, we showed that C646 (i) radiosensitized NSCLC cell lines of major histological types; (ii) enhanced mitotic catastrophe after IR; and (iii) suppressed the phosphorylation of CHK1 and after IR. Mitotic catastrophe is one of the typical mechanisms of cell death in cancer cells induced by IR [23]. Therefore, the enhancement of mitotic catastrophe by C646 is a reasonable and attractive radiosensitization strategy that should be carefully validated in future in vivo studies.
Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgments This work was supported by the Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan for Scientific Research on Innovative Areas (22131006); from the Japan Society for the Promotion of Science for Young Scientists (B) KAKENHI (23701110); and from the National Cancer Center Research and Development Fund. This work was also supported by the Program for Cultivating Global Leaders in Heavy Ion Therapeutics and Engineering, and by the Program for Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.radonc.2014. 03.015. References [1] Rossetto D, Truman AW, Kron SJ, et al. Epigenetic modifications in doublestrand break DNA damage signaling and repair. Clin Cancer Res 2010;15: 4543–52. [2] Oike T, Ogiwara H, Torikai K, et al. Garcinol, a histone acetyltransferase inhibitor, radiosensitizes cancer cells by inhibiting non-homologous end joining. Int J Radiat Oncol Biol Phys 2012;84:815–21. [3] Sandur SK, Deorukhkar A, Pandey MK, et al. Curcumin modulates the radiosensitivity of colorectal cancer cells by suppressing constitutive and inducible NF-jB activity. Int J Radiat Oncol Biol Phys 2009;75:534–42. [4] Sun Y, Jiang X, Chen S, et al. Inhibition of histone acetyltransferase activity by anacardic acid sensitizes tumor cells to ionizing radiation. FEBS Lett 2006;580: 4353–6. [5] Ogiwara H, Ui A, Otsuka A, et al. Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors. Oncogene 2011;5: 2135–46. [6] Bowers EM, Yan G, Mukherjee C, et al. Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor. Chem Biol 2010;17:471–82. [7] Russo AL, Kwon HC, Burgan WE, et al. In vitro and in vivo radiosensitization of glioblastoma cells by the poly (ADP-ribose) polymerase inhibitor E7016. Clin Cancer Res 2009;15:607–12. [8] Gupta S, Koru-Sengul T, Arnold SM, et al. Low-dose fractionated radiation potentiates the effects of cisplatin independent of the hyper-radiation sensitivity in human lung cancer cells. Mol Cancer Ther 2011;10: 292–302. [9] Cariveau MJ, Stackhouse M, Cui XL, et al. Clofarabine acts as radiosensitizer in vitro and in vivo by interfering with DNA damage response. Int J Radiat Oncol Biol Phys 2008;70:213–20. [10] Eom YW, Kim MA, Park SS, et al. Two distinct modes of cell death induced by doxorubicin: apoptosis and cell death through mitotic catastrophe accompanied by senescence-like phenotype. Oncogene 2005;24:4765–77. [11] Bolderson E, Richard DJ, Zhou BB, et al. Recent advances in cancer therapy targeting proteins involved in DNA double-strand break repair. Clin Cancer Res 2009;15:6314–20.
Please cite this article in press as: Oike T et al. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.015
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[12] Kousholt AN, Fugger K, Hoffmann S, et al. CtIP-dependent DNA resection is required for DNA damage checkpoint maintenance but not initiation. J Cell Biol 2012;197:869–76. [13] Shibata A, Barton O, Noon AT, et al. Role of ATM and the damage response mediator proteins 53BP1 and MDC1 in the maintenance of G2/M checkpoint arrest. Mol Cell Biol 2010;30:3371–83. [14] Menzel T, Nahse-Kumph V, Kousholt AN, et al. A genetic screen identifies BRCA2 and PALB2 as key regulators of G2 checkpoint maintenance. EMBO Rep 2011;12:705–12. [15] Hurley PJ, Bunz F. ATM and ATR, components of an integrated circuit. Cell Cycle 2007;6:414–7. [16] Balasubramanyam K, Altaf M, Varier RA, et al. Polysoprenylated Benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression. J Biol Chem 2008;279: 33716–26. [17] Karamouzis MV, Konstantinopoulos PA, Papavassiliou AG. Roles of CREBbinding protein (CBP) p300 in respiratory epithelium tumorigenesis. Cell Res 2007;17:324–32.
[18] Huhn D, Bolck HA, Sartori AA. Targeting DNA double-strand signalling and repair: recent advance in cancer therapy. Swiss Med Wkly 2013;143: w13837. [19] Ogiwara H, Kohno T. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes. PLoS One 2012;7:e52810. [20] Bucher N, Britten CD. G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer. Br J Cancer 2008;98:523–8. [21] Bodfold DC, Kasper LH, Fukuyama T, Brindle PK. Target gene context influences the transcriptional requirement for the KAT3 family of CBP and p300 histone acetyltransferases. Epigenetics 2010;5:9–15. [22] Jung SY, Malovannaya A, Wei J, O’Malley BW, Qin J. Proteomic analysis of steady-state nuclear hormone receptor coactivator complexes. Mol Endocrinol 2005;19:2451–65. [23] Wouters BG. Cell death after irradiation: how, when and shy cells die. In: Joiner M, van der Kogel A, editors. Basic clinical radiobiology. Philadelphia: Hodder Arnold; 2009. p. 27–40.
Please cite this article in press as: Oike T et al. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.015
Contents lists available at ScienceDirect
Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
Original article
C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe Takahiro Oike a,b, Mayumi Komachi a, Hideaki Ogiwara b, Napapat Amornwichet a, Yuka Saitoh a, Kohta Torikai c, Nobuteru Kubo a, Takashi Nakano a, Takashi Kohno b,⇑ a c
Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi; b Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku; Gunma University Heavy Ion Medical Center, Maebashi, Japan
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 3 March 2014 Accepted 18 March 2014 Available online xxxx Keywords: Radiosensitizer p300 Histone acetyltransferase Mitotic catastrophe G2 checkpoint
a b s t r a c t Background and purpose: Chromatin remodeling through histone modifications, including acetylation, plays an important role in the appropriate response to DNA damage induced by ionizing radiation (IR). Here we investigated the radiosensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase, and explored the underlying mechanisms. Materials and methods: A549, H157 and H460 human non-small cell lung carcinoma (NSCLC) cells, and HFL-III human lung fibroblasts were assessed by clonogenic survival assay. Apoptosis and necrosis were assessed by annexin V staining. Senescence was assessed by Senescence-associated b-galactosidase staining. Mitotic catastrophe was assessed by evaluating nuclear morphology with DAPI staining. Cell cycle profiles were analyzed by flow cytometry. Protein expression was analyzed by immunoblotting. Results: C646 sensitized A549, H460 and H157 cells to IR with a dose enhancement ratio at 10% surviving fraction of 1.4, 1.2 and 1.2, respectively. C646 did not radiosensitize HFL-III cells. In A549 cells, but not in HFL-III cells, C646 (i) enhanced mitotic catastrophe but not apoptosis, necrosis, or senescence after IR; (ii) increased the hyperploid cell population after IR; and (iii) suppressed the phosphorylation of CHK1 after IR. Conclusions: C646 radiosensitizes NSCLC cells by enhancing mitotic catastrophe through the abrogation of G2 checkpoint maintenance. Ó 2014 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2014) xxx–xxx
Ionizing radiation (IR)-induced DNA damage leads to the generation of DNA double-strand breaks (DSBs), resulting in cell death. A critical component of the DNA damage response (DDR) after IR is the induction of dynamic changes in chromatin structure called chromatin remodeling [1]. Post-translational histone modifications play critical roles in chromatin remodeling after IR by mediating the recognition and signaling of DNA damage, repair of DNA damage sites, and the regulation of cell cycle checkpoints. Therefore, compounds that specifically target the enzymes responsible for these histone modifications have high potential as radiosensitizing agents. Histone acetyltransferases (HATs) catalyze the acetylation of histone proteins, which is a process required for chromatin remodeling at DSB sites. We and others have previously demonstrated that natural compounds with multi-HAT-inhibitory activity such as garcinol, curcumin and anacardic acid have a radiosensitizing effect in vitro [2–4]. Moreover, we have shown that siRNA-mediated ⇑ Corresponding author. Address: Division of Genome Biology, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. E-mail address: [email protected] (T. Kohno).
ablation of the HATs p300 and CBP sensitizes cancer cells to IR [5]. These data indicate that the inhibition of HATs is a promising strategy for radiosensitization. However, radiosensitization by selective small molecule inhibitors targeting a specific HAT, which could have a significant clinical potential, has not been well investigated. Recently, Bowers et al. showed that the small molecule compound C646 has selective inhibitory activity against p300 [6]. In that study, an in silico virtual ligand screening based on the Xray structure of the HAT active site of p300 that included approximately 500,000 commercially available small molecules identified C646 as a competitive inhibitor of p300 that antagonizes acetylCoA, a substrate of p300 for acetylation, and C646 was shown to suppress histone acetylation in human cells in vitro. These findings prompted us to investigate the effect of C646 on radiosensitization of human cancer cells. Here, we show that C646 radiosensitizes non-small cell lung carcinoma (NSCLC) cells but not non-cancerous cells, and that C646 suppresses the phosphorylation of the checkpoint kinase CHK1 after IR in cancer cells, leading to the enhancement of mitotic catastrophe.
http://dx.doi.org/10.1016/j.radonc.2014.03.015 0167-8140/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Oike T et al. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.015
2
Radiosensitization by a p300 inhibitor
Materials and methods Materials The human NSCLC cell lines A549 (adenocarcinoma), H157 (squamous cell carcinoma) and H460 (large cell carcinoma), and human lung fibroblast lines HFL-III and MRC-5, were cultured in RPMI-1640 supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. C646 was purchased from R&D (Minneapolis, MN, USA). Dimethyl sulfoxide (DMSO) purchased from Sigma (St Louis, MO, USA) was used as a solvent for the drugs. Antibodies were purchased from Epitomics (Burlingame, CA, USA) (Chk1, 1740-1), Cell Signaling Technology (Danvers, MA, USA) (Phospho-Chk1 Ser345, 133D3; Phospho-Chk2 Thr68, C13C1; b-actin, 3700P), and BD (Franklin Lakes, NJ, USA) (Chk2, 611571). Irradiation Cells received IR using a 60Co c-ray source (Atomic Energy of Canada, Ltd., Ontario, Canada) with a dose rate of 9.1 Gy/min.
using Prolong Gold (Life Technologies, Carlsbad, CA, USA). Confocal images were taken using an inverted microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 100 oil lens. Images were acquired using ZEN Software (2008, Carl Zeiss). Two or more distinct nuclear lobes within a single cell were scored as positive for mitotic catastrophe [7]. The percentage of cells undergoing mitotic catastrophe was quantified by counting at least 300 cells in each experimental condition. Cell cycle analysis Cells were exposed (or not) to C646 at 37 °C for 2 h, and subjected (or not) to IR. After an additional incubation for 72 h, the cells were trypsinized, centrifuged, washed in PBS, and fixed in ice-cold 70% ethanol. The cells were then re-centrifuged and incubated with PBS containing 200 lg/mL RNaseA and 5 lg/mL propidium iodide (PI), and cell cycle distribution was analyzed by Guava easyCyte HT. Immunoblot analysis
Clonogenic survival assay
Immunoblot analysis was performed as described previously [5].
Cells were seeded in 6-well plates, incubated at 37 °C for 4–10 h for attachment, and exposed (or not) to C646. After incubation for 2 h, the cells were subjected (or not) to IR and incubated for 10 days for colony formation. The cells were fixed with methanol and stained with crystal violet. Colonies of at least 50 cells were counted. The surviving fraction was normalized to the corresponding controls. The dose required to reduce the surviving fraction to 10% (D10) of the irradiated cells was calculated by using the linearquadratic model as described previously [2].
Experiments were performed at least in triplicate. The significance of differences was determined by the unpaired Student’s t-test and Welch’s test in the cases where the standard deviations (SDs) of two populations were equal or not equal, respectively, using StatMateIII ver. 3.17 software (ATMS, Tokyo, Japan). A P value 4 N DNA) cell population, consistent with the induction of mitotic catastrophe [10]. Meanwhile, combined treatment did not significantly increase the hyperploid cell population in HFL-III cells. Taken together, these data indicate that the phenotype of cancer cells (but not that of non-cancerous cells) increases their susceptibility to C646-induced mitotic catastrophe after IR. C646 suppresses phosphorylation of CHK1 after IR in A549 cells To explore the mechanisms underlying the enhancement of mitotic catastrophe by C646 in irradiated cells, we examined the effect of C646 on the phosphorylation of the checkpoint kinases CHK1 and CHK2, which is critical for the activation and maintenance of DNA damage checkpoints in response to IR (Fig. 4) [11–15]. The kinetics of phosphorylated CHK1 pSer345 (p-CHK1) expression in A549 and HFL-III cells after IR (D10) were comparable; p-CHK1 expression increased at 30 min after IR and was sustained for 120 min. Interestingly, in the presence of C646 (IC50), p-CHK1 levels in A549 cells (but not those in HFL-III cells) were significantly reduced at 60 min and 120 min after IR. The kinetics of phosphorylated CHK2 pThr68 (p-CHK2) expression after IR were also comparable between the two cell lines; however, in these cases the kinetics were not significantly affected by C646 treat-
Fig. 2. Radiosensitizing effect of C646 assessed by clonogenic survival assay. (a) A549, (b) H157, (c) H460, and (d) HFL-III cells. Average ± SD is shown.
Please cite this article in press as: Oike T et al. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.015
4
Radiosensitization by a p300 inhibitor
Fig. 3. Effect of C646 on cell death in irradiated A549 (a) and HFL-III (b) cells. Treatment with IR and C646 was performed at D10 and IC50 in each cell, respectively. Induction of apoptosis and necrosis was assessed by annexin V staining 72 h after IR. Induction of senescence was assessed by SA-b-Gal staining 6 days after IR. Induction of mitotic catastrophe (MC) was assessed based on the morphology of DAPI-stained nuclei 72 h after IR. Average ± SD is shown. ⁄, P < 0.05; , P < 0.01. Note that the percentages of senescent cells are shown in different scales from the others.
Fig. 4. Effect of C646 on the phosphorylation of CHK1 and CHK2 in irradiated A549 (a) and HFL-III (b) cells. Treatment with IR and C646 was performed at D10 and IC50 in each cell, respectively. The cells harvested at the indicated time points were analyzed by immunoblot with antibodies against CHK1, CHK2, p-CHK1, p-CHK2, and b-actin (loading control). Representative immunoblot images are shown (upper panels). The lower panels show the relative band intensities for p-CHK1 and CHK1 (left hand panel in A and B) and for p-CHK2 and CHK2 (right hand panel in A and B). The intensities of the p-CHK1 bands relative to the CHK1 bands at the same time points are shown after normalization to samples treated with IR alone and harvested at 60 min (the time at which the greatest band intensity was observed). The intensities of the p-CHK2 bands relative to the CHK2 bands at the same time points are shown after normalization to samples treated with IR alone and harvested at 30 min post-RT (the time at which the greatest band intensity was observed). Data are expressed as the mean ± SD. ⁄P < 0.05; P < 0.01.
ment. Taken together, these results suggest that C646 suppresses CHK1 phosphorylation in irradiated cancer cells, which may then alter the cell cycle checkpoint and lead to premature entry into mitosis and the enhancement of mitotic catastrophe.
Discussion In the present study, we showed that C646, a selective small molecule inhibitor of p300, radiosensitized NSCLC cell lines of
Please cite this article in press as: Oike T et al. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.015
T. Oike et al. / Radiotherapy and Oncology xxx (2014) xxx–xxx
three different histological types, but not a non-cancerous cell line. C646 enhanced IR-induced mitotic catastrophe and suppressed the phosphorylation of CHK1 after IR in A549 cells but not in HFL-III cells. Targeting HATs critical for chromatin remodeling in the DDR after IR is a potential radiosensitizing strategy [1]. Therefore, we and others have investigated the radiosensitizing effect of several natural compounds with multiple HAT-inhibitory activities such as garcinol, curcumin and anacardic acid, and showed that they sensitized cancer cells to IR in vitro [2–4]. Garcinol inhibits p300, CBP and PCAF [16]; curcumin inhibits p300 and CBP [17]; and anacardic acid inhibits p300 and TIP60 [4,17]. In the present study, C646 was shown to sensitize NSCLC cells to IR (Fig. 2a–c). Together, these findings demonstrate that p300 is a candidate target for radiosensitization in cancer cells. However, the effects of C646 on proteins other than p300 have not been fully elucidated [6]. Thus, the possibility that C646 may target other proteins in addition to p300 for radiosensitization should be further pursued. The radiosensitizing effect of C646 was not evident in noncancerous HFL-III cells (Fig. 2d), which was consistent with our previous data showing that garcinol, a specific inhibitor of CBP, p300 and PCAF HATs, had no radiosensitizing activity in non-cancerous cells [2]. It is considered that, in the process of malignant transformation from normal cells, cancer cells acquire aberrations in DDR-related factors leading to genomic instability which contributes to the higher vulnerability of cancer cells to IR than normal cells [18]. This may in part explain the cancer-specific radiosensitizing effect by HAT inhibition since the present and previous studies suggested the involvement of HAT in DDR including non-homologous end joining, homologous recombination and cell cycle checkpoints [2,19]. The present study suggests that the type of IR-mediated cell death in cancer and normal cells is different, i.e., IR mainly induced mitotic catastrophe in A549 cells, but induced senescence in the majority of HFL-III cells (Fig. 3). This may also reflect the differences in HAT-mediated radiosensitization in these two cell lines. However, the mechanisms underlying the stronger radiosensitizing effect of HAT inhibition in cancer cells than in normal cells have not been fully elucidated. Since sufficient anti-tumor effect with limited toxicity to normal tissues is an essential factor for the clinical application of radiosensitizers, the tumor-specific radiosensitizing effect of C646 should be further investigated in animal models. Our results suggest that the radiosensitizing effect of C646 is mainly mediated by the enhancement of mitotic catastrophe after IR. G2 checkpoints activated after IR allow the repair of DNA damage before it is replicated and passed on to daughter cells, and abrogation of the G2 checkpoint forces the cells with prematurely repaired DNA damage into M phase, leading to mitotic catastrophe [20]. CHK1 and CHK2 are key signal transducers in the DNA damage checkpoint signaling pathway. Especially, the phosphorylation of CHK1 at Ser345 by ATR (ataxia telangiectasia mutated and Rad3 related) has a pivotal role in the activation and maintenance of the G2 checkpoint [12–15]. In the present study, C646-treated A549 cells (but not C646-treated HFL-III cells) showed a significant reduction in p-CHK1 expression at 60 min and 120 min after IR (Fig. 4). These results indicate that the C646-induced enhancement of mitotic catastrophe in irradiated cancer cells can be attributed, at least in part, to the suppression of the phosphorylation of CHK1 after IR, which may abrogate G2 checkpoint. However, the mechanisms by which C646 affects the phosphorylation of CHK1 after IR remain unknown. Since p300 has been shown to interact with more than 400 proteins [21], it is difficult to identify the proteins responsible for these mechanisms. 53BP1 and MDC1, which are involved in the maintenance of the G2 checkpoint [13] and interact with p300 [22], can be possible candidates. Besides, p300-mediated down-regulation of BRCA1 in cancer cells may, in
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part, be involved in these mechanisms, since BRCA1 acts upstream of the ATR-CHK1 signaling pathway [19]. Further investigation on this issue is underway. In conclusion, we showed that C646 (i) radiosensitized NSCLC cell lines of major histological types; (ii) enhanced mitotic catastrophe after IR; and (iii) suppressed the phosphorylation of CHK1 and after IR. Mitotic catastrophe is one of the typical mechanisms of cell death in cancer cells induced by IR [23]. Therefore, the enhancement of mitotic catastrophe by C646 is a reasonable and attractive radiosensitization strategy that should be carefully validated in future in vivo studies.
Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgments This work was supported by the Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan for Scientific Research on Innovative Areas (22131006); from the Japan Society for the Promotion of Science for Young Scientists (B) KAKENHI (23701110); and from the National Cancer Center Research and Development Fund. This work was also supported by the Program for Cultivating Global Leaders in Heavy Ion Therapeutics and Engineering, and by the Program for Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation.
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Please cite this article in press as: Oike T et al. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.015
