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DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA... Background: Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. Methods: A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2’-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of gH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Results: Pretreatment with psammaplin A, 5-aza-2’-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of gH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Conclusions: Psammaplin A, 5-aza-2’-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair. Keywords: Cancer, Epigenetics, DNA methylation, DNA methyltransferase inhibitor, Radiosensitization Background DNA methylation is controlled by DNA methyltrans- Epigenetic alteration is one of the most important gene ferase (DNMT), an enzyme that catalyzes the transfer of regulatory mechanisms. Unlike genetic alterations, epi- a methyl moiety from S-adenosyl-l-methionine to the 5- genetic events are not changes in gene function that postion of cytosines in the CpG dinucleotide [1]. occur in conjunction with DNA sequence changes. DNMT overexpression has been detected in a variety of Recently, epigenetic studies have been conducted in malignancies, including lung, prostate, and colorectal many different aspects of biology, and particularly in the tumors [2-4]. Because DNA methylation is a reversible biochemical cancer field. DNA methylation and histone modifica- tions are two principal factors in epigenetic phenomena. process, DNMT may be a viable target for the treatment These two mechanisms perform a crucial function in of cancer. Since two cytidine analogues, 5-azacytidine carcinogenesis and tumor progression. and 5-aza-2’deoxycytidine, have been reported in the 1980s, several DNMT inhibitors are currently under investigation for their possible utility in treating a variety * Correspondence: ihkim@snu.ac.kr 1 of tumors [5-7]. Department of Radiation Oncology, Seoul National University College of Medicine, Seoul, Republic of Korea Full list of author information is available at the end of the article © 2012 Kim et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kim et al. Radiation Oncology 2012, 7:39 Page 2 of 10 http://www.ro-journal.com/content/7/1/39 It has become widely accepted that histone modification pretreated with epigenetic drugs before irradiation for 18 and DNA methylation are intricately interrelated in terms hours. Based on these results, all subsequent experiments of affecting chromatin structure and gene expression [8]. were done with DNMT inhibitors added before radiation Because these two parameters have long been implicated for 18 hours [10]. To cancel out additive cytotoxicity of in the regulation of cellular radioresponse, histone deace- DNMT inhibitor and reveal synergism with radiation, we tylase (HDAC) inhibitors and DNMT inhibitors might be normalized surviving fractions of irradiated cells by using considered potential targets for radiosensitization. Actu- corresponding plating efficacies of unirradiated cells ally, several studies have reported that HDAC inhibitors within respective groups. The cells were incubated for 14 to 21 days to allow for the formation of colonies. The such as trichostatin A induce radiosensitization [9-11]. However, relatively little information is currently available colonies formed were fixed with methanol and stained concerning the use of DNMT inhibitors in this context with 0.5% crystal violet; the number of colonies contain- [12,13]. This allows us to evaluate the functions of DNMT ing at least 50 cells was determined, and the surviving inhibitors as radiosensitizing agents. fraction (SF) was then calculated. Each point on the sur- We tried to assess the influence of a variety of DNMT vival curves represents the mean surviving fraction from inhibitors on radiosensitivity in two human cancer cell at least three dishes. lines of different histologic origins, and to elucidate the mechanisms relevant to those influences. Flow cytometric analysis The effects of DNMT inhibitors on cell cycles were ana- Methods lyzed via flow cytometry. All centrifugation procedures Cell culture and DNMT inhibitors were conducted for 4 min at 1500 rpm. The cells were In this study, two different cancer cell lines were chosen: treated with RPMI or an IC s concentration of DNMT A549, a human lung cancer cell line harboring wild-type inhibitors for 18 hours. The cells were sham-irradiated or p53, and U373MG, a human glioblastoma cell line harbor- irradiated with 6 Gy of 6 MV x-ray and were collected at ing inactive mutant p53. The A549 and U373MG cell lines 0, 2, 6, 12 and 24 hours after radiation. The cells were har- were purchased from the Korean Cell Line Bank. Cells vested at the indicated times and fixed in 2 mL of 80% were cultured at 37°C in water saturated with 5% CO . ethanol for 24 hours for fixation. The fixed cells were then The cultures were maintained in RPMI media (Welgene, washed twice in PBS, and suspended in PBS on ice for Daegu, Korea), supplemented with 10% fetal bovine serum 5 min. After centrifugation, the cell pellets were washed and 12.5 μg/ml of gentamicin. and resuspended in 5 μg/ml propidium iodide (Molecular 5-azacytidine, 5-aza-2’-deoxycytidine, zebularine, hydra- Probes, Eugene, OR, USA) and 0.1% RNase A (Sigma). At lazine, epigallocatechin gallate (EGCG), and psammaplin least 1 × 10 events were counted. A were obtained from Sigma Chemical Co. (St. Louis, MO, USA), and dissolved as concentrated stock solutions Western blot for DNMT1, DNMT3A/3B, and cleaved in DMSO, stored at -20°C, and diluted in the respective caspase-3 culture media at the time of use. Control cells were treated Cell lysates were prepared in cell lysis buffer (iNtRON with media containing an equal concentration of the drug Biotechnology, Seoul, Korea). The total cellular proteins carrier, DMSO. (50 μg) were separated on SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corp., Bedford, MA, Clonogenic assay USA). The membranes were blocked with blocking solu- Cells were trypsinized from the exponentially growing tion in 5% nonfat dry milk (25 mM Tris, pH 7.5; 0.15 M monolayer cultures. The appropriate numbers of cells NaCl; 0.05% Tween) for 1 hour and probed with primary were seeded into T25 flasks, and then incubated for rabbit polyclonal IgG antibody at a dilution of 1:1,000 24 hours prior to treatment. To compare the combined overnight. The antibody for DNMT1 was obtained from cytotoxic effect of DNMT inhibitors and radiation with Abcam (Cambridge,UK),and DNMT3A,DNMT3Band that of radiation alone, radiation was administered with 6 cleaved caspase-3 antibody were purchased from Cell MV of x-rays from a linear accelerator (Clinac 2100 C or Signaling Technology (Beverly, MA, USA). The mem- Clinac 21EX, Varian Medical systems, Palo Alto, CA, branes were incubated with blocking solution containing USA) with graded doses of x-rays. In combined treat- a dilution of HRP-conjugated goat anti-rabbit IgG as a ment, DNMT inhibitors (5-azacytidine, 5-aza-2’-deoxycy- secondary antibody (Santa Cruz, Biotechnology, CA, tidine, zebularine, hydralazine, EGCG and psammaplin USA) at 1:2,000 for 2 hours. Western blot protein detec- A) were administered for 18 hours prior to radiation. tion was conducted using the ECL kit (Intron Biotechnol- Ourpreviousstudies forepigentics, in whichHDAC ogy, Seongnam, Korea) according to the manufacturers’ inhibitors had been used, showed that the greatest degree recommendations. As a control, monoclonal antibody of radiosensitization was observed when cells are against actin (Santa Cruz) was utilized. Kim et al. Radiation Oncology 2012, 7:39 Page 3 of 10 http://www.ro-journal.com/content/7/1/39 Immunocytochemistry Table 1 IC values for six DNMT inhibitors Cells were grown and treated in tissue culture chamber Drugs Cell lines slides (Nalge Nunc International, Naperville, IL, USA). A549 U373MG At the specified times, the medium was aspirated and 5-azacytine 3 uM 3 uM the cells were fixed in 4% paraformaldehyde for 10 min- 5-aza-2’-deoxycytidine 300 nM 100 nM utes at room temperature. The paraformaldehyde was Zebularine 800 uM ND* aspirated, and the cells were treated for 15 min with a Hydralazine 2 uM 20 uM 0.2% NP40/PBS solution. The cells were then washed Epigallocatechin gallate 15 uM 5 uM twice in PBS, and anti-gH2AX antibody (Cell Signaling Psammaplin A 5 ug/ml 5 ug/ml Technology) was added at a dilution of 1:200 in 1% *ND; not determined, IC value in U373MG, 800 uM, was used in subsequent bovine serum albumin and incubated overnight at 4°C. experiments in A549 cells The cells were again washed twice in PBS prior to 1 hour of incubation in the dark with an FITC-labeled because no significant inhibition of cell growth was secondary antibody (Invitrogen, Camarillo, CA, USA) at observed at any dose. Thus, IC value in U373MG, 800 a dilution of 1:50 in 1% bovine serum albumin. The sec- uM, was used in subsequent experiments in A549 cells. ondary antibody solution was then aspirated and the cells were washed twice in PBS, followed by 30 minutes Effects of DNMT inhibitors on radiosensitivity of A549 of incubation in the dark with 4’,6diamindino-2-pheny- and U373MG cell lines lindole (1 μg/mL) in PBS and two subsequent washings. Survival curves of A549 and U373MG cells treated with The coverslips were then mounted with antifade solu- DNMT inhibitors and radiation were compared with tion (Vector Laboratories, Burlingame, CA, USA). Slides those of cells treated with radiation alone. Among six were examined with a Leica DMRXA fluorescent micro- DNMT inhibitors, psammaplin A, 5-aza-2’-deoxycyti- scope (Leica, Wetzlar, Germany). The images were cap- dine, and zebularine pretreatment significantly enhanced tured using a Photometrics Sensys CCD camera (Leica) radiation cell killing in both A549 and U373MG cells and imported into the IP Labs image analysis software lines (Figure 1A and 1B). Using the dose required to package (Leica). For each treatment condition, the num- generate a SF of 0.5 as a reference, the dose enhance- bers of gH2AX foci were counted in 50 cells. Cells were ment ratios (DER) were estimated. The DERs of psam- classified positive (i.e., containing radiation-induced maplin A, 5-aza-2’-deoxycytidine, and zebularine for the gH2AX foci) when more than five foci were detected A549 cell line were 1.29, 1.96, and 1.69, respectively and [14]. for U373MG were 1.29, 1.55, and 2.13, respectively. Statistics Effects of DNMT inhibitors on DNMT expression Kaleidagraph version 3.51 (Synergy Software, Reading, DNMT1, DNMT3A, and DNMT3B are the three main PA, USA) was utilized to fit the survival data of irra- functional methyltransferases responsible for the estab- diated cells into a linear quadratic (LQ) model and to lishment and maintenance of DNA methylation patterns estimate the a and b values. The LQ model was defined in mammals. The effects of DNMT inhibitors on the as follows: levels of DNMT expression were analyzed via Western blotting using specific antibodies against DNMT1, - (αd+βd2) SF = e DNMT3A, and DNMT3B. Western blot analysis revealed a drastic depletion of DNMT1 and DNMT3A Differences in mean values between groups were com- by psammaplin A, 5-aza-2’-deoxycytidine, and zebular- pared using Student t-test. Probability values of p < 0.05 ine in both A549 and U373MG cell lines. However, no were regarded as statistically significant. depletion of DNMT3B by the three DNMT inhibitors was observed in either of the cell lines (Figure 2). These Results results indicate that DNMT inhibitors induce selective Cytotoxic effect and determination of IC s of DNMT demethylation in each of the evaluated tumor cell lines. inhibitors Increasing concentrations of six DNMT inhibitors Mechanisms of radiosensitization reduced the viability of two cell lines. The 50% inhibi- Cell cycle and apoptosis were evaluated by flow cytome- tory concentrations (IC s) were determined after an 18- try. Both cell lines evidenced a G2/M delay after radia- hour exposure to six DNMT inhibitors, and those con- tion treatment alone. Although the effects of combining centration were used in subsequent experiments. The DNMT inhibitors and radiation vary between cell lines, IC sofDNMTinhibitorsare showninTable 1.IC 50 50 we noted no significant differences in cell cycle phase value of zebularine in A549 cells could not be obtained Kim et al. Radiation Oncology 2012, 7:39 Page 4 of 10 http://www.ro-journal.com/content/7/1/39 Figure 2 Methylation status was determined after exposure to DNMT inhibitors using Western blot analysis of DNMT1, 3A/3B. The cells were treated with DNMT inhibitors for 18 hours. A drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza- 2’deoxycytidine, and zebularine in both A549 and U373MG cell lines was observed. However, there was no depletion of DNMT3B by the three DNMT inhibitors in either of the cell lines. Each blot is representative of two independent experiments, with actin used as a loading control. Figure 1 The effects of DNMT inhibitors on tumor cell radiosensitivity. Survival curves of (A) A549 cells and (B) U373MG distinguished after the 6 Gy of radiation. The gH2AX cells treated with the respective DNMT inhibitors prior to radiation expression of cells treated with radiation alone was and radiation were compared with those of radiation alone. Points, mean for three independent experiments; bars, SE. compared with those treated with a combination of DNMT inhibitors and radiation. Although gH2AX foci expression was shown to be reduced in cells treated distribution patterns between cells treated with radiation with radiation alone over time, gH2AX foci levels in the alone and those treated with a combination of radiation cells exposed to DNMT inhibitors prior to radiation combined with DNMT inhibitors (Figure 3). However, remained constant over a 12-hour time course in A549 in the A549 cells, radiation-induced G2/M arrest was cells and over a 24-hour time course in U373MG cells abrogated by zebularine pretreatment at 6-12 hours, but (Figure 7). These results implicate an inhibition of the this abrogation disappeared at 24 hours (Figure 3 and DNA damage repair process as the possible mechanism 4). underlying the effects of DNMT inhibitors on Psammaplin A pretreatment increased the sub-G1 radiosensitization. fraction of A549 cells, as compared to cells exposed to radiation alone (Table 2). Western blotting also revealed Discussion an increased expression of cleaved caspase-3 in psam- Recently, histone modifications and DNA methylation, maplin A pretreated A549 cells (Figure 5). However, both of which are prominent epigenetic mechanisms, psammaplin A exerted no effect on the apoptotic induc- have been evaluated with a view toward enhancing the tion of U373MG cell lines. Pretreatment with other radiosensitivity of tumor cells via the regulation of chro- DNMT inhibitors did not influence the expression of matin structure modifications and the expression of cleaved caspase-3 in either of the cell lines. genes involved in cell cycle checkpoints, apoptosis, and gH2AX has been identified as a marker of DNA dou- DNA repair. A number of investigators have previously ble-strand break (DSB) [15]. Immunocytochemical ana- reported that several HDAC inhibitors exert direct cyto- lysis using the anti-gH2AX antibodies was conducted in toxic effects, and can sensitize tumor cells to radiother- order to determine the effects of DNMT inhibitors on apy [14,16-19]. Kim et al. also demonstrated that DNA repair. As shown in the representative micro- trichostatin A, which is the most potent HDAC inhibi- graphs in Figure 6, gH2AX foci could be clearly tor identified thus far, enhanced radiosensitivity in a Kim et al. Radiation Oncology 2012, 7:39 Page 5 of 10 http://www.ro-journal.com/content/7/1/39 Figure 3 Influence of DNMT inhibitors on cell cycle phase distributions of A549 and U373MG cells. Cell cycle phase was measured using flow cytometry at 12 hours after 6 Gy of radiation. Columns, proportion of cell cycle phase; bars, SE. variety of human cancer cell lines [11,20]. Unlike the radioresponse, it is critically important to evaluate the HDAC inhibitors, however, little information is cur- effects of DNMT inhibitors on radiosensitization. In rently available regarding the effects of DNMT inhibi- order to assess this possibility, we employed a total of six tors on radiosensitization [12,13]. DNMT inhibitors. Among them, 5-aza2’-deoxycytidine, There are two classes of DNMT inhibitors: the nucleo- zebularine, and psammaplin A were shown to exert a side analogues and the non-nucleoside analogues. Several radiosensitizing effect in both A549 and U373MG cells. compounds are currently being evaluated in preclinical Although some differences in the degrees of radiosensiti- and clinical trials for the treatment of solid and hemato- zation were detected according to the different DNMT logical malignancies. Contrary to their effect in hemato- inhibitors and cell lines used, these results demonstrate logic malignancies, the clinical effects of these that some DNMT inhibitors may prove to be useful compounds against solid tumors have yet to be evaluated radiation sensitizers in human cancer cells. [21]. Therefore, the use of epigenetic therapies in combi- 5-aza-2’-deoxycytidine, along with 5-azacytidine, is the nation with other regimens, such as conventional che- first DNMT inhibitor reported, and evidences clinical motherapeutics or radiotherapy, should be considered. efficacy in cases of myelodysplastic syndrome and acute As DNA methylation is intricately interrelated with chro- myelogenous leukemia. Several previous studies have matin structure and gene expression, parameters long demonstrated that 5-aza-2’-deoxycytidine exerts a radio- thought to be involved in the regulation of cellular sensitizing effect, with or without HDAC inhibitors, in a variety of cancer cell lines [13,22,23]. Recently, the radio- sensitization effect of 5-azacytidine was also referenced by Hofstetter et al., who demonstrated that 5-azacyti- dine-induced genomic hypomethylation induces enhanced radiation sensitivity in colorectal carcinoma [24]. These results are logical consequences, considering that 5-azacytidine has a molecular structure and exerts clinical effects similar to those of 5-aza-2’-deoxycytidine. However, in this study, 5-azacytidine did not sensitize either of the tested cancer cell types to radiotherapy. 5- azacytidine may exert differing effects on radiosensitivity according to cell type, and the IC of 5-azacytidine var- ies among different cell lines. Although this may explain, at least in part, the different results detected in our study, we did not successfully generate a definitive explanation for the absence of any detectable radiosensitizing effect of 5-azacytidine. Because very few studies have addressed Figure 4 The effect of zebularine on radiation-induced G2/M the role of 5-azacytidine as a radiosensitizer, further stu- arrest in A549 cells. A549 cells were treated with 800 uM dies will be required to resolve this issue. zebularine for 18 hours and then irradiated with 6 Gy. A549 cells were accumulated in G2/M phase after radiation. This radiation- Zebularine, another 5-azacytidine derivative, was also induced G2/M arrest was abrogated by zebularine pretreatment at shown in this study to induce radiosensitivity. Zebularine 6-12 hours, but this abrogation disappeared at 24 hours. has been demonstrated to have anticancer properties, Kim et al. Radiation Oncology 2012, 7:39 Page 6 of 10 http://www.ro-journal.com/content/7/1/39 Table 2 Apoptotic rates measured by the sub-G1 portion of flow cytometry in A549 and U373MG cells Control/drug 0 h 2 h 6 h 12 h 24 h A549 IR 1.11 ± 0.93 1.55 ± 0.82 3.01 ± 1.93 1.72 ± 0.83 1.20 ± 0.99 1.03 ± 0.33 PSA + IR 2.61 ± 1.05 1.53 ± 0.73 3.29 ± 1.71 8.02 ± 2.49 9.02 ± 3.20 7.72 ± 2.16 IR 0.53 ± 0.10 1.23 ± 1.07 0.98 ± 0.37 0.70 ± 0.06 1.13 ± 0.88 0.89 ± 0.12 DAC + IR 0.43 ± 0.10 1.20 ± 0.70 0.54 ± 0.02 0.57 ± 0.09 0.53 ± 0.07 1.05 ± 0.14 IR 0.59 ± 0.16 0.94 ± 0.60 0.61 ± 0.15 0.86 ± 0.33 1.77 ± 0.66 1.66 ± 0.14 ZEB + IR 0.84 ± 0.32 1.00 ± 0.61 0.97 ± 0.49 1.65 ± 0.52 1.62 ± 0.14 2.13 ± 0.05 U373MG IR 0.95 ± 0.10 0.61 ± 0.23 0.80 ± 0.18 0.71 ± 0.09 1.39 ± 0.20 1.68 ± 0.55 PSA + IR 0.71 ± 0.19 0.67 ± 0.13 0.55 ± 0.06 0.63 ± 0.05 0.89 ± 0.16 1.57 ± 0.61 IR 0.94 ± 0.05 0.64 ± 0.13 0.61 ± 0.05 0.64 ± 0.05 0.99 ± 0.20 1.68 ± 0.65 DAC + IR 0.77 ± 0.04 0.83 ± 0.01 0.58 ± 0.05 0.81 ± 0.08 0.93 ± 0.12 1.45 ± 0.37 IR 1.58 ± 0.25 1.39 ± 0.34 1.35 ± 0.01 1.12 ± 0.21 1.78 ± 0.34 2.33 ± 0.35 ZEB + IR 0.95 ± 0.19 1.90 ± 1.21 2.14 ± 1.48 1.06 ± 0.01 1.28 ± 0.15 4.59 ± 1.23 Abbreviations: IR = irradiation, PSA = psammplin A, DAC = 5-aza-2’-deoxycytidine, ZEB = zebularine which can cause re-expression of the epigenetically been confirmed. As psammaplin A has not yet been the silenced tumor suppressor p16 in solid malignancies. subject of clinical trials, further clinical research should However, its clinical development is limited by its higher be conducted to determine its efficacy when adminis- dosage and poor bioavailability, as observed previously in tered in combination with radiotherapy. rats, mice, and monkeys [6,25,26]. Owing to the relatively Three different DNMTs exist, all of which are viable tar- high toxicityof thesenucleosideanaloguecompounds, gets for DNMT inhibitors. DNMT1, the most abundant of non-nucleoside DNMT inhibitors are currently being the three, is responsible for methylation during DNA investigated for their possible use as anticancer agents. replication (maintenance methyltransferase). Other known The natural marine product, psammaplin A, was initi- methyltransferases include DNMT3A and DNMT3B, which exhibit identical preferences for hemi-methylated ally isolated from the Psammaplysilla sponge in 1987. Since that time, psammaplin A has been shown to exert potent cytotoxicity against several cancer cell lines, via the selective induction of genes associated with cell cycle arrest and apoptosis [27,28]. Because psammaplin A has already been shown to exhibit potent histone dea- cetylase inhibitory activity, this drug is a promising radiosensitizing agent [28]. This is the first study in which the radiosensitizing effects of psammaplin A have Figure 5 Western blot analysis of cleaved caspase-3.Thecells were treated with a combination of three different DNMT inhibitors Figure 6 Radiation-induced gH2AX foci.Representative and 6 Gy of radiation. Increased cleaved caspase-3 protein level was micrographs obtained from (A) control cells and (B) cells that had observed in cells treated with a combination of psammaplin A and received 6 Gy of radiation 1 hour earlier. (A) top panel, (B) bottom 6 Gy of radiation in the A549 cells. panel. Kim et al. Radiation Oncology 2012, 7:39 Page 7 of 10 http://www.ro-journal.com/content/7/1/39 Figure 7 Influence of DNMT inhibitors on radiation-induced gH2AX foci. A549 and U373MG cells growing in chamber slides were exposed to DNMT inhibitors for 18 hours, irradiated, and fixed at specific times for immunocytochemical analyses of nuclear gH2AX foci. Open columns, data from cells receiving radiation alone; grey columns, data from cells that were exposed to psammaplin A and radiation; filled columns, data from cells that were exposed to 5-aza-2’-deoxycytidine and radiation; hatched columns, data from cells that were exposed to zebularine and radiation. Cells with more than five foci per nucleus were classified as positive for radiation-induced gH2AX. Foci were evaluated in 50 nuclei. Bars, SE. *p < 0.01 as determined by a logistic regression compared with radiation alone (6 Gy) group. and non-methylated DNA, and have thus been classified employed in this study, we were unable to ascertain as de novo methyltransferases. However, it has been whether this selective inhibition of DNMT isoforms was demonstrated that DNMT1 and DNMT3A/3B do not a function of the concentration of the drug. appear to be completely distinct in their activities [29]. In addition to the physical modification of chromatin This supposition is based on two lines of evidence: first, structure, cellular processes that can affect intrinsic despite being DNMT1-deficient, a colon adenocarcinoma radiosensitivity were evaluated in this study. Cell cycle, cell line was shown to be capable of retaining 80% of its apoptosis, and DNA repair have all been shown to influ- methylation level while replicating, which implies that ence radiosensitivity. DNMT3 may perform a function in the maintenance of The proportion of cells in the G2/M phase detected in methylation [30]; secondly, forced DNMT1 overexpression this study was not significantly altered by pretreatment in cancer cell lines does, indeed, induce de novo methyla- with DNMT inhibitors, and no abrogation of radiation- tion [31]. Gene silencing of DNMT, which functions as an induced G2/M arrest was noted except in the zebularine- oncogene, has been proposed as a good cancer treatment treated A549 cell line. Radiation-induced G2/M arrest was strategy [32]. In this study, three different DNMT inhibi- abrogated as the result of zebularine pretreatment at 6-12 tors, all of which demonstrated radiosensitizing effects, hours, but this effect disappeared at 24 hours in the A549 depleted DNMT1 and DNMT3A, but no such DNMT3B cell line. In another study, zebularine was not found to depletion was noted in the A549 and U373MG cell lines. result in the abrogation of radiation-induced G2/M arrest Depletion of DNMT, along with several members of the in a U251 glioblastoma cell line [12]. Thus, although there structural maintenance of chromatin proteins (SMCs), appears to be some relationship between cell lines and SMC-associated protein, and heterochromatin proteins, radiation-induced G2/M arrest, it is less likely that the was reported to correlate with chromatin decondensation radiosensitization induced by the DNMT inhibitors [33]. Considering that the effect of chromatin compaction employed in this study is the result of a general inhibition on protection of DNA against radiation-induced DSBs has of G2 checkpoint activation. been relatively well-established [34,35], the relaxation of Apoptosis has previously been regarded as a potential chromatin structure via the downregulation of DNMT mechanism for radiosensitization. Different results have with DNMT inhibitors may be a possible mechanism for been reported regarding the role of apoptosis as a radio- radiosensitization. sensitizing mechanism induced by DNMT inhibitors. Selective inhibition of DNMT isoforms shown in this Dote et al. previously reported that a combination of study may be attributable, in part, to the doses of DNMT radiation and zebularine did not significantly increase inhibitors. Beaulieu et al. previously reported that Wes- the sub-G1 population (apoptotic cells) [12]. On the tern blot analysis with anti-DNMT1, DNMT3A, or other hand, Qui et al. demonstrated that 5-aza-2’-deoxy- DNMT3B antibodies treated with increasing doses of cytidine induces radiosensitization in certain gastric can- DNMT inhibitors effected a selective dose-dependent cer cell lines via induced increases in the apoptotic rate, inhibition of the target isoform of DNMT [36]. However, as evidenced by enhanced expression of the p53, as only the IC values of the DNMT inhibitors were RASSF1, and DAPK gene families [23]. In this study, the 50 Kim et al. Radiation Oncology 2012, 7:39 Page 8 of 10 http://www.ro-journal.com/content/7/1/39 addition of 5-aza-2’-deoxycytidine or zebularine to further investigations are warranted to determine radiation did not increase the apoptosis rate in either of whether or not alterations in the methylation patterns the cell lines. However, exposure to psammaplin A in of a specific gene or set of genes involved in DNA repair the A549 cell line induced a significant increase in might be modulated by DNMT inhibitors, and that apoptotic death. Psammaplin A has been reported to these changes might contribute to the observed exert cytotoxic effects on cancer cells via the selective enhancements of radiosensitivity. Several remain to be determined in future studies. induction of apoptosis-related genes [27]. This effect of First, a unified treatment schedule - specifically, the psammalin A may result in an increase in radiation- administration of DNMT inhibitors 18 hours prior to induced apoptosis. However, in this study, radiation- induced apoptosis was noted only in the A549 cell line, radiation - was employed in this study, and thus the and not in the U373MG cell line. This finding may be optimal treatment schedule of DNMT inhibitors and attributable, at least in part, to p53 expression status. radiation remains to be established. In other studies, The U373MG cell line, which contains mutated p53, DNMT inhibitors were administered for different treat- might be comparatively resistant to radiation-induced ment durations, i.e. 2 ~ 48 hours prior to radiation, con- apoptosis, considering that apoptosis is mediated by the sidering several factors such as drug half-lives and the p53 protein. expression of radiosensitivity-related genes [22,23]. DNA repair is another process involved in the deter- Further investigations into the optimal treatment sche- mination of cellular radiosensitivity. The activation of dule of DNMT inhibitors and radiation for clinical DNA repair of cancer cells after sublethal DNA damage applications will be necessary in the future. induced by radiation might be one of the most impor- Second, another important issue will involve assess- tant factors in resistance. The expression of gH2AX has ments of the synergistic effects of DNMT inhibitors and been recently identified as a sensitive indicator of radia- HDAC inhibitors on radiosensitivity. Although some tion-induced DSBs [37]. In this study, gH2AX expres- previous studies have reported that the combined gene sion in cells treated with a combination of DNMT silencing reversal effect was superior to that of treat- inhibitors (5-aza-2’deoxycytidine and psammaplin A, as ment with a single agent [32,39], only a few studies have well as zebularine) was found to be similar to the results thus far evaluated the influence of this combined effect achieved with radiation treatment at 1 hour after initial on increased radiosensitivity. Third, the mechanisms treatment, but was significantly greater over time. This underlying DNMT inhibitor-induced radiosensitzations need further investigation. We speculate that suppres- finding is generally consistent with the results obtained sion of DNMTs by the DNMT inhibitors was associated in other previous studies, in which zebularine and 5- aza-2’-deoxycytidine were employed as radiosensitizing with enhanced radiosensitivity through the change in agents [12,22]. Dote et al. evaluated the expression of DNA structure. However, the relationship between gH2AX foci after exposure to 2 Gy with and without DNA methylation and cellular radiosensitivity is to be zebularine pretreatment. Whereas zebularine had no elucidated in the future study. effect on radiation-induced gH2AX foci at 1 hour, the number of gH2AX per cell was significantly greater in Conclusions the zebularine-treated cells at 24 hours after irradiation, Taken together, our study indicate that psammaplin A, which suggested the possible presence of unrepaired 5-aza-2’-deoxycytidine, and zebularine have the potential DNA damage [12]. DNA methylation is intimately asso- to increase radiosensitivity in lung cancer A549 and ciated with histone deacetylases in terms of the epige- glioblastoma U373MG cells, most probably by modulat- netic regulation of gene expression. Actually, HDAC ing the impairment of the DNA repair process. Further inhibitors such as LBH589 and MS-275 have been investigations will be required to identify other addi- shown to enhance radiosensitivity through similar tional mechanisms associated with radiosensitivity, and mechanisms as those of the DNMT inhibitors. These to confirm the synergistic effects on radiosensitivity with HDAC inhibitors prolonged gH2AX expression, suggest- other epigenetic drugs, such as the HDAC inhibitors. ing an inhibition of DNA repair [14,38]. A previous pre- Also, future studies should be conducted to determine clinical study demonstrated that an HDAC inhibitor definitively whether the combination of DNMT inhibi- downmodulated the expression of DNA-PK and Rad51, tors and radiation has real potential as a clinical strategy which participated in the recovery of DSB, thereby abro- for the treatment of cancer. gating key cellular pathways involved in DNA DSB repair [17]. These results indicate that the impairment Acknowledgements of DNA DSB repair maybeone of themostcrucial This work was supported by grant No. M2070202001-07 N0202-00111 from mechanisms underlying enhanced radiation responses in the 2008 National Nuclear R&D Program of the Korean Science & Engineering Foundation (KOSEF) and SNUH CRI grant (0420100900). epigenetic phenomena. Based on this assumption, Kim et al. Radiation Oncology 2012, 7:39 Page 9 of 10 http://www.ro-journal.com/content/7/1/39 17. Chinnaiyan P, Vallabhaneni G, Armstrong E, Huang SM, Harari PM: Author details Modulation of radiation response by histone deacetylase inhibition. Int J Department of Radiation Oncology, Seoul National University College of Radiat Oncol Biol Phys 2005, 62(1):223-229. Medicine, Seoul, Republic of Korea. Cancer Research Institute, Seoul National 18. Munshi A, Kurland JF, Nishikawa T, Tanaka T, Hobbs ML, Tucker SL, Ismail S, University College of Medicine, Seoul, Republic of Korea. Department of Stevens C, Meyn RE: Histone deacetylase inhibitors radiosensitize human Radiation Oncology, Seoul National University Bundang Hospital, Seongnam, melanoma cells by suppressing DNA repair activity. Clin Cancer Res 2005, Republic of Korea. Institute of Radiation Medicine, Medical Research Center, 11(13):4912-4922. Seoul National University, Seoul, Republic of Korea. 19. Zhang Y, Jung M, Dritschilo A: Enhancement of radiation sensitivity of human squamous carcinoma cells by histone deacetylase inhibitors. Authors’ contributions Radiat Res 2004, 161(6):667-674. HJK, IHK, Idea and study design, JHK, IAK Analysis and development of 20. Kim IA, No M, Lee JM, Shin JH, Oh JS, Choi EJ, Kim IH, Atadja P, methods. HJK, Manuscript writing. DYP, Technique work. All authors, Review and final approval. Bernhard EJ: Epigenetic modulation of radiation response in human cancer cells with activated EGFR or HER-2 signaling: potential role of Competing interests histone deacetylase 6. Radiother Oncol 2009, 92(1):125-132. The authors declare that they have no competing interests. 21. Aparicio A, Eads CA, Leong LA, Laird PW, Newman EM, Synold TW, Baker SD, Zhao M, Weber JS: Phase I trial of continuous infusion 5-aza-2’- Received: 5 September 2011 Accepted: 20 March 2012 deoxycytidine. Cancer Chemother Pharmacol 2003, 51(3):231-239. Published: 20 March 2012 22. De Schutter H, Kimpe M, Isebaert S, Nuyts S: A systematic assessment of radiation dose enhancement by 5-Aza-2’-deoxycytidine and histone deacetylase inhibitors in head-and-neck squamous cell carcinoma. Int J References Radiat Oncol Biol Phys 2009, 73(3):904-912. 1. Robertson KD: DNA methylation, methyltransferases, and cancer. 23. Qiu H, Yashiro M, Shinto O, Matsuzaki T, Hirakawa K: DNA Oncogene 2001, 20(24):3139-3155. methyltransferase inhibitor 5-aza-CdR enhances the radiosensitivity of 2. Manoharan M, Ramachandran K, Soloway MS, Singal R: Epigenetic targets gastric cancer cells. Cancer Sci 2009, 100(1):181-188. in the diagnosis and treatment of prostate cancer. Int Braz J Urol 2007, 24. Hofstetter B, Niemierko A, Forrer C, Benhattar J, Albertini V, Pruschy M, 33(1):11-18. Bosman FT, Catapano CV, Ciernik IF: Impact of genomic methylation on 3. Lin RK, Hsu HS, Chang JW, Chen CY, Chen JT, Wang YC: Alteration of DNA radiation sensitivity of colorectal carcinoma. Int J Radiat Oncol Biol Phys methyltransferases contributes to 5’CpG methylation and poor 2010, 76(5):1512-1519. prognosis in lung cancer. Lung Cancer 2007, 55(2):205-213. 25. Cheng JC, Yoo CB, Weisenberger DJ, Chuang J, Wozniak C, Liang G, 4. Eads CA, Danenberg KD, Kawakami K, Saltz LB, Danenberg PV, Laird PW: Marquez VE, Greer S, Orntoft TF, Thykjaer T, et al: Preferential response of CpG island hypermethylation in human colorectal tumors is not cancer cells to zebularine. Cancer Cell 2004, 6(2):151-158. associated with DNA methyltransferase overexpression. Cancer Res 1999, 26. 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Camphausen K, Cerna D, Scott T, Sproull M, Burgan WE, Cerra MA, Fine H, deacetylase (HDAC) inhibitor LBH589 increases duration of gamma- Tofilon PJ: Enhancement of in vitro and in vivo tumor cell H2AX foci and confines HDAC4 to the cytoplasm in irradiated non-small radiosensitivity by valproic acid. Int J Cancer 2005, 114(3):380-386. cell lung cancer. Cancer Res 2006, 66(23):11298-11304. Kim et al. Radiation Oncology 2012, 7:39 Page 10 of 10 http://www.ro-journal.com/content/7/1/39 39. Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB: Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 1999, 21(1):103-107. doi:10.1186/1748-717X-7-39 Cite this article as: Kim et al.: DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity. Radiation Oncology 2012 7:39. 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DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

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Copyright © 2012 by Kim et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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Abstract

Background: Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. Methods: A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2’-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of gH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Results: Pretreatment with psammaplin A, 5-aza-2’-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of gH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Conclusions: Psammaplin A, 5-aza-2’-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair. Keywords: Cancer, Epigenetics, DNA methylation, DNA methyltransferase inhibitor, Radiosensitization Background DNA methylation is controlled by DNA methyltrans- Epigenetic alteration is one of the most important gene ferase (DNMT), an enzyme that catalyzes the transfer of regulatory mechanisms. Unlike genetic alterations, epi- a methyl moiety from S-adenosyl-l-methionine to the 5- genetic events are not changes in gene function that postion of cytosines in the CpG dinucleotide [1]. occur in conjunction with DNA sequence changes. DNMT overexpression has been detected in a variety of Recently, epigenetic studies have been conducted in malignancies, including lung, prostate, and colorectal many different aspects of biology, and particularly in the tumors [2-4]. Because DNA methylation is a reversible biochemical cancer field. DNA methylation and histone modifica- tions are two principal factors in epigenetic phenomena. process, DNMT may be a viable target for the treatment These two mechanisms perform a crucial function in of cancer. Since two cytidine analogues, 5-azacytidine carcinogenesis and tumor progression. and 5-aza-2’deoxycytidine, have been reported in the 1980s, several DNMT inhibitors are currently under investigation for their possible utility in treating a variety * Correspondence: ihkim@snu.ac.kr 1 of tumors [5-7]. Department of Radiation Oncology, Seoul National University College of Medicine, Seoul, Republic of Korea Full list of author information is available at the end of the article © 2012 Kim et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kim et al. Radiation Oncology 2012, 7:39 Page 2 of 10 http://www.ro-journal.com/content/7/1/39 It has become widely accepted that histone modification pretreated with epigenetic drugs before irradiation for 18 and DNA methylation are intricately interrelated in terms hours. Based on these results, all subsequent experiments of affecting chromatin structure and gene expression [8]. were done with DNMT inhibitors added before radiation Because these two parameters have long been implicated for 18 hours [10]. To cancel out additive cytotoxicity of in the regulation of cellular radioresponse, histone deace- DNMT inhibitor and reveal synergism with radiation, we tylase (HDAC) inhibitors and DNMT inhibitors might be normalized surviving fractions of irradiated cells by using considered potential targets for radiosensitization. Actu- corresponding plating efficacies of unirradiated cells ally, several studies have reported that HDAC inhibitors within respective groups. The cells were incubated for 14 to 21 days to allow for the formation of colonies. The such as trichostatin A induce radiosensitization [9-11]. However, relatively little information is currently available colonies formed were fixed with methanol and stained concerning the use of DNMT inhibitors in this context with 0.5% crystal violet; the number of colonies contain- [12,13]. This allows us to evaluate the functions of DNMT ing at least 50 cells was determined, and the surviving inhibitors as radiosensitizing agents. fraction (SF) was then calculated. Each point on the sur- We tried to assess the influence of a variety of DNMT vival curves represents the mean surviving fraction from inhibitors on radiosensitivity in two human cancer cell at least three dishes. lines of different histologic origins, and to elucidate the mechanisms relevant to those influences. Flow cytometric analysis The effects of DNMT inhibitors on cell cycles were ana- Methods lyzed via flow cytometry. All centrifugation procedures Cell culture and DNMT inhibitors were conducted for 4 min at 1500 rpm. The cells were In this study, two different cancer cell lines were chosen: treated with RPMI or an IC s concentration of DNMT A549, a human lung cancer cell line harboring wild-type inhibitors for 18 hours. The cells were sham-irradiated or p53, and U373MG, a human glioblastoma cell line harbor- irradiated with 6 Gy of 6 MV x-ray and were collected at ing inactive mutant p53. The A549 and U373MG cell lines 0, 2, 6, 12 and 24 hours after radiation. The cells were har- were purchased from the Korean Cell Line Bank. Cells vested at the indicated times and fixed in 2 mL of 80% were cultured at 37°C in water saturated with 5% CO . ethanol for 24 hours for fixation. The fixed cells were then The cultures were maintained in RPMI media (Welgene, washed twice in PBS, and suspended in PBS on ice for Daegu, Korea), supplemented with 10% fetal bovine serum 5 min. After centrifugation, the cell pellets were washed and 12.5 μg/ml of gentamicin. and resuspended in 5 μg/ml propidium iodide (Molecular 5-azacytidine, 5-aza-2’-deoxycytidine, zebularine, hydra- Probes, Eugene, OR, USA) and 0.1% RNase A (Sigma). At lazine, epigallocatechin gallate (EGCG), and psammaplin least 1 × 10 events were counted. A were obtained from Sigma Chemical Co. (St. Louis, MO, USA), and dissolved as concentrated stock solutions Western blot for DNMT1, DNMT3A/3B, and cleaved in DMSO, stored at -20°C, and diluted in the respective caspase-3 culture media at the time of use. Control cells were treated Cell lysates were prepared in cell lysis buffer (iNtRON with media containing an equal concentration of the drug Biotechnology, Seoul, Korea). The total cellular proteins carrier, DMSO. (50 μg) were separated on SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corp., Bedford, MA, Clonogenic assay USA). The membranes were blocked with blocking solu- Cells were trypsinized from the exponentially growing tion in 5% nonfat dry milk (25 mM Tris, pH 7.5; 0.15 M monolayer cultures. The appropriate numbers of cells NaCl; 0.05% Tween) for 1 hour and probed with primary were seeded into T25 flasks, and then incubated for rabbit polyclonal IgG antibody at a dilution of 1:1,000 24 hours prior to treatment. To compare the combined overnight. The antibody for DNMT1 was obtained from cytotoxic effect of DNMT inhibitors and radiation with Abcam (Cambridge,UK),and DNMT3A,DNMT3Band that of radiation alone, radiation was administered with 6 cleaved caspase-3 antibody were purchased from Cell MV of x-rays from a linear accelerator (Clinac 2100 C or Signaling Technology (Beverly, MA, USA). The mem- Clinac 21EX, Varian Medical systems, Palo Alto, CA, branes were incubated with blocking solution containing USA) with graded doses of x-rays. In combined treat- a dilution of HRP-conjugated goat anti-rabbit IgG as a ment, DNMT inhibitors (5-azacytidine, 5-aza-2’-deoxycy- secondary antibody (Santa Cruz, Biotechnology, CA, tidine, zebularine, hydralazine, EGCG and psammaplin USA) at 1:2,000 for 2 hours. Western blot protein detec- A) were administered for 18 hours prior to radiation. tion was conducted using the ECL kit (Intron Biotechnol- Ourpreviousstudies forepigentics, in whichHDAC ogy, Seongnam, Korea) according to the manufacturers’ inhibitors had been used, showed that the greatest degree recommendations. As a control, monoclonal antibody of radiosensitization was observed when cells are against actin (Santa Cruz) was utilized. Kim et al. Radiation Oncology 2012, 7:39 Page 3 of 10 http://www.ro-journal.com/content/7/1/39 Immunocytochemistry Table 1 IC values for six DNMT inhibitors Cells were grown and treated in tissue culture chamber Drugs Cell lines slides (Nalge Nunc International, Naperville, IL, USA). A549 U373MG At the specified times, the medium was aspirated and 5-azacytine 3 uM 3 uM the cells were fixed in 4% paraformaldehyde for 10 min- 5-aza-2’-deoxycytidine 300 nM 100 nM utes at room temperature. The paraformaldehyde was Zebularine 800 uM ND* aspirated, and the cells were treated for 15 min with a Hydralazine 2 uM 20 uM 0.2% NP40/PBS solution. The cells were then washed Epigallocatechin gallate 15 uM 5 uM twice in PBS, and anti-gH2AX antibody (Cell Signaling Psammaplin A 5 ug/ml 5 ug/ml Technology) was added at a dilution of 1:200 in 1% *ND; not determined, IC value in U373MG, 800 uM, was used in subsequent bovine serum albumin and incubated overnight at 4°C. experiments in A549 cells The cells were again washed twice in PBS prior to 1 hour of incubation in the dark with an FITC-labeled because no significant inhibition of cell growth was secondary antibody (Invitrogen, Camarillo, CA, USA) at observed at any dose. Thus, IC value in U373MG, 800 a dilution of 1:50 in 1% bovine serum albumin. The sec- uM, was used in subsequent experiments in A549 cells. ondary antibody solution was then aspirated and the cells were washed twice in PBS, followed by 30 minutes Effects of DNMT inhibitors on radiosensitivity of A549 of incubation in the dark with 4’,6diamindino-2-pheny- and U373MG cell lines lindole (1 μg/mL) in PBS and two subsequent washings. Survival curves of A549 and U373MG cells treated with The coverslips were then mounted with antifade solu- DNMT inhibitors and radiation were compared with tion (Vector Laboratories, Burlingame, CA, USA). Slides those of cells treated with radiation alone. Among six were examined with a Leica DMRXA fluorescent micro- DNMT inhibitors, psammaplin A, 5-aza-2’-deoxycyti- scope (Leica, Wetzlar, Germany). The images were cap- dine, and zebularine pretreatment significantly enhanced tured using a Photometrics Sensys CCD camera (Leica) radiation cell killing in both A549 and U373MG cells and imported into the IP Labs image analysis software lines (Figure 1A and 1B). Using the dose required to package (Leica). For each treatment condition, the num- generate a SF of 0.5 as a reference, the dose enhance- bers of gH2AX foci were counted in 50 cells. Cells were ment ratios (DER) were estimated. The DERs of psam- classified positive (i.e., containing radiation-induced maplin A, 5-aza-2’-deoxycytidine, and zebularine for the gH2AX foci) when more than five foci were detected A549 cell line were 1.29, 1.96, and 1.69, respectively and [14]. for U373MG were 1.29, 1.55, and 2.13, respectively. Statistics Effects of DNMT inhibitors on DNMT expression Kaleidagraph version 3.51 (Synergy Software, Reading, DNMT1, DNMT3A, and DNMT3B are the three main PA, USA) was utilized to fit the survival data of irra- functional methyltransferases responsible for the estab- diated cells into a linear quadratic (LQ) model and to lishment and maintenance of DNA methylation patterns estimate the a and b values. The LQ model was defined in mammals. The effects of DNMT inhibitors on the as follows: levels of DNMT expression were analyzed via Western blotting using specific antibodies against DNMT1, - (αd+βd2) SF = e DNMT3A, and DNMT3B. Western blot analysis revealed a drastic depletion of DNMT1 and DNMT3A Differences in mean values between groups were com- by psammaplin A, 5-aza-2’-deoxycytidine, and zebular- pared using Student t-test. Probability values of p < 0.05 ine in both A549 and U373MG cell lines. However, no were regarded as statistically significant. depletion of DNMT3B by the three DNMT inhibitors was observed in either of the cell lines (Figure 2). These Results results indicate that DNMT inhibitors induce selective Cytotoxic effect and determination of IC s of DNMT demethylation in each of the evaluated tumor cell lines. inhibitors Increasing concentrations of six DNMT inhibitors Mechanisms of radiosensitization reduced the viability of two cell lines. The 50% inhibi- Cell cycle and apoptosis were evaluated by flow cytome- tory concentrations (IC s) were determined after an 18- try. Both cell lines evidenced a G2/M delay after radia- hour exposure to six DNMT inhibitors, and those con- tion treatment alone. Although the effects of combining centration were used in subsequent experiments. The DNMT inhibitors and radiation vary between cell lines, IC sofDNMTinhibitorsare showninTable 1.IC 50 50 we noted no significant differences in cell cycle phase value of zebularine in A549 cells could not be obtained Kim et al. Radiation Oncology 2012, 7:39 Page 4 of 10 http://www.ro-journal.com/content/7/1/39 Figure 2 Methylation status was determined after exposure to DNMT inhibitors using Western blot analysis of DNMT1, 3A/3B. The cells were treated with DNMT inhibitors for 18 hours. A drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza- 2’deoxycytidine, and zebularine in both A549 and U373MG cell lines was observed. However, there was no depletion of DNMT3B by the three DNMT inhibitors in either of the cell lines. Each blot is representative of two independent experiments, with actin used as a loading control. Figure 1 The effects of DNMT inhibitors on tumor cell radiosensitivity. Survival curves of (A) A549 cells and (B) U373MG distinguished after the 6 Gy of radiation. The gH2AX cells treated with the respective DNMT inhibitors prior to radiation expression of cells treated with radiation alone was and radiation were compared with those of radiation alone. Points, mean for three independent experiments; bars, SE. compared with those treated with a combination of DNMT inhibitors and radiation. Although gH2AX foci expression was shown to be reduced in cells treated distribution patterns between cells treated with radiation with radiation alone over time, gH2AX foci levels in the alone and those treated with a combination of radiation cells exposed to DNMT inhibitors prior to radiation combined with DNMT inhibitors (Figure 3). However, remained constant over a 12-hour time course in A549 in the A549 cells, radiation-induced G2/M arrest was cells and over a 24-hour time course in U373MG cells abrogated by zebularine pretreatment at 6-12 hours, but (Figure 7). These results implicate an inhibition of the this abrogation disappeared at 24 hours (Figure 3 and DNA damage repair process as the possible mechanism 4). underlying the effects of DNMT inhibitors on Psammaplin A pretreatment increased the sub-G1 radiosensitization. fraction of A549 cells, as compared to cells exposed to radiation alone (Table 2). Western blotting also revealed Discussion an increased expression of cleaved caspase-3 in psam- Recently, histone modifications and DNA methylation, maplin A pretreated A549 cells (Figure 5). However, both of which are prominent epigenetic mechanisms, psammaplin A exerted no effect on the apoptotic induc- have been evaluated with a view toward enhancing the tion of U373MG cell lines. Pretreatment with other radiosensitivity of tumor cells via the regulation of chro- DNMT inhibitors did not influence the expression of matin structure modifications and the expression of cleaved caspase-3 in either of the cell lines. genes involved in cell cycle checkpoints, apoptosis, and gH2AX has been identified as a marker of DNA dou- DNA repair. A number of investigators have previously ble-strand break (DSB) [15]. Immunocytochemical ana- reported that several HDAC inhibitors exert direct cyto- lysis using the anti-gH2AX antibodies was conducted in toxic effects, and can sensitize tumor cells to radiother- order to determine the effects of DNMT inhibitors on apy [14,16-19]. Kim et al. also demonstrated that DNA repair. As shown in the representative micro- trichostatin A, which is the most potent HDAC inhibi- graphs in Figure 6, gH2AX foci could be clearly tor identified thus far, enhanced radiosensitivity in a Kim et al. Radiation Oncology 2012, 7:39 Page 5 of 10 http://www.ro-journal.com/content/7/1/39 Figure 3 Influence of DNMT inhibitors on cell cycle phase distributions of A549 and U373MG cells. Cell cycle phase was measured using flow cytometry at 12 hours after 6 Gy of radiation. Columns, proportion of cell cycle phase; bars, SE. variety of human cancer cell lines [11,20]. Unlike the radioresponse, it is critically important to evaluate the HDAC inhibitors, however, little information is cur- effects of DNMT inhibitors on radiosensitization. In rently available regarding the effects of DNMT inhibi- order to assess this possibility, we employed a total of six tors on radiosensitization [12,13]. DNMT inhibitors. Among them, 5-aza2’-deoxycytidine, There are two classes of DNMT inhibitors: the nucleo- zebularine, and psammaplin A were shown to exert a side analogues and the non-nucleoside analogues. Several radiosensitizing effect in both A549 and U373MG cells. compounds are currently being evaluated in preclinical Although some differences in the degrees of radiosensiti- and clinical trials for the treatment of solid and hemato- zation were detected according to the different DNMT logical malignancies. Contrary to their effect in hemato- inhibitors and cell lines used, these results demonstrate logic malignancies, the clinical effects of these that some DNMT inhibitors may prove to be useful compounds against solid tumors have yet to be evaluated radiation sensitizers in human cancer cells. [21]. Therefore, the use of epigenetic therapies in combi- 5-aza-2’-deoxycytidine, along with 5-azacytidine, is the nation with other regimens, such as conventional che- first DNMT inhibitor reported, and evidences clinical motherapeutics or radiotherapy, should be considered. efficacy in cases of myelodysplastic syndrome and acute As DNA methylation is intricately interrelated with chro- myelogenous leukemia. Several previous studies have matin structure and gene expression, parameters long demonstrated that 5-aza-2’-deoxycytidine exerts a radio- thought to be involved in the regulation of cellular sensitizing effect, with or without HDAC inhibitors, in a variety of cancer cell lines [13,22,23]. Recently, the radio- sensitization effect of 5-azacytidine was also referenced by Hofstetter et al., who demonstrated that 5-azacyti- dine-induced genomic hypomethylation induces enhanced radiation sensitivity in colorectal carcinoma [24]. These results are logical consequences, considering that 5-azacytidine has a molecular structure and exerts clinical effects similar to those of 5-aza-2’-deoxycytidine. However, in this study, 5-azacytidine did not sensitize either of the tested cancer cell types to radiotherapy. 5- azacytidine may exert differing effects on radiosensitivity according to cell type, and the IC of 5-azacytidine var- ies among different cell lines. Although this may explain, at least in part, the different results detected in our study, we did not successfully generate a definitive explanation for the absence of any detectable radiosensitizing effect of 5-azacytidine. Because very few studies have addressed Figure 4 The effect of zebularine on radiation-induced G2/M the role of 5-azacytidine as a radiosensitizer, further stu- arrest in A549 cells. A549 cells were treated with 800 uM dies will be required to resolve this issue. zebularine for 18 hours and then irradiated with 6 Gy. A549 cells were accumulated in G2/M phase after radiation. This radiation- Zebularine, another 5-azacytidine derivative, was also induced G2/M arrest was abrogated by zebularine pretreatment at shown in this study to induce radiosensitivity. Zebularine 6-12 hours, but this abrogation disappeared at 24 hours. has been demonstrated to have anticancer properties, Kim et al. Radiation Oncology 2012, 7:39 Page 6 of 10 http://www.ro-journal.com/content/7/1/39 Table 2 Apoptotic rates measured by the sub-G1 portion of flow cytometry in A549 and U373MG cells Control/drug 0 h 2 h 6 h 12 h 24 h A549 IR 1.11 ± 0.93 1.55 ± 0.82 3.01 ± 1.93 1.72 ± 0.83 1.20 ± 0.99 1.03 ± 0.33 PSA + IR 2.61 ± 1.05 1.53 ± 0.73 3.29 ± 1.71 8.02 ± 2.49 9.02 ± 3.20 7.72 ± 2.16 IR 0.53 ± 0.10 1.23 ± 1.07 0.98 ± 0.37 0.70 ± 0.06 1.13 ± 0.88 0.89 ± 0.12 DAC + IR 0.43 ± 0.10 1.20 ± 0.70 0.54 ± 0.02 0.57 ± 0.09 0.53 ± 0.07 1.05 ± 0.14 IR 0.59 ± 0.16 0.94 ± 0.60 0.61 ± 0.15 0.86 ± 0.33 1.77 ± 0.66 1.66 ± 0.14 ZEB + IR 0.84 ± 0.32 1.00 ± 0.61 0.97 ± 0.49 1.65 ± 0.52 1.62 ± 0.14 2.13 ± 0.05 U373MG IR 0.95 ± 0.10 0.61 ± 0.23 0.80 ± 0.18 0.71 ± 0.09 1.39 ± 0.20 1.68 ± 0.55 PSA + IR 0.71 ± 0.19 0.67 ± 0.13 0.55 ± 0.06 0.63 ± 0.05 0.89 ± 0.16 1.57 ± 0.61 IR 0.94 ± 0.05 0.64 ± 0.13 0.61 ± 0.05 0.64 ± 0.05 0.99 ± 0.20 1.68 ± 0.65 DAC + IR 0.77 ± 0.04 0.83 ± 0.01 0.58 ± 0.05 0.81 ± 0.08 0.93 ± 0.12 1.45 ± 0.37 IR 1.58 ± 0.25 1.39 ± 0.34 1.35 ± 0.01 1.12 ± 0.21 1.78 ± 0.34 2.33 ± 0.35 ZEB + IR 0.95 ± 0.19 1.90 ± 1.21 2.14 ± 1.48 1.06 ± 0.01 1.28 ± 0.15 4.59 ± 1.23 Abbreviations: IR = irradiation, PSA = psammplin A, DAC = 5-aza-2’-deoxycytidine, ZEB = zebularine which can cause re-expression of the epigenetically been confirmed. As psammaplin A has not yet been the silenced tumor suppressor p16 in solid malignancies. subject of clinical trials, further clinical research should However, its clinical development is limited by its higher be conducted to determine its efficacy when adminis- dosage and poor bioavailability, as observed previously in tered in combination with radiotherapy. rats, mice, and monkeys [6,25,26]. Owing to the relatively Three different DNMTs exist, all of which are viable tar- high toxicityof thesenucleosideanaloguecompounds, gets for DNMT inhibitors. DNMT1, the most abundant of non-nucleoside DNMT inhibitors are currently being the three, is responsible for methylation during DNA investigated for their possible use as anticancer agents. replication (maintenance methyltransferase). Other known The natural marine product, psammaplin A, was initi- methyltransferases include DNMT3A and DNMT3B, which exhibit identical preferences for hemi-methylated ally isolated from the Psammaplysilla sponge in 1987. Since that time, psammaplin A has been shown to exert potent cytotoxicity against several cancer cell lines, via the selective induction of genes associated with cell cycle arrest and apoptosis [27,28]. Because psammaplin A has already been shown to exhibit potent histone dea- cetylase inhibitory activity, this drug is a promising radiosensitizing agent [28]. This is the first study in which the radiosensitizing effects of psammaplin A have Figure 5 Western blot analysis of cleaved caspase-3.Thecells were treated with a combination of three different DNMT inhibitors Figure 6 Radiation-induced gH2AX foci.Representative and 6 Gy of radiation. Increased cleaved caspase-3 protein level was micrographs obtained from (A) control cells and (B) cells that had observed in cells treated with a combination of psammaplin A and received 6 Gy of radiation 1 hour earlier. (A) top panel, (B) bottom 6 Gy of radiation in the A549 cells. panel. Kim et al. Radiation Oncology 2012, 7:39 Page 7 of 10 http://www.ro-journal.com/content/7/1/39 Figure 7 Influence of DNMT inhibitors on radiation-induced gH2AX foci. A549 and U373MG cells growing in chamber slides were exposed to DNMT inhibitors for 18 hours, irradiated, and fixed at specific times for immunocytochemical analyses of nuclear gH2AX foci. Open columns, data from cells receiving radiation alone; grey columns, data from cells that were exposed to psammaplin A and radiation; filled columns, data from cells that were exposed to 5-aza-2’-deoxycytidine and radiation; hatched columns, data from cells that were exposed to zebularine and radiation. Cells with more than five foci per nucleus were classified as positive for radiation-induced gH2AX. Foci were evaluated in 50 nuclei. Bars, SE. *p < 0.01 as determined by a logistic regression compared with radiation alone (6 Gy) group. and non-methylated DNA, and have thus been classified employed in this study, we were unable to ascertain as de novo methyltransferases. However, it has been whether this selective inhibition of DNMT isoforms was demonstrated that DNMT1 and DNMT3A/3B do not a function of the concentration of the drug. appear to be completely distinct in their activities [29]. In addition to the physical modification of chromatin This supposition is based on two lines of evidence: first, structure, cellular processes that can affect intrinsic despite being DNMT1-deficient, a colon adenocarcinoma radiosensitivity were evaluated in this study. Cell cycle, cell line was shown to be capable of retaining 80% of its apoptosis, and DNA repair have all been shown to influ- methylation level while replicating, which implies that ence radiosensitivity. DNMT3 may perform a function in the maintenance of The proportion of cells in the G2/M phase detected in methylation [30]; secondly, forced DNMT1 overexpression this study was not significantly altered by pretreatment in cancer cell lines does, indeed, induce de novo methyla- with DNMT inhibitors, and no abrogation of radiation- tion [31]. Gene silencing of DNMT, which functions as an induced G2/M arrest was noted except in the zebularine- oncogene, has been proposed as a good cancer treatment treated A549 cell line. Radiation-induced G2/M arrest was strategy [32]. In this study, three different DNMT inhibi- abrogated as the result of zebularine pretreatment at 6-12 tors, all of which demonstrated radiosensitizing effects, hours, but this effect disappeared at 24 hours in the A549 depleted DNMT1 and DNMT3A, but no such DNMT3B cell line. In another study, zebularine was not found to depletion was noted in the A549 and U373MG cell lines. result in the abrogation of radiation-induced G2/M arrest Depletion of DNMT, along with several members of the in a U251 glioblastoma cell line [12]. Thus, although there structural maintenance of chromatin proteins (SMCs), appears to be some relationship between cell lines and SMC-associated protein, and heterochromatin proteins, radiation-induced G2/M arrest, it is less likely that the was reported to correlate with chromatin decondensation radiosensitization induced by the DNMT inhibitors [33]. Considering that the effect of chromatin compaction employed in this study is the result of a general inhibition on protection of DNA against radiation-induced DSBs has of G2 checkpoint activation. been relatively well-established [34,35], the relaxation of Apoptosis has previously been regarded as a potential chromatin structure via the downregulation of DNMT mechanism for radiosensitization. Different results have with DNMT inhibitors may be a possible mechanism for been reported regarding the role of apoptosis as a radio- radiosensitization. sensitizing mechanism induced by DNMT inhibitors. Selective inhibition of DNMT isoforms shown in this Dote et al. previously reported that a combination of study may be attributable, in part, to the doses of DNMT radiation and zebularine did not significantly increase inhibitors. Beaulieu et al. previously reported that Wes- the sub-G1 population (apoptotic cells) [12]. On the tern blot analysis with anti-DNMT1, DNMT3A, or other hand, Qui et al. demonstrated that 5-aza-2’-deoxy- DNMT3B antibodies treated with increasing doses of cytidine induces radiosensitization in certain gastric can- DNMT inhibitors effected a selective dose-dependent cer cell lines via induced increases in the apoptotic rate, inhibition of the target isoform of DNMT [36]. However, as evidenced by enhanced expression of the p53, as only the IC values of the DNMT inhibitors were RASSF1, and DAPK gene families [23]. In this study, the 50 Kim et al. Radiation Oncology 2012, 7:39 Page 8 of 10 http://www.ro-journal.com/content/7/1/39 addition of 5-aza-2’-deoxycytidine or zebularine to further investigations are warranted to determine radiation did not increase the apoptosis rate in either of whether or not alterations in the methylation patterns the cell lines. However, exposure to psammaplin A in of a specific gene or set of genes involved in DNA repair the A549 cell line induced a significant increase in might be modulated by DNMT inhibitors, and that apoptotic death. Psammaplin A has been reported to these changes might contribute to the observed exert cytotoxic effects on cancer cells via the selective enhancements of radiosensitivity. Several remain to be determined in future studies. induction of apoptosis-related genes [27]. This effect of First, a unified treatment schedule - specifically, the psammalin A may result in an increase in radiation- administration of DNMT inhibitors 18 hours prior to induced apoptosis. However, in this study, radiation- induced apoptosis was noted only in the A549 cell line, radiation - was employed in this study, and thus the and not in the U373MG cell line. This finding may be optimal treatment schedule of DNMT inhibitors and attributable, at least in part, to p53 expression status. radiation remains to be established. In other studies, The U373MG cell line, which contains mutated p53, DNMT inhibitors were administered for different treat- might be comparatively resistant to radiation-induced ment durations, i.e. 2 ~ 48 hours prior to radiation, con- apoptosis, considering that apoptosis is mediated by the sidering several factors such as drug half-lives and the p53 protein. expression of radiosensitivity-related genes [22,23]. DNA repair is another process involved in the deter- Further investigations into the optimal treatment sche- mination of cellular radiosensitivity. The activation of dule of DNMT inhibitors and radiation for clinical DNA repair of cancer cells after sublethal DNA damage applications will be necessary in the future. induced by radiation might be one of the most impor- Second, another important issue will involve assess- tant factors in resistance. The expression of gH2AX has ments of the synergistic effects of DNMT inhibitors and been recently identified as a sensitive indicator of radia- HDAC inhibitors on radiosensitivity. Although some tion-induced DSBs [37]. In this study, gH2AX expres- previous studies have reported that the combined gene sion in cells treated with a combination of DNMT silencing reversal effect was superior to that of treat- inhibitors (5-aza-2’deoxycytidine and psammaplin A, as ment with a single agent [32,39], only a few studies have well as zebularine) was found to be similar to the results thus far evaluated the influence of this combined effect achieved with radiation treatment at 1 hour after initial on increased radiosensitivity. Third, the mechanisms treatment, but was significantly greater over time. This underlying DNMT inhibitor-induced radiosensitzations need further investigation. We speculate that suppres- finding is generally consistent with the results obtained sion of DNMTs by the DNMT inhibitors was associated in other previous studies, in which zebularine and 5- aza-2’-deoxycytidine were employed as radiosensitizing with enhanced radiosensitivity through the change in agents [12,22]. Dote et al. evaluated the expression of DNA structure. However, the relationship between gH2AX foci after exposure to 2 Gy with and without DNA methylation and cellular radiosensitivity is to be zebularine pretreatment. Whereas zebularine had no elucidated in the future study. effect on radiation-induced gH2AX foci at 1 hour, the number of gH2AX per cell was significantly greater in Conclusions the zebularine-treated cells at 24 hours after irradiation, Taken together, our study indicate that psammaplin A, which suggested the possible presence of unrepaired 5-aza-2’-deoxycytidine, and zebularine have the potential DNA damage [12]. DNA methylation is intimately asso- to increase radiosensitivity in lung cancer A549 and ciated with histone deacetylases in terms of the epige- glioblastoma U373MG cells, most probably by modulat- netic regulation of gene expression. Actually, HDAC ing the impairment of the DNA repair process. Further inhibitors such as LBH589 and MS-275 have been investigations will be required to identify other addi- shown to enhance radiosensitivity through similar tional mechanisms associated with radiosensitivity, and mechanisms as those of the DNMT inhibitors. These to confirm the synergistic effects on radiosensitivity with HDAC inhibitors prolonged gH2AX expression, suggest- other epigenetic drugs, such as the HDAC inhibitors. ing an inhibition of DNA repair [14,38]. A previous pre- Also, future studies should be conducted to determine clinical study demonstrated that an HDAC inhibitor definitively whether the combination of DNMT inhibi- downmodulated the expression of DNA-PK and Rad51, tors and radiation has real potential as a clinical strategy which participated in the recovery of DSB, thereby abro- for the treatment of cancer. gating key cellular pathways involved in DNA DSB repair [17]. These results indicate that the impairment Acknowledgements of DNA DSB repair maybeone of themostcrucial This work was supported by grant No. M2070202001-07 N0202-00111 from mechanisms underlying enhanced radiation responses in the 2008 National Nuclear R&D Program of the Korean Science & Engineering Foundation (KOSEF) and SNUH CRI grant (0420100900). epigenetic phenomena. Based on this assumption, Kim et al. Radiation Oncology 2012, 7:39 Page 9 of 10 http://www.ro-journal.com/content/7/1/39 17. Chinnaiyan P, Vallabhaneni G, Armstrong E, Huang SM, Harari PM: Author details Modulation of radiation response by histone deacetylase inhibition. Int J Department of Radiation Oncology, Seoul National University College of Radiat Oncol Biol Phys 2005, 62(1):223-229. Medicine, Seoul, Republic of Korea. Cancer Research Institute, Seoul National 18. Munshi A, Kurland JF, Nishikawa T, Tanaka T, Hobbs ML, Tucker SL, Ismail S, University College of Medicine, Seoul, Republic of Korea. 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Radiation OncologySpringer Journals

Published: Mar 20, 2012

References