The Inhibitor of Histone Deacetylases Sodium Butyrate Enhances the Cytotoxicity of Mitomycin C

Published OnlineFirst August 13, 2012; DOI: 10.1158/1535-7163.MCT-12-0193

Molecular Cancer Therapeutic Discovery Therapeutics

Anastas Gospodinov, Stanislava Popova, Ivelina Vassileva, and Boyka Anachkova

Abstract The use of histone deacetylase inhibitors has been proposed as a promising approach to increase the cell killing effect of DNA damage–inducing drugs in chemotherapy. However, the molecular mechanism of their action remains understudied. In the present article, we have assessed the effect of the histone deacetylase inhibitor sodium butyrate on the DNA damage response induced by the crosslinking agent mitomycin C. Sodium butyrate increased mitomycin C cytotoxicity, but did not impair the repair pathways required to remove mitomycin C-induced lesions as neither the rate of nucleotide excision repair nor the homologous recombination repair rate were diminished. Sodium butyrate treatment abrogated the S-phase cell-cycle

checkpoint in mitomycin C-treated cells and induced the G2–M checkpoint. However, sodium butyrate treatment alone resulted in accumulation of reactive oxygen species, double-strand breaks in DNA, and apoptosis. These results imply that the accumulation of reactive oxygen species–mediated increase in DNA lesion burden may be the major mechanism by which sodium butyrate enhances the cytotoxicity of mitomycin C. Mol Cancer Ther; 11(10); 1–11. 2012 AACR.

Introduction In relation to the DNA damage response, different Histone deacetylase inhibitors (HDACi) are seen as studies reported effects of the HDACi on DNA repair promising epigenetic drugs that are used in mono and pathways and induction of apoptosis that could potenti- combination cancer therapy, but it is still unclear what ate the DNA damaging agents used in cancer therapy. mechanisms cause their clinical effects (1). The initial Treatment with HDACi resulted in defects in double- rationale of using HDACi in anticancer therapy was to strand break (DSB) repair by both homologous recombi- induce global transition to euchromatic state and thus nation (HR) repair (8) and nonhomologous end joining express heterochromatin-silenced genes (2). HDACs, (NHEJ; refs. 8, 9). The effect on NHEJ, which is the however, have broad spectrum of targets that include principal DSB repair pathway in mammals, was attribut- histones as well as nonhistone proteins, and their inhibi- ed to downregulation of Ku70 and Ku86 (10) or to the tion results in similarly broad range of effects — from disruption of critical deacetylation events at the break complex transcription-associated effects to roles on pro- sites (9). Another study found that sodium butyrate atten- tein stability and protein–protein interactions (3). HDACi uated nucleotide excision repair (NER) and enhanced cell- affect cell-cycle distribution and activate cell-cycle check- killing effect of psoralen-induced lesions (11). HDACi points. Thus, cells treated with sodium butyrate displayed induce cell death directly via various other mechanisms such as hyperacetylation of promoters of TRAIL, DR5, G1 arrest due to disruption of the Rb-mediated signaling and suppressed expression of cyclins required for S-phase FAS, and FASL death receptor pathway members (12, 13) or production of proapoptotic reactive oxygen species entry (4). In addition to the G1 block, HDACi reduced the (ROS) and oxidative injury (14–17). expression of cyclin B1, a key cyclin for G2–M transition An important way by which HDACi increase the cyto- and resulted in G2 block (5). In some cases, the HDACi- induced growth arrest has been shown to be irreversible toxic effect of DNA damaging agents is by canceling the and tightly linked to the induction of p21 (6, 7). cell-cycle checkpoints, which allows entry of cells with damaged DNA in S-phase or mitosis (18, 19) and greatly facilitates their demise. Recently, it has been reported that Authors' Afffiliation: Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria sodium butyrate could abrogate the G1–S checkpoint activated by cisplatin, force cells in S-phase with damaged Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). DNA, and thus, promote cell killing (20). On the other hand, sodium butyrate was not able to abrogate the G2 Corresponding Author: Boyka Anachkova, Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. Block 21, 1113 phase checkpoint induced in gamma-irradiated HeLa Sofia, Bulgaria, Phone: 3592-979-3680; Fax: 3592-872-3507; E-mail: cells (8) indicating that different cell-cycle arrest points [email protected] have different sensitivities to HDACi modulation. doi: 10.1158/1535-7163.MCT-12-0193 The aim of the present investigation was to study the 2012 American Association for Cancer Research. effect of HDAC inhibition on the cytotoxicity of an agent

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that induces DNA interstrand crosslinks and block cells in generate reactive DNA alkylating species and when acti- S-phase. We attempted a systematic examination of the vated, it alkylates guanine at the N2-position to form DNA effect of the HDACi sodium butyrate on mitomycin C monoadducts and DNA intra- and interstrand crosslink (MMC)-induced DNA damage response (Fig. 1A). Sodi- adducts (21). MMC-induced lesions activate the S-phase um butyrate is the sodium salt of butyric acid, which is a 4 DNA damage checkpoint (22) and are repaired by NER carbon normal fatty acid and is a natural metabolite in (23) and HR (24). many organisms including bacteria populating the gas- Our observations indicated that sodium butyrate trointestinal tract. MMC is used as a chemotherapeutic increased MMC cytotoxicity. Treatment with sodium agent to treat upper gastrointestinal, breast and bladder butyrate did not impair the repair pathways required to tumors. MMC is metabolized by reductive enzymes to remove MMC-induced lesions as neither the rate of NER, nor HR repair rate were diminished. Sodium butyrate was able to abrogate the MMC-induced S-phase checkpoint and cells arrested in G2, which could in part explain the enhanced cytotoxicity of cells treated with both agents. Sodium butyrate treatment induced time-dependent accumulation of ROS, DNA damage, and massive apoptosis that could be inhibited by a ROS scavenger. We conclude that the mechanism by which sodium butyrate enhances the cytotoxicity of mitomycin C is complex and a major factor of this enhancement is the ROS-mediated overall increase in the DNA lesion burden induced by MMC. Materials and Methods Cell lines, culture conditions, transfection, and cell treatment HeLa and HCT-116 cells were obtained from the Amer- ican Type Culture Collection and were grown in Dulbec- co’s Modiﬁed Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS), and penicillin/strepto- mycin and pyruvate. The male mouse ES cell line containing direct repeat GFP (DR-GFP) construct integrated in the hprt locus (clone 18-1/10) were obtained from the laboratory of Dr. Zdenko Herceg (International Agency for Research on Cancer, Lyon, France) in 2011. The cell line has been described previously (25) and was not authenti- cated in our laboratory. ES cells were initially grown on "feeders" and routinely on gelatin in DMEM supplemented with 20% FCS, leukemia inhibitory factor, penicillin/strep- tomycin, glutamine, pyruvate, and nonessential amino acids in 5% CO2. Transfection of plasmids was carried out using Lipofectamine 2000, following the manufacturer’s recommendations. To inhibit HDACs, cells were treated with either 5 mmol/L sodium butyrate or 5 mmol/L vorinostat (suberoylanilide hydroxamic acid) added to the culture medium and incubated for the indicated times.

Host cell reactivation assay, homologous recombination assay, and cell-cycle analysis Figure 1. Survival of cells treated with MMC in the presence or absence of Host cell reactivation assay, using UV-irradiated plas- sodium butyrate. A, structural formula of sodium butyrate and mitomycin mids, was carried out as described earlier (26). Brieﬂy, plas- C. B, HCT-116 cells were treated with sodium butyrate for different time periods. Total protein extracts were analyzed by Western blotting using mid DNA was dissolved in Tris-EDTA buffer (10 mmol/L an antibody against acetylated histone H4. Actin was used as a loading Tris–HCl, pH 8, and 1 mmol/L EDTA) poured in Petri control. C, survival of HCT-116 cells treated with different mitomycin C dishes to form 1 to 2 mm thick layer and UV irradiated for concentrations in the presence (light columns) or absence (dark columns) 2 and 3 minutes with a UV lamp (emission maximum of 5 mmol/L sodium butyrate. The results from the clonogenic assay are 2 1 expressed as percentage of the untreated control. The results are the 254 nm and exposure rate of 8.3 J.m s ). Damaged and mean of 3 independent experiments, error bars show deviation from the control undamaged pEGFP-C1 were introduced into cells mean (s.d.m). using Lipofectamine 2000 transfection reagent.

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For the HR assay, the I-SceI expression vector incubated in PBS with 10 mmol/L 20,70-dichlorodihydro- pCBASce34 was transfected into ES cells using Lipofec- fluorescein diacetate (H2DCFDA) for 15 minutes at room tamine 2000. Plasmid pCAGGS (without the I-SceI gene) temperature. Samples were then washed 3 times (5 min- and GFP-expressing plasmid pEGFP-C1 were used for utes each), resuspended in the same buffer, and after 30 mock and transfection efficiency controls, respectively. minutes, incubation in the dark at room temperature was Flow cytometry analysis was conducted 48 hours after analyzed by fluorescence-activated cell sorting (FACS). transfection using a FACScalibur apparatus with Cell- For fluorescent imaging, cells were grown on cover slips, quest software (Becton Dickinson). For analysis of GFP treated and processed as above, and examined under a expression, cells were harvested by trypsinization and fluorescent microscope. analyzed by a FACScalibur apparatus with Cellquest software (Becton Dickinson). Results To analyze cell-cycle profiles, cells were harvested by trypsinization and fixed in 70% ethanol. Before analysis, Sodium butyrate enhances cytotoxicity of cells were resuspended in PBS, treated with RNAse A mitomycin C (20 mg/mL) and stained with propidium iodide (20 mg/ We first checked the effect of sodium butyrate on mL), and analysis conducted by a FACScalibur apparatus histone H4 acetylation using an acetyl-H4–specific anti- with Cellquest software (Becton Dickinson). body. Western blot analysis of total extracts from cells To measure bromodeoxyuridine (BrdUrd) incorpo- treated for 8, 16, and 24 hours with 5 mmol/L sodium ration, cells were cultured in medium containing 15 butyrate or left untreated as a control indicated that mmol/L BrdUrd for 45 minutes, harvested, washed in acetylation strongly increased in a time-dependent man- PBS, fixed in 70% ice-cold ethanol, and stored at 20C. ner (Fig. 1B). To assess the effect of sodium butyrate on To denature DNA, fixed cells were resuspended in solu- mitomycin C (MMC) cytotoxicity, we conducted clono- tion containing 2N HCl and 0.5% Triton X-100 and incu- genic survival assay. HCT-116 cells were pretreated with 5 bated for 30 minutes at room temperature. To neutralize mmol/L sodium butyrate for 6 hours to induce histone hyperacetylation before addition of MMC for additional the acid, cells were resuspended in 0.1 mol/L Na2B4O7 and then blocked in 2% bovine serum albumin (BSA) in 18 hours without removal of sodium butyrate from the PBS – 0.1% Tween-20. Cells were incubated with mouse medium. Following treatment, cells were washed, plated anti-BrdUrd antibody (Becton Dickinson cat# 347580) at a density of 500 cells/sq cm, and left to grow in fresh diluted at a ratio of 1:50, rinsed, and incubated with an medium. Colony formation was assessed 9 days later. The anti-mouse fluorescein isothiocynate (FITC)-conjugated result showed that 5 mmol/L sodium butyrate alone secondary antibody. RNA was digested with 20 mg/mL caused about 20% reduction in the viability of the cells, RNAse A and stained with propidium iodide (5 mg/mL). 50 nmol/L MMC caused 50% reduction, whereas the combined effect of the 2 agents was stronger than that of Western blotting and immunofluorescence either one alone and reached over 90% reduction in microscopy viability (Fig. 1C). Similar results were obtained with Protein lysates were resolved on SDS-PAGE (6% or HeLa cells (data not shown). 12.5%, as appropriate) and blotted on nitrocellulose mem- brane (Bio-Rad). The primary antibodies used were Sodium butyrate does not impair nucleotide excision mouse anti-RAD51 antibody (Abcam #14B4), mouse repair rates anti-phospho-H2AX antibody (1:1000; Abcam #18311), NER is a major pathway that deals with MMC-induced rabbit anti-phospho-ATM (1:1,000; Abcam #81292), ace- lesions as it removes DNA adducts and intrastrand cross- tyl-H4 (Lys5/8/12/16) antibody (Upstate, cat# 17-229), links and participates in the repair of interstrand cross- and mouse anti-actin (1:10,000; Sigma). Proteins were links (ICL) caused by the drug. To assess efficiency of visualized using Li-cor Odyssey IR imaging system with NER, we used the host cell reactivation assay of in vitro appropriate IRDye-labeled secondary antibodies (Li-cor). UV-damaged plasmids. UV induces cyclobutane pyrim- For immunofluorescence, cellswere grownoncoverslips, idine dimers and 6-4 photoproducts that are repaired only washed in PBS, fixed with methanol for 5 minutes at 20C, by NER. pEGFP-C1 was irradiated with 1 or 1.5 kJ/m2 UV washed with PBS, and blocked in 3% BSA in PBS for 1 hour. to obtain 0.8 to 1 lesion per kbp (27, 28) and transfected in Staining was done using mouse anti-RAD51 antibody HeLa cells. After transfection, cells were cultured for 6 (Abcam #14B4) diluted 1:100 overnight at 4C. Secondary hours in fresh medium and for 24 hours in the presence or FITC-conjugated anti-mouse IgG (Sigma) were used at absence of sodium butyrate and expression efficiency of 1:200 dilution for 1 hour at room temperature, and after 3 GFP assessed using flow cytometry analysis (Fig. 2A and times for 5-minute washes with PBS, slides were mounted B). In HeLa cells, there were insignificant fluctuations in using ProLong Gold mounting medium (Invitrogen). the repair efficiency between sodium butyrate-treated and -untreated cells (Fig. 2C). Similar outcome was ROS assay observed when the experiment was carried out with the After experimental manipulation, floating and attached cell line HCT-116 (not shown). These results suggest that cells were harvested and pooled, washed once in PBS, and treatment with 5 mmol/L sodium butyrate did not

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Figure 2. Effect of sodium butyrate on the efficiency of nucleotide excision repair. A, The pEGFP plasmid was subjected to different doses of UV radiation (1 or 1.5 kJ/m2) under in vitro conditions. HeLa cells were transfected with the damaged plasmids or with the undamaged plasmid. Transfected cells were cultured for 24 hours in the presence (þ SB) or absence (SB) of 5 mmol/L sodium butyrate. The percentage of GFP- positive cells was assessed by flow cytometry analysis. B, FACS analysis of HeLa cells not transfected with the pEGFP plasmid. C, efficiency of NER in HeLa cells in the presence or absence of sodium butyrate. The ratio of the GFP-positive fraction in the population of cells transfected with the damaged plasmids (second and third row in A) to the GFP-positive fraction in the population of cells transfected with the undamaged plasmid (first row in A) was used as a measure of repair efficiency. Dark columns, control cells; light columns, cells treated with 5 mmol/L sodium butyrate.

significantly change NER capacity as measured by the transfected with the I-SceI restrictase expression plasmid host cell reactivation assay. to the expression efficiency of the control, it was seen that sodium butyrate treatment did not result in HR repair Effect of sodium butyrate on homologous rate defect, but instead stimulated HR repair (with about recombination repair rates 40%; Fig. 3C). That could be attributed to higher acces- Homologous recombination repair is essential for ICL sibility of both the restrictase and the HR repair machin- repair. To measure the effect of sodium butyrate on HR ery to DNA under the conditions of histone hyperace- repair rate, we took advantage of the DR-GFP construct tylation. integrated in the hprt locus of mouse ES cells (25, 29; Fig. To further confirm these results, we checked Rad51 3A). Expression of I-SceI endonuclease induces a single recruitment into nuclear foci after MMC treatment under DSB in an out-of-frame GFP reporter, which if repaired by the conditions of HDAC inhibition. Rad51 functions as a HR with the tandem GFP fragment, restores a functional helical nucleoprotein filament loaded on the single- 0 GFP gene and its expression can be detected by flow stranded DNA 3 - overhangs that carries out the search cytometry analysis. To evaluate the effect of sodium for and exchange with the homologous duplex and thus, butyrate on HR repair, ES cells were transfected with I- constitutes the core of the HR repair reaction (30). Treat- SceI expression vector and treated with 5 mmol/L sodium ment of HeLa cells with 5 mmol/L MMC for 18 hours butyrate or left untreated as control. After 48 hours of followed by immunofluorescent staining resulted in treatment, cells were collected and the size of the GFP clearly visible Rad51 foci in both sodium butyrate -treated population was measured by flow cytometry analysis and -untreated cells (Fig. 3D). There were no apparent (Fig. 3B Top). In parallel, cells were transfected with defects in Rad51 foci formation in sodium butyrate-trea- pEGFP-C1 plasmid to assess possible transcriptional ted cells, confirming the result obtained using the DR- effect of sodium butyrate on GFP expression, as GFP GFP reporter. Taken together, the data about the rates of expression in that plasmid is driven by the same (CMV) NER and HR repair in sodium butyrate-treated cells promoter as that of the I-SceI–containing plasmid (Fig. 3B indicate that sodium butyrate did not compromise the Bottom). After normalization of the GFP-positive cells repair of ICLs.

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Figure 3. Effect of sodium butyrate on the efficiency of HR repair. A, a scheme of the DR-GFP construct for assessment of the efficiency of HR repair. The upstream GFP open reading frame (Sce-GFP) is disrupted by the insertion of a recognition site for the I-SceI restrictase. The downstream GFP gene (iGFP) lacks a functional promoter and thus cannot be expressed. B, first row, ES cells carrying the DR-GFP construct were transfected with a plasmid encoding the I-SceI restrictase and subjected to sodium butyrate treatment (5 mmol/L SB). Second row, ES cells were transfected with the pEGFP plasmid and subjected to sodium butyrate treatment (5 mmol/L SB). GFP expression efficiency was analyzed by flow cytometry analysis and the percentages correspond to the fraction of GFP-positive cells. C, efficiency of HR repair in ES cells in the presence (5 mmol/L) or absence (0 mmol/L) of sodium butyrate. The ratio of the GFP-positive fraction in the population of cells transfected with the restrictase-encoding plasmid (in B, first row) to the GFP- positive fraction in the population of cells transfected with the pEGFP plasmid (second row in B) was used as a measure of repair efficiency. D, HeLa cells were treated with mitomycin C or sodium butyrate or a combination of both agents. Control cells were cultured in the absence of mitomycin C and sodium butyrate. Rad51 foci formation was assessed by immunofluorescence with an antibody against Rad51.

Effect of sodium butyrate on cell-cycle distribution, pared with those treated with MMC only. This suggests DNA damage signaling, and apoptosis that upon HDAC inhibition, the S-phase checkpoint was To check whether sodium butyrate affected DNA dam- abrogated and cells accumulated in G2, which implied age signaling in the MMC-treated cells, we analyzed their activation of the G2–M checkpoint (Fig. 4A and B). cell-cycle distribution. HeLa cells were treated with 5 To further conﬁrm these results, we checked BrdUrd mmol/L sodium butyrate for 6 hours before the addition incorporation in HeLa cells that were either treated for 6 of 5 mmol/L MMC and incubated for additional 18 hours. hours with 2 mmol/L MMC or with 2 mmol/L MMC and 5 Following treatment, cells were analyzed by FACS. The mmol/L sodium butyrate simultaneously (Fig. 4C). Stain- obtained results indicated that MMC treatment alone ing with an anti-BrdUrd antibody followed by FACS caused S-phase accumulation, consistent with earlier analysis indicated that in MMC-treated cells, DNA syn- reports on the effect of MMC (22). Treatment with sodium thesis was strongly inhibited compared with the untreat- butyrate blocked cells in G1 and G2, causing depletion of S- ed control, and the fraction of cells that were BrdUrd phase cells, which has also been described (31–33). In cells positive was diminished more than 3.5-fold. Treatment treated with both agents, S-phase cells were diminished, with both agents resulted in an increase in the population whereas the G2 fraction was increased over 5-fold, com- of replicating cells compared with the cells treated only

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Figure 4. Cell-cycle distribution and DNA damage signaling in cells treated with sodium butyrate, MMC, and a combination of the drugs. A, HeLa cells were treated with 5 mmol/L sodium butyrate for 24 hours, 5 mmol/L MMC for 18 hours, a combination of the drugs (MMC added during the last 18 hours of the 24-hour sodium butyrate treatment), or left untreated as a control. After propidium iodide staining, cells were analyzed by FACS analysis. B, cell-cycle distribution of cells treated as in A. Light gray bars, percentage of G1 cells; dark gray bars, S-phase cells; gray bars, G2 cells; dotted bar, sub-G1 fraction. Data are medium of two 2 independent experiments s.d.m. C, scatter plots showing BrdUrd incorporation and DNA content (stained with propidium iodide) in control cells, cells treated for 6 hours with 5 mmol/L mitomycin C or a combination of 5 mmol/L mitomycin C and 5 mmol/L sodium butyrate. Indicated are the percentages of replicating cells. D, phosphorylated ATM in cells treated with sodium butyrate for the indicated times and with 5 mmol/L mitomycin C for the last 18 hours of sodium butyrate treatment or with MMC only. E, levels of phosphorylated ATM in cells treated with sodium butyrate for the indicated time periods. F, HeLa cells were treated with 5 mmol/L of sodium butyrate for 24 hours, 5 mmol/L MMC for 18 hours, and a combination of the drugs (MMC added during the last 18 hours of sodium butyrate treatment) or left untreated as control. Total proteins extracts were analyzed by Western blot analysis for the accumulation of the p85 PARP apoptotic fragment. G, HCT-116 cells were treated and analyzed as in F.

with MMC to reach a value that was slightly lower than ylated active form of ATM checkpoint kinase by Western that of the control. These data clearly showed that sodium blot analysis. HeLa cells were treated with 5 mmol/L butyrate impaired activation of the S-phase checkpoint in sodium butyrate for 24 and 48 hours or were left untreated cells treated with MMC. and 5 mmol/L MMC was added during the last 18 hours of To test the G2–M DNA damage signaling in a more treatment (Fig. 4D). In parallel, cells were treated with direct way, we checked the abundance of the phosphor- sodium butyrate only for 24 and 48 hours and left

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untreated as controls (Fig. 4E). Western blot analyses analysis of DSB formation at the cellular level. The com- indicated that cells treated with sodium butyrate dis- parison between the pattern of g-H2AX foci formation in played increased amounts of phosphorylated ATM, irre- MMC- and SB-treated cells showed that these formed as spective of whether they were or were not treated with early as 8 hours after treatment with either of the agents MMC. These results are consistent with the FACS data (Fig. 5C). However, the density of foci formation in sodi- and indicate a shift to G2–M ATM-dependent checkpoint um butyrate-treated cells was lower than in MMC-treated activation in cells treated with MMC and sodium cells. The appearance of g-H2AX foci in sodium butyrate- butyrate. treated cells argues that sodium butyrate induces DSB As treatment with sodium butyrate induced DNA formation. damage signaling and appearance of sub-G1 cells (Fig. 4A and B), we next checked whether the reduction in Induction of apoptosis in HDACi-treated cells is due viability in sodium butyrate and MMC-treated cells was a to DSB formation by ROS result of apoptosis. To this end, we looked for the accu- It has been reported that sodium butyrate may cause mulation of the p85 fragment of PARP that is a marker significant deregulation of the activities of enzymes that characteristic for ongoing apoptosis (34). The result indi- cope with ROS inside the cell (35). To test the possibility cated that the treatment of HeLa and HCT-116 cells with that the sodium butyrate-induced DSBs are ROS-depen- either of the drugs induced apoptosis, sodium butyrate dent, we treated HeLa cells with sodium butyrate for 8 being the more potent proapoptotic agent. At 24 hours of and 24 hours and then stained the cells with the cell- treatment, the combined effect of sodium butyrate and permeant dye 20,70-dichlorodihydrofluorescein diacetate, MMC was stronger than that of either of the drugs alone which is a chemically reduced form of fluorescein and is (Fig. 4F and G). widely used as an indicator for ROS. FACS analysis and To extend these observations we analyzed the phos- fluorescent microscopy indicated that after 8 hours in phorylation of the histone variant H2AX, which is the sodium butyrate, cells were already showing increased principle chromatin substrate of ATM and is widely used staining. At 24 hours of treatment, the median value of as an indicator for the formation of DSBs. Cells treated the fluorescence has increased nearly 2-fold and much with 5 mmol/L of sodium butyrate for 24 and 48 hours, more brightly stained cells were observed under the with or without addition of MMC exhibited increased microscope (Fig. 6A and B). Similar results were obtained amount of g-H2AX (Fig. 5A and B), strongly suggesting using the HCT-116 cell line. To verify that increased accumulation of DSBs in the cells treated with the HDACi. intracellular concentration of ROS led to apoptosis, we H2AX is phosphorylated in broad chromatin regions used dithiothreitol (DTT), a substance known to be an surrounding the DSBs that can be visualized as nuclear effective scavenger of ROS and checked apoptosis induc- foci by immunofluorescent microscopy, which allows tion in HeLa cells treated with sodium butyrate, DTT, or both agents simultaneously. FACS analysis indicated that while apoptotic sub-G1 population was readily observed in sodium butyrate-treated cells, it was absent when the cells were treated with a combination of sodium butyrate and DTT (Fig. 6C). This result strongly suggested that apoptosis in sodium butyrate-treated cells depended on the generation of ROS. To further these observations, we checked accumulation of the p85 PARP fragment as a marker of apoptotic induction. As expected, the p85 PARP fragment, while abundant in sodium butyrate-treated cells, was absent when DTT was added (Fig. 6D). Taken together, our results indicate that sodium butyrate enhances the cell killing effect of the crosslinking agent MMC mostly as a result of increased ROS accumulation in the cells and induction of DSBs and apoptosis. In addition, we used another structurally unrelated to sodium butyrate HDACi vorinostat (Supplementary Fig. S1A) to test whether the observed effects were specific to sodium butyrate or common to different classes of Figure 5. H2AX phosphorylation in sodium butyrate treated cells. A, HDACi. Treatment with 5 mmol/L vorinostat resulted in levels of phosphorylated H2AX in HCT116 cells treated with 5 mM sodium histone hyperacetylation, appearance of DSBs as judged butyrate. B, H2AX phosphorylation in HCT116 cells treated with sodium by accumulation of phosphorylated H2AX, and an butyrate for the indicated time periods and with mitomycin C for the last 18 hours of sodium butyrate treatment or with MMC only. C, HeLa cells increase of p85 PARP, indicative of apoptosis induction were treated with 5 mM of MMC or 5 mM sodium butyrate for the times (Supplementary Fig. S1B). These changes were concom- indicated, fixed, and stained with an antibody against g-H2AX. itant with increase of ROS production (Supplementary

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Figure 6. Induction of ROS in sodium butyrate-treated HeLa cells. A, cells were treated with 5 mmol/L sodium butyrate for 8 or 24 hours or left untreated as a control. Live cells were stained with 10 mmol/L 20,70- dichlorodihydrofluorescein diacetate, harvested, and analyzed by flow cytometry analysis. To compare fluorescence intensity of the different populations, data were normalized taking the maximal Y-value as 100%. Black line, untreated control (NT); gray lines, following treatment with sodium butyrate. B, fluorescent images of cells treated as in A. C, cells were treated with 5 mmol/L of sodium butyrate for 48 hours or with a combination of 5 mmol/L of sodium butyrate and 2 mmol/L DTT. Cells were collected, stained with propidium iodide, and analyzed by FACS. D, cells were treated with 5 mmol/L sodium butyrate, 2 mmol/L DTT, and combination of both for 24 hours or left untreated as a control. Total protein extracts were analyzed by Western blot analysis for the accumulation of the p85 PARP apoptotic fragment.

Fig. S1C) that was observed in cells following staining affected the efficiency of repair of MMC-induced DNA with 20,70-dichlorodihydrofluorescein diacetate, indicat- lesions, it could have significant effect on MMC cytotox- ing that both sodium butyrate and vorinostat induced icity. However, we did not detect differences in NER similar effects. capacity between sodium butyrate-treated and -untreated cells. A previous report found reduced NER rate after Discussion sodium butyrate treatment (11). The discrepancy could be Here, we have shown that the combined effect of the due to differences in the experimental design (plasmid HDACi sodium butyrate and the DNA crosslinking agent host cell reactivation vs. cell-based assay) or the cell types MMC on reduction of cell viability was stronger than that used. Regarding the repair by HR, sodium butyrate treat- of either one of the agents alone. If sodium butyrate ment resulted in a higher HR repair rate (Fig. 3B). This is in

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agreement with previous data that histone acetylation at (44). To choose between the hypotheses of elevated lipid lesion sites was needed for efficient HR repair (25, 36, 37). source and transcriptional perturbation leading to ROS The result from the reporter assay (Fig. 3C) was strength- production, we repeated some of the experiments with ened by the fact that Rad51 foci formation after induction vorinostat. Vorinostat is an HDAC inhibitor that is of ICLs by MMC was also not impaired in cells treated structurally unrelated to sodium butyrate, but also inhi- with sodium butyrate (Fig. 3D). Taken together, the data bits class I and class II HDACs. The application of about the rates of NER and HR repair indicated that the vorinostat resulted in phosphorylation of H2AX and increased cytotoxicity of ICL lesions in sodium butyrate- p85 PARP accumulation that was concomitant with ROS treated cells did not result from changes in their repair production in the treated cells. These results favor the capacity. latter hypothesis and indicate that ROS-mediated apo- Another possible cause for increased sensitivity of ptosis is a common effect of HDAC inhibition. The sodium butyrate-treated cells to MMC could be the abro- results also suggest that the data obtained with sodium gation of DNA damage–induced checkpoint signaling. butyrate as a model compound could be extended to The distribution of the cells in the cell cycle under con- clinically applicable HDACi. ditions of hyperacetylation of chromatin showed that the HDACi are an emerging new class of agents that MMC-induced ATR-dependent block of the cells in S- could enhance the effectiveness of cancer therapy. It phase was abrogated. This was confirmed by the has been reported that different HDACi sensitize tumor increased rate of BrdUrd incorporation in MMC-damaged cells towards the killing effect of many anticancer cells that were treated with sodium butyrate. The obser- agents (11, 45–47) and this effect has been attributed vation is in line with a recent report that HDAC inhibition to interference with DNA repair pathways (8, 11, 48) or in yeast specifically counteracted activation of the yeast abrogation of the cell-cycle checkpoints (18–20). Our ATR ortholog, Mec1 (38). However, here we observed a results suggest that the accumulation of intracellular shift in cell-cycle signaling from S-phase ATR-dependent ROS-mediated increase in the lesion burden may be the block of the cells to ATM-dependent blockage in G2–M major mechanism by which sodium butyrate enhances phase of the cell cycle. That is why we do not think that the cytotoxicity of MMC-treated cells. The results also perturbations of cell-cycle signaling could be the reason explain the selectivity of HDACi to tumor cells, as for the sensitization of MMC-treated cells by sodium normal, not transformed human fibroblasts treated with butyrate. In search for another reason for the increased HDACi accumulate thioredoxin, a natural ROS scaven- apoptosis rate, we treated cells with sodium butyrate only ger, making the normal cells less sensitive to HDACi and found phosphorylation of H2AX, indicating the pres- (49, 50). ence of DSBs. Taken together, our results underscore the importance These results raised the question about the cause of of understanding the mechanistic effect of each mod- DSBs production. Because there were data about ulator of the DNA damage response to use the best HDACi-mediated ROS generation (15), we also tested regimen of combination with genotoxic drugs to manip- this possibility. The results showed that sodium buty- ulate the clinically desired outcome of treatment of rate treatment resulted in a time-dependent accumula- tumor cells. tion of ROS. The apoptotic effect caused by sodium butyrate was largely abolished in the presence of the Disclosure of Potential Conflicts of Interest ROS scavenger DTT (Fig. 6C and D). This suggested No potential conflicts of interest were disclosed. that apoptosis induction under our conditions was mostly due to deregulation of ROS metabolism in sodi- Authors' Contributions um butyrate-treated cells and together with other Conception and design: B. Anachkova reported proapoptotic effects of ROS (39–42) may be Acquisition of data (provided animals, acquired and managed patients, the primary contributor to sodium butyrate -mediated provided facilities, etc.): A. Gospodinov, S. Popova, I. Vassileva Analysis and interpretation of data (e.g., statistical analysis, biostatis- cytotoxicity. tics, computational analysis): A. Gospodinov, S. Popova, B. Anachkova One explanation for the production of ROS by sodium Writing, review, and/or revision of the manuscript: A. Gospodinov, B. Anachkova butyrate could be that the high concentration of sodium Administrative, technical, or material support (i.e., reporting or orga- butyrate would enhance the synthesis of medium and nizing data, constructing databases): I. Vassileva long chain fatty acids and the increased lipid concen- Study supervision: B. Anachkova tration may induce ROS. On the other side, a recent proteomic study of HDACi-treated cells found altered Grant Support expression levels of enzymes associated with energy This work was supported by grant DO 02-232 awarded to B. Anachkova and A.Gospodinov by the Bulgarian National Science Fund. metabolism, antioxidative stress, and cellular redox The costs of publication of this article were defrayed in part by the control, which suggests an excessive production of ROS payment of page charges. This article must therefore be hereby marked under conditions of hyperacetylation of chromatin (43). advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. In addition, HDAC inhibition has led to increased tran- TRAIL scription of the gene that resulted in accelerated Received February 27, 2012; revised July 13, 2012; accepted July 15, 2012; caspase activation and promoted mitochondrial damage published OnlineFirst August 13, 2012.

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The Inhibitor of Histone Deacetylases Sodium Butyrate Enhances the Cytotoxicity of Mitomycin C

Anastas Gospodinov, Stanislava Popova, Ivelina Vassileva, et al.

Mol Cancer Ther Published OnlineFirst August 13, 2012.

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