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Enhancement of Radiation Response in Osteosarcoma and Rhabomyosarcoma Cell Lines by Histone Deacetylase Inhibition
Int. J. Radiation Oncology Biol. Phys., Vol. 78, No. 1, pp. 237–245, 2010 Copyright Ó 2010 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/$–see front matter
doi:10.1016/j.ijrobp.2010.03.010
BIOLOGY CONTRIBUTION
ENHANCEMENT OF RADIATION RESPONSE IN OSTEOSARCOMA AND RHABOMYOSARCOMA CELL LINES BY HISTONE DEACETYLASE INHIBITION CLAUDIA BLATTMANN, M.D.,* SUSANNE OERTEL, M.D.,y VOLKER EHEMANN, PH.D.,z MARKUS THIEMANN, PH.D.,y PETER E. HUBER, M.D.,yx MARC BISCHOF, M.D.,* OLAF WITT, M.D.,*k HEDWIG E. DEUBZER, M.D.,*k ANDREAS E. KULOZIK, M.D., PH.D.,* JU¨RGEN DEBUS, M.D., PH.D.,y y AND KLAUS-J. WEBER, PH.D. *Department of Pediatric Oncology, Hematology, Immunology and Pulmology, yDepartment of Radiation Oncology, and zInstitute of Pathology, University of Heidelberg, Germany; xDepartment of Radiation Oncology, German Cancer Research Center, Heidelberg, Germany; and kCCU Pediatric Oncology, German Cancer Research Center, Heidelberg, Germany Purpose: Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. Methods and Materials: Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. Results: SAHA induced an inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). Conclusion: Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs. Ó 2010 Elsevier Inc. Sarcoma, histone deacetylase inhibition, suberoylanilide hydroxamic acid (SAHA), radiosensitization.
potent anticancer activities in a wide variety of tumor models. HDACIs can induce growth arrest, apoptosis, and differentiation of transformed cells with low toxicity to normal cells (2). SAHA has already been approved for the treatment of refractory cutaneous T-cell lymphoma (3). HDACIs promote hyperacetylation of histones, which is a key posttranslational modification of many proteins responsible for regulating critical intracellular pathways (4). HDACIs influence cell cycle, cell differentiation, apoptosis, angiogenesis, cell motility, aggresome formation, and immune function by epigenetic and nonepigenetic mechanisms. Modulation of chromatin structure and gene expression influences radiation response. In vitro and in vivo studies have shown that HDAC inhibitors
INTRODUCTION Local tumor control plays a key role in the therapy of osteosarcoma (OS) and rhabdomyosarcoma (RMS). Surgery is still the ‘‘gold standard,’’ but in nonresectable tumors radiation is the only alternative. Unfortunately sarcomas, especially OS, are rather radiation resistant. Very high radiation doses are necessary to achieve local tumor control, which often leads to high rates of late side effects (1). Radiosensitization by new substances such as histone deacetylase inhibitors (HDACIs) with low toxicity to normal cells could widen the therapeutic options. HDACIs such as suberoylanilide hydroxamic acid (SAHA) have recently entered clinical trials because of their Reprint requests to: Claudia Blattmann, M.D., Department of Pediatric Oncology, Hematology, Immunology, University Children’s Hospital, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany. Tel: +49(0)62215632343; Fax: +49(0)6221566059; E-mail: claudia. [email protected] C. Blattmann and S. Oertel are co–first authors of this work.
Conflict of interest: none. Acknowledgments—We thank our excellent laboratory technicians Sylvia Trinh and Ludmilla Frick as well as Gabriele Becker (German Cancer Research Center) for their great work. We also thank the Dietmar Hopp Stiftung for the generous funding. Received Nov 2, 2009, and in revised form March 1, 2010. Accepted for publication March 19, 2010. 237
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are able to enhance the radiation sensitivity of various cell lines in culture and in tumor xenograft models (5, 6, 7, 8). The aim of this study was to investigate the potential therapeutic value of combining SAHA and photon radiation (XRT) in OS and RMS cell lines and to analyze potential mechanisms of interaction. METHODS AND MATERIALS Cell lines Human OS (KHOS-24OS, SAOS2), human RMS (A-204, RD), and human osteoblast (hFOB 1.19) cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD). Human fibroblasts (NHDFc) were obtained from Promocell (Heidelberg, Germany). KHOS-24OS and RD cell lines were maintained in complete culture medium (Dulbecco’s Modified Eagle Medium [DMEM]) supplemented with 10% fetal calf serum (FCS). A-204 cell line was maintained in complete culture medium (McCoy) supplemented with 10% FCS, SAOS2 was maintained in complete culture medium (MyCoy) supplemented with 15% FCS. All culture media were purchased from the ATCC.
Chemicals SAHA was obtained from Alexis Biochemicals (Lo¨rrach, Germany), M344 and valproate (VPA) from Calbiochem Merck (Germany). Primary monoclonal mouse antibody against Rad51 and Ku80 was obtained from Abcam (Cambridge, UK). Primary monoclonal mouse antibodies against b-actin and histone H3 as well as secondary antibody of Western blot experiments were purchased from Cell Signaling Technology (Danvers, MA).
Clonogenic assay Exponentially growing tumor cells were plated in T25 culture bottles and incubated with medium containing 0 to 1 mmol/l SAHA. Incubation of SAHA started 24 h before XRT. Incubation was stopped after 10 to 12 days. Monolayers were stained with 0.5% Crystal Violet for 10 min. Plates were stained with 0.1 mol/l sodium citrate (pH 4) in ethanol 100% (3:1) for another 10 min. Afterward, plates were dried for 48 to 72 h, and colonies were counted visually. Survival was defined as the ability of cells to form colonies ($50 cells).
Flow cytometry cell-cycle analysis and apoptosis Cells were seeded in T25 culture plates at a density of 1 106 cells per plate 48 h before XRT. Then, 24 h before XRT, 0 to 1 mmol/l SAHA was added. Next, 24, 48, and 72 h after XRT, cells and medium were harvested, centrifuged (800 g), and the cell pellet divided for cell cycle and apoptosis analysis. Flow cytometry analyses were performed using a PAS II flow cytometer (Partec, Munster, Germany) equipped with mercury lamp 100 W and filter combination for 2, 4-diamidino-2-phenylindole (DAPI)–stained single cells. From native sampled probes, the cells were isolated with 2.1% citric acid/ 0.5% Tween 20 according to the method for high-resolution DNA and cell-cycle analyses at room temperature by slightly shaking (9, 10). Staining of the cell suspension was performed using phosphate buffer (7.2 g Na2HPO4 2 H2O in 100 ml H2O distilled, pH 8.0) containing 2, 4-diamidino-2phenylindole (DAPI). Each histogram represents 30,000 cells for measuring DNA index and cell cycle. For histogram analysis, we used the Multicycle program (Phoenix Flow Systems, San Diego, CA) (9).
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For detection of apoptotic cells, a FACS Calibur flow cytometer (Becton Dickinson Cytometry Systems, San Jose, CA) was used with filter combinations for propidium iodide. For analyses and calculations, the Cellquest program (Becton Dickinson Cytometry Systems, San Jose, CA) was used. Each histogram represents 10,000 cells. After preparation according to Nicoletti with modifications measurements were acquired in Fl-2 in logarithmic mode and calculated by setting gates over the first three decades to detect apoptotic cells (10).
Immunoblot analysis After treatment, cells were lysed with lysis buffer (0.5 mol/l Tris/ Cl, pH 6.8, SDS, 87% glycerin, DTT 1 mol/l ad Aqua 100 ml). Afterward, 1 ml lysate was incubated with 1 ml benzonase for 15 min at 37 C. A 40-mg quantity of protein extracts was electrophoresed onto a 12% polyacrylamide gel (Pierce Biotechnology, Rockford, IL) under reducing conditions. The separated proteins were transferred onto nitrocellulose membranes (Amersham Pharmacia Bioscience, Piscataway, NJ). The membranes were then incubated for 45 min in blocking buffer (Tris-buffered saline with 0.1% Tween [TBS-T] and 5% nonfat dry milk), followed by incubation with specific primary antibodies at 1:1,000 dilution for 24 h at 4 C or for 1 h at room temperature. After being washed three times with TBS-T buffer, the membrane was incubated with antimouse IgG secondary antibody (Cell Signaling Technology, Danvers, MA) at 1:1,000 dilution for 1 h at room temperature. The signals were visualized with the ECL+detection system and autoradiography.
Statistical analysis All experiments were performed at least twice. In addition, each experiment was done in duplicate. Combination studies were evaluated using Student’s t test, with the resulting p value representing a two-sided test of statistical significance.
RESULTS SAHA enhances radiosensitivity in OS and RMS cell lines but not in normal cells The main aim of this study was to examine the potency of HDACIs such as SAHA to enhance radiosensitivity in OS and RMS cell lines. We first determined the effect of SAHA alone on clonogenic survival of two OS (KHAOS24OS, SAOS2) and two RMS (A-204, RD) cell lines. As shown in Fig. 1, SAHA inhibits clonogenic survival in a dose-dependent manner in both tumor entities after treatment of cells for 5 days. Susceptibility varies between cells, as has been shown for other entities (11). We determined LD50s of various SAHA doses (0.2 mmol/l (RD), 0.5 mmol/l (SAOS2, A-204), and 1 mmol/l (KHAOS-24OS)). These concentrations correspond well to clinically achievable plasma concentrations shown in Phase I studies of SAHA in adult patients (12). Next, we investigated a potential radiosensitizing effect of SAHA on the OS and RMS cell lines. To this aim, cells were pretreated with SAHA for 24 h at their respective LD50 doses followed by various radiation doses. Our results demonstrate that 24-h pretreatment with SAHA significantly reduces clonogenic survival (1- to 2-log decrease) after XRT exposure in all sarcoma cell lines but not in the normal cells such as fibroblasts and osteoblasts. The dose enhancement factors at 10%
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Fig. 1. Suberoylanilide hydroxamic acid (SAHA) inhibits clonogenic survival in osteosarcoma (KHOS-24OS and SAOS2) and rhabdomyosarcoma (A-204 and RD) cell lines in a dose-dependent manner.
survival in the experiment presented are 1.9 for the KHAOS24OS, 1.5 for the SAOS2, 1.6 for the A-204, and 1.5 for the RD cell line. Data are shown in Fig. 2. Significantly shorter pretreatment with SAHA (2 h before or immediately after radiation) showed no radiosensitizing effect (data not shown). SAHA affects the cell cycle SAHA treatment of cancer cell lines has been shown to alter the cell cycle by inducing a G0/1 cell cycle block by upregulating the cyclin-dependent kinase inhibitor p21 (13, 14). We investigated the effect of SAHA, XRT, or combination therapy on the cell cycle by flow cytometry analysis. Cell cycle analysis revealed two mainly aneuploid (>75%) cell lines (KHOS-24OS and RD) as well as two pseudodiploid cell lines (A-204, SAOS2). After treatment with SAHA, all cell lines showed a shift into G0/1 arrest by 5% to 10%. Use of XRT alone, as wells as combination therapy with SAHA, induced a G2/M-arrest by 10% to 50% (Fig. 3). SAHA enhances radiation induced apoptosis in all cell lines Having shown that SAHA, both alone and in combination with XRT, induces inhibition of clonogenic survival, we next investigated whether OS and RMS cells would undergo apoptosis under these conditions. All cell lines were incubated with different doses of SAHA 24 h before XRT. Apoptosis was measured using the Nicoletti method 24 h, 48 h, and 72 h after XRT. In the OS cell lines KHAOS-24OS and SAOS-24OS, SAHA at 0.5 respectively 1 mmol/l (added 24 h before XRT) and XRT showed a synergistic effect inducing apoptosis in up to 40% of the cells, whereas XRT alone did not change the apoptosis rate. In the RMS cell lines, there was also a marginal induction of apoptosis but significantly less than in the osteosarcoma cell lines (Fig. 4; example data shown for one RMS and one OS cell line). A maximum of apoptosis was measured 48 h after radiation. The incubation with SAHA only 2 h before or immediately after radio-
therapy could not induce apoptosis, corresponding to our findings in the clonogenic assay. SAHA attenuates key proteins involved in the repair of DNA double-strand breaks To examine potential underlying mechanisms of the radiosensitizing effect of HDACIs, we investigated expression of total and acetylated histone H3 and DNA–double strand break (DSB) repair proteins such as Rad51 and Ku80. Both Rad51 and ku80 play a critical role in the repair of DNADSBs. Our results show that SAHA enhances histone H3 acetylation and reduces expression of Rad51 and Ku80 measured 24 h after radiation exposure when added to the cell culture 24 h before radiation exposure (Fig. 5; example data shown for one RMS and one OS cell line). Total H3 expression was essentially unchanged by SAHA treatment. Other HDAC inhibitors have comparable radiosensitizing effects SAHA is a pan-HDACI inhibiting all HDAC family members investigated. We were interested to determine whether other pan-HDACI such as M344 or Class I inhibitors such as VPA would show the same radiosensitizing effect as SAHA. To the KHAOS-24OS cell line, 1 mmol/l M344 and 1 mmol/l VPA were added 24 h before XRT. Results show a dose-dependent decrease of clonogenic survival as well as potent induction of apoptosis measured 48 h after radiation (Figs. 6 and 7). DISCUSSION Radiotherapy is an important alternative in the therapy of nonresectable sarcoma; however new strategies to enhance local control and reduce toxic effects on the normal tissue are needed.
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Fig. 2. Pretreatment for 24 h with suberoylanilide hydroxamic acid (SAHA) significantly reduces clonogenic survival (1- to 2-log decrease) after photon radiation (XRT) exposure in osteosarcoma (KHOS-24OS, SAOS2) and rhabdomyosarcoma (A-204, RD) cell lines. A 24-h pretreatment with 1 mmol/l of SAHA did not influence clonogenic survival after XRT exposure to the osteoblast (hFOB 1.19) and human fibroblasts (NHDFc) cell line.
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Fig. 3. Cell cycle analysis revealed two mainly aneuploid (>75%) cell lines (KHOS-24OS, RD) and two pseudodiploid cell lines (A-204, SAOS2). After treatment with suberoylanilide hydroxamic acid (SAHA), most cell lines showed a slight shift into G0/1 arrest by 5% to 10%. In all cell lines, photon radiation (XRT) alone, as well as combination therapy with SAHA + XRT, induced a G2/M-arrest by 10% to 50% as expected. For KHOS-24OS and RD, cell cycle analysis of the aneuploid cell fraction is shown.
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Fig. 4. In the osteosarcoma cell lines (KHAOS-24OS, SAOS2), apoptosis was induced when suberoylanilide hydroxamic acid (SAHA) was added 24 h before photon radiation (XRT). Already 1 mmol/l SAHA induced potent apoptosis measured 48 h after XRT. In the two rhabdomyosarcoma cell lines, there was no induction of aptoptosis measured 24 and 48 h after photon radiation (XRT) when suberoylanilide hydroxamic acid (SAHA 0.5 mmol/l) was added 24 h before (example data are shown for KHAOS24OS and A-204).
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Fig. 6. Pretreatment for 24 h with other histone deacetylase inhibitors (HDACIs) such as valproate (VPA) 1 mmol/l and M344 (1 mmol/l) have comparable effect on clonogenic survival like suberoylanilide hydroxamic acid (SAHA) after 6-Gy photon radiation (XRT) exposure in the osteosarcoma cell line KHAOS-24OS.
Fig. 5. Suberoylanilide hydroxamic acid (SAHA) attenuates radiation-induced DNA repair protein expression (ku80, Rad51) and acetylation of histone H3. KHOS-24OS and A-204 cells were treated with vehicle control (C), 0.5 (A-204) to 1 mmol/l (KHOS24OS) SAHA (S), 6 Gy radiation (X), or a combination (SX). In combination studies, cells were pretreated with SAHA 24 h before radiation exposure. Western blot analysis was performed on whole-cell lysates 24 h after radiation using antibodies against b-actin, Rad51, ku80, acetylated, and total histone H3.
In this study, we evaluated the capacity of HDACIs such as SAHA to modulate radiation response by enhancing radiation-induced cell death in OS and RMS cell lines in clinically achievable concentrations. We found that SAHA enhanced radiosensitivity in all four RMS and OS cell lines studied but not in normal cells such as fibroblasts and osteoblasts. SAHA did not affect the radiation-induced cell cycle block but did induce apoptosis in both tumor entities. Other
HDACIs such as M344 and VPA also resulted in increased apoptosis when administered with radiation. Furthermore, we demonstrated a decreased expression of key proteins involved in DNA-DSB repair through SAHA as a possible underlying mechanism. SAHA is a prototype of a family of hybrid polar compounds, with a longer half-life, lower toxicity, and greater stability than earlier HDACIs inducing growth arrest in transformed cells. Furthermore, it is a hydroxamic acid derivate that potently inhibits both Class I and Class II HDACIs, and has been approved in the United States by the Food and Drug Administration for the treatment of relapsed and refractory cutaneous T-cell lymphoma (15). Manifold effects of HDACIs such as SAHA modify a large number of proteins that play a role in the regulation of oncogene pathways. Therefore, HDACIs are potential anticancer agents (16, 17). SAHA is known to induce differentiation and/or apoptosis in transformed cells in culture (18, 19, 20). SAHA is considered suitable for combination therapy with XRT because of its relevant biological effects on cellular damage response processes and because of its favorable toxicity profile in normal cells. Pretreatment with SAHA to sensitize cells to XRT has been shown to modify transcription rates of genes involved in DNA damage repair in human prostate and glioma cell lines (4). Furthermore, a combination of SAHA and XRT showed prolonged expression of gH2AX in melanoma cells, consistent with the inhibition of DNA-DSB repair (21, 22). The potential influence of HDACIs on chromatin structure and acetylation status on radiation-induced DNA damage and repair has also been shown by other authors (23, 24, 25), but the mechanisms of action of HDACIs still have to be defined.
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Fig. 7. Apoptosis in osteosarcoma cell line (KHAOS-24OS) measured 48 h after photon radiation (XRT) and 24 h before treatment with suberoylanilide hydroxamic acid (SAHA) 1 mmol/l, valproate (VPA) 1 mmol/l, or M344 1 mmol/l.
A critical event in determining radiosensitivity is the repair of DNA-DSBs. gH2AX expression has been established as a sensitive indicator of DSB induced by ionizing radiation. The ubiquitous histone H2AX becomes rapidly phosphorylated (then called gH2AX) at sites of radiation-induced DNA-DSBs (26, 27). Recent data have shown that the phosphorylation status of H2AX correlates with the repair of DNA-DSBs (28, 29) and also with the radiosensitivity of tumor cells (30). A radiosensitizing mechanism involving DNA repair by pretreatment with SAHA could be its effect on the transcription rate of genes involved in DNA damage repair. However HDACIs are directly or indirectly involved in numerous important cell pathways, including control of gene expression, regulation of cell proliferation, differentiation, migration, and death (16). As a consequence, HDACIs can have multiple mechanisms of inducing radiosensitization. In lung cancer and melanoma cells, SAHA had a strong inhibitory effect on the nonhomologous end-joining pathway after radiation, apparently due to reduced expression of DNA-repair related genes (6). A SAHA-induced decrease in expression of DNA damage repair proteins like DNAPK and Rad51 were seen in human prostate and human glioma cells (7). Our results demonstrate the capacity of SAHA to down-regulate the expression of the DNA-DSB repair proteins Ku80 and Rad51 in sarcoma cell lines. These proteins are critical components of nonhomologous endjoining and homologous recombination, respectively, during repair of DNA-DSBs (31). The inhibition of the expression of
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both genes has previously been shown to increase radiosensitivity (32, 33). The number and type of genes the transcription of which is altered by HDACI is determined by the duration of exposure of the cells to the inhibitor, by the particular HDACI to which the cells are exposed, and by the type of cell (34). The basis for the gene selective action of HDACI in altering transcription is not well understood, and may be determined in part by the composition of the transcription factor protein complexes (35). For example, the activation of apoptotic pathways are common and well-recognized mechanisms of HDACIinduced cell death. HDACI are able to upregulate proapoptotic proteins and to decrease antiapoptotic proteins (36, 37). Furthermore, some authors report the influence of HDACIs on the p53 tumor suppressor pathway, which regulates the transcription of target genes such as p21/ CDKN1A, BAX, FAS, PUMA, BCL-2, and hTERT (38). In our analysis, apoptosis could be induced by the combination therapy of SAHA and XRT in all investigated tumor cell lines. In the OS cell lines, the combination of radiation with SAHA increased apoptosis in a supra-additive manner, and was then responsible for approximately 40% to 50% of cell kill. We further examined the alteration of the cell cycle induced by XRT, SAHA, or the combination therapy. It is well known that cell cycle is altered by XRT as well as by HDACIs such as SAHA (36, 39, 40). Most HDACIs studied to date can induce a G0/1-phase arrest but can also result in a G2/M-phase arrest, although the latter is a much rarer event. This is most often associated with the p53-independent induction of CDKN1A (encoding p21waf/CIP1) (14). In this report, G2/M cell cycle arrest was observed in all cells exposed to XRT alone or combination therapy with SAHA, whereas SAHA alone induced a G0/1 arrest in the KHOS-24OS, A-204, and RD cell line but not in the SAOS2 cell line. Finally, we investigated the effect of other pan-HDACIs such as M344 and VPA to the KHAOS-24OS cell line. Consistent with their similar modes of action, HDACIs seem to have a common toxicity profile, although idiosyncratic side effects of particular HDACIs have been noted and may relate to differences in chemical structure. An individual ‘‘match’’ to particular tumors or genetic profiles could be relevant in the future. Our data demonstrate similar radiosensitizing effects of M344 and VPA on clonogenic survival and apoptosis in comparison to SAHA. Lopez et al. investigated the novel histone deacetylase inhibitor PCI-24781 alone and in combination with conventional chemotherapy in soft tissue sarcoma, and showed that PCI-24781 has a significant antiproliferative activity in vitro, including alteration of the cell cycle and proapoptotic effects (41). However, OS and RMS are tumor entities that are often associated with multidrug resistance, especially in cases of recurrence. Okada et al. demonstrated that the HDACIs FK228 and apicidin exhibited strong resistance in doxorubicin-resistant clones of OS and Ewing’s family in tumors expressing P-glycoprotein (P-gp) and multidrug resistance–associated protein 1 (MRP1) (42). Therefore,
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the authors suggest clarifying the expression of P-gp and MRP1 in patients with such tumors. CONCLUSION In conclusion, our results demonstrate the capacity of HDACIs such as SAHA to significantly enhance radiation response in both OS as well as RMS cell lines, and sup-
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port the evidence showing that HDACIs are promising substances in multimodal cancer treatment. In all investigated sarcoma cell lines, SAHA induced apoptosis as well as a decrease in expression of DNA repair proteins, suggesting underlying key mechanisms for radiosensitization. Therefore the presented data will be reassessed in vivo by the authors in the near future.
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34. Ungerstedt JS, Sowa y, Xu WS, et al. Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors. Proc Natl Acad Sci U S A 2005;102:673–678. 35. Gui CY, Ngo L, Xu WS, et al. Histone deacetylase (HDAC) inhibitor activation of p21WAF1 involves changes in promotorassociated proteins, including HDAC1. Proc Natl Acad Sci U S A 2004;101:1241–1246. 36. Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 2006;5: 769–784. 37. Rosato RR, Maggio SC, Almenara JA, et al. The histone deacetylase inhibitor LAQ824 induces human leukemia cell death through a process involving XIAP down-regulation, oxidative injury, and the acid sphingolyelinase-dependent generation of ceramide. Mol Pharmacol 2006;69:216–225. 38. Condorelli F, Gnemmi A, Vallario A, et al. Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells. Br J Pharmacol 2008;153:657–668.
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39. Arnold NB, Arkus N, Gunn J, et al. The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces growth inhibition and enhances gemcitabine-induced cell death in pancreatic cancer. Clin Cancer Res 2007;13:18–26. 40. Theron T, Binder A, Verheye-Dua F, et al. The role of G2-block abrogation, DNA double-strand break repair and apoptosis in the radiosensitization of melanoma and squamous cell carcinoma cell lines by pentoxifylline. Int J Radiat Biol 2000;76: 1197–1208. 41. Okada T, Tanaka K, Nakatani F, et al. Involvement of P-glycoprotein and MRP1 in resistance to cyclic tetrapeptide subfamiliy of histone deacetylase inhibitors in the drug-resistant osteosarcoma and Ewing’s sarcoma cells. Int J Cancer 2006; 118:90–97. 42. Okada T, Tanaka K, Nakatani F, et al. Involvement of P-glycoprotein and MRP1 in resistance to cyclic tetrapeptide subfamiliy of histone deacetylase inhibitors in the drug-resistant osteosarcoma and Ewing’s sarcoma cells. Int J Cancer 2006;118:90–97.
doi:10.1016/j.ijrobp.2010.03.010
BIOLOGY CONTRIBUTION
ENHANCEMENT OF RADIATION RESPONSE IN OSTEOSARCOMA AND RHABOMYOSARCOMA CELL LINES BY HISTONE DEACETYLASE INHIBITION CLAUDIA BLATTMANN, M.D.,* SUSANNE OERTEL, M.D.,y VOLKER EHEMANN, PH.D.,z MARKUS THIEMANN, PH.D.,y PETER E. HUBER, M.D.,yx MARC BISCHOF, M.D.,* OLAF WITT, M.D.,*k HEDWIG E. DEUBZER, M.D.,*k ANDREAS E. KULOZIK, M.D., PH.D.,* JU¨RGEN DEBUS, M.D., PH.D.,y y AND KLAUS-J. WEBER, PH.D. *Department of Pediatric Oncology, Hematology, Immunology and Pulmology, yDepartment of Radiation Oncology, and zInstitute of Pathology, University of Heidelberg, Germany; xDepartment of Radiation Oncology, German Cancer Research Center, Heidelberg, Germany; and kCCU Pediatric Oncology, German Cancer Research Center, Heidelberg, Germany Purpose: Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. Methods and Materials: Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. Results: SAHA induced an inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). Conclusion: Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs. Ó 2010 Elsevier Inc. Sarcoma, histone deacetylase inhibition, suberoylanilide hydroxamic acid (SAHA), radiosensitization.
potent anticancer activities in a wide variety of tumor models. HDACIs can induce growth arrest, apoptosis, and differentiation of transformed cells with low toxicity to normal cells (2). SAHA has already been approved for the treatment of refractory cutaneous T-cell lymphoma (3). HDACIs promote hyperacetylation of histones, which is a key posttranslational modification of many proteins responsible for regulating critical intracellular pathways (4). HDACIs influence cell cycle, cell differentiation, apoptosis, angiogenesis, cell motility, aggresome formation, and immune function by epigenetic and nonepigenetic mechanisms. Modulation of chromatin structure and gene expression influences radiation response. In vitro and in vivo studies have shown that HDAC inhibitors
INTRODUCTION Local tumor control plays a key role in the therapy of osteosarcoma (OS) and rhabdomyosarcoma (RMS). Surgery is still the ‘‘gold standard,’’ but in nonresectable tumors radiation is the only alternative. Unfortunately sarcomas, especially OS, are rather radiation resistant. Very high radiation doses are necessary to achieve local tumor control, which often leads to high rates of late side effects (1). Radiosensitization by new substances such as histone deacetylase inhibitors (HDACIs) with low toxicity to normal cells could widen the therapeutic options. HDACIs such as suberoylanilide hydroxamic acid (SAHA) have recently entered clinical trials because of their Reprint requests to: Claudia Blattmann, M.D., Department of Pediatric Oncology, Hematology, Immunology, University Children’s Hospital, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany. Tel: +49(0)62215632343; Fax: +49(0)6221566059; E-mail: claudia. [email protected] C. Blattmann and S. Oertel are co–first authors of this work.
Conflict of interest: none. Acknowledgments—We thank our excellent laboratory technicians Sylvia Trinh and Ludmilla Frick as well as Gabriele Becker (German Cancer Research Center) for their great work. We also thank the Dietmar Hopp Stiftung for the generous funding. Received Nov 2, 2009, and in revised form March 1, 2010. Accepted for publication March 19, 2010. 237
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are able to enhance the radiation sensitivity of various cell lines in culture and in tumor xenograft models (5, 6, 7, 8). The aim of this study was to investigate the potential therapeutic value of combining SAHA and photon radiation (XRT) in OS and RMS cell lines and to analyze potential mechanisms of interaction. METHODS AND MATERIALS Cell lines Human OS (KHOS-24OS, SAOS2), human RMS (A-204, RD), and human osteoblast (hFOB 1.19) cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD). Human fibroblasts (NHDFc) were obtained from Promocell (Heidelberg, Germany). KHOS-24OS and RD cell lines were maintained in complete culture medium (Dulbecco’s Modified Eagle Medium [DMEM]) supplemented with 10% fetal calf serum (FCS). A-204 cell line was maintained in complete culture medium (McCoy) supplemented with 10% FCS, SAOS2 was maintained in complete culture medium (MyCoy) supplemented with 15% FCS. All culture media were purchased from the ATCC.
Chemicals SAHA was obtained from Alexis Biochemicals (Lo¨rrach, Germany), M344 and valproate (VPA) from Calbiochem Merck (Germany). Primary monoclonal mouse antibody against Rad51 and Ku80 was obtained from Abcam (Cambridge, UK). Primary monoclonal mouse antibodies against b-actin and histone H3 as well as secondary antibody of Western blot experiments were purchased from Cell Signaling Technology (Danvers, MA).
Clonogenic assay Exponentially growing tumor cells were plated in T25 culture bottles and incubated with medium containing 0 to 1 mmol/l SAHA. Incubation of SAHA started 24 h before XRT. Incubation was stopped after 10 to 12 days. Monolayers were stained with 0.5% Crystal Violet for 10 min. Plates were stained with 0.1 mol/l sodium citrate (pH 4) in ethanol 100% (3:1) for another 10 min. Afterward, plates were dried for 48 to 72 h, and colonies were counted visually. Survival was defined as the ability of cells to form colonies ($50 cells).
Flow cytometry cell-cycle analysis and apoptosis Cells were seeded in T25 culture plates at a density of 1 106 cells per plate 48 h before XRT. Then, 24 h before XRT, 0 to 1 mmol/l SAHA was added. Next, 24, 48, and 72 h after XRT, cells and medium were harvested, centrifuged (800 g), and the cell pellet divided for cell cycle and apoptosis analysis. Flow cytometry analyses were performed using a PAS II flow cytometer (Partec, Munster, Germany) equipped with mercury lamp 100 W and filter combination for 2, 4-diamidino-2-phenylindole (DAPI)–stained single cells. From native sampled probes, the cells were isolated with 2.1% citric acid/ 0.5% Tween 20 according to the method for high-resolution DNA and cell-cycle analyses at room temperature by slightly shaking (9, 10). Staining of the cell suspension was performed using phosphate buffer (7.2 g Na2HPO4 2 H2O in 100 ml H2O distilled, pH 8.0) containing 2, 4-diamidino-2phenylindole (DAPI). Each histogram represents 30,000 cells for measuring DNA index and cell cycle. For histogram analysis, we used the Multicycle program (Phoenix Flow Systems, San Diego, CA) (9).
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For detection of apoptotic cells, a FACS Calibur flow cytometer (Becton Dickinson Cytometry Systems, San Jose, CA) was used with filter combinations for propidium iodide. For analyses and calculations, the Cellquest program (Becton Dickinson Cytometry Systems, San Jose, CA) was used. Each histogram represents 10,000 cells. After preparation according to Nicoletti with modifications measurements were acquired in Fl-2 in logarithmic mode and calculated by setting gates over the first three decades to detect apoptotic cells (10).
Immunoblot analysis After treatment, cells were lysed with lysis buffer (0.5 mol/l Tris/ Cl, pH 6.8, SDS, 87% glycerin, DTT 1 mol/l ad Aqua 100 ml). Afterward, 1 ml lysate was incubated with 1 ml benzonase for 15 min at 37 C. A 40-mg quantity of protein extracts was electrophoresed onto a 12% polyacrylamide gel (Pierce Biotechnology, Rockford, IL) under reducing conditions. The separated proteins were transferred onto nitrocellulose membranes (Amersham Pharmacia Bioscience, Piscataway, NJ). The membranes were then incubated for 45 min in blocking buffer (Tris-buffered saline with 0.1% Tween [TBS-T] and 5% nonfat dry milk), followed by incubation with specific primary antibodies at 1:1,000 dilution for 24 h at 4 C or for 1 h at room temperature. After being washed three times with TBS-T buffer, the membrane was incubated with antimouse IgG secondary antibody (Cell Signaling Technology, Danvers, MA) at 1:1,000 dilution for 1 h at room temperature. The signals were visualized with the ECL+detection system and autoradiography.
Statistical analysis All experiments were performed at least twice. In addition, each experiment was done in duplicate. Combination studies were evaluated using Student’s t test, with the resulting p value representing a two-sided test of statistical significance.
RESULTS SAHA enhances radiosensitivity in OS and RMS cell lines but not in normal cells The main aim of this study was to examine the potency of HDACIs such as SAHA to enhance radiosensitivity in OS and RMS cell lines. We first determined the effect of SAHA alone on clonogenic survival of two OS (KHAOS24OS, SAOS2) and two RMS (A-204, RD) cell lines. As shown in Fig. 1, SAHA inhibits clonogenic survival in a dose-dependent manner in both tumor entities after treatment of cells for 5 days. Susceptibility varies between cells, as has been shown for other entities (11). We determined LD50s of various SAHA doses (0.2 mmol/l (RD), 0.5 mmol/l (SAOS2, A-204), and 1 mmol/l (KHAOS-24OS)). These concentrations correspond well to clinically achievable plasma concentrations shown in Phase I studies of SAHA in adult patients (12). Next, we investigated a potential radiosensitizing effect of SAHA on the OS and RMS cell lines. To this aim, cells were pretreated with SAHA for 24 h at their respective LD50 doses followed by various radiation doses. Our results demonstrate that 24-h pretreatment with SAHA significantly reduces clonogenic survival (1- to 2-log decrease) after XRT exposure in all sarcoma cell lines but not in the normal cells such as fibroblasts and osteoblasts. The dose enhancement factors at 10%
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Fig. 1. Suberoylanilide hydroxamic acid (SAHA) inhibits clonogenic survival in osteosarcoma (KHOS-24OS and SAOS2) and rhabdomyosarcoma (A-204 and RD) cell lines in a dose-dependent manner.
survival in the experiment presented are 1.9 for the KHAOS24OS, 1.5 for the SAOS2, 1.6 for the A-204, and 1.5 for the RD cell line. Data are shown in Fig. 2. Significantly shorter pretreatment with SAHA (2 h before or immediately after radiation) showed no radiosensitizing effect (data not shown). SAHA affects the cell cycle SAHA treatment of cancer cell lines has been shown to alter the cell cycle by inducing a G0/1 cell cycle block by upregulating the cyclin-dependent kinase inhibitor p21 (13, 14). We investigated the effect of SAHA, XRT, or combination therapy on the cell cycle by flow cytometry analysis. Cell cycle analysis revealed two mainly aneuploid (>75%) cell lines (KHOS-24OS and RD) as well as two pseudodiploid cell lines (A-204, SAOS2). After treatment with SAHA, all cell lines showed a shift into G0/1 arrest by 5% to 10%. Use of XRT alone, as wells as combination therapy with SAHA, induced a G2/M-arrest by 10% to 50% (Fig. 3). SAHA enhances radiation induced apoptosis in all cell lines Having shown that SAHA, both alone and in combination with XRT, induces inhibition of clonogenic survival, we next investigated whether OS and RMS cells would undergo apoptosis under these conditions. All cell lines were incubated with different doses of SAHA 24 h before XRT. Apoptosis was measured using the Nicoletti method 24 h, 48 h, and 72 h after XRT. In the OS cell lines KHAOS-24OS and SAOS-24OS, SAHA at 0.5 respectively 1 mmol/l (added 24 h before XRT) and XRT showed a synergistic effect inducing apoptosis in up to 40% of the cells, whereas XRT alone did not change the apoptosis rate. In the RMS cell lines, there was also a marginal induction of apoptosis but significantly less than in the osteosarcoma cell lines (Fig. 4; example data shown for one RMS and one OS cell line). A maximum of apoptosis was measured 48 h after radiation. The incubation with SAHA only 2 h before or immediately after radio-
therapy could not induce apoptosis, corresponding to our findings in the clonogenic assay. SAHA attenuates key proteins involved in the repair of DNA double-strand breaks To examine potential underlying mechanisms of the radiosensitizing effect of HDACIs, we investigated expression of total and acetylated histone H3 and DNA–double strand break (DSB) repair proteins such as Rad51 and Ku80. Both Rad51 and ku80 play a critical role in the repair of DNADSBs. Our results show that SAHA enhances histone H3 acetylation and reduces expression of Rad51 and Ku80 measured 24 h after radiation exposure when added to the cell culture 24 h before radiation exposure (Fig. 5; example data shown for one RMS and one OS cell line). Total H3 expression was essentially unchanged by SAHA treatment. Other HDAC inhibitors have comparable radiosensitizing effects SAHA is a pan-HDACI inhibiting all HDAC family members investigated. We were interested to determine whether other pan-HDACI such as M344 or Class I inhibitors such as VPA would show the same radiosensitizing effect as SAHA. To the KHAOS-24OS cell line, 1 mmol/l M344 and 1 mmol/l VPA were added 24 h before XRT. Results show a dose-dependent decrease of clonogenic survival as well as potent induction of apoptosis measured 48 h after radiation (Figs. 6 and 7). DISCUSSION Radiotherapy is an important alternative in the therapy of nonresectable sarcoma; however new strategies to enhance local control and reduce toxic effects on the normal tissue are needed.
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Fig. 2. Pretreatment for 24 h with suberoylanilide hydroxamic acid (SAHA) significantly reduces clonogenic survival (1- to 2-log decrease) after photon radiation (XRT) exposure in osteosarcoma (KHOS-24OS, SAOS2) and rhabdomyosarcoma (A-204, RD) cell lines. A 24-h pretreatment with 1 mmol/l of SAHA did not influence clonogenic survival after XRT exposure to the osteoblast (hFOB 1.19) and human fibroblasts (NHDFc) cell line.
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Fig. 3. Cell cycle analysis revealed two mainly aneuploid (>75%) cell lines (KHOS-24OS, RD) and two pseudodiploid cell lines (A-204, SAOS2). After treatment with suberoylanilide hydroxamic acid (SAHA), most cell lines showed a slight shift into G0/1 arrest by 5% to 10%. In all cell lines, photon radiation (XRT) alone, as well as combination therapy with SAHA + XRT, induced a G2/M-arrest by 10% to 50% as expected. For KHOS-24OS and RD, cell cycle analysis of the aneuploid cell fraction is shown.
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Fig. 4. In the osteosarcoma cell lines (KHAOS-24OS, SAOS2), apoptosis was induced when suberoylanilide hydroxamic acid (SAHA) was added 24 h before photon radiation (XRT). Already 1 mmol/l SAHA induced potent apoptosis measured 48 h after XRT. In the two rhabdomyosarcoma cell lines, there was no induction of aptoptosis measured 24 and 48 h after photon radiation (XRT) when suberoylanilide hydroxamic acid (SAHA 0.5 mmol/l) was added 24 h before (example data are shown for KHAOS24OS and A-204).
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Fig. 6. Pretreatment for 24 h with other histone deacetylase inhibitors (HDACIs) such as valproate (VPA) 1 mmol/l and M344 (1 mmol/l) have comparable effect on clonogenic survival like suberoylanilide hydroxamic acid (SAHA) after 6-Gy photon radiation (XRT) exposure in the osteosarcoma cell line KHAOS-24OS.
Fig. 5. Suberoylanilide hydroxamic acid (SAHA) attenuates radiation-induced DNA repair protein expression (ku80, Rad51) and acetylation of histone H3. KHOS-24OS and A-204 cells were treated with vehicle control (C), 0.5 (A-204) to 1 mmol/l (KHOS24OS) SAHA (S), 6 Gy radiation (X), or a combination (SX). In combination studies, cells were pretreated with SAHA 24 h before radiation exposure. Western blot analysis was performed on whole-cell lysates 24 h after radiation using antibodies against b-actin, Rad51, ku80, acetylated, and total histone H3.
In this study, we evaluated the capacity of HDACIs such as SAHA to modulate radiation response by enhancing radiation-induced cell death in OS and RMS cell lines in clinically achievable concentrations. We found that SAHA enhanced radiosensitivity in all four RMS and OS cell lines studied but not in normal cells such as fibroblasts and osteoblasts. SAHA did not affect the radiation-induced cell cycle block but did induce apoptosis in both tumor entities. Other
HDACIs such as M344 and VPA also resulted in increased apoptosis when administered with radiation. Furthermore, we demonstrated a decreased expression of key proteins involved in DNA-DSB repair through SAHA as a possible underlying mechanism. SAHA is a prototype of a family of hybrid polar compounds, with a longer half-life, lower toxicity, and greater stability than earlier HDACIs inducing growth arrest in transformed cells. Furthermore, it is a hydroxamic acid derivate that potently inhibits both Class I and Class II HDACIs, and has been approved in the United States by the Food and Drug Administration for the treatment of relapsed and refractory cutaneous T-cell lymphoma (15). Manifold effects of HDACIs such as SAHA modify a large number of proteins that play a role in the regulation of oncogene pathways. Therefore, HDACIs are potential anticancer agents (16, 17). SAHA is known to induce differentiation and/or apoptosis in transformed cells in culture (18, 19, 20). SAHA is considered suitable for combination therapy with XRT because of its relevant biological effects on cellular damage response processes and because of its favorable toxicity profile in normal cells. Pretreatment with SAHA to sensitize cells to XRT has been shown to modify transcription rates of genes involved in DNA damage repair in human prostate and glioma cell lines (4). Furthermore, a combination of SAHA and XRT showed prolonged expression of gH2AX in melanoma cells, consistent with the inhibition of DNA-DSB repair (21, 22). The potential influence of HDACIs on chromatin structure and acetylation status on radiation-induced DNA damage and repair has also been shown by other authors (23, 24, 25), but the mechanisms of action of HDACIs still have to be defined.
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Fig. 7. Apoptosis in osteosarcoma cell line (KHAOS-24OS) measured 48 h after photon radiation (XRT) and 24 h before treatment with suberoylanilide hydroxamic acid (SAHA) 1 mmol/l, valproate (VPA) 1 mmol/l, or M344 1 mmol/l.
A critical event in determining radiosensitivity is the repair of DNA-DSBs. gH2AX expression has been established as a sensitive indicator of DSB induced by ionizing radiation. The ubiquitous histone H2AX becomes rapidly phosphorylated (then called gH2AX) at sites of radiation-induced DNA-DSBs (26, 27). Recent data have shown that the phosphorylation status of H2AX correlates with the repair of DNA-DSBs (28, 29) and also with the radiosensitivity of tumor cells (30). A radiosensitizing mechanism involving DNA repair by pretreatment with SAHA could be its effect on the transcription rate of genes involved in DNA damage repair. However HDACIs are directly or indirectly involved in numerous important cell pathways, including control of gene expression, regulation of cell proliferation, differentiation, migration, and death (16). As a consequence, HDACIs can have multiple mechanisms of inducing radiosensitization. In lung cancer and melanoma cells, SAHA had a strong inhibitory effect on the nonhomologous end-joining pathway after radiation, apparently due to reduced expression of DNA-repair related genes (6). A SAHA-induced decrease in expression of DNA damage repair proteins like DNAPK and Rad51 were seen in human prostate and human glioma cells (7). Our results demonstrate the capacity of SAHA to down-regulate the expression of the DNA-DSB repair proteins Ku80 and Rad51 in sarcoma cell lines. These proteins are critical components of nonhomologous endjoining and homologous recombination, respectively, during repair of DNA-DSBs (31). The inhibition of the expression of
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both genes has previously been shown to increase radiosensitivity (32, 33). The number and type of genes the transcription of which is altered by HDACI is determined by the duration of exposure of the cells to the inhibitor, by the particular HDACI to which the cells are exposed, and by the type of cell (34). The basis for the gene selective action of HDACI in altering transcription is not well understood, and may be determined in part by the composition of the transcription factor protein complexes (35). For example, the activation of apoptotic pathways are common and well-recognized mechanisms of HDACIinduced cell death. HDACI are able to upregulate proapoptotic proteins and to decrease antiapoptotic proteins (36, 37). Furthermore, some authors report the influence of HDACIs on the p53 tumor suppressor pathway, which regulates the transcription of target genes such as p21/ CDKN1A, BAX, FAS, PUMA, BCL-2, and hTERT (38). In our analysis, apoptosis could be induced by the combination therapy of SAHA and XRT in all investigated tumor cell lines. In the OS cell lines, the combination of radiation with SAHA increased apoptosis in a supra-additive manner, and was then responsible for approximately 40% to 50% of cell kill. We further examined the alteration of the cell cycle induced by XRT, SAHA, or the combination therapy. It is well known that cell cycle is altered by XRT as well as by HDACIs such as SAHA (36, 39, 40). Most HDACIs studied to date can induce a G0/1-phase arrest but can also result in a G2/M-phase arrest, although the latter is a much rarer event. This is most often associated with the p53-independent induction of CDKN1A (encoding p21waf/CIP1) (14). In this report, G2/M cell cycle arrest was observed in all cells exposed to XRT alone or combination therapy with SAHA, whereas SAHA alone induced a G0/1 arrest in the KHOS-24OS, A-204, and RD cell line but not in the SAOS2 cell line. Finally, we investigated the effect of other pan-HDACIs such as M344 and VPA to the KHAOS-24OS cell line. Consistent with their similar modes of action, HDACIs seem to have a common toxicity profile, although idiosyncratic side effects of particular HDACIs have been noted and may relate to differences in chemical structure. An individual ‘‘match’’ to particular tumors or genetic profiles could be relevant in the future. Our data demonstrate similar radiosensitizing effects of M344 and VPA on clonogenic survival and apoptosis in comparison to SAHA. Lopez et al. investigated the novel histone deacetylase inhibitor PCI-24781 alone and in combination with conventional chemotherapy in soft tissue sarcoma, and showed that PCI-24781 has a significant antiproliferative activity in vitro, including alteration of the cell cycle and proapoptotic effects (41). However, OS and RMS are tumor entities that are often associated with multidrug resistance, especially in cases of recurrence. Okada et al. demonstrated that the HDACIs FK228 and apicidin exhibited strong resistance in doxorubicin-resistant clones of OS and Ewing’s family in tumors expressing P-glycoprotein (P-gp) and multidrug resistance–associated protein 1 (MRP1) (42). Therefore,
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the authors suggest clarifying the expression of P-gp and MRP1 in patients with such tumors. CONCLUSION In conclusion, our results demonstrate the capacity of HDACIs such as SAHA to significantly enhance radiation response in both OS as well as RMS cell lines, and sup-
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port the evidence showing that HDACIs are promising substances in multimodal cancer treatment. In all investigated sarcoma cell lines, SAHA induced apoptosis as well as a decrease in expression of DNA repair proteins, suggesting underlying key mechanisms for radiosensitization. Therefore the presented data will be reassessed in vivo by the authors in the near future.
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