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Celecoxib Enhances the Radiosensitizing Effect of 7-Hydroxystaurosporine (UCN-01) in Human Lung Cancer Cell Lines
International Journal of
Radiation Oncology biology
physics
www.redjournal.org
Biology Contribution
Celecoxib Enhances the Radiosensitizing Effect of 7-Hydroxystaurosporine (UCN-01) in Human Lung Cancer Cell Lines Young-Mee Kim, Ph.D., In-Hye Jeong, M.Sc., and Hongryull Pyo, M.D., Ph.D. Department of Radiation Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Received Oct 31, 2011, and in revised form Oct 31, 2011. Accepted for publication Jan 2, 2012
Summary The combination of physiologically achievable low concentrations of UCN-01 and celecoxib could induce synergistic cytotoxicity and radiosensitizing effects in in vitro and in vivo systems. The cooperative radiosensitizing effect seemed to be related to a cooperative G2 checkpoint-inhibiting activity by the drug combination. This combined strategy may be useful in clinical applications of UCN-01 for radiotherapy of cancer patients.
Purpose: 7-Hydroxystaurosporine (UCN-01), a Chk1-specific inhibitor, showed promising in vitro and in vivo chemo- or radiosensitizing activity. However, there have been concerns about its limited therapeutic efficacy and risk of side effects. A method of enhancing the treatment efficacy of UCN-01 while not increasing its side effects on normal tissue may therefore be required to apply this drug in clinical settings. Celecoxib is a cyclooxygenase-2 (COX-2)-specific inhibitor that downregulates ataxia telangiectasia and rad3-related (ATR) protein, an upstream kinase of Chk1. In this study, we investigated whether the addition of celecoxib can potentiate the radiosensitizing effect of UCN-01. Methods and Materials: The cooperative radiosensitizing effects and the underlying molecular mechanisms of UCN-01 plus celecoxib were determined by clonogenic assay, tumor growth delay assay, flow cytometry, and Western blotting. Synergism of the three agents combined (UCN-01 plus celecoxib plus radiation) were evaluated using median drug effect analysis and drug-independent action model analysis. Results: The combination of UCN-01 and celecoxib could induce synergistic cytotoxicity and radiosensitizing effects in in vitro and in vivo systems. The combination of both drugs also cooperatively inhibited IR-induced G2/M arrest, and increased the G2 to mitotic transition. Conclusions: Combined treatment with UCN-01 and celecoxib can exert synergistically enhanced radiosensitizing effects via cooperative inhibition of the ionizing radiationactivated G2 checkpoint. We propose that this combination strategy may be useful in clinical applications of UCN-01 for radiotherapy of cancer patients. Ó 2012 Elsevier Inc. Keywords: Celecoxib, Chk1, COX-2, Radiosensitization, UCN-01
Reprint requests to: Hongryull Pyo, M.D., Ph.D., Department of Radiation Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. Tel: 82-2-3410-2438; Fax: 82-2-3410-2619; E-mail: [email protected] This work was supported by Samsung Biomedical Research Institute grant SBRI C-B1-134-1 and Korean Health Technology R and D Project, Int J Radiation Oncol Biol Phys, Vol. 83, No. 3, pp. e399ee407, 2012 0360-3016/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ijrobp.2012.01.001
Ministry of Health and Welfare, Republic of Korea, grant 2010-1037-000. Y.M. Kim and I.H. Jeong contributed equally to this article. Conflict of interest: The authors have no conflicts of interest to disclose. Supplementary material for this article can be found at www.redjournal.org.
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Introduction
for 72 h at 37 C. To determine radiosensitizing effects, the cells were pre-incubated with UCN-01 or celecoxib at indicated concentrations for 4 h. Cells were then exposed to graded doses of IR at a dose rate of 200 cGy/min using 4-MV Clinac iX (Varian Co.), further incubated for 68 h at 37 C, and incubated with drug-free medium for an additional 96 h. The surviving fraction was calculated as follows: mean number of colonies / (number of cells inoculated plating efficiency), where plating efficiency is defined as the mean number of colonies / number of cells inoculated for untreated controls.
Ionizing radiation (IR) induces DNA double-strand breaks and typically activates the G2 checkpoint, which arrests cells in the G2 phase. This arrest provides time to repair DNA damage before mitosis via a signaling cascade from ATR/ATM to Chk1/Chk2 to Cdc25A/-B/-C to the Cdk1/cyclin B1 complex (1). Especially Chk1 plays a key role in this DNA checkpoint pathway (2). The G2 checkpoint abrogation has been regarded as an attractive radiosensitizing method, and a number of G2 checkpoint-inhibiting agents have been developed and tested to enhance the effect of IR (3). 7-Hydroxystaurosporine (UCN-01) is a first generation Chk1 inhibitor that has been evaluated in clinical trials (4, 5). The plasma half-life of UCN-01 in humans is very long (300e1,200 h) due to its avid binding to human a-1 acid-glycoprotein, and, thus, free UCN-01 concentrations are low (110 nM in saliva) (1, 6). The low concentration of free UCN-01 may result in low therapeutic efficacy, and the long half-life of UCN-01 may cause side effects (1, 6). A method of enhancing the treatment efficacy of UCN-01 while not increasing its side effects on normal tissue may therefore be required to apply this drug in clinical settings. We previously reported that celecoxib, a cyclooxygenase-2 (COX-2)-specific inhibitor, downregulates transcription of ATR, providing a possible explanation for its radiosensitizing effect (7). Celecoxib may also therefore act as a G2 checkpoint inhibitor, dependent on or independent of COX-2 expression levels (8). We hypothesized that addition of celecoxib may be able to potentiate the radiosensitizing effect of UCN-01 because celecoxib downregulates ATR and UCN-01 inhibits Chk1, and then their combination may result in a bigger radiosensitizing effect. In this study, we investigated whether combined treatment of UCN-01 at a very low concentration with celecoxib at a clinically achievable concentration could induce cooperative radiosensitizing effects in human lung cancer cells and human tumortransplanted mice. We also investigated the mechanisms underlying this cooperative effect.
Radiation enhancement ratio Radiation enhancement ratios (RERs) for UCN-01 or celecoxib or the combination of both drugs were determined at surviving fractions (SFs) of 0.1 (SF0.1) and 0.25 (SF0.25), using data from clonogenic survival assays as described previously (8).
Synergy analysis Synergy for the three agent combinations (UCN-01 plus celecoxib plus IR) was analyzed using two different and representative synergy analysis methods: median-drug effect analysis (9) and drugindependent action model analysis (10). The basic concepts of these two analyses are identical to mode I and mode II lines of an isobologram, so these methods can also substitute for the complicated three-dimensional isobolographic analysis for the combination of more than two agents. In median drug effect analysis, a combination index (CI) of <1 is synergistic, a CI of 1 is additive, and a CI of >1 is antagonistic (9). In independent action model analysis, the observed SF (OSF) was obtained from the results of clonogenic assays after treatment with UCN-01 and celecoxib with IR, and the expected (additive) SF (ESF) was calculated using an independent action model with data from clonogenic assays after treatment with UCN01 alone, celecoxib alone, or IR alone, followed by statistical comparison of OSF and ESF as previously described (8).
Flow cytometry analysis
Methods and Materials Reagents Celecoxib was provided by Pfizer Inc. (NY). UCN-01 was purchased from Calbiochem (Darmstadt, Germany).
Cell culture The A549 human lung carcinoma cell line, NCI-H460 large-cell lung carcinoma cell line, and BEAS-2B, an immortalized normal bronchial epithelial cell line, were purchased from American Type Culture Collection (ATCC, Manassas, VA). The VMRC-LCD human lung carcinoma cell line was acquired from the Japanese Cancer Research Resources Bank (Osaka, Japan). Cells were cultured in the recommended medium.
In vitro clonogenic assay for cytotoxicity and radiosensitizing effect after radiation Clonogenic assay was performed as previously described (8). To measure cytotoxicity, we exposed the cells to a vehicle (0.1% dimethylsulfoxide [DMSO]) or to graded doses of UCN-01 or celecoxib
To analyze the cell cycle, we exposed cells to the appropriate concentrations of UCN-01 and celecoxib or vehicle (0.1% DMSO) for 4 h and then to 6 Gy IR and further incubated the cells for 6 h at 37 C. The number of cells in each cell cycle was measured as previously described (11).
Western blot analysis Western blotting was performed as previously described (7). Antibodies were a polyclonal rabbit antibody to phospho-Ser345Chk1, Chk1, phospho-Tyr15-Cdk1, Cdk1 (Cell Signaling), phospho-Ser15-histone H3, histone H3 (Millipore), and a monoclonal mouse antibody to b-actin (Sigma).
In vivo tumor growth delay assay All mouse experiments were carried out following protocols approved by the Laboratory Animal Research Center at Samsung Medical Center. The human tumor xenograft was performed as previously described (12). For tumor growth delay assay, tumorbearing mice received celecoxib (15 mg/kg), UCN-01 (2.5 mg/kg), or a drug combination (celecoxib plus UCN-01) intraperitoneally (i.p.) for 5 consecutive days. From the second day of drug treatment,
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Fig. 1. Cooperative cytotoxicity by combined treatment with UCN-01 and celecoxib in lung cancer cell lines and a normal bronchial epithelial cell line. (A) Cells were treated with UCN-01, celecoxib, or a combination of the two drugs at the indicated concentrations for 72 h, and cell survival was monitored by clonogenic assay. (B and C) Synergy of the combination of UCN-01 and celecoxib was determined by CI values according to median drug effect analysis (B) and the ESF/OSF ratio, according to the independent action model (C). Underlined numbers at the top of panel C are the ESF/OSF ratios. All experiments were performed in triplicate, and error bars represent SEM. The symbols (), z) are p < 0.05; UCN-01 plus celecoxib vs. UCN-01 or celecoxib, ESF vs. OSF, respectively. mice were irradiated with 3.3 Gy on the tumor-bearing right hind leg for 3 consecutive days (total dose of 9.9 Gy) using 4-MV Clinac iX. Tumor volume was determined as previously described (12).
Statistical analysis Data from the clonogenic assay were calculated as means standard error of the mean (SEM). Data were analyzed using Student’s t-test to compare the two groups.
Results Combined treatment with UCN-01 and celecoxib induces cooperative cytotoxicity in lung cancer cells The cytotoxicity of UCN-01 in a normal cell line (BEAS-2B) and in three lung cancer cell lines was measured by clonogenic assay,
and the inhibition concentrations (IC) for clonogenic death were determined for UCN-01 (data not shown). The clinically achievable plasma concentration of celecoxib is approximately 5 to 10 mM (13). To investigate combined cytotoxicity effect by UCN-01 and celecoxib, cells were treated with UCN-01 at IC5 (NCI-H460 and BEAS-2B cells, 40 nM) or IC10 (A549 cells, 5 nM; VMRCLCD cells, 30 nM) and 5 mM celecoxib for 72 h, and SFs were determined by clonogenic assay. The combined treatments of UCN-01 and celecoxib significantly increased cell death in all tested cancer cells in a more than additive manner but not in a normal cell line (Fig. 1A). To quantitatively evaluate the cooperative cytotoxic effect of UCN-01 and celecoxib, we further analyzed clonogenic assay data using two different analytic methods: CI values from median-drug effect analysis and the comparison of OSF and ESF, using an independent action model. Results showed that CIs values of UCN-01 plus celecoxib were <1 in all tested cancer cells, but the CI value for BEAS-2B cells was 1 (additive) (Fig. 1B). Analysis by the independent action model also showed that ratios of ESF/
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Fig. 2. Addition of celecoxib synergistically potentiates the radiosensitizing effect of UCN-01 in cancer cells but not in normal cells. Cells were pre-incubated with UCN-01 at IC10 (5 nM for A549 cells [A] and 30 nM for VMRC-LCD cells [B]) or IC5 (40 nM for NCIH460 [C] and BEAS-2B cells [D]), 5 mM celecoxib, or the combination of both drugs for 4 h and then exposed to graded doses of IR; SF was determined by clonogenic assay. SFs of the combination groups were normalized by those of groups exposed to drug alone or drug combination. Error bars represent SEM, which was calculated after pooling the results of three independent experiments. Symbols are p < 0.05; UCN-01 plus celecoxib plus IR vs. IR alone, UCN-01 plus IR, or celecoxib plus IR. OSF in all tested cancer cells were >1 but almost 1 in normal cells (Fig. 1C). These results indicate that combined treatment with UCN-01 and celecoxib at low concentrations could synergistically enhance cell death in cancer cells but not in normal cells.
Celecoxib enhances the radiosensitizing effects of UCN-01 in lung cancer cells To investigate whether celecoxib could potentiate the radiosensitizing effect by UCN-01, we incubated cells with celecoxib (5 mM) and/or UCN-01 (IC5 or IC10) for 4 h and then exposed them to graded doses of IR, and performed the clonogenic assay. Treatment with celecoxib plus IR showed no or slight radiosensitizing effects in the tested cancer cell lines, while treatment
with UCN-01 plus IR showed mild radiosensitizing effects at these low concentrations. However, treatments with UCN-01 plus celecoxib plus IR cooperatively enhanced the radiosensitizing effects in all tested cancer cells (Fig. 2AeC). In contrast, no radiosensitizing effect was found in the normal cell line treated with either one drug or a combination of drugs (Fig. 2D). These results suggest that celecoxib potentiates radiosensitizing effect by UCN01 in lung cancer cell lines.
Combined treatment with UCN-01 and celecoxib increases RER compared to treatment with either drug alone To evaluate the cooperative radiosensitizing effect conferred by the two-drug combination treatment, we measured RERs for
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Fig. 3. Analyses of the RERs and synergy after triple (UCN-01 plus celecoxib plus IR) combined treatment in three lung cancer cell lines and a normal cell line. RERs for UCN-01, celecoxib, or the combination of both drugs were determined at SF0.1 (A) and SF0.25 (B), using data from clonogenic survival assays shown in Figure 2, as described in “Materials and Methods.” Synergy values of combined treatment with UCN-01, celecoxib, and IR determined by CI values according to median drug effect analysis (C) and the ratio of ESF/OSF according to the independent action model are shown as numbers above each bar (D). Symbols are p < 0.05; ), UCN-01 plus celecoxib plus IR versus UCN-01 plus IR; or celecoxib plus IR, y, ESF vs. OSF.
UCN-01, celecoxib, or the combination of both drugs at SF0.1 or SF0.25 (Fig. 2). The RERs for the combined treatment of UCN-01 and celecoxib in the tested cancer cell lines were 1.45w1.55 at SF0.1 and 1.58w1.85 at SF0.25, while those for treatment with either drug alone were 1.01w1.20 and 1.03w1.34, respectively (Fig. 3A and B). In contrast, RERs for the combined drug treatment in the normal cell line were between 1.02 and 1.03 (Fig. 3). These results indicate that the addition of celecoxib can significantly enhance the radiosensitization of cancer cells by UCN-01 while not affecting normal cell radiosensitivity.
Synergy analyses of cytotoxicity by triple combined treatment with UCN-01, celecoxib, and IR To further quantitatively evaluate whether the triple-combination treatment (UCN-01 plus celecoxib plus IR) enhances tumor cell killing synergistically, we estimated synergy by using a mediandrug effect analysis and an independent action model analysis. CIs of all tested cancer cell lines after triple-combination treatment were <1, and the ESF/OSF ratios at 2 Gy or 4 Gy IR in each cancer cell line were between 2.21 and 4.20, which indicates
a synergistic interaction among agents (Fig. 3C and D). However, triple-combination treatment in a normal cell line showed no cooperative effects, with CIs that were 1 and a ESF/OSF ratio that was almost 1. These results support the conclusion that combined treatment with UCN-01 plus celecoxib can synergistically enhance the effect of radiation in cancer cells but not in normal cells and result in potentiating tumor cell killing.
Combined treatment with UCN-01 and celecoxib cooperatively inhibits IR-induced G2/M arrest Next, to determine whether combined drug treatment induces cooperative inhibition of G2 arrest, we treated cells with UCN-01 or celecoxib alone or with a combination of both drugs with or without IR. The G2 checkpoint is usually activated immediately after DNA damage and lasts for several hours (w6 h). We confirmed that both G2 checkpoint activation and G2 arrest can be seen at 6 h after IR exposure in our system (data not shown). The G2 population was significantly decreased in the combination drug treatment group compared to that in groups receiving either drug alone after IR exposure in only cancer cell lines (Fig. 4) but not in
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Fig. 4. Combined treatment with UCN-01 and celecoxib cooperatively inhibits IR-induced G2/M arrest in VMRC-LCD and NCI-H460 cells. VMRC-LCD (A) cells and NCI-H460 (B) cells were incubated with 30 or 40 nM UCN-01 or 5 mM celecoxib or a combination of both drugs for 4 h and then exposed or not to 6 Gy IR. Cells were left to recover for 6 h, and levels of each cell cycle phase were determined by fluorescence-activated cell sorter analysis. The control was treated with 0.1% DMSO. Histograms are representative of three independent experiments, and all three experiments showed identical results. Arrows (/) indicate that the G2/M population decreased by treatment with UCN-01 plus celecoxib with IR.
Volume 83 Number 3 2012 normal cells (see supplementary Fig. E1). Treatment with either drug alone did not result in decreased G2 arrest due to the very low concentrations. These results indicate that UCN-01 plus celecoxib can cooperatively inhibit IR-induced G2/M arrest compared to treatment with UCN-01 alone and suggest that the cooperative radiosensitizing effect shown in the clonogenic assay may be associated with the cooperative inhibition of G2/M arrest after combined administration of both drugs.
Synergistic radiosensitization by UCN-01 and celecoxib e405 To further investigate the cooperative inhibition of G2/M arrest by UCN-01 plus celecoxib at the molecular level, we assessed levels of pY15-Cdk1(a molecular marker for G2 phase cells) and pS10-histone H3 (a molecular marker for mitotic cells) (14). Treatment with either drug alone plus IR did not affect levels of pY15-Cdk1, but combined treatment with both drugs plus IR significantly decreased the pY15-Cdk1 level (Fig. 5A and B). Treatment with UCN-01 plus IR increased the pS10-histone H3
Fig. 5. Combined treatment with UCN-01 and celecoxib cooperatively increased the G2 to mitosis transition after IR treatment in cancer cells. Cells were pretreated with 30 (VMRC-LCD) or 40 (BEAS-2B) nM UCN-01 or 5 mM celecoxib or a combination of both drugs for 4 h and then exposed or not to 6 Gy IR. Cells were left to recover for 6 h, and levels of the indicated proteins (phospho-Y15-cdk1, cdk1, phospho-S10-histone H3, histone H3, b-actin) were determined by Western blot analysis (A, C, E). The relative phosphorylation levels of cdk1 and histone H3 were quantified using Multi Gauge version 3.0 software (FUJI Photo Film Co., Japan) (B, D, F). Error bars represent SEM. Symbols ()) are p < 0.05; UCN-01 plus celecoxib plus IR vs. IR alone, UCN-01 plus IR, or celecoxib plus IR. Control Z 0.1% DMSO only; IR Z 6 Gy radiation; U Z 30 or 40 nM UCN-01; C Z 5 mM celecoxib; U plus C Z UCN-01 plus celecoxib; U plus C plus IR Z UCN-01 plus 5 celecoxib plus radiation.
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level, but treatment with celecoxib plus IR did not lead to an increase compared to IR alone. Addition of celecoxib to UCN-01 in the IR-exposed group increased the level of pS10-histone H3 more significantly than treatment with UCN-01 alone (Fig. 5C and D). In contrast, BEAS-2B normal cells showed increased pY15Cdk1 and decreased pS10-histone H3 levels by treatment with UCN-01 plus celecoxib plus IR (Fig. 5E and F). These results suggest that combined treatment with UCN-01 and celecoxib can cooperatively cause increased G2 to mitotic transition in cancer cells but not normal cells, and may therefore be able to enhance the radiosensitizing effect.
Triple combined treatment with UCN-01, celecoxib, and IR synergistically delays in vivo tumor growth Finally, we examined whether the cooperative radiosensitizing effects conferred by combined treatment with UCN-01 and celecoxib in vitro could also effectively delay in vivo tumor growth. The tumor-bearing mice were treated with celecoxib (15 mg/kg), UCN-01 (2.5 mg/kg), or a combination of both drugs with IR exposure (3.3 Gy 3 fractions). Combined treatment with both drugs with IR significantly delayed tumor growth compared to either drug alone with IR, without weight loss (Fig. 6). These results suggest that UCN-01 and celecoxib also cooperatively enhance the radiosensitizing effect in vivo.
Discussion UCN-01, a Chk1-specific inhibitor, showed promising in vitro and in vivo antineoplastic, chemo- or radiosensitizing activities (2). However, although the plasma concentration of UCN-01 is greater than 30 mM (4), there have been concerns about its limited therapeutic efficacy and risk of side effects (5). A method of enhancing the treatment efficacy of UCN-01 while not increasing its side effects on normal tissue may therefore be required to apply this drug in clinical settings. In this study, we investigated whether celecoxib can potentiate the antineoplastic or radiosensitizing effect of UCN-01. Combined treatment with UCN-01 and celecoxib cooperatively enhanced tumor cell killing and synergistically enhanced radiosensitization in all tested lung cancer cells but not in normal cells (Figs. 1 and 2). The drug combination showed significantly enhanced RERs (1.58e1.85 at SF0.25) only in cancer cells. Two different synergy analyses also concordantly showed synergistic cytotoxicity or radiosensitization with treatment of the UCN-01 plus celecoxib plus IR combination in all tested cancer cells but not in normal cells (Fig. 3C and D). We also found that UCN-01 and celecoxib could cooperatively enhance tumor growth delay by radiotherapy in an in vivo xenograft system (Fig. 6). Taken together, the data indicate that combined treatment with UCN-01 and celecoxib could radiosensitize cancer cells in a cooperative manner in in vivo as well as in vitro systems. Combined treatment with UCN-01 and celecoxib cooperatively decreased the population of G2/M phase cells after IR exposure compared to those in the groups treated with IR and either drug alone (Fig. 4). The G2 to mitotic transition of irradiated cells was also cooperatively increased by the combined drug treatment (Fig. 5). We recently showed that celecoxib could inhibit ATR transcription and that inhibition of ATR increased radiosensitivity (7). These findings suggest that Chk1 inhibition by UCN-01 and
Fig. 6. Combined treatment with UCN-01 and celecoxib effectively delayed tumor growth in vivo. (A) Tumor-bearing mice received celecoxib (15 mg/kg) or UCN-01 (2.5 mg/kg) or a drug combination (celecoxib plus UCN-01) i.p. for 5 consecutive days with or without irradiation of tumors (3.3 Gy 3 fractions) for 3 consecutive days starting from the second day of drug treatment. The control group received an equal volume of DMSO i.p. (B) Changes in body weight of mice in each group. Error bars represent SEM. ), p < 0.05 for a comparison of IR alone with UCN-01 plus IR or celecoxib plus IR at 57 days; z, p < 0.05 for a comparison of celecoxib plus UCN-01 plus IR with UCN-01 plus IR or celecoxib plus IR at 57 days.
ATR downregulation by celecoxib may cooperatively abrogate the G2 checkpoint pathway and may lead to synergistic radiosensitization in cancer cells.
Conclusions In conclusion, we found that a physiologically achievable concentration of celecoxib combined with a nontoxic low concentration of UCN-01 could induce synergistic cytotoxicity and radiosensitizing effects in in vitro and in vivo systems in cancer cells but not in normal cells. The cooperative radiosensitizing effect seemed to be related to a cooperative G2 checkpoint-inhibiting activity by the drug combination. Further
Volume 83 Number 3 2012 studies are underway to understand the precise molecular mechanisms of the combined drug effect with radiation.
References 1. Sampath D, Cortes J, Estrov Z, et al. Pharmacodynamics of cytarabine alone and in combination with 7-hydroxystaurosporine (UCN-01) in AML blasts in vitro and during a clinical trial. Blood 2006;107: 2517e2524. 2. Dai Y, Chen S, Shah R, et al. Disruption of Src function potentiates Chk1-inhibitor-induced apoptosis in human multiple myeloma cells in vitro and in vivo. Blood 2011;117:1947e1957. 3. Sausville EA, Arbuck SG, Messmann R, et al. Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J Clin Oncol 2001;19:2319e2333. 4. Dudgeon C, Wang P, Sun X, et al. PUMA induction by FoxO3a mediates the anticancer activities of the broad-range kinase inhibitor UCN-01. Mol Cancer Ther 2011;9:2893e2902. 5. Fracasso PM, Williams KJ, Chen RC, et al. A phase 1 study of UCN01 in combination with irinotecan in patients with resistant solid tumor malignancies. Cancer Chemother Pharmacol 2010;67:1225e1237. 6. Edelman MJ, Bauer KS Jr., Wu S, et al. Phase I and pharmacokinetic study of 7-hydroxystaurosporine and carboplatin in advanced solid tumors. Clin Cancer Res 2007;13:2667e2674.
Synergistic radiosensitization by UCN-01 and celecoxib e407 7. Kim YM, Lee EJ, Park SY, et al. Cyclooxygenase-2 up-regulates ataxia telangiectasia and Rad3 related through extracellular signal-regulated kinase activation. Mol Cancer Res 2009;7: 1158e1168. 8. Park JS, Jun HJ, Cho MJ, et al. Radiosensitivity enhancement by combined treatment of celecoxib and gefitinib on human lung cancer cells. Clin Cancer Res 2006;12:4989e4999. 9. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58:621e681. 10. Berenbaum MC. A method for testing for synergy with any number of agents. J Infect Dis 1978;137:122e130. 11. Jun HJ, Kim YM, Park SY, et al. Modulation of ionizing radiationinduced G2 arrest by cyclooxygenase-2 and its inhibitor celecoxib. Int J Radiat Oncol Biol Phys 2009;75:225e234. 12. Pyo H, Choy H, Amorino GP, et al. A Selective cyclooxygenase-2 inhibitor, NS-398, enhances the effect of radiation in vitro and in vivo preferentially on the cells that express cyclooxygenase-2. Clin Cancer Res 2001;7:2998e3005. 13. Davies NM, McLachlan AJ, Day RO, et al. Clinical pharmacokinetics and pharmacodynamics of celecoxib: a selective cyclo-oxygenase-2 inhibitor. Clin Pharmacokinet 2000;38:225e242. 14. On KF, Chen Y, Ma HT, et al. Determinants of mitotic catastrophe on abrogation of the G2 DNA damage checkpoint by UCN-01. Mol Cancer Ther 2011;10:784e794.
Radiation Oncology biology
physics
www.redjournal.org
Biology Contribution
Celecoxib Enhances the Radiosensitizing Effect of 7-Hydroxystaurosporine (UCN-01) in Human Lung Cancer Cell Lines Young-Mee Kim, Ph.D., In-Hye Jeong, M.Sc., and Hongryull Pyo, M.D., Ph.D. Department of Radiation Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Received Oct 31, 2011, and in revised form Oct 31, 2011. Accepted for publication Jan 2, 2012
Summary The combination of physiologically achievable low concentrations of UCN-01 and celecoxib could induce synergistic cytotoxicity and radiosensitizing effects in in vitro and in vivo systems. The cooperative radiosensitizing effect seemed to be related to a cooperative G2 checkpoint-inhibiting activity by the drug combination. This combined strategy may be useful in clinical applications of UCN-01 for radiotherapy of cancer patients.
Purpose: 7-Hydroxystaurosporine (UCN-01), a Chk1-specific inhibitor, showed promising in vitro and in vivo chemo- or radiosensitizing activity. However, there have been concerns about its limited therapeutic efficacy and risk of side effects. A method of enhancing the treatment efficacy of UCN-01 while not increasing its side effects on normal tissue may therefore be required to apply this drug in clinical settings. Celecoxib is a cyclooxygenase-2 (COX-2)-specific inhibitor that downregulates ataxia telangiectasia and rad3-related (ATR) protein, an upstream kinase of Chk1. In this study, we investigated whether the addition of celecoxib can potentiate the radiosensitizing effect of UCN-01. Methods and Materials: The cooperative radiosensitizing effects and the underlying molecular mechanisms of UCN-01 plus celecoxib were determined by clonogenic assay, tumor growth delay assay, flow cytometry, and Western blotting. Synergism of the three agents combined (UCN-01 plus celecoxib plus radiation) were evaluated using median drug effect analysis and drug-independent action model analysis. Results: The combination of UCN-01 and celecoxib could induce synergistic cytotoxicity and radiosensitizing effects in in vitro and in vivo systems. The combination of both drugs also cooperatively inhibited IR-induced G2/M arrest, and increased the G2 to mitotic transition. Conclusions: Combined treatment with UCN-01 and celecoxib can exert synergistically enhanced radiosensitizing effects via cooperative inhibition of the ionizing radiationactivated G2 checkpoint. We propose that this combination strategy may be useful in clinical applications of UCN-01 for radiotherapy of cancer patients. Ó 2012 Elsevier Inc. Keywords: Celecoxib, Chk1, COX-2, Radiosensitization, UCN-01
Reprint requests to: Hongryull Pyo, M.D., Ph.D., Department of Radiation Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. Tel: 82-2-3410-2438; Fax: 82-2-3410-2619; E-mail: [email protected] This work was supported by Samsung Biomedical Research Institute grant SBRI C-B1-134-1 and Korean Health Technology R and D Project, Int J Radiation Oncol Biol Phys, Vol. 83, No. 3, pp. e399ee407, 2012 0360-3016/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ijrobp.2012.01.001
Ministry of Health and Welfare, Republic of Korea, grant 2010-1037-000. Y.M. Kim and I.H. Jeong contributed equally to this article. Conflict of interest: The authors have no conflicts of interest to disclose. Supplementary material for this article can be found at www.redjournal.org.
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Introduction
for 72 h at 37 C. To determine radiosensitizing effects, the cells were pre-incubated with UCN-01 or celecoxib at indicated concentrations for 4 h. Cells were then exposed to graded doses of IR at a dose rate of 200 cGy/min using 4-MV Clinac iX (Varian Co.), further incubated for 68 h at 37 C, and incubated with drug-free medium for an additional 96 h. The surviving fraction was calculated as follows: mean number of colonies / (number of cells inoculated plating efficiency), where plating efficiency is defined as the mean number of colonies / number of cells inoculated for untreated controls.
Ionizing radiation (IR) induces DNA double-strand breaks and typically activates the G2 checkpoint, which arrests cells in the G2 phase. This arrest provides time to repair DNA damage before mitosis via a signaling cascade from ATR/ATM to Chk1/Chk2 to Cdc25A/-B/-C to the Cdk1/cyclin B1 complex (1). Especially Chk1 plays a key role in this DNA checkpoint pathway (2). The G2 checkpoint abrogation has been regarded as an attractive radiosensitizing method, and a number of G2 checkpoint-inhibiting agents have been developed and tested to enhance the effect of IR (3). 7-Hydroxystaurosporine (UCN-01) is a first generation Chk1 inhibitor that has been evaluated in clinical trials (4, 5). The plasma half-life of UCN-01 in humans is very long (300e1,200 h) due to its avid binding to human a-1 acid-glycoprotein, and, thus, free UCN-01 concentrations are low (110 nM in saliva) (1, 6). The low concentration of free UCN-01 may result in low therapeutic efficacy, and the long half-life of UCN-01 may cause side effects (1, 6). A method of enhancing the treatment efficacy of UCN-01 while not increasing its side effects on normal tissue may therefore be required to apply this drug in clinical settings. We previously reported that celecoxib, a cyclooxygenase-2 (COX-2)-specific inhibitor, downregulates transcription of ATR, providing a possible explanation for its radiosensitizing effect (7). Celecoxib may also therefore act as a G2 checkpoint inhibitor, dependent on or independent of COX-2 expression levels (8). We hypothesized that addition of celecoxib may be able to potentiate the radiosensitizing effect of UCN-01 because celecoxib downregulates ATR and UCN-01 inhibits Chk1, and then their combination may result in a bigger radiosensitizing effect. In this study, we investigated whether combined treatment of UCN-01 at a very low concentration with celecoxib at a clinically achievable concentration could induce cooperative radiosensitizing effects in human lung cancer cells and human tumortransplanted mice. We also investigated the mechanisms underlying this cooperative effect.
Radiation enhancement ratio Radiation enhancement ratios (RERs) for UCN-01 or celecoxib or the combination of both drugs were determined at surviving fractions (SFs) of 0.1 (SF0.1) and 0.25 (SF0.25), using data from clonogenic survival assays as described previously (8).
Synergy analysis Synergy for the three agent combinations (UCN-01 plus celecoxib plus IR) was analyzed using two different and representative synergy analysis methods: median-drug effect analysis (9) and drugindependent action model analysis (10). The basic concepts of these two analyses are identical to mode I and mode II lines of an isobologram, so these methods can also substitute for the complicated three-dimensional isobolographic analysis for the combination of more than two agents. In median drug effect analysis, a combination index (CI) of <1 is synergistic, a CI of 1 is additive, and a CI of >1 is antagonistic (9). In independent action model analysis, the observed SF (OSF) was obtained from the results of clonogenic assays after treatment with UCN-01 and celecoxib with IR, and the expected (additive) SF (ESF) was calculated using an independent action model with data from clonogenic assays after treatment with UCN01 alone, celecoxib alone, or IR alone, followed by statistical comparison of OSF and ESF as previously described (8).
Flow cytometry analysis
Methods and Materials Reagents Celecoxib was provided by Pfizer Inc. (NY). UCN-01 was purchased from Calbiochem (Darmstadt, Germany).
Cell culture The A549 human lung carcinoma cell line, NCI-H460 large-cell lung carcinoma cell line, and BEAS-2B, an immortalized normal bronchial epithelial cell line, were purchased from American Type Culture Collection (ATCC, Manassas, VA). The VMRC-LCD human lung carcinoma cell line was acquired from the Japanese Cancer Research Resources Bank (Osaka, Japan). Cells were cultured in the recommended medium.
In vitro clonogenic assay for cytotoxicity and radiosensitizing effect after radiation Clonogenic assay was performed as previously described (8). To measure cytotoxicity, we exposed the cells to a vehicle (0.1% dimethylsulfoxide [DMSO]) or to graded doses of UCN-01 or celecoxib
To analyze the cell cycle, we exposed cells to the appropriate concentrations of UCN-01 and celecoxib or vehicle (0.1% DMSO) for 4 h and then to 6 Gy IR and further incubated the cells for 6 h at 37 C. The number of cells in each cell cycle was measured as previously described (11).
Western blot analysis Western blotting was performed as previously described (7). Antibodies were a polyclonal rabbit antibody to phospho-Ser345Chk1, Chk1, phospho-Tyr15-Cdk1, Cdk1 (Cell Signaling), phospho-Ser15-histone H3, histone H3 (Millipore), and a monoclonal mouse antibody to b-actin (Sigma).
In vivo tumor growth delay assay All mouse experiments were carried out following protocols approved by the Laboratory Animal Research Center at Samsung Medical Center. The human tumor xenograft was performed as previously described (12). For tumor growth delay assay, tumorbearing mice received celecoxib (15 mg/kg), UCN-01 (2.5 mg/kg), or a drug combination (celecoxib plus UCN-01) intraperitoneally (i.p.) for 5 consecutive days. From the second day of drug treatment,
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Fig. 1. Cooperative cytotoxicity by combined treatment with UCN-01 and celecoxib in lung cancer cell lines and a normal bronchial epithelial cell line. (A) Cells were treated with UCN-01, celecoxib, or a combination of the two drugs at the indicated concentrations for 72 h, and cell survival was monitored by clonogenic assay. (B and C) Synergy of the combination of UCN-01 and celecoxib was determined by CI values according to median drug effect analysis (B) and the ESF/OSF ratio, according to the independent action model (C). Underlined numbers at the top of panel C are the ESF/OSF ratios. All experiments were performed in triplicate, and error bars represent SEM. The symbols (), z) are p < 0.05; UCN-01 plus celecoxib vs. UCN-01 or celecoxib, ESF vs. OSF, respectively. mice were irradiated with 3.3 Gy on the tumor-bearing right hind leg for 3 consecutive days (total dose of 9.9 Gy) using 4-MV Clinac iX. Tumor volume was determined as previously described (12).
Statistical analysis Data from the clonogenic assay were calculated as means standard error of the mean (SEM). Data were analyzed using Student’s t-test to compare the two groups.
Results Combined treatment with UCN-01 and celecoxib induces cooperative cytotoxicity in lung cancer cells The cytotoxicity of UCN-01 in a normal cell line (BEAS-2B) and in three lung cancer cell lines was measured by clonogenic assay,
and the inhibition concentrations (IC) for clonogenic death were determined for UCN-01 (data not shown). The clinically achievable plasma concentration of celecoxib is approximately 5 to 10 mM (13). To investigate combined cytotoxicity effect by UCN-01 and celecoxib, cells were treated with UCN-01 at IC5 (NCI-H460 and BEAS-2B cells, 40 nM) or IC10 (A549 cells, 5 nM; VMRCLCD cells, 30 nM) and 5 mM celecoxib for 72 h, and SFs were determined by clonogenic assay. The combined treatments of UCN-01 and celecoxib significantly increased cell death in all tested cancer cells in a more than additive manner but not in a normal cell line (Fig. 1A). To quantitatively evaluate the cooperative cytotoxic effect of UCN-01 and celecoxib, we further analyzed clonogenic assay data using two different analytic methods: CI values from median-drug effect analysis and the comparison of OSF and ESF, using an independent action model. Results showed that CIs values of UCN-01 plus celecoxib were <1 in all tested cancer cells, but the CI value for BEAS-2B cells was 1 (additive) (Fig. 1B). Analysis by the independent action model also showed that ratios of ESF/
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Fig. 2. Addition of celecoxib synergistically potentiates the radiosensitizing effect of UCN-01 in cancer cells but not in normal cells. Cells were pre-incubated with UCN-01 at IC10 (5 nM for A549 cells [A] and 30 nM for VMRC-LCD cells [B]) or IC5 (40 nM for NCIH460 [C] and BEAS-2B cells [D]), 5 mM celecoxib, or the combination of both drugs for 4 h and then exposed to graded doses of IR; SF was determined by clonogenic assay. SFs of the combination groups were normalized by those of groups exposed to drug alone or drug combination. Error bars represent SEM, which was calculated after pooling the results of three independent experiments. Symbols are p < 0.05; UCN-01 plus celecoxib plus IR vs. IR alone, UCN-01 plus IR, or celecoxib plus IR. OSF in all tested cancer cells were >1 but almost 1 in normal cells (Fig. 1C). These results indicate that combined treatment with UCN-01 and celecoxib at low concentrations could synergistically enhance cell death in cancer cells but not in normal cells.
Celecoxib enhances the radiosensitizing effects of UCN-01 in lung cancer cells To investigate whether celecoxib could potentiate the radiosensitizing effect by UCN-01, we incubated cells with celecoxib (5 mM) and/or UCN-01 (IC5 or IC10) for 4 h and then exposed them to graded doses of IR, and performed the clonogenic assay. Treatment with celecoxib plus IR showed no or slight radiosensitizing effects in the tested cancer cell lines, while treatment
with UCN-01 plus IR showed mild radiosensitizing effects at these low concentrations. However, treatments with UCN-01 plus celecoxib plus IR cooperatively enhanced the radiosensitizing effects in all tested cancer cells (Fig. 2AeC). In contrast, no radiosensitizing effect was found in the normal cell line treated with either one drug or a combination of drugs (Fig. 2D). These results suggest that celecoxib potentiates radiosensitizing effect by UCN01 in lung cancer cell lines.
Combined treatment with UCN-01 and celecoxib increases RER compared to treatment with either drug alone To evaluate the cooperative radiosensitizing effect conferred by the two-drug combination treatment, we measured RERs for
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Fig. 3. Analyses of the RERs and synergy after triple (UCN-01 plus celecoxib plus IR) combined treatment in three lung cancer cell lines and a normal cell line. RERs for UCN-01, celecoxib, or the combination of both drugs were determined at SF0.1 (A) and SF0.25 (B), using data from clonogenic survival assays shown in Figure 2, as described in “Materials and Methods.” Synergy values of combined treatment with UCN-01, celecoxib, and IR determined by CI values according to median drug effect analysis (C) and the ratio of ESF/OSF according to the independent action model are shown as numbers above each bar (D). Symbols are p < 0.05; ), UCN-01 plus celecoxib plus IR versus UCN-01 plus IR; or celecoxib plus IR, y, ESF vs. OSF.
UCN-01, celecoxib, or the combination of both drugs at SF0.1 or SF0.25 (Fig. 2). The RERs for the combined treatment of UCN-01 and celecoxib in the tested cancer cell lines were 1.45w1.55 at SF0.1 and 1.58w1.85 at SF0.25, while those for treatment with either drug alone were 1.01w1.20 and 1.03w1.34, respectively (Fig. 3A and B). In contrast, RERs for the combined drug treatment in the normal cell line were between 1.02 and 1.03 (Fig. 3). These results indicate that the addition of celecoxib can significantly enhance the radiosensitization of cancer cells by UCN-01 while not affecting normal cell radiosensitivity.
Synergy analyses of cytotoxicity by triple combined treatment with UCN-01, celecoxib, and IR To further quantitatively evaluate whether the triple-combination treatment (UCN-01 plus celecoxib plus IR) enhances tumor cell killing synergistically, we estimated synergy by using a mediandrug effect analysis and an independent action model analysis. CIs of all tested cancer cell lines after triple-combination treatment were <1, and the ESF/OSF ratios at 2 Gy or 4 Gy IR in each cancer cell line were between 2.21 and 4.20, which indicates
a synergistic interaction among agents (Fig. 3C and D). However, triple-combination treatment in a normal cell line showed no cooperative effects, with CIs that were 1 and a ESF/OSF ratio that was almost 1. These results support the conclusion that combined treatment with UCN-01 plus celecoxib can synergistically enhance the effect of radiation in cancer cells but not in normal cells and result in potentiating tumor cell killing.
Combined treatment with UCN-01 and celecoxib cooperatively inhibits IR-induced G2/M arrest Next, to determine whether combined drug treatment induces cooperative inhibition of G2 arrest, we treated cells with UCN-01 or celecoxib alone or with a combination of both drugs with or without IR. The G2 checkpoint is usually activated immediately after DNA damage and lasts for several hours (w6 h). We confirmed that both G2 checkpoint activation and G2 arrest can be seen at 6 h after IR exposure in our system (data not shown). The G2 population was significantly decreased in the combination drug treatment group compared to that in groups receiving either drug alone after IR exposure in only cancer cell lines (Fig. 4) but not in
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Fig. 4. Combined treatment with UCN-01 and celecoxib cooperatively inhibits IR-induced G2/M arrest in VMRC-LCD and NCI-H460 cells. VMRC-LCD (A) cells and NCI-H460 (B) cells were incubated with 30 or 40 nM UCN-01 or 5 mM celecoxib or a combination of both drugs for 4 h and then exposed or not to 6 Gy IR. Cells were left to recover for 6 h, and levels of each cell cycle phase were determined by fluorescence-activated cell sorter analysis. The control was treated with 0.1% DMSO. Histograms are representative of three independent experiments, and all three experiments showed identical results. Arrows (/) indicate that the G2/M population decreased by treatment with UCN-01 plus celecoxib with IR.
Volume 83 Number 3 2012 normal cells (see supplementary Fig. E1). Treatment with either drug alone did not result in decreased G2 arrest due to the very low concentrations. These results indicate that UCN-01 plus celecoxib can cooperatively inhibit IR-induced G2/M arrest compared to treatment with UCN-01 alone and suggest that the cooperative radiosensitizing effect shown in the clonogenic assay may be associated with the cooperative inhibition of G2/M arrest after combined administration of both drugs.
Synergistic radiosensitization by UCN-01 and celecoxib e405 To further investigate the cooperative inhibition of G2/M arrest by UCN-01 plus celecoxib at the molecular level, we assessed levels of pY15-Cdk1(a molecular marker for G2 phase cells) and pS10-histone H3 (a molecular marker for mitotic cells) (14). Treatment with either drug alone plus IR did not affect levels of pY15-Cdk1, but combined treatment with both drugs plus IR significantly decreased the pY15-Cdk1 level (Fig. 5A and B). Treatment with UCN-01 plus IR increased the pS10-histone H3
Fig. 5. Combined treatment with UCN-01 and celecoxib cooperatively increased the G2 to mitosis transition after IR treatment in cancer cells. Cells were pretreated with 30 (VMRC-LCD) or 40 (BEAS-2B) nM UCN-01 or 5 mM celecoxib or a combination of both drugs for 4 h and then exposed or not to 6 Gy IR. Cells were left to recover for 6 h, and levels of the indicated proteins (phospho-Y15-cdk1, cdk1, phospho-S10-histone H3, histone H3, b-actin) were determined by Western blot analysis (A, C, E). The relative phosphorylation levels of cdk1 and histone H3 were quantified using Multi Gauge version 3.0 software (FUJI Photo Film Co., Japan) (B, D, F). Error bars represent SEM. Symbols ()) are p < 0.05; UCN-01 plus celecoxib plus IR vs. IR alone, UCN-01 plus IR, or celecoxib plus IR. Control Z 0.1% DMSO only; IR Z 6 Gy radiation; U Z 30 or 40 nM UCN-01; C Z 5 mM celecoxib; U plus C Z UCN-01 plus celecoxib; U plus C plus IR Z UCN-01 plus 5 celecoxib plus radiation.
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level, but treatment with celecoxib plus IR did not lead to an increase compared to IR alone. Addition of celecoxib to UCN-01 in the IR-exposed group increased the level of pS10-histone H3 more significantly than treatment with UCN-01 alone (Fig. 5C and D). In contrast, BEAS-2B normal cells showed increased pY15Cdk1 and decreased pS10-histone H3 levels by treatment with UCN-01 plus celecoxib plus IR (Fig. 5E and F). These results suggest that combined treatment with UCN-01 and celecoxib can cooperatively cause increased G2 to mitotic transition in cancer cells but not normal cells, and may therefore be able to enhance the radiosensitizing effect.
Triple combined treatment with UCN-01, celecoxib, and IR synergistically delays in vivo tumor growth Finally, we examined whether the cooperative radiosensitizing effects conferred by combined treatment with UCN-01 and celecoxib in vitro could also effectively delay in vivo tumor growth. The tumor-bearing mice were treated with celecoxib (15 mg/kg), UCN-01 (2.5 mg/kg), or a combination of both drugs with IR exposure (3.3 Gy 3 fractions). Combined treatment with both drugs with IR significantly delayed tumor growth compared to either drug alone with IR, without weight loss (Fig. 6). These results suggest that UCN-01 and celecoxib also cooperatively enhance the radiosensitizing effect in vivo.
Discussion UCN-01, a Chk1-specific inhibitor, showed promising in vitro and in vivo antineoplastic, chemo- or radiosensitizing activities (2). However, although the plasma concentration of UCN-01 is greater than 30 mM (4), there have been concerns about its limited therapeutic efficacy and risk of side effects (5). A method of enhancing the treatment efficacy of UCN-01 while not increasing its side effects on normal tissue may therefore be required to apply this drug in clinical settings. In this study, we investigated whether celecoxib can potentiate the antineoplastic or radiosensitizing effect of UCN-01. Combined treatment with UCN-01 and celecoxib cooperatively enhanced tumor cell killing and synergistically enhanced radiosensitization in all tested lung cancer cells but not in normal cells (Figs. 1 and 2). The drug combination showed significantly enhanced RERs (1.58e1.85 at SF0.25) only in cancer cells. Two different synergy analyses also concordantly showed synergistic cytotoxicity or radiosensitization with treatment of the UCN-01 plus celecoxib plus IR combination in all tested cancer cells but not in normal cells (Fig. 3C and D). We also found that UCN-01 and celecoxib could cooperatively enhance tumor growth delay by radiotherapy in an in vivo xenograft system (Fig. 6). Taken together, the data indicate that combined treatment with UCN-01 and celecoxib could radiosensitize cancer cells in a cooperative manner in in vivo as well as in vitro systems. Combined treatment with UCN-01 and celecoxib cooperatively decreased the population of G2/M phase cells after IR exposure compared to those in the groups treated with IR and either drug alone (Fig. 4). The G2 to mitotic transition of irradiated cells was also cooperatively increased by the combined drug treatment (Fig. 5). We recently showed that celecoxib could inhibit ATR transcription and that inhibition of ATR increased radiosensitivity (7). These findings suggest that Chk1 inhibition by UCN-01 and
Fig. 6. Combined treatment with UCN-01 and celecoxib effectively delayed tumor growth in vivo. (A) Tumor-bearing mice received celecoxib (15 mg/kg) or UCN-01 (2.5 mg/kg) or a drug combination (celecoxib plus UCN-01) i.p. for 5 consecutive days with or without irradiation of tumors (3.3 Gy 3 fractions) for 3 consecutive days starting from the second day of drug treatment. The control group received an equal volume of DMSO i.p. (B) Changes in body weight of mice in each group. Error bars represent SEM. ), p < 0.05 for a comparison of IR alone with UCN-01 plus IR or celecoxib plus IR at 57 days; z, p < 0.05 for a comparison of celecoxib plus UCN-01 plus IR with UCN-01 plus IR or celecoxib plus IR at 57 days.
ATR downregulation by celecoxib may cooperatively abrogate the G2 checkpoint pathway and may lead to synergistic radiosensitization in cancer cells.
Conclusions In conclusion, we found that a physiologically achievable concentration of celecoxib combined with a nontoxic low concentration of UCN-01 could induce synergistic cytotoxicity and radiosensitizing effects in in vitro and in vivo systems in cancer cells but not in normal cells. The cooperative radiosensitizing effect seemed to be related to a cooperative G2 checkpoint-inhibiting activity by the drug combination. Further
Volume 83 Number 3 2012 studies are underway to understand the precise molecular mechanisms of the combined drug effect with radiation.
References 1. Sampath D, Cortes J, Estrov Z, et al. Pharmacodynamics of cytarabine alone and in combination with 7-hydroxystaurosporine (UCN-01) in AML blasts in vitro and during a clinical trial. Blood 2006;107: 2517e2524. 2. Dai Y, Chen S, Shah R, et al. Disruption of Src function potentiates Chk1-inhibitor-induced apoptosis in human multiple myeloma cells in vitro and in vivo. Blood 2011;117:1947e1957. 3. Sausville EA, Arbuck SG, Messmann R, et al. Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J Clin Oncol 2001;19:2319e2333. 4. Dudgeon C, Wang P, Sun X, et al. PUMA induction by FoxO3a mediates the anticancer activities of the broad-range kinase inhibitor UCN-01. Mol Cancer Ther 2011;9:2893e2902. 5. Fracasso PM, Williams KJ, Chen RC, et al. A phase 1 study of UCN01 in combination with irinotecan in patients with resistant solid tumor malignancies. Cancer Chemother Pharmacol 2010;67:1225e1237. 6. Edelman MJ, Bauer KS Jr., Wu S, et al. Phase I and pharmacokinetic study of 7-hydroxystaurosporine and carboplatin in advanced solid tumors. Clin Cancer Res 2007;13:2667e2674.
Synergistic radiosensitization by UCN-01 and celecoxib e407 7. Kim YM, Lee EJ, Park SY, et al. Cyclooxygenase-2 up-regulates ataxia telangiectasia and Rad3 related through extracellular signal-regulated kinase activation. Mol Cancer Res 2009;7: 1158e1168. 8. Park JS, Jun HJ, Cho MJ, et al. Radiosensitivity enhancement by combined treatment of celecoxib and gefitinib on human lung cancer cells. Clin Cancer Res 2006;12:4989e4999. 9. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58:621e681. 10. Berenbaum MC. A method for testing for synergy with any number of agents. J Infect Dis 1978;137:122e130. 11. Jun HJ, Kim YM, Park SY, et al. Modulation of ionizing radiationinduced G2 arrest by cyclooxygenase-2 and its inhibitor celecoxib. Int J Radiat Oncol Biol Phys 2009;75:225e234. 12. Pyo H, Choy H, Amorino GP, et al. A Selective cyclooxygenase-2 inhibitor, NS-398, enhances the effect of radiation in vitro and in vivo preferentially on the cells that express cyclooxygenase-2. Clin Cancer Res 2001;7:2998e3005. 13. Davies NM, McLachlan AJ, Day RO, et al. Clinical pharmacokinetics and pharmacodynamics of celecoxib: a selective cyclo-oxygenase-2 inhibitor. Clin Pharmacokinet 2000;38:225e242. 14. On KF, Chen Y, Ma HT, et al. Determinants of mitotic catastrophe on abrogation of the G2 DNA damage checkpoint by UCN-01. Mol Cancer Ther 2011;10:784e794.