Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells

Biochemical and Biophysical Research Communications 394 (2010) 774–779

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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells Shu-Jun Chiu a,b,*, Ching-Hui Hsaio a, Ho-Hsing Tseng a, Yu-Han Su a, Wen-Ling Shih c, Jeng-Woei Lee a, Jennifer Qiu-Yu Chuah a a b c

Department of Life Science, Tzu Chi University, Hualien, Taiwan Institute of Radiation Sciences, Tzu Chi Technology College, Hualien, Taiwan Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung, Taiwan

a r t i c l e

i n f o

Article history: Received 7 March 2010 Available online 17 March 2010 Keywords: Rosiglitazone Radiosensitivity Human colorectal cancer cells

a b s t r a c t Combined-modality treatment has improved the outcome in cases of various solid tumors, and radiosensitizers are used to enhance the radiotherapeutic efficiency. Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptors c used in the treatment of type-2 diabetes, has been shown to reduce tumor growth and metastasis in human cancer cells, and may have the potential to be used as a radiosensitizer in radiotherapy for human colorectal cancer cells. In this study, rosiglitazone treatment significantly reduced the cell viability of p53-wild type HCT116 cells but not p53-mutant HT-29 cells. Interestingly, rosiglitazone pretreatment enhanced radiosensitivity in p53-mutant HT-29 cells but not HCT116 cells, and prolonged radiation-induced G2/M arrest and enhanced radiation-induced cell growth inhibition in HT-29 cells. Pretreatment with rosiglitazone also suppressed radiation-induced H2AX phosphorylation in response to DNA damage and AKT activation for cell survival; on the contrary, rosiglitazone pretreatment enhanced radiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT29 cells. In addition, pretreatment with a pan-caspase inhibitor, zVAD-fmk, attenuated the levels of caspase-3 activation and PARP cleavage in radiation-exposed cancer cells in combination with rosiglitazone pretreatment. Our results provide proof for the first time that rosiglitazone suppresses radiation-induced survival signals and DNA damage response, and enhances the radiation-induced apoptosis signaling cascade. These findings can assist in the development of rosiglitazone as a novel radiosensitizer. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Colon cancer, the second leading cause of cancer death in the USA [1], has become a common malignancy in Asia with the recent changes in diet. Radiotherapy is a standard therapy in the adjuvant treatment of resected colon and rectum cancers [2]. Functional mutations or loss of p53 lead to the promotion of cell proliferation, survival, genomic instability, and increased resistance to radiotherapy and chemotherapy in several tumor cell lines [3]; moreover, up to 50% of human tumors carry a mutant p53 gene. HT-29, a p53mutant human colorectal cancer cell line, is highly resistant to radiation. Therefore, radiosensitizers are used to enhance the radiotherapeutic efficiency in p53-mutant human colorectal cancer carcinoma cells. Research into enhancement of the efficacy of radiotherapy has been focused on the use of conventional chemotherapeutic agents to improve the therapeutic index of radiotherapy [4]. Rosiglitazone * Corresponding author at: Department of Life Science, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien 970, Taiwan. Fax: +886 3 8572526. E-mail address: [email protected] (S.-J. Chiu). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.068

is a member of a class of drugs called thiazolidinediones (TZDs), anti-diabetic drugs that include pioglitazone, rosiglitazone, ciglitazone, and trioglitazone, which have been shown to suppress tumor development in several in vitro and in vivo models [5]. Rosiglitazone inhibits endothelial proliferation and angiogenesis in addition to metastasis in a murine mammary tumor cell line LMM3 [6,7], and previous reports have indicated that rosiglitazone enhances the chemosensitivity of human cancer cells to 5-FU, carboplatin, tumor necrosis factor-a and TRAIL [8–11]. Among the proposed mechanisms of the anti-tumor effects of rosiglitazone, apoptosis induction in colorectal cancer cells [12] and cell-cycle arrest [13] have been reported. As treatment of locally advanced gastrointestinal carcinoma by surgery combined with chemotherapy and radiotherapy remains insufficient, rosiglitazone-modulated cell-cycle arrest and apoptosis may provide a novel basis for the development of chemo-radiotherapy of cancers. However, the use of rosiglitazone as a potential radiosensitizer in radiotherapy for human colorectal cancer cells requires further investigation. Cellular radiosensitivity is governed by the efficient repair of radiation-induced DNA damage in cells. Recent studies have revealed that the suppression of DNA repair capacity forms part of

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the mechanism responsible for the effect of radiosensitizers such as LY294002 [14], gefitinib [15], and roscovitine [16] in human cancer cells. Phosphorylation of H2AX at serine 139, forming cH2AX, is a response of mammalian cells to DNA double-strand breaks (DSBs) induced by ionizing radiation and a variety of genotoxic drugs, along with the production of several other repair proteins to facilitate DNA repair [17,18]. c-H2AX is regarded as a checkpoint maintenance factor, dephosphorylation of which enables resumption of the cell cycle after the DNA damage is repaired [19,20]. Therefore, c-H2AX expression was determined in this study and used as a measure of radiation-induced DSBs, and the effects of tumor-cell radiosensitivity were assessed using a clonogenic survival assay. The counteraction of cell survival and cell death signaling pathways determines the fate of cancer cells in response to radiotherapy. The phosphoinositide 3-kinase (PI3K)/AKT and RAS-RAFMEK-ERK signaling cascades play a role in the regulation of cell survival and cell proliferation [21], and the PI3K/AKT-pathway is one of the radiation resistance mechanisms present in human cancer cells [22,23]. Studies have linked activation of the ERK1/2, ERK5, and AKT pathways to the protection of cells from toxic stresses, which leads to chemo- and radio-resistance [24–26], and their suppression is expected to increase radiosensitivity. In this study, we found that rosiglitazone pretreatment prior to radiation exposure enhanced the radiosensitivity of HT-29 cancer cells through suppression of radiation-induced phosphorylation of H2AX and AKT, and enhanced radiation-induced apoptosis. To our knowledge, this is the first report linking rosiglitazone to enhancement of radiosensitivity. 2. Materials and methods

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medium and further incubated for 7–10 days post-irradiation. The cells were subsequently fixed in 1% crystal violet (containing 30% ethanol) and visualized by an inverted phase-contrast microscope. Twenty random fields were selected, and isolated clusters of more than 50 cells were counted as a single colony. The relative surviving cell fraction was calculated by dividing the number of colonies of treated cells by that of the control. 2.4. Cell cycle analysis Cells were seeded at a density of 5  105 cells per 60-mm culture dish and incubated for 24 h, then pretreated with or without rosiglitazone and irradiated by X-ray (2 and 4 Gy for DNA damage-mediated cell cycle perturbation and cell survival signaling pathways, respectively). After treatment, cells were trypsinized and fixed with ice-cold 70% ethanol at 20 °C overnight. Fixed cells were subsequently stained with 20 lg/ml PI staining buffer (containing 1% triton X-100 and 100 lg/ml RNase A) for 30 min and then the samples were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA). DNA histograms were plotted to calculate the percentage of cells in the different cell cycle phases using ModFit LT software (Ver. 2.0, Becton Dickinson). 2.5. Cell number assay HT-29 cells were plated at a density of 2  106 cells per 100mm Petri dish in 10 ml culture medium for 24 h before treatment. Cells were then pretreated with or without 10 lM rosiglitazone for 24 h prior to 4-Gy X-ray irradiation. After irradiation, cells were washed twice with PBS and recultured in complete medium for various lengths of time before being counted using a hemocytometer.

2.1. Cell culture and reagents 2.6. Western blot analysis p53-wild type human HCT116 colorectal cancer cells (kindly provided by Dr. B. Vogelstein of Johns Hopkins University) were routinely maintained in McCoy’s 5A medium (Sigma, St. Louis, MO). p53-mutant human HT-29 colorectal cancer cells were cultured in RPMI 1640 medium (Invitrogen). Complete medium was additively supplemented with 10% fetal bovine serum. Rosiglitazone was purchased from TOCRIS Bioscience (Ellisville, MO); propidium iodide (PI) from Sigma Chemical Co.; crystal violet from Showa Chemical Co. (Okinawa, Japan); anti-phospho-ERK1/2 (Thr-202/Tyr-204), ERK, anti-phospho-AKT (Ser-473), AKT, caspase-8 and -9, and PARP from Cell Signaling Technology, Inc. (Beverly, MA); anti-phospho-histone H2AX (Ser-139) and anti-actin from Upstate (Lake Placid, NY) and anti-caspase-3 from Imgenex (San Diego, CA).

Total cellular protein extracts were prepared as described in our previous study [18]. Briefly, equal amounts of total protein (20– 60 lg/well) were subjected to electrophoresis using 10–12% sodium dodecyl sulfate-polyacrylamide gels. Following electrophoretic transfer of proteins onto polyvinylidene fluoride membranes, the proteins were sequentially hybridized with primary antibody, followed by a horseradish peroxidase-conjugated secondary antibody. The protein bands were visualized on X-ray film using the ECL detection system (ImmobilonTM Western Chemiluminescent HRP Substrate, WBKLS0500, Millipore) and a gel-digitizing software, Un-Scan-It gel (ver. 5.1; Silk Scientific, Inc., Orem, UT, USA), was used to quantify the relative intensity of each band on the X-ray film. 2.7. Statistical analysis

2.2. X-ray irradiation Irradiation was produced by an X-ray machine (RS2000, RAD Source Technologies, Inc.) operating at 160 kvp and 25 mA; the dose rate at a source–subject distance of 38 cm was 1.83 Gy/min. The machine output was routinely calibrated using an air ionization chamber. Briefly, cells were replenished with fresh medium before irradiation, then treated with X-ray irradiation immediately at room temperature. 2.3. Clonogenic survival assay Cells were seeded at a density of 10,000 cells per well in 2 ml of culture medium in 6-well plates for 20–24 h before treatment. Cells were then treated with various concentrations of rosiglitazone and exposed to a distinct dose of X-ray irradiation. After X-ray exposure, cells were immediately replaced in complete

All data are represented as the mean ± standard error of the mean (SEM) of at least three independent experiments. Statistical analysis was performed by one-way analysis of variance, and further post hoc testing was performed using the statistical software GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA, USA). A p value of <0.05 was considered statistically significant. 3. Results 3.1. Effects of rosiglitazone treatment on cell viability and cell cycle progression in human colorectal cancer cells To evaluate whether rosiglitazone has the ability to induce cell death of human colorectal cancer cells, we examined the cell viability in rosiglitazone-treated cells by MTT assay. Rosiglitazone treatment (5–80 lM) significantly reduced the cell viability of

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p53-wild type HCT116 cells in a concentration-dependent manner but not p53-mutant HT-29 cells (Fig. 1A). The cell cycle progression of cells treated with rosiglitazone was determined by flow cytometry analysis. Interestingly, rosiglitazone treatment (5–80 lM, 24 h) reduced the number of cells in the G0/G1 phase and elevated the number in the G2/M phase in HT-29 cells but not HCT116 cells (Fig. 1B and C).

shows a comparison of the cell cycle phases of HT-29 cells with or without rosiglitazone pretreatment (5 and 10 lM, 24 h) followed by 4-Gy irradiation with a post-irradiation period of 6–24 h. Cell cycle progression was not affected after rosiglitazone treatment (Fig. 3A, left); however, the progression of cells from the G0/G1 phase to the S phase was significantly delayed and the numbers of cells in the G2/M phases were increased in irradiated HT-29 cells after rosiglitazone pretreatment (Fig. 3A, right). In addition, cell proliferation was inhibited by 4-Gy irradiation combined with rosiglitazone pretreatment (10 lM, 24 h) after 1–9 days (Fig. 3B).

3.2. Radiosensitization induced by rosiglitazone pretreatment in human HT-29 colorectal cancer cells

3.4. Rosiglitazone pretreatment suppresses phosphorylation of H2AX and enhances caspase-dependent apoptosis induced by irradiation in HT-29 cells

To explore whether rosiglitazone enhances radiosensitivity in HT-29 cells, the surviving cell fractions of human colorectal cancer cells pretreated with rosiglitazone after exposure to 0–6 Gy X-ray irradiation were determined by clonogenic assay. The radiation sensitivity is expressed as the surviving fraction at 2 Gy (SF2). As shown in Fig. 2, p53-wild type HCT116 cells were more susceptible to irradiation than p53-mutant HT-29 cells, and the SF2 of p53wild type HCT116 and p53-mutant HT-29 cells after irradiation alone was 0.318 and 0.556, respectively. The survival fraction was significantly decreased after rosiglitazone pretreatment (5 and 10 lM, 24 h) in HT-29 cells, and pretreatment with 10 lM rosiglitazone for 24 h significantly reduced the survival fraction of p53-mutant HT-29 cells (54%) as compared with p53-wild type HCT116 cells after 2-Gy irradiation (Fig. 2A and B).

The molecular pathways of rosiglitazone in modulating radiosensitivity were investigated using Western blot analysis. The levels of c-H2AX were transiently increased in HT-29 cells after 6-Gy of irradiation (Fig. 4A). The protein level of c-H2AX was not increased in HT-29 cells by rosiglitazone treatment alone; however, pretreatment with rosiglitazone (10 lM, 24 h) suppressed radiation-induced c-H2AX in irradiated HT-29 cells (Fig. 4A). Furthermore, the effect of rosiglitazone, radiation, and a combination of both on apoptosis induction was examined. Both death receptor and mitochondrial pathways initiator caspases, caspase-8 and -9, and effector caspase-3 were measured by Western blot, and rosiglitazone pretreatment (5–40 lM, 24 h) was found to significantly elevate irradiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT-29 cells (Fig. 4B). In addition, pretreatment with an apoptosis pan-caspase inhibitor, z-VAD-fmk, remarkably reduced caspase-3 activation and PARP cleavage in irradiated HT29 cells pretreated with rosiglitazone (Fig. 4C).

3.3. Rosiglitazone pretreatment prolongs radiation-induced G2/M arrest and enhances radiation-induced cell growth inhibition in HT-29 cells Cell cycle perturbation and growth inhibition induced by treatment with either rosiglitazone, radiation, or a combination of both were analyzed using flow cytometry and cell number assay. Fig. 3A

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Rosiglitazone (μM, 24 h) Fig. 1. Cell viability and cell cycle progression in human colorectal cancer cells treated with rosiglitazone. A, cell viability was assessed by MTT assay after rosiglitazone treatment. B and C, cell cycle progression was assessed by flow cytometry analysis. The populations of G0/G1, S, G2/M and sub-G1 cells were quantified. p < 0.01 () indicates a significant difference between rosiglitazone-treated and -untreated samples.

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Fig. 2. Radiosensitization effects of rosiglitazone in human colorectal cancer cells with irradiation treatment. A, p53-wild type HCT116 and B, p53-mutant HT-29 cells were pretreated with or without rosiglitazone (5 and 10 lM) for 24 h, then exposed to 0–6 Gy X-ray irradiation. The surviving cell fractions were determined by clonogenic survival assay after 7–10 days post-irradiation. Ros: rosiglitazone.

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Fig. 3. Effects of rosiglitazone pretreatment on radiation-induced cell-cycle arrest and cell growth inhibition in HT-29 cells. A, cells were pretreated with or without rosiglitazone, then exposed to 4 Gy X-ray irradiation. After a 0–24-h post-irradiation period, the populations of G0/G1, S, and G2/M cells were analyzed using flow cytometry. B, after rosiglitazone, irradiation, and combination treatment, the total cell numbers were determined after a 1–9-day post-irradiation period by cell growth assay. p < 0.01 () indicates a significant difference between untreated and rosiglitazone-treated samples; p < 0.01 (##) indicates a significant difference between irradiated cells with and without rosiglitazone pretreatment. Ros: rosiglitazone.

3.5. Pretreatment with rosiglitazone enhances the radiosensitivity of irradiated HT-29 cells via the inhibition of phosphorylation of AKT As shown in Fig. 4D, the levels of phospho-AKT and phosphoERK1/2 were elevated by 2-Gy irradiation alone but were not affected by rosiglitazone treatment (10 lM for 24 h) alone in HT29 cells. Interestingly, the level of phospho-AKT in HT-29 cells pretreated with rosiglitazone was reduced after exposure to 2-Gy

irradiation; on the contrary, rosiglitazone pretreatment did not reduce the radiation-induced phosphorylation of ERK in HT-29 cells. 4. Discussion In this study, we found that p53-mutant HT-29 cells were more resistant to rosiglitazone treatment (5–80 lM) than p53-wild type

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actin Fig. 4. Effects of rosiglitazone pretreatment on radiation-induced c-H2AX, caspase-mediated apoptosis, phospho-ERK and phospho-AKT in HT-29 cells. A, the levels of cH2AX were ascertained by Western blot analysis in irradiated cells with or without rosiglitazone pretreatment (10 lM, 24 h) followed by a 1–12-h post-irradiation duration. B, the levels of active caspase-8, -9, and -3 and PARP cleavage were determined by Western blot analysis in 6-Gy irradiated cells with or without rosiglitazone pretreatment (0–40 lM, 24 h). C, cells were pretreated with a pan-caspase inhibitor, z-VAD-fmk, followed by rosiglitazone treatment and 6-Gy irradiation. The levels of active caspase-3 and PARP cleavage were ascertained by Western blot analysis. Analysis was performed after rosiglitazone pretreatment, 6-Gy irradiation, or a combination of both treatments. D, the levels of phospho-ERK1/2, phospho-AKT, total ERK and AKT were analyzed by Western blot analysis in cells pretreated with or without rosiglitazone (10 lM, 24 h) followed by 2-Gy irradiation after 15–60 min post-irradiation. Ros: rosiglitazone.

HCT116 cells. Rosiglitazone (40–80 lM) was observed to significantly increase cytotoxicity in HCT116 but not HT-29 cells, and HCT116 cells were found to be more susceptible to irradiation than HT-29 cells. HT-29 cells were chemo- and radio-resistant in response to rosiglitazone and X-ray radiation treatment; however, the radiosensitivity of HT-29 cells was significantly enhanced by rosiglitazone pretreatment. This was the first study to demonstrate that rosiglitazone exhibits the capability to enhance the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells. We found that rosiglitazone treatment arrested p53-mutant HT-29 cells in the G2/M phase. Mammalian cells exhibit significant radiosensitivity variation as the cell cycle progresses; i.e., cells in the G2/M phase are the most radiosensitive [27]. Therefore, the accumulation of cells in the most radiosensitive G2/M phase caused by rosiglitazone pretreatment is one of the mechanisms responsible for the radiosensitization effect in HT-29 cells. It has been reported that rosiglitazone reduced the protein levels of cyclin D1 and D3 and increased the cyclin-dependent kinase inhibitors p21 and p27 [5,13]. The G2/M cell-cycle arrest may be a result of the inhibition of CDK2/4 and CDK2 by kinase inhibitors; however, the molecular mechanisms of the cell-cycle arrest induced by rosiglitazone treatment require further investigation. Phosphorylation of H2AX was assessed as an indication of the radiation-induced DNA damage response. We observed transient phosphorylation of c-H2AX, which achieved a peak value at 1 h

post-irradiation in irradiated HT-29 cells. c-H2AX is rapidly phosphorylated after DNA DSBs are created following irradiation, and several other repair proteins are recruited to facilitate DNA repair [17,28], which then dissipate over time in correlation with the rejoining of DNA breaks and resumption of cell cycle progression [19,20]. However, rosiglitazone pretreatment suppressed radiation-induced c-H2AX phosphorylation and increased radiation-induced caspase-mediated apoptosis in irradiated HT-29 cells. Our results suggest that rosiglitazone inhibits the repair of DNA DSBs after IR treatment, decreasing the survival of these cells in part by the induction of apoptosis. Activation of the caspase cascade is a key element in the apoptotic process. It has been reported that rosiglitazone sensitizes MDA-MB-231 breast cancer cells to the anti-tumor effects of tumor necrosis factor-alpha [29]. However, rosiglitazone protects the mitochondrial membrane potential and prevent apoptosis by upregulating anti-apoptotic Bcl-2 family proteins in neuroblastoma cells [30]. Rosiglitazone treatment has also been shown to confer resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3epsilon transcription [31]. The results of the present study show that rosiglitazone enhanced radiation-induced activation of caspase-8, -9 and -3 in HT-29 cells, indicating that rosiglitazone enhanced irradiated HT-29 cell apoptosis, which might be mediated via both death receptor (mediated by caspase-8) and mitochondrial (mediated by caspase-9) pathways. Therefore, abundant irreparable DNA damage is accumu-

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lated in irradiated HT-29 cells pretreated with rosiglitazone, augmenting the radiosensitivity through enhancement of induction of caspase-mediated apoptosis, which can be blocked by pan-caspase inhibitor z-VAD-fmk. These findings indicate that rosiglitazone pretreatment inhibits radiation-induced c-H2AX expression and enhances apoptosis in irradiated HT-29 cells. We found that irradiation alone induced significant phosphorylation of AKT and ERK1/2 in HT-29 cells; however, phosphorylation of AKT but not ERK1/2 was significantly suppressed when X-ray exposure was combined with rosiglitazone pretreatment in HT-29 cells. ERK1/2 signaling has often been suggested to play no role in controlling radiosensitivity [32]; moreover, in some cell lines, inhibition of ERK1/2 has been linked to protection from radiation toxicity [21,33]. This is consistent with our finding that rosiglitazone pretreatment did not reduce the radiation-induced phosphorylation of ERK in HT-29 cells. It has been shown that AKT activation inhibits cell death during radiation-induced apoptosis [34]. Rosiglitazone induced apoptosis in human hepatocellular carcinoma cells via inhibition of AKT activation [35]. In addition, blockade of AKT activation by PI3K inhibitor enhances radiosensitivity through the derangement of radiation-induced apoptosis [36]. Accordingly, we suggest that the suppression of radiation-induced phosphorylation of AKT following rosiglitazone pretreatment could contribute to the enhancement of radiosensitivity via promoting apoptosis in HT-29 cells. In conclusion, we found that rosiglitazone enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells by causing the accumulation of cells in the radiosensitive G2/M phase and suppressing the cellular DNA repair capacity, thereby increasing radiation-induced DSBs. Rosiglitazone pretreatment augmented radiation-induced caspase activation and shut down prosurvival signals AKT induced by irradiation. These findings will assist in providing a molecular basis for the development of rosiglitazone as a novel radiosensitizer for human colon cancer cells, and in vivo animal model tests should be conducted for clinical applications.

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Acknowledgment This work was supported by a grant from the National Science Council, Taiwan, NSC (96-2321-B-320-001-MY3).

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