Synergistic effects of meloxicam and conventional cytotoxic drugs in human MG-63 osteosarcoma cells

Biomedicine & Pharmacotherapy 61 (2007) 338e346 www.elsevier.com/locate/biopha

Original article

Synergistic effects of meloxicam and conventional cytotoxic drugs in human MG-63 osteosarcoma cells T. Naruse*, Y. Nishida, N. Ishiguro Department of Orthopaedic Surgery, Nagoya University School and Graduate School of Medicine, 65-Tsurumai, Showa, Nagoya, 466-8550, Japan Received 7 February 2007; accepted 12 February 2007 Available online 9 March 2007

Abstract Cyclooxygenase-2 (COX-2) inhibitors have been shown to exert inhibitory effects on many types of malignant tumors and several groups have suggested that COX-2 inhibitors enhance the cytotoxic effects of other anti-cancer agents. We previously reported that meloxicam has an anti-tumorigenic effect on COX-2-expressing osteosarcoma cells. In the current study, we evaluated the synergy between meloxicam and cisplatin (CDDP), doxorubicin (DXR) and 4-hydroperoxy ifosfamide (4OOH-IFM), using the human osteosarcoma cell line, MG-63. Cytotoxicity was determined using 3-(4,50 -dimethylthiazol-2-yl)-2,50 -diphenyltetrazolium bromide (MTT) assays, and isobolographic analysis was used to evaluate any synergy. Apoptotic activity was determined by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), and by evaluating Bax and Bcl-2 expression levels using real-time RT-PCR and western blotting analysis. Cell cycling was evaluated by flow cytometry. The cytotoxic effects of CDDP and DXR were enhanced synergistically in the presence of meloxicam and were partially due to an increase in apoptosis. By contrast, meloxicam enhanced neither the cytotoxic nor the apoptotic activity of 4OOH-IFM. Combining meloxicam with DXR significantly up-regulated Bax expression, whereas it down-regulated Bcl-2 expression in combination with CDDP. Furthermore, the number of cells in the G2/M phase was significantly increased in DXR-treated samples by the addition of meloxicam, but not in CDDP-treated or 4OOH-IFM-treated samples. These results suggest a potential clinical application of meloxicam in combination with cytotoxic drugs in patients with COX-2-positive osteosarcoma. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Cyclooxygenase-2; Meloxicam; Osteosarcoma

1. Introduction Cyclooxygenase (COX) has two isoforms: COX-1 and COX-2. The latter is an inducible enzyme, the expression of which is usually associated with inflammatory disease. However many investigators have demonstrated constitutive expression of COX-2 associated with carcinogenesis in a variety of malignant tumors [1e3]. These findings have raised the possibility that COX-2 inhibitors might also act as tumor suppressors. Previous publications have shown that many types of COX-2 inhibitors and other non-steroidal anti-inflammatory

* Corresponding author: Tel.: þ81 52 741 2111x5095; fax: þ81 52 744 2260. E-mail address: [email protected] (T. Naruse). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.02.011

drugs (NSAIDs) have anti-tumor effects [4e6]. Osteosarcomas have been reported to express COX-2 constitutively [7], including our previous study, which demonstrated relative over-expression of COX-2 in human osteosarcoma cell line, MG-63 [8]. But few reports have been published concerning the effects of COX-2 inhibitors on this type of tumor [8,9]. Meloxicam was developed in 1977 as a new type of NSAID, and was categorized as a preferential COX-2 inhibitor; the concentration of meloxicam causing 80% inhibition (IC80) of COX-2 is approximately the same as the concentration of meloxicam causing 25% inhibition (IC25) of COX-1. Several authors have reported inhibitory effects of meloxicam on colorectal cancer [10], non-small-cell lung cancer [11], and osteosarcoma cells [8]. However, significant effects on tumors have only been seen at high doses of meloxicam or other NSAIDs, and their efficacy has been low compared with

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traditional anti-cancer agents. It would therefore be impractical to use these NSAIDs alone as therapeutic agents for osteosarcoma patients. Osteosarcoma is the most common primary malignant bone cancer [12], and is characterized as a heterogeneous tumor. Improvements in the prognosis for osteosarcoma patients have mainly been achieved as a result of the introduction of chemotherapy. However, many patients still develop widespread metastasis and have a poor prognosis. Therefore, new strategies that enhance the efficacy of chemotherapeutic agents could help to further improve prognoses. Several reports have suggested that synergistic effects can be achieved by combining COX-2 inhibitors or other NSAIDs with traditional cytotoxic agents for some types of cancer [13e15]. The detailed mechanisms of these synergistic effects are still controversial. To our knowledge, there are no published reports demonstrating synergistic cytotoxic effects on osteosarcoma cells elicited by anti-cancer agents in combination with COX-2 inhibitors. Yet, if such effects could be demonstrated for osteosarcoma, they might provide a new strategy for improving the prognosis for this tumor, and could reduce the doses of anti-cancer drugs needed and thus the occurrence of complications. To this end, the current study evaluated the synergistic effects of meloxicam combined with several anti-cancer agents on the human osteosarcoma cell line, MG-63, in vitro. 2. Materials and methods 2.1. Drug preparation Stock solutions of meloxicam (kindly provided by Boehringer Ingelheim Pharmaceuticals, Ingelheim, Germany), cisplatin (CDDP; Bristol Pharmaceuticals K.K., Japan), and doxorubicin (DXR; Kyouwahakkou, Japan) were prepared and diluted in culture medium to the required final concentrations for experiments. Stock solutions of meloxicam and DXR were stored at 4  C, and the CDDP stock solution was stored at room temperature. The activated form of ifosfamide, 4hydroperoxy ifosfamide (4OOH-IFM), was kindly provided by Shionogi, Japan, and was stored at 20  C and dissolved immediately before the experiments. 2.2. Cell culture The human osteosarcoma cell line, MG-63, was purchased from the American Type Culture Collection (Manassus, VA), and grown as monolayers in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Aldrich, MO) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate. The cultures were maintained in a humidified atmosphere with 5% CO2 at 37  C. 2.3. Assay of cell viability MG-63 cells were seeded in 96-well plates at 5  103 cells/ well and allowed to adhere for 12 h. The sub-confluent cell

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cultures were exposed to medium containing 10e750 mM meloxicam, 1e10 mg/ml CDDP, 0.5e2.5 mg/ml DXR, or 1e 10 mg/ml 4OOH-IFM for 24 h. Cell viability was determined using 3-(4,50 dimethylthiazol-2-yl)-2,50 -diphenyltetrazolium bromide (MTT) assays (Boehringer Mannheim, Germany). Doseeresponse curves for each drug alone were constructed. To evaluate any synergistic effects between the anti-cancer agents and meloxicam, sub-confluent cells were exposed to 1 mg/ml or 5 mg/ml CDDP, 0.5 mg/ml or 0.75 mg/ml DXR, or 1 mg/ml or 5 mg/ml 4OOH-IFM, for 24 h in the presence or absence of 30 mM or 50 mM meloxicam, and cell viability was measured using MTT. The half-maximal inhibitory concentration (IC50) was determined for each drug, and any synergy between meloxicam and each cytotoxic agent was evaluated by isobolographic analysis [16,17]. 2.4. Apoptosis assessment by TUNEL TdT terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) was used to evaluate the induction of apoptosis by each anti-cancer agent, both alone and in combination, with meloxicam. The MG-63 cells were seeded on chamber slides (Becton Dickinson Labware, NJ) and allowed to adhere for 12 h. On the basis of the isobolographic analysis, the following concentrations of the anti-cancer drugs were used for TUNEL: 5 mg/ml CDDP, 0.75 mg/ml DXR, or 5 mg/ml 4OOH-IFM. Cells were incubated with the drug in the presence or absence of 50 mM meloxicam for 12 h, fixed with 4% paraformaldehyde, and processed for TUNEL using an in situ cell death-detection kit, POD (Roche Diagnostics, PA). Cells with fluorescein-stained nuclei were counted in four different fields using a fluorescence microscope at a magnification of 400, and the percentage of positive cells was calculated to give an apoptotic index. 2.5. Real time RT-PCR analysis To investigate the mechanism by which each anti-cancer agent, either alone or in combination with meloxicam, induced apoptosis, Bax and Bcl-2 mRNA levels were determined by semi-quantitative real time RT-PCR. MG-63 cells were seeded on six-well plates and allowed to adhere for 12 h. The subconfluent cells were exposed to 5 mg/ml CDDP, 0.75 mg/ml DXR or 5 mg/ml 4OOH-IFM, in the presence or absence of 50 mM meloxicam for 6 h or 12 h. Total cellular RNA was isolated from each well using Trizol reagent (Life Technologies, NY), and subsequently 0.5 mg total cellular RNA was converted into complementary DNA (cDNA) using Molony murine leukemia virus reverse transcriptase in the presence of 0.15 mM oligo d(T)16. The following primer pairs were used: Bax sense 50 AAT CCC CGA TTC ATC TAC CC-30 and antisense 50 -TCA CAC CTG TAA TCC CAG CA-30 (predicted PCR product of 271 base pairs (bp)); Bcl-2 sense 50 -GGA TGC CTT TGT GGA ACT GT-30 and antisense 50 -AGC CTG CAG CTT TGT TTC AT-30 (predicted PCR product of 236 bp); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense 50 -TTA CTC CTT GGA GGC CAT GTG GGC-30 and antisense

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50 -ACT GCC ACC CAG AAG ACT GTG GAT GG-30 (predicted PCR product of 465 bp). Assuming the efficiency of reverse transcription was the same in each reaction, cDNA levels were taken as representative of the original messenger RNA (mRNA) levels. cDNA was subjected to real-time RT-PCR for the semi-quantitative determination of Bax or Bcl-2 mRNA levels using a LightCycler (Roche Molecular Biochemicals, Germany). The mRNA levels were expressed relative to GAPDH mRNA levels in the same sample. Pre-diluted cDNA samples were included in each experiment to obtain a standard curve from which the Bax, Bcl-2, and GAPDH PCR products were calculated. The SYBR Green fluorescence was proportional to the concentration of the product; it was measured once per cycle and was immediately displayed on a computer screen, permitting real-time monitoring of the PCR. The mRNA levels were calculated for each sample from the standard curve using the LightCycler Data Analysis software (Roche Molecular Biochemicals, Germany). The relative levels of Bax and Bcl-2 mRNA in samples were expressed as the percentage of the GAPDH mRNA level in the same sample. 2.6. Western blot analysis In addition to assessing mRNA expression for Bax and Bcl2 by real-time RT-PCR, protein expression levels in MG-63 cells were analyzed by western blot analysis. Sub-confluent cells were lysed after 6 h or 12 h exposure to each drug, as described above, and protein was extracted using CelLyticÔ-M (Sigma Aldrich). Protein concentrations were measured using the BCA protein assay (BioRad Protein Assay, BioRad Laboratories, CA), and 100 mg protein per lane was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (HybondÔ-P, Amersham Biosciences, UK). The membranes were incubated in 10% non-fat dried milk for 30 min, and then probed with primary antibody for 1 h at 37  C. The primary antibodies used were rabbit anti-human Bax polyclonal antibody (Santa Cruz Biotechnology, CA; 1:100) and mouse anti-human Bcl2 monoclonal antibody (Oncogene, CA; 1:50). After rinsing with phosphate-buffered saline (PBS) containing 0.1% Tween-20 (Sigma Aldrich), the membranes were incubated for 1 h at 37  C with either horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse immunoglobulin G (IgG; Vector Laboratories, CA;1:400) as secondary antibodies for detecting Bax or Bcl-2, respectively. The membranes were washed again, visualized with enhanced chemiluminescence

reagents (ECLÔ western blotting detection reagents; American Biosciences, UK) and immediately photographed with a charge-coupled device (CCD) digital scan camera (Cool Saver, Rise & ATTO Corporation, Japan). The membranes were also probed with a mouse anti-human b-actin monoclonal antibody (Sigma, MI; 1:5000). 2.7. Cell-cycle analysis The effects of each anti-cancer agent, with or without meloxicam, on the MG-63 cell cycle were evaluated by flow cytometry. Sub-confluent cell layers were exposed to 5 mg/ ml CDDP, 0.75 mg/ml DXR, or 5 mg/ml 4OOH-IFM in the presence or absence of 50 mM meloxicam for 12 h. Cells were trypsinized, suspended in PBS, and stained with propidium iodide (PI) using the CycleTESTÔ PLUS DNA reagent kit (Becton Dickinson Immunocytometry Systems, CA). The DNA content of the stained cells was immediately analyzed using a FACSCalibur (Becton Dickinson, NJ). At least 10,000 cells were counted. The percentages of cells in G0/ G1 phase, S phase, and G2/M phase were calculated using ModFit LT software (Verity Software House, CA). 2.8. Statistical analysis All quantitative experiments were performed at least three times. Analysis of variance (ANOVA), followed by the Bonferroni-Dunn post-hoc test, was used to assess differences between means. p < 0.05 was considered statistically significant. 3. Results 3.1. Synergistic effects on cell viability As shown in Table 1, all three anti-cancer drugs and meloxicam reduced MG-63 cell viability in a dose-dependent manner after 24 h. Based on these data, each anti-cancer drug was used in subsequent experiments at concentrations that would cause similar levels of cytotoxicity, inhibiting cell viability by 10e20% to plot IC50-isobologram. The IC50 values  standard deviation (SD) for each drug at 24 h were as follows: 718.71  7.25 mM for meloxicam, 8.57  0.02 mg/ml for CDDP, 1.86  0.06 mg/ml for DXR, and 7.29  0.01 mg/ml for 4OOH-IFM. Doses of 30 mM and 50 mM meloxicam reduced MG-63 cell viability by w5.12% ( p ¼ 0.0918) and 6.97% ( p ¼ 0.0121), respectively. As low concentrations of

Table 1 Inhibitory concentration of each drug at 24 h

Meloxicam CDDP DXR 4OOH-IFM

IC10

IC20

IC30

IC40

IC50

69.35  1.1 2.16  0.04 0.53  0.02 0.80  0.03

243.40  5.8 4.99  0.02 0.81  0.03 3.75 0 0.04

478.66  4.2 6.19  0.03 0.96  0.02 5.57  0.02

607.40  6.6 7.38  0.01 1.35  0.05 6.43  0.02

718.7  7.25 8.57  0.02 1.82  0.06 7.29  0.01

Meloxicam: mM. CDDP, DXR and 4OOH-IFM: mg/ml.

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meloxicam are most practical for clinical use, we aimed to use as the lowest possible dose in these experiments. In preliminary studies, 20 mM meloxicam had no effect on the level of cytotoxicity induced by the anti-cancer drugs (data not shown). For the analysis of synergy, meloxicam was therefore used at concentrations of 30 mM and 50 mM. Combining 30 mM meloxicam with 1 mg/ml or 5 mg/ml CDDP significantly reduced MG-63 cell viability by w10.8% ( p < 0.0001) or w15.5% ( p ¼ 0.0032), respectively (data not shown). The effect on cell viability was greater using the higher (50 mM) concentration of meloxicam, with 1 mg/ml or 5 mg/ml CDDP reducing cell viability by w15.5% ( p < 0.0001) or w20.6% ( p ¼ 0.0001), respectively (Fig. 1A). Similarly, combining 30 mM meloxicam with 0.5 mg/ml or 0.75 mg/ml

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DXR reduced cell viability by w6.0% ( p ¼ 0.0012) or w9.6% ( p ¼ 0.0004), respectively (data not shown). Combining 50 mM meloxicam with 0.5 mg/ml or 0.75 mg/ml DXR reduced cell viability significantly by w9.1% ( p ¼ 0.0001) or w13.7% ( p < 0.0001), respectively (Fig. 1B). By contrast, the cytotoxic effect of 4OOH-IFM was not enhanced in the presence of 30 mM (data not shown) or 50 mM meloxicam (Fig. 1C). Isobolographic analysis revealed evident synergy between CDDP and meloxicam, especially at 50 mM (Fig. 2A). There was also synergy between DXR and meloxicam; however, the effect was slightly weaker than for the CDDP and meloxicam combination (Fig. 2B). The combined effects of 4OOHIFM and meloxicam were only additive or possibly inhibitory (Fig. 2C). As the maximal reduction in MG-63 cell viability was seen when 50 mM meloxicam was combined with 5 mg/ml CDDP, 0.75 mg/ml DXR, or 5 mg/ml 4OOH-IFM, these concentrations were used for subsequent experiments. 3.2. TUNEL assay To determine the mechanism by which meloxicam acted synergistically with the anticancer drugs to reduce osteosarcoma cell viability, we calculated apoptotic indices using TUNEL data (Table 2). The average percentage of apoptotic cells increased significantly, by 10.7% ( p ¼ 0.029), after exposure to 50 mM meloxicam alone, compared with the control. Apoptosis was also increased in cultures treated with 50 mM meloxicam in the presence of CDDP and DXR, by 15.9% ( p < 0.0001) and 6.5% ( p ¼ 0.0114), respectively. By contrast, meloxicam had no significant effect on apoptosis in cells that were also treated with 4OOH-IFM. 3.3. Expression of Bax and Bcl-2 mRNAs and protein

Fig. 1. Effects of anti-cancer drugs on MG-63 cell viability in the presence of 50 mM meloxicam. Cytotoxicity was determined using MTT assays after 24 h treatment with 1 mg/ml or 5 mg/ml CDDP (A), 0.5 mg/ml or 0.75 mg/ml DXR (B), and 1 mg/ml or 5 mg/ml 4OOH-IFM (C). Each experiment was performed in triplicate and the bars represent the mean  SD. An asterisk indicates statistically significant differences between groups (p < 0.01).

To investigate the mechanism by which apoptosis was induced by combinations of meloxicam and the anticancer drugs, we evaluated the expression levels of Bax and Bcl-2 mRNAs and protein. Real-time RT-PCR showed that combining DXR and meloxicam increased the expression of Bax mRNA in MG-63 cells significantly at 6 h, compared with DXR alone ( p ¼ 0.0098; Fig. 3A). Although all three anticancer agents down-regulated Bcl-2 mRNA expression markedly after 6 h when used alone, the addition of meloxicam did not make an appreciable difference (Fig. 3B). At 12 h, Bax mRNA expression was significantly up-regulated in cells treated with meloxicam, compared with the control ( p ¼ 0.0009; Fig. 3C). Bax mRNA expression was up-regulated in the presence of 4OOH-IFM after 12 h, compared with the control, with or without meloxicam ( p < 0.0001 and p ¼ 0.0011, respectively), However there was no significant difference in Bax expression between cultures treated with 4OOH-IFM in the presence and absence of meloxicam (Fig. 3C). Combining CDDP with meloxicam significantly down-regulated Bcl-2 expression in MG-63 cells, compared with CDDP alone ( p ¼ 0.0261), after 12 h (Fig. 3D).

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Fig. 2. Isobolograms evaluating the synergy between 50 mM meloxicam and the anti-cancer drugs CDDP (A), DXR (B), and 4OOH-IFM (C). The mode lines were drawn based on the dose-response curves for each drug (Table 1), and the points plotted on the isobolograms were based on the results of cytotoxicity assays using MG-63 cells (Fig. 1).

Fig. 3. Expression levels of Bax (A and C) and Bcl-2 (B and D) mRNAs. Total cellular RNA was isolated from MG-63 cells treated with 5 mg/ml CDDP, 0.75 mg/ml DXR, or 5 mg/ml 4OOH-IFM in the presence or absence of 50 mM meloxicam, and subjected to real time RT-PCR after 6 h (A and B) or 12 h (C and D). Each experiment was performed in triplicate, and the bars represent the mean  SD. Statistically significant differences between groups are indicated by *(p < 0.05) and **(p < 0.01). Statistically significant differences from the control group are indicated by þ(p < 0.05) and þþ (p < 0.01).

Table 2 Apoptotic index at 12 h

Without meloxicam With meloxicam

Control (%)

CDDP (%)

DXR (%)

4OOH-IFM (%)

2.7  3.8(%) 13.4  1.0*

18.5  3.8 34.4  4.0**

22.9  2.1 29.4  3.0*

28.16.6 27.27.5

Meloxicam, 50 mM; CDDP, 5 mg/ml; DXR, 0.75 mg/ml; 4OOH-IFM, 5 mg/ml. *Statistically significant differences compared with the groups treated without meloxicam ( p < 0.05). **Statistically significant differences compared with the groups treated without meloxicam ( p < 0.01).

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As shown in Fig. 4, all three anti-cancer drugs inhibited Bcl-2 expression at the protein level, with or without meloxicam. The expression of Bax protein in MG-63 cells was higher in cultures treated with DXR and meloxicam together, compared with DXR alone, at 6 h. At 12 h, Bax protein levels were also raised in cells treated with meloxicam alone and with 4OOH-IFM, in the presence or absence of meloxicam, compared with controls. The expression of Bcl-2 protein was lower in MG-63 cells treated with CDDP and meloxicam together than in those treated with CDDP alone, at 12 h. These results for protein expression were in agreement with those for mRNA expression (Fig. 3).

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Table 3 Cell cycle distribution at 12 h

Control þmeloxicam CDDP þmeloxicam DXR þmeloxicam 4OOH-IFM þmeloxicam

G0/G1(%)

S(%)

G2/M(%)

57.3  4.5 50.7  0.5** 48.0  2.7 50.5  0.2* 33.1  1.3 40.0  1.3** 35.7  0.5 39.0  0.3**

33.1  2.6 37.2  0.4** 48.9  3.6 48.3  0.1 60.0  2.8 46.3  2.8** 50.0  1.1 47.1  0.5

9.6  1.9 12.1  0.2** 0.7 0.4 1.5  0.3 7.0  1.9 14.1  1.9** 14.4  0.8 14.0  0.3

Meloxicam, 50 mM; CDDP, 5 mg/ml; DXR, 0.75 mg/ml; 4OOH-IFM, 5 mg/ml. *Statistically significant differences compared with the samples treated without meloxicam ( p < 0.05). **Statistically significant differences compared with the samples treated without meloxicam ( p < 0.01).

3.4. Effects on the cell cycle The results of analysis for cell cycle were summarized in Table 3. The number of cells in the S and G2/M phases in cultures treated with 50 mM meloxicam were significantly higher ( p ¼ 0.0020 and p ¼ 0.0001, respectively) than in the controls, although not markedly. A drop in the number of cells in the G0/G1 phase was also observed ( p < 0.0001). Treatment with all three anti-cancer agents resulted in a marked accumulation of cells in the S phase compared with the control (CDDP, p < 0.0001; DXR, p < 0.0001; 4OOH-IFM, p < 0.0001). CDDP and DXR treatment significantly lowered the number of cells in the G2/M phase compared with the control ( p < 0.0001 and p ¼ 0.0196, respectively), whereas 4OOHIFM treatment significantly increased the number of cells

in G2/M phase compared with the control ( p < 0.0001). The most remarkable change caused by combining meloxicam with anti-cancer agents was seen in DXR-treated samples. Compared with DXR alone, the addition of meloxicam markedly increased the number of cells in the G0/G1 phase from 33.1% to 40.0% ( p < 0.0001), and in the G2/M phase from 7.0% to 14.1% ( p < 0.0001), while the number of cells in the S phase decreased from 60.0% to 46.3% ( p < 0.0001). By contrast, adding meloxicam to CDDP or 4OOH-IFM generated significant, but smaller, increases in the number of cells in the G0/G1 phase ( p ¼ 0.0231 and p ¼ 0.0056, respectively). Furthermore, the numbers of cells in the S and G2/M phases in CDDP-treated or 4OOH-IFM-treated samples were not affected significantly by the addition of meloxicam.

4. Discussion

Fig. 4. Expression levels of Bax and Bcl-2 proteins on western blots. Proteins were isolated from MG-63 cells incubated with 5 mg/ml CDDP, 0.75 mg/ml DXR, or 5 mg/ml 4OOH-IFM in the presence or absence of 50 mM meloxicam after 6 h (A) or 12 h (B).

There is ample evidence of the involvement of COX-2 in carcinogenesis for many different types of malignant tumor [1e3]. The efficacy of COX-2 inhibitors against tumor cells has been described by many authors [4e6]. We previously demonstrated that meloxicam, which is a preferential COX-2 inhibitor, reduced cell viability and induced apoptosis in osteosarcoma cell lines through both COX-2-dependent and COX2-independent pathways [8]. In the current study, we showed that meloxicam acted synergistically with both CDDP and DXR, when either cytotoxicity or the induction of apoptosis was measured. By contrast, neither the cytotoxicity nor apoptosis induced by 4OOH-IFM was enhanced significantly by meloxicam. These results suggested that the synergistic effects on cytotoxicity seen with meloxicam might be dependent upon the enhancement of apoptosis. Other authors have reported synergistic effects for combinations of COX-2 inhibitors or other NSAIDs with traditional anti-cancer agents, and have demonstrated that the synergy was generated predominantly by the enhancement of apoptosis [15,18e20]. CDDP in combination with JTE-522, which is a selective COX-2 inhibitor, has been reported to act synergistically on bladder cancer cells by activating caspase-3, and as a result of the intracellular accumulation of CDDP. Furthermore the down-regulation of Bcl-2 expression by JTE-522 has also

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been shown to enhance apoptosis [14]. A combination of oxaliplatin (a third-generation platinum compound) and etodolac down-regulated survivin (an anti-apoptotic protein) in colon carcinoma cells, and led to the cells undergoing apoptosis [20]. The viability of gastric cancer cells has also been reported to be suppressed by a combination of CDDP and JTE-522, which acted together synergistically both in vitro and in vivo [21]. We have demonstrated that a combination of CDDP and meloxicam down-regulated the expression of Bcl-2 in osteosarcoma cells, and enhanced apoptosis. By contrast, several authors have argued that the synergistic effects of combinations of CDDP with COX-2 inhibitors or other NSAIDs are mainly due to the intracellular accumulation of CDDP [14,22,23]. The inhibition of CDDP efflux might be one of the mechanisms by which COX-2 inhibitors function. The mechanism by which anti-cancer agents and COX-2 inhibitors generate synergy seems to depend on the cell type studied, and there might be different types of as yet unidentified modulators in different types of tumor cell. Several groups have reported that the synergy observed between DXR and COX-2 inhibitors is the result of an inhibition of the expression of the MDR1 gene or MRP, and leads to an intracellular accumulation of DXR [24e26]. DXR and nimesulide have been found to activate caspase-3 and to induce peroxisome proliferator-activated receptor-g (PPARg) expression in non-small-cell lung cancer cells [18]. Celecoxib was found to enhance the cytotoxicity induced by DXR not only by inhibiting P-glycoprotein (P-gp) but also by elevating nitric oxide (NO) levels, leading to the accumulation of p53 in breast cancer cells, and to apoptosis in vivo [13]. In this study, 50 mM meloxicam alone up-regulated Bax expression and, in combination with DXR, both markedly up-regulated Bax expression at the transcriptional level and increased apoptosis, providing a mechanism for the generation of synergy. Since preliminary studies had shown that MG-63 cells were sensitive to each of the anti-cancer drugs used, we assumed that P-gp or MRP and the intracellular accumulation of the drugs were unlikely to play a great role in the mechanism underlying synergy; therefore, we did not look at the intracellular accumulation of the cytotoxic agents in this study. However, MG-63 cells have been shown to express MRP at both the protein and mRNA levels [27], so the possibility that it has a role in the synergy between the drugs deserves further analysis. Synergy was not evident when cells were treated with 4OOH-IFM in combination with meloxicam. In addition, to our knowledge there are no previous reports on synergistic effects between ifosfamide and COX-2 inhibitors. We assumed that the lack of synergy might reflect shared or overlapping pathways causing apoptosis induced by these two drugs. Real-time RT-PCR and western blot analysis revealed significant up-regulation of Bax expression by 4OOH-IFM alone in MG-63 cells compared with the control. There are few previous reports that discuss the induction of apoptosis by ifosfamide. Both 4OOH-IFM and cyclophosphamide have been reported to induce caspase-dependent apoptosis, especially involving caspase-9 [28,29], suggesting that ifosfamide might induce apoptosis predominantly through a mitochondrial

pathway. This supports our suggestion that 4OOH-IFMinduced apoptosis was generated by Bax up-regulation at the transcriptional level in MG-63 cells, at least partially. At 50 mM, meloxicam also increased the expression of Bax in MG-63 cells, and this behavior was similar to that observed in the 4OOH-IFM treated samples. The dominant initiator of meloxicam-induced Bax up-regulation in MG-63 cells was unknown; however, there is a possibility that the same initiator was stimulated by 4OOH-IFM. The use of common or overlapping pathways by 4OOH-IFM and meloxicam to induce apoptosis could explain their failure to have synergistic effects on cytotoxicity. By contrast, CDDP or DXR alone did not increase Bax expression significantly compared with the control, suggesting that they induced apoptosis by a Bax-independent mechanism. Previously, some groups have reported that CDDP might induce apoptosis in different types of malignant tumor cells by diverse mechanisms, such as G2-phase and S-phase arrest [30], up-regulation of GADD153 mRNA expression [29], and the involvement of cMyc [31]. Others have described the involvement of caspase-9, caspase-3, and caspase-8 activation, and polyADP-ribose polymerase (PARP) cleavage in CDDP-induced apoptosis, and, furthermore, have suggested a role for the redistribution of Bax, although changes in its expression level were not discussed [32]. DXR has been reported to induce apoptosis in malignant tumor cells by G2-phase arrest [33], G2-phase or early S-phase arrest [34], elevation of NO levels and p53 accumulation [13], down-regulation of Bcl-XL mRNA and up-regulation of Bcl-XS mRNA [35], and activation of CD95 (Fas) [36]. Although the precise mechanism by which CDDP and DXR induce apoptosis is still unknown and remains controversial, our results indicate that separate pathways might induce apoptosis by a Bax-dependent mechanism in the case of meloxicam, and by a Bax-independent mechanism in the case of CDDP and DXR in MG-63 cells. Promotion of these separate apoptotic mechanisms could explain the enhancement of apoptosis and synergistic effects on cytotoxicity with combinations of meloxicam and CDDP or DXR. Meloxicam modulated Bax and Bcl-2 expression in different ways, in combination with CDDP and DXR. When meloxicam was administrated with CDDP, Bcl-2 was downregulated, whereas administration with DXR caused Bax up-regulation. Our previous report suggested that 100 mM meloxicam up-regulated Bax in MG-63 cells, but had no significant effect on Bcl-2 expression [8]. These results were unexpected and difficult to explain. To explain this unusual modulation of Bax and Bcl-2, we hypothesized that an unknown meloxicam-reactive modulator existed in MG-63 cells, which could regulate Bcl-2 family molecules. DXR in combination with meloxicam markedly altered the MG-63 cell cycle in the S and G2/M phases. This modulator might respond to the accumulation of cells in the S or G2/M phases induced by meloxicam, and induce Bax up-regulation. By contrast, the S and G2/M populations were not affected in CDDPtreated samples, suggesting that additional stimulation by meloxicam, which did not affect the cell cycle, might influence

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this modulator and so lead to Bcl-2 down-regulation. In addition, the reason why meloxicam caused cell accumulation in the S and G2/M phases is unknown. Most COX-2 inhibitors and other NSAIDs are reported to arrest the cell cycle in the G0/G1 phase, due to the involvement of cyclin D1 [37,38]. There are few reports of NSAIDs inducing arrest in the S or G2 phase, with only high concentrations of aspirin reported to have this effect [39]. Furthermore, the accumulation of cells in the G0/G1 phase was induced by meloxicam only in the presence of the anti-cancer drugs, and especially in combination with DXR, which was difficult to explain. Clarifying the precise mechanism by which changes in Bcl-2 expression, the cell cycle, and their interactions are induced by combinations of anti-cancer agents and meloxicam will require further investigation of up-stream signaling cascades. The synergy between CDDP and meloxicam might have been greater than that between DXR and meloxicam due to the involvement of Bcl-2 modulation when CDDP and meloxicam were combined. Many authors have demonstrated that drug-induced apoptosis in several types of malignant tumor cells, mediated through various pathways, could be strongly inhibited by Bcl-2 [40e43]. The down-regulation of Bcl-2 could result in a strongly enhanced chemosensitivity in cancer cells. Furthermore, quercetin (an Akt/PKB inhibitor) has been reported to enhance CDDP-induced apoptosis in head and neck cancer cells, accompanied by the down-regulation of Bcl-2 and Bcl-XL [44]. This observation supports an important role for Bcl-2 in CDDP-induced apoptosis, and suggests that the role of Bcl-2 might be dominant to that of Bax in the modulation of drug-induced apoptosis, especially in response to CDDP. We believe that this suggestion might explain how CDDP-induced apoptosis generated by the down-regulation of Bcl-2 could exceed DXR-induced apoptosis generated by the up-regulation of Bax in MG-63 cells. On the contrary to our previous [8] and the current study using MG-63 cells, a recent study demonstrated that artificial over-expression of COX-2 in osteosarcoma cells (U2-OS and Saos-2) decreased their proliferation [45]. These discrepancies suggested a possibility that the effects of COX-2 expression in osteosarcoma cells might be cell-type specific. Another possibility is that artificially over-expressed COX-2 might have different interpretation compared with spontaneously over-expressed COX-2. Further investigations are required to establish better understanding of the interpretations of COX2 over-expression in osteosarcoma. In conclusion, we propose that the synergy observed in the cytotoxic effects of meloxicam with CDDP and DXR in the human osteosarcoma cell line, MG-63, was generated by an enhancement of apoptosis. Although the exact mechanisms by which the down-regulation of Bcl-2 by CDDP and the up-regulation of Bax by DXR bring about changes in the cell cycle and apoptosis require further investigation, the results raise the possibility of enhancing the effects of chemotherapeutic agents and may herald an important development for improving the prognosis for osteosarcoma patients. The concentration of meloxicam used in this study was higher than that used clinically; therefore, novel approaches to reduce

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the drug dose, while preserving its anti-tumor effects, need to be developed.

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