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COX-2 specific inhibitors enhance the cytotoxic effects of pemetrexed in mesothelioma cell lines
Lung Cancer 67 (2010) 160–165
Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
COX-2 specific inhibitors enhance the cytotoxic effects of pemetrexed in mesothelioma cell lines Sara L. O’Kane a , Gina L. Eagle a , John Greenman a , Michael J. Lind a,b , Lynn Cawkwell a,∗ a b
Postgraduate Medical Institute of the University of Hull and the Hull York Medical School, University of Hull, UK Centre for Oncology and Haematology, Castle Hill Hospital, Hull and East Yorkshire NHS Trust, Hull, UK
a r t i c l e
i n f o
Article history: Received 19 February 2009 Received in revised form 7 April 2009 Accepted 17 April 2009 Keywords: Cisplatin COX-2 Cytotoxicity Mesothelioma Pemetrexed Vinorelbine
a b s t r a c t Background: Although malignant pleural mesothelioma is a rare tumour, its incidence is increasing. The prognosis remains very poor with an average survival of 10 months from diagnosis. The choice of chemotherapy regimens for mesothelioma patients is limited and new approaches are required. COX-2 inhibition induces apoptosis in a variety of tumour cell lines. The cytotoxic effect of conventional drugs may be enhanced by the addition of a COX-2 inhibitor. In order to identify possible new therapeutic approaches we aimed to determine whether the addition of COX-2 inhibitors would enhance the cytotoxic effect of chemotherapeutic agents in mesothelioma cell lines. Materials and methods: Three mesothelioma cell lines MSTO-211H, NCI-H2052 and NCI-H2452 were utilised. Using the COX-2 positive A549 lung cancer cell line as control, all cell lines were assayed using an MTT assay with non-specific COX-2 inhibitors (sulindac and flurbiprofen), specific COX-2 inhibitors (DuP-697 and NS-398), and chemotherapeutic agents (cisplatin, vinorelbine and pemetrexed). Results: All cell lines exhibited COX-2 expression by western blotting using two antibodies. The addition of either DuP-697 or NS-398 increased the sensitivity to pemetrexed in all cell lines. Conclusion: These findings suggest that the design of novel pemetrexed-containing combination regimens with increased cytotoxicity may be feasible. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Malignant pleural mesothelioma (MPM) is a rare but highly aggressive disease which is increasing in incidence throughout the world. The prognosis for patients with malignant mesothelioma remains very poor with only 10% surviving more than 3 years and a median survival of 10 months from diagnosis even with radical surgery. Therapeutic options remain very limited with radical surgery only being applicable to a very few early stage fit patients [1] and producing only a few long-term survivors. Chemotherapy has been limited to use in palliative therapy of the disease and as an adjunct to radical surgery. A number of cytotoxic compounds have been assessed and typically give tumour response rates in the range of 10–30% when used as single agents [2–4]. Combination regimens have been investigated in order to increase the benefit. Cisplatin and carboplatin are among the most active single drugs and usually included in combination therapy regimens [4,5]. Vinorelbine, a semisynthetic vinca alkaloid derived from vinblastine, has also attracted interest. It is a tubulin-binding agent
∗ Corresponding author at: Research Laboratories, Daisy Building, Castle Hill Hospital, Hull HU16 5JQ, UK. Tel.: +44 1482 461850; fax: +44 1482 461874. E-mail address: [email protected] (L. Cawkwell). 0169-5002/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2009.04.008
which inhibits mitosis. A phase II trial of single agent vinorelbine demonstrated response in 24% of MPM patients and symptom improvement in over 47% with low toxicity [6]. In combination with cisplatin, vinorelbine achieved a response rate of 29.6% [7]. Currently the recommended systemic therapy for mesothelioma is cisplatin plus pemetrexed based on the phase III EMPHACIS trial which achieved a response rate of 41% with a median survival of 12.1 months in 456 patients [8,9]. Pemetrexed (LY 231514) is a structural analogue of methotrexate and lometrexol which is retained at high concentrations within the cell by irreversible polyglutamation [10,11] and demonstrated activity in MPM as a single agent in Phase II trials [12]. It is a multitargeted antifolate that acts by inhibiting thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase. The interruption of both purine and pyrimidine synthesis induces arrest at the G1/S checkpoint in the cell cycle with ensuing cytotoxicity [13,14]. Cyclooxygenase-2 (COX-2) is an inducible enzyme which catalyses the conversion of arachidonic acid to prostaglandins in response to proinflammatory or mitogenic signals. COX-2 is overexpressed in many solid tumours and inhibition of this enzyme in vitro using specific COX-2 inhibitors has shown that COX-2 is a potential target for novel cancer therapies [15–17]. The use of COX-2 inhibitors in vitro has been demonstrated to produce a wide range of cellular effects including the induction of apoptosis, reduction in cell proliferation,
S.L. O’Kane et al. / Lung Cancer 67 (2010) 160–165
inhibition of angiogenesis and, importantly, enhancement of the cytotoxic effects of anticancer agents [18,19]. However, the possibility of enhancing the cytotoxic effects of chemotherapeutic agents for mesothelioma through the addition of a COX-2 inhibitor has not been investigated. In MPM COX-2 has been shown to be overexpressed in 59–100% of tumour samples [20–22], suggesting that COX-2 may be a potential target for novel therapies for this disease. The COX-2 inhibitor celecoxib has previously been demonstrated to have a cytotoxic effect in malignant mesothelioma cell lines [23]. To further this work we have assessed the effect of COX-2 inhibitors using a clinically achievable low dose range on three mesothelioma cell lines: NCI-H2452, NCI-H2052, and MSTO-211H. In order to identify new therapeutic approaches we further assessed the enhanced effects of chemotherapeutic agents when used in combination with COX-2 inhibitors in these cell lines. 2. Materials and methods 2.1. Cell lines The mesothelioma cell lines MSTO-211H, NCI-H2052 and NCIH2452 were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium. The lung cancer cell line (A549), which was selected as a positive control for COX-2 expression [24,25], was obtained from the European Collection of Cell Cultures (ECACC) and maintained in RPMI 1640 medium. The medium was supplemented with 10% (v/v) foetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. 2.2. COX-2 protein expression status Western blotting was performed as described previously [26]. In short, at approximately 70–80% cell confluence protein lysates were generated in 1 ml of Laemmli buffer [62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 2% SDS, 5% -mercaptoethanol, 1% protease inhibitor mix, 0.00125% bromophenol blue]. Three biological replicates, extracted on different days, were obtained for all samples. Following transfer to nitrocellulose membranes and blocking in 5% non-fat milk the samples were probed with two primary antibodies against COX-2. These were #SC-19999 (Santa Cruz Biotechnology) and #ab52237 (Abcam) which were both applied at a final concentration of 1:200 for 2–3 h. Visualisation was achieved using the SuperSignal® West Pico Chemiluminescent Substrate (Pierce). 2.3. MTT assay The non-specific COX-2 inhibitors sulindac (#1707) and flurbiprofen (#1769) and the specific COX-2 inhibitors DuP-697 (#1430) and NS-398 (#0942) were obtained from Tocris Bioscience and were reconstituted as stock solutions of 0.5 M using DMSO. A dilution series (0, 1, 2.5, 5, 10, 25, 50, 100, and 250 M) was prepared for analysis in the MTT assay. Cisplatin (#P4394, Sigma–Aldrich) was obtained in lyophilised form and reconstituted in distilled water. Vinorelbine (#V2264, Sigma–Aldrich) was used at 10 mg/ml in 0.9% saline. Pemetrexed (Eli Lilly) was obtained in lyophilised form and reconstituted in phosphate buffered saline (PBS). Cells in media were treated with a range of concentrations in triplicate in 96 well tissue culture plates and cell proliferation assessed using a standard MTT assay. To ensure cells were in exponential growth, an optimised cell plating density for each cell line was used (5000 cells per well for NCI-H2052 and NCI-H2452; 10,000 cells per well for MSTO-211H and A549). To ensure any effect observed was resulting from the addition of the test drugs, the cells were incubated for 24 h, 48 h and 72 h after the addition of the vehicle (DMSO, distilled water, PBS or 0.9% saline) and an MTT assay was completed. Each
161
experiment was conducted three times and all assays were performed with 24, 48 and 72 h incubation periods. Where relevant the IC50 values were calculated using linear regression. The results was converted into the log10 value and plotted onto a graph using Microsoft Excel. A best fit line was applied and the resulting equation from the line was used to calculate the IC50 value. A mean value was calculated to indicate the IC50 value representative of the replicates of the experiment. The standard error between the replicates was calculated using Microsoft Excel. 3. Results 3.1. Protein expression status of COX-2 Fig. 1 demonstrates the expression of COX-2 using two antibodies on triplicate samples extracted from each cell line. As expected, the known positive control A549 cells exhibit clear expression of COX-2. In addition, all three mesothelioma cell lines demonstrate COX-2 positivity in all replicates and with both antibodies tested. 3.2. MTT assays: COX-2 inhibitors A range of concentrations (0–250 M) of each COX-2 inhibitor was assayed over four incubation times (2, 24, 48 and 72 h). Maximal effects were seen at 72 h and the IC50 values within the dose range at this time point are shown in Table 1. Fig. 2 illustrates the effect of the specific COX-2 inhibitor DuP-697. 3.3. MTT assays: COX-2 inhibitors and chemotherapeutic agent combinations A range of clinically achievable concentrations of single agent cisplatin (0–100 M; Fig. 3), pemetrexed (0–10 M; Fig. 4) and vinorelbine (0–100 nM) were assayed over four incubation times (2, 24, 48 and 72 h). Maximal effects were seen at 72 h. The cell lines demonstrated a variety of effects with the addition of a specific COX-2 inhibitor (10 M DuP-697 or 10 M NS-398) to the cisplatin dose (Fig. 3; Table 2). An enhanced cytotoxic effect was observed in the A549 lung cancer cell line (3.7-fold decrease in IC50 with the addition of DuP-697; 3.1-fold decrease in IC50 with the addition of NS-398) but not in any of the three mesothelioma cell lines. The effects of the specific COX-2 inhibitors were assessed at 1 M, 10 M and 100 M of inhibitor. There was additional benefit with the addition of 10 M compared to 1 M, but no additional benefit was observed with increasing the concentration to 100 M. The results of combinations of vinorelbine and the specific COX2 inhibitors DuP-697 and NS-398 are shown in Table 2. No additive effects were observed in any cell line. The results of combinations of pemetrexed and the specific COX2 inhibitors DuP-697 and NS-398 are shown in Fig. 4 and Table 2. All cell lines demonstrated increased sensitivity to pemetrexed with the addition of either specific COX-2 inhibitor. MSTO-211H cells demonstrated a 26.8-fold decrease in IC50 with the addition of 10 M NS-398 and a 12-fold decrease with the addition of Table 1 The IC50 values obtained from three COX-2 positive mesothelioma cell lines and the control COX-2 positive A549 lung cancer cell line after a 72 h treatment with selective (S) COX-2 inhibitors DuP-697 and NS-398 and non-selective (NS) COX-2 inhibitors flurbiprofen and sulindac.
DuP-697 (S) NS-398 (S) Flurbiprofen (NS) Sulindac (NS)
NCI-H2052
MSTO-211H
NCI-H2452
A549
86 M – – –
231 M – 155 M –
– – 152 M –
169 M – – –
(–) Did not reach an IC50 within the dose range (0–250 M) used.
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Fig. 1. Western blot demonstrating positive expression of COX-2 (arrowed) in positive control A549 cells and in all three mesothelioma cell lines using two anti-COX-2 antibodies. Upper panel shows the results with antibody #SC-19999 (Santa Cruz) and the lower panel shows the findings with antibody #ab52237 (Abcam). For each cell line, protein extracts were prepared independently on three separate days, denoted by (a)–(c). The expected band for COX-2 of approximately 70 kDa is seen in all samples. However, there is a persistent additional band, estimated to be approximately 85 kDa, seen in all A549 replicates when using the #SC-19999 antibody.
10 M DuP-697. NCI-H2052 cells demonstrated greater than 4-fold decrease in IC50 for both inhibitors. NCI-H2452 cells demonstrated a 6.5-fold decrease in IC50 with the addition of 10 M NS-398 and a 7.4-fold decrease with the addition of 10 M DuP-697. A549 cells demonstrated a 6.7-fold decrease in IC50 with the addition of 10 M NS-398 and an 8.7-fold decrease with the addition of 10 M DuP697. The effect of specific COX-2 inhibitors was assessed at both 10 M and 100 M of inhibitor but there was no statistical advantage to the increased concentration of inhibitor.
4. Discussion
Fig. 2. The cytotoxic effect of the specific COX-2 inhibitor DuP-697 on three COX-2 positive mesothelioma cell lines (MSTO-211H, NCI-H2052, NCI-H2452) and a COX-2 positive lung cancer cell line (A549). Each data point is the mean of nine replicates produced using the MTT assay after 72 h and error bars represent the standard error of the mean. Treatment durations of 2, 24, 48 and 72 h were assessed but the maximal effect was seen at 72 h. A significant cytotoxic effect was not produced in the NCIH2452 cells (IC50 not reached), however IC50 values were attained for NCI-H2052 (86 M), A549 (169 M) and MSTO-211H (231 M).
Two COX-2 antibodies were utilised to assess protein expression status of the mesothelioma cell lines, which were all found to exhibit COX-2 positivity. There have been no previous studies in the literature describing the COX-2 protein expression status assessed using these antibodies in the three mesothelioma cell lines cell lines employed in this study. Our MTT results indicate that the specific COX-2 inhibitor DuP-697 inhibited the growth of two of the mesothelioma cell lines and the A549 control cells.
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Fig. 3. The cytotoxic effect of single agent cisplatin and the combination of cisplatin and 10 M DuP-697 or 10 M NS-398 (specific COX-2 inhibitors) after 72 h. Three COX-2 positive mesothelioma cell lines (MSTO-211H, NCI-H2052 and NCI-H2452) and a COX-2 positive lung cancer cell line (A549) were assayed using an MTT assay. Treatment durations of 2, 24, 48 and 72 h were assessed but the maximal effect was seen at 72 h. Each data point is the mean of nine replicates produced using the MTT assay and error bars represent the standard error of the mean. The IC50 values are shown in Table 2.
The addition of either of the specific COX-2 inhibitors (DuP697, NS-398) to cisplatin or vinorelbine did not enhance the cytotoxic effects in any of the three mesothelioma cell lines. In fact the IC50 values increased when these inhibitors were added to cisplatin in NCI-H2452 cells and to vinorelbine in MSTO-211H cells (Table 2). However, the combination of either
of the specific COX-2 inhibitors DuP-697 or NS-398 with pemetrexed enhanced the cytotoxicity of this drug by at least 4-fold in all cell lines tested. To our knowledge this is only the second report of a COX inhibitor demonstrating synergy with a chemotherapeutic agent in mesothelioma. The non-selective COX inhibitor piroxicam has previously been demonstrated to
Fig. 4. The cytotoxic effect of single agent pemetrexed and the combination of pemetrexed and 10 M DuP-697 or 10 M NS-398 (specific COX-2 inhibitors) after 72 h. Three COX-2 positive mesothelioma cell lines (MSTO-211H, NCI-H2052 and NCI-H2452) and a COX-2 positive lung cancer cell line (A549) were assayed using an MTT assay. Treatment durations of 24, 48 and 72 h were assessed but the maximal effect was seen at 72 h. Each data point is the mean of nine replicates produced using the MTT assay and error bars represent the standard error of the mean. The IC50 values are shown in Table 2.
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Table 2 Summary of MTT assay data showing IC50 values obtained from three COX-2 positive mesothelioma cell lines and the control COX-2 positive A549 lung cancer cell line after a 72 h treatment with three chemotherapeutic agents with and without the addition of a selective COX-2 inhibitor (DuP-697 or NS-398). See also Figs. 3 and 4. Possible additive combinations are highlighted in bold. MSTO-211H
NCI-H2052
Cisplatin
Alone +10 M DuP-697 +10 M NS-398
3.4 M 2.2 M 4.3 M
2.2 M 2.2 M 1.5 M
Vinorelbine
Alone +10 M DuP-697 +10 M NS-398
7.9 nM >100 nM >100 nM
1.6 nM 1.2 nM 1.4 nM
Pemetrexed
Alone +10 M DuP-697 +10 M NS-398
1.608 M 0.134 M 0.06 M
enhance the cytotoxic effects of cisplatin in mesothelioma cells [27,28]. The treatment options for mesothelioma remain limited and novel chemotherapeutic regimens remain under investigation. Pemetrexed is currently the drug of choice in this disease and the possibility of enhancing the cytotoxic effects of pemetrexed with the addition of a cell pathway inhibitor is an attractive goal. Some COX-2 inhibitors are known to be associated with significant cardiovascular risks and therefore the choice of agent for testing in clinical trials will be important. However, the risks associated with short-term use as a therapeutic strategy in mesothelioma, a disease currently with dire prognosis, may be lower (or more acceptable) than that for long-term use as a chemoprevention strategy. To understand the side effects of COX-2 inhibitors, and to possibly design improved drugs, it is important to investigate their underlying cellular effects. Several studies have reported that different members of the COX-2 inhibitor drug class demonstrate significant differences in their cellular mechanism of action and there is a growing body of evidence that some COX-2 inhibitors do not exert their effects via COX-2 [29,30]. Further in vitro work is required to establish the mechanism of action of the active agents which we have identified and we are currently investigating this using proteomic-based strategies. Very little is known about the full mechanism of action of DuP-697 [31] but there have been several studies on NS-398 [32–36]. Several compounds which are structurally related to COX-2 inhibitors, but devoid of the ability to inhibit the COX-2 enzyme, for example dimethyl-celecoxib (DMC) and R-flurbiprofen, have been shown to promote apoptosis and these would be anticipated to have fewer cardiovascular toxicities. The analysis of additional compounds, such as these, in our in vitro model may reveal further candidates to progress towards clinical trials. Acknowledgments Gina Eagle is funded by Yorkshire Cancer Research. Funding for parts of this study was provided by the Colt Foundation and Eli Lilly, who also supplied the pemetrexed. The study sponsors played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.
0.204 M <0.05 M <0.05 M
[4] [5]
[6]
[7]
[8]
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[13]
[14]
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[21]
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[24]
NCI-H2452
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4.0 M 26.1 M 31.2 M
24.6 M 6.6 M 7.9 M
0.95 nM 1.8 nM 1.2 nM 1.081 M 0.147 M 0.167 M
8.3 nM 12.3 nM 13.6 nM 5.275 M 0.603 M 0.792 M
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Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
COX-2 specific inhibitors enhance the cytotoxic effects of pemetrexed in mesothelioma cell lines Sara L. O’Kane a , Gina L. Eagle a , John Greenman a , Michael J. Lind a,b , Lynn Cawkwell a,∗ a b
Postgraduate Medical Institute of the University of Hull and the Hull York Medical School, University of Hull, UK Centre for Oncology and Haematology, Castle Hill Hospital, Hull and East Yorkshire NHS Trust, Hull, UK
a r t i c l e
i n f o
Article history: Received 19 February 2009 Received in revised form 7 April 2009 Accepted 17 April 2009 Keywords: Cisplatin COX-2 Cytotoxicity Mesothelioma Pemetrexed Vinorelbine
a b s t r a c t Background: Although malignant pleural mesothelioma is a rare tumour, its incidence is increasing. The prognosis remains very poor with an average survival of 10 months from diagnosis. The choice of chemotherapy regimens for mesothelioma patients is limited and new approaches are required. COX-2 inhibition induces apoptosis in a variety of tumour cell lines. The cytotoxic effect of conventional drugs may be enhanced by the addition of a COX-2 inhibitor. In order to identify possible new therapeutic approaches we aimed to determine whether the addition of COX-2 inhibitors would enhance the cytotoxic effect of chemotherapeutic agents in mesothelioma cell lines. Materials and methods: Three mesothelioma cell lines MSTO-211H, NCI-H2052 and NCI-H2452 were utilised. Using the COX-2 positive A549 lung cancer cell line as control, all cell lines were assayed using an MTT assay with non-specific COX-2 inhibitors (sulindac and flurbiprofen), specific COX-2 inhibitors (DuP-697 and NS-398), and chemotherapeutic agents (cisplatin, vinorelbine and pemetrexed). Results: All cell lines exhibited COX-2 expression by western blotting using two antibodies. The addition of either DuP-697 or NS-398 increased the sensitivity to pemetrexed in all cell lines. Conclusion: These findings suggest that the design of novel pemetrexed-containing combination regimens with increased cytotoxicity may be feasible. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Malignant pleural mesothelioma (MPM) is a rare but highly aggressive disease which is increasing in incidence throughout the world. The prognosis for patients with malignant mesothelioma remains very poor with only 10% surviving more than 3 years and a median survival of 10 months from diagnosis even with radical surgery. Therapeutic options remain very limited with radical surgery only being applicable to a very few early stage fit patients [1] and producing only a few long-term survivors. Chemotherapy has been limited to use in palliative therapy of the disease and as an adjunct to radical surgery. A number of cytotoxic compounds have been assessed and typically give tumour response rates in the range of 10–30% when used as single agents [2–4]. Combination regimens have been investigated in order to increase the benefit. Cisplatin and carboplatin are among the most active single drugs and usually included in combination therapy regimens [4,5]. Vinorelbine, a semisynthetic vinca alkaloid derived from vinblastine, has also attracted interest. It is a tubulin-binding agent
∗ Corresponding author at: Research Laboratories, Daisy Building, Castle Hill Hospital, Hull HU16 5JQ, UK. Tel.: +44 1482 461850; fax: +44 1482 461874. E-mail address: [email protected] (L. Cawkwell). 0169-5002/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2009.04.008
which inhibits mitosis. A phase II trial of single agent vinorelbine demonstrated response in 24% of MPM patients and symptom improvement in over 47% with low toxicity [6]. In combination with cisplatin, vinorelbine achieved a response rate of 29.6% [7]. Currently the recommended systemic therapy for mesothelioma is cisplatin plus pemetrexed based on the phase III EMPHACIS trial which achieved a response rate of 41% with a median survival of 12.1 months in 456 patients [8,9]. Pemetrexed (LY 231514) is a structural analogue of methotrexate and lometrexol which is retained at high concentrations within the cell by irreversible polyglutamation [10,11] and demonstrated activity in MPM as a single agent in Phase II trials [12]. It is a multitargeted antifolate that acts by inhibiting thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase. The interruption of both purine and pyrimidine synthesis induces arrest at the G1/S checkpoint in the cell cycle with ensuing cytotoxicity [13,14]. Cyclooxygenase-2 (COX-2) is an inducible enzyme which catalyses the conversion of arachidonic acid to prostaglandins in response to proinflammatory or mitogenic signals. COX-2 is overexpressed in many solid tumours and inhibition of this enzyme in vitro using specific COX-2 inhibitors has shown that COX-2 is a potential target for novel cancer therapies [15–17]. The use of COX-2 inhibitors in vitro has been demonstrated to produce a wide range of cellular effects including the induction of apoptosis, reduction in cell proliferation,
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inhibition of angiogenesis and, importantly, enhancement of the cytotoxic effects of anticancer agents [18,19]. However, the possibility of enhancing the cytotoxic effects of chemotherapeutic agents for mesothelioma through the addition of a COX-2 inhibitor has not been investigated. In MPM COX-2 has been shown to be overexpressed in 59–100% of tumour samples [20–22], suggesting that COX-2 may be a potential target for novel therapies for this disease. The COX-2 inhibitor celecoxib has previously been demonstrated to have a cytotoxic effect in malignant mesothelioma cell lines [23]. To further this work we have assessed the effect of COX-2 inhibitors using a clinically achievable low dose range on three mesothelioma cell lines: NCI-H2452, NCI-H2052, and MSTO-211H. In order to identify new therapeutic approaches we further assessed the enhanced effects of chemotherapeutic agents when used in combination with COX-2 inhibitors in these cell lines. 2. Materials and methods 2.1. Cell lines The mesothelioma cell lines MSTO-211H, NCI-H2052 and NCIH2452 were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium. The lung cancer cell line (A549), which was selected as a positive control for COX-2 expression [24,25], was obtained from the European Collection of Cell Cultures (ECACC) and maintained in RPMI 1640 medium. The medium was supplemented with 10% (v/v) foetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. 2.2. COX-2 protein expression status Western blotting was performed as described previously [26]. In short, at approximately 70–80% cell confluence protein lysates were generated in 1 ml of Laemmli buffer [62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 2% SDS, 5% -mercaptoethanol, 1% protease inhibitor mix, 0.00125% bromophenol blue]. Three biological replicates, extracted on different days, were obtained for all samples. Following transfer to nitrocellulose membranes and blocking in 5% non-fat milk the samples were probed with two primary antibodies against COX-2. These were #SC-19999 (Santa Cruz Biotechnology) and #ab52237 (Abcam) which were both applied at a final concentration of 1:200 for 2–3 h. Visualisation was achieved using the SuperSignal® West Pico Chemiluminescent Substrate (Pierce). 2.3. MTT assay The non-specific COX-2 inhibitors sulindac (#1707) and flurbiprofen (#1769) and the specific COX-2 inhibitors DuP-697 (#1430) and NS-398 (#0942) were obtained from Tocris Bioscience and were reconstituted as stock solutions of 0.5 M using DMSO. A dilution series (0, 1, 2.5, 5, 10, 25, 50, 100, and 250 M) was prepared for analysis in the MTT assay. Cisplatin (#P4394, Sigma–Aldrich) was obtained in lyophilised form and reconstituted in distilled water. Vinorelbine (#V2264, Sigma–Aldrich) was used at 10 mg/ml in 0.9% saline. Pemetrexed (Eli Lilly) was obtained in lyophilised form and reconstituted in phosphate buffered saline (PBS). Cells in media were treated with a range of concentrations in triplicate in 96 well tissue culture plates and cell proliferation assessed using a standard MTT assay. To ensure cells were in exponential growth, an optimised cell plating density for each cell line was used (5000 cells per well for NCI-H2052 and NCI-H2452; 10,000 cells per well for MSTO-211H and A549). To ensure any effect observed was resulting from the addition of the test drugs, the cells were incubated for 24 h, 48 h and 72 h after the addition of the vehicle (DMSO, distilled water, PBS or 0.9% saline) and an MTT assay was completed. Each
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experiment was conducted three times and all assays were performed with 24, 48 and 72 h incubation periods. Where relevant the IC50 values were calculated using linear regression. The results was converted into the log10 value and plotted onto a graph using Microsoft Excel. A best fit line was applied and the resulting equation from the line was used to calculate the IC50 value. A mean value was calculated to indicate the IC50 value representative of the replicates of the experiment. The standard error between the replicates was calculated using Microsoft Excel. 3. Results 3.1. Protein expression status of COX-2 Fig. 1 demonstrates the expression of COX-2 using two antibodies on triplicate samples extracted from each cell line. As expected, the known positive control A549 cells exhibit clear expression of COX-2. In addition, all three mesothelioma cell lines demonstrate COX-2 positivity in all replicates and with both antibodies tested. 3.2. MTT assays: COX-2 inhibitors A range of concentrations (0–250 M) of each COX-2 inhibitor was assayed over four incubation times (2, 24, 48 and 72 h). Maximal effects were seen at 72 h and the IC50 values within the dose range at this time point are shown in Table 1. Fig. 2 illustrates the effect of the specific COX-2 inhibitor DuP-697. 3.3. MTT assays: COX-2 inhibitors and chemotherapeutic agent combinations A range of clinically achievable concentrations of single agent cisplatin (0–100 M; Fig. 3), pemetrexed (0–10 M; Fig. 4) and vinorelbine (0–100 nM) were assayed over four incubation times (2, 24, 48 and 72 h). Maximal effects were seen at 72 h. The cell lines demonstrated a variety of effects with the addition of a specific COX-2 inhibitor (10 M DuP-697 or 10 M NS-398) to the cisplatin dose (Fig. 3; Table 2). An enhanced cytotoxic effect was observed in the A549 lung cancer cell line (3.7-fold decrease in IC50 with the addition of DuP-697; 3.1-fold decrease in IC50 with the addition of NS-398) but not in any of the three mesothelioma cell lines. The effects of the specific COX-2 inhibitors were assessed at 1 M, 10 M and 100 M of inhibitor. There was additional benefit with the addition of 10 M compared to 1 M, but no additional benefit was observed with increasing the concentration to 100 M. The results of combinations of vinorelbine and the specific COX2 inhibitors DuP-697 and NS-398 are shown in Table 2. No additive effects were observed in any cell line. The results of combinations of pemetrexed and the specific COX2 inhibitors DuP-697 and NS-398 are shown in Fig. 4 and Table 2. All cell lines demonstrated increased sensitivity to pemetrexed with the addition of either specific COX-2 inhibitor. MSTO-211H cells demonstrated a 26.8-fold decrease in IC50 with the addition of 10 M NS-398 and a 12-fold decrease with the addition of Table 1 The IC50 values obtained from three COX-2 positive mesothelioma cell lines and the control COX-2 positive A549 lung cancer cell line after a 72 h treatment with selective (S) COX-2 inhibitors DuP-697 and NS-398 and non-selective (NS) COX-2 inhibitors flurbiprofen and sulindac.
DuP-697 (S) NS-398 (S) Flurbiprofen (NS) Sulindac (NS)
NCI-H2052
MSTO-211H
NCI-H2452
A549
86 M – – –
231 M – 155 M –
– – 152 M –
169 M – – –
(–) Did not reach an IC50 within the dose range (0–250 M) used.
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Fig. 1. Western blot demonstrating positive expression of COX-2 (arrowed) in positive control A549 cells and in all three mesothelioma cell lines using two anti-COX-2 antibodies. Upper panel shows the results with antibody #SC-19999 (Santa Cruz) and the lower panel shows the findings with antibody #ab52237 (Abcam). For each cell line, protein extracts were prepared independently on three separate days, denoted by (a)–(c). The expected band for COX-2 of approximately 70 kDa is seen in all samples. However, there is a persistent additional band, estimated to be approximately 85 kDa, seen in all A549 replicates when using the #SC-19999 antibody.
10 M DuP-697. NCI-H2052 cells demonstrated greater than 4-fold decrease in IC50 for both inhibitors. NCI-H2452 cells demonstrated a 6.5-fold decrease in IC50 with the addition of 10 M NS-398 and a 7.4-fold decrease with the addition of 10 M DuP-697. A549 cells demonstrated a 6.7-fold decrease in IC50 with the addition of 10 M NS-398 and an 8.7-fold decrease with the addition of 10 M DuP697. The effect of specific COX-2 inhibitors was assessed at both 10 M and 100 M of inhibitor but there was no statistical advantage to the increased concentration of inhibitor.
4. Discussion
Fig. 2. The cytotoxic effect of the specific COX-2 inhibitor DuP-697 on three COX-2 positive mesothelioma cell lines (MSTO-211H, NCI-H2052, NCI-H2452) and a COX-2 positive lung cancer cell line (A549). Each data point is the mean of nine replicates produced using the MTT assay after 72 h and error bars represent the standard error of the mean. Treatment durations of 2, 24, 48 and 72 h were assessed but the maximal effect was seen at 72 h. A significant cytotoxic effect was not produced in the NCIH2452 cells (IC50 not reached), however IC50 values were attained for NCI-H2052 (86 M), A549 (169 M) and MSTO-211H (231 M).
Two COX-2 antibodies were utilised to assess protein expression status of the mesothelioma cell lines, which were all found to exhibit COX-2 positivity. There have been no previous studies in the literature describing the COX-2 protein expression status assessed using these antibodies in the three mesothelioma cell lines cell lines employed in this study. Our MTT results indicate that the specific COX-2 inhibitor DuP-697 inhibited the growth of two of the mesothelioma cell lines and the A549 control cells.
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Fig. 3. The cytotoxic effect of single agent cisplatin and the combination of cisplatin and 10 M DuP-697 or 10 M NS-398 (specific COX-2 inhibitors) after 72 h. Three COX-2 positive mesothelioma cell lines (MSTO-211H, NCI-H2052 and NCI-H2452) and a COX-2 positive lung cancer cell line (A549) were assayed using an MTT assay. Treatment durations of 2, 24, 48 and 72 h were assessed but the maximal effect was seen at 72 h. Each data point is the mean of nine replicates produced using the MTT assay and error bars represent the standard error of the mean. The IC50 values are shown in Table 2.
The addition of either of the specific COX-2 inhibitors (DuP697, NS-398) to cisplatin or vinorelbine did not enhance the cytotoxic effects in any of the three mesothelioma cell lines. In fact the IC50 values increased when these inhibitors were added to cisplatin in NCI-H2452 cells and to vinorelbine in MSTO-211H cells (Table 2). However, the combination of either
of the specific COX-2 inhibitors DuP-697 or NS-398 with pemetrexed enhanced the cytotoxicity of this drug by at least 4-fold in all cell lines tested. To our knowledge this is only the second report of a COX inhibitor demonstrating synergy with a chemotherapeutic agent in mesothelioma. The non-selective COX inhibitor piroxicam has previously been demonstrated to
Fig. 4. The cytotoxic effect of single agent pemetrexed and the combination of pemetrexed and 10 M DuP-697 or 10 M NS-398 (specific COX-2 inhibitors) after 72 h. Three COX-2 positive mesothelioma cell lines (MSTO-211H, NCI-H2052 and NCI-H2452) and a COX-2 positive lung cancer cell line (A549) were assayed using an MTT assay. Treatment durations of 24, 48 and 72 h were assessed but the maximal effect was seen at 72 h. Each data point is the mean of nine replicates produced using the MTT assay and error bars represent the standard error of the mean. The IC50 values are shown in Table 2.
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Table 2 Summary of MTT assay data showing IC50 values obtained from three COX-2 positive mesothelioma cell lines and the control COX-2 positive A549 lung cancer cell line after a 72 h treatment with three chemotherapeutic agents with and without the addition of a selective COX-2 inhibitor (DuP-697 or NS-398). See also Figs. 3 and 4. Possible additive combinations are highlighted in bold. MSTO-211H
NCI-H2052
Cisplatin
Alone +10 M DuP-697 +10 M NS-398
3.4 M 2.2 M 4.3 M
2.2 M 2.2 M 1.5 M
Vinorelbine
Alone +10 M DuP-697 +10 M NS-398
7.9 nM >100 nM >100 nM
1.6 nM 1.2 nM 1.4 nM
Pemetrexed
Alone +10 M DuP-697 +10 M NS-398
1.608 M 0.134 M 0.06 M
enhance the cytotoxic effects of cisplatin in mesothelioma cells [27,28]. The treatment options for mesothelioma remain limited and novel chemotherapeutic regimens remain under investigation. Pemetrexed is currently the drug of choice in this disease and the possibility of enhancing the cytotoxic effects of pemetrexed with the addition of a cell pathway inhibitor is an attractive goal. Some COX-2 inhibitors are known to be associated with significant cardiovascular risks and therefore the choice of agent for testing in clinical trials will be important. However, the risks associated with short-term use as a therapeutic strategy in mesothelioma, a disease currently with dire prognosis, may be lower (or more acceptable) than that for long-term use as a chemoprevention strategy. To understand the side effects of COX-2 inhibitors, and to possibly design improved drugs, it is important to investigate their underlying cellular effects. Several studies have reported that different members of the COX-2 inhibitor drug class demonstrate significant differences in their cellular mechanism of action and there is a growing body of evidence that some COX-2 inhibitors do not exert their effects via COX-2 [29,30]. Further in vitro work is required to establish the mechanism of action of the active agents which we have identified and we are currently investigating this using proteomic-based strategies. Very little is known about the full mechanism of action of DuP-697 [31] but there have been several studies on NS-398 [32–36]. Several compounds which are structurally related to COX-2 inhibitors, but devoid of the ability to inhibit the COX-2 enzyme, for example dimethyl-celecoxib (DMC) and R-flurbiprofen, have been shown to promote apoptosis and these would be anticipated to have fewer cardiovascular toxicities. The analysis of additional compounds, such as these, in our in vitro model may reveal further candidates to progress towards clinical trials. Acknowledgments Gina Eagle is funded by Yorkshire Cancer Research. Funding for parts of this study was provided by the Colt Foundation and Eli Lilly, who also supplied the pemetrexed. The study sponsors played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.
0.204 M <0.05 M <0.05 M
[4] [5]
[6]
[7]
[8]
[9] [10] [11] [12]
[13]
[14]
[15] [16] [17] [18]
[19]
[20]
[21]
[22]
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24.6 M 6.6 M 7.9 M
0.95 nM 1.8 nM 1.2 nM 1.081 M 0.147 M 0.167 M
8.3 nM 12.3 nM 13.6 nM 5.275 M 0.603 M 0.792 M
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