Cyclooxygenase inhibition and hyperthermia for the potentiation of the cytotoxic response in ovarian cancer cells

Gynecologic Oncology 104 (2007) 443 – 450 www.elsevier.com/locate/ygyno

Cyclooxygenase inhibition and hyperthermia for the potentiation of the cytotoxic response in ovarian cancer cells Amber P. Barnes a , Brigitte E. Miller b , Gregory L. Kucera c,⁎ a

c

Department of Internal Medicine Section on Molecular Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA b Department of Obstetrics and Gynecology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA Department of Internal Medicine Section on Hematology and Oncology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA Received 12 June 2006 Available online 22 September 2006

Abstract Objectives. The progression of chemotherapy-resistant cancer confers poor prognosis and decreases overall survival in ovarian cancer patients. Adjuvants to traditional chemotherapy regimens have become attractive modalities for the clinical treatment of refractory or resistant ovarian cancer. We evaluated whether the addition of NSAID to hyperthermic chemotherapy would increase cytotoxicity in cisplatin- and taxane-treated ovarian cancer cells. Methods. Western blot analysis was utilized to determine COX-2 protein expression levels in the 2008, cisplatin-sensitive, and C13⁎, cisplatinresistant, cell lines. PGE2 levels were determined and analyzed as a function of cyclooxygenase activity by LC/MS/MS. Cells were treated with cisplatin, docetaxel or paclitaxel in combination with either NS-398 or sulindac sulfide at 37°C, 41°C or 43°C. Cell viability was determined by a MTS cell proliferation assay. Results. Both cell lines expressed COX-2 protein, and NS-398 and sulindac sulfide effectively blocked PGE2 production. The addition of a NSAID to cisplatin treatment in 2008 and C13⁎ cells offered enhanced cytotoxicity and this effect was further enhanced at 41°C. In docetaxeltreated 2008 cells, both NS-398 and sulindac sulfide offered enhanced cell kill; however, this result was not observed in paclitaxel-treated cells. Hyperthermia appeared to play no additional role in taxane cytotoxicity enhancement, however no antagonism was detected. Conclusions. Our results suggest that the combination treatment (cisplatin or docetaxel in combination with NSAID) cause a dose-dependent enhancement of cytotoxicity. Hyperthermia may improve the results of intraperitoneal cisplatin therapy, thus warranting further evaluation in clinical studies. © 2006 Elsevier Inc. All rights reserved. Keywords: Ovarian cancer; NSAID; Cisplatin; Paclitaxel; Docetaxel; Hyperthermia; Cyclooxygenase

Introduction Ovarian cancer, mostly of the epithelial origin, is the deadliest malignancy of the female reproductive system. Diagnosis is made only after the tumor has reached advanced stages. The inability to diagnose early symptoms and the absence of effective screening methods are the primary causes of late diagnosis and decreased survival for ovarian cancer patients. Current treatment includes aggressive tumor reductive surgery (TRS) followed by platinum based combination chemotherapy, using cisplatin or carboplatin combined with a taxane such as ⁎ Corresponding author. Fax: +1 336 716 0255. E-mail address: [email protected] (G.L. Kucera). 0090-8258/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2006.08.008

paclitaxel or docetaxel. Patients, after adequate TRS to a residual tumor volume of less than 1cm, also benefit from intraperitoneal drug administration leading to increased progression free interval as well as improved overall survival. A recent study completed by the Gynecology Oncology Group (GOG) reported a median survival time of 65 months for patients with stage III ovarian cancer treated with TRS followed by intravenous and intraperitoneal combination chemotherapy with cisplatin and paclitaxel [1]. This is among the longest survival times reported in a phase III study for this patient population. Unfortunately, 70% of all initial responders to chemotherapy relapse and subsequently require additional therapy to treat their disease [2]. It is during second- or third-line therapy that many ovarian cancer patients develop resistance to platinum drugs, thus

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limiting further treatment options and decreasing overall survival rates. Ways to overcome drug resistance include the development of new chemotherapeutic compounds, addition of translational agents which modify signal transduction pathways or investigation of new methods for drug administration. Cisplatin resistance is a poor prognostic factor for ovarian cancer patients as it also decreases the response rate to other compounds. Resistance to cisplatin is not completely understood; however, it is thought to occur through many different mechanisms. Up-regulation of the COX enzymes in ovarian tumor cells has been implicated to play a key role in platinum drug resistance [3–7]. The Cox gene encodes for two forms of the protein. These isoforms, COX-1 and COX-2, are also known as prostaglandin G/H synthase or prostaglandin endoperoxide synthase [11]. COX is an integral membrane protein located primarily in the endoplasmic reticulum containing both cyclooxygenase and peroxidase activities. This bi-functional protein catalyzes the rate-limiting step in the formation of prostaglandins by converting arachidonic acid, a 20-carbon fatty acid liberated from membrane phospholipids by PLA2, to prostaglandin G2 (PGG2) and secondly by converting PGG2 to PGH2. PGH2 is an unstable intermediate and a substrate for several downstream isomerases that produce the bioactive prostanoids. Enhanced Cox-2 gene expression is thought to be associated with cisplatin resistance and the promotion of tumor progression. Furthermore, taxanes which are often used in firstand second-line therapy have been shown to induce Cox-2 expression [8]. As a result, inhibitors of the COX enzymes may increase the efficacy of this drug class. Inhibitors of the COX enzymes, nonsteroidal anti-inflammatory drugs (NSAIDs), have been shown in some tumor types to enhance the efficacy of platinum drugs [9,10]. Therefore, this phenomenon deserves further study to improve the response of patients with ovarian cancer when treated with NSAIDS in combination with taxane containing chemotherapy regimens. Mild hyperthermia (39–43°C) has successfully been utilized in combination with chemotherapy to increase cellular sensitivity to anticancer drugs [12–17] mainly using an intraperitoneal approach. However, systemic drug administration under hyperthermic conditions has also been reported. As an adjunct to traditional therapy, hyperthermia has been shown to enhance cisplatin cytotoxicity. The interaction between heat and chemotherapeutic agents results in increased drug uptake by accelerating the primary step in a drug's efficacy and increasing the intracellular drug concentration [18]. Therefore, the combination of hyperthermia and anti-cancer drugs may reduce the required effective dose of the anti-cancer drug, and it could enhance the response rates in ovarian cancer cells. In this study we hypothesized that the addition of NSAID and/or hyperthermia to chemotherapy drug treatment of ovarian cancer cells would potentiate cytotoxicity. As an endpoint to our studies, we evaluated whether the combination drug treatment in the presence of hyperthermia resulted in a decrease in the IC50 of the chemotherapy drug and if this translated into a synergistic interaction. Our study shows that while hyperthermia potentiates cisplatin toxicity, mild elevated temperature does not enhance cytotoxicity in taxane-treated cells. Impor-

tantly, however, hyperthermia is not antagonistic to taxane therapy alone. Secondly, we compare the effectiveness of NSAIDs to enhance cytotoxicity with and without hyperthermic drug application. We evaluate the effect of a COX-2 specific inhibitor (NS-398) and a non-selective COX inhibitor (sulindac sulfide) on the cytotoxicity of cisplatin, docetaxel and paclitaxel in ovarian cancer cell lines in the presence and absence of hyperthermia at 37°C, 41°C and 43°C. Our studies are important in the clinical setting since a potential caveat to intraperitoneal hyperthermic chemotherapy (IPHC) in gynecologic oncology patients is the formation of adhesions in the peritoneal cavity from the treatment itself. Greene et al. [19] demonstrated that treatment with Celecoxib, a COX-2 specific NSAID, significantly reduced the number of adhesions following surgery in mice. They proposed that the mechanism for the reduction of intraabdominal adhesion formation was primarily due to antiangiogenic properties of the NSAIDs tested. Our findings may lend credence to the use of COX inhibitors with cisplatin in combination with taxanes to treat refractory chemotherapy resistant ovarian cancer by IPHC. Materials and methods Materials MTS reagent, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbomethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt was purchased from Promega Corporation (Madison, WI), phenazine methosulfate (PMS), cisplatin and paclitaxel were purchased from Sigma-Aldrich (St. Louis, MO). Docetaxel was a kind gift of Aventis Pharma. N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), sulindac sulfide and tetradecanoyl phorbol 13-acetate (TPA) were purchased from Biomol (Plymouth Meeting, PA). Prostaglandin E2-d4 (PGE2-d4) was purchased from Cayman Chemical (Ann Arbor, MI).

Cell culture 2008 and C13⁎ cells (Wake Forest University Comprehensive Cancer Center's Tissue Culture Core Lab, originally obtained from ATCC) were maintained in RPMI-1640 medium (Invitrogen, Life Technologies, Carlsbad, CA) supplemented with 10% FBS. The parental cell line, 2008, is cisplatin sensitive while its derivative, C13⁎, possesses approximately a 4-fold resistance to cisplatin. Cells were maintained in log phase and kept in a humidified atmosphere of 5% CO2/95% air at 37°C (unless otherwise specified). The culture medium contained penicillin (100units/ml), streptomycin (100μg/ml) and 2mM L-glutamine. Cells were periodically tested and deemed to be free of mycoplasma contamination.

MTS assay The CellTiter 96® Aqueous Non-radioactive Cell Proliferation Assay (Promega life Sciences, Madison, WI) was used according to the manufacturer's instructions. The MTS assay tests cellular viability and mitochondrial function in the cell cultures. Briefly, cells were grown in T-25 tissue culture flasks. The cells were harvested by treating the flasks with 0.025% trypsin and 0.25mM EDTA for 5min. Once the cells detached from the flask, they were washed, counted and an aliquot was placed in each well of a 96-well cell culture cluster plate (Costar, Fisher Scientific, Pittsburgh, PA) in a total volume of 100μl, and the cells were allowed to attach overnight. The cells were exposed for 30 min to hyperthermic temperature prior to drug exposure. Following drug treatment, the cells were re-exposed to the hyperthermic temperature and then they were allowed to incubate for the remainder of 72h at 37°C. All drug stocks were dissolved in dimethyl sulfoxide (DMSO) and frozen at −80°C until use.

A.P. Barnes et al. / Gynecologic Oncology 104 (2007) 443–450 Working dilutions of drugs were made with culture medium, and the concentration of DMSO (drug vehicle) was kept at or below 0.1% in all experiments. Following the drug treatment, 20 μl MTS solution was added to each well and the plates were incubated for 3h at 37°C. The absorbency of the product formazan, which is considered to be directly proportional to the number of living cells in the culture, was measured at 490nm using a Precision Microplate Reader (Molecular Devices, Sunnyvale, CA). The optical densities of the drug treated cells were compared to untreated control cells and a percent of control value was determined by dividing the optical density of the treated well by the optical density of the control well. The data were plotted in GraphPad Prism (GraphPad Software, San Diego, CA), and the sigmoidal dose– response non-linear regression analysis was used to determine IC50 values. To obtain a Combination Index (CI) for drug interaction in combination cancer chemotherapy, the effect of each drug treatment was entered into CalcuSyn software (Biosoft, Cambridge, UK) for analysis. The cytotoxic effects of a given drug combination are described by the equation fa/fu = (D/Dm)m where fa is the fraction of the cells affected, fu is the fraction of the cells unaffected (1 − fa), D is the dose of drug, Dm is the dose of the drug to cause the median effect and m is the slope of the median–effect curve. The CI is derived from this relationship with a CI <1 indicating synergism, a CI = 1 indicating additive effects and a CI >1 indicating antagonism. PGE2 Mass Spectrometry Analysis-Mass spectrometry analysis of PGE2 concentrations was performed using the Yang method [20]. Briefly, cells were plated in 100mm cell culture dishes (Costar, Fisher Scientific, Pittsburgh, PA) at a density of 5 × 106 cells/dish and allowed to attach overnight. Cells were treated with drug for 4h, washed with ice cold phosphate buffered saline (PBS), and harvested by scraping the cells into PBS. Following centrifugation at 300×g for 5min at 4°C, the pellets were drained and frozen at − 80°C until further processing. To measure the PGE2 concentration, the cell pellets were resuspended in PBS with CaCl2 (1 mM), and an internal standard was added to each sample (100ng/ml PGE2-d4). A lipid extraction was performed by adding hexane:ethyl acetate (1:1 v/v) and vortex mixed for 2min. Samples were centrifuged (1800×g for 10min at 4°C) and the upper organic layer was then transferred to a glass tube on ice. Following two more extractions, the organic layers were pooled and the solvent was evaporated under a gentle stream of nitrogen at room temperature. Prior to LC/MS/MS analysis [20], samples were reconstituted in a methanol:10mM ammonium acetate buffer, pH 8.5 (25:75). Reversed-phase electrospray ionization mass spectrometry was performed by the Wake Forest University School of Medicine Mass Spectrometry Core Laboratory. All extraction procedures were performed in low light to prevent the photodegradation of the PGE2.

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differences. Quantification was conducted via densitometry (Bio-Rad Quantity One Software).

Statistical analysis Data are expressed as means ± standard deviation (S.D.) and are analyzed with regard to statistical significance using a Student's t-test where indicated. A p value < 0.05 was considered to be significant.

Results Increased COX-2 protein expression has been implicated as a resistance factor for many cancer cell types. We analyzed the COX-2 protein levels in the ovarian cancer cell lines C13⁎ and 2008, and they are presented in Fig. 1. It is evident from this figure that both cell lines express this protein whether cells were in the presence or absence of hyperthermia. Furthermore, the resistant cell line, C13⁎, expresses more COX-2 protein than its cisplatin sensitive counterpart, 2008. Since both of these cell lines possess COX-2 protein, we analyzed PGE2 production by mass spectrometry. PGE2 is a downstream product of the COX enzymes and it is used as a measure of COX activity. PGE2 levels were determined as a function of COX activity in untreated, TPA-stimulated and NSAID-treated cells. TPA is a potent tumor promoter that stimulates PGE2 production in cells and serves as a positive control for PGE2 production. The PGE2 levels are presented in Fig. 2. It is evident from this figure that a reduction in the PGE2 levels was observed in the NS-398- and

Western blotting Cells were plated in 100mm cell culture dishes (Costar, Fisher Scientific, Pittsburgh, PA) at a density of 3 × 106 cells/dish and allowed to attach overnight. Cells were treated with drug for 24 h, washed with ice cold PBS, and harvested on ice by scraping the cells into lysis buffer (50mM Tris–HCl, 100 mM NaCl, 2mM EDTA, 20mM β-glycerophosphate, 0.1% sodium dodecyl sulfate, 0.5% sodium desoxycholate, 0.5% NP-40, and 0.5% Triton-X 100) containing proteinase inhibitors (1mM sodium vanadate, 0.05M sodium fluoride, 1mM phenylmethylsulfonylflouoride, 0.01mg/ml aprotinin, and 0.01mg/ml leupeptin). The samples were sonicated, spun at high speed for 10min in a microfuge at 4°C, and the supernatant was transferred to a fresh tube. The protein concentration in the cell lysates was determined using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Proteins (40μg) were denatured and fractionated on a 15% polyacrylamide gel, and then transferred to nitrocellulose membranes after electrophoresis. The membranes were incubated overnight at 4°C in blocking solution (PBS containing 5% nonfat, dried milk) followed by a 3h incubation with antiCOX-2 antibodies. A goat polyclonal anti-human COX-2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at a dilution of 1:1000. The membranes were washed 4 times and incubated with a horseradish peroxidaseconjugated anti-goat immunoglobulin as a secondary antibody for 1h at a 1:10,000 dilution. After 4 additional washes, the membranes were developed with an enhanced chemiluminescence system (Pierce, Rockford, IL) and exposed to Kodak Biomax MR film. The membranes were also probed with an anti-actin antibody (Abcam, Inc. Cambridge, MA) to normalize sample loading

Fig. 1. Ovarian cancer cells express COX-2 protein. (A) Western immunoblot analysis documenting COX-2 expression in C13⁎ and 2008 cell lysates treated at normal (37°C) or hyperthermic temperatures (41°C) for 1.5 h. Cellular protein lysate (40 μg) was loaded in each well and subjected to electrophoresis. AntiCOX-2 and anti-actin antibodies were used to visualize the respective proteins. (B) Relative densitometric units of the COX-2 bands are shown following normalization to the actin loading controls.

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Fig. 2. NSAIDs block PGE2 production in ovarian cancer cell lines. PGE2 production in (A) C13⁎ and (B) 2008 cells was effectively blocked by treatment with NSAIDs. Cells were incubated for 4h with either 10 μM NS-398 (NS) or 10μM sulindac sulfide (SS) to block PGE2 production or 100nM TPA to induce PGE2 production. An internal standard was added to each sample as described in Materials and methods. Prostaglandins were extracted with hexane:ethyl acetate (1:1) and analyzed by LC/MS/MS.

the sulindac sulfide-treated cells as compared to control cells. Therefore, the NSAIDs we chose efficiently block COX-2 activity in the cells used for the following experiments. To determine the dose of NSAID that did not affect cell viability in a MTS cytotoxicity assay, cells were treated with NS-398 or sulindac sulfide and dose–response curves were plotted (data not shown). Neither NSAID caused a decrease in cell viability below approximately 100μM. The concentrations chosen for the subsequent experiments were below the level where cytotoxicity was observed. The cytotoxicity of cisplatin was determined in the C13⁎ and 2008 cell lines, at 37°C, 41°C and 43°C (Fig. 3). In Table 1, the IC50 values were compared. There was no significant enhancement of cisplatin cytotoxicity when cells were exposed to hyperthermia in either cell line; therefore, hyperthermia alone does not enhance cisplatin's cytotoxicity in these ovarian cancer cell lines. The cytotoxicity of docetaxel and paclitaxel was also evaluated in the 2008 cell line at 37°C, 41°C and 43°C, (Fig. 4) and the IC50 values were compared in Table 2. Again, there was no enhanced taxane cytotoxicity with increased temperature. Data for C13⁎ cells is not shown for docetaxel and paclitaxel cytotoxicity since a dose-dependent response was not observed

Fig. 3. Hyperthermia alone does not potentiate cisplatin cytotoxicity. Dose– response curves for (A) C13⁎ and (B) 2008 cells treated with cisplatin at 37°C (■), 41°C (▾) or 43°C (●). Points represent the mean ± S.D. of 3 experiments. Error bars are shown when the error is greater than the size of the symbol.

when this cell line was treated with paclitaxel; therefore, comparisons would not be possible with docetaxel. Adjuvants to traditional chemotherapy regimens have become attractive modalities for the clinical treatment of refractory or resistant cancers. We hypothesize that the addition of NSAID to hyperthermic chemotherapy would increase in vitro cytotoxicity and would ultimately result in an enhancement of the clinical response. Combination drug cytotoxicity assays were performed to specifically answer the question of whether the addition of NSAID to cisplatin chemotherapy enhances cytotoxicity and to determine if the addition of hyperthermia to this combination treatment has an added effect. Our data demonstrate that the addition of NS-398 increases Table 1 Hyperthermia causes no significant cytotoxic enhancement of cisplatin cytotoxicity Cell line

Temperature (°C)

Cisplatin (μM)

C13⁎

37 41 42 37 41 43

27.1 ± 5.2 22.7 ± 2.2 32.4 ± 3.7 6.2 ± 3.4 7.0 ± 2.3 5.0 ± 2.2

2008

IC50 values for cisplatin treated C13⁎ and 2008 cells at 37°C, 41°C and 43°C (IC50 ± S.D., n = 3). IC50 values were compared using a Student's t-test. p > 0.05, and the values were not statistically different within the same cell line at the different temperatures.

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when combined with docetaxel. This emphasizes the importance of inhibiting both COX-1 and COX-2 enzymes in order to achieve a greater cytotoxic response at the lower taxane doses. Specifically, docetaxel + 15μM sulindac sulfide resulted in a dose-dependent cytotoxicity enhancement and decreased the IC50 value by 4.6-fold while a similar response (4.8-fold decrease in the IC50 value) was observed with docetaxel + 40μM NS-398. This leads to the conclusion that in combination with docetaxel, both the specific and the non-specific COX inhibitors offer enhanced cell kill. Surprisingly, the combination treatment of 2008 cells with paclitaxel and NSAIDs did not significantly enhance cytotoxicity, nor did hyperthermia enhance cell killing with this taxane (data not shown). This observation may be explained by the fact that although the taxanes share similar mechanisms of action, they exhibit differences in their molecular pharmacology, pharmacokinetics and pharmacodynamic profiles. Therefore, these factors may account for the differences observed between the taxanes and their cytotoxicities [21]. Discussion

Fig. 4. Hyperthermia does not potentiate taxane cytotoxicity in the 2008 cell line. Using a MTS assay, the cytotoxicity of docetaxel and paclitaxel treatment were determined in the ovarian cancer cell line, 2008, at 37°C, 41°C and 43°C. Shown are dose–response curves for (A) docetaxel and (B) paclitaxel treated 2008 ovarian cancer cells at 37°C (■), 41°C (●) or 43°C (□). Points represent the mean ± S.D. of 3 experiments. Error bars are shown when the error is greater than the size of the symbol.

cytotoxicity in both cell lines (Fig. 5). A significant increase in cytotoxicity was achieved when 20μM NS-398 was added to 1 μM or 10 μM cisplatin at each temperature in both cell lines (p < 0.001). Treatment of 2008 cells with 1μM cisplatin + 20 μM NS-398 caused a 1.8-fold increase in cytotoxicity at 37°C and a 3.3-fold increase at 41°C. Interestingly, a better response was observed with the combination drug treatment in the cisplatinresistant C13⁎ cell line. Treatment of C13⁎ cells with 1 μM cisplatin + 20 μM NS-398 caused a 2.9-fold increase in cytotoxicity at 37°C and a 3.4-fold increase at 41°C. Therefore, the addition of 20 μM NS-398 to 1μM cisplatin achieved at least the same level of cytotoxicity as a 10-fold higher dose of cisplatin alone in both cell lines. This suggests that the addition of NSAID may allow for a reduction of the cisplatin dose needed to achieve a similar cytotoxic affect in patients. Additionally, co-treatment with moderate hyperthermia enhances the synergistic affect of the combined cisplatin and NS-398 treatment. We did not observe additional cytotoxicity above 41°C (Fig. 5); therefore, 41°C is the optimal hyperthermic dose to enhance drug synergy in the C13⁎ and 2008 cells. Similarly, a dose-dependent enhancement of docetaxel induced cytotoxicity was observed when 2008 cells were treated with a taxane in combination with NS-398 or sulindac sulfide (Fig. 6). However, a lower dose of sulindac sulfide was able to achieve the same cytotoxicity as higher doses of NS-398

A combination approach of multiple therapeutic agents has become the standard of care for the treatment of ovarian cancer, initially and after recurrence. Although cisplatin and taxanes are currently administered by intraperitoneal and systemic therapy, the addition of NSAIDs to this multi-modal treatment approach has not been fully evaluated. The addition of NSAIDs to various chemotherapies has proven to offer a beneficial effect in several different cancer cell types including cancers of the bone [22], cervix [23], head and neck [24] and lung [25,26]. Our present study is aimed at determining the potential of NSAIDs in increasing the cytotoxicity of cisplatin or taxanes in ovarian cancer cells. Using a MTS assay, we determined that the addition of a NSAID to cisplatin treatment offered enhanced cytotoxicity. This effect was further enhanced at 41°C, but not at 43°C. Next, we evaluated this effect on taxane treated cells. Although a similar effect was not observed in cells treated with paclitaxel, the addition of NSAIDs to docetaxel treatment likewise significantly enhanced cytotoxicity. Finally, we tried to determine if moderate hyperthermia would potentiate this combination effect, by treating cells at 37°C, 41°C and 43°C. When the drugs were administered to the cells for 1.5 h at an elevated temperature and then returned to 37°C for the remainder of the 72 h incubation, no statistical difference in IC50s was observed between the different temperatures. Longer Table 2 Hyperthermia causes no significant cytotoxic enhancement of taxane cytotoxicity Temperature (°C)

Docetaxel (nM)

Paclitaxel (nM)

37 41 43

12.0 ± 4.3 11.8 ± 2.5 12.5 ± 8.1

38.8 ± 20.8 23.8 ± 7.2 32.5 ± 2.4

IC50 values for docetaxel and paclitaxel treated 2008 cells at 37°C, 41°C and 43°C are reported (mean ± S.D., n = 3). The means of 41°C and 43°C treated groups were compared to 37°C and there was no statistical significance between the treatment groups (p > 0.05) using a Student's t-test.

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Fig. 5. Effects of COX-2 inhibition on cisplatin sensitive and resistant ovarian cancer cells in the presence of hyperthermia. Shown are results from MTS assays of (A) 4-fold cisplatin resistant C13⁎ cells and (B) cisplatin sensitive 2008 cells treated with a combination of cisplatin and NS-398. “Percent of Control” refers to control cells at each given temperature that received no drug treatment. There was no significant difference between controls at each temperature. Values shown represent the mean ± S.D. of three separate experiments. Means of combination treatments were compared to cisplatin treatment alone at the given dose using a Student's t-test. Significant differences are indicated by asterisks, p < 0.001.

times of hyperthermic treatment were not evaluated due to the fact that clinical treatments for more than 1.5 h would be difficult. In vitro experiments have reported that the addition of hyperthermia to cisplatin treatment causes a beneficial cytotoxic effect [18,27,28]. Although hyperthermia allows for increased drug accumulation due to enhanced cellular permeability; hyperthermia inhibits taxane related cell cycle effects and thus inhibits taxane-induced cytotoxicity [29]. Our data corroborates with others [29,30] to conclude that elevated temperature does not cause enhanced taxane cytotoxicity; and this result remains unchanged even in the presence of NSAID treatment. These findings refute reports by others that show hyperthermic enhancement of paclitaxel and docetaxel-induced cytotoxicity [31–33]. Mohamed et al. [44] found that moderate hyperthermia increased docetaxel's cytotoxicity, but paclitaxel did not show enhancement. Although it is not understood how hyperthermia interacts with paclitaxel, possible explanations offered by Mohamed et al. include that in the presence of hyperthermia, the synthesis of heat-shock proteins, such as hsp74, may be inhibited by paclitaxel, resulting in structural modifications of the mitotic spindle, and an inhibition of paclitaxel induced cytotoxicity. It is known that both docetaxel and paclitaxel are heat stable drugs [30]; therefore, drug instability is not likely a cause for this difference. The concentration of NS-398 used in these experiments that produced a significant positive result in the combination assays is above the IC50 value reported to inhibit the COX-2 enzyme for assays conducted with purified, recombinant COX (0.1 μM) [34]. Therefore, we hypothesize that the concentrations of NS-

398 used are inhibiting COX-2 as well as reducing the enzymatic activity of COX-1. The reported IC50 value for NS-398's inhibition of COX-1 is > 100 μM in purified, recombinant human COX enzyme [34]; therefore, at high doses of NS-398 (i.e. 40μM), the observed effect could be a result of a combination of COX-2 and COX-1 inhibition. Our results are consistent with Altorki et al. [35] who have shown enhanced taxane cytotoxicity when used in combination with NSAIDs. However, Munkarah et al. have reported that the combination of NSAIDs with paclitaxel (either in tandem or pretreatment with NSAID) inhibited the apoptotic effects of paclitaxel [36]. A possible reason for these opposing results is the difference in treatment schedules and regimens. In our experience simultaneous exposure with taxane and NSAID produced synergy when the cells were treated with NS-398 and cisplatin while either pre or post cisplatin treatment with NSAID did not have an enhanced cytotoxic effect (data not shown). Our results suggest that the combination treatment (cisplatin or docetaxel in combination with NSAID) caused a dose-dependent enhancement of cytotoxicity. Although hyperthermia appears to play no additional role in taxane enhancement, in our experience no antagonism was detected with the addition of hyperthermia to a chemotherapy regimen including taxanes and NSAIDs. Since the addition of hyperthermia showed enhancement in cisplatin + NSAIDtreated cells, this could be an important finding since most patients are initially treated with a combination of platinum and taxane drugs. In addition, NSAIDs have been shown to prevent adhesion formation following surgical trauma to the peritoneum [37,38]; therefore NSAIDs may offer additional

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Fig. 6. Effects of combination taxane + NSAID treatment in 2008 cell line. Dose–response curves for combination treatment of cells with either (A) docetaxel and NS398, (B) docetaxel and sulindac sulfide, (C) paclitaxel and NS-398 or (D) paclitaxel and sulindac sulfide are shown above. Cells were dosed with serial dilutions of taxane and then treated at 37°C with either 1μM (□), 5μM (▴), 10μM (▾), 15 μM (○), 20μM (♦), 40μM (●), or no NSAID (■). Points represent the mean ± S.D. of 3 experiments. Error bars are shown when the error is greater than the size of the symbol. A combination index (CI) was calculated using the CalcuSyn dose effect analysis program to determine positive or negative interactions. A CI < 1 indicating synergy was observed at the taxane dose range shown between the vertical dashed lines.

anti-inflammatory benefits that do not have a direct effect on the cancer cell. In addition, others have shown that NSAIDs offer an anti-proliferative effect on many cancer cell types [39–41]. Our results indicate that the addition of a NSAID to cisplatin or taxane therapy is beneficial to enhance cytotoxicity as well as to reduce the chemotherapy dose necessary to achieve a cytotoxic response. Also, hyperthermia in combination with NSAIDs may lead to further increased antiproliferative effects in cisplatin treated cells. Clearly, there are limitations of cell culture systems for predicting the utility of a given treatment response in patients with cancer; however, cell culture systems have proven to be a useful model and can provide insight into molecular mechanisms involved in a disease state, such as cancer. Due to an increased risk of serious adverse cardiovascular effects of several COX-2 inhibitors on the market, there is a need for further research to determine which NSAIDs are best suited for administration with chemotherapy in cancer patients. Since we observed a beneficial effect of the non-selective COX inhibitor, sulindac sulfide, when in combination with docetaxel treatment, our findings would suggest that a non-selective COX inhibitor lacking in major side effects such as sulindac or indomethacin would be best suited for this combination therapy. Additionally, hyperthermia in combination with NSAIDs may lead to further increased anti-proliferative effects

without being antagonistic to the cellular effects of taxanes. Together, these data suggest that the addition of COX inhibitors to cisplatin and docetaxel chemotherapy should be further evaluated in clinical studies; in addition, hyperthermia may improve the results of intra-peritoneal cisplatin therapy in this setting. Acknowledgments Partial funding for this project was provided by a pilot grant from the Wake Forest University Comprehensive Cancer Center and by Aventis Pharmaceuticals. We would also like to acknowledge the Wake Forest University Comprehensive Cancer Center Tissue Culture Core Laboratory. References [1] Armstrong DK, Bundy B, Wenzel L, Huang HQ, Baergen R, Lele S, et al. Intraperitoneal cisplatin and paclitaxel in ovarian cancer. N Engl J Med 2006;354:34–43. [2] Muggia F. Recent updates in the clinical use of platinum compounds for the treatment of gynecologic cancers. Semin Oncol 2004;31(Suppl 14): 17–24. [3] Hinz B, Brune K. Cyclooxygenase—2–10years later. J Pharmacol Exp Ther 2002;300(2):367–75. [4] Gupta RA, Tejada LV, Tong BJ, Das SK, Morrow JD, Dey SK, et al.

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