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Clinical Cancer Research Vol. 12, 6133-6143, October 15, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Piroxicam and Cisplatin in a Mouse Model of Peritoneal Mesothelioma

Enrico P. Spugnini1, Irene Cardillo2, Alessandra Verdina2, Stefania Crispi5, Silvia Saviozzi8, Raffaele Calogero8, Angela Nebbioso6, Lucia Altucci6, Giancarlo Cortese1, Rossella Galati2, Jeremy Chien9, Viji Shridhar9, Bruno Vincenzi3, Gennaro Citro1, Francesco Cognetti4, Ada Sacchi2 and Alfonso Baldi7

Authors' Affiliations: 1 SAFU Department, CRS and 2 Laboratory D, Department for the Development of Therapeutic Programs, CRS, Regina Elena Cancer Institute; 3 Section of Oncology, Campus BioMedico University; 4 Division of Medical Oncology A, Regina Elena National Cancer Institute, Rome, Italy; 5 Gene Expression Core-Human Molecular Genetics Laboratory, Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Naples, Italy; 6 Department of General Pathology and Oncology, "Centro Sperimentale S. Andrea delle Dame"; 7 Section of Pathology, Department of Biochemistry and Biophysics, Second University of Naples, Naples, Italy; 8 Department of Clinical and Biological Science, University of Turin, Turin, Italy; and 9 Department of Laboratory Medicine and Experimental Pathology, Mayo Clinic Cancer Center, Rochester, Minnesota

Requests for reprints: Alfonso Baldi, Section of Pathology, Department of Biochemistry, II University of Naples, Via L. Armanni, 5, 80138 Naples, Italy. Phone: 39-815-666003; Fax: 39-815-569693; E-mail: alfonsobaldi{at}tiscali.it.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The aim of the present study was to evaluate the effects of piroxicam, a widely used nonsteroidal anti-inflammatory drug, alone and in combination with cisplatin (CDDP), on cell growth of mesothelioma cells.

Experimental Design: Cell proliferation, cell cycle analysis, and microarray technology were done on MSTO-211H and NCI-H2452 cells treated with piroxicam. Moreover, the effects of piroxicam and CDDP on tumor growth and survival of mouse xenograft models of mesothelioma were determined.

Results: Piroxicam treatment of MSTO-211H and NCI-H2452 cells resulted in a significant inhibition of proliferation. Cell cycle analysis revealed that there was an increase in the rate of apoptosis in MSTO-211H cells and an increase in the cells accumulating in G2-M in NCI-H2452. Moreover, a marked tumor growth inhibition and an extended survival of mice treated with a combination of piroxicam and CDDP in MSTO-211H cell–induced peritoneal mesotheliomas was observed. Last, GeneChip array analysis of MSTO-211H mesothelioma cell line revealed that piroxicam treatment caused up-regulation of metabolic pathway–associated genes and down-regulation of genes related to RNA processing apparatus. Of note, epidermal growth factor receptor, one of the new biological targets of chemotherapy for mesothelioma, was down-regulated and HtrA1, a serine protease recently shown to be an endogenous mediator of CDDP cytotoxicity, was up-regulated following piroxicam treatment both in vitro and in vivo.

Conclusion: These data suggest that piroxicam sensitizes mesothelioma cells to CDDP-induced cytotoxicity by modulating the expression of several target genes. Therefore, piroxicam in combination with CDDP might potentially be useful in the treatment of patients with mesothelioma.

Malignant mesothelioma (MM) is a rare disease that affects serosal surfaces in humans. Epidemiologic and clinicopathologic studies have shown a correlation between exposure to asbestos and development of pleural mesothelioma (1). Although it has been proposed that alteration of the regulatory proteins of cell cycle could play a role in the progression of this deadly disease (2), the exact mechanism whereby asbestos induces mesothelioma remains unknown (3). Due to the low incidence of the disease, only few randomized studies have been done (reviewed in ref. 4). Moreover, the reported response rates to the different therapeutic protocols ranged from 10% to 45% with no clear advantage in terms of poor survival that is between 4 and 9 months (4). Various drugs have been tested in different combinations thus far; among the most commonly used are doxorubicin, cyclophosphamide, cisplatin (CDDP), carboplatin, gemcitabine, and pemetrexed (5–9). Of interest, the combination of these drugs does not seem to provide any clear advantage over monotherapy (4).

Several articles reported the anticancer effects of a number of nonsteroidal anti-inflammatory drugs (NSAID) in cell cultures, animal models, and humans (10–16) through various mechanisms: direct inhibition of growth, induction of apoptosis, arrest of angiogenesis, and decreased production of carcinogenic and immunosuppressive molecules such as malondialdehyde (17). Among NSAIDs, aspirin, piroxicam, sulindac, and celecoxib have been shown to be highly effective chemopreventive agents (18, 19). At the molecular level, these effects may be, at least in part, due to cyclooxygenase-2 (COX-2) inhibition. COX-2 overexpression seen in many solid tumors has been correlated with a worse prognosis in colorectal cancer, non–small-cell lung cancer, gastric cancer, and mesothelioma (20–26). In fact, NSAIDs that selectively inhibit COX-2 have been shown to reduce tumor formation in vivo, and inhibition of colony formation in vitro (10). To date, there are no reports on the possible role of NSAID piroxicam as a chemopreventive agent in human MM or the molecular mechanisms involved in this activity.

The aim of the present study was, therefore, to determine the effects of piroxicam in vitro on cell growth of mesothelioma cell lines, and to evaluate the antitumor potential of this drug in vivo, alone or in combination with CDDP. For in vivo experiments, we used a mesothelioma flank tumor model (27, 28) and a mesothelioma orthotopic tumor model (29).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Piroxicam (FELDENE; Pfizer, New York, NY) was a 60 mmol/L injectable solution; CDDP (Pharmacia-Italia, Nerviano, MI, Italy) was supplied as a 50 mmol/L injectable solution.

Cell lines
The human mesothelioma cell lines MSTO-211H and NCI-H2452 were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured as monolayers in flasks using American Type Culture Collection complete growth medium in a humidified atmosphere containing 5% CO2 at 37°C.

Software for statistical analyses
SPSS software (version 10.00, Chicago, IL) was used for statistical analyses.

Cell treatment with piroxicam and CDDP
Cells were seeded 16 hours in complete growth medium. Twenty-four hours later, the cells were treated with different concentrations of piroxicam and CDDP as indicated in each experiment. Controls were untreated.

Cell growth and viability
Cells were seeded in a 96-well plate (104 per well) 16 hours before treatment with piroxicam and CDDP. Cell viability was determined using Cell Proliferation kit (Roche Molecular Biochemicals, Indianapolis, IN) after 48 hours of treatment. Experiments were repeated in quadruplicate and, in each experiment, concentration values were quantified five times and averaged. Sheffe test and two-way ANOVA were used to compare proliferation rates among the different experimental conditions (multiple comparisons). Two-tailed P values were considered significant when ≤0.05.

Cell cycle analysis
Unsynchronized cells in the mid log phase were seeded at a density of 106 per T25 flask. After 16 hours, cells were treated with piroxicam, as described in the previous section. At 48 hours, adherent and floating cells were harvested, resuspended in staining solution containing propidium iodide (50 µg/mL), sodium citrate (0.1%), NP40 (0.1%) in PBS 1x, and incubated for 30 minutes in the dark. Cell cycle distribution of 20,000 cells were analyzed with a FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) by ModFit version 3 Technology (Verity, Topsham, ME). Cells in sub-G1 were considered apoptotic. The experiments were repeated thrice.

Western blotting
Cell lysates were prepared by treating cells or tissue with ice-cold lysis buffer (Roche Applied Science, Mannheim, Germany) for 20 minutes followed by centrifugation at 4°C for 15 minutes. Proteins (35 µg) were separated on 10% SDS-PAGE gels and then transferred on polyvinylidene difluoride membrane. Membranes were incubated with COX-2 monoclonal antibody (Cayman Chemical, Ann Arbor, MI) diluted 1:1,000 or with anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:1,000, to normalize the sample loading. Horseradish peroxidase–conjugated secondary antibodies from Santa Cruz were used at 1:3,000 dilution. Ovine COX-2 standard from Cayman Chemical and PC3 lysate were used as positive controls (30). Antibody reaction was visualized using ECL Western blotting detection reagents (Amersham-Pharmacia, Uppsala, Sweden). The experiments were done in duplicate.

Prostaglandin E2 assay
Prostaglandin E2 levels were detected in medium from cell culture by using the Correlate-EIA High Sensitivity Prostaglandin E2 Enzyme Immunoassay kit from Assay Designs (Ann Arbor, MI).

Animal models
Female nude mice (6-8 weeks old; weight 18-25 g) were obtained from Harlan Laboratories (Milan, Italy). Mice were housed in the animal facility of the Regina Elena Cancer Institute for 2 weeks before each experiment; animals had ad libitum water and food. Each experiment used 10 mice per treatment arm and was repeated twice for confirmation of the obtained data. The ethical committee of the Cancer Institute approved all the experimental protocols that were done in accordance with Italian regulations (116/92) and with the Guide for the Care and Use of Laboratory Animals.

Mesothelioma flank tumor model
MSTO-211H cells (2.5 x 106) in 0.2 mL complete medium were injected in the dorsum of 40 mice. One week later, animals were randomly allocated to one of the following groups: (a) control; (b) i.p. piroxicam, 0.3 mg/kg daily for 15 days (5 days per week); (c) CDDP 3.3 mg/kg, administered i.p. for the first 3 days; and (d) combination of piroxicam and CDDP following the schedules above. Mice were weighed and calipered thrice every week and were sacrificed when they met the established criteria for minimizing pain and suffering. Tumor size was assessed by using the formula ({pi} x long axis x short axis x short axis) / 6. Tumor volumes at a given time were analyzed by comparing the different groups in each experiment by using ANOVA with appropriate post hoc testing (Wilcoxon test) when significant differences (P < 0.05) were found. The growth curves were designed considering the mean tumor volume at any different time points. Cutoff point for statistical significance was <0.05.

Mesothelioma orthotopic tumor model
MSTO-211H tumor cells (2.5 x 106) in 0.2 mL complete medium were injected in the peritoneal cavity of 40 mice. One week after the inoculation, mice were randomized to each of treatment groups described above (n = 10 per group) and followed prospectively according to the schedules illustrated above. Mice studied in these experiments were also weighed thrice weekly and were sacrificed when they met the established criteria for minimizing pain and suffering. Survival was calculated from the time of inoculation to the last date of follow-up by means of the Kaplan-Meier estimate and prognosis was compared using the log-rank test. Cutoff point for statistical significance was <0.05.

Histology and immunohistochemistry
For histology, stainings with H&E and hematoxylin/Van Gieson were used. For immunohistochemistry, tissue sections were heated twice in a microwave oven for 5 minutes each at 700 W in citrate buffer (pH 6) and then processed with the streptavidin-biotin-immunoperoxidase method (Universal kit, DAKO, Carpinteria, CA). Rabbit anti-human FVIII-RA polyclonal antibody and anti-CD31 monoclonal antibody from DAKO were used at a 1:100 dilution. Anti-HtrA1 (high-temperature requirement protein A1) polyclonal antibody was used as previously described (31). Diaminobenzidine was used as the final chromogen, and hematoxylin as the nuclear counterstain. Negative control were done leaving out the primary antibody. Positive controls consisted of tissues previously shown to express the antigen of interest. Epidermal growth factor receptor (EGFR) protein expression was detected by the EGFR pharmDx from DAKO. Two observers (E.P.S. and A.B.), blinded to treatment conditions, evaluated the staining pattern of the proteins separately and quantitated the protein expression in each specimen by scanning the entire section and estimating the number of vessels or positive cells at the high-power field 10 x 20. The level of concordance, expressed as the percentage of agreement between the observers, was 94%. In the remaining specimens, the score was obtained after collegial revision and agreement. The U–Mann-Whitney test was used to assess relationship between ordinal data. Two-tailed P value was considered significant when ≤0.05.

Terminal Deoxyribonucleotide Transferase–Mediated Nick-End Labeling assay
Terminal deoxyribonucleotide transferase–mediated nick-end labeling (TUNEL) reaction was done using the peroxidase-based Apoptag kit (Oncor, Gaithersburg, MD). One hundred random fields (x250) per section were analyzed (12.5 mm2). The level of concordance, expressed as the percentage of agreement between the two observers, was 100%. The U Mann-Whitney test was used to assess relationship between ordinal data. Two-tailed P value was considered significant when ≤0.05.

GeneChip array analysis
GeneChip array sample preparation. MSTO-211H cells (3 x 106) were seeded in T75 flasks and then treated with piroxicam for 48 hours. Total RNA was extracted and purified using the RNeasy Midi kit (Qiagen, Hilden, Germany). All the analyses were done with chips, kits, and instrumentation from Affymetrix (Santa Clara, CA). Nine HGU133plus2 array chips were hybridized with biotinylated cRNA (15 µg/chip, 16 hours, 45°C). A GeneChip Fluidics station 400 was used to wash and stain the arrays. The chips were scanned with a specific scanner to generate digitized image data files.

GeneChip array data analysis. Two prototypic situations (control, untreated cell line, and treated, piroxicam-treated cell line) were analyzed by GCOS (Affymetrix) to generate background-normalized image data (CEL files; Tables 1 and 2 ). Microarray quality control and statistical validation was done using Bioconductor (32). The presence of hybridization/construction artifacts was evaluated with the fitPLM function (Bioconductor package affyPLM). The probe (PM) intensity distribution was evaluated using hist function (Bioconductor package affy). Probe set intensities were obtained by means of GCRMA (33) and normalization was done according to quantiles method (34). The HGU133plus2 54645 probe sets were filtered to have an interquantile range for each probe set >0.4 (35). This filtering yielded 11,926 probe sets. The identification of differentially expressed genes associated to piroxicam effects was addressed using linear modeling approach (36). For assessing differential expression, an empirical Bayesian method (37) was used together with false-discovery rate correction of the P value (38) to moderate the SE values of the estimated log-fold changes. We applied the false-discovery rate criterion (P ≤ 0.05) to balance type I and type II error rates and a fold change threshold of |log2(fc)| ≥ 1. The search of overrepresented gene ontology (GO) Biological Process themes was done using Bioconductor GOstats package (32). Gene annotation was done using Bioconductor annaffy library and hgu133plus2 annotation package (version 1.8.4).


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  		Table 1. Gene ontology terms of the biological process class found enriched within the set of down-modulated genes

 

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  		Table 2. Gene ontology terms of the biological process class found enriched within the set of up-modulated genes

 
Quantitative real-time reverse transcription-PCR
Primers were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA) and, when possible, the same coding target region identified by the Affymetrix probe was amplified; otherwise, primers were designed on the coding sequence (Table 3 ). The specificity of each target amplicon was assessed by dissociation curve analysis and all amplicons were spanning over exon-exon regions to avoid genomic amplification. Quantitative PCR was done on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) in 384-well plates assembled by Biorobot 8000 (Qiagen) using a final volume of 20 µL and the following cycle conditions: 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. All quantitative PCR mixtures contained 1 µL cDNA template (corresponding to 20 ng retrotranscribed total RNA), 1x Sybr Green PCR-Master-Mix (2x; Applied Biosystems) and 150 µmol/L of each target-specific primer. For each experiment, a no-template reaction was included as negative control. The expression of each target gene was evaluated by a relative quantification approach (–{Delta}{Delta}Ct method; ref. 39), using ß-actin (BACT), polymerase RNA II polypeptide B (POLR2B, 140 kDa), and NH2-terminal asparagine amidase (NTAN1) as multiple internal references. Internal controls were selected within not differentially expressed genes in this experiment. BACT, POLR2B, and NTAN1 were high-, medium-, and low-expressed genes, respectively, and characterized by an interquantile range <0.1. The untreated cell line was used as calibrator and target genes were accepted as differential expressed when the {Delta}{Delta}Ct absolute value was >1, which correspond a 2-fold change in transcript abundance.


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  		Table 3. Primer sequences of target and housekeeping genes used in qPCR validation

 

   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effect of piroxicam on cell growth and cell cycle regulation of MSTO-211H and NCI-H2452 mesothelioma cell lines. We assessed the effects of piroxicam on cell viability of two human mesothelioma cell lines (MSTO-211H and NCI-H2452). Tumor cells were treated with drug concentrations ranging from 12 to 1,000 µmol/L. Compared with untreated cells, both cell lines showed a dose-dependent reduction in proliferation levels when treated with piroxicam. In particular, the MSTO-211H cell line showed a inhibitory concentration of 50% for cell proliferation (IP50) of 760 µmol/L, whereas NCI-H2452 reached an IP50 with 680 µmol/L (Fig. 1, left ).


Figure 1
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  		Fig. 1. Effect of piroxicam on cell proliferation and on cell cycle regulation in MSTO-211H and NCI-H2452. Left, increasing concentrations of piroxicam (from 12 to 1,000 µmol/L) reduce mesothelioma cell proliferation expressed as percentage of control (CTRL). Right, MSTO-211H and NCI-H2452 were treated as indicated in the figure and then analyzed to determine cell cycle distribution (expressed as percentage of total cells). *, P < 0.0001 versus control; bullet, P = 0.003 versus control.

 
We used flow cytometry to analyze in greater detail the mechanisms by which piroxicam affects the proliferation of mesothelioma cells. Cell cycle analysis on MSTO-211H showed an increase of the sub-G1 fraction, which is an index of apoptosis. On the other hand, in NCI-H2452, a decrease in the S phase accompanied by an increased G2-M fraction suggested a G2 block of the cell cycle and this was confirmed by the fact that there was only a slight increase of apoptosis in these cells (Fig. 1, right).

Subcutaneous tumor growth of MSTO-211H cells is reduced by the combination of i.p. administered piroxicam plus CDDP. Figure 2 illustrates the in vivo growth in volume of mesotheliomas induced by s.c. injection of 2.5 x 106 MSTO-211H cells between the scapulae of nude mice. Tumor growth progressed in all experimental groups. In the control group, tumors reached large volumes over a 25-day period (3.03 ± 0.61 mm3). Monotherapy with piroxicam failed to significantly slow the tumor progression, allowing for tumor growth at 25 days to 2.10 ± 0.44 mm3. At the end of the 25-day follow-up period, CDDP exerted a significant reduction in the volume of the mesothelioma xenografts compared with the control group (on average, 1.77 ± 0.31 mm3), which is nearly 50% smaller, with P < 0.04. An even greater growth inhibition was obtained by the combination of the two treatments (piroxicam plus CDDP: 1.19 ± 0.17 mm3 at 25 days), with P < 0.007. The average tumor volume in this group was already significantly smaller also at 20 days (P < 0.04). To note, all mice assigned to the various treatments groups showed the same range of weight (data not shown), providing no immediate evidence for overt toxic effect of the two drugs.


Figure 2
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  		Fig. 2. Effect of piroxicam and CDDP on s.c. tumor growth. Mice were inoculated s.c. with MSTO-211H cells 1 week before starting treatment with piroxicam, CDDP, or the combination and then they were calipered (tumor volumes were expressed in mm3) for 25 days. *, P < 0.04 versus untreated; bullet, P = 0.007 versus untreated.

 
Piroxicam plus CDDP diminish mesothelioma growth in vivo via their proapoptotic action. Histopathologic and immunohistochemical analyses of the explanted mesothelioma xenografts were done to characterize features that could help explain the enhanced antitumor effect attained by piroxicam/CDDP combination therapy. Microscopic examination by H&E and hematoxylin-Van Gieson stainings of the xenografts revealed no significant differences in the number and size of vessels in the different treated animals (data not shown). This was confirmed by immunohistochemical staining of lesions with endothelial markers, such as CD31 and FVIII-RA, which gave similar results (data not shown). Therefore, a reduction in tumor vascularity was not an apparent mechanism for the observed benefits of dual therapy. However, TUNEL staining (Fig. 3 ) showed that the number of apoptotic cells was significantly different in the four group of tumors. Interestingly, treatment with piroxicam alone caused only a small increase in apoptotic cells, with respect to the control. A representative pair of tissue sections illustrating this increase in apoptosis in the piroxicam/CDDP combination therapy group compared with CDDP alone is shown in Fig. 3.


Figure 3
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  		Fig. 3. TUNEL staining of s.c. tumors. Right, typical staining of sections obtained from CDDP-treated tumor (C) and from CDDP + piroxicam–treated tumor (C+P); left, U–Mann-Whitney test was done on TUNEL-positive cells to assess relationship between data. *, P < 0.0001 versus untreated; bullet, P < 0.0001 versus piroxicam.

 
Piroxicam increases survival of CDDP-treated mice with orthotopic mesothelioma. Figure 4 presents Kaplan-Meier survival curves for the four experimental groups in mice in which orthotopic mesotheliomas were induced via i.p. injection of 2.5 x 106 MSTO 211H tumor cells. The analysis shows that the group receiving combination therapy i.p. had again the best response, attaining twice as long a survival time (P < 0.0001). Consistent with the previous sets of experiments, animals treated with piroxicam did not achieve better survival time than controls. Of note, comparable with what is seen in humans, mice experienced tumor cell growth along the serosal surfaces of the peritoneum, leading to the formation of small nodules and local inspissations that ultimately induced the formation of a large plaque similar to the mesothelial cuirass described in terminal patients (not shown). Also, in this case, toxic effects of the drugs were not evidenced (data not shown).


Figure 4
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  		Fig. 4. Effect of piroxicam and CDDP on survival of mice with orthotopic MM. Mice were inoculated i.p. with MSTO-211H cells 1 week before starting treatment with piroxicam, CDDP, or the combination. P < 0.0001 versus untreated.

 
Effect of CDDP and of piroxicam plus CDDP on proliferation of MSTO-211H cells. To confirm that the effect of piroxicam plus CDDP combination is the result of a specific action on cell proliferation, we treated MSTO-211H cells with various concentrations of CDDP ranging from 0.1 to 10 µg/mL alone, or in combination with piroxicam with concentration that resulted in 25% and 50% inhibition of cell proliferation. IP50 was 7.8 x 10–4 mol/L for piroxicam and 4.2 µg/mL for CDDP and IP25 was 3.1 x 10–4 mol/L for piroxicam and 1.5 µg/mL for CDDP. The results show the dose-dependent response of cell to CDDP (Fig. 5A ) and the synergistic effect of the two drugs (Fig. 5B). For example, the concentrations of piroxicam and CDDP, administered to give a proliferation inhibition of 25%, when combined, resulted in 42% inhibition of proliferation. A further increase of piroxicam concentration (from IP25 to IP50), maintaining CDDP at IP25 dose, produced a greater inhibition (54%). Interestingly, this inhibition is quite similar to that achieved increasing only CDDP dose.


Figure 5
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  		Fig. 5. Effect of CDDP and of piroxicam plus CDDP on cell proliferation of MSTO-211H. A, increasing concentrations of CDDP (from 0.01 to 10 µg/mL) reduce mesothelioma cell proliferation expressed as percentage of control. B, effect of different doses of CDDP and piroxicam administered alone or in combination on cell proliferation. Percentage indicates the inhibitory concentration for cell proliferation used. *, P < 0.0001 versus control; +, P < 0.0001 versus control, P50%, C50%, C25%, P25% + C25%, P50% + C25%; #, P < 0.0001 versus control, P25%, C25%.

 
COX-2 and prostaglandin E2 protein expression levels in the MSTO-211H cell line. Because piroxicam is a COX inhibitor and COX-2 is normally overexpressed in many tumor cell lines, we assessed COX-2 protein levels in MSTO-211H cells. As shown in Fig. 6 , MSTO-211H expressed very low level of COX-2. As positive control, we used COX-2 ovine standard and human prostate cancer cell line (PC3) lysate that express COX-2 (31).


Figure 6
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  		Fig. 6. COX-2 level in MSTO-211H cell line and in proteic extract of MSTO-211H–derived tumor. Ovine COX-2 standard and PC-3 (human prostate cancer cell line) lysate was used as positive control.

 
The low expression of COX-2 was further confirmed by the lack of detectable levels of prostaglandin E2 in cell medium analyzed (detection limit for the used kit was 8 to 10 pg/mL; data not shown).

GeneChip array analysis of piroxicam treatment in vitro of MSTO-211H. To better characterize at the transcriptional level the effects of piroxicam treatment in vitro on MSTO-211H cells, transcriptomic GeneChip array analyses were done. Two prototypic situations were investigated: control, untreated MSTO-211H cells, and treated, MSTO-211H cells treated with 780 µmol/L piroxicam for 48 hours. The quality of the total RNA extracted was assayed by Bioanalyzer (Agilent Technologies, Palo Alto, CA). Three biological replicas were generated for all prototypic situations. These replicas were used to synthesize biotinylated cRNAs for hybridization on six HGU133plus two arrays (Affymetrix), containing 54,675 probe sets associated with 46,241 EntrezGene identifiers. To remove nonsignificant probe sets (i.e., not expressed and those not changing), we applied an interquantile range filter yielding 11,926 probe sets. We used a linear model analysis (37) to explain known sources of variation and test the hypothesis that there is a change of expression of a gene between control versus treated. Linear model analysis indicated that using a minimum change criterion of |log2(fc)|≥1 and a false-discovery rate criterion of P ≤ 0.05, 3,405 probe sets were differentially expressed. These probe sets correspond to 2,583 unique expressed genes. Of these, 1,897 genes were up-regulated and 730 genes were down-regulated.

GO provided a restricted vocabulary as well as clear indications of the relationships between biological terms and genes (40). The GO consortium produces three independent ontologies for gene products. The three ontologies form the basis for the description of the molecular function, biological process, and cellular component of gene products. Having partitioned the genes into up- and down-modulated upon treatment with piroxicam, we investigated whether these gene groups were associated with common functions or processes using the GO annotations. Overrepresented GO classes were identified by means of the hypergeometric distribution (40). Concerning the analysis of the biological process ontology, we observed that the main GO classes found enriched in the down-regulation group were related to the RNA processing apparatus, as the up-regulated genes were instead mainly associated with metabolic pathways (Tables 1 and 2).

To identify patterns of gene expression associated with the inhibition of proliferation levels and the molecular action of the piroxicam, a transcriptomic GeneChip array analysis was done. The gene list obtained from the comparison between control and treated cells were filtered to exclude genes that were underexpressed or overexpressed at low levels. To extract a set of biologically interesting genes, we analyzed the GO classes with P < 0.001. The choice was done looking mainly to genes that could be possibly related to the phenotypic effect resulting from piroxicam treatment. A subset of genes from this list was selected for functional validation. Quantitative reverse transcription-PCR (RT-PCR) was done on the selected group of up- and down-regulated genes identified by GeneChip array analysis, using specific primers (Table 3).

As shown in Table 4 , the different expression of the great majority of the genes analyzed was confirmed by quantitative RT-PCR in MSTO-211H cells.


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  		Table 4. Quantitative RT-PCR validation of differential gene expression detected by GeneChip array analysis on MSTO-211H cells

 
HtrA1 and EGFR are two targets of the piroxicam antitumoral activity in vivo. To analyze the in vivo effects of piroxicam treatment, we determined the differential expression of two genes, HtrA1 (up-regulated in vitro by piroxicam) and EGFR (down-regulated in vitro by piroxicam), at the protein level by immunohistochemistry in the xenografts. The differential expression revealed by in vitro GeneChip array analysis were validated at protein level in vivo for both HtrA1 and EGFR. An evident up-regulation of HtrA1 was confirmed in tumors grown s.c., compared with untreated controls (Fig. 7A, left ). To note, CDDP alone did not provoke a significant up-regulation of HtrA1; consequently, the combination of piroxicam plus CDDP did not show an effect superior to the action of piroxicam alone for the expression of HtrA1. Alternatively, a strong EGFR down-regulation was registered in tumors grown s.c., compared with untreated controls (Fig. 7B, right). In this case, CDDP alone was not able to significantly down-regulate EGFR with respect to the control; therefore, the combination of piroxicam plus CDDP displayed a significant down-regulation of EGFR with respect to the effect of piroxicam alone. To note, an even higher difference in expression with respect to controls were observed in ortothopic tumors (data not shown).


Figure 7
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  		Fig. 7. Staining for HtrA1 (A) and EGFR (B) of tumor explants. Above, U–Mann-Whitney test done on positive cells to assess relationship between data. *, P < 0.0001 versus untreated; bullet, P < 0.0001 versus CDDP; #, P = 0.005 versus piroxicam. Below, typical staining of sections obtained from untreated and piroxicam-treated tumors.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The objective of our study was to examine the chemopreventive efficacy of the NSAID piroxicam in experimental models of MM. The rationale for examining the potential efficacy of piroxicam as a MM chemopreventive agent was based on several lines of evidence. First, recent data suggest that piroxicam can prevent or reduce the development of various human tumors (41). Second, although virtually every chemotherapeutic agents has been tested in MM patients, few drugs have consistently yielded response rate >20% (4). Third, based on the observation that COX-2 overexpression in MM is a strong prognostic factor in MM, which contributes independently to the other clinical and histopathologic factors in determining a short survival (10), NSAIDs with COX-2 inhibitory activity are becoming more and more appealing as potential therapeutic tools in clinical cancer research.

As a first line strategy, we did an in vitro study to evaluate the effects of piroxicam in two mesothelioma cell lines. Our data suggest that piroxicam has both antiproliferative and proapoptotic effects depending on the cell lines used, a finding that is consistent with data from the literature showing that piroxicam may target multiple component of the molecular machinery regulating cell cycle, also independently of its COX inhibitor activity (42, 43). Interestingly, the flow cytometry analysis of the treated cells revealed that the effects on cell proliferation in the two cell lines were achieved in two different ways: mostly by a proapoptotic action on MSTO-211H cells and by a G2 block of the cell cycle in NCI-H2452.

We then designed an in vivo animal trial of piroxicam alone and in association with CDDP, a chemotherapeutic agent routinely used for MM treatment. We used the MSTO-211H cells that are tumorigenic in nude mice using two different models. In the first model, cells were s.c. inoculated and growth of these xenografts was measured. In the second model, cells were injected i.p. Cells grew along the serosal surfaces forming a large plaque similar to the mesothelial cuirass described in terminal patients. For in vivo trials, the i.p. rather than the oral administration route was chosen to achieve a more homogeneous treatment. It is well known that using a medicated chow or administering a drug by gavage may lead to less uniform daily doses. The dose of piroxicam was mediated from studies in humans and dogs (42). Consistent with our working hypothesis, the s.c. study evidenced a higher inhibitory capacity of the combination of piroxicam and CDDP over piroxicam and CDDP as single agents. This beneficial effect of combination therapy was confirmed in the ortothopic model of MM, in which a prolonged survival of mice treated with the combination therapy was achieved compared with the effect of CDDP alone. Interestingly, piroxicam alone had no significant effects either on tumor growth and animal survival with respect to controls.

This synergistic effect of the combination of piroxicam and CDDP was then also confirmed in vitro on MSTO-211H cells, where piroxicam and CDDP, administered to give a proliferation inhibition of 25%, resulted in a 42% inhibition of proliferation when combined.

Finally, based on the fact that in MSTO-211H cells, the level of COX-2 is very low and prostaglandin E2 is undetectable, we assumed that piroxicam in these cells exerts its antiproliferative activity via COX/prostaglandin E2–independent mechanisms. These data confirm recent reports that some of the antiproliferative and antineoplastic effects of NSAIDs are independent of the inhibition of COX enzymes (43).

We took advantage of the Affymetrix technology to dissect further the molecular targets of this drug in mesothelioma cells. To the best of our knowledge, this is the first report on the effects of piroxicam on tumor cells by the use of GeneChip array. Interestingly, in the down-regulation cluster, the GO analysis found enriched genes related to the RNA-processing apparatus, whereas the up-regulated genes were mainly associated with metabolic pathways. We validated the expression of a subset of both up- and down-regulated genes by quantitative RT-PCR (see Table 4). Of note, most of the up-regulated genes selected have been shown to have an antiproliferative effect on tumor cells. Among them, BTG2 is one of five members of a newly identified family of antiproliferative genes. BTG2 was first described as an immediate early gene whose expression is induced in response to mitogenic as well as differentiative and antiproliferative factors (44). COX17, a cytochrome c oxidase assembly protein, has been proposed as therapeutic target in lung cancer (45). The fragile histidine triad (FHIT) gene, a frequent target of deletions in lung cancer, and PMS2, a mismatch repair protein, have been shown to enhance paclitaxel- and CDDP-induced apoptosis in cancer cells (46, 47). Finally, the human HtrA1, a member of the HtrA family of serine proteases, has been characterized for its effects as a tumor suppressor–like protein (48). Interestingly, recent data from our research group show that HtrA1 acts as an endogenous modulator of CDDP-induced cytotoxicity (49). Conversely, EGFR is among the down-regulated genes. Overexpression of EGFR is commonly detected in MM and at least one phase II trial has evaluated the efficacy of ZD1839 (Iressa, AstraZeneca, Basiglio, Italy) in MM patients (50). The fact that, at least for HtrA1 and EGFR, these different modulations were confirmed also in vivo on the experimental tumors, further strengthens the biological significance of these data. Although EGFR was initially identified using microarray approach and it is clearly modulated at protein level as shown by the immunohistochemistry, we could not validate it using quantitative RT-PCR with probes mapping into the translated region. This discrepancy between microarray and quantitative RT-PCR could be related to the different location of the GeneChip detection probes that are placed in the 3' end nontranslated region of the EGFR.

Piroxicam is a widely used, well-tolerated, easily administrable medication that could be readily associated not only to CDDP but also to a broad spectrum of chemotherapy and immunotherapy agents in multiagent protocols for mesothelioma. In particular, piroxicam has already been shown to exert a synergistic effect with CDDP in different in vitro and in vivo models of spontaneous tumors in companion animals (51). Our lines of evidence concur to show that piroxicam exerts its growth-inhibitory functions through multiple pathways. Moreover, they provide a rationale for the synergistic effects seen when using piroxicam in combination with CDDP. Our findings make it plausible to hypothesize that piroxicam sensitize mesothelioma cells to the CDDP-induced cytotoxicity by up-regulating the expression of endogenous effectors of this drug, such as HtrA1 or FHIT, and down-regulating important regulators of cell growth, such as EGFR.

An important caveat is that this protocol was ineffective at controlling bulky neoplasms in our mice; however, our animals received only pulsed therapy. It is conceivable that, by adopting this scheme in a dose-intensity protocol, oncologists might be able to achieve extended tumor control, especially considering that this approach could be associated to aggressive pleurectomy. Because the best results have been achieved with the combination of piroxicam and CDDP, it is also possible that addition of other drugs to the treatment regimen may help achieve an additional increase in the antitumor efficacy of this therapy.

In conclusion, our findings reveal novel, previously unappreciated synergistic effects of piroxicam with an established antineoplastic agent, CDDP, in a model of MM, the current prognosis of which remains very unfavorable. Formal verification of these findings in a randomized clinical trial setting seems, therefore, warranted, even if the cardiac risks associated with COX inhibitors should be considered.


   Acknowledgments
 
We thank Drs. A. Abbate (University of Virginia, Charlottesville, VA) and A. Iannaccone (University of Tennessee, Knoxville, TN) for critical reading of the manuscript, and Istituto di Medicine Sociale for its continuous support.


   Footnotes
 
Grant support: Associazione Italiana Ricerca sul Cancro, SUN, and FUTURA-onlus (A. Baldi); grant 2005 of the Italian Ministry of Health (E.P. Spugnini and G. Cortese); PRIN 2004055579_003, the French-Italian Vinci program, the Association Francaise pour la Recherche contre le Cancer, and the European Community (EPITRON contract no. 518417; L. Altucci).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: E.P. Spugnini and I. Cardillo contributed equally to this work.

Received 5/ 1/06; revised 7/10/06; accepted 7/25/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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