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Ann Thorac Surg 2002;74:1648-1652
© 2002 The Society of Thoracic Surgeons

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Original article: general thoracic

The role of COX-2 inhibitors in lung cancer

^a Department of Cardiothoracic Surgery, Cork University Hospital, Wilton, Cork, Ireland
^b Department of Academic Surgery, Cork University Hospital and University College Cork, Wilton, Cork, Ireland
^c Department of Surgery, Cork University Hospital and University College Cork, Wilton, Cork, Republic of Ireland

Accepted for publication June 28, 2002.

* Address reprint requests to Dr Redmond, Department of Academic Surgery, Cork University Hospital, Wilton, Cork, Republic of Ireland
e-mail: redmondhp{at}shb.ie

	Abstract

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BACKGROUND: Human lung cancer is a major cause of death worldwide with few known effective therapeutic modalities. The isoenzyme cyclooxygenase 2 (COX-2) is an inducible inflammatory enzyme with increased activity evidenced in lung carcinoma. The objective was to determine the effect of a selective COX-2 inhibitor on proliferation and apoptosis rates in the Lewis lung (3LL) tumor cell line in vitro.

METHODS: First, 1 x 10⁴ 3LL cells were plated in a 96-well plate. Cells were incubated for 24 hours with either a control or increasing doses of rofecoxib (0.1 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1.0 mmol/L, and 2.5 mmol/L) in complete Dulbecco’s Modified Eagle’s Medium culture medium. Cell proliferation was measured using BrdU enzyme-linked immunosorbent assay. Next, 1 x 10⁶ 3LL cells were similarly treated. Cells were permeabilized, immunostained with propidium iodide and apoptotic rates were measured using flow cytometry. Then, 5 x 10⁴ cells were plated on a 24-well plate. Cells were incubated for 24 hours with either control or increasing doses of rofecoxib (0.1 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1.0 mmol/L, and 2.5 mmol/L) in complete culture medium. Supernatant was collected and lactate dehydrogenase was measured for cell necrosis using a cytotoxicity detection kit.

RESULTS: The selective COX-2 inhibitor rofecoxib resulted in a dose-dependent increase in apoptosis and dose and time-dependent growth inhibition in cell proliferation. However, rofecoxib did not cause cell necrosis.

CONCLUSIONS: There was a significant decrease in proliferation and increase in apoptosis of 3LL tumor cells when treated with the highly selective COX-2 inhibitor rofecoxib. COX-2 inhibitors may have a potential role to play in the treatment of lung carcinoma.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Human lung cancer is a major cause of death worldwide. The Irish National Central Statistics Office reports lung carcinoma to be the leading cause of cancer death in Ireland, with figures comparable with those in the United States (incidence 20.8% of all cancer-related deaths). Despite the availability of various therapeutic modalities, mortality is high. Although sequential high resolution computed tomography has been recommended by the Early Lung Cancer Action Project [1] there is as of yet no internationally recognized effective screening program. Also its natural history encourages early spread of the disease, with late diagnosis lending to low cure rates. Therefore there is a need to develop new, minimally toxic therapeutic agents for the definitive treatment of lung cancer. These agents may provide an adjunct to the palliative care of inoperable elderly patients with metastatic disease as well as a possible form of chemoprevention in high-risk patients, namely, heavy smokers, elderly persons with a previous history of smoking, and patients with premalignant conditions of the lung.

Inflammatory cytokines, eicosanoids, and growth factors are thought to play a critical role in initiation and maintenance of cancer cell survival and growth [2]. One of these mediators, prostaglandin E₂ (PGE₂), is produced in large quantities by different tumors [3]. PGE₂ is produced from arachidonic acid with the help of cyclooxygenase (COX) enzyme. COX catalyzes the synthesis of PGH₂ from arachiodonic acid and the microsomal form of human prostaglandin E synthase (mPGES), an inducible enzyme, converts the PGH₂ to PGE₂ [4]. It is found in the form of two different types of isoenzymes, COX-1 and COX-2. The former is constitutively expressed in most tissues whereas the latter is primarily induced in inflammatory cells and in human tumors by cytokines, growth factors, oncogenes, and tumor promoters [5]. Increased COX-2 expression has been found in tumor cells of various cancers such as breast, colon, prostate, and lung cancer [6, 7]. COX-2 overexpression was found not only in lung cancer but also in premalignant conditions of the lung such as atypical adenomatous hyperplasia and cuboidal cell hyperplasia [8].

Both isoenzymes can be inhibited by classic nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and indomethacin. Unfortunately the prolonged use of NSAIDs can result in gastrointestinal toxicity that ranges from mild dyspepsia to bleeding, perforation, and even death. This gastrointestinal toxicity of NSAIDS results from a nonspecific inhibition of the cyclooxygenase enzyme. COX-1 synthesizes prostacyclin that is cytoprotective for the gastric mucosa [9]. Such tissue specificity identifies the importance of selective drug therapy. An agent that inhibits the cyclooxygenase enzyme obliterates such side effects as gut toxicity [10]. It has been shown that inhibition of COX-2 in colonic cancers has yielded successful results in decreasing the rate of death when used as a chemopreventive agent (in animal models) by both decreasing tumor growth and metastasis [11]. Several population-based studies have shown a 40% to 50% decrease of relative risk for colorectal cancer in humans who regularly use aspirin and other NSAIDS [12].

COX-2 overexpression promotes and maintains tumor growth by increasing resistance to the apoptosis signal [13], inducing angiogenesis [14], and reducing the immune body response against the tumor [15]. COX-2 inhibitors have shown their antitumor effects by increasing apoptosis [13], antiangiogenic activities [14], and restoring the host immune system [15]. When used as a form of adjunct therapy, COX-2 inhibitors also increased the sensitivity of tumor cells to other anticancer agents and radiation [16]. This synergy promotes its use in combined regimes. Familial adenomatous polyposis (FAP) is a genetic premalignant disease of the colon. Blockade of COX-2 either by gene deletion or by pharmacologic inhibition of enzyme activity suppresses intestinal polyp formation [17]. COX-2 inhibitor has been licensed in the United States for treating FAP.

In the present study we determined the antitumorigenic effects after specific pharmacologic inhibition of COX-2 in a murine Lewis lung (3LL) cells. We investigated whether the highly selective COX-2 inhibitor rofecoxib can inhibit proliferation of cancer cells and induce apoptosis. We report here that rofecoxib induced a significant dose-dependent increase in apoptosis and dose- and time-dependent growth inhibition in cell proliferation.

	Material and methods

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Cell culture
The murine Lewis lung (3LL) carcinoma cell line was a generous gift from Dr. Alan Alfieri (Albert Einstein College of Medicine, Department of Pathology, New York, New York). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) culture medium supplemented with 10% fetal calf serum (FCS), penicillin (100 U/mL), streptomycin sulfate (100 µg/ml), and 2.0 mmol/L glutamine. Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere and subcultured by trypsinization with 0.05% trypsin-0.02% ethylenediamine tetraacetic acid (EDTA) when cells became confluent.

Agents
Rofecoxib was purchased from Merck & Co (Whitehouse Station, NJ). Dimethylsulfoxide (DMSO) was used as a solvent and concentrated drug stocks were diluted in DMEM medium before addition to cell cultures. The DMSO concentration in all cultures was 0.01%. Propidium iodide was purchased from Sigma Chemicals (St. Louis, MO).

In vitro proliferation assay
The cell proliferation enzyme-linked immunosorbent assay (ELISA), BrdU kit (Roche Molecular Biochemicals, Mannheim, Germany) was used to determine cell proliferation. This colorimetric immunoassay is designed to quantitate cell proliferation based on the measurement of BrdU (5-bromo-2'-deoxyuridine) incorporation during DNA synthesis in proliferating cells. Cells were plated at 1 x 10⁴ cells per well in a 100 µL volume in 96-well plates (MTP) and incubated at 37°C in a humidified 5% CO₂ atmosphere for 6 hours. All supernatants were removed and fresh medium (DMEM) was added as a control along with various doses of rofecoxib as the test substance. The various concentrations of rofecoxib used were 50, 100, 200, 500, and 1,000 mol/L; 1.25, 2.5, 5, 10, and 20 µmol/L; and 0.1, 0.25, 0.5, 1.0, & 2.5 mmol/L. Cells were incubated at 37°C in a humidified 5% CO₂ atmosphere for 24 hours. Cells were labeled with BrdU labeling solution as indicated at 14 hours and cultured for further 10 hours. After 24 hours incubation at 37°C, BrdU assay was conducted and the absorbance of the sample was measured at 450 nm in an ELISA reader. The 3LL cells were also treated with rofecoxib for different time periods including 12, 18, 24, and 72 hours. Results are expressed as the mean ± standard deviation.

Apoptosis determination
Aliquots of 0.25 mL of 3LL cells (5 x 10⁵ cells per tube) in 17 x 100 mm polypropylene tubes (Falcon, Lincoln Park, NJ) were treated with 0.25 mL volume of fresh medium (DMEM) as a control or 0.1, 0.25, 0.5, 1.0, and 2.5 mmol/L of rofecoxib in DMEM medium. Apoptosis in 3LL cells was measured according to the percentage of cells with hypodiploid DNA by propidium iodide staining using the technique describe by Wang and associates [18]. After 24 hours incubation, cell suspensions were transferred into flow tubes, centrifuged at 300g for 5 minutes and supernatants were removed. Cell pellets were gently resuspended in 0.5 mL flourochrome solution (50 µg/mL propidium iodide, 3.4 mmol/L sodium citrate, 1 mmol/L Tris, 0.1 mmol/L EDTA, 0.1% Triton X-100) and incubated at 4°C for 6 hours in the dark before they were analyzed by FACScan Flow Cytometry (Becton Dickinson, Mountain View, CA). The forward scatter and side scatter of 3LL particles were simultaneously measured. The PI fluorescence of individual nuclei with an acquisition of fluorescence channel (FL)-2 was plotted against forward scatter, and the data were registered on logarithmic scale. The minimum number of 10,000 events collected and analyzed on software Lysis II. Apoptotic nuclei were distinguished by their diploid DNA content of normal 3LL cell nuclei. The 3LL debris was excluded from analysis by raising the forward threshold. All measurements were performed at the same instruments settings.

Necrosis
A cytotoxicity detection kit (Roche Molecular Biochemicals, Mannheim, Germany) was used to determine cell death caused by necrosis. This colorimetric assay is conducted to quantitate cytotoxicity/cytolysis based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the supernatant. Cells were plated at 5 x 10⁴ cells/well in a 0.5 mL volume in 24-well plates and incubated at 37°C in a humidified 5% CO₂ atmosphere for 6 hours. All supernatants were removed and fresh medium (DMEM) was added as a control or 0.1, 0.25, 0.5, 1.0, and 2.5 mmol/L of rofecoxib in DMEM medium and incubated at 37°C in a humidified 5% CO₂ atmosphere for 24 hours. After 24 hours incubation the supernatants were collected and transferred to Enttdof tubes (SARSTEDT, Germany). These were spun at 500g for 10 minutes at 4°C and supernatant were transferred to new tubes. A volume of 100 µL per well of supernatant was obtained in a 96-well plate, 100 µL of reaction mixture was added to each well and incubated for 30 minutes at room temperature in the dark. After 30 minutes incubation the absorbance of the samples was measured at 490 nm using microtiter plate (ELISA) reader.

Statistical analysis
Results are presented as the mean ± standard deviation. Statistical analysis was performed using analysis of variance (ANOVA). Statistical significance was accepted at a p value less than 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Treatment with rofecoxib causes a dose- and time-dependent growth inhibition in cell proliferation. There was no inhibitory effect on 3LL cells when treated with low-dose rofecoxib (50, 100, 200, 500, and 1000 mol/L; and 1.25, 2.5, 5.0, 10, and 20 µmol/L; data not shown) for 24 hours. However, increasing doses of the COX-2 inhibitor resulted in a significant dose-dependent decrease in cell proliferation; cell growth was 66% at 1 mmol/L rofecoxib, 62% at 0.25 mmol/L, 56% at 0.5 mmol/L, 53% at 1.0 mmol/L, and only 25% at 2.5 mmol/L when compared with vehicle control. Its inhibitory effect ranged from 33% to 74% (Fig 1).

View larger version (14K): [in this window] [in a new window]   Fig 1. Rofecoxib dose-dependently inhibits growth of Lewis lung cancer (3LL) cancer cells in vitro. Cell proliferation in 3LL cells is shown after treatment with various concentrations of rofecoxib for 24 hours at 37°C in a humidified 5% CO2 atmosphere. Cell proliferation was assessed as described in Material and Methods. Data are expressed as the mean ± SD and are representative of six separate experiments. Each experiment was carried out in triplicate. The statistical significance was compared with control, *p < 0.05. Diamonds indicate cell proliferation; squares indicate growth inhibition. (CONT = control.)

View larger version (20K): [in this window] [in a new window]   Fig 2. Rofecoxib time-dependently inhibits cellular proliferation. Cell proliferation in Lewis lung cancer cells is shown after treatment with various concentrations of rofecoxib for different time periods at 37°C in a humidified 5% CO2 atmosphere. Cell proliferation was assessed as described in Material and Methods. Data are expressed as the mean ± SD and are representative of six separate experiments. Each experiment was carried out in triplicate. The statistical significance was compared with control, *p < 0.05. Diamonds = 12 hours; squares = 18 hours; triangles = 24 hours; X = 72 hours.

View larger version (29K): [in this window] [in a new window]   Fig 3. Rofecoxib induces dose-dependent apoptosis. Induction of apoptosis in Lewis lung cancer cells is shown after treatment with various concentrations of rofecoxib for 24 hours at 37°C in a humidified 5% CO2 atmosphere. Cell apoptosis was assessed according to the percent of the cells with hypodiploid DNA by flow cytometry as described in Material and Methods. Data are expressed as the mean ± SD and are representative of six separate experiments. Each experiment was carried out in triplicate. The statistical significance was compared with control, *p < 0.05. Shaded bars indicate percent apoptosis.

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Arachidonic acid is metabolized to give prostaglandins, prostacyclins, and thromboxanes by cyclooxygenase. Two forms of the enzyme, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), have been identified as the constitutive and inducible forms, respectively [5, 6]. Several epidemiologic studies have reported that inhibitors of cyclooxygenase enzyme such as aspirin and NSAIDs reduce the risk of colorectal, esophagus, stomach, prostate, breast, and lung cancer, and COX-2 was expressed in these cancers [7, 8, 17]. Animal studies support the hypothesis that inhibition of COX-2 is one important target for NSAIDs in the treatment and prevention of cancer, which may mechanistically be facilitated by activation of host immune function, promotion of tumor cell apoptosis, and inhibition of angiogenesis [13–15]. Nonspecific inhibition of the enzyme produces mild to severe gastrointestinal toxicity that could outweigh the benefits of its cancer-preventive effect [11].

Various studies have shown increased expression of COX-2 in different cancers including lung cancer [6, 7]. The inhibition of this enzyme in colonic cancer has reduced the rate of tumor growth and metastasis in vitro & in vivo (animal model) when used as a therapeutic and chemo-preventive agent [11]. With an increase in the incidence of lung cancer and diagnosis occurring primarily at inoperable stage, there is a need to develop a therapeutic agent with greater potency and lesser toxicity. Cyclooxygenase inhibitors have this potential either alone or in combination with other therapeutic modalities [16].

On the basis of these studies, we tried to determine the effects of the highly selective COX-2 inhibitor rofecoxib on lung cancer. Initially we used the murine Lewis lung carcinoma cell line to assess in vitro any inhibitory effects of COX-2 inhibitor on tumor growth. Results revealed a reduction in tumor cell proliferation and an increase in apoptosis. These initial results favor our hypothesis about the role of COX-2 inhibitors in lung cancer. To validate these results, we are further studying the effects of cyclooxygenase inhibition on various human lung cancer cell lines in vitro and in vivo (animal model). Recent studies have shown overexpression of the COX-2 enzyme in nonsmall cell lung carcinomas, metastatic lung cancer, and premalignant conditions of lung [8], therefore these preliminary results could be encouraging for the development of future therapies of the lung cancer. Further studies need to be conducted to validate the role of COX-2 inhibition for therapeutic, chemopreventive, palliative, and adjuvant treatment of lung cancer.

In summary, rofecoxib can significantly attenuate growth of murine Lewis lung carcinoma cells (3LL) in vitro. It causes considerable dose- and time-dependent inhibition of proliferation in cancerous cells and similarly induces dose-dependent apoptosis. There is no compromise in cell viability because of necrosis. It is also now identified that at concentrations 0.1 mmol/L rofecoxib causes substantial growth inhibition and apoptosis in vitro.

Despite therapeutic efforts lung cancer remains the major cause of cancer-related death in the world [1]. Although immunologic-based therapies have shown some success for other malignancies, lung cancer has been largely unresponsive. The present findings are therefore of potential importance. The recent development of highly selective COX-2 inhibitors like rofecoxib can be expected to have a potential role in the phenotyping and subsequent staging, treatment, and prevention of lung cancer.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
We would like to thank Trendy Wu and S. Blanckson for their technical support.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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