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Carcinogenesis Advance Access originally published online on November 21, 2007
Carcinogenesis 2008 29(2):371-380; doi:10.1093/carcin/bgm265
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Licofelone, a dual COX/5-LOX inhibitor, induces apoptosis in HCA-7 colon cancer cells through the mitochondrial pathway independently from its ability to affect the arachidonic acid cascade

Simona Tavolari, Massimiliano Bonafè1, Marina Marini2, Carla Ferreri3, Giovanna Bartolini, Elisa Brighenti, Sonia Manara3, Vittorio Tomasi, Stefan Laufer4 and Tiziana Guarnieri*

Department of Experimental Evolutionary Biology, University of Bologna, via F. Selmi 3, Bologna 40126, Italy
1 Center for Applied Biomedical Research, Sant'Orsola University Hospital, Bologna 40126, Italy
2 Department of Histology, Embryology and Applied Biology, University of Bologna, Bologna 40126, Italy
3 Institute for the Organic Synthesis and Photoreactivity, Consiglio Nazionale delle Ricerche, Bologna 40129, Italy
4 Department of Pharmaceutical-Medicinal Chemistry, Eberhard Karls Universität Tübingen, Tübingen 72076, Germany

* To whom correspondence should be addressed. Tel: +39 051 2094253; Fax: +39 051 2094286; Email: tiziana.guarnieri{at}unibo.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Nowadays, no data are available concerning the potential use of dual cyclooxygenase (COX)/5-lipoxygenase (LOX) inhibitors as anticancer agents in colon cancer treatment. Here, we report, for the first time, that the dual COX/5-LOX inhibitor licofelone triggers apoptosis in a dose- and time-dependent manner in HCA-7 colon cancer cells. Induction of apoptosis was related to the recruitment of the intrinsic mitochondrial apoptotic pathway, as shown by loss in mitochondrial membrane potential, cytochrome c release, caspase-9 and 3 activation and poly-(ADP-ribose)polymerase-1 cleavage. Moreover, licofelone induced the cleavage of the full-length p21Bax into p18Bax, a more potent inducer of the apoptotic process than the uncleaved form. Pre-treatment of HCA-7 cells with the pan-caspase inhibitor z-VAD-fmk significantly blocked licofelone-induced apoptosis, confirming that this process occurred primarily in a caspase-dependent pathway. We also present evidences that licofelone was able to affect the arachidonic acid (AA) cascade, as it blocked the activity of 5-LOX and COX enzymes, and it induced, through the phosphorylation of cytoplasmic phospholipase A2 (cPLA2), the release of unesterified AA from HCA-7 membrane phospholipids. However, apoptosis induction was not related to the ability of licofelone to affect the AA cascade, since neither exogenous prostaglandin E2 and leukotriene B4 addition, nor pharmacological inhibition of cPLA2, was able to rescue HCA-7 cells from apoptosis. Even if further studies are needed to clarify the mechanism of licofelone-induced apoptosis, this study suggests that this drug, as well as similar dual COX/5-LOX inhibitors, may represent a novel and promising approach in colon cancer treatment.

Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; cPLA2, cytoplasmic phospholipase A2; FFA, free fatty acid; GC, gas chromatography; JNK, c-jun N-terminal kinase; LOX, lipoxygenase; LTB4, leukotriene B4; MAFP, methyl arachidonyl fluorophosphate; MAPK, mitogen-activated protein kinase; NSAID, non-steroidal anti-inflammatory drug; PARP-1, poly-(ADP-ribose)polymerase-1; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; PLA2, phospholipase A2


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Colon cancer is the second leading cause of cancer-related mortality in Western population and its pathogenesis, together with its chemoprevention, are currently area of intense investigation. Recent studies regarding the relationship between arachidonic acid (AA) cascade and carcinogenesis are revealing novel molecular targets for cancer treatment. AA is an essential polyunsaturated fatty acid, typically esterified at the sn-2 position of cellular phospholipids and hydrolyzed by phospholipase A2 (PLA2) enzymes (1,2). Once released, unesterified AA is mainly metabolized by two important classes of enzymes: cyclooxygenases (COXs) and lipoxygenases (LOXs). The involvement of COX-2 and its metabolite prostaglandin E2 (PGE2) in colon carcinogenesis is well documented. A large body of studies indicate that PGE2 can increase the proliferation, the motility and the metastatic potential of tumour cells, can promote tumour angiogenesis, induce local immunosuppression and inhibit apoptosis (3). On the other hand, COX-2 can contribute to colon carcinogenesis through mechanisms that are independent from PGE2 action. COX-2 peroxidase activity can in fact transform many procarcinogens into ultimate carcinogens that, in turn, are able to activate many genes involved in cell proliferation (4). Moreover, through the reduction of the levels of unesterified AA, which has a proapoptotic action (5), COX-2 can promote tumour cell survival.

More recently, an involvement of 5-LOX enzyme and its products, in particular leukotriene B4 (LTB4), in the development and progression of colon cancer, has emerged (6,7). Similarly to COX-2, the expression and activity of 5-LOX have been found to be up-regulated during colon carcinogenesis (8) and closely related to tumour size, depth and vessel invasion (6). Even if the exact mechanism connecting 5-LOX to cancer aetiology remains to be fully elucidated, it seems likely that COX-2 and 5-LOX may represent an integrated system that regulates the proliferative, metastatic and proangiogenic potential of cancer cells. Both enzymes, in fact, induce cell cycle progression and block apoptosis, enhance cancer cells chemoresistance and stimulate angiogenesis, with a convergent targeting on vascular endothelial growth factor expression and release (9). The frequent co-expression of these two enzymes and the striking analogy of their biological functions suggest that molecules which are able to block equally well both COX-2 and 5-LOX pathways, may represent a novel and promising alternative in colon cancer treatment.

To date, various structural families of dual COX/LOX inhibitors have been designed and several compounds are currently undergoing clinical development as anti-inflammatory drugs (10), but no data are available concerning their potential use as anticancer agents in colon cancer treatment. On this basis, we decided to evaluate the possible anti-proliferative effect of the dual COX/5-LOX inhibitor licofelone {[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid, Figure 1A}, in a human colon cancer line expressing both COX and 5-LOX enzymes. Among dual COX/LOX inhibitors, licofelone is the molecule in the most advanced phase of clinical trials (phase III) as anti-inflammatory drug (11), and its safety and efficacy, in comparison with the non-steroidal anti-inflammatory drugs (NSAIDs) naproxen and rofecoxib, are reviewed (12,13). This drug is a potent, competitive and well-balanced inhibitor of the 5-LOX and COX pathways; because of its conformational similarity to AA, licofelone is able, in fact, to bind the active sites of both classes of enzymes, thus blocking their catalytic activity (14).


Figure 1
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  		Fig. 1. Licofelone decreases HCA-7 cells viability in a time- and dose-dependent manner. (A) Chemical structure of licofelone. (B) Western blotting analysis of 5-LOX, COX-2 and COX-1 enzymes in Caco-2, LS174T and HCA-7 human colon cancer cell lines. Representation of three separate experiments with similar findings. (C) Evaluation of HCA-7 cell viability after 24 and 48 h of treatment with licofelone (100–150 µM), naproxen (0.5–1.5 mM) and celecoxib (100–150 µM), determined by trypan blue exclusion assay, as previously described in the Materials and Methods. Results are expressed as percentage mean ± SD of three independent experiments. Asterisks indicate significance, with *P < 0.05 (significant) and **P < 0.001 (very significant). (D) Light microscope examination of HCA-7 cell morphology after 24 h treatment with licofelone 100–150 µM (bar scale, 100 µm).

 
In this paper we show, for the first time, that licofelone is able to trigger apoptosis in HCA-7 colon cancer cells, through the recruitment of the intrinsic mitochondrial apoptotic pathway and independently from its ability to affect the AA cascade. Taken together, these data suggest that this drug, as well as similar dual COX/5-LOX inhibitors, may represent a really interesting therapeutic approach for colon cancer treatment.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Reagents and antisera
Licofelone (Merckle–ratiopharm, GmbH, Ulm, Germany) and celecoxib were a generous gift of Prof. Stefan Laufer (Department of Pharmaceutical-Medicinal Chemistry, Eberhard Karls Universität Tübingen, Germany); naproxen was purchased from Sigma Chemical Co. (Darmstadt, Germany). The pan-caspase inhibitor z-VAD-fmk was from R&D Systems (Minneapolis, MN); methyl arachidonyl fluorophosphate (MAFP), UO126, SB203587 and SP600125 were from Biomol (Plymouth Meeting, PA), whereas bromoenol lactone and p-bromophenacyl bromide were purchased from Sigma Chemical Co. Primary antibody anti-β-actin (1:1000) and anti-caspase-9 (1:300) were from Sigma Chemical Co.; anti-COX-1 (1:250), anti-COX-2 (1:250) and anti-caspase-3 (1:1000) were from Cayman Chemical Company (Ann Arbor, MI); anti-Bcl-2 (1:500) was from Trevigen (Gaithersburg, MD); anti-Bax (Ab-1, 150–165, 1:2000) was from Calbiochem (Darmstadt, Germany); anti-5-LOX (1:250) was from BD Transduction Laboratories (Lexington, KY); anti-poly-(ADP-ribose)polymerase-1 (PARP-1) (1:500) was from Santa Cruz Biotechnology (Santa Cruz, CA); anti-cytoplasmic phospholipase A2 (cPLA2) (1:500) was from Chemicon International (Temecula, CA) and anti-phospho-cPLA2 Ser505 (1:1000) was from Cell Signaling Technology (MA, USA). Monoclonal and polyclonal horseradish peroxidase-conjugated secondary antibodies (1:1000) were from Sigma Chemical Co.

Cells and cell culture
The human colon cancer cell lines LS174T and CACO-2 were a generous gift of Prof. F.Dall'Olio (Department of Experimental Pathology, University of Bologna, Italy), whereas HCA-7 cells were obtained from European Collection of Cell Cultures. All the cell lines were cultured in Dulbecco's modified Eagle's Medium with 4.5 g/l glucose (Cambrex Bioscience, Milan, Italy), supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Cambrex Bioscience), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma Chemical Co.). Cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2 and routinely passaged using trypsin–EDTA 0.025% (Sigma Chemical Co.).

Treatment with drugs
For treatment with licofelone, celecoxib and naproxen, exponentially growing cells were trypsinized and seeded in complete medium for 24 h. At the end of incubation, fresh medium, alone or supplemented with the drugs, was added and completely replaced every 48 h. Naproxen was directly dissolved in culture medium, whereas licofelone and celecoxib were dissolved in dimethyl sulfoxide (Sigma Chemical Co.) and diluted into the medium to obtain the required final concentration before each experiment. To control for a possible effect of dimethyl sulfoxide, control samples were treated in parallel with an equivalent concentration of this solvent.

Cell viability assay
The effect of licofelone, celecoxib and naproxen on HCA-7 cell viability was determined by trypan blue exclusion assay. Briefly, HCA-7 were plated on a six-well plate at the density of 6 x 105 and treated with the drugs for 24 and 48 h, respectively. At the end of incubation, cells were harvested by trypsinization, stained with trypan blue solution (0.04% wt/vol) and counted in a Neubauer haemocytometer chamber (Sigma Chemical Co.). Three plates were assayed for each condition and each experiment was repeated in triplicate.

Nuclear morphological changes
Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by the DNA-binding fluorochrome bis-benzimide stain (Hoechst 33342, Sigma Chemical Co.). Briefly, HCA-7 were seeded on a six-well plate at the density of 6 x 105 and cultured in the presence or absence of licofelone for the indicated times. At the end of incubation, 1 µg/ml Hoechst 33342 was added to each well for 30 min at 37°C in the dark. Cells were then washed with phosphate-buffered saline (PBS) and promptly observed under a fluorescence photomicroscope (Nikon Eclipse 90i, Nikon Instruments Italia, Firenze, Italy).

Flow cytofluorimetric analysis
For the analysis of DNA content, HCA-7 were seeded on a six-well plate at the density of 6 x 105 and then incubated with licofelone for 24 and 48 h, respectively. At the end of incubation, cells were harvested by trypsinization, washed in PBS and incubated at 4°C for 16 h in the DNA-staining solution, containing 0.1% Triton X-100, 0.1% sodium citrate and 50 µg/ml propidium iodide. DNA content analysis was performed by FACSAriaTM cell sorter (Becton Dickinson, Franklin Lakes, NJ).

Mitochondrial membrane potential ({Delta}{psi}m) measurement
Tetramethyl rhodamine methyl ester (Invitrogen–Molecular Probes, Milan, Italy) is a fluorescent probe suitable for cytofluorimetric measurements of changes in {bigtriangleup}{psi}m (15). Briefly, HCA-7 were seeded on a six-well plate at the density of 6 x 105 and treated with licofelone for 12 h. Staining was performed by incubating cells in a 10 µM tetramethyl rhodamine methyl ester solution in culture medium at 37°C for 30 min. Cells were washed and immediately assessed by FACSAriaTM cell sorter (Becton Dickinson). Morphological evaluation of changes in {bigtriangleup}{psi}m was carried out by the cationic fluorochrome JC-1 (Invitrogen–Molecular Probes), as described previously (16). In cells with normal mitochondrial function, JC-1 forms aggregates that fluoresce red; when mitochondrial membrane potential decreases, as occurs when mitochondrial pores are formed during the apoptotic process, JC-1 becomes monomeric and fluoresces green. Briefly, HCA-7 cells were seeded onto ethanol-sterilized glass coverslips in a six-well plate at the density of 6 x 105 and then incubated with licofelone for 12 h. At the end of incubation, cells were stained with 2.5 µg/ml JC-1 for 30 min at 37°C in the dark, washed and promptly viewed under a fluorescence photomicroscope (Nikon Eclipse 90i, Nikon Instruments Italia).

Cytochrome c release from mitochondria
The concentration of cytochrome c inside or outside mitochondria was measured by ELISA (Assay Designs, Ann Arbor, MI). Briefly, HCA-7 were seeded at the density of 7 x 106 in a 75 cm2 flask and treated with licofelone for 12 h. At the end of incubation, cytoplasmatic and mitochondrial fractions were obtained according to the manufacturer's instructions. Fractions were then run in the assay and values are expressed as nanogram per milligram of total protein from each fraction.

Western blotting analysis
Western blotting analysis was carried out as described previously (17). Briefly, cells were seeded in a 25 cm2 flask at the density of 1.5 x 106 and grown in complete medium for 24 h. Cells were then treated with licofelone for the indicated times and, at the end of incubation, scraped from flasks and lysed in a buffer containing 10 mM Tris, 1% sodium dodecyl sulphate, 1 mM Na-orthovanadate and a protease inhibitor cocktail (Roche, Penzberg, Germany). After addition of sodium dodecyl sulphate–polyacrylamide gel electrophoresis buffer and boiling, 50 µg of denaturated proteins were separated in pre-casted gels 4–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA), transferred to nitrocellulose paper and probed with antibodies. Detection was performed by using the enhanced chemiluminescence procedure developed by Amersham Biosciences (Little Chalfont, UK) and band intensities were determined using a densitometric image analysis software (Image Master, Pharmacia Biotech, Uppsala, Sweden). Afterwards, membranes were re-blotted with anti-β-actin antibody for normalization and equal protein loading.

Enzyme immunoassays
Determination of PGE2 and LTB4 levels in HCA-7 culture medium was evaluated by ELISA (Cayman Chemical Company). Briefly, cells were seeded in a 24-well plate at the density of 1.2 x 105 cells per well and exposed to licofelone in serum-free medium for 24 h. The harvested medium was centrifuged at 500g for 5 min (4°C) to remove floating cells and the supernatant was collected and assayed following the customer's instructions. Results are expressed as picogram per millilitre of medium. Determinations were carried out on three similarly treated wells and the medium from each experiment was assayed at three different dilutions.

Release of unesterified AA from HCA-7 membrane phospholipids
For the determination of unesterified AA release from HCA-7 membrane phospholipids, cells were seeded in a 25 cm2 flask at the density of 1.5 x 106, grown in complete medium for 24 h and then treated with licofelone for 24 h. At the end of incubation, membrane pellets were obtained by centrifugation of HCA-7 cells washed twice with PBS. Each sample contained a final concentration of 3 x 106 cells/ml PBS and the experiment was repeated for four times. Phospholipids extraction and transesterification to obtain the fatty acid residues as the corresponding methyl esters were performed, and the analysis of the fatty acid composition was carried out according to the published procedures (18). The evaluation of AA concentration in control and treated samples was carried out by using a commercially available methyl arachidonate standard as the external reference in the gas chromatography (GC) analysis.

Statistical evaluation
Results are expressed as mean or percentage mean ± SD and reproducibility was confirmed at least in three separate experiments. Data were analysed by Student's t-test in Excel program and a P value < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Licofelone decreases HCA-7 viability through the induction of apoptosis
To test the putative anti-proliferative effect of the dual COX/5-LOX inhibitor licofelone, preliminary experiments were carried out in order to select a human colon cancer cell line expressing both COX and 5-LOX enzymes. HCA-7, CACO-2 and LS174T cell lines, which were available in our laboratory, were analysed for this purpose. As shown in Figure 1B, the level of the constitutively expressed COX-1 protein was similar in all the three cell lines tested and the highest amounts of COX-2 and 5-LOX enzymes were observed in HCA-7 cells, which were consequently chosen for further studies. Since this was the first study to assess the possible effect of licofelone on human colon cancer cell viability, increasing concentrations of this drug, ranging from 1 µM to 1 mM, were tested for 24 and 48 h, respectively. As shown in Figure 1C, we observed that licofelone, starting from the dose of 100 µM, significantly decreased HCA-7 cell viability in a time- and dose-dependent manner half maximal inhibitory concentration (IC50) 48 h: 72 ± 3.6 µM. This effect was much more potent than naproxen (IC50 48 h: 1.45 ± 0.07 mM) and similar to that observed for celecoxib (IC50 48 h: 78 ± 3.9 µM). It is worth underlining that, as we found a relevant cytotoxic effect at doses of licofelone >150 µM (data not shown), we decided not to go beyond this dose in our subsequent experiments.

We also observed that licofelone, after 24 h incubation, deeply affected HCA-7 morphology, as treated cells became rounded, detached from the culture flask and showed membrane blebbing (Figure 1D), suggesting that an apoptotic pathway did occur. To test this hypothesis, we performed flow cytofluorimetric analysis that revealed, after 24 and 48 h of incubation, a substantial time- and dose-dependent increase in the hypodiploid (subG1) peak, which represents the fraction of fragmented apoptotic cells (Figure 2A). An increase in typical apoptotic figures, such as unfragmented or irregularly fragmented pycnotic nuclei, was observed in treated cells also upon Hoechst 33342 staining (Figure 2B). To study the possible involvement of caspase activation in licofelone-induced apoptosis, we firstly examined, by western blotting, the cleavage of caspase-3. This enzyme is, in fact, one of the most important executioner of the apoptotic process and its activation, which depends on the proteolytic cleavage of procaspase-3 to a smaller and enzymatically active form, is responsible for the subsequent PARP-1 cleavage (19). As shown in Figure 2C, we found that licofelone 150 µM induced, starting from 16 h treatment onward, a strong and time-dependent cleavage of the 34 kDa proenzyme caspase-3 into its active 17 kDa form. Caspase-3 activation was rapidly followed by PARP-1 cleavage, seen as a typical 89 kDa band, which was almost complete after 24 h of treatment (Figure 2D). These results were in agreement with Hoechst 33342 staining, which displayed typical apoptotic figures not earlier of 16 h of treatment (Figure 2E). To better understand the role of caspase activation in licofelone-induced apoptosis, HCA-7 cells were pre-treated for 2 h with the pan-caspase inhibitor z-VAD-fmk (25 µM) and then co-treated with z-VAD-fmk (25 µM) and licofelone 150 µM for further 24 h. As shown in Figure 2F, treatment of HCA-7 cells with licofelone alone resulted in~53% of apoptosis, whereas co-treatment with z-VAD-fmk resulted only in 10% of apoptotic cells, suggesting that, in our in vitro model, licofelone-induced apoptosis occurred primarily in a caspases-dependent manner.


Figure 2
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  		Fig. 2. Licofelone induces apoptosis in HCA-7 cells through caspases activation. (A) Flow cytofluorimetric analysis of DNA content in HCA-7 cells, after 24 and 48 h incubation with licofelone 100–150 µM, as previously described in the Materials and Methods. (B) Fluorescence microscopy analysis of HCA-7 cells incubated for 24 and 48 h with licofelone 100–150 µM and stained with 1 µg/ml Hoechst 33342, as previously described in the Materials and Methods (bar scale, 20 µm). (C and D) Time course of caspase-3 activation and PARP-1 cleavage, evaluated by western blotting, in HCA-7 cells treated with licofelone 150 µM. Representation of three separate experiments with similar findings. Histograms show full-length and cleaved caspase-3 and PARP-1 levels normalized to the corresponding β-actin level, as previously described in Materials and Methods. Asterisks indicate significance, with *P < 0.05 (significant) and **P < 0.001 (very significant). (E) Fluorescence microscopy analysis of HCA-7 cells treated for the indicated times with licofelone 150 µM and stained with 1 µg/ml Hoechst 33342 (bar scale, 10 µm). (F) Evaluation of the percentage of apoptotic cells in HCA-7 pre-treated for 2 h with the pan-caspase inhibitor z-VAD-fmk (25 µM) and then co-treated with z-VAD-fmk (25 µM) and licofelone 150 µM for further 24 h. Apoptosis was evaluated by flow cytofluorimetric analysis, as previously described in the Materials and Methods. Asterisks indicate significance, with **P < 0.001 (very significant).

 
Licofelone triggers apoptosis through the recruitment of the intrinsic mitochondrial pathway
Next, we were interested in determining the possible mechanism of licofelone-induced apoptosis. Many 5-LOX and COX inhibitors, alone or in association, have been reported to induce apoptosis through the intrinsic mitochondrial pathway (20,21), commonly characterized by early events, such as loss of {bigtriangleup}{psi}m and cytochrome c release from mitochondria to cytoplasm. On this basis, we decided to analyse the status of HCA-7 mitochondria after treatment with licofelone. As shown in Figure 3A (panels a–d), cytofluorimetric analysis of tetramethyl rhodamine methyl ester-stained cells revealed, after 12 h of treatment with licofelone, a dose-dependent loss in {bigtriangleup}{psi}m (~90% at the dose 150 µM). Fluorescence microscopy analysis of JC-1-stained cells confirmed the loss in {bigtriangleup}{psi}m, as shown by the red/green fluorescence shift in treated cells (Figure 3A, panels e–h). It is worth underlining that, at this time, we did not observe any nuclear hallmark of apoptosis, thus suggesting that mitochondrial dysfunction was an event that preceded DNA fragmentation. Loss in {bigtriangleup}{psi}m was associated to a dose-dependent release of cytochrome c from mitochondria to cytoplasm, as shown by its decrease in the mitochondrial fraction and its accumulation in the soluble cytoplasmic fraction after 12 h of treatment with licofelone (Figure 3B). As shown in Figure 4A, caspase-9, which is known to bind to cytochrome c–Apaf-1 complex, was cleaved and activated starting from 12 h of treatment onward, concurrent with the release of cytochrome c from HCA-7 mitochondria. The release of cytochrome c into the cytosol prompted us to analyse the involvement of upstream regulators of mitochondrial membrane perturbations, such as the proapoptotic and anti-apoptotic members of the Bcl-2 family, p21Bax and bcl-2. Accordingly, we evaluated their levels in HCA-7 cells treated for 12 h with increasing doses of licofelone. As shown in Figure 4B, while we did not detect any significant change in bcl-2 expression at all the doses tested, we observed, by using an antibody directed against the 150–165 amino acid sequence of human Bax, a dose-dependent cleavage of the full-length p21Bax into p18Bax fragments in the whole HCA-7 cells lysate. Such cleavage, due to the removal of the 33 N-terminal amino acids during apoptosis, has been shown to be a more potent inducer of the apoptotic process than p21Bax in several cell lines (22).


Figure 3
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  		Fig. 3. Licofelone causes the loss in {bigtriangleup}{psi}m and induces cytochrome c release from mitochondria to cytoplasm in HCA-7 cells. (A) Flow cytofluorimetric (panels a–d) and fluorescence microscopy (panels e and f) analysis of loss in {bigtriangleup}{psi}m in HCA-7 cells, after 12 h of treatment with licofelone 100–150 µM, as previously described in the Materials and Methods (bar scale, 5 µm). (B) Evaluation of cytochrome c level in the cytoplasmic and mitochondrial fraction, in HCA-7 cells treated for 12 h with licofelone 100–150 µM. Results are expressed as mean ± SD of three independent experiments. Asterisks indicate significance, with **P < 0.001 (very significant).

 


Figure 4
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  		Fig. 4. Licofelone induces caspase-9 activation and the cleavage of p21Bax into p18Bax. (A) Time course of caspase-9 activation, evaluated by western blotting, in HCA-7 cells treated with licofelone 150 µM. Representation of three separate experiments with similar findings. Histograms show full-length and cleaved caspase-9 levels normalized to the corresponding β-actin level, as previously described in Materials and Methods. Asterisks indicate significance, with *P < 0.05 (significant) and **P < 0.001 (very significant). (B) Western blotting analysis of bcl-2 and Bax expression in HCA-7 cells treated for 12 h with licofelone 100–150 µM. Note that anti-Bax detected both the full-length p21Bax and the cleaved form p18Bax. Histograms show bcl-2, full-length and cleaved Bax levels, normalized to the corresponding β-actin level, as previously described in the Materials and Methods. Representation of three separate experiments with similar findings. Asterisks indicate significance, with *P < 0.05 (significant) and **P < 0.001 (very significant).

 
Licofelone-induced apoptosis is not related to its ability to affect the AA cascade
As both PGE2 and LTB4 have been shown to promote colon cancer cell proliferation and to act as anti-apoptotic agents (23–25), we first decided to verify whether licofelone-induced apoptosis was related to a reduction of these two AA metabolites in HCA-7 culture medium. As shown in Figure 5A, after 24 h treatment, licofelone reduced in a dose-dependent manner the level of LTB4 (the 5-LOX metabolite) and strongly inhibited PGE2 (the COX metabolite) production at all the doses tested. This effect was not due to a down-regulation of COX and 5-LOX protein levels (data not shown) and confirmed the ability of licofelone to bind the active sites of both classes of enzymes, thus blocking their catalytic activity (14). We next treated HCA-7 cells for 48 h with licofelone 150 µM in association with exogenous PGE2 and LTB4, in a concentration range mimicking their endogenous production. We found that, neither the addition of PGE2 nor LTB4, alone or in association (data not shown), were able to reverse licofelone-induced apoptosis, suggesting that this effect was not related to the ability of this drug to decrease PGE2 and LTB4 levels in HCA-7 culture medium (Figure 5B).


Figure 5
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  		Fig. 5. Licofelone-induced apoptosis is independent from the decrease of LTB4 and PGE2 levels in HCA-7 culture medium. (A) Evaluation of LTB4 and PGE2 levels in HCA-7 culture medium after 24 h treatment with licofelone 100–150 µM. Results are expressed as mean ± SD of three independent experiments. Asterisks indicate significance, with *P < 0.05 (significant) and **P < 0.001 (very significant). (B) Evaluation of the percentage of apoptotic cells in HCA-7 treated for 48 h with licofelone 150 µM, in association with increasing concentrations of exogenous PGE2 or LTB4. Results are expressed as percentage mean ± SD of three independent experiments. Asterisks indicate significance, with **P < 0.001 (very significant).

 
Next, since it has been reported that accumulation of intracellular unesterified AA is a common mechanism that links inhibitors of AA metabolism to induction of apoptosis (5,26,27), we were interested in evaluating whether licofelone could trigger apoptosis by this pathway. In resting cells, the levels of unesterified AA are maintained very low because it is rapidly esterified, typically at the sn-2 position of membrane phospholipids (1). However, following the activation of PLA2 enzymes, phospholipids are hydrolyzed at the sn-2 position and unesterified AA is released from membranes, thus becoming available for the biosynthesis of eicosanoids (28). To evaluate the effect of licofelone on AA release, a different approach from the detection of free fatty acids (FFAs) released in the cellular aqueous medium was followed. FFAs can in fact be identified by GC analysis only after esterification using diazomethane, a toxic and explosive gas not easily obtainable in biochemistry laboratories (29). Moreover, it is well known that FFAs cannot be detected and quantified directly by the GC methodology alone, since large errors in the manipulation steps cannot be avoided. In addition, the concentration of FFAs under normal conditions is very low, sometimes below detection limits, thus making untreated cells not very useful as controls. On this basis, we evaluated the cell membrane compartment and the fatty acid residues present in membrane phospholipids, which can be easily transformed for GC analysis by the transesterification procedure under mild conditions (30). As shown in Figure 6A, we found that, after 24 h of treatment with licofelone, the level of membrane-bound AA was reduced in a dose-dependent manner, indicating that a progressive release of unesterified AA from HCA-7 membrane phospholipids has occurred. Among the several types of PLA2 enzymes, cPLA2 is the most well-characterized isoform and it is widely expressed in colon carcinomas (2,31). It is now recognized that this enzyme, which exhibits a significant selectivity towards phospholipids bearing AA at the sn-2 position, is a central regulator of stimulus-coupled cellular AA mobilization (2). Accordingly, we investigated whether licofelone-induced AA release may be related to cPLA2 activation. As shown in Figure 6B, we found that this drug induced, in a time-dependent manner and without changing total protein level, the phosphorylation of cPLA2 at Ser505, reaching the maximum after 24 h of treatment. On the other hand, licofelone did not elicit any significant effect on secretory PLA2 and inducible PLA2 (data not shown), indicating that, in our experimental model, the release of unesterified AA from HCA-7 membrane phospholipids was related to cPLA2 activity. Next, as cPLA2 phosphorylation can be catalyzed by several mitogen-activated protein kinases (MAPKs) (2), such as extracellular signal-regulated kinase 1/2, p38 and c-jun N-terminal kinase (JNK), we explored the effects of the mitogen-activated protein kinase/extracellular signal regulated kinase inhibitor UO126, the p38 MAPK inhibitor SB203580 and the JNK inhibitor SP600125. As shown in Figure 6C, whereas UO126 was not effective, SB203580 and SP600125 significantly decreased cPLA2 phosphorylation. This effect was increased when they were used in association, thus suggesting, as already shown by Han et al. (32), the concurrent involvement of p38 MAPK and JNK in cPLA2 phosphorylation. Finally, in order to clarify the role of cPLA2 activation in licofelone-induced apoptosis, we treated HCA-7 cells with MAFP, a cPLA2 and iPLA2 inhibitor. As shown in Figure 6D, treatment with MAFP failed to decrease the percentage of apoptotic events in licofelone-treated cells, suggesting that cPLA2 activation and subsequent AA release were not directly involved in licofelone-induced apoptosis. We also tested the effect on apoptosis of the selective iPLA2 inhibitor bromoenol lactone and of the sPLA2 inhibitor p-bromophenacyl bromide; as expected, they were not effective in decreasing the percentage of apoptotic cells (Figure 6E and F).


Figure 6
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  		Fig. 6. Licofelone-induced apoptosis is not related to cPLA2 activation and AA release from HCA-7 membrane phospholipids. (A) Evaluation of AA levels in HCA-7 membrane phospholipids after 24 h incubation with licofelone 100–150 µM, as previously described in the Materials and Methods. Results are expressed as percentage mean ± SD of four independent experiments. Asterisks indicate significance, with *P < 0.05 (significant). (B) Phospho-cPLA2 and total cPLA2 western blotting analysis in HCA-7 cells exposed for different times to licofelone 150 µM. The histograms show phospho-cPLA2 levels normalized for the corresponding total cPLA2 levels. Representation of three separate experiments with similar findings. Asterisks indicate significance, with *P < 0.05 (significant) and **P < 0.001 (very significant). (C) Phospho-cPLA2 and total cPLA2 western blotting analysis in HCA-7 cells pre-treated for 30 min with UO126 (10 µM), SB203580 (10 µM) and SP600125 (2 µM), before co-treatment with licofelone 150 µM for further 24 h. The histograms show phospho-cPLA2 levels normalized for the corresponding total cPLA2 levels. Representation of three separate experiments with similar findings. Asterisks indicate significance, with * P < 0.05 (significant) and **P < 0.001 (very significant). (D–F) Evaluation of the percentage of apoptotic cells in HCA-7 pre-treated for 2 h with MAFP (10–100 µM), bromoenol lactone (BEL, 1–25 µM) and p-bromophenacyl bromide (BPB, 1–25 µM), before co-treatment with licofelone 150 µM for further 24 h. Results are expressed as percentage mean ± SD of three independent experiments. Asterisks indicate significance, with **P < 0.001 (very significant).

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The loss of cell ability to self-destroy by an apoptotic process is considered a pivotal factor for colon carcinogenesis (33). Thereby, molecules which are able to restore the equilibrium between proliferation and apoptosis in colonic epithelial cells and/or to promote apoptosis in cancer cells could be of relevant therapeutic interest. Epidemiological and clinical studies, as well as animal models of colon carcinogenesis, have clearly demonstrated that NSAIDs may be effective against the development and progression of colorectal cancer (34). However, the frequent and often severe gastrointestinal side effects limit their use as anticancer agents (35). Although the introduction of selective COX-2 inhibitors seemed promising, it is now well established that these compounds are not devoid of serious side effects, above all on the cardiovascular system (36). Therefore, novel molecules that maintain the same effectiveness of NSAIDs or selective COX-2 inhibitors, associated to a more favourable toxicological profile, may open a promising approach in colon cancer treatment. In this regard, the new class of anti-inflammatory drugs named dual COX/LOX inhibitors, and in particular, dual COX/5-LOX inhibitors, may represent a good alternative. Both COX and 5-LOX inhibitors, alone or in association, have been reported in fact to induce apoptosis in many cancer cell types (7,26,37,38); moreover, the concurrent inhibition of 5-LOX and COX enzymes has the potential to offer clinically relevant advantages in terms of overall safety (39).

To date, no data are available concerning the potential use of dual COX/5-LOX inhibitors as anticancer drugs in colon cancer treatment. In this regard, in the present study we have shown, for the first time, that the dual COX/5-LOX inhibitor licofelone possesses an anti-proliferative effect on the human colon cancer cell line HCA-7. We demonstrated in fact that this drug strongly decreases HCA-7 cell viability in a time- and dose-dependent manner, with an effectiveness much higher than naproxen and similar to that observed for the COX-2 selective inhibitor celecoxib. The decrease in cell viability was associated to the induction of apoptosis, as shown by the fact that, when exposed to licofelone, HCA-7 cells displayed the typical hallmarks of apoptotic death, such as membrane blebbing, unfragmented or irregularly fragmented pycnotic nuclei, caspase-3 activation and PARP-1 cleavage. Treatment with licofelone was also associated to mitochondrial dysfunction, an effect that is related to the recruitment of the intrinsic apoptotic pathway (40). This pathway is in fact characterized by early mitochondrial events, such as the loss in {bigtriangleup}{psi}m and cytochrome c release from mitochondria to cytosol. As the level of cytochrome c in cytosol increases, caspase-9, which binds to cytochrome c–Apaf-1 complex, is cleaved and activated, thus leading to the activation of the executioner caspase-3 and subsequent PARP-1 cleavage. Consistent with these reports, we observed that licofelone induced, in a dose-dependent manner, the loss in {bigtriangleup}{psi}m and cytochrome c release from mitochondria, which was associated to caspase-9 activation, an event that preceded caspase-3 activation and PARP-1 cleavage. The involvement of caspase activation in licofelone-induced apoptosis was further investigated using the pan-caspase inhibitor z-VAD-fmk. Co-treatment of HCA-7 cells with licofelone and z-VAD-fmk resulted in a significant decrease in the percentage of apoptotic cells, thus indicating that licofelone-induced apoptosis occurred primarily in a caspase-dependent manner.

The Bcl-2 protein family plays an important role in regulating cytochrome c release during apoptosis (41). In particular, proteolytic cleavage of full-length p21Bax into p18Bax has been reported to occur in tumour cells treated with various chemotherapeutic agents that are able to activate the intrinsic apoptotic pathway (42). In agreement with these data we found, after treatment with licofelone, a dose-dependent decrease of full-length p21Bax, that was associated to the concurrent appearance of the cleaved form p18Bax, which has been reported to be more potent in disrupting mitochondrial integrity and inducing apoptotic cell death with respect to p21Bax (43,44). It has been hypothesized, in fact, that the removal of the 33 N-terminal amino acids from p21Bax may expose the hydrophobic BH3 domain (amino acids 59–73), involved in dimer formation and required for the proapoptotic function (45). Such a conformational change may facilitate Bax oligomerization and potential formation of death pores in the outer mitochondrial membrane that, in agreement with what we observed after treatment with licofelone, leak cytochrome c into the cytosol to initiate caspase activation. Taken together, these data suggest that disruption of {bigtriangleup}{psi}m could be a possible, but not necessarily the only, mechanism of licofelone-induced apoptosis in HCA-7 cells.

An aberrant AA metabolism has been related to carcinogenic processes and its modulation has been suggested to be an important strategy for cancer prevention and treatment. In this regard, we investigated whether licofelone could be able to affect the AA cascade and also evaluated its possible involvement in HCA-7 apoptosis induction. We found that licofelone not only was able to block the activity of the AA-metabolizing enzymes 5-LOX and COX (as shown by the strong inhibition of LTB4 and PGE2 production), but also induced, through cPLA2 phosphorylation, the release of unesterified AA from HCA-7 membrane phospholipids. However, we ascertained that apoptosis induction was not related to the ability of licofelone to affect the AA cascade, as neither the exogenous addition in culture medium of the two AA metabolites, LTB4 and PGE2, nor the pharmacological inhibition of cPLA2, rescued HCA-7 cells from apoptosis. The observation that cPLA2 phosphorylation was significantly blocked following treatment with the inhibitors of p38 MAPK and JNK, also called stress-activated kinases, led us to hypothesize a possible involvement of oxidative stress in licofelone-induced apoptosis. Even if this hypothesis will be closely examined in a next paper, preliminary data obtained in our laboratory have shown, following treatment with licofelone, an early increase in reactive oxygen species production, parallel to p38 MAPK and JNK activation (data not shown). The presence of oxidative stress could explain, at least in part, the reason why, although we observed cPLA2 activation and unesterified AA release following treatment with licofelone, pharmacological inhibition with MAFP failed to reverse HCA-7 cell apoptosis. Oxidative stress is in fact a phenomenon acting on a broad spectrum of molecular targets, including lipids, proteins and nucleic acids, thus making, as we hypothesize in our case, PLA2 inhibition not a sufficient countermeasure to block oxidative stress-induced cell damages.

The concentrations of licofelone required to induce apoptosis in our in vitro model are higher than those reached in plasma samples (46). In this regard, it is worth reminding that cell culture systems are extremely simplified experimental models, where it is not possible to reproduce the complexity of the human organism as a whole. In vivo tumour growth is in fact determined by the interaction between factors intrinsic to tumour cells, extracellular matrix, stromal cells and other host factors. All these elements, not reproducible in culture systems, may be responsible of the discrepancy between the doses of an agent which are necessary to elicit an effect in in vitro and in vivo studies. Furthermore, in vitro studies have to be terminated within few days and, therefore, high concentrations of an agent may be required to obtain a detectable effect. In contrast, in vivo studies are usually conducted for longer time periods, thus allowing to achieve an effect at doses that may be significantly lower than those employed in in vitro studies. Moreover, it is important to underline that, in vivo, many NSAIDs can exert their anti-tumoural effects not only by directly inhibiting colon cancer cells growth, but also by affecting angiogenesis (47). In this regard, preliminary studies obtained in our laboratory have shown that licofelone is able to affect also angiogenesis in vitro and at doses significantly lower than those employed in this study (data not shown). This observation reinforces the hypothesis that the high concentration of licofelone needed to induce apoptosis in HCA-7 cells could be due, at least in part, to the fact that, in vitro, the concurrent anti-angiogenic effect (which probably occurs in vivo) cannot be elicited.

In summary, results obtained in our study have shown, for the first time, that licofelone is able to trigger apoptosis in colon cancer cells, suggesting that this drug, as well as similar dual COX/5-LOX inhibitors, may represent a novel and promising approach in colon cancer chemoprevention and therapy.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Basic Research Investment Fund to S.T., E.B., V.T. and T.G.: Alma Mater Studiorum—Università di Bologna, Fundamental Oriented Research to G.B.; Marie Curie Training Network Contract (HPRN-CT-2002-00184) to C.F. and S.M.; Relevant National Interest Programs to M.M.; Cornelia and Roberto Pallotti Foundation and Alma Mater Studiorum—Università di Bologna (Fundamental Oriented Research) to M.B.


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received July 23, 2007; revised October 26, 2007; accepted November 12, 2007.


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