Abstract
Background and Purpose
The pathogenesis of chronic rhinosinusitis with nasal polyposis is unknown. Chronic inflammation along with local tissue hypoxia may effect polyp’s growth. Activation of Cycloxygenases may also be involved. COX-2 up-regulates in response to different stimuli including hypoxia. Its activation is associated with enhanced cell proliferation. Histologically, besides inflammatory cells, increased stromal fibrosis is seen in nasal polyposis. The aims of this study were to test whether hypoxia amplifies nasal polyp fibroblasts proliferation, whether treatment with various COX inhibitors could influence fibroblasts, and whether this effect may be modulated in response to different oxygenation conditions.
Materials and Methods
Polyp fibroblasts were incubated under hypoxic or normoxic conditions with or without NSAIDs at different concentrations for 12 or 24 hours. Cell proliferation was quantified using BrdU ELISA. Metabolic activity was evaluated using MTT assay. Cell death was measured using Annexin V staining and FACS scan.
Results
No significant difference was found between proliferation of fibroblasts treated under hypoxia or normoxia. Cells incubated with indomethacin proliferated in a slightly enhanced manner compared with non-treated cells. Celecoxib inhibited fibroblast proliferation ( P < .001) but did not influence cell survival. Metabolic activity of cells treated with celecoxib was significantly reduced ( P < .003), unlike cells treated with indomethacin or rofecoxib.
Conclusion
Hypoxia does not affect fibroblasts proliferation. It may contribute to nasal polyposis pathogenesis in other ways. The anti-proliferative effect of celecoxib may be associated with cell cycle arrest rather than with pro-apoptotic activity. Celecoxib may be considered for treating nasal polyposis.
1
Introduction
Nasal polyposis (NP) is the most severe form of chronic rhinosinusitis (CRS) . The stroma of nasal polyps is composed mainly of fibroblasts and extra cellular matrix and is infiltrated by many inflammatory cells, predominantly eosinophils .
Numerous inflammatory mediators and differentiation factors are involved in the growth of nasal polyps. Vascular endothelial growth factor (VEGF) has been associated with the development of edema . TGF-β induces fibroblasts proliferation and fibrosis. IL-8, RANTES, GM-CSF, IgE, IL-1 and eotaxin affect the growth of nasal polyps . Furthermore, prostanoids play a key role in nasal mucosal inflammation .
The pathogenesis of NP has not been completely understood yet. Chronic inflammation plays an important role, but the trigger is still unknown .
Medical treatment of NP is usually based on the use of topical and occasionally systemic steroids. Other optional drugs are anti-microbials, anti-histamines, anti-congestives and immunomodulatory drugs. Surgery is reserved for severe cases and for patients who have failed medical therapy. Recurrence is common, and many patients require further medical treatment and additional procedures . Better understanding of the causes and aggravating factors effecting NP, may provide better treatment options and prevention of recurrence.
Nasal polyps usually originate and recur in and around the middle nasal meatus, which receives only a poor blood supply compared with other parts of the nasal cavity. Polyps rarely occur in the inferior meatus, although this meatus is located in an inferior-anterior part of the nasal cavity, where it is much more vulnerable to harmful stimuli than the middle meatus (located in a more superior-posterior part) . These findings suggest that local tissue hypoxia may exist in the site of origin of nasal polyps, and thus may be involved in their growth.
Another characteristic of nasal polyps is substantial tissue edema. This is attributed to local production of VEGF, a potent vascular permeability agent. Hypoxia is considered to be one of the most effective inducers of VEGF production . Hypoxia-inducible-factor 1 (HIF-1) is a transcription factor responsible for VEGF gene expression. Hypoxia inhibits HIF-1a subunit degradation and gives rise to elevated levels of HIF-1 and VEGF. HIF-1a expression was demonstrated to be increased significantly in nasal polyps. These findings support the hypothesis that hypoxia prevails in nasal polyps and may be involved in their pathogenesis . Chronic sinus pathologies are also associated with a low oxygen tension. Chronic exposure to even moderate hypoxia may result in inflammation . Low oxygen levels were measured within the sinus cavities of patients suffering from CRS, which correlated to sinus opacities on computed tomography scans . Another evidence supporting the hypothesis that hypoxia exists in sinus disease, is the colonization of anaerobic bacteria in CRS .
Fibroblasts are key cells in nasal polyp architecture. They are stimulated by pro-inflammatory cytokines and by eosinophils’ secreted growth factors . Stimulated fibroblasts contribute to the inflammatory process by releasing inflammatory mediators . Hypoxia has been associated with increased fibroblasts’ proliferation in several tissues. Vascular adventitial fibroblasts, obtained from pulmonary arteries, proliferate in response to hypoxia. The pathological endpoint is pulmonary hypertension . Hypoxia is also involved in the pathogenesis of peritoneal adhesions . To the best of our knowledge, besides one study , no manuscript has been published regarding the effect of hypoxia on nasal polyp fibroblasts’ proliferation.
Nasal polyps’ tissue is infiltrated by many activated inflammatory cells which release a variety of pro-inflammatory mediators including cytokines, leukotrienes, histamine and prostaglandins (PGs). Therefore, anti-inflammatory drugs are being studied as potential treatment for NP. Corticosteroids, which possess powerful anti-inflammatory qualities, are the most effective medical treatment for NP . Another treatment is macrolid antibiotics, which do not only decrease the virulence of colonizing bacteria, but also have anti-inflammatory activities . Other (still experimental) treatments are intranasal lysine acetylsalicylate (LAS), found to reduce nasal polyps’ recurrence after surgical resection (both in aspirin tolerant and intolerant patients) , and high dose ibuprofen, reported beneficial for children with cystic fibrosis .
Arachidonic acid metabolism is considered to play a key role in the pathogenesis of NP . Prostaglandins (PGs) and Leukotrienes (LTs) are the main products of this pathway. PGs are generated through the cycloxygenase (COX) pathway of arachidonic acid metabolism. COX exists in two isoforms: COX-1, which is constitutively expressed in most tissues, and COX-2, which is inducible by a variety of agents such as inflammation and tissue damage. Altered regulation of both isoenzyems has been associated with the development of airway inflammation and NP . COX dysregulation is also associated with aspirin intolerance, which is often accompanied by asthma and NP .
Previous studies revealed conflicting evidence regarding COX-1 and COX-2 expression and regulation in nasal polyps. Some studies have demonstrated COX-2 up-regulation and abnormal induction in nasal polyps . Others claimed that COX-2 is actually down-regulated . Even COX-1, usually considered a constitutively expressed enzyme, was found to be dysregulated in nasal polyps’ tissue .
The inducible enzyme COX-2 stimulates cell proliferation and promotes neovascularization and mitogenesis. Elevated levels of COX-2 were found in endothelial cells , adhesion fibroblasts , rheumatoid synovial tissue and in other proliferating tissues. Over-expression of COX-2 was also found in human malignancies . COX inhibitors, especially COX-2 selective inhibitors, have been shown to exert inhibitory effects on malignant tumors cells . , rheumatoid arthritis synovial fibroblasts , Tenon’s capsule fibroblasts and normal skin fibroblasts . Some COX inhibitors exhibited an inhibitory effect on the proliferation of nasal polyp derived fibroblasts .
COX-2 is augmented in response to tissue damage and to inflammatory stimuli such as hypoxia. Peritoneal fibroblasts , synoviocytes and pulmonary fibroblasts demonstrate COX-2 over-expression under oxygen-deprived conditions and proliferate in an enhanced manner, suggesting a correlation between hypoxia, COX-2 augmentation, and proliferation. In the current study, we assessed whether hypoxia amplifies nasal polyp fibroblasts proliferation, whether treatment with COX inhibitors could influence fibroblasts, and whether this effect may be modulated in response to different oxygenation conditions.
2
Materials and methods
2.1
Subjects
In order to reduce confounding factors, we chose to focus in this study only on non-smoking, non-allergic/non-atopic, aspirin tolerant patients. Only patients with negative history for allergy/atopy, negative signs in the physical examination, and negative skin prick tests were included in the study. Patients with an antrochoanal polyp or with other diseases related to NP such as cystic fibrosis, primary ciliary dyskinesia, and allergic fungal sinusitis, were excluded. CRS with NP was diagnosed on the basis of history and findings at anterior rhinoscopy, nasal endoscopy and sinus computed tomography. All patients suffered from symptoms related to CRS and NP for more than 3 months and were offered surgery after failure of medical treatment. None had used topical or oral steroids within 6 weeks. The study was approved and conducted according to guidelines of the Internal Review Board (Helsinki Committee).
2.2
Materials
Dulbecco’s Modified Eagle’s Medium (DMEM) (Biological Industries)
Antibiotics solution (penicillin + streptomycin) (Biological Industries)
Fetal Calf Serum (FCS) (Biological Industries)
Collagenase type-I (Sigma)
Hyaluronidase (Sigma)
DNase (Sigma)
Trypsin/EDTA (Biological Industries)
PBS (Biological Industries)
Trypan blue solution (0.4%) (Sigma)
Indomethacin (Sigma)
Rofecoxib (VIOXX 25 mg) (Merck Sharp & Dohme, Israel)
Celecoxib (Sigma)
Celecoxib (CELCOX 200mg) (TRIMA Israel Pharmaceutical Products, Israel)
BrdU ELISA (CHEMICON International, Cat No. 2752)
MTT assay (Sigma)
Annexin V-FITC (IQ Products, Groningen, Netherlands)
Propidium Iodide (PI) (Sigma)
2.3
Preparations
Cell culture medium was prepared using Dulbecco’s Modified Eagle’s Medium (DMEM), 100mg/ml (Penicillin/Streptomycin) and heat inactivated Fetal Calf Serum (FCS). Indomethacin was prepared as a stock solution of 1mg/ml ethanol. This solution was diluted with DMEM containing 2% FCS to final concentrations of 10 -5 M, 10 -6 M, 10 -7 M and 10 -8 M.
Rofecoxib was crushed, dissolved in acetone and filtered through a 150-micron nylon mesh. A stock solution of 12.5mg/ml acetone was made, and diluted as mentioned previously. Celecoxib solution was prepared from two different sources. Celecoxib (TRIMA) was used for assessing proliferation. Initially, the gelatin coating of a 200mg pill was removed. A stock solution of 100mg/ml DMSO was prepared, filtered through a 150-micron nylon mesh and sonicated. Celecoxib (Sigma) was used for assessing metabolic activity and detection of apoptosis. A stock solution of 1mg/ml DMSO was prepared. Solutions were diluted as described above. Ethanol, acetone and DMSO were diluted in a similar way as control solutions.
2.4
Cell culture
Nasal polyps were washed twice in a medium containing 2% FCS and cut into small fragments. The fragments were digested by incubation with an enzymatic cocktail containing collagenase type-I (6mg/g tissue), hyaluronidase (3mg/g tissue), and DNase (100 μg/g tissue) in culture medium, in 37°C with shaking, for 90 minutes. The digested tissue was washed with medium and filtered through a 150-micron nylon mesh. Filtered fragments were cultured with medium containing10% FCS at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . Medium was changed twice a week. Once confluence was reached, cells were harvested with 0.25% trypsin/0.02% EDTA, and divided into subcultures. Cells were used after 4 to 8 passages (previous studies have shown that multiple passaging did not impact the function or viability of fibroblasts ). Cells viability was assessed by trypan blue exclusion.
2.5
Hypoxic culture conditions
Cells were seeded in 96 well plates, 5⁎10 4 cells per well, and incubated overnight with 200 μl of medium containing 10% FCS until sub-confluence was reached. Cells were rinsed and medium changed to a medium containing 2% FCS (serum starvation). Plates were incubated under different oxygenation conditions.
For hypoxic conditions plates were placed in a closed chamber at 37°C with continuous flow of a gas mixture of 95% N 2 and 5% CO 2 . During the experiment the oxygen percentage in the medium was monitored with a dissolved oxygen meter (Mettler, Toledo, USA) and was measured ≤ 3%. Other plates were placed in normoxia (21% oxygen) and incubated at 37°C in a humidified incubator. Cell proliferation was assessed after 12 and 24 hours of incubation under the selected conditions.
2.6
Cell proliferation assay
Fibroblasts proliferation was assessed using BrdU ELISA. The assay was performed according to the manufacturer’s instructions. Cells were seeded as previously mentioned, 20 μl/well of BrdU (1:500) was added, and cells were further incubated at normoxic and hypoxic conditions. At the endpoint of the incubation, medium was withdrawn, 200 μl/well of fixing solution was added, and the plates incubated at room temperature (R.T.) for 30min. Thereafter, the solution was aspirated and the wells rinsed three times with a washing solution and eventually dried on a paper towel. Anti- BrdU monoclonal antibodies were then added, 100 μl/well (1:200), and the wells were incubated for 1 hour at R.T. Medium was removed and wells rinsed three times with a washing solution. Goat anti- Mouse IgG peroxidase conjugate was diluted 1:2000 and filtered using 0.22 μm syringe filter. 100 μl/well solution was added and incubated for 30 min. at R.T. Medium was removed and wells rinsed three times with washing solution. 100 μl/well Tetramethylbenzidine (TMB) peroxidase substrate was added and incubated for 30min. at R.T. in the dark. Positive wells were colored in blue and the color intensity was correlated with the degree of BrdU incorporation in proliferating cells. 100 μl/well of stop solution was then added. Positive wells changed their color from blue to yellow. BrdU absorbance was measured at 405nm with a reference wavelength at 530nm using an ELISA plate reader.
The experiments were carried out in triplicates. Some of the wells were used as controls. These included blank wells containing no cells and background wells containing cells not treated with BrdU reagent.
2.7
COX inhibitors treatment
Cells were seeded in 96 well plates as previously mentioned and incubated for 24 hours until sub-confluence was reached. Medium was removed, cells rinsed twice with medium containing 2% FCS and different treatments were added as follows.
Cells were treated with celecoxib and indomethacin. 200 μl of a treatment solution was added to each well. Parallel plates were put both in normoxic or hypoxic conditions. Cell proliferation under the different conditions at different time points, was assessed by BrdU ELISA cell proliferation assay, as previously described.
2.8
Cell metabolic activity
Cells were seeded in 96 well plates and incubated for 24 hours until sub-confluence was reached. Medium was removed; cells rinsed twice with medium containing 2% FCS and different treatments of indomethacin, celecoxib and rofecoxib were added. 200 μl of a treatment solution was added to each well. Plates were incubated for 12 and 24 hours. Three different cell lines were tested in these experiments. The experiments were carried out in triplicates.
Cells viability/metabolic activity was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and conducted according to the manufacturer’s instructions. Briefly, 10 μl/well of reagent was added after ending incubation. Plates were further incubated for 2 hours at 37°C. Thereafter, 100 μl of DMSO was added to each well. The plates were incubated in the dark for 2 more hours. Optical density of each well at 570nm was measured using a microplate reader.
2.9
Detection of apoptosis
Nasal polyp fibroblasts were seeded in 24 well plates, 2 × 10 5 cells per well, with 1ml of medium containing 10% FCS. Plates were incubated until sub-confluence was reached. Wells were rinsed twice with medium containing 2% FCS and treatments were added. The cells were treated with 600 μl of indomethacin and celecoxib at different concentrations. Cell lines originating from two patients were used in this experiment. The experiment was carried out in duplicates.
Apoptotic cells were detected by Flurescence Activated Cell Sorter (FACS) scan using annexin V staining. Cells were harvested using 0.25% trypsin/0.02% EDTA, washed with medium containing 2% FCS and suspended with ice cold annexin buffer. They were then transferred into 96 U-shaped well plates. Plates were centrifuged at 150g for 5min at -4°C, and rinsed twice with annexin buffer. 100 μl of annexin buffer and 2 μl of annexin were added to each well. Plates were incubated in ice for 15min in the dark. Wells were rinsed twice more with annexin buffer. 5 μl of Propidium Iodide (PI) were added to each well before analyzing the cells by FACS. Cells were defined as either apoptotic cells (annexin V positive and PI negative) or necrotic cells (annexin V and PI positive). Definition was also made between living cells (anexin V and PI negative) and dead cells (Anexin V negative and PI positive).
2.10
Statistical analysis
The experiments compared groups of cell cultures from all donors under different treatments. Once we have compared all samples individually (after 4 to 8 passages in cell cultures), and found similar behavior, each sample was addressed individually in the final statistical analysis, assuming the source of the cells being insignificant in their new micro-environments. Since the experiments were carried out in duplicates or triplicates, and since each group included 2–3 cell lines outgrown from each donor, 20–45 observations in each group were available for the final statistical analysis.
In order to compare cell proliferation under different treatments and oxygen conditions, each treatment group was analyzed using ANOVA variation analysis, both parametric and non-parametric (Kruskal-Wallis Test), with multiple comparisons and adjustment to significance levels according to Dunnelt. When unequal variations were encountered, average comparison was performed according to the adjustment of Brown-Forsythe. Comparison between different treatment and concentrations effect in normoxia and hypoxia was done using T-test and Mann-Whitney Test. P < .05 was considered as statistically significant. Two-way ANOVA model was applied in order to assess the effect of the concentration, the time, and the interaction between them on cell metabolism. Non-parametric ANOVA (Kruskal-Wallis Test) was applied in order to assess dose effect of Celecoxib on cell death.
2
Materials and methods
2.1
Subjects
In order to reduce confounding factors, we chose to focus in this study only on non-smoking, non-allergic/non-atopic, aspirin tolerant patients. Only patients with negative history for allergy/atopy, negative signs in the physical examination, and negative skin prick tests were included in the study. Patients with an antrochoanal polyp or with other diseases related to NP such as cystic fibrosis, primary ciliary dyskinesia, and allergic fungal sinusitis, were excluded. CRS with NP was diagnosed on the basis of history and findings at anterior rhinoscopy, nasal endoscopy and sinus computed tomography. All patients suffered from symptoms related to CRS and NP for more than 3 months and were offered surgery after failure of medical treatment. None had used topical or oral steroids within 6 weeks. The study was approved and conducted according to guidelines of the Internal Review Board (Helsinki Committee).
2.2
Materials
Dulbecco’s Modified Eagle’s Medium (DMEM) (Biological Industries)
Antibiotics solution (penicillin + streptomycin) (Biological Industries)
Fetal Calf Serum (FCS) (Biological Industries)
Collagenase type-I (Sigma)
Hyaluronidase (Sigma)
DNase (Sigma)
Trypsin/EDTA (Biological Industries)
PBS (Biological Industries)
Trypan blue solution (0.4%) (Sigma)
Indomethacin (Sigma)
Rofecoxib (VIOXX 25 mg) (Merck Sharp & Dohme, Israel)
Celecoxib (Sigma)
Celecoxib (CELCOX 200mg) (TRIMA Israel Pharmaceutical Products, Israel)
BrdU ELISA (CHEMICON International, Cat No. 2752)
MTT assay (Sigma)
Annexin V-FITC (IQ Products, Groningen, Netherlands)
Propidium Iodide (PI) (Sigma)
2.3
Preparations
Cell culture medium was prepared using Dulbecco’s Modified Eagle’s Medium (DMEM), 100mg/ml (Penicillin/Streptomycin) and heat inactivated Fetal Calf Serum (FCS). Indomethacin was prepared as a stock solution of 1mg/ml ethanol. This solution was diluted with DMEM containing 2% FCS to final concentrations of 10 -5 M, 10 -6 M, 10 -7 M and 10 -8 M.
Rofecoxib was crushed, dissolved in acetone and filtered through a 150-micron nylon mesh. A stock solution of 12.5mg/ml acetone was made, and diluted as mentioned previously. Celecoxib solution was prepared from two different sources. Celecoxib (TRIMA) was used for assessing proliferation. Initially, the gelatin coating of a 200mg pill was removed. A stock solution of 100mg/ml DMSO was prepared, filtered through a 150-micron nylon mesh and sonicated. Celecoxib (Sigma) was used for assessing metabolic activity and detection of apoptosis. A stock solution of 1mg/ml DMSO was prepared. Solutions were diluted as described above. Ethanol, acetone and DMSO were diluted in a similar way as control solutions.
2.4
Cell culture
Nasal polyps were washed twice in a medium containing 2% FCS and cut into small fragments. The fragments were digested by incubation with an enzymatic cocktail containing collagenase type-I (6mg/g tissue), hyaluronidase (3mg/g tissue), and DNase (100 μg/g tissue) in culture medium, in 37°C with shaking, for 90 minutes. The digested tissue was washed with medium and filtered through a 150-micron nylon mesh. Filtered fragments were cultured with medium containing10% FCS at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . Medium was changed twice a week. Once confluence was reached, cells were harvested with 0.25% trypsin/0.02% EDTA, and divided into subcultures. Cells were used after 4 to 8 passages (previous studies have shown that multiple passaging did not impact the function or viability of fibroblasts ). Cells viability was assessed by trypan blue exclusion.
2.5
Hypoxic culture conditions
Cells were seeded in 96 well plates, 5⁎10 4 cells per well, and incubated overnight with 200 μl of medium containing 10% FCS until sub-confluence was reached. Cells were rinsed and medium changed to a medium containing 2% FCS (serum starvation). Plates were incubated under different oxygenation conditions.
For hypoxic conditions plates were placed in a closed chamber at 37°C with continuous flow of a gas mixture of 95% N 2 and 5% CO 2 . During the experiment the oxygen percentage in the medium was monitored with a dissolved oxygen meter (Mettler, Toledo, USA) and was measured ≤ 3%. Other plates were placed in normoxia (21% oxygen) and incubated at 37°C in a humidified incubator. Cell proliferation was assessed after 12 and 24 hours of incubation under the selected conditions.
2.6
Cell proliferation assay
Fibroblasts proliferation was assessed using BrdU ELISA. The assay was performed according to the manufacturer’s instructions. Cells were seeded as previously mentioned, 20 μl/well of BrdU (1:500) was added, and cells were further incubated at normoxic and hypoxic conditions. At the endpoint of the incubation, medium was withdrawn, 200 μl/well of fixing solution was added, and the plates incubated at room temperature (R.T.) for 30min. Thereafter, the solution was aspirated and the wells rinsed three times with a washing solution and eventually dried on a paper towel. Anti- BrdU monoclonal antibodies were then added, 100 μl/well (1:200), and the wells were incubated for 1 hour at R.T. Medium was removed and wells rinsed three times with a washing solution. Goat anti- Mouse IgG peroxidase conjugate was diluted 1:2000 and filtered using 0.22 μm syringe filter. 100 μl/well solution was added and incubated for 30 min. at R.T. Medium was removed and wells rinsed three times with washing solution. 100 μl/well Tetramethylbenzidine (TMB) peroxidase substrate was added and incubated for 30min. at R.T. in the dark. Positive wells were colored in blue and the color intensity was correlated with the degree of BrdU incorporation in proliferating cells. 100 μl/well of stop solution was then added. Positive wells changed their color from blue to yellow. BrdU absorbance was measured at 405nm with a reference wavelength at 530nm using an ELISA plate reader.
The experiments were carried out in triplicates. Some of the wells were used as controls. These included blank wells containing no cells and background wells containing cells not treated with BrdU reagent.
2.7
COX inhibitors treatment
Cells were seeded in 96 well plates as previously mentioned and incubated for 24 hours until sub-confluence was reached. Medium was removed, cells rinsed twice with medium containing 2% FCS and different treatments were added as follows.
Cells were treated with celecoxib and indomethacin. 200 μl of a treatment solution was added to each well. Parallel plates were put both in normoxic or hypoxic conditions. Cell proliferation under the different conditions at different time points, was assessed by BrdU ELISA cell proliferation assay, as previously described.
2.8
Cell metabolic activity
Cells were seeded in 96 well plates and incubated for 24 hours until sub-confluence was reached. Medium was removed; cells rinsed twice with medium containing 2% FCS and different treatments of indomethacin, celecoxib and rofecoxib were added. 200 μl of a treatment solution was added to each well. Plates were incubated for 12 and 24 hours. Three different cell lines were tested in these experiments. The experiments were carried out in triplicates.
Cells viability/metabolic activity was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and conducted according to the manufacturer’s instructions. Briefly, 10 μl/well of reagent was added after ending incubation. Plates were further incubated for 2 hours at 37°C. Thereafter, 100 μl of DMSO was added to each well. The plates were incubated in the dark for 2 more hours. Optical density of each well at 570nm was measured using a microplate reader.
2.9
Detection of apoptosis
Nasal polyp fibroblasts were seeded in 24 well plates, 2 × 10 5 cells per well, with 1ml of medium containing 10% FCS. Plates were incubated until sub-confluence was reached. Wells were rinsed twice with medium containing 2% FCS and treatments were added. The cells were treated with 600 μl of indomethacin and celecoxib at different concentrations. Cell lines originating from two patients were used in this experiment. The experiment was carried out in duplicates.
Apoptotic cells were detected by Flurescence Activated Cell Sorter (FACS) scan using annexin V staining. Cells were harvested using 0.25% trypsin/0.02% EDTA, washed with medium containing 2% FCS and suspended with ice cold annexin buffer. They were then transferred into 96 U-shaped well plates. Plates were centrifuged at 150g for 5min at -4°C, and rinsed twice with annexin buffer. 100 μl of annexin buffer and 2 μl of annexin were added to each well. Plates were incubated in ice for 15min in the dark. Wells were rinsed twice more with annexin buffer. 5 μl of Propidium Iodide (PI) were added to each well before analyzing the cells by FACS. Cells were defined as either apoptotic cells (annexin V positive and PI negative) or necrotic cells (annexin V and PI positive). Definition was also made between living cells (anexin V and PI negative) and dead cells (Anexin V negative and PI positive).
2.10
Statistical analysis
The experiments compared groups of cell cultures from all donors under different treatments. Once we have compared all samples individually (after 4 to 8 passages in cell cultures), and found similar behavior, each sample was addressed individually in the final statistical analysis, assuming the source of the cells being insignificant in their new micro-environments. Since the experiments were carried out in duplicates or triplicates, and since each group included 2–3 cell lines outgrown from each donor, 20–45 observations in each group were available for the final statistical analysis.
In order to compare cell proliferation under different treatments and oxygen conditions, each treatment group was analyzed using ANOVA variation analysis, both parametric and non-parametric (Kruskal-Wallis Test), with multiple comparisons and adjustment to significance levels according to Dunnelt. When unequal variations were encountered, average comparison was performed according to the adjustment of Brown-Forsythe. Comparison between different treatment and concentrations effect in normoxia and hypoxia was done using T-test and Mann-Whitney Test. P < .05 was considered as statistically significant. Two-way ANOVA model was applied in order to assess the effect of the concentration, the time, and the interaction between them on cell metabolism. Non-parametric ANOVA (Kruskal-Wallis Test) was applied in order to assess dose effect of Celecoxib on cell death.
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