Research Article |
Corresponding author: Lyubomir Marinov ( lubomir.t.marinov@gmail.com ) Academic editor: Plamen Peikov
© 2024 Lyubomir Marinov, Ani Georgieva, Reneta Toshkova, Ivanka Kostadinova, Iliya Mangarov, Tanya Toshkova-Yotova, Irina Nikolova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Marinov L, Georgieva A, Toshkova R, Kostadinova I, Mangarov I, Toshkova-Yotova T, Nikolova I (2024) The effects of meloxicam, lornoxicam, ketoprofen, and dexketoprofen on human cervical, colorectal, and mammary carcinoma cell lines. Pharmacia 71: 1-12. https://doi.org/10.3897/pharmacia.71.e113677
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Targeting the inflammation-related molecules with nonsteroidal anti-inflammatory drugs (NSAIDs) represents a promising approach for cancer prevention/therapy. We evaluated the in vitro anticancer effects of meloxicam, lornoxicam, ketoprofen, and dexketoprofen on the proliferation, migration, and apoptosis of human cervical, colorectal, and mammary carcinoma cells. The antiproliferative activity and cytotoxicity of tested NSAIDs on HeLa, HT-29, and MCF-7 cell lines were assessed by the MTT test. The apoptosis-inducing potential was analyzed by fluorescent staining with acridine orange/ethidium bromide and DAPI. Migration activity was assessed by a wound-healing scratch assay. The tested NSAIDs reduced the viability of the used tumor cell lines. The cytomorphological analysis revealed reduced cell density and mitotic activity and the presence of cells with morphological features of early and late apoptosis. Significant inhibition of the migration capacity was established as well. In conclusion, NSAIDs could be candidates for the development of new pharmacological strategies for the treatment and prevention of cancer.
anti-proliferative activity, NSAIDs, meloxicam, lornoxicam, ketoprofen, dexketoprofen
The association between cancer and inflammation has long been known. However, the possible role of nonsteroidal anti-inflammatory drugs (NSAIDs) in the induction and progression of cancer remains unclear. Some studies revealed a reduced risk of breast, prostate, colorectal, ovarian, and head and neck cancer after NSAIDs therapy, while others demonstrated no association between cancer and NSAID use (
NSAIDs are the most prescribed drugs for the treatment of inflammation, fever, and pain. Their main mechanism of action is the inhibition of cyclooxygenase (COX), an enzyme responsible for the synthesis of prostanoids (
The antiproliferative effect of ketoprofen on the expression of the HE4 gene and viability of the A2780 human ovarian cancer cell line was demonstrated by
The present study aimed to evaluate in vitro anticancer effects of the following NSAIDs: meloxicam, lornoxicam, ketoprofen, and dexketoprofen on cell viability, proliferation, migration, and apoptosis of the human tumor cell lines HeLa, HT-29, and MCF-7. The signal transduction changes were outside the scope of the present study but be the subject of a future study together with the interaction of those NSAIDs with antineoplastic agents.
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-Invitrogen (UK). Dimethyl sulfoxide (DMSO) and trypsin were obtained from AppliChem (Germany); thiazolyl blue tetrazolium bromide (MTT) was from Sigma-Aldrich Chemie GmbH (Germany). The antibiotics (penicillin and streptomycin) were from Lonza (Belgium). All other chemicals of the highest purity commercially available were purchased from local agents and distributors. All sterile plastic ware was purchased from Orange Scientific (Belgium).
Meloxicam (Melbek 15 mg/1.5 mL); lornoxicam (Xefo 4 mg/ml; 2.0 mL); ketoprofen (Profenid 100 mg powder and solvent for solution for injection), and dexketoprofen (Dexofen inject – 50 mg/2 mL solution for injection) were used in the experiments. Solutions of NSAIDs were prepared according to the instructions for each preparation and dilutions with a complete cell culture medium were made to obtain the concentrations desired for the different in vitro assays.
Permanent human tumor cell lines – HeLa (cervical cancer), HT-29 (colorectal adenocarcinoma), MCF-7 (adenocarcinoma of the mammary gland) and BALB/3T3 (mouse embryonal fibroblasts) purchased from the American Type Culture Collection (Rockville, MD, USA) were used. HeLa, HT-29 and MCF-7 cells were used in our study as models of some of the most common types of oncologic diseases and BALB/3T3 cells were used as a nontumor control. The cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37 °C in a humidified 5% CO2-incubator (Thermo Scientific, HEPA Class 100). Confluent cell monolayers were trypsinized (a mixture of 0.05% trypsin – 0.02% ethylenediaminetetraacetic acid) and cells were used at the exponentially growing phase.
The effects of the NSAIDs on cell viability and proliferation were evaluated using a standard colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay (
Cells in a concentration of 104 cells/well were seeded in 100 μL complete culture medium in 96-well flat-bottom culture plates and pre-cultured for a day before the beginning of treatment. The sub-confluent cells were then exposed for 24 and 48 h to eight different concentrations of the NSAIDs ranging from 3.125 to 800 μg/mL. Each concentration was applied in 6 replicates. Cells grown in a medium without any addition of compounds were used as a control. At the end of the incubation periods (24 and 48 h), 100 μL of MTT (5 mg/mL in PBS) was added to each well, and plates were cultivated at 37 °C for 3 h in the dark. The MTT-containing medium was removed and 100 μL DMSO: ethanol (1:1) was added to each well, and the plates were shaken for 5 min. The optical densities (OD) were measured at 570 nm with a reference wavelength of 630 nm using an ELISA spectrophotometer (TECAN, SunriseTM, Grödig/Salzburg, Austria). The percentage of cell viability was calculated as follows:
Cell viability (%) = OD of treated cells/OD of untreated cells × 100
Inhibitory concentration IC50, resulting in a 50% reduction in cell viability, as compared to controls was determined. All assays were performed in triplicate.
The tumor cells were grown on sterile cover glasses placed on the bottom of 24-well plates (2.0 × 105 cells/well) for 24 h in a CO2 incubator to form a cell monolayer. The next day, the NSAID solutions with appropriate concentrations, chosen based on the results of the cell viability assays were added. Meloxicam and lornoxicam were applied at concentrations of 400 μg/mL and dexketoprofen at concentrations of 800 μg/mL for all cell lines. Ketoprofen was used in a concentration of 200 μg/mL for the MCF-7 cells and 800 μg/mL for the HeLa and HT-29 cells. Tumor cells from the respective cell line, cultured only in medium served as controls. After 24 h of incubation, the coverslips were removed and washed twice with phosphate-buffered saline (PBS). Equal volumes of fluorescent dyes containing AO (10 μg/mL in PBS) and EtBr (10 μg/mL in PBS) were added to the cells. Freshly stained cells were placed on a glass slide and examined immediately under a fluorescence microscope (Leica DM 5000B, Wetzlar, Germany) before the fluorescent color started to fade.
The cells were seeded, treated, and cultured as described in the previous paragraph. After incubation, the glass lamellae were washed twice with phosphate-buffered saline (PBS) to remove non-adhered tumor cells. DAPI staining of the cells was performed after fixation with 3% paraformaldehyde according to the instructions of the manufacturer‘s protocol. Samples of treated and untreated cells were coated with Mowiol, mounted on slides, and stored in the dark until examination with a fluorescence microscope (Leica DM 5000B, Wetzlar, Germany).
To investigate the migration of tumor cells treated with NSAIDs, a wound-healing scratch assay was performed as previously described (
The data were analyzed by the one-way analysis of variance (ANOVA) using GraphPad PRISM (Version 5) and presented as mean ± standard deviation (SD). Values of P < 0.05 were considered statistically significant. Nonlinear regression (curve fit) analysis (GraphPad Prism) was applied to determine the concentrations inducing 50% inhibition of the cell growth (IC50 values).
The NSAIDs meloxicam, lornoxicam, ketoprofen, and dexketoprofen induced statistically significant and concentration-dependent reduction in the viability of the cervical, colorectal, and mammary carcinoma cell lines HeLa, MCF-7, and HT-29, respectively (Fig.
The treatment of HeLa and HT-29 tumor cells with meloxicam for 24 h induced significant antiproliferative and cytotoxic effects at all tested concentrations higher than 6.3 μg/mL. At the highest concentration used in the experiments, the viability of HeLa and HT-29 cells was 79% and 75% compared to the control, respectively. The inhibitory effects enhanced at 48 h of treatment and statistical significance of the differences between the control and treated cells was established at all used concentrations. The cell viability values of the cervical and colorectal carcinoma cells recorded at 48 h of exposure ranged from 83% to 43% and from 87% to 47%, respectively. Meloxicam significantly inhibited the proliferation of MCF-7 tumor cells at concentrations higher than 50 μg/mL at 24 h and higher than 100 μg/mL at 48 h. The mean cell viability at 24 h ranged between 84.86 ± 6.65% and 70.14 ± 3.54%, and between 87.35 ± 3.03% and 82.64 ± 2.36% at 48 h.
Lornoxicam induced a statistically significant reduction of the viability of HeLa tumor cells only at concentrations of 200 μg/mL and 400 μg/mL after 24 h and at all tested concentrations after 48 h of exposure. The cell viability values for 24 h were 88.36 ± 0.30% and 79.95 ± 0.53%, and at 48 h were in the range between 87.91 ± 2.12% and 40.69 ± 2.45%. The antiproliferative effect of lornoxicam in HT-29 cells was observed at concentrations of 100 μg/mL, 200 μg/mL and 400 μg/mL at both time intervals, with corresponding values of 92.58 ± 2.72%, 82.50 ± 2.99%, and 70.38 ± 2.09% at 24 h and 77.24 ± 4.49%; 52.09 ± 2.58%, and 38.93 ± 3.10% at 48 h. Lornoxicam showed no statistically significant effect on the cell viability of MCF-7 cells at all applied concentrations and time intervals.
A statistically significant cytotoxic effect of ketoprofen against HeLa tumor cells was observed at concentrations of 200 μg/mL and 400 μg/mL at 24 h (78.15 ± 4.39 % and 49.90 ± 2.16%, respectively), and all concentrations higher than 25 μg/mL at 48 h (with values from 85.60 ± 3.12% to 22.73 ± 0.93%). Ketoprofen significantly decreased the viability of HT-29 tumor cells at all concentrations studied. The mean values of the cell viability recorded at 24 h were in the range from 87.03 ± 1.75% to 55.54 ± 5.13% and at 48 h – from 82.92 ± 4.40% to 24.62 ± 2.24%. The effect was strongest at a concentration of 800 µg/ml at 24 and 48 h. In MCF-7 tumor cells, ketoprofen was effective at concentrations higher than 25 μg/mL at 24 h with cell viability values of 84.92 ± 6.54% to 30.41 ± 2.53% and at concentrations higher than 50 μg/mL at 48 h with values from 78.12 ± 5.30% to 20.50 ± 2.35%, with a dose-dependent effect.
Only the concentration of 800 µg/mL of dexketoprofen inhibited the proliferation of HeLa cells at 24 h (75.69 ± 2.82%). At 48 h, concentrations of 400 µg/mL and 800 µg/mL were effective with values of 85.74 ± 13.53% and 44.84 ± 7.74%, respectively. High concentrations (of 200 μg/mL, 400 μg/mL, and 800 μg/mL) at 24 h and all concentrations at 48 h of dexketoprofen reduced the viability of HT-29 and MCF-7 cells. The lowest values of HT-29 cell viability were observed at a dose of 800 μg/mL – 79.39 ± 2.66% and 32.82 ± 3.87% at 24 and 48 h, respectively. For MCF-7 cells, the values were 46.32 ± 4.55% and 45.98 ± 3.87% at 24 and 48 h).
The cytotoxicity of the tested drugs was also assessed on the nontumor cell line BALB/3T3. As evident from Fig.
The half maximum inhibitory concentrations (IC50) (µg/mL) of the tested NSAIDs, calculated by nonlinear regression analysis of the dose-response curves (percentage of living cells relative to the concentration of the corresponding NSAIDs) were determined. The data are presented in Table
Inhibitory concentrations IC50 (µg/mL) of the NSAIDs meloxicam, lornoxicam, ketoprofen, and dexketoprofen, established by MTT test after exposure of human tumor cells and nontumorigenic cells for 24 and 48 h.
Cell lines | Lornoxicam | Meloxicam | Ketoprofen | Dexketoprofen | ||||
---|---|---|---|---|---|---|---|---|
24 h | 48 h | 24 h | 48 h | 24 h | 48 h | 24 h | 48 h | |
HeLa | >400 (1.1 mM) | 235.6 (0.6 mM) | >400 (1.4 mM) | 529.3 (1.5 mM) | 852.5 (3.1 mM) | 455.9 (1.8 mM) | >800 (3.1 mM) | 744.6 (2.9 mM) |
HT-29 | >400 (1.1 mM) | 258.8 (0.7 mM) | >400 (1.4 mM) | 973.9 (2.8 mM) | >800 (3.1 mM) | 489.4 (1.9 mM) | >800 (3.1 mM) | 541.6 (2.1 mM) |
MCF-7 | >400 (1.1 mM) | >400 (1.4 mM) | >400 (1.4 mM) | >400 (1.4 mM) | 241.2 (0.9 mM) | 360.9 (1.4 mM) | 818.7 (3.2 mM) | 917.0 (3.6 mM) |
BALB/3T3 | >400 (1.1 mM) | >400 (1.4 mM) | >400 (1.4 mM) | >400 (1.4 mM) | >800 (3.1 mM) | >800 (3.1 mM) | >800 (3.1 mM) | >800 (3.1 mM) |
The IC50 value is the concentration required to reduce cell viability by 50%; therefore, the lower the IC50 concentration, the higher the cytotoxicity of the tested compound. The lowest IC50 values were observed in MCF-7 cells at 24 h and 48 h after treatment with ketoprofen, and in Hela and HT-29 cells at 48 h after treatment with lornoxicam. The remaining IC50 values were higher than the maximal concentration used in the present study (Table
The blood plasma concentration of ketoprofen in therapeutic doses according to
Cytomorphological changes at the cell and nucleus level that occurred after treatment with NSAIDs visualized by live/dead AO/EtBr staining are presented in Fig.
Fluorescence microscopy of human tumor cells HeLa, HT-29, and MCF-7 treated with the NSAIDs meloxicam, lornoxicam, ketoprofen, and dexketoprofen. Control (a–c); HeLa cells (a, d, g, j, m); HT-29 cells (b, e, h, k, n); MCF-7 cells (c, f, i, l, o); Meloxicam (d–f); Lornoxicam (g–i); Ketoprofen (j–l); Dexketoprofen (m–o); Lens 40X; AO/EtBr staining.
Control HeLa tumor cells were slightly elongated in shape, monolayered, uniformly green in color, with one or more bright green nuclei, and perinuclear orange-colored granules (Fig.
Control-untreated HT-29 tumor cells were round, with monolayer and three-dimensional growth (glandular-like structures), uniformly green with one or more light green nuclei and light orange granules in the cytoplasm (Fig.
Control untreated MCF-7 tumor cells were elongated, uniformly green in color with several light green nuclei and light orange granules in the cytoplasm, and with monolayer growth (Fig.
To assess the nuclear morphology of NSAID-treated and untreated (control) tumor cells, fluorescent dye staining with 4‘,6-diamino-2-phenylindole (DAPI) was applied. The DAPI molecule can cross the intact cytoplasmic membrane, making it a suitable agent for studying the nuclear morphology of both living and fixed cells. The results are presented in Fig.
Fluorescence microscopy of human tumor cells HeLa, HT-29, and MCF-7 treated with the NSAIDs meloxicam, lornoxicam, ketoprofen, and dexketoprofen. Control (a–c); HeLa cells (a, d, g, j, m); HT-29 cells (b, e, h, k, n); MCF-7 cells (c, f, i, l, o); Meloxicam (d–f); Lornoxicam (g–i); Ketoprofen (j–l); Dexketoprofen (m–o); Lens 40X; DAPI staining.
The nuclei of control untreated HeLa tumor cells were intact, approximately uniform in shape and size, with smooth edges and homogeneously distributed chromatin (Fig.
Control HT-29 tumor cells had intact nuclei, uniform in shape and size, with smooth edges, and evenly distributed nuclear chromatin (Fig.
Control MCF-7 tumor cells had intact nuclei, uniform in shape and size, with smooth edges, and uniformly distributed nuclear chromatin, with one to several nucleoli in the nucleus. There were nuclei of cells in mitosis (Fig.
The migration of human tumor cells treated with NSAIDs was studied using an in vitro wound healing method in which scrape wounds were induced in monolayer cell culture. The results are presented in Fig.
The migration of HeLa tumor cells cultivated in the presence of NSAIDs was inhibited with the best effect for meloxicam and dexketoprofen at 24 h (39.59 ± 3.37% and 45.17 ± 2.41% respectively). At the same time 88.92±1.35% of the wound area was filled in control cultures. At 48 and 72 h the highest inhibitory effect was observed for dexketoprofen (65.26 ± 2.09% and 81.04 ± 1.94% respectively). The decrease in the HeLa cell migration capacity in the presence of lornoxicam was much weaker. At 24 h, the wound was filled to 82.18 ± 1.93%. Ketoprofen did not affect the migration of HeLa cells and the values were the same as in the control (100% wound healing).
Statistically significant inhibition of HT-29 cell migration was observed after treatment with lornoxicam, ketoprofen, and dexketoprofen. In the control cell cultures, the migration percentages for the three-time intervals were 14.05 ± 0.66%, 25.81 ± 0.90%, and 27.43 ± 0.50%. For lornoxicam, the migration percentages were 12.34 ± 9.57%, 18.28 ± 0.99%, and 18.61 ± 0.62% at 24, 48 and 72 h, respectively. The effect of ketoprofen and dexketoprofen was well expressed in all three time intervals with corresponding values of 5.87 ± 0.7%; 6.10 ± 1.67%; 9.65 ± 0.95% and 7.50 ± 1.76%; 15.60 ± 1.90%; 17.98 ± 2.9%.
All four NSAIDs used showed statistically significant and time-dependent inhibition of MCF-7 cell migration at all time intervals studied. The migration percentages of the untreated cell cultures were 50.75 ± 0.65%, 86.49 ± 0.65%, and 100% for 24 h, 48 h, and 72 h. The highest inhibition was reported after treatment with lornoxicam (the migration percentages for the three time intervals were 3.85 ± 1.77%, 7.77 ± 1.49%, and 9.06 ± 1.59%, respectively). Approximately 5–6-fold lower inhibition of MCF-7 cell migration was observed for meloxicam (15.03 ± 0.54%; 39.75 ± 2.47%; 58.85 ± 2.98%, at the same time intervals). After treatment with dexketoprofen and ketoprofen, the highest inhibition of migration was established at 24 h – 20.21 ± 2.687 and 40.42 ± 1.61, respectively.
Meloxicam, lornoxicam, ketoprofen, and dexketoprofen inhibited the migration of the tumor cells. According to the results of the wound healing assay, the most sensitive to the NSAID treatment was MCF-7, followed by HeLa and HT-29 cells.
The results of the study showed that NSAIDs altered the migration of treated tumor cells. MCF-7 cells were the most sensitive to the NSAIDs, followed by HeLa and HT-29 cells. Dexketoprofen inhibited the migration of HeLa, MCF-7, and HT-29 tumor cell lines at 24, 48, and 72 h with the strongest effect in HeLa carcinoma cells. Lornoxicam decreased cell migration of the three lines, but at different time intervals (in HeLa at 24h and 48h; in HT-29 at 48 and 72 h) and it was the most active for MCF-7 tumor cells. Meloxicam inhibited the migration of HeLa and MCF-7 tumor cells and did not affect the migration of HT-29 cells. Ketoprofen was ineffective in HeLa cells but affected the migration of MCF-7 and HT-29 cells in the three-time intervals. NSAID-induced inhibition of tumor cell migration was the most pronounced in mammary carcinoma cells. The oxicams, meloxicam, and lornoxicam, induced stronger inhibition of MCF-7 tumor cell migration than the profens, ketoprofen, and dexketoprofen. In the present study, the cell viability after treatment with NSAIDs showed different degrees of reduction. Ketoprofen and dexketoprofen decreased the viability of the three tumor cell lines with the strongest effect on MCF-7. Lornoxicam and meloxicam had a more pronounced inhibitory effect on HT-29 and HeLa cells and had a little (meloxicam) and no effect (lornoxicam) on MCF-7. The lowest IC50 values were observed in MCF-7 cells at 24 and 48 h after treatment with ketoprofen and in Hela and HT-29 cells at 48 h after treatment with lornoxicam. Based on these findings, it could be concluded that breast cancer cells were more sensitive to the cytotoxic effects of profens, while the viability of cervical and colon carcinoma cells was more significantly affected by the oxicams. The IC50 values of the tested NSAIDs in the three tumor lines were higher than those obtained with diclofenac treatment in our previous studies and comparable to those found for metamizole in the same human tumor lines (
The obtained results for ketoprofen are also supported by
IC50=241.2 µg/mL at 24 h; IC50=360.9 µg/mL at 48 h, followed by HT-29 IC50=489.4 µg/mL at 48 h and HeLa – IC50=852.5 µg/mL and IC50=455.9 µg/mL at 24 and 48 h respectively.
A reduction of the tumor’s weight in ketoprofen-treated Wistar rats with ovarian cancer was shown and this effect was probably associated with an increase of apoptosis, inhibition of angiogenesis, reduction of cell proliferation, and biological modification of the tumor by inhibiting COX (
The in vitro antitumor effects of COX inhibitors may differ significantly between cancer cell lines, even when possessing similar COX-2 selectivity (
The NSAIDs meloxicam, lornoxicam, ketoprofen, and dexketoprofen exerted significant anticancer effects in in vitro models of some of the most common types of human epithelial neoplasms, expressed by decreased cell viability, inhibition of tumor cell proliferation and migration, and induction of apoptotic cell death. The different tumor cell lines showed different sensitivity towards the tested NSAIDs. Based on our results, it could be concluded that breast cancer cells were more sensitive to the cytotoxic effects of propionic NSAIDs, while the viability of cervical and colon carcinoma cells was more significantly affected by the oxicams. NSAID-induced inhibition of tumor cell migration was most pronounced in mammary carcinoma cells. The oxicams meloxicam and lornoxicam induced stronger inhibition of MCF-7 tumor cell migration than the propionic derivatives ketoprofen and dexketoprofen. The results of performed fluorescent microscopy studies indicated that the apoptotic morphological alterations were most clearly expressed in NSAID-treated breast cancer cells. The present results indicate that NSAIDs have anticancer potential and could be used together with conventional chemotherapeutics to develop new pharmacological strategies for the treatment and prevention of cancer.