Int J Cancer Manag. 2017 April; 10(4):e7565. doi: 10.5812/ijcm.7565. Published online 2017 April 22. Research Article Anti-Proliferative Effects of Piroxicam and Nimesulide on A431 Human Squamous Carcinoma Cell Line Faezeh Khodaie,1 Yalda Khazaei-Poul,1 and Taraneh Moini-Zanjani1,* 1Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran *Corresponding author: Taraneh Moini-Zanjani, Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Koodakyar Street, Daneshjoo boulevard, Velenjak, Tehran, IR Iran. Tel/Fax: +98-21-22439969, Tel: +98-9123497094, E-mail: [email protected] Received 2016 June 15; Accepted 2017 March 08. Abstract Background: Skin cancer is one of the most common types of cancer worldwide and non-steroidal anti-inflammatory drugs (NSAIDs) have been proposed for prevention and treatment of a variety of cancers. Objectives: In this study we aimed to evaluate the cytotoxic effects of piroxicam (a non-selective cyclooxygenase (COX) inhibitor) and nimesulide (a highly selective COX-2 inhibitor) on A431 human squamous carcinoma cell line. Methods: Squamous carcinoma cell line (A431) was cultured in RPMI medium containing 10% FBS and penicillin-streptomycin at 37°C and 5% CO2. Cells were treated with different concentrations of piroxicam and nimesulide (100 - 1000 µmol/L) for 24, 48 and 72 hours (h). Anti-proliferative effects were determined using MTT colorimetric assay. Results: Piroxicam and nimesulide reduced cell viability in a time and concentration dependent manner. The most cytotoxic effect was produced in 72 hours incubation time. The IC50 value of nimesulide was significantly lower than piroxicam in 24 and 72 hours, but not in 48 hours treatment duration. Conclusions: This study demonstrates that the administration of a highly selective COX-2 inhibitor could probably be more effec- tive than a non-selective NSAID in reducing cancer cells proliferation and that COX-2 can possibly play an important role in skin cancer development. Keywords: Piroxicam, Nimesulide, Squamous Cell Carcinoma 1. Background zyme. Cyclooxygenase has two isoforms: cyclooxygenase 1 (COX-1) which has an important role in homeostasis, and Skin cancer is one of the most common types of can- cyclooxygenase 2 (COX-2) which is induced by inflamma- cer worldwide and squamous cell carcinoma (SCC) is the tory mediators (12). NSAIDs inhibit COX enzyme and nu- second most common form of skin cancer after basal merous studies have demonstrated their anti-proliferative cell carcinoma (BCC) (1-3). Treatments used for SCC in- effects on various cancer cell lines (13-19). Their mechanism clude surgical excision, topical and systemic chemother- of action is not well understood but COX dependent and apy (4). Some chemotherapeutic agents have been used independent pathways have been suggested to have an im- to treat SCC such as cisplatin, doxorubicin, 5-fluorouracil, portant role in tumor genesis (20). vincristine and bleomycin. Unfortunately, these drugs tar- Piroxicam is a non-selective COX inhibitor and its anti- get all cells with high proliferation rate in the body like proliferative effects on human breast, lung, urinary blad- bone marrow and hair follicles and cause many signifi- der, skin melanoma and colon cancer cell lines have been cant side effects (5). Other drugs have been demonstrated investigated (13-17). On the other hand, nimesulide is a to have anti-proliferative properties in cancer cells in ad- highly selective COX-2 inhibitor and its cytotoxic effects dition to their conventional therapeutic effects including have been evaluated in some cancer cell lines such as head some antibiotics like doxycycline and ciprofloxacin, some and neck squamous cell carcinoma, colon carcinoma and herbal extracts and non-steroidal anti-inflammatory drugs pancreatic cancer (21-23). (NSAIDs) (6-10). Chronic exposure to ultraviolet B (UVB) radiation is a 2. Objectives recognized etiology for skin cancer, one possible mech- anism is thought to be through an increase in the pro- Based on this background, the aim of the present study duction of prostaglandins (PGs) in the keratinocytes in re- was to evaluate the anti-proliferative effects of piroxicam sponse to UVB irradiation (11). Prostaglandins are synthe- and nimesulide on human squamous carcinoma cell line sized from arachidonic acid by cyclooxygenase (COX) en- A431and to compare the effectiveness of a non-selective Copyright © 2017, International Journal of Cancer Management. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. Khodaie F et al. with a highly selective COX-2 inhibitor on this cancer cell the plates were read in an absorbance reader (ELx800, line. BioTek, Winooski, VT, USA) at 590 nm. The concentration of drug needed to reduce proliferation of cancer cells by 50% was determined as inhibitory concentration 50 (IC ). 3. Methods 50 3.1. Reagents 3.4. Statistical Analysis Piroxicam, nimesulide and MTT (3-(4, 5- Data were analyzed using GraphPad Prism software dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro- (version 6) and Microsoft Excel (2013). Data were expressed mide) tetrazolium were obtained from Sigma-Aldrich as mean ± SEM within 95% confidence intervals. Determi- (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), nation of IC50 values were performed using nonlinear re- penicillin-streptomycin and trypan blue were pur- gression analysis. The mean differences were compared by chased from Merck (Darmstadt, Germany). Heat inac- the analysis of variance (ANOVA) and P value < 0.05 was tivated fetal bovine serum (FBS), RPMI 1640, trypsin- considered significant. ethylenediaminetetraacetic acid (trypsin-EDTA) and phosphate buffered saline (PBS) were provided from Gibco 4. Results (New York, USA). Cytotoxic effects of piroxicam and nimesulide on A431 3.2. Cell Culture squamous carcinoma cells were determined using MTT The human squamous carcinoma cell line A431 (Pas- colorimetric assay as described above. Cell viability was teur Institute, Tehran, Iran) was cultured in RPMI 1640 considered as 100% in controls to compare the cytotoxicity medium containing 10% FBS, 100 U/mL penicillin and 100 effects of treatment groups. Since there was no significant µg/mL streptomycin in a humidified and 5% CO2 incubator difference between DMSO with CTCM containing controls, at 37°C. Cell density was monitored by an inverted micro- treatment groups were compared to 0.25% DMSO control. scope until approximately 80% of confluency was attained. Piroxicam caused a decrease in cell proliferation in a Then the cells were trypsinized and centrifuged at 1500 time (24, 48 and 72 hours), and concentration (100 - 1000 rpm for 10 minutes. Cell count and viability were assessed µM/L) dependent manner (Figure 1A). A significant reduc- using the trypan blue staining assay. Cells stained with try- tion in cell viability was observed from 500 µM/L of pirox- pan blue (0.5% in NaCl) were transferred to the Neubauer icam in 24 hours treatment (P < 0.05); however, in 48 and chamber and counted by light microscope. Cell viability 72 hours treatment, the concentration of 100 µM/L of drug was calculated as approximately 98% (dark blue colored caused significant cytotoxic effects (P < 0.05 and P < 0.001, cells were defined as dead cells). respectively). Figure 1B shows the comparison of piroxicam IC50 val- 3.3. Cell Treatment and MTT Assay ues at different time points in cell proliferation assay, The anti-proliferative effects of piroxicam and nime- which demonstrates a significant decrease in IC50 values af- sulide were evaluated using the MTT assay (24). For this ter 48 and 72 hours compared to 24 hours piroxicam treat- purpose, 5 × 103 cells per well were seeded in triplicate in a ment (P < 0.01 and P < 0.001, respectively). Besides, IC50 96-well plate containing 200 µL of complete tissue culture value was significantly lower in 72 hours than 48 hours medium (CTCM) and were incubated for 24 hours (h) prior treatment (P < 0.05). to treatment. The drugs were dissolved in 100% DMSO In contrast to piroxicam, the minimum concentration as stock solution and then diluted with RPMI medium of nimesulide which significantly decreased A431 cell pro- with the final DMSO concentration of 0.25%. Drug solu- liferation was 250 µM/L in 24, 48 and 72 hours treatment (P tions were prepared freshly before the test. The A431 cells < 0.001) (Figure 2A). were treated with different concentrations (100, 250, 500, Figure 2B shows the IC50 values of nimesulide in differ- 750 and 1000 µM/L) of piroxicam and nimesulide for 24, ent time points. In 72 hours, the IC50 value was lower com- 48 and 72 hours. Cells treated with drug vehicle (DMSO pared to 24 and 48 hours treatment (P < 0.001 and P < 0.05, 0.25%) served as control. To determine the DMSO cytotox- respectively). However, there was no significant difference icity, a control group containing only CTCM was added. Af- between the IC50 value of 48 hours compared to 24 hours ter incubation, cells were washed with PBS and then incu- treatment assay (P > 0.05). bated with 100 µL of MTT (5 mg/mL dissolved in PBS) at The comparison of IC50 values between piroxicam and 37°C for 4 hours. The viable cells metabolized MTT to for- nimesulide is shown in Figure 3. The IC50 value of nime- mazan crystals and after dissolving the crystals in DMSO, sulide is significantly lower than piroxicam at 24 and 72 2 Int J Cancer Manag. 2017; 10(4):e7565. Khodaie F et al. A A 24 h 100 24 h 100 48 h 48 h 75 72 h 75 72 h 50 50 25 25 Cell Viability (% of Control) Cell Viability (% of Control) 0 0 250 500 750 1000 0 0 250 500 750 1000 Concentration, µM/L Concentration, µM/L B 900 B 500 800 700 400 600 500 300 400 300 200 200 Concentration, µmol/L Concentration, µmol/L 100 100 Time, h 0 Time, h 24 48 72 0 24 48 72 Piroxicam IC50 691.4 454.4 338.8 Nimesulide IC50 426.4 349.4 265.6 Figure 1.