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Abstract. – OBJECTIVE: Osteosarcoma (OS), an aggressive malignancy, is the most common primary bone tumor in children. Nonsteroidal an-ti-inflammatory drugs (NSAIDs) are widely used to reduce pain and inflammation. NSAIDs have shown to be toxic to certain malignancies such as colorectal, breast, and pancreatic cancers, but are not well-studied in OS. The purpose of this study is to assess whether ketorolac induces apoptosis in OS cells, compare this to indometh-acin, which has been shown to inhibit OS prolif-eration, and explore the underlying mechanism.

MATERIALS AND METHODS: A rat OS cell line (UMR-108) was exposed to various concen-trations of ketorolac and indomethacin. Cell vi-ability, cytotoxicity, apoptosis induction, DNA fragmentation and the expression of apopto-sis-related markers were examined by MTT as-say, colony formation assay, flow cytometry, agarose gel electrophoresis, and Western blot respectively.

RESULTS: The results indicated that ketoro-lac and indomethacin could induce apoptosis of rat OS cells in a dose- and time-dependent man-ner. Apoptosis was confirmed by cell morphology and annexin positivity. The molecular data showed that NSAIDs affected expression of Bcl-2, survivin, and Poly (ADP-ribose) polymerase-1 (PARP).

CONCLUSIONS: These findings demonstrat-ed that NSAIDs induced apoptosis in rat OS cells in vitro. Further research focusing on the poten-tial cytotoxicity of NSAIDs in vivo is needed.

Key Words:Osteosarcoma, Non-Steroidal Anti-Inflammatory Drugs

(NSAIDs), Ketorolac, Indomethacin, Apoptosis.

Introduction

Osteosarcoma (OS) is the most common pri-mary malignant bone tumor and typically affects

the metaphyses of the distal femur, proximal tibia, or humerus in children and adolescents1,2. Stan-dard treatment for OS includes chemotherapy and surgery. Even though there has been a significant improvement in mortality, metastatic disease still occurs, which is the major cause of mortality in patients with OS3,4. Currently, the prognosis of patients with OS is 75% to 77% when no disea-se is detected. Without chemotherapy, the 5-year survival rate is no more than 20%5-9. Unfortu-nately, survival rates have remained unchanged over the past several decades as no new effective drug regimen has been developed. Non-steroidal anti-inflammatory drugs (NSAIDs) are frequent-ly used to reduce postoperative pain and chronic inflammation in orthopaedic patients worldwi-de10. NSAIDs block the synthesis of prostaglan-dins, particularly prostaglandin E2 (PGE2) in the cyclooxygenase (COX) pathway11. PGE2 has been shown to promote tumor cell growth and invasive-ness and to contribute to tumor angiogenesis12,13. NSAIDs can cause antitumor activity in different cell populations by inhibiting their growth capa-city and reducing cell migration, which may re-sult in a potential benefit of these drugs in cancer treatments13-15. Multiple types of sarcomas, inclu-ding OS, Ewing’s sarcoma and chondrosarcoma, express cyclooxygenase-2 (COX-2)16-18. Inhibition of COX-2 with NSAIDs such as meloxicam and celecoxib, have been shown to be toxic to OS cel-ls in vitro19-21. Indomethacin blocks the activity of both cyclooxygenase-1 (COX-1) and COX-2, although its greatest effect is on COX-1. Indo-methacin has been shown to have an effect on the osteoblastic proliferation of the human MG-63 OS cell line10. In addition to this, preclinical stu-dies22-25 have established that indomethacin has significant efficacy against colon, breast and lung

European Review for Medical and Pharmacological Sciences 2019; 23: 2681-2690

L.M. ZUCKERMAN1, W.L. FRAMES1, J.G. ELSISSY1, T.G. SHIELDS1, R. DE NECOCHEA-CAMPION3, H.R. MIRSHAHIDI2, N.L. WILLIAMS1, S. MIRSHAHIDI3

1Department of Orthopaedic Surgery, Loma Linda University Medical Center, Loma Linda, CA, USA2Division of Hematology/Oncology, Loma Linda University School of Medicine, Loma Linda, CA, USA3Loma Linda Cancer Center Biospecimen Laboratory, Loma Linda University Medical Center, Loma Linda, CA, USA

Corresponding Author: William L. Frames, MD; email: wframes@llu.edu

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cancers. Ketorolac is a non-selective COX inhi-bitor of COX-1 and COX-2, has potent analgesic affects, and is given perioperatively, intraopera-tively and postoperatively to reduce moderate to severe surgical and cancer related pain26. Despite a lack of inhibition of serotonin and opioid recep-tors, ketorolac treatment confers opioid level pain relief more potent than many opioids27. Ketorolac is non-habit forming and patients do not develop tolerance. Ketorolac’s cost effectiveness relative to opioids or steroids, makes it a popular choice for alternative short term pain relief 28. It has been suggested that there is an association between the use of ketorolac and better outcomes after ma-stectomy for breast cancer and with ovarian and lung cancer29-31. However, the effect of ketorolac has not been studied in OS. This study investi-gated the effects of ketorolac and indomethacin and the underlying antitumor mechanisms in rat OS cells.

Materials and Methods

Cell Culture and ReagentsA rat OS cell line UMR-108 (ATCC CRL-1663)

was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) for this study. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicil-lin G, 100 mg/mL streptomycin and 1% nonessen-tial amino acids. Cell cultures were maintained in a humidified incubator at 37°C with 5% CO2. Ke-torolac (30 mg/ml) and indomethacin (2 mg/ml) were purchased from McKesson Medical-Surgi-cal Inc. (Chino, CA, USA).

Cell Viability AssayThe UMR-108 cell line was seeded in triplicate

at a density of 4000 cells/well in 96-well plates. After cell confluence reached 80%, the cells were treated at various time points between 24 and 74 hours at 37°C with various concentrations of keto-rolac and indomethacin. The untreated group was placed in phosphate-buffered saline (PBS) with a pH of 5.5 and 100% ethanol in order to control for the pH and microenvironment of the treated cells. Cell viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT reagent, Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s pro-tocol. The plates were read, and the absorbance at 570 nm was measured on a microplate reader

(Bio-Rad Model 3550, Hercules, CA, USA). The background death of the untreated group was subtracted from the treated groups. The results were expressed as a percentage of the untreated control (% of control). Each experiment was done in triplicate.

Microscopic Observation of Cell Morphology

Morphologic changes in tumor cells were exa-mined by phase-contrast photomicrographs at 24, 48 and 72 hours after exposure to various concen-trations of ketorolac (0.93, 1.87, 3.75 mg) and in-domethacin (0.125, 0.250, 0.500, 1 mg). Apoptotic cells were characterized by cell shrinkage and de-tachment from the plates.

Clonogenic Survival AssayUMR-108 cells were exposed to various con-

centrations of ketorolac (0.93, 1.87, 3.75 mg) and indomethacin (0.125, 0.250, 0.500, 1 mg) for 24 and 48 hours. Then, 1000 cells from each time point were seeded in 6-well plates. After 12-14 days the colonies were stained with crystal vio-let, and the number of colonies was counted. Each experiment was done in triplicate.

Analysis of Apoptosis and Cell CycleApoptotic cells were evaluated by an An-

nexin-V (Ann) and propidium iodide (PI) apop-tosis detection kit (BD Biosciences, Franklin La-kes, NJ, USA). 1x105 cells were seeded in 6-well plates, and when confluence reached 80%, they were exposed to medications at various concen-trations. After 24 and 72 hours, both floating and attached cells were harvested and washed. Flow cytometry with FITC-conjugated Ann/PI double staining was used to assess the number of apopto-tic cells. Samples were analyzed by flow cytome-try (MACSQuant, Miltenyi Biotech, Auburn, CA, USA). The results were displayed as 4 quadrants, in which the lower right quadrant represented the apoptosis rate during the early stages, the upper right quadrant indicated advanced apoptosis rate, the upper left quadrant represented dead cells, and the lower left quadrant represented living cells. The apoptotic rate was calculated as early apop-tosis (Ann+/PI-) and late apoptosis (Ann+/PI+) using the FlowJo software (TreeStar, Ashland, OR, USA). This experiment was repeated 3 times.

DNA Fragmentation on Agarose GelsUMR-108 cells were plated in 12-well plates,

and after 24 hours cells were exposed to keto-

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rolac (0.93, 1.87, 3.75 mg) and indomethacin (0.125, 0.250, 0.500, 1 mg) for 48 hours. All of the cells, including floating cells, were harvested by trypsinization and washed with Dulbeccò s Phosphate Buffered Saline. The genomic DNA was extracted with a DNA extraction kit (Qui-ck-g DNA miniprep kit, Zymo Research, Irvine, CA, USA) as described by the manufacturer. The DNA from each group was measured by Nano-Vue (GE Healthcare, Chicago, IL, USA) and 0.4 μg was then applied to 1.5% agarose gels contai-ning μg/ml ethidium bromide. A DNA molecular marker (1 kb DNA step ladder, Promega, Ma-dison, WI, USA) with the DNA from apoptotic UMR-108 cells induced by H2O2 (0.4 mM) was used as a positive control. The DNA fragmenta-tion pattern was examined in photographs taken under UV illumination.

Western Blot AnalysisUMR-108 cells were exposed to ketorolac (0.93,

1.87, 3.75 mg) and indomethacin (0.125, 0.250, 0.500, 1 mg) for 48 and 72 hours. Treated cells were harvested, washed and resuspended in NP-40 lysis buffer. Whole cell lysates were homoge-nized and the protein concentrations were deter-mined by using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (20 µg) from each sam-ple were loaded and separated through 10-12% sodium dodecyl sulfate polyacrylamide (SDS-PA-GE) gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bille-rica, MA, USA). The membranes were blocked with 5% BSA in PBST buffer for 1 hour at room temperature and incubated with primary antibo-dies overnight at 4°C. The following antibodies were used; Bcl-2, Poly(ADP-ribose) polymera-se-1 (PARP), caspase-3, β-Actin (Cell Signaling Technologies, Danvers, MA, USA) and survivin (Novus, Littleton, CO, USA). After washing with PBST, membranes were incubated with horsera-dish peroxidase (HRP) conjugated anti-rabbit IgG antibody (Cell Signaling Technologies, Beverly, MA, USA) for 1 hour at room temperature. The bands were visualized by an enhanced chemilu-minescence kit (Thermo Fisher Scientific, Wal-tham, MA, USA) according to the manufacturer’s instructions. The data was normalized to corre-sponding values of β-actin densitometry.

Statistical AnalysisEach assay was performed at least three times

as independent experiments. Statistical analyses

were performed with ANOVA and done with Pri-sm 5.01 software (GraphPad Software, La Jolla, CA, USA). A p-value of

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NSAIDs Induced Apoptosis in Tumor Cells Quantitative analysis showed that total apop-

totic cells (Ann+) increased significantly when cells were exposed to the different doses of both ketorolac and indomethacin (Figure 4). The total apoptotic cells significantly increased from 10% to 32% after 24 hours and from 27% to 87% after 72 hours of exposure to ketorolac. At 24 hours, the apoptotic cells increased from 7% to 48% and from 16% to 79% after 72 hours of exposure to indomethacin. This was again found to occur in a time- and dose-dependent manner when compa-red to untreated tumor cells.

NSAIDs Induced Apoptosis is Characterized by the Degradation of DNA

One of the hallmarks of apoptosis is the degra-dation of nuclear DNA into nucleosomal units, which is demonstrated as a ‘DNA ladder’ by gel 32. We further ascertained the drug-induced apop-tosis by DNA gel electrophoresis at 48 hours. As shown in Figure 5, both drugs significantly incre-ased the intensity of the fluorescence of ethidium bromide. A significant increase in the DNA frag-mentation occurred in a dose-dependent manner.

NSAIDs Modulate the Expression of Apoptosis Related Proteins

To further elucidate the mechanisms of ke-torolac- and indomethacin-induced apoptosis in OS cells, we evaluated the changes in expression of proteins involved in apoptosis such as Bcl-2, survivin, caspase-3 and PARP by Western blot analysis. UMR-108 cells were exposed to various

doses of ketorolac and indomethacin. After 48 and 72 hours, total protein was isolated. The densito-metry analysis demonstrated that treatment with ketorolac and indomethacin significantly reduced the level of Bcl-2 in UMR-108 cells in a dose-de-pendent manner (Figure 6A). The time-dependent effect was more pronounced at 72 hours when compared to 48 hours (***p

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reducing cell migration, which inhibits metasta-sis33. It has also been shown that NSAIDs are ca-spase inhibitors34. The results of this study showed that ketorolac and indomethacin affect caspase-3 in OS. Caspase-3 is involved in both the intrinsic and extrinsic apoptotic pathway35. When caspa-se-3 is activated, PARP cleavage occurs. The cle-avage of PARP causes the enzyme to lose much of its DNA binding capacity36,37. This degradation of PARP also affects DNA repair38. These results demonstrate that the effect of NSAID’s on OS is likely in the apoptotic pathway. These findings are furthered by the effect of NSAID’s on survivin in this study. Survivin inhibits both the intrinsic and extrinsic apoptotic pathways39. It has been shown

that DNA damage results in down-regulation of survivin and apoptosis40. Survivin has also been found to target caspase-3 which further disrupts the apoptotic pathway41. Bcl-2 was also affected by both NSAIDs in this study. Bcl-2 has a diffe-rent role in apoptosis compared to survivin. Whe-reas survivin inhibits caspases, which complete apoptosis, Bcl-2 prevents the initiation of apop-tosis42. Bcl-2 protects cell membrane integrity by interacting with mitochondrial proteins to prevent the opening of the mitochondrial permeability transition pore and the release of apoptogenic fac-tors43. Scholars10,17 have shown that COX inhibi-tion in certain malignancies results in impaired tumor cell proliferation, migration, and metasta-

Figure 2. NSAIDs decrease colony formation capacity of OS tumor cells. UMR-108 cells were exposed to ketorolac and indo-methacin at various concentrations for A) 24 hours and B) 48 hours. Colonies were stained with crystal violet after 12-14 days, and the number of colonies was counted. Error bars indicate the standard deviation of the mean of three independent experiments performed in triplicate (***p

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Figure 3. NSAIDs induce abnormal morphological changes in OS tumor cells. UMR-108 cells were treated with ketoroloc and indomethacin at various concentrations. Morphologic changes in tumor cells were examined by phase-contrast photomicrograph after 24, 48 and 72 hours. Cells exhibited abnormal morphology characterized by cellular shrinkage, turning round, floating and eventually death when compared to untreated tumor cells.

Figure 4. Flow cytometric analysis of NSAIDs on OS tumor cells. UMR-108 cells were exposed to ketorolac and indometha-cin at various for 24 and 72 hours. Both floating and attached cells were harvested and then washed. Flow cytometry with FITC conjugated Annexin-V/propidium iodide double staining was used to assess the number of apoptotic cells and compared to the untreated group using the FlowJo software. Error bars indicate the standard deviation of the mean of three independent experi-ments performed in triplicate (***p

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Figure 5. Effect of NSAIDs on the DNA fragmentation in OS tumor cells. UMR-108 cells were exposed to ketorolac and indo-methacin at various concentrations for 48 hours. The genomic DNA was applied to 1.5% agarose gels containing mg/ml ethidium bromide. DNA step ladder (1 kb) and the DNA from apoptotic UMR-108 cells induced by H2O2 (0.4 mM) were used as a positive control. The DNA fragmentation pattern was examined in photographs taken under UV illumination. Error bars indicate the stan-dard deviation of the mean of three independent experiments performed in triplicate (***p

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tic potential. Our data supports and adds to these findings. The NSAIDs were found to act in both a time- and dose-dependent fashion to inhibit co-lony formation, induce abnormal cell morphology and induce apoptosis. This was observed direct-ly by the analysis of the cell shape, as well as by DNA fragmentation assay and cell viability stu-dies. The demonstration that the NSAIDs affect proliferation, migration, and induce apoptosis can carry clinical relevance and potentially help to direct future studies. A weakness of the study includes the fact that this is an in vitro study. The behavior of medications in the in vivo setting can-not be assumed based on in vitro studies. Further in vivo studies should be performed in order to evaluate the effect as well as proper dosing of the medications in order to achieve a similar effect. The use of a cell line is both an advantage and disadvantage. The cell line allows for consistency and reproducibility of the findings. The problem with a cell line, in regards to OS, is that OS has multiple subtypes44. These subtypes have varying responses to chemotherapy and differing survival rates45. This heterogeneity and varying response to treatment between patients make it difficult to fully extrapolate the results to a clinical setting. NSAIDs are well studied, inexpensive and readily available medications with known side effect pro-files10. Due to this, translational research can be performed without having to develop new medi-cations or testing for toxicity. Targeted therapies could be developed based on the mechanism of action found and be utilized as well. The medica-tions also help decrease inflammation and pain, which can assist the cancer-related pain that OS patients endure.

Conclusions

The findings of this study support the role for evaluating NSAIDs in the clinical setting. If a translatable effect is demonstrated, the medica-tions could be considered as both a neoadjuvant and adjuvant treatment.

Conflict of InterestThe Authors declare that they have no conflict of interest.

DisclosuresLee M. Zuckerman - consultant to NuVasive Specialized Orthopedics.

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