Non-steroidal anti-inflammatory drugs are safe with respect to the transcriptome of human dermal fibroblasts

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) provide important benefits to millions of patients, but are associated with a number of serious adverse events. These adverse drug reactions are an important clinical issue and a serious public health risk. While most unfortunate responses in human to NSAIDs are mild and may disappear after decreasing the dose or withdrawal of the drug, some of them can produce serious outcomes. Currently, little is known regarding the effects of NSAIDs on global RNA expression in normal, non-transformed cells. Therefore, in this report, the effect of NSAIDs, COX-nonspecific and COX-2-specific inhibitors, indomethacin and nimesulide respectively, commonly used medications worldwide for the reduction of pain, fever, inflammation and stiffness, on transcriptomic signature of human dermal fibroblasts was investigated. A total of 3803 differentially expressed genes with a fold change greater than or equal to 1.3 and below than or equal to 0.7 for whole genome transcripts, with a P value of < 0.05 were identified in response to all applied conditions. We found that although the total number of deregulated genes was relatively high at such criteria, changes in fibroblast transcriptome profile after treatment at selected experimental conditions were however smallish, as the selected drugs slightly modulate transcriptome with only a few genes with expression altered a bit more than twice. Nevertheless, transcriptomic data has its own limitations and it cannot reflect all post-transcriptional changes, which in turn may cause same risks, especially for a long time of medication.

Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) are primarily used as analgesics, antipyretics and anti-inflammatory agents, which are among the most widely used medications worldwide (Conaghan, 2012). It is estimated that everyday about 30 million people consume NSAIDs (Sostres et al., 2013). The main mechanism of action of non-steroidal anti-inflammatory drugs is reduction of prostaglandins’ (PGs) biosynthesis by inhibition the activity of the cyclooxygenase (COX) enzymes. The production of prostaglandins is catalyzed by two COX isoenzymes, commonly referred to as COX-1 and COX-2 (Su and O’Connor, 2013). COX-1 is constitutively expressed in many tissues and its metabolic products are considered to be involved in cellular housekeeping functions. In contrast to COX-1, the expression of COX-2 is highly inducible by various inflammatory mediators and mitogens (Bernardi et al., 2006). Interestingly, COX-2 is significantly up-regulated in a variety of neurological disorders (e.g. Alzheimer's and Parkinson's diseases) and numerous types of human tumors, including colorectal, gastric, lung and breast cancers (Ettarh et al., 2010, Gasparini et al., 2004, Tian et al., 2012, Yang et al., 2016). Currently, a growing amount of evidence from numerous in vitro and in vivo experiments suggests positive effects of NSAIDs, used in combination with other therapies, in achieving additive or synergistic benefits in numerous cancers (Huerta et al., 2015, Liu et al., 2015, Özalp et al., 2012, Stolfi et al., 2013, Trask et al., 2004). However, the exact mechanism underlying cancer preventive and cancer therapeutic actions of NSAIDs is still largely unknown (Hilovska et al., 2014). Moreover, considering NSAIDs with other agents as a potential adjunctive approach for therapy of neuronopathic forms of lysosomal storage disorders (LSDs) appears reasonable (Jeyakumar et al., 2004, Mozolewski et al., 2017, Smith et al., 2009, Williams et al., 2014).

So far, the results from transcriptomic studies reported in the literature concern mostly human cancer cells treated with a high (typically used in preclinical studies) doses of cyclooxygenases inhibitors. Currently, little is known regarding the effects of NSAIDs on global RNA expression in normal, non-transformed cells. Therefore, the purpose of this study was to examine the effect of COX-nonspecific and COX-2-specific inhibitors, indomethacin (2-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetic acid) and nimesulide (N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide) respectively. The concentrations of indomethacin (2 and 10 µmol/l) and nimesulide (5 and 25 µmol/l) used in this work are close to clinically relevant molar doses (Dingle, 1999, Liu et al., 2015, Miura et al., 2004). We used transcriptomic approach to examine global gene expression changes, as well as cell viability and proliferation test to study development of fibroblasts exposed to different concentrations of tested compounds. We also analyzed cell cycle to investigate whether tested drugs can change DNA content in human dermal fibroblasts. Moreover, we tested whether the cell cycle is affected after treatment with selected NSAIDs in cells of patients suffering from mucopolysaccharidoses (MPS), according to recent studies concerning specific disturbances in the cell cycle of MPS type II fibroblasts (Moskot et al., 2016).