Indomethacin and ibuprofen induce Hsc70 nuclear localization and activation of the heat shock response in HeLa cells Lucio Lagunas, a C. Matthew Bradbury, b Andrei Laszlo, a Clayton R. Hunt, a and David Gius b, * a Division of Radiation and Cancer Biology, Department of Radiation Oncology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO, USA b Radiation Oncology Branch, Radiation Oncology Sciences Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Received 25 November 2003 Abstract It has been established that non-steroidal anti-inflammatory drugs (NSAIDs), such as sodium salicylate, sulindac, ibuprofen, and indomethacin, induce anti-inflammatory and anti-proliferative effects independent of cyclooxygenase. These cyclooxygenase-inde- pendent pharmacodynamic effects appear to regulate several signaling pathways involving proliferation, apoptosis, and heat shock response. However, the mechanisms of these actions remain an area of ongoing investigation. Hsc70 is a cytoplasmic chaperone protein involved in folding and trafficking of client proteins to different subcellular compartments, plays roles in signal transduction and apoptosis processes, and translocates to the nucleus following exposure to heat shock. Since NSAIDs induce some aspects of the heat shock response, we hypothesized that they may also induce Hsc70 nuclear translocation. Western immunoblotting and indirect cellular immunofluorescence showed that indomethacin and ibuprofen induce Hsc70 nuclear translocation at concentrations pre- viously shown to induce HSF DNA-binding activity. Chemical inhibition of both p38 MAPK and Erk42/44 had no effect on locali- zation patterns. In addition, while indomethacin has been shown to behave as an oxidative stressor, the radical scavenging agent, N - acetyl cysteine, did not inhibit nuclear translocation. These results indicate that induction of the heat shock response by NSAIDs occurs at concentrations fivefold greater than those required to inhibit cyclooxygenase activity, suggesting a cyclooxygenase-in- dependent mechanism, and in the presence or absence of kinase inhibitors and a free radical scavenger, suggesting independence of Erk42/44 or p38 MAPK activities and intracellular oxidoreductive state. Published by Elsevier Inc. Keywords: Indomethacin; Ibuprofen; Hsc70; Nuclear localization Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of pharmacological agents that are traditionally used for their anti-cyclooxygenase properties in the treatment of inflammation and other associated illnesses [5,8,28,36]. Recently, however, it has been demonstrated that several NSAIDs, such as sulindac, ibuprofen, and indomethacin, exhibit anti-inflammatory as well as anti- proliferative effects independent of cyclooxygenase activity [38]. For example, NSAIDs affect multiple in- tracellular signaling pathways that: (1) inhibit the induction of NF-jB [3,41]; (2) induce injury-response genes in plants [26]; and (3) sensitize tumors to the cy- totoxicity of ionizing radiation [3,34]. Interestingly, the concentrations of NSAIDs necessary to initiate each one of these processes are similar and much greater than those reported for the inhibition of cyclooxygenase ac- tivity, suggesting a unique cyclooxygenase-independent pharmacodynamic effect of NSAIDs on specific intra- cellular processes. The cellular stress induced by exposure to elevated temperature, a phenomenon known as hyperthermia, also has profound effects on many aspects of cellular biochemistry, morphology, and physiology [9,10,24,30, 31,35]. The response to hyperthermia is evolutionarily well conserved across all species from bacteria to * Corresponding author. Fax: 1-301-480-5439. E-mail address: giusd@mail.nih.gov (D. Gius). 0006-291X/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.bbrc.2003.12.018 Biochemical and Biophysical Research Communications 313 (2004) 863–870 BBRC www.elsevier.com/locate/ybbrc