Celecoxib transiently inhibits cellular protein synthesis
Introduction
Celecoxib is widely prescribed under the trade name Celebrex® for relief of symptoms of osteoarthritis and rheumatoid arthritis and was also approved as an adjunct to standard care for patients with familial adenomatous polyposis (FAP). It is suspected that this drug might be useful for the prevention and treatment of colorectal and possibly other types of cancer, and several clinical trials are ongoing to confirm this expectation. In addition, celecoxib has demonstrated potent anti-cancer activity in various animal tumor models in the laboratory [1]. Despite these promising results, however, the underlying molecular mechanisms by which celecoxib exerts its anti-tumor potential are not completely understood. Particularly intriguing are a number of reports describing potent anti-proliferative and pro-apoptotic effects of this drug in the absence of any apparent involvement of COX-2 (see Ref. [2], for a review).
When the antitumor effects of celecoxib are studied in cell culture in vitro, concentrations in the range of 30–100 μM are generally required in order to achieve substantial growth inhibition or induction of apoptosis in tumor cells. On occasion, lower concentrations of the drug might be effective as well, although in those cases reduced serum concentrations in the cell culture growth medium or longer incubation times seem to be required [3], [4], [5], [6]. It has been well established that celecoxib and other coxibs are able to inhibit their main target, COX-2, at sub-micromolar concentrations in cell culture [7], [8], [9]. Remarkably, however, despite efficient inhibition of COX-2, cell proliferation and survival is not affected at these low concentrations. Quite in contrast, much higher concentrations are required to achieve antitumor effects in vitro. This discrepancy suggests that it is very unlikely that those effects of celecoxib that are observed only in the 30–100 μM range are related to the inhibition of COX-2. And indeed, when proper controls were being included in high-dosage in vitro experiments with celecoxib, it generally turned out that the respective effects did not involve the inhibition of COX-2 [2], [10], [11].
Those in vitro effects of celecoxib that are only observed in the 30–100 μM range are generally discarded as artifacts of the cell culture setting and not considered relevant for the in vivo setting, because such elevated drug concentrations cannot be achieved in the serum of patients or animals [12]. However, there are several recently published examples that clearly demonstrate that specific drug-induced processes, which only take place at 30–100 μM in vitro, can also be detected in tumor tissues from celecoxib-treated experimental animals [13], [14], [15]. For example, a minimum of 40–60 μM of celecoxib is required to inhibit the expression of the anti-apoptotic protein survivin and noticeably stimulate apoptosis in glioblastoma cell lines in culture. Nonetheless, and even though drug concentrations below 40 μM are ineffective in vitro, down-regulation of survivin expression and increased apoptosis can also be detected in xenograft glioblastoma tissue from celecoxib-treated animals, clearly indicating that the in vitro and in vivo processes are congruent in this case [15]. Although there is as yet no explanation as to this conundrum between effective in vitro and in vivo concentrations, such results caution against the prevalent tendency of minimizing those drug outcomes that were obtained at elevated celecoxib concentrations in vitro.
Several recent reports have indicated that treatment of cells with celecoxib leads to the activation of the endoplasmic reticulum (ER) stress response (ESR) [16], [17], [18], [19], [20], [21], [22], [23]. One of the features of ESR is a transient inhibition of overall cellular protein synthesis, which is achieved through the inactivation of eukaryotic translation initiation factor 2 alpha (eIF2α) [24], [25]. The intensity of ESR-induced translational attenuation can be relatively weak or very strong, depending on the ESR-inducing insult. Naturally, any pharmacologic intervention that interferes substantially with overall protein synthesis may have profound consequences for other processes that are affected by fluctuations in this basic cellular function. Therefore, we have investigated general protein translation in response to treatment of cultured cells with celecoxib. We found that commonly used concentrations of this drug severely (>90%) impaired cellular translation, and this took place similarly in cells expressing or not expressing COX-2 protein. Inhibition of translation involved phosphorylation (i.e., inactivation) of eIF2α, revealing a prominent role of ESR in generating this outcome. We investigated cyclin D protein as an example of a previously reported down-regulated target of celecoxib and found that mere inhibition of protein translation could account for the rapid down-regulation of this crucial cell cycle-regulator. Thus, our results reveal the inactivation of eIF2α as a critical mechanism mediating celecoxib's inhibitory effect on the cellular protein synthesis machinery, and indicate that inhibition of translation should be considered as a potentially significant factor during the interpretation of results obtained from the use of this drug in cell culture.
Section snippets
Materials
All coxibs and NSAIDs were obtained and used as described previously [15]. The synthesis of 2,5-dimethyl-celecoxib (DMC) was described in Ref. [26]. 35S-methionine (10 μCi/μl; 540 Ci/mmol) was purchased from MP Biomedicals, LLC (Solon, OH).
Cell lines and culture conditions
HeLa cervix carcinoma and PC-3 pancreatic carcinoma cell lines were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA). The glioblastoma cell line U251 was provided by Frank B. Furnari (Ludwig Institute of Cancer Research, La Jolla, CA).
Celecoxib transiently inhibits general translation
When studied in cell culture, celecoxib is most commonly used at concentrations ranging from 10 to 100 μM. In the vast majority of reported cases, the observed drug effects become most apparent towards the upper limit of this concentration range. To determine whether such concentrations might impinge on general protein synthesis, we treated the human glioblastoma cell line U251 with increasing concentrations of celecoxib and determined the ongoing rate of translation via the incorporation of 35
Discussion
Investigations with the use of elevated concentrations (10–100 μM) of celecoxib in vitro have been met with skepticism, because such high concentrations cannot be achieved in vivo. Nonetheless, it is remarkable that several of celecoxib's in vitro effects, which can only be detected at 30–100 μM, can also be verified in xenograft tumor tissue in vivo, where drug concentrations remain well below 5 μM. For instance, inhibition of 3-phosphoinositide-dependent protein kinase-1 (PDK1), down-regulation
Acknowledgements
We thank Frank B. Furnari, Guido Eibl, as well as members of the laboratories of Randal J. Kaufman and David Ron for providing cell lines. We are grateful to Bangyan Stiles for antibodies. The USC Glioma Research Group is acknowledged for stimulating discussions. Funding for this project was received from the Margaret E. Early Medical Research Trust (to AHS).
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Cited by (25)
Differential effects of cyclooxygenase-2 (COX-2) inhibitors on endoplasmic reticulum (ER) stress in human coronary artery endothelial cells
2022, Vascular PharmacologyCitation Excerpt :This work as well as prior research in our laboratory utilized primary HCAEC [16]. Since celecoxib has been reported to induce a transient decrease in protein synthesis as well as ER stress in tumor cells [27], we examined the effect of celecoxib and rofecoxib on both long-term (24-h) and short-term (6-h) tunicamycin treatment in HepG2 hepatoblastoma cells (Fig. 5). These studies demonstrated that celecoxib, but not rofecoxib, induced ER stress at 6-h but not at 24-h. Furthermore, both celecoxib and rofecoxib had no effect on tunicamycin-induced ER stress in these cells.
Glioblastoma and chemoresistance to alkylating agents: Involvement of apoptosis, autophagy, and unfolded protein response
2018, Pharmacology and TherapeuticsCitation Excerpt :GRP78 regulates UPR activation in the ER lumen by binding to the luminal domain of IRE1, PERK and ATF6 and keeping them in an inactive state. GRP78 is normally expressed at low levels in the normal adult brain (Pyrko, Kardosh, & Schonthal, 2008). However, higher grade gliomas, including GB, exhibit high expression of GRP78 (Lee et al., 2008; Prabhu, Sarcar, Kahali, Shan, & Chinnaiyan, 2012; Pyrko et al., 2008).
Targeting apoptosis pathways by Celecoxib in cancer
2013, Cancer LettersCitation Excerpt :At the same time, phospho-eIF2α stimulates the translation of a specific set of mRNAs, such as the transcriptional regulator activating transcription factor 4 (ATF4), resulting in the accumulation of proteins involved in cellular protection against injury, e.g. the ER chaperone GRP78 (glucose-regulated 78 kDa protein)/BiP, and apoptosis regulation, e.g. the transcription factor c/EBP homologous transcription factor (CHOP)/GADD153 (growth arrest and DNA-damage associated protein) [71,72]. The Celecoxib-induced ER-stress response involved the accumulation of ATF4, GRP78/BiP and CHOP/GADD153, a transient inhibition of protein translation, and an increase in the concentrations of cytosolic Ca2+, resulting in the induction of apoptosis [47,69,73]. In addition to the upregulated expression of pro-apoptotic CHOP/GADD153, the increase in the concentrations of cytosolic Ca2+ may contribute to the pro-apoptotic effects of Celecoxib by triggering the activation of Ca2+-sensitive proteases, endonucleases, and caspase-4 [46,70,73,74].
Anti-apoptotic Bcl-2 fails to form efficient complexes with pro-apoptotic Bak to protect from Celecoxib-induced apoptosis
2011, Biochemical PharmacologyCitation Excerpt :Celecoxib and the related OSU-03012 could also interfere with the PKB/Akt survival pathway [29,30]. Moreover, Celecoxib, but not the other coxibes Rofecoxib and Valdecoxib, can inhibit protein translation transiently with subsequent downregulation of short-lived proteins [31]. Previous results in our lab revealed that Celecoxib facilitated a rapid downregulation of the anti-apoptotic Mcl-1.
ERK/ribosomal S6 kinase (RSK) signaling positively regulates death receptor 5 expression through co-activation of CHOP and Elk1
2010, Journal of Biological ChemistryCitation Excerpt :Thus, blockade of ATF4 increase abolishes not only CHOP induction, but also DR5 up-regulation in cells exposed to celecoxib, indicating that celecoxib induces CHOP and DR5 expression through an ATF4-dependent mechanism. The effects of celecoxib on induction of ER stress, including CHOP and up-regulation of DR5, have been demonstrated in several previous studies (23, 25, 33, 37–40, 47). CHOP-dependent DR5 induction by celecoxib was also documented (23).
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Both authors contributed equally to this work.