Elsevier

Biochemical Pharmacology

Volume 75, Issue 2, 15 January 2008, Pages 395-404
Biochemical Pharmacology

Celecoxib transiently inhibits cellular protein synthesis

https://doi.org/10.1016/j.bcp.2007.08.029Get rights and content

Abstract

To uncover the full spectrum of its pharmacological activities, the selective COX-2 inhibitor celecoxib is routinely being used at concentrations of up to 100 μM in cell culture. At these elevated concentrations, several COX-2-independent effects were identified, although many details of these events have remained unclear. Here, we report a COX-2-independent effect of celecoxib that might have profound consequences for the interpretation of previous results obtained at elevated concentrations of this drug in vitro. We found that celecoxib rapidly inhibits general protein translation at concentrations as low as 30 μM. This appears to be a consequence of endoplasmic reticulum (ER) stress and entails the phosphorylation and inactivation of eukaryotic translation initiation factor 2 alpha (eIF2α). These effects were not achieved by other coxibs (rofecoxib, valdecoxib) or traditional NSAIDs (indomethacin, flurbiprofen), but were mimicked by the COX-2-inactive celecoxib analog, 2,5-dimethyl-celecoxib (DMC), indicating COX-2 independence. Considering the obvious impact of blocked translation on cellular function, we provide evidence that this severe inhibition of protein synthesis might suffice to explain some of the previously reported COX-2-independent effects of celecoxib, such as the down-regulation of the essential cell cycle regulatory protein cyclin D, which is a short-lived protein that rapidly disappears in response to the inhibition of protein synthesis. Taken together, our findings establish ER stress-induced inhibition of general translation as a critical outcome of celecoxib treatment in vitro, and suggest that this effect needs to be considered when interpreting observations from the use of this drug in cell culture.

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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    Both authors contributed equally to this work.

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