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Department of Molecular Microbiology and Immunology, Keck School of Medicine, K. Norris Jr. Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90089
Letter
With great interest did I read the article published in Cancer Research by Swamy et al. (1) , who investigated the effects of celecoxib on the expression and subcellular localization of the tumor suppressor p53. These authors report that treatment of the colon cancer cell lines HT-29 and HCT-116 with 100 µM celecoxib increases the accumulation of p53 in the nucleus. They ascribe this effect to inhibition of the cyclooxygenase (COX)-2 enzyme, which is a well-known target of celecoxib and inhibited by celecoxib, and they conclude that "Celecoxib protects p53 function by inhibiting COX-2 activity and production of PGs."
However, when viewing the presented data in the background of the available literature, I would suggest rather that the observed effects of celecoxib are quite likely independent of COX-2.
First, it is intriguing that the effects of celecoxib are only observed at 100 µM, but not at lower concentrations. It has been published by others that in vitro half-maximal inhibition (IC50) of COX-2 is achieved at low nanomolar concentrations (2)
and that even in whole cells, an efficient reduction of prostaglandin (PG) E2 production occurs at celecoxib concentrations of 
1 µM (3, 4, 5)
. In the case of HT-29 cells, it has been reported that 0.001 µM celecoxib reduced the elevated PGE2 levels induced by the ionophore A23187 by >50% (6)
. This same group further demonstrated that 50 µM celecoxib potently induced apoptosis in these cells and that, at higher concentrations (100 µM), some necrosis occurred as well (6)
. Performing experiments with the same cell line, others concluded that induction of apoptosis by 100 µM celecoxib was COX-2 independent (7)
. In general, it appears that at higher concentrations (>10 µM), celecoxib exerts its effects via targets other than COX-2, and a role for several other proteins has been suggested (see Refs. 8
and 9
for reviews). In this regard, 3-phosphoinositide-dependent protein kinase-1 (PDK1) has recently been identified as an additional direct target of celecoxib (10)
. The IC50 for inhibition of purified PDK1 is in the low micromolar range, whereas significantly higher concentrations of celecoxib are required for its inhibition in cell culture. Taken together, these reports suggest that cellular responses that are observed at high celecoxib concentrations (i.e., those above 10 µM), but not at low concentrations (i.e., those below 10 µM), are more likely due to COX-2-independent effects of this drug.
Second, there is another line of evidence that suggests that the effects observed by Swamy et al. (1)
were independent of COX-2. The current excitement about celecoxib and its potential use in cancer therapy was generated in large part by the observation that COX-2 is found to be elevated in many tumors and tumor-derived cell lines. However, in this regard, the choice of the two colon carcinoma cell lines, HT-29 and HCT-116, by Swamy et al. (1)
was somewhat unfortunate. As reported by Sheng et al. (11)
, HCT-116 cells lack COX-2 protein. Furthermore, these cells "produced no detectable PGE2, 6K-PGF1
, PGF2, PGD2, or TXB2 ... indicating a total absence of COX activity." (11)
. Thus, any responses to celecoxib in these cells are quite unlikely to be due to the inhibition of COX-2. Similarly, COX activity in HT-29 cells is problematic as well. Although expressed at high levels, COX-2 has been shown to be enzymatically inactive in these cells (12)
. Even though this latter finding may seem in conflict with the report mentioned further above with the use of A23187 (6)
, it is possible that this ionophore might generate particular conditions in these cells. In any case, the use of HT-29 cells to study COX-2 function appears problematic. Thus, taken together, these considerations would further argue that the effects of celecoxib observed by Swamy et al. (1)
were in fact independent of COX-2.
As an alternative, one could consider another potential target of celecoxib, namely, PDK1, as a more plausible candidate to mediate the observed effects of celecoxib on the nuclear accumulation of active p53. The inhibition of PDK1 by celecoxib in HT-29 cells has been shown to inactivate the Akt/protein kinase B survival pathway (10) . A series of recent reports (for a review, see Ref. 13 ) revealed that elevated Akt activity reduces the transcriptional activity of p53 via phosphorylation of Mdm2, which is a ubiquitin ligase that plays a central role in regulating the stability of p53. In response to direct phosphorylation by Akt, Mdm2 translocates to the nucleus, where it promotes p53 degradation. Conversely, the down-regulation of Akt/protein kinase B activity by increased PTEN activity protects p53 from Mdm2 and results in increased p53 levels and activity (14) . Thus, one could envision that celecoxib might affect p53 levels and activity via the inhibition of PDK1, which would result in reduced Akt activity, retention of Mdm2 in the cytoplasm, and, as a consequence, increased levels and activity of p53 in the nucleus. The use of high concentrations (100 µM) of celecoxib to achieve inhibition of PDK1 in HT-29 cells (10) , which is the same concentration used by Swamy et al. (1) that resulted in nuclear localization of active p53, would be in further support of such a scenario.
Received 10/ 6/03. Revised 10/31/03. Accepted 12/10/03.
REFERENCES
Chemoprevention Program, Institute For Cancer Prevention, American Health Foundation-Cancer Center, One Dana Road, Valhalla, NY 10595
We thank Axel Schönthal for his interest in and suggestions regarding our paper (1) . Our study regarding the effect of celecoxib on p53 localization and activity focused on the potential effect of electrophilic prostaglandins, known to be produced by cyclooxygenase (COX) enzymes, because COX-2 activities are elevated in colon cancer, and celecoxib is a selective inhibitor of this enzyme (1 2 3) . We selected colon cancer cell lines with different p53 status (HCT-116 is p53 wild type, and HT-29 is p53 mutant) to address whether p53 functionality influenced the effect of celecoxib on p53 cellular localization. Our results indicate that it does not (1) .
Some of the published observations suggest that although COX-2 levels are increased in HT-29 cells, the enzyme is nevertheless inactive, even though it becomes active when the HT-29-derived clone is transfected into HCT-116 cells (4) . We would argue that it is uncertain that COX-2 is completely inactive in HT-29 but that COX-2 may have a relatively reduced activity due to some nongenetic mechanism (4 , 5) . Accordingly, a number of recent investigations involving COX-2 expression and activity have used HT-29 cells (5 6 7 8) . Similarly, it is equivocal that HCT-116 cells completely lack COX-2 because there has been no mechanistic determination made to date (9 10 11) . As such, low levels of COX-2 and prostaglandins could be further reduced and account for the effect of high celecoxib levels on p53 in these cell lines. As we describe in the paper, electrophilic prostaglandins impede the nuclear localization and therefore the activity of p53 as a transcription factor. Our results provide evidence that this effect can be prevented through the selective inhibition of COX-2 by celecoxib (1) . This particular effect may be influenced by unknown interactions of celecoxib with other cellular pathways; however, this remains to be shown. It was also noted that levels of celecoxib such as those used in our study induced apoptosis in HT-29 cells in a previous study by Yamazaki et al. (12) . However, no significant apoptosis was observed in our assays. We attribute this apparent discrepancy to the short-term nature of our assays and measurements, which were not designed to address the induction of apoptosis by celecoxib.
We do not disagree with evidence showing that alternative (non-COX-2) pathways are also likely to contribute to the inhibitory effects of celecoxib on tumor cell growth. The effect of celecoxib on p53 localization may also be, in part, COX-2 independent, but we tend to disagree with the notion suggested by Schönthal (13) that inhibition of the phosphatidylinositol 3'-kinase/Akt pathway accounts for the effect that we observed. If this were the case, then we should have observed a noticeable increase in total cellular p53 levels after celecoxib treatment because p53 accumulates as a consequence of decreased Akt and a subsequent decrease in MDM2-mediated degradation of p53 (14 , 15) . In fact, p53 levels were moderately reduced after our treatments with celecoxib, although a significant percentage was redistributed in the nucleus, as we have described previously (1) . Furthermore, to address whether alternative pathways contribute in a significant way to the effects of celecoxib on tumor inhibition and molecular targets, we would argue that selective inhibitors of these alternative pathways be used in direct comparison with celecoxib.
Received 1/30/04. Accepted 2/19/04.
REFERENCES
This article has been cited by other articles:
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  A. H. Schonthal, C. R. Herzog, M. V. Swamy, and C. V. Rao Correspondence re: M. V. Swamy et al., Inhibition of COX-2 in Colon Cancer Cell Lines by Celecoxib Increases the Nuclear Localization of Active p53. Cancer Res 2003;63:5239-42. Cancer Res., April 15, 2004; 64(8): 2937 - 2938. [Full Text] [PDF]
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