Potential Misidentification of Cyclooxygenase-2 by Western Blot Analysis and Prevention Through the Inclusion of Appropriate Controls

Abstract

Cyclooxygenase (COX)-2 plays an important role in the development of cancer and has been recognized as a potential therapeutic target. Because nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the activity of this enzyme, the potential efficacy of such drugs for purposes of cancer prevention or therapy is an area of intense research. Therefore, it is of critical importance to unequivocally determine the expression levels of COX-2 protein in tumor cells. In this regard, there are several conflicting reports in the literature where the same type of tumor cell lines were reported as COX-2 positive and as COX-2 negative. We found that during Western blot analysis of COX-2 positive and COX-2 negative cells, different antibodies to COX-2 protein are able to generate strong signals, which are false-positives and can be confused with COX-2. Thus, we believe that some of the conflicting reports on COX-2 expression in tumor cell lines could be the result of improper interpretation of the Western blot signals. Here, we present some of these pitfalls and suggest the inclusion of appropriate controls to unequivocally identify COX-2 protein levels.

Neuronal differentiation by indomethacin and IBMX inhibits proliferation of small cell lung cancer cells in vitro

“…3D and H) [23]. Furthermore, the marked sensitivity of H82 cells for indomethacin treatment can be explained by the high level of COX-2 expression in H82 cells being compared with that of SW2 cells (COX-2 is expressed in the non-glycosylated form, which has been reported to migrate at 64 kDa [24,25], Fig. 5), whereas COX-1 expression showed no differences (Fig.…”

Aggravated Endoplasmic Reticulum Stress as a Basis for Enhanced Glioblastoma Cell Killing by Bortezomib in Combination with Celecoxib or Its Non-Coxib Analogue, 2,5-Dimethyl-Celecoxib

“…LN229 and U251 were obtained from Frank B. Furnari (Ludwig Institute of Cancer Research, La Jolla, CA). COX-2 expression levels and drug effects on prostaglandin production in these cells have been published elsewhere (17,19).…”

“…The underlying molecular mechanisms by which celecoxib exerts its antitumor effects are not entirely understood, in particular due to an increasing number of reports describing potent antiproliferative and proapoptotic effects of this drug in the absence of any apparent involvement of COX-2 (15)(16)(17)(18)(19)(20)(21)(22). One of several lines of evidence excluding the potential contribution of COX-2 came from the use of 2,5-dimethyl-celecoxib (DMC), a close structural analogue of celecoxib that lacks the ability to inhibit COX-2 activity (23).…”

Induction of Apoptosis by Celecoxib in Cell Culture: An Uncertain Role for Cyclooxygenase-2

“…For example, as shown in Fig. 1, BxPC-3 and MIA-PaCA-2 pancreatic carcinoma cells responded nearly identically to these drugs, even though COX-2 protein is absent in the former and was expressed at very high levels in the latter (5).…”