The anti-proliferative potency of celecoxib is not a class effect of coxibs.

Celecoxib, a COX-2 (cyclooxygenase-2)-selective inhibitor (coxib), is the only NSAID (non-steroidal anti-inflammatory drug) that has been approved for adjuvant treatment of patients with familial adenomatous polyposis. To investigate if the anti-proliferative effect of celecoxib extends to other coxibs, we compared the anti-proliferative potency of all coxibs currently available (celecoxib, rofecoxib, etoricoxib, valdecoxib, lumiracoxib). Additionally, we used methylcelecoxib (DMC), a close structural analogue of celecoxib lacking COX-2-inhibitory activity. Due to the fact that COX-2 inhibition is the main characteristic of these substances (with exception of methylcelecoxib), we conducted all experiments in COX-2-overexpressing (HCA-7) and COX-2-negative (HCT-116) human colon cancer cells, in order to elucidate whether the observed effects after coxib treatment depend on COX-2 inhibition. Cell survival was assessed using the WST proliferation assay. Apoptosis and cell cycle arrest were determined using flow cytometric and Western blot analysis. The in vitro results were confirmed in vivo using the nude mouse model. Among all coxibs tested, only celecoxib and methylcelecoxib decreased cell survival by induction of cell cycle arrest and apoptosis and reduced the growth of tumor xenografts in nude mice. None of the other coxibs (rofecoxib, etoricoxib, valdecoxib, lumiracoxib) produced anti-proliferative effects, indicating the lack of a class effect and of a role for COX-2. Our data emphasize again the outstanding anti-proliferative activity of celecoxib and its close structural analogue methylcelecoxib in colon carcinoma models in vitro and in vivo.

p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells.

S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53(wt)) or being p(HCT-116 p53(-/-)), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53(-/-) xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53(wt) cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53(wt) cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75(NTR), p53 and Bax.

Evidence of COX-2 independent induction of apoptosis and cell cycle block in human colon carcinoma cells after S- or R-ibuprofen treatment.

Ibuprofen belongs to the 2-aryl propionic-acid derivatives and consists of two enantiomers, of which S-ibuprofen is a potent cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitor whereas the R-enantiomer is two to three orders of magnitude less potent to inhibit cyclooxygenases. Beside its positive effects on inflammation and pain several animal studies have shown that ibuprofen also inhibits tumor initiation and proliferation but the molecular mechanisms are not fully understood. To investigate to which extent the antiproliferative effect of ibuprofen depends on COX-inhibition we tested both enantiomers in different human colon carcinoma cell lines (HCA-7 express COX-1, COX-2 and produce high prostaglandin E2 level; HCT-15 express only COX-1 and produce nearly no prostaglandin E2). S- and R-ibuprofen reduced concentration dependently cell survival in both cell lines to a similar extent and caused a G0/G1 phase block as well as apoptosis. The cell cycle block was accompanied by a down regulation of cyclin A and B and an increase of the cell cycle inhibitory protein p27Kip-1. HCA-7 cells were less sensitive against the antiproliferative effects of ibuprofen enantiomers which was probably due to lower ibuprofen concentrations in this cell type. Also in the nude mice model both enantiomers inhibited tumor growth of HCA-7 and HCT-15 xenografts to a similar extent. However, in mice about 54% of R-ibuprofen was unidirectionally inverted to S-ibuprofen, thus the observed antitumorigenic effect of R-ibuprofen in vivo cannot solely be assigned to this enantiomer. In conclusion our data indicate that S- and R-ibuprofen show similar antiproliferative effects in human colon carcinoma cell lines irrespective of its COX-inhibiting potencies.

Targeting the beta-catenin/APC pathway: a novel mechanism to explain the cyclooxygenase-2-independent anticarcinogenic effects of celecoxib in human colon carcinoma cells.

Celecoxib, a cyclooxygenase-2 (COX-2) selective nonsteroidal anti-inflammatory drug, is a new anticarcinogenic agent. Its antitumor effects depend on the one hand on its COX-2-inhibiting potency, but on the other hand on COX-2-independent mechanisms, which until now have not been fully understood. Here, we investigated whether celecoxib has an impact on the APC/beta-catenin pathway, which has been shown to play a pivotal role in the development of various cancers, especially of the colon. After only 2 h of treatment of human Caco-2 colon carcinoma cells with 100 muM celecoxib, we observed a rapid translocation of beta-catenin from its predominant membrane localization to the cytoplasm. Inhibition of the glycogen-synthase-kinase-3beta (GSK-3beta) by LiCl prevented this celecoxib-induced translocation, suggesting that phosphorylation of beta-catenin by the GSK-3beta kinase was essential for this release. Furthermore, the cytosolic accumulation was accompanied by a rapid increase of beta-catenin in the nuclei, starting already 30 min after celecoxib treatment. The DNA binding activity of beta-catenin time dependently decreased 2 h after celecoxib treatment. After this cellular reorganization, we observed a caspase- and proteasome-dependent degradation of beta-catenin after 8 h of drug incubation. Celecoxib-induced beta-catenin degradation was also observed in various other tumor cell lines (HCT-116, MCF-7, and LNCAP) but was not seen after treatment of Caco-2 cells with either the anticarcinogenic nonsteroidal anti-inflammatory drug R-flurbiprofen or the highly COX-2-selective inhibitor rofecoxib. These findings indicate that the anticarcinogenic effects of celecoxib can be explained, at least partly, by an extensive degradation of beta-catenin in human colon carcinoma cells.

Induction of apoptosis by R-flurbiprofen in human colon carcinoma cells: involvement of p53.

R-flurbiprofen, a non cyclooxygenase inhibiting non-steroidal anti-inflammatory drug (NSAID), has been found to inhibit tumor growth in various animal models. In vitro experiments have shown that this effect is based on the induction of a cell cycle block and apoptosis. Cell cycle inhibition has been explained by activation of the c-Jun-N-terminal kinase (JNK) and downregulation of cyclin D1 expression. However, the molecular mechanism leading to apoptosis is unknown. Here, we show that treatment of the human colon carcinoma cell line HCT116 with different concentrations of R-flurbiprofen leads to an accumulation of p53 protein which is accompanied by an increase in phosphorylated p53 at serine 15. Mutation of serine 15 to alanine by site directed mutagenesis and overexpression of the mutated p53 gene in HCT116 cells, revealed that these cells are significantly less sensitive to apoptosis induced by R-flurbiprofen than pcDNA control cells, as measured by PARP-cleavage and flow cytometry. By contrast, no difference was detected between HCT116p53ser15ala cells and HCT116 pcDNA cells with respect to induction of a cell cycle block after R-flurbiprofen treatment. Moreover, in nude mice HCT116p53ser15ala overexpressing xenografts were significantly less sensitive to R-flurbiprofen than HCT116 pcDNA control xenografts. In conclusion, we were able to show that induction of apoptosis in HCT116 cells after R-flurbiprofen treatment is at least partly dependent on the tumor suppressor gene p53 and that mutation of p53 at serine 15 impairs the apoptotic potency of R-flurbiprofen.