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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 20, 2003 as doi:10.1096/fj.02-0919fje. |
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pharmazentrum frankfurt, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt, 60590 Frankfurt/Main, Germany
2Correspondence: pharmazentrum frankfurt, Institut für Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe Universität, Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany. E-mail: groesch{at}em.uni-frankfurt.de
SPECIFIC AIMS
Flurbiprofen is a racemic nonsteroidal anti-inflammatory drug known to prevent tumor development and growth in APC-min, TRAMP mice, and rats, but the molecular mechanisms contributing to the anticarcinogenic effect of flurbiprofen are unknown. In contrast to its optical antipode, R-flurbiprofen is not a COX inhibitor at therapeutically relevant concentrations. The aim of the present study was therefore to identify potential pathways that may contribute to the anticarcinogenic effects of R-flurbiprofen and its COX-inhibiting counterpart, S-flurbiprofen.
PRINCIPAL FINDINGS
1. S- and R-flurbiprofen affected cell survival and induced apoptosis in colon carcinoma cell lines with equal efficiency
To estimate the cytotoxicity of S- and R-flurbiprofen, we assessed cell survival of three different colon carcinoma cell lines in the colony-forming assay. The IC50 values for S-flurbiprofen were 448 ± 7 µM, 296 ± 27 µM, and 403 ± 11 µM for Caco-2, HT-29, and HCT-15 cells, respectively. The IC50 values for R-flurbiprofen were 432 ± 11 µM, 261 ± 4 µM, and 410 ± 5 µM for Caco-2, HT-29, and HCT-15 cells, respectively. HT-29 cells were more sensitive toward R- and S-flurbiprofen than Caco-2 and HCT-15 cells. There was no significant difference between R- and S-flurbiprofen (P=1 for all three cell lines).
We determined the percentage of cells in the sub-G1 phase that corresponded to the apoptotic fraction by flow cytometry. As shown in Fig. 1
, treatment of Caco-2, HT-29, and HCT-15 cells with S- and R-flurbiprofen increased the percentage of cells in the sub-G1 fraction in a concentration-dependent manner.
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2. S- and R-flurbiprofen induced a G0/G1 cell cycle arrest in colon cancer cell lines and influenced the expression level of cell cycle regulating proteins
Since apoptosis often occurs as a consequence of a cell cycle block, we checked whether the observed cytotoxicity of S- and R-flurbiprofen was mediated through an alteration of cell cycle progression. HCT-15, Caco-2, and HT-29 cells were synchronized by incubation in FCS-free medium for 48 h, treated with increasing concentrations (300, 500, 800, 1000 µM) of S- and R-flurbiprofen for 20–24 h, or left untreated.
Treatment of cells with S- and R-flurbiprofen caused a concentration-dependent inhibition of cell cycle progression from G0/G1 to S and G2/M phase in all three cell lines (Fig. 1)
. Accordingly, we investigated the expression level of different proteins by means of Western blot analysis that are known to be involved in regulation of cell cycle transition from the G1 to S phase (cyclins D and A and the CDKIs p21Waf1, p27Kip1, and p16INK4). Control cells were equally synchronized. Treatment with R- and S-flurbiprofen (800 µM) caused a decrease of cyclin D1 expression after 16–24 h whereas cyclin D1 expression in control cells increased. Cyclin A expression decreased in all three cell lines in a time-dependent manner after treatment of cells with 800 µM S- or R-flurbiprofen. Western blot analysis of various CDKIs (p21Waf1, p27Kip1, and p16INK4) revealed no detectable changes.
3. S- and R-flurbiprofen activated c-Jun-N-terminal kinase (JNK) and the transcription activator protein-1(AP-1)
JNK signaling has been shown to be involved in cell cycle control and induction of apoptosis. We determined phospho-JNK levels by means of Western blot analysis using a specific phospho-JNK antibody. Treatment with S- or R-flurbiprofen caused a phosphorylation of JNK1 within 10–30 min in all three cell lines. Activation of JNK1 was associated with an increase of nuclear expression of c-Fos (Fig. 2
A) and JunB (Fig. 2B
) at 30 min to 6 h after starting S- or R-flurbiprofen treatment. In contrast, nuclear expression of c-Jun remained unchanged (data not shown). In parallel, S- or R-flurbiprofen treatment resulted in an increase of the DNA binding activity of nuclear protein extracts to a radioactively labeled oligonucleotide that corresponds to the consensus sequence of AP-1 (Fig. 2C
). To identify the components of the AP-1 DNA binding complex, we performed supershift experiments with antibodies directed against various AP-1 family members. In HCT-15 control extracts, supershifts occurred with antibodies directed against phospho c-Jun, c-Fos, and JunB. In cells treated with 800 µM S- or R-flurbiprofen, there was no phospho c-Jun supershift but a clear supershift for JunB and a weak supershift with the c-Fos antibody (Fig. 2)
. Supershift experiments for other potential members of AP-1 (FosB, Fra-1, Fra-2) were not performed. Hence, we cannot exclude that additional members are involved in flurbiprofen-activated AP-1 complexes. However, the data suggest that flurbiprofen treatment causes a change of the AP-1 protein complex composition from c-Jun to JunB.
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To find out whether activation of JNK was essential for S- and R-flurbiprofen-induced cell cycle block and cell death, we assessed whether the effects of S- and R-flurbiprofen were inhibited in the presence of SP600125, a specific competitive inhibitor at the ATP binding site of JNK. EMSA showed that pretreatment of cells with 10 µM SP600125 prevented the activation of AP-1 after S- or R-flurbiprofen treatment. Pretreatment of the cells with 10 µM SP600125 reduced the S- or R-flurbiprofen-induced G1 phase block as well as the reduction of cyclin D1 expression. However, SP600125 had no influence on S- or R-flurbiprofen-induced increases of sub-G1 cells, indicating that S- and R-flurbiprofen-induced apoptosis was independent of JNK activation.
4. R-flurbiprofen inhibited growth of HCT-15 tumors in nude mice
Although in vitro effects of R- and S-flurbiprofen had been indistinguishable, only R-flurbiprofen significantly inhibited HCT-15 tumor growth in vivo whereas HT-29 tumors were resistant toward S-and R-flurbiprofen treatment in vivo. Plasma concentrations of S- and R-flurbiprofen after a daily treatment with 15 mg/kg S-flurbiprofen were 202 ± 46 µM and 7 ± 2 µM, respectively. Mouse plasma concentrations after treatment with 15 mg/kg R-flurbiprofen were 192 ± 33 µM for the R-enantiomer and 30 ± 4 µM for the S-enantiomer. The minor amount of R-flurbiprofen in plasma samples of mice treated with S-flurbiprofen was probably caused by an impurity of the substance used but may also be explained by different pharmacokinetics of R-flurbiprofen compared with the S-enantiomer. Meanwhile, R-flurbiprofen is unidirectionally inverted to S-flurbiprofen in mice, which explains the relative high amount of S-flurbiprofen in R-flurbiprofen treated mice.
CONCLUSIONS AND SIGNIFICANCE
In the present study we show that induction of apoptosis and alteration of cell cycle regulatory proteins are major mechanisms accounting for the anti-proliferative effects of R- and S-flurbiprofen. We observed that S- and R-flurbiprofen activate c-Jun-N-terminal kinase 1 (JNK1) and the transcription factor AP-1. Activation of AP-1 was associated with nuclear translocation of c-Fos and JunB whereas nuclear c-Jun expression remained unchanged. It has been shown that JunB could be stabilized by JNK and that activated JNK is able to enhance the transcriptional activity of JunB through its phosphorylation at Thr102 and Thr104. It is known that c-Jun enhances cell proliferation through inhibition of tumor suppressor gene expression and induction of cyclin D1 transcription. These actions are antagonized by JunB, which up-regulates tumor suppressor genes and represses cyclin D1. Thus, inhibition of cyclin D1 promotor by a relative increase of activated JunB in nuclear AP-1 complexes is a possible mechanism causing inhibition of cell cycle progression. In line with this assumption, we found that all effects after treatment with flurbiprofen enantiomers—activation of AP-1, repression of cyclin D1, and the G1 cell cycle block—were largely reversed when the cells were pretreated with a specific JNK inhibitor, suggesting that JNK activation is the primary mechanism underlying the cell cycle inhibitory effects of R- and S-flurbiprofen. Nevertheless, in addition to the transcriptional changes, cyclin D1 expression can be suppressed by an enhanced cyclin D1 mRNA turnover or facilitated degradation of cyclin D1 protein via the proteasome pathway. So we could not exclude the possibility that these mechanisms might also contribute to the low cyclin D1 protein levels after flurbiprofen treatment. A facilitated degradation might also explain the observed decrease of cyclin A because cyclin A has been shown to be transcriptionally up-regulated by c-Fos and JunB.
However, inhibition of JNK had no effect on R- and S-flurbiprofen-induced apoptosis, although activated JNK has been implicated in the induction of apoptosis either by directly activating the mitochondrial death pathway or by activation of c-Jun. Hence, the effects of R– and S-flurbiprofen cannot be attributed solely to the activation of JNK signaling. Thus, additional mechanisms remain to be identified that may account for the observed induction of apoptosis after flurbiprofen treatment.
In vitro we did not observe any differences between R- and S-flurbiprofen. Both enantiomers were equally potent, the effects followed an identical time course and occurred in all three cell lines. Surprisingly, in vivo only R-flurbiprofen was able to inhibit the growth of HCT-15 tumors in nude mice whereas HT-29 tumor growth was not influenced although plasma concentrations in flurbiprofen-treated mice (
190 µM) were in the range of the IC50 values of the HT-29 survival assay. This suggests that 1) certain differences between R- and S-flurbiprofen only become operative in vivo and 2) the chemopreventive effects of flurbiprofen in vivo are probably restricted to a certain subset of tumors so that the efficiency of these substances could be variable in different patients.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0919fje; doi: 10.1096/fj.02-0919fje 
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