Induction of spermidine/spermine N1-acetyltransferase (SSAT) by aspirin in Caco-2 colon cancer cells

Abstract

Epidemiological, experimental and clinical results suggest that aspirin and other NSAIDs (non-steroidal anti-inflammatory drugs) inhibit the development of colon cancer. It has been shown that the NSAID sulindac induces apoptosis and suppresses carcinogenesis, in part, by a mechanism leading to the transcriptional activation of the gene encoding SSAT (spermidine/spermine N1-acetyltransferase), a rate-limiting enzyme in polyamine catabolism. In the present study, we show that a variety of NSAIDs, including aspirin, sulindac, ibuprofen and indomethacin, can induce SSAT gene expression in Caco-2 cells. Aspirin, at physiological concentrations, can induce SSAT mRNA via transcriptional initiation mechanisms. This induction leads to increased SSAT protein levels and enzyme activity. Promoter deletion analysis of the 5' SSAT promoter-flanking region led to the identification of two NF-kappaB (nuclear factor kappaB) response elements. Electrophoretic mobility-shift assays showed binding of NF-kappaB complexes at these sequences after aspirin treatment. Aspirin treatment led to the activation of NF-kappaB signalling and increased binding at these NF-kappaB sites in the SSAT promoter, hence providing a potential mechanism for the induction of SSAT by aspirin in these cells. Aspirin-induced SSAT ultimately leads to a decrease in cellular polyamine content, which has been associated with decreased carcinogenesis. These results suggest that activation of SSAT by aspirin and different NSAIDs may be a common property of NSAIDs that plays an important role in their chemopreventive actions in colorectal cancer.

Figure 1. Various SSAT promoter–reporter constructs

(A) Putative transcription factor binding sites in the SSAT 5′ promoter flanking region as determined by TESS analysis. NF-κB, PPRE and PRE are labelled. (B) Schematic representation of the various SSAT promoter–reporter constructs cloned into the pGL2-basic luciferase reporter plasmid, which were used in the present study, with three putative NF-κB sites (w, w₂ and w₃) and the +1 site marked.

Figure 2. Various NSAIDs induce SSAT RNA expression in Caco-2 cells

Caco-2 cells were treated with different concentrations of NSAIDs or their vehicle (V; open bar) for 48 h, harvested, and total RNA was extracted and used for Northern blotting. SSAT expression was normalized to the GAPDH expression in these treatments. Fold induction was calculated by dividing normalized values of the sample with the control value. The result shown is an average from three different experiments. NSAIDs used were Sul (sulindac), So (sulindac sulphone), Indo (indomethacin), acetylsalicylic acid (ASA) and Ibu (ibuprofen). *P<0.05 compared with the vehicle-treated control.

Figure 3. Aspirin induces SSAT expression in Caco-2 cells

(A) Effect on SSAT promoter activity. Caco-2 cells were grown overnight, transfected with the Full-SSAT-luc reporter–promoter construct and then treated for 48 h with either vehicle (open bar) or aspirin (20 or 100 μM; all closed bars). Relative luciferase units (RLU) were calculated after normalizing to the protein and β-galactosidase activities in the cell lysates. Fold induction was calculated after dividing the RLU of the sample by RLU of vehicle. (B) Effect on SSAT mRNA expression. Caco-2 cells were grown overnight and then treated for 48 h with either vehicle (open bar) or aspirin (20 or 100 μM; all closed bars). Cells were harvested, total RNA extracted and used for quantitative RT–PCR. SSAT expression was normalized to the GAPDH expression in these treatments. Fold induction was calculated by dividing normalized values of the sample with the control value. The result is an average from three different experiments. (C) Effect on SSAT protein. Immunoblotting for SSAT protein in Caco-2 cells grown overnight and then treated with either vehicle (V) or different concentrations of aspirin (ASA; 100 μM, 1 mM and 4 mM) for 48 h. BENS (10 μM)-treated A549 lung cancer cell protein extract [BENS, N¹,N¹¹-bis(ethyl)norspermine] was used as a positive control. (D) Effect on SSAT enzyme activity. Caco-2 cells were grown overnight and then treated with either vehicle (open bar) or 20 or 100 μM aspirin (all closed bars) for 48 h. Cells were then harvested and SSAT enzyme activity was measured. The result shown is an average from three different experiments. *P<0.05 compared with the vehicle control.

Figure 4. Mapping of a putative aspirin responsive element in the SSAT promoter

(A) Caco-2 cells were grown overnight, transfected with either Full-SSAT-luc or 197-SSAT-luc and then treated with either vehicle (V; open bars) or various concentrations of aspirin (ASA; all closed bars) for 48 h. (B) Caco-2 cells were grown overnight, transfected with a SSAT deletion reporter construct and then treated for 48 h with either vehicle (open bars) or 100 μM aspirin (closed bars). Relative luciferase units (RLU) were calculated after normalizing to the protein and β-galactosidase activities, and fold induction was calculated. Normalized luciferase activities are shown as mean±S.D. (n=3) and are expressed as fold inductions relative to the activity in the presence of vehicle alone; *P<0.05 compared with vehicle.

Figure 5. NF-κB, but not PPAR, is induced by aspirin in Caco-2 cells

Caco-2 cells were grown overnight and transfected with either the NF-κB₂ Luc reporter construct or the control NF-κB₂-luc reporter construct along with the β-gal plasmid. The cells were then treated for 48 h with either vehicle (V; open bars) or 100 μM aspirin (ASA; closed bars). Inset: NF-κB₂-luc reporter construct. Relative luciferase units (RLU) were calculated after normalizing to the protein and β-galactosidase activities in the cell lysates. Normalized luciferase activities are shown as mean±S.D. (n=3) and are expressed as fold inductions relative to the activity in the presence of vehicle alone; *P<0.05 compared with vehicle.

Figure 6. Increased binding of NF-κB complexes to the SSAT promoter

(A) Oligonucleotides containing the NF-κB-wild-1 (w) or NF-κB-mut-1 (m) sequence were ³²P-labelled (‘Hot Probe’) and incubated with 10 μg of nuclear extract, isolated from Caco-2 cells treated with different concentrations of aspirin (ASA) for 48 h. The unlabelled wild probe (‘Cold Probe’) was used at 100-fold molar excess for the competition reaction. Anti-p50 antibody was used for the supershift reactions. The specific-band shift and the supershifted bands are indicated. The specific-band shift intensity was quantified and then normalized to the vehicle-treated specific-band shift and shown as specific-band density. (B) Oligonucleotides containing the NF-κB-wild-3 (w₃) or NF-κB-mut-3 (m₃) sequence were ³²P-labelled and incubated with 10 μg of nuclear extract, isolated from Caco-2 cells treated with different concentrations of aspirin for 48 h. The unlabelled wild probe was used at 100-fold molar excess for the competition reaction. Anti-p50 antibody was used for the supershift reactions. The specific-band shift and the supershifted bands are indicated. The specific-band shift intensity was quantified and then normalized to the vehicle-treated specific-band shift and shown as specific-band density. Each experiment was performed four times, the specific-band intensities were quantified and statistics were done using Student's t test; *P<0.05 compared with vehicle.