Expression of interleukin-8, heme oxygenase-1 and vascular endothelial growth factor in DLD-1 colon carcinoma cells exposed to pyrrolidine dithiocarbamate

Markus Hellmuth, Christian Wetzler, Marcel Nold, Jae-Hyung Chang, Stefan Frank, Josef Pfeilschifter and Heiko Mühl,1

Pharmazentrum frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Interleukin (IL)-8, heme oxygenase-1 (HO-1), and vascular endothelial growth factor (VEGF) appear to be critically involved in immune responses associated with inflammation, infection and tumor growth. Regulation of these mediators was studied in the human colon carcinoma cell line DLD-1. Here we report that pyrrolidine dithiocarbamate (PDTC) not only augmented tumor necrosis factor-{alpha}-induced release of IL-8, but also mediated IL-8 expression as a single stimulus. Mutational analysis of the IL-8 promotor and electrophoretic mobility shift analysis revealed that activation of the transcription factor activator protein-1 (AP-1) and a constitutive nuclear factor-{kappa}B (NF-{kappa}B) binding activity in DLD-1 cells were mandatory for PDTC-induced IL-8 expression. Besides IL-8, PDTC also upregulated the expression of HO-1 and VEGF in these cells. Induction of IL-8 by PDTC was not restricted to DLD-1 cells, but was observed in Caco-2 colon carcinoma cells and in peripheral blood mononuclear cells. PDTC is currently advocated for use as a chemotherapeutic drug in the treatment of certain malignancies, among them colorectal cancer. Induction of IL-8, HO-1 and VEGF may affect therapeutic applications of this agent.

Abbreviations: AP-1, activator protein-1; EMSA, electrophoretic mobility shift assay; HO-1, heme oxygenase-1; IL, interleukin; NF-{kappa}B, nuclear factor-{kappa}B; PBMC, peripheral blood mononuclear cells; PDTC, pyrrolidine dithiocarbamate; VEGF, vascular endothelial growth factor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pyrrolidine dithiocarbamate (PDTC) is a thiol-containing agent, which mediates a variety of effects on cell physiology. Actions of PDTC are probably related to the characteristics of this synthetic compound as an antioxidant, metal chelator (1) and zinc ionophore (2). The most prominent and pharmacologically widely used property of PDTC is its capacity to inhibit activation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) in several biological systems (3,4). Consistent with this activity, use of dithiocarbamates such as PDTC has been proposed for the treatment of endotoxic shock (5), acquired immunodeficiency syndrome (6) and diabetic retinopathy (7). PDTC has also been described as an inducer of apoptotic cell death in certain cell types, among them colon carcinoma cells (8). Accordingly, PDTC enhances 5-fluorouracil-induced growth inhibition of colon carcinoma cells (8). Based on these functions, PDTC is currently being advocated for treatment of human malignancies such as colorectal cancer (8,9).

The CXC-chemokine interleukin(IL)-8 is expressed in response to pro-inflammatory cytokines (10) and cellular stress (11–14), and is recognized as a major activator of neutrophil function. Release of IL-8 is therefore believed to be of pivotal importance in the pathogenesis of neutrophil-mediated acute inflammation (15). Accordingly, blockage of IL-8 action efficiently reduces the severity of disease in rabbit models of reperfusion injury, acute dermatitis, acute arthritis, respiratory distress syndrome and immune complex-mediated glomerulonephritis (16). Moreover, in a model of gouty synovitis development of neutrophilic inflammation was impaired in knockout mice deficient for the murine homolog of the IL-8 receptor CXCR-2 (17). Another crucial function of IL-8 is its role as a mediator of angiogenesis, which locates this chemokine at the interface of inflammation and tumor biology. Accordingly, expression of IL-8 has been determined to play a significant role in mediating human tumorigenesis (18). In addition to IL-8, two other proteins were investigated in the present study: the angiogenic growth factor vascular endothelial growth factor (VEGF) and the stress protein heme oxygenase-1 (HO-1). Upregulation of both proteins has been associated with tumor growth (19–21). Here we report on the unexpected finding that PDTC as a single stimulus induces expression of IL-8, as well as HO-1, and VEGF in DLD-1 colon carcinoma cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
PDTC was from Calbiochem-Novabiochem GmbH (Bad Soden, Germany). Lipopolysaccharide (LPS, 026B6), and Polymyxin B (PmxB) were purchased from Sigma (Deisenhofen, Germany). Tumor necrosis factor-{alpha} (TNF{alpha}) was kindly provided by the Knoll AG (Ludwigshafen, Germany). IL-1ß was purchased from Cell Concepts (Umkirch, Germany).

Cell culture of DLD-1 and Caco-2 colon carcinoma cells
Human DLD-1 and Caco-2 colon carcinoma/epithelial cells were obtained from the Centre for Applied Microbiology and Research (Salisbury, UK) and the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were maintained in DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% heat-inactivated FCS (Gibco BRL, Eggenstein, Germany). For the experiments, confluent cells on polystyrene plates (Greiner, Frickenhausen, Germany) were washed with PBS and incubated in the aforementioned medium.

Isolation and cultivation of peripheral blood mononuclear cells (PBMC)
The study protocol and consent documents were approved by the Ethik Kommission of the Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main. Healthy volunteers abstained from using any drugs during 2 weeks before the study. PBMC were isolated as described previously (22) and cultivated in RPMI 1640 (Gibco BRL) supplemented with 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin and 1% (v/v) heat-inactivated human AB serum (Sigma). PBMC were resuspended at 3x106 cells/ml and were seeded in round-bottom polypropylene-tubes at 1 ml. All incubations of either DLD-1 cells or PBMC were performed at 37°C and 5% CO2.

IL-8 determination by ELISA
Cell-free culture supernatants were analyzed for IL-8 protein content by ELISA using commercial kits according to the manufacturer’s instructions (Pharmingen, Hamburg, Germany).

RNase protection assay for analysis of IL-8, HO-1, VEGF and GAPDH mRNA accumulation
RNA was isolated using Trizol-Reagent (Gibco BRL) according to the manufacturer’s instructions. Ten micrograms of total RNA were used for RNase protection assay, performed as recently described (23). Briefly, DNA probes were cloned into the transcription vector pBluescript II KS (+) (Stratagene, Heidelberg, Germany). After linearization, an antisense transcript was synthezised in vitro with T3 or T7 RNA polymerase and [{alpha}-32P]UTP (800 Ci/mmol). RNA samples were hybridized at 42°C overnight with 100 000 c.p.m. of the labeled antisense transcript. Hybrids were digested with RNase A and T1 for 1 h at 30°C. Under these conditions every single mismatch was recognized by the RNases. Protected fragments were separated on a 5% (w/v) polyacrylamide/8 M urea gels and analyzed using a PhosphoImager (Fuji, Straubenhardt, Germany). The individual gene expression of IL-8, HO-1 or VEGF was evaluated on the basis of the GAPDH housekeeping gene expression. The cDNA probes were cloned by RT–PCR and correspond to nucleotides (nt) 148–302 (for GAPDH), nt 146–433 (for IL-8), nt 706–949 (for HO-1) and nt 339–498 (for VEGF) of the published sequences (24–27).

Detection of HO-1 protein by immunoblotting
After stimulation, DLD-1 cells were treated with lysis buffer (300 mM NaCl, 50 mM Tris–HCl pH 7.6, 0.5% Triton X-100) supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany). For detection of HO-1, 50 µg of total proteins were separated by 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto Immobilon membrane (Millipore, Bedford, UK). HO-1 was detected using a primary rabbit polyclonal antibody (Stressgene, Hamburg, Germany), horseradish peroxidase-labeled secondary antibodies (Bio-Rad, Munich, Germany), and a chemiluminescence detection kit (Amersham Pharmacia Biotech, Freiburg, Germany) according to the manufacturers’ instructions.

Plasmid constructs, transient transfection experiments, reporter assays and mutational analysis
The human IL-8 promotor 5'-flanking region from –558 to +98 bp was amplified by pfu-polymerase (Stratagene, Gebouw California, The Netherlands) from human genomic DNA using the oligonucleotides 5'-ctt cac tct gtt aac tag cat ta-3' (–558/–535) as forward and 5'-aca cac agt gag aat ggt tcc t-3' (+76/+98 bp) as reverse primer, respectively. The resulting PCR product was cloned into pGL3-promotor vector containing a luc+ transcriptional unit. Constructs were verified by sequencing. Mutations were performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Amsterdam) according to the manufacturer’s instructions. For the mutations of the activator protein-1 (AP-1), NF-{kappa}B and C/EBPß sites (28) the following primer sequences were used: AP-1 sequence (forward) 5'-aag tgt gat atc tca ggt ttg ccc tga-3', (reverse) 5'-caa acc tga gat atc aca ctt cct a-3'; NF-{kappa}B sequence (forward) 5'-ttg caa atc gtt tta att taa tct gac ata a-3', (reverse) 5'-ttc att atg tca gat taa att aaa cga ttt-3'; C/EBPß sequence (forward) 5'-gcc atc agc tac gag tcg tg-3', (reverse) 5'-gaa att cca cga ctc gta gct-3'. Mutations were checked by sequencing using the automated sequence analyzer ABI 310 Genetic Analyzer (PE Applied Biosystems, Weiterstadt, Germany). Luciferase expression contructs (750 ng DNA/35 mm well) were transfected into DLD-1 cells using Fugen-6 Transfection Reagent (Roche) according to the manufacturer’s instructions. For control of transfection efficiency, 500 ng of pCMV-ß galactosidase DNA was cotransfected. After a 36 h transfection period, the cells were washed twice with PBS. DLD-1 cells were then either kept as unstimulated control or stimulated with PDTC or TNF{alpha}. After 8 h, cells were harvested and luciferase activity was determined using the Promega Luciferase Assay System (Promega, Madison, WI).

Electrophoretic mobility shift assay (EMSA)
Preparation of crude extracts from DLD-1 cells was performed as described previously (29). Consensus oligonucleotides used in the binding reactions were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Sequences of the double-stranded oligonucleotides are as follows, NF-{kappa}B: wild-type (wt) 5'-agt tga ggg gac ttt ccc agg c-3'; mutated 5'-agt tga ggc gac ttt ccc agg c-3'. AP-1: wt 5'-cgc ttg atg act cag ccg gaa-3'. Complementary oligonucleotides were end labeled by T4 polynucleotide kinase (MBI Fermentas, St Leon-Roth, Germany) using [{gamma}-32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech, Braunschweig, Germany). Binding reactions were performed for 35 min on ice with 10 {gamma} of protein in 20 µl of binding buffer containing 4% Ficoll, 20 mM HEPES pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.25 mg/ml BSA, 2 µg of poly(dI–dC) and 20 000–25 000 d.p.m. of 32P-labeled oligonucleotide. For AP-1 super-shift analysis nuclear proteins were pre-incubated for 15 min at room temperature with a polyclonal anti-c-jun antibody (Santa Cruz). DNA–protein complexes were separated from unbound oligonucleotide by electrophoresis through a 4% polyacrylamide gel using 0.5x TBE buffer. Thereafter, gels were fixed and analyzed by PhosphoImager analysis (Fuji). Competition experiments were performed by coincubation with a 100-fold excess (20 pmol) of unlabeled double-stranded oligonucleotide in the DNA–protein binding reaction.

Detection of cell death by analysis of cytosolic oligosome-bound DNA and release of lactate dehydrogenase (LDH) activity
Cytosolic oligonucleosome-bound DNA was quantified using an ELISA according to the manufacturer’s instructions (Boehringer Mannheim, Mannheim, Germany). Absorbance values (OD405/495 nm) give a relative measure of ongoing DNA fragmentation, a common marker for cell death by apoptosis. Release of LDH was determined using an assay kit according to the manufacturer’s instructions (Boehringer Mannheim).

S-Nitrosoglutathione (GSNO) synthesis
GSNO was synthezised as described previously (30).

Statistics
For experiments using DLD-1 cells data are shown as means ± SD. For experiments with PBMC data are shown as means ± SEM. Data are presented either as pg/ml, as ng/ml or as fold-induction compared with unstimulated control and were analyzed by unpaired Student’s t-test (DLD-1 cell experiments) or paired Student’s t-test (PBMC experiments) on raw data using Sigma Plot (Jandel Scientific).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PDTC efficiently augments TNF{alpha}-induced IL-8 release and mediates production of IL-8 as a single stimulus in DLD-1 cells
While studying the expression of IL-8 in DLD-1 colon carcinoma cells, we investigated the effects of PDTC, a compound that is widely used as inhibitor of NF-{kappa}B activation. Unexpectedly, PDTC not only efficiently augmented TNF{alpha}-induced IL-8 (Figure 1AGo), but was also able to mediate release of IL-8 from DLD-1 cells as a single stimulus (Figure 1A and BGo). Similar data were obtained using Caco-2 colon carcinoma cells (data not shown). Under these experimental conditions, induction of IL-8 was not associated with cytotoxicity as assessed by a LDH assay and by quantitative analysis of DNA fragmentation, a common marker of apoptotic cell death (data not shown). Time-course analysis revealed that PDTC induced a continuous release of IL-8 from the cells that started after 4 h of incubation. Most of IL-8 was released between hours 8 and 24 of stimulation. After 24 h, a plateau of IL-8 concentration in the culture supernatants was reached (Figure 1Go).



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Fig. 1. PDTC enhances TNF{alpha}-induced release of IL-8 from DLD-1 cells and likewise induces IL-8 release as single stimulus. (A) DLD-1 cells were incubated as unstimulated control, stimulated with TNF{alpha} (50 ng/ml), with PDTC (50 µM) or with TNF{alpha} (50 ng/ml) in combination with PDTC (50 µM). (B) DLD-1 cells were incubated as unstimulated control or stimulated with the indicated concentrations of PDTC. After 8 h, cell-free supernatants were assayed for IL-8 protein content by ELISA. Data are expressed as mean IL-8 concentrations ± SD (n = 5). **P < 0.01 compared with untreated control; #P < 0.05 compared with PDTC alone. (C) DLD-1 cells were incubated as unstimulated control (closed circles) or stimulated with PDTC at 50 µM (open circles) or 200 µM (closed triangles). After the indicated time periods, cell-free supernatants were assayed for IL-8 protein content by ELISA. Data are expressed as mean IL-8 concentrations ± SD (n = 4). **P < 0.01 compared with untreated control.

 
PDTC induces gene expression of IL-8, VEGF and HO-1 in DLD-1 cells
To further characterize PDTC-induced activation of DLD-1 cells, mRNA accumulation in response to the agent was investigated by RNase protection assay. In addition to IL-8, expression levels of two additional markers of cellular activation were determined: VEGF and HO-1. mRNA induction by PDTC was observed for all three genes investigated (Figure 2Go). In order to confirm that upregulation of mRNA levels translated into protein expression, immunoblotting for HO-1 was performed. As shown in Figure 3Go, PDTC was a strong inducer of HO-1 in DLD-1 cells. As a positive control, DLD-1 cells were exposed to the nitric oxide donor GSNO, which was identified previously as mediator of HO-1 expression in rat renal mesangial cells (31) and human HaCaT keratinocytes (Wetzler et al., unpublished observations). In contrast, stimulation of DLD-1 cells with the pro-inflammatory cytokine IL-1ß was not sufficient to trigger induction of HO-1, which is in accord with a recent publication on regulation of HO-1 in HaCaT keratinocytes (32).



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Fig. 2. PDTC induces gene expression of IL-8, HO-1 and VEGF in DLD-1 cells. DLD-1 cells were incubated as unstimulated control or stimulated with the indicated concentrations of PDTC for 8 h. Thereafter, PDTC-induced IL-8 (A), HO-1 (B) and VEGF (C) mRNA accumulation was evaluated by RNase protection assay. One representative of three independently performed experiments is shown. Relative mRNA expression of this same experiment was quantified by PhosphoImager (Fuji) analysis of the radiolabeled gel. P denotes probe.

 


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Fig. 3. PDTC induces expression of HO-1 protein in DLD-1 cells. DLD-1 cells were incubated as unstimulated control or stimulated with PDTC (50 µM), IL-1ß (50 ng/ml) or GSNO (500 µM). After 8 h, cells were harvested and homogenates were assayed for the presence of HO-1 protein by immunoblotting.

 
PDTC upregulates IL-8 promotor activity in DLD-1 cells through an NF-{kappa}B- and AP-1-dependent mechanism
Gene induction of proteins such as intercellular adhesion molecule-1 (33), ß2-integrin (34), stromelysin (35) or glutathione S-transferase (36) by PDTC is mediated by activation of the transcription factor AP-1. In keeping with these previous observations, we observed activation of AP-1 by PDTC by performing EMSA analysis (Figure 4AGo). In order to further investigate whether activation of the IL-8 promotor was responsible for induction by PDTC, we transiently transfected the luciferase reporter gene under the control of an IL-8 promotor fragment (–558 to +98 nt) (10) into DLD-1 cells. To evaluate a potential role for the AP-1 binding site (–127 to –120 nt) in the IL-8 promotor, a site-directed mutation of this site was performed. In addition, a NF-{kappa}B mutant (–80 to –70) was generated in the same context of the IL-8 promotor fragment (–558 to +98 nt). As a positive control for activation of the IL-8 promotor, DLD-1 cells were stimulated with TNF{alpha}. As expected, TNF{alpha} induction of IL-8 promotor activity was abrogated by mutation of the NF-{kappa}B binding site in the IL-8 promotor fragment (Figure 4BGo). In agreement with PDTC-induced IL-8 mRNA accumulation and protein release, promotor activity was significantly upregulated by the agent (Figure 4BGo). Similar data were obtained using Caco-2 colon carcinoma cells (data not shown). Mutational analysis revealed that both AP-1 and NF-{kappa}B were necessary for induction of IL-8 by PDTC (Figure 4BGo). It is well described that AP-1 and NF-{kappa}B can synergistically activate the IL-8 promotor (10). Therefore, we investigated the status of NF-{kappa}B activation in DLD-1 cells. A specific DNA binding complex was strongly upregulated in response to TNF{alpha}, which was used as a positive control in these experiments. In accord with a previous report (37), constitutive NF-{kappa}B binding activity was observed in DLD-1 cells. This constitutive NF-{kappa}B binding activity was still detectable in the presence of PDTC (Figure 4CGo). It has been reported previously that PDTC is able to activate the transcription factor CCAAT/enhancer binding protein ß (C/EBPß) (38). In addition, C/EBPß has been identified as co-stimulus for optimal IL-8 induction (10). To evaluate a potential role for the C/EBPß binding site (–94 to –81 nt) in PDTC-induced IL-8 promotor activation, a site-directed mutation was performed, in the same context of the IL-8 promotor fragment (–558 to +98 nt). As shown in Figure 4BGo, mutation of this site only partially reduced PDTC-induced IL-8 promotor activity.



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Fig. 4. PDTC upregulates IL-8 promotor activity in DLD-1 cells through an NF-{kappa}B- and AP-1-dependent mechanism. (A) Detection of PDTC-induced AP-1 DNA binding activity in DLD-1 cells by EMSA analysis. DLD-1 cells were incubated as unstimulated control, or stimulated with PDTC (200 µM) for the indicated time periods. Thereafter, nuclear extracts were prepared and EMSA analysis was performed using 32P-labeled AP-1 consensus oligonucleotide. Experiments were performed in the presence or absence of an anti-c-jun antibody. The position of a supershifted band is indicated by an arrow. One representative of three independently performed experiments is shown. (B) 500 ng/well of ß-Gal-constructs and 500 ng/well of one of the indicated luciferase-IL-8-promotor-constructs were used to transiently transfect DLD-1 cells. After 36 h, transfected cells were either maintained as unstimulated control, or treated with PDTC (50 µM) (upper panel), or with TNF{alpha} (50 ng/ml) (lower panel) for an additional 8 h incubation period. Thereafter, cells were harvested and luciferase- and ß-Gal-assays were performed, respectively. Data are expressed as mean fold-luciferase induction ± SD obtained from five independently performed experiments. *P < 0.05 compared to unstimulated control. A 1-fold induction was assigned to the luciferase activity versus ß-Gal activity in the respective unstimulated transfected DLD-1 cells. (C) Detection of constitutive DNA binding activity of NF-{kappa}B in DLD-1 cells by EMSA. DLD-1 cells were incubated as unstimulated control, or stimulated with TNF{alpha} (50 ng/ml), or PDTC (50 µM). After 1 h, nuclear extracts were prepared and EMSA analysis was performed using 32P-labeled NF-{kappa}B consensus oligonucleotide, excess cold NF-{kappa}B consensus oligonucleotide, and excess cold mutated NF-{kappa}B consensus oligonucleotide. One representative of two independently performed experiments is shown.

 
PDTC augments release of IL-8 from PBMC
We also studied the effect of PDTC on IL-8 release from PBMC. As shown in Figure 5AGo, basal release of IL-8 from PBMC was significantly augmented by incubation with PDTC. In order to control for a potential LPS contamination of the agent, experiments using the inhibitor of LPS action PmxB were performed. Upregulation of IL-8 release was not affected by pre-treatment of control medium and PDTC with PmxB (final concentration 2 µg/ml) (Figure 5AGo). Under these same experimental conditions LPS (10 ng/ml)-induced release of IL-8 from PBMC was efficiently abrogated by pre-treatment with PmxB (Figure 5BGo). LPS at 100 ng/ml did not mediate release of IL-8 from DLD-1 cells (data not shown).



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Fig. 5. PDTC augments release of IL-8 from PBMC. PBMC were incubated as unstimulated control (A and B), or stimulated with PDTC (50 µM) (A), or with LPS (10 ng/ml) (B). Cells were also incubated as PmxB-pretreated (final concentration 2 µg/ml) control (A and B), or stimulated with PmxB-pretreated (final concentration: 2 µg/ml) PDTC (50 µM) (A), or with PmxB-pretreated (final concentration 2 µg/ml) LPS (10 ng/ml) (B). After 24 h, cell-free supernatants were determined by ELISA. Data are expressed as means ± SEM (n = 4). *P < 0.05 compared with PmxB-pre-treated control; **P < 0.01 compared with untreated or PmxB-pre-treated control.

 

    Discussion
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 Materials and methods
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 References
 
IL-8 and VEGF are two mediators of angiogenesis and tumor progression, which are coexpressed in a variety of human tumors, including colorectal cancer (39,40). Primarily, due to its capability of inducing apoptosis in certain cancer cells, use of PDTC as an anticancer drug has been proposed (8,9,41). In the present study we investigated the effects of PDTC on the expression of IL-8, VEGF and HO-1. As for IL-8 and VEGF, induction and activity of HO-1 has been associated with growth of solid tumors (20,21). Here, we report that expression of this particular set of genes is induced by PDTC in human DLD-1 colon carcinoma cells. Induction of HO-1 by PDTC agrees with recent data from rat aortic smooth muscle cells (42). In addition, we demonstrate that PDTC can efficiently enhance TNF{alpha}-mediated release of IL-8 from DLD-1 cells. At first sight these observations appear unexpected as IL-8 is an NF-{kappa}B-dependent gene (10) and NF-{kappa}B activation can be inhibited significantly by treatment with PDTC in certain cell types (3,4). However, in human colon carcinoma cells, inhibition of NF-{kappa}B activation by PDTC was not observed (8,43). In leukemic promonocytic U937 cells, PDTC not only was unable to inhibit TNF{alpha}-induced NF-{kappa}B activation, but actually enhanced TNF{alpha}-mediated {kappa}B-dependent gene induction (44). Moreover, TNF{alpha}-induced NF-{kappa}B binding activity could only be partially suppressed (50% inhibition) by PDTC at 100 µM in SW620 human colon carcinoma cells (45). In keeping with previous reports, we confirm constitutive NF-{kappa}B binding activity in human colon carcinoma cells as seen in DLD-1 cells (37) or SW48 cells (46). This constitutive activity was not reduced by treatment with PDTC in the present study. Molecular cooperation particularly between the transcription factors NF-{kappa}B and AP-1 has been identified as an essential prerequisite for optimal gene induction of IL-8 in epithelial cells (47). As PDTC is a well-characterized activator of AP-1 (33–36,42,48), we investigated the importance of the binding sites for these two transcription factors with regard to PDTC-induced IL-8 by mutational analysis of the IL-8 promotor. The NF-{kappa}B binding site (–80 to –70 nt) as well as the AP-1 binding site (–127 to –120 nt) (10) both turned out to be necessary for PDTC-induced IL-8. The data imply that PDTC-induced AP-1 cooperates with constitutive NF-{kappa}B activity in DLD-1 cells, which then drives synergistic expression of IL-8. In addition, mutational analysis of the C/EBPß binding site (–94 to –81 nt) suggested that PDTC-induced activation of C/EBPß (38) contributes to a certain degree to IL-8 promotor activation by this agent. This observation is in keeping with previous reports demonstrating the capability of C/EBPß to enhance NF-{kappa}B dependent IL-8 expression (49).

Induction of IL-8 by PDTC was not restricted to colon carcinoma cells but was also detectable in human PBMC, where monocytes are regarded as the major source of IL-8 (50). Accordingly, PDTC has been shown previously to activate AP-1 in monocytic cells (44). In the present study, PBMC were isolated from whole blood by density separation over a Ficoll-Hypaque gradient, which is standard methodology. This procedure is inevitably associated with activation of PBMC, as shown by induction of TNF{alpha} mRNA expression (51), a process probably mediated by activation of NF-{kappa}B. Therefore, upregulation of IL-8 in PBMC is in keeping with the hypothesis that PDTC-stimulated AP-1 mediates IL-8 production in pre-activated cells. Augmentation of IL-8 release from PBMC by PDTC was however modest compared with inflammatory stimuli such as IL-1ß or LPS (Figure 5Go). The present observation that IL-8 can be induced by PDTC as a single stimulus appears to be of particular significance in the context of transformed colon carcinoma cells in which NF-{kappa}B is not (8,43, present data) or only partially (45) modulated by PDTC. Thus, our data do not necessarily contradict the anti-inflammatory potential of PDTC (5,52).

In the present study, we did not further investigate the molecular basis of PDTC-induced VEGF and HO-1 gene activation. However, in similarity to IL-8, expression of VEGF (40,53) and HO-1 (54) can be induced by activation of AP-1 in cancer cells. AP-1 in particular mediates PDTC-induced HO-1 gene induction in the murine transformed macrophage-like cell line RAW 264.7 (42).

Recently, it has been reported that NF-{kappa}B binding activity is augmented in human colorectal cancer (46). The present data imply that exposure of these tumor tissues to PDTC during chemotherapeutic intervention may mediate expression of pro-angiogenic IL-8 and VEGF, as well as HO-1 in colon carcinoma cells. A cellular response that could adversely affect the efficacy of PDTC in cancer therapy.


    Notes
 
1 To whom correspondence should be addressed Email: h.muehl{at}em.uni-frankfurt.de Back


    Acknowledgments
 
We thank Drs S.Harder, J.Graff and U.Klinkhardt for their help obtaining heparinized blood. We also thank Stefanie Garkisch and Sonja Höfler for technical assistance. This work was supported by the Riese-Stiftung and the Klein-Stiftung.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 26, 2001; revised March 22, 2002; accepted April 23, 2002.