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Carcinogenesis Advance Access originally published online on November 13, 2007
Carcinogenesis 2008 29(4):779-789; doi:10.1093/carcin/bgm248
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Curcumin downregulates the inflammatory cytokines CXCL1 and -2 in breast cancer cells via NF{kappa}B

Beatrice E. Bachmeier1,5,*, Isabelle V. Mohrenz1, Valentina Mirisola2,3, Erwin Schleicher4, Francesco Romeo5,6, Clara Höhneke1, Marianne Jochum1, Andreas G. Nerlich7 and Ulrich Pfeffer2

1 Department of Clinical Chemistry and Clinical Biochemistry, Ludwig-Maximilians-University Munich, D-80336 München, Germany
2 Functional Genomics, National Cancer Research Institute, Largo Rosanna Benzi 10, 16132 Genoa, Italy
3 CNR–IEIIT, Torre di Francia, Via de Marini 6, 16149 Genoa, Italy
4 Department of Medicine IV, University of Tübingen, Hoppe-Seyler-Str. 3, 72076 Tübingen, Germany
5 Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genoa, Italy
6 University of Genoa, Via Balbi 2 – 16126 Genoa, Italy
7 Institute of Pathology, Academic Hospital Munich-Bogenhausen, Englschalkingerstr. 77, 80927 Munich, Germany

* To whom correspondence should be addressed: Tel: +49 89 5160 2650; Fax: +49 89 5160 4740; Email: bachmeier{at}med.uni-muenchen.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
The dietary antioxidant Curcumin has been proposed for cancer chemoprevention since it induces apoptosis and inhibits the formation of breast cancer metastases. Curcumin acts through the inhibition of phosphorylation of the inhibitor of kappa B (I{kappa}B), which in turn reduces the nuclear translocation of nuclear factor kappa B (NF{kappa}B), an inflammation- and cell survival-related transcription factor. However, it is not clear whether the strong antimetastatic effect can exclusively be explained by inhibition of NF{kappa}B. Here, we addressed the effects of Curcumin (IC50 = 17 µM) in MDA-MB-231 breast cancer cells using microarray gene expression analyses. Among the 62 genes whose expression was significantly altered, we found the two inflammatory cytokines CXCL1 and -2 (Gro{alpha} and -β) that were downregulated. Further validation of the microarray results by quantitative real-time reverse transcription–polymerase chain reaction, western blots and enzyme-linked immunosorbent assay revealed that Curcumin impairs transcription of CXCL1 and -2 >24 h and reduces the corresponding proteins. Using small interfering RNA techniques, we elucidated the underlying molecular mechanism revealing that reduction of CXCL1 and -2 messenger RNA levels is NF{kappa}B dependent and requires intact I{kappa}B{alpha} expression. Moreover, CXCL1 and -2 silencing leads to downregulation of several metastasis-promoting genes among which we found the cytokine receptor CXCR4. We therefore suggest that the decrease of CXCL1 and -2 mediated by Curcumin is involved in the inhibition of metastasis.

Abbreviations: HMOX1, hemeoxygenase-1; I{kappa}B, inhibitor of kappa B; mRNA, messenger RNA; NF{kappa}B, nuclear factor kappa B; PTGS2, prostaglandin-endoperoxide synthase 2; qRT–PCR, quantitative real-time reverse transcription–polymerase chain reaction; siRNA, small interfering RNA


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Curcumin (diferuloylmethane) is an antioxidant polyphenol from the plant turmeric (Curcuma longa) and is commonly used as a spice component. In traditional Indian medicine, Curcumin has been used to treat inflammation (1). Curcumin exerts antiproliferative and proapoptotic effects against various tumors in vitro (2–4) and in vivo, and it has been found to suppress carcinogenesis of the breast (5) and other organs (6–8). We and others have reported evidence that Curcumin acts via the inhibition of nuclear factor kappa B (NF{kappa}B)-dependent transcription in as much as it impairs phosphorylation of the inhibitor of kappa B (I{kappa}B), thereby preventing its proteolytic degradation (9,10). Inhibition of NF{kappa}B activation by Curcumin can explain the apoptotic effects of the polyphenol but many other effects including the strong antimetastatic effects that we have recently described (9) are not necessarily or exclusively linked to NF{kappa}B action. On the other hand, Curcumin’s well-established effects on NF{kappa}B do not allow the simple conclusion that the expression of all NF{kappa}B target genes will actually be affected by the drug in a given cell type. As for most transcription factors, the extent of induction by NF{kappa}B greatly varies for different target genes probably as a consequence of number and location of NF{kappa}B response elements in their promoters and of interactions with other transcriptional regulators. It is therefore probable that different target genes will differently respond to the inhibition of NF{kappa}B in various cell types, especially when the inhibition is not complete as it is the case for Curcumin.

For this reason, we wished to analyze the complete scale of Curcumin effects on gene transcription in the breast cancer cell line MDA-MB-231. We recently demonstrated that the metastatic potential of these cells is strongly reduced upon treatment with Curcumin in a murine model of hematogenous metastasis: 68% of the mice treated had no or few metastases as opposed to 17% of the untreated mice (9). Metastasis requires a tight interaction of the invasive tumor cell with the microenvironment at the site of the primary tumor as well as in the target tissue. In fact, many of the genes that have been identified by whole transcriptome analyses as being involved in metastasis act in cell-to-cell communication. The inflammatory cytokine, CXCL1, for example, has been identified as being overexpressed by breast cancer cells with an elevated potential to metastasize to the lung (11–13). Tumor derived prometastatic stimuli like CXCL1 provoke infiltration of inflammatory cells which in turn promote angiogenesis thus providing the route for metastatic dissemination. Chemoprevention of metastasis could therefore also act via the regulation of cell communication molecules produced by the tumor cell.

Most cancers are surgically removed shortly after diagnosis. At that point, either (micro-) metastases have already formed or they never will. Prevention of metastasis must therefore address growth of micro-metastases to clinically overt ones. Inhibition of the formation rather than the growth of already existing ones could be an important prevention target, yet would have to be performed in high-risk patients who do not yet have a clinically manifest disease. We need to understand the molecular mechanisms of action of preventive drugs in specific tumor types in order to understand which patients may benefit from such a strategy. Dietary compounds like Curcumin, which lack toxicity, are particularly suited for the development of long-term prevention in healthy high-risk patients.

For this reason, we wished to obtain a complete picture of Curcumin-responsive genes in breast cancer cells and therefore performed gene expression profiling of Curcumin-treated MDA-MB-231 breast cancer cells using complex microarrays. We show for the first time that Curcumin significantly reduces the messenger RNA (mRNA) and protein expression of the inflammatory cytokines CXCL1 and -2 (GRO{alpha} and -β) and describe the underlying regulation mechanisms. We suggest that downregulation of these cytokines can contribute to the inhibition of metastases observed in the mouse model.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell culture conditions
We obtained the estrogen receptor-negative cell line MDA-MB-231 (14,15) (referred to as 231 cells) from American Type Culture Collection. MDA-MB-231 cells injected into the mammary fat pad of nude mice result in the formation of tumors and distant metastases in lungs, brain and lymph nodes of most mice (16).

Cells were grown at 37°C in a humidified atmosphere of 5% CO2 in modified eagle's medium (Eagle’s) with Earle’s salts supplemented with 5% heat inactivated fetal calf serum, 1% L-glutamine solution (200 mM), 1% sodium pyruvate solution (100 mM), non-essential amino acids and vitamins. Medium was changed every 2 days.

Curcumin treatment of cells
Curcumin was purchased from Fluka (Buchs, Switzerland). Our own high-performance liquid chromatography analysis revealed that the preparation contained {approx}82.38% Curcumin, {approx}15.03% desmethoxycurcumin and {approx}2.59% bisdesmethoxycurcumin. The polyphenol was dissolved in 0.5 M NaOH as a 25 mM stock solution and stored at –20°C. For the use in cell culture, a 2.5 mM solution in sterile phosphate-buffered saline was prepared. Curcumin was applied at an end concentration of 25 µM in all assays. For microarray analysis, MDA-MB-231 cells were treated 6 h and for expression data evaluation and kinetic analysis 2, 4, 6, 15 and 24 h with 25 µM Curcumin. Further time points have not been analyzed due to the strong apoptotic effect of the polyphenol, an MDA-MB-231 cells, which we previously published (9).

Preparation of conditioned media
Cell culture supernatants of Curcumin and mock-treated MDA-MB-231 cells were collected and centrifuged 15 min at 4000g. The supernatants were used for western blots and enzyme-linked immunosorbent assay analyses.

Preparation of cellular extracts
Cells were washed three times with phosphate-buffered saline and collected by scraping and centrifuged. Lysis buffer (10 mM Na3PO4, 0.4 M NaCl and 0.2% Triton X-100) was added to the pellets and the mixture was sonicated. After centrifugation for 15 min at 15 000g, the supernatant containing the soluble proteins was collected and either analyzed immediately or stored at –20°C.

Determination of protein concentration
Protein concentrations were determined by the bicinchoninic acid protein assay (Pierce, Oud-Beijerland, Netherlands) with bovine serum albumin as standard.

Preparation of RNA, cDNA and cRNA
Total RNAs were isolated from cells treated with 25 µM Curcumin for several time periods and from cells silenced with specific small interfering RNA (siRNA) oligos or from control cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Thereafter, oligo dT-primed cDNAs were synthesized using the SuperScript® III First-Strand Synthesis SuperMix (Invitrogen, Irvine, CA) following the manufacturer’s instructions. For microarray analysis, double-stranded cDNAs were synthesized using the Custom SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) and extracted with phenol–chloroform–isoamyl alcohol (25:24:1), ethanol precipitated and used to prepare cRNAs using the Bioarray High Yield RNA Transcription Kit (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. cRNAs were purified using the RNeasy Mini Kit (Qiagen), controlled by agarose gel electrophoresis and subjected to fragmentation for 35 min at 94°C in fragmentation buffer (40 mM Tris–acetate pH 8.1, 100 mM CH3COOH and 30 mM Mg(CH3COO)2 x 4H2O).

GeneChip microarray analysis and data normalization
Labeled cRNA was used for screenings of GeneChip Human Genome U133plus2 arrays (Affymetrix). The experiment consisted of three biological replicates. Hybridization and scanning were performed on the Affymetrix platform. Data were normalized following the GC robust microarray analysis procedure (17) of Bioconductor 1.4 (18) (http://www.bioconductor.org). Statistically significant expression changes were determined using permutation tests [significance Analysis of Microarrays (19); http://www-stat.stanford.edu/~tibs/SAM/]. Genes regulated at least 2-fold in comparison with untreated controls were considered. The delta value was set to return a median false significant number <1. Annotations were obtained through the DAVID database (http://www-stat.stanford.edu/~tibs/SAM/) (20).

Quantitative real-time reverse transcription–polymerase chain reaction
Expression data validation was performed by quantitative real-time reverse transcription–polymerase chain reaction (qRT–PCR) using RNA extracted from drug- or mock-treated cells; analysis of transcript expression after gene silencing was performed from cells transfected with siRNAs or a non-silencing fluorescein-labeled control (Qiagen). RNA was extracted from cells using the RNeasy Protect Mini Kit (Qiagen) according to the recommendation of the manufacturer and reverse transcribed as above with oligo dT primers in 20 µl final volume. All primers for the genes tested were designed using primer3 software (21) with a temperature optimum of ~60°C and a product length of 100–150 nucleotides (see primer list, supplementary Table, available at Carcinogenesis Online). Real-time PCR was performed on an I-Cycler (Bio-Rad, Hercules, CA) using iQ Supermix (Bio-Rad) supplemented with 10 nM fluorescein (Bio-Rad), 0.1x Sybr-Green I (Sigma–Aldrich, Buchs, Switzerland), 2.5 µl of cDNA (5x diluted), 3 pmol sense and antisense primers in a final reaction volume of 25 µl. After an initial denaturation step of 3 min during which the well factor was measured, 50 cycles of 15 s at 95°C followed by 30 s at 60°C were performed. Fluorescence was measured during the annealing step in each cycle. After amplification, melting curves with 80 steps of 15 s and 0.5°C increase were performed to monitor amplicon identity. Amplification efficiency was assessed for all primer sets utilized in separate reactions, and primers with efficiencies >94% were used. Expression data were normalized on glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) and on RNA polymerase II gene expression data obtained in parallel. Relative expression values with standard errors and statistical comparisons (unpaired two-tailed t-test) were obtained using Qgene software (22). Expression changes were calculated using the mean value of normalizations obtained using GAPDH and RNA polymerase II genes as references.

Gene silencing
RNA interference was used to generate specific knockdowns of NF{kappa}B and I{kappa}B{alpha} mRNA transcripts in the human breast cancer cell line MDA-MB-231. siRNAs [r(GAUCAAUGGCUACACAGGA) d(TT) and r(UCCUGUGUAGCCAUUGAUC) d(TT)] targeted to NF{kappa}B and [r(GAAAAGGCACUGACCAUGG) d(TT) and r(CCAUGGUCAGUGCCUUUU d(TT)] named I{kappa}B{alpha}1 as well as [r(CCAGCCAGAAAUUGCUGAG d(TT) and r(CUCAGCAAUUUCUGGCUGG d(TT)] named I{kappa}B{alpha}2 targeted to I{kappa}B{alpha} were synthesized and annealed (Qiagen). Predesigned annealed double-stranded siRNAs targeted to CXCL1 and CXCL2 were synthesized and purchased by Ambion (Applied Biosystems, Darmstadt, Germany). A non-silencing fluorescein labeled siRNA (Qiagen) was used as control for transfection efficiency as well as for monitoring the effect of silencing during all experiments. Cell cultures with at least 90% transfection efficiency were used for further studies. Transfection of MDA-MB-231 cells (40% confluency) with siRNA was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the recommendations of the manufacturer. Briefly, the transfection reagent was preincubated with the siRNA Oligos either targeted to NF{kappa}B, I{kappa}B{alpha}, CXCL1 and CXCL2 or to an irrelevant control 30 min prior to the application to the cells.

Western blots
Conditioned media from Curcumin-treated (6, 15 and 24 h) and non-treated control cells as well as conditioned media from p65-, I{kappa}B{alpha}-, CXCL1- or CXCL2-silenced and non-silenced control cells were analyzed using antibodies against CXCL1 and -2 (both from Dianova, Hamburg, Germany). Cellular extracts from p65 I{kappa}B{alpha}-, CXCL1- or CXCL2-silenced and non-silenced control cells were analyzed using a p65, CXCR4 or I{kappa}B{alpha} antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Equal amounts of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the intracellular amount of β-actin was analyzed as loading control (antibody from Sigma, Deisenhofen, Germany). For conditioned media, the amount of protein blotted onto the membranes was visualized with Ponceau red before blocking. Following electrophoretic separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, proteins were electroblotted on nitrocellulose membranes (Whatman, Brentford, UK). The membranes were blocked in 5% non-fat milk (Merck, Darmstadt, Germany) overnight at 4°C. The first antibody was incubated for 1 h at room temperature. Thereafter, membranes were washed in tris buffered saline with Tween buffer, and a further incubation was carried out with a peroxidase-conjugated antibody (Dako, Hamburg, Germany) for 1 h at room temperature. The enhanced chemiluminescence system was used for visualization of the protein bands as recommended by the manufacturer (GE Healthcare, Little Chalfont, UK). Semi-quantitative evaluation of the bands was performed by densitometric analysis with the ImageJ software provided by the National Institutes of Health (http://rsb.info.nih.gov/ij/).

CXCL1 enzyme-linked immunosorbent assay
CXCL1 was quantified in conditioned media of Curcumin-treated and non-treated MDA-MB-231 cells using the Quantikine Human CXCL1/GRO{alpha} Immunoassay (R&D Systems, Minneapolis, MN) according to the recommendations of the manufacturer.

Promoter analysis
Genomic sequences containing the coding sequences for CXCL1 and -2 have been analyzed for the presence of NF{kappa}B element using the P-Match algorithm (23) in the sequences 5000 nts up- and downstream of the transcription start site.

Data analysis
Statistical significance was assessed by comparing mean (±SD) values, which were normalized to the control group with Student’s t-test for independent groups. One-way analysis of variance was used to test for statistical significance (P < 0.05), and when significance was determined, Bonferroni’s multiple comparison test was performed post hoc, as indicated in the figure legends. Statistical analysis was performed using the Prism software (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Curcumin alters the expression of various genes in human breast cancer cells
We used 25 µM Curcumin [IC50 = 17 µM, (24)] for the treatment of the human breast cancer cell line MDA-MB-231, since we previously published that this concentration induces apoptosis and inhibits expression and activity of several transcription factors (9). In addition, dose-dependent effects in the range of 1–50 µM had been reported for breast cancer cell lines with strongest effects between 20 and 50 µM (25).

Analysis of the gene expression profile in the human breast cancer cell line MDA-MB-231 treated with Curcumin for 6 h revealed that 62 probe sets were statistically significant as tested using a permutation analysis [significance analysis of microarrays (19)] with a false discovery rate of 0%, q value = 0, fold change threshold = 2 (Table I). Of these probe sets, 37 were significantly downregulated and 25 were upregulated. These probe sets correspond to 57 independent transcripts. Annotations were obtained from Affymetrix and/or DAVID for 45 of these genes (Table I). The genes which were induced >3-fold by Curcumin treatment are hemeoxygenase-1 (HMOX1) (11-fold) and GCLM (3.8-fold; three hits) whereas several genes were significantly reduced e.g. EGR1 (5.5-fold; two hits), prostaglandin-endoperoxide synthase 2 (PTGS2/COX2) (3.7-fold; two hits) and the chemokines CXCL1 (2.9-fold) and CXCL2 (5.3-fold).


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  		Table I. Gene expression after Curcumin treatment in human breast cancer cells

 
We have recently described Curcumin as an inhibitor of NF{kappa}B activation in the human breast cancer cell line MDA-MB-231 (9). We therefore examined whether the genes that we found to be regulated by Curcumin in these cells were known NF{kappa}B targets. As NF{kappa}B has no entry in the gene ontology annotation database, we based our analysis on a list of NF{kappa}B-related genes that we obtained from the website http://people.bu.edu/gilmore/nf-kb/. NF{kappa}B targets are indicated in Table I in italics.

The differences in gene expression found by microarray analyses were validated by using qRT–PCR. We chose genes that were >3-fold regulated according to our microarray results (see also Table I). Among the genes that were >3-fold upregulated (1) by Curcumin treatment, we chose HMOX1 and GCLM which are both NF{alpha}B target genes. The gene that was found to be most strongly downregulated by the polyphenol was the transcription factor EGR1 whose expression has very recently been shown to be reduced by Curcumin in colon cancer cells in vitro (26). Furthermore, we selected the chemotactic cytokines CXCL2 and CXCL1, which belong to the genes on which only little is known in the context of breast cancer and whose regulation mechanisms are not fully understood. Finally, we selected PTGS2/COX2 which is well known to be downregulated by Curcumin, but turned out to be only slightly and temporarily diminished in MDA-MB-231 cells treated with the polyphenol.

Although some variation concerning the degree of regulation was observed, the data obtained with microarrays were substantially confirmed for all genes by qRT–PCR (Figure 1).


Figure 1
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  		Fig. 1. qRT–PCR validation of microarray data. cDNA from Curcumin- or mock-treated MDA-MB-231 cells from three independent experiments was used for qRT–PCR validation of the gene expression data obtained from microarray analyses. The microarray data were substantially confirmed by qRT–PCR: PTGS2/COX2, CXCL1, CXCL2 and EGR1 were downregulated, and GCLM and HMOX1 were upregulated in MDA-MB-231 cells treated for 6 h with 25 µM Curcumin. Data were presented as relative expression in Curcumin-treated cells as compared with mock-treated cells (logarithmic scale).

 
In summary, microarrays together with qRT–PCR validation results clearly demonstrate that PTGS2/COX2, CXCL1, CXCL2 and EGR1 were downregulated, whereas GCLM and HMOX1 were upregulated in MDA-MB-231 cells treated for 6 h with Curcumin.

These and all following results are representative for measurements accomplished with MDA-MB-231 cells from three different experiments.

Kinetics of gene expression in Curcumin-treated breast cancer cells
To investigate the dynamics of the transcriptional changes caused by Curcumin in MDA-MB-231 cells, a time course analysis was performed. The breast cancer cells were treated with 25 µM Curcumin for different time periods and total RNA was extracted at different time points (6, 15 and 24 h). The impact of Curcumin on the expression of the six genes selected before (see section above) was monitored over time by qRT–PCR and the results are illustrated in Figure 2.


Figure 2
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  		Fig. 2. Kinetics of gene expression in Curcumin-treated breast cancer cells. MDA-MB-231 cells were treated with 25 µM Curcumin and total RNA was extracted after 2, 4, 6, 15 and 24 h. (a) Expression of PTGS2/COX2, EGR, HMOX1 and GCLM was determined by qRT–PCR. While transcription levels of PTGS2/COX2 and EGR were downregulated, expression of HMOX1 and GCLM revealed a strong upregulation. (b) Expression of CXCL1, -2 and NF{kappa}B (p65) was downregulated upon Curcumin treatment from early time points on (2 h). *P < 0.05; **P < 0.01 and ***P < 0.001 (analysis of variance with Bonferroni’s multiple comparison test).

 
In accordance with the results obtained by microarray and qRT–PCR validation analyses, gene expressions of PTGS2/COX2 and EGR were downregulated and that of HMOX1 and GCLM were induced by Curcumin treatment. Additionally, it became evident that the change in gene expression of ERG1, HMOX1 and GCLM was robust throughout the observed time course, whereas the effect of the polyphenol on the expression of PTGS2/COX2 was only transient and much weaker than expected: transcription levels of this gene were reduced after 15 h of treatment, but the effect was abrogated after 24 h (Figure 2a). The strong effect of Curcumin on transcription of CXCL1 and CXCL2 is also confirmed by real-time PCR (Figure 2b). Interestingly, regulation of these two cytokines parallels the kinetics of p65/NF{kappa}B transcription from very early time points on (Figure 2b).

Curcumin impairs the expression of CXCL1 and -2 proteins
Since it was not known previously that Curcumin impairs the expression of the inflammatory cytokines CXCL1 and -2, we investigated in detail whether the Curcumin-mediated downregulation of the two cytokines that we discovered by microarray and qRT–PCR analyses at the level of transcription could be verified on the protein level. Well in accordance with our previous results on mRNA expression, CXCL1 and -2 protein concentrations were reduced in conditioned media of Curcumin-treated MDA-MB-231 cells as confirmed by western blots (Figure 3a) with densitometrical quantification. The decrease of CXCL1 amounts in cell culture supernatants was 3.3-fold after 6 h of treatment with Curcumin and 4.2-fold after 24 h of treatment (Figure 3a, left panel). The effect of the polyphenol on the expression of CXCL2 in the breast cancer cell line was similar though weaker: after 6 h treatment with the polyphenol, the decrease was 1.8-fold with a further decline to 2.0-fold at 24 h (Figure 3a, right panel). To assure that equal amounts of protein were subjected to the gels, we monitored the intensities of the bands on the nitrocellulose membranes by Ponceau red staining (Figure 3a, ‘loading control’). Well in line with our western blot results, enzyme-linked immunosorbent assay analyses of conditioned media from MDA-MB-231 cells treated with Curcumin revealed an ~3.5-fold diminished CXCL1 concentration after 6 h with enduring effect up to 24 h (Figure 3b).


Figure 3
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  		Fig. 3. Curcumin impairs the expression of CXCL1 and -2 proteins. (a) Western blots of conditioned media from MDA-MB-231 cells treated for 6, 15 and 24 h with Curcumin reveal a downregulation of CXCL1 and CXCL2 expression when compared with non-treated controls (ctrl). This effect was quantified by subsequent densitometry which indicates that the differences in expression were up to ~4-fold for CXCL1 and up to 2.5-fold for CXCL2. To verify that equal amounts of protein were loaded on the gel and subsequently blotted to the membrane, we visualized the protein bands by Ponceau staining. (b) Quantification of CXCL1 by enzyme-linked immunosorbent assay demonstrated that Curcumin downregulates the expression of this chemokine up to ~4-fold, which correlates very well with our data obtained from western blotting.

 
CXCL1/2 expression in breast cancer cells is regulated via the NF{kappa}B pathway
We asked whether the effect of Curcumin on the expression of the two cytokines depends on NF{kappa}B in particular since Tian et al. (27) described this transcription factor to be a regulator of CXCL2. Our own analyses of the CXCL1 and -2 promoters revealed 7 and 13 perfectly conserved NF{kappa}B elements, respectively, in this region, 1 and 2 of which within 1000 nts upstream (–1000) and 500 nts downstream (+500) of the transcription start site. We also analyzed the promoter region of PTGS2/COX2, a classic NF{kappa}B target, which contains six NF{kappa}B elements, none of which in the region between –1000 and +500. It is therefore probable that the two cytokine genes are regulated by NF{kappa}B.

In this context, we modulated the NF{kappa}B pathway in MDA-MB-231 cells by specifically silencing the gene transcription of p65 by RNA interference. As determined by qRT–PCR 6 and 24 h after transfection, we achieved knock-down efficiencies of 62 and 77%, respectively, for p65 as compared with control cells transfected with non-target-directed siRNA (Figure 4a). As a consequence of p65 silencing, expression of the inflammatory cytokines CXCL1 and CXCL2 was downregulated to 28 and 48%, respectively, after 6 h and both to ~50% after 24 h.


Figure 4
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  		Fig. 4. CXCL1/2 is regulated via the NF{kappa}B pathway. p65 silencing downregulates CXCL1 and -2 expression in MDA-MB-231 cells—(a) p65 was silenced in MDA-MB-231 cells and quantitative RT–PCR analysis was performed to monitor the effects of p65 knockdown on the expression of CXCL1 and -2. All data are shown as fraction of values in non-silenced control cells. *P < 0.05; **P < 0.01 and ***P < 0.001 (analysis of variance with Bonferroni’s multiple comparison test). (b) Western blots of cellular extracts from p65-silenced MDA-MB-231 cells (left panel) revealed a 3.3-fold reduction of p65 protein (indicated with an arrow, densitometric data not shown) 24 h after transfection with siRNAs (p65-si) when compared with controls (nonsi). Equal intensities of the bands specific for β-actin (indicated with an arrow) indicate that equal amounts of total proteins were loaded on each lane (left lower panel). Conditioned media from MDA-MB-231 cells carrying the specific p65 knockdowns show an ~3-fold decline of secreted CXCL1 and CXCL2 (middle and right upper panels) followed by densitometric evaluation of the intensities of the bands (middle and right lower panels; ***P < 0.001). Regulation of CXCL1/2 by Curcumin requires intact I{kappa}B{alpha} expression—(c) Gene silencing of I{kappa}B{alpha} increases NF{kappa}B activity resulting in overexpression of the NF{kappa}B target genes CXCL1 and CXCL2. As determined by qRT–PCR, the use of oligo 1 (I{kappa}B{alpha}-si 1) halved the transcription levels of I{kappa}B{alpha}, whereas the application of oligo 2 (I{kappa}B{alpha}-si 2) resulted in a silencing effect of ~30%, 24 h after transfection. Accordingly, CXCL1 and CXCL2 levels increased ~5-fold using I{kappa}B{alpha}-si 1 and ~3-fold using I{kappa}B{alpha}-si 2 when compared with control cells that were transfected with a non-target-directed siRNA (nonsi). *P < 0.05; **P < 0.01 and ***P < 0.001 (analysis of variance with Bonferroni’s multiple comparison test). (d) Western blots of cellular extracts and conditioned media from I{kappa}B{alpha}-silenced MDA-MB-231 cells reveal a reduction of I{kappa}B{alpha} 15 h after transfection with I{kappa}B{alpha}-si 1 when compared with controls (nonsi); equal intensities of the bands specific for β-actin (indicated with an arrow) reveal that equal amounts of total proteins were loaded on each lane (left panel). Accordingly, concentration of CXCL1 and CXCL2 in conditioned media of I{kappa}B{alpha}-silenced cells increased as evidenced here by western blots (middle and right upper panels) followed by densitometric evaluation of the bands (middle and right lower panels). ***P < 0.001 (analysis of variance with Bonferroni’s multiple comparison test).

 
To verify these findings at the protein level, we performed western blotting analysis of lysates of p65 knocked down MDA-MB-231 cells. Consistent with our mRNA data, cellular extracts of p65-silenced MDA-MB-231 revealed a 3.3-fold reduction of p65 protein (65 kDa) 24 h after transfection with siRNAs when compared with control cells that were transfected with non-target-directed siRNA. As a loading control, intracellular β-actin levels were monitored (Figure 4b, left panel).

Culture supernatants from MDA-MB-231 cells carrying the specific p65 knockdowns revealed an ~3.5-fold decline of secreted CXCL1 (Figure 4b, middle) and an ~2-fold decline of secreted CXCL2 as determined by western blotting (Figure 4b, right panel) and densitometric evaluation.

These experiments clearly demonstrate that knocking down the proinflammatory transcription factor NF{kappa}B results in a diminished expression of the two inflammatory chemokines.

Regulation of CXCL1/2 by Curcumin requires intact I{kappa}B{alpha} expression
After we had shown that NF{kappa}B is the key regulator in the molecular mechanism of Curcumin-mediated reduction of CXCL1 and -2, we wished to study the contribution of I{kappa}B{alpha}, which acts as an inhibitor of cellular translocation and activation of p65. We therefore designed two siRNA oligos directed to I{kappa}B{alpha} and established conditions in the human breast cancer cell line MDA-MB-231 to specifically silence the transcription of this gene by RNA interference (Figure 4c). As determined by qRT–PCR, we achieved different knock-down efficiencies of I{kappa}B{alpha} depending on the siRNA utilized. With the use of oligo 1 (I{kappa}B{alpha}-si 1), I{kappa}B{alpha} transcription levels could be halved, whereas the application of oligo 2 (I{kappa}B{alpha}-si 2) resulted in a silencing effect of ~30%, 24 h after transfection. Accordingly to the knock-down efficiencies of I{kappa}B{alpha}, CXCL1 and CXCL2 mRNA expression levels increased ~5-fold using I{kappa}B{alpha}-si 1 and ~3-fold using I{kappa}B{alpha}-si 2 when compared with control cells that were transfected with a non-target-directed siRNA (nonsi). The rise in CXCL1 and CXCL2 expression can be attributed to the increased activation of p65, which is caused by the lack of its inhibitor, I{kappa}B{alpha}.

Well in line with the results from qRT–PCR, protein levels of CXCL1 and CXCL2 augmented when I{kappa}B{alpha} was silenced (Figure 4d). Western blots from cellular extracts of I{kappa}B{alpha}-silenced MDA-MB-231 revealed a diminished expression of the corresponding protein 15 h after transfection with I{kappa}B{alpha}-si 1 when compared with control cells that were transfected with a non-target-directed siRNA (nonsi). As a loading control intracellular β-actin levels were monitored (left panel). Accordingly, CXCL1 and CXCL2 concentrations in the corresponding conditioned media were elevated ~2.5-fold as evidenced by densitometry of the western blot bands (middle and left panels).

CXCL1 and -2 silencing inhibits the metastatic potential of breast cancer cells
We furthermore asked whether downregulation of the two cytokines can affect the metastatic potential of the cells. To answer this question, we analyzed the expression of a series of metastasis-related genes in CXCL1- and CXCL2-silenced MDA-MB-231 cells. We chose genes whose functional involvement has been shown through the analysis of highly metastatic subclones of MDA-MB-231 cells with pulmonary tropism (12). Here, we demonstrate that silencing of the two cytokines is followed by downregulation of the metastasis-related genes SPARC, CXCR4, PTGS2/COX2, ANGPT, ALDH, EREG and EFEMP as evidenced by quantitative qRT–PCR (Figure 5a; CXCL1 silencing: left panel; CXCL2 silencing: right panel). Downregulation of the metastatic potential of breast cancer cells is in general more pronounced after CXCL1 silencing (left panel).


Figure 5
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  		Fig. 5. Expression of metastasis-related genes after gene silencing of CXCL1 and CXCL2. (a) Silencing of the two cytokines is followed by downregulation of the metastasis-related genes SPARC, CXCR4, PTGS2/COX2, ANGPT, ALDH, EREG and EFEMP as evidenced by quantitative RT–PCR (CXCL1 silencing: left panel; CXCL2 silencing: right panel). Downregulation of the metastatic potential of breast cancer cells is in general more pronounced after CXCL1 silencing (left panel). (b) Western blots of CXCL1- or CXCL2-silenced MDA-MB-231 cells (right panel; lanes indicated with CXCL1-si and CXCL2-si, respectively) demonstrate that CXCR4 expression is abrogated in lysates from MDA-MB-231 cells 15 h after transfection with the according oligo when compared with cells transfected with a non-silencing oligo (lanes indicated with nonsi). As a loading control, intracellular β-actin levels were monitored (right panel). Silencing of I{kappa}B{alpha} increases the metastatic potential of MDA-MB-231 breast cancer cells. (c) Knockdown of I{kappa}B{alpha} reduces mRNA expression levels of the metastasis-related genes PTGS2/COX2, SPARC and ANGPT (P < 0.001) when compared with control cells that were transfected with a non-target-directed siRNA (nonsi).

 
Among the genes that were downregulated by silencing of CXCL1 or -2 equally was CXCR4, which has recently been implicated to promote metastasis in breast cancer (28). Our western blot results of CXCL1- or CXCL2-silenced MDA-MB-231 cells (Figure 5b, right panel; lanes indicated with CXCL1-si and CXCL2-si, respectively) demonstrate that CXCR4 expression was abrogated in lysates from MDA-MB-231 cells 15 h after transfection with the specific oligo when compared with cells transfected with a non-silencing oligo (lanes indicated with nonsi). Intracellular β-actin levels were monitored as a loading control (right panel).

Silencing of I{kappa}B{alpha} increases metastatic potential of MDA-MB-231 cells
In order to further investigate the involvement of NF{kappa}B in regulating the metastatic potential of breast cancer cells through CXCL1 and -2, we complemented the list of genes altered after I{kappa}B{alpha} silencing (see also Regulation of CXCL1/2 by Curcumin requires intact I{kappa}B{alpha} expression; Figure 4c and d) with the metastasis-related genes PTGS2/COX2, SPARC and ANGPT.

Accordingly to the knockdown of I{kappa}B{alpha}, mRNA expression levels of all three genes increased statistically highly significantly (P < 0.001) using both silencer oligos (I{kappa}B{alpha}-si 1 or I{kappa}B{alpha}-si 2, respectively) when compared with control cells that were transfected with a non-target-directed siRNA (nonsi). As for CXCL1 and CXCL2 before, the rise in the expression of the metastasis-related genes PTGS2/COX2, SPARC and ANGPT can be attributed to the increased activation of NF{kappa}B, which is caused by the lack of its inhibitor I{kappa}B{alpha}, leading to elevated metastatic potential of the breast cancer cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
The dietary polyphenol Curcumin is an interesting candidate for chemoprevention since much evidence on its efficacy and the absence of undesired side effects has accumulated. Curcumin preparations used for in vitro and in vivo studies normally also contain desmethoxycurcumin and bisdesmethoxycurcumin, and, in the liver, curcumin is metabolized to tetrahydrocurcumin, hexahydrocurcumin, curcumin glucuronide and curcumin sulfate (29). It is not clear which of these compounds is actually biologically active. We recently showed strong effects on hematogenous metastasis in the mouse fed with a 1% Curcumin diet (9).

Bland chemoprevention, especially in terms of secondary prevention, will probably be able to retard but not to definitively inhibit tumor progression. In order to observe an effect using compounds like Curcumin in clinical trials, the identification of potential responders would greatly increase the power of such trials. Potential responders are those patients whose tumors expose molecular characteristics that match the molecular effects of Curcumin. For this reason, we wished to understand the effects of Curcumin on breast cancer cells in detail.

We had recently shown that Curcumin strongly prevents the formation of hematogenous metastases in vivo after intracardial injection of the metastatic human breast cancer cell line MDA-MB-231 into nude mice (9). In addition, extensive in vitro studies by us and others had previously identified the transcription factor NF{kappa}B to be the main target of Curcumin (9,30) and other natural occurring chemopreventive polyphenols like epigallocatechin-gallate (31). However, downregulation of NF{kappa}B-related genes by Curcumin alone, which leads, as is known, to induction of apoptosis, cannot explain the antimetastatic properties of this compound.

In order to investigate the antimetastatic potential of the polyphenol, we used microarray gene expression profiling to identify all genes whose expression is altered in MDA-MB-231 cells after 6 h of Curcumin treatment, where the apoptotic effect is still low [(9) and B.E.Bachmeier, I.V.Mohrenz, V.Mirisola, E.Schleicher, F.Romeo, C.Höhneke, M.Jochum, A.G.Nerlich and U.Pfeffer, unpublished data]. Among the 62 genes that were statistically significantly regulated by Curcumin, we found expression of the two chemotactic cytokines, CXCL1 and -2, the transcription factor, EGR1, and the prostaglandin synthase, PTGS2/COX2, to be reduced by the polyphenol. Interestingly, the strength of downregulation of CXCL1 and -2 was much more potent than that of PTGS2/COX2, which is described in literature to be strongly inhibited by Curcumin (32). In other tumor types, the effect of the polyphenol on the expression of PTGS2/COX2 seems to be more potent since very recently it has been shown that induction of PGE2 synthesis and downregulation of PTGS2/COX2 by Curcumin inhibits cell survival and induces apoptosis in colorectal adenocarcinoma cell lines (33). It is unlikely that the remote degree of PTGS2/COX2 regulation that we have observed in breast cancer cells has a major impact on tumor progression.

Six genes, whose syntheses were >3-fold altered as evidenced by microarray results, were selected for validation of their expression change by qRT–PCR. The qRT–PCR results agreed with those obtained from expression arrays. In detail, we found the expression of EGR1, which belongs to the group of metastasis-associated genes, to be reduced in Curcumin-treated cells. This is well in line with other reports that demonstrated that Curcumin inhibited the activity of the transcription factor EGR1 and thereby growth of human colon cancer cells (26). Recent work has shown that EGR1 expression is significantly higher in gastric cancer tissues than in normal mucosa and has a significant role in carcinogenesis and in cancer progression, especially metastasis (34).

HMOX1 and GCLM were the most upregulated genes by the polyphenol in the human breast cancer cell line MDA-MB-231. They are known to be linked to antioxidant responses. The highly inducible enzyme, HMOX1, metabolizes heme, thereby protecting a variety of cells against oxidative stress and apoptosis. Curcumin has been shown to induce antioxidant response element-mediated gene expression of HMOX1 and GCLM in human monocytes (35) or hepatocytes (36) by a mechanism that is directed by Nrf2, phospho kinase C and p38 induction. Nevertheless, it had been shown that induction of HMOX1 by Curcumin did not protect breast cancer cells from apoptosis (37), in line with our observations.

We identify the two proinflammatory cytokines CXCL1 and -2 as major targets of Curcumin. Most probably, the two cytokines exert overlapping if not identical functions as shown in several cellular systems (38–42). The results presented here clearly demonstrate for the first time that the chemopreventive polyphenol downregulates mRNA expression and protein secretion of the two chemotactic cytokines in the metastatic breast cancer cell line MDA-MB-231 and give insight into the underlying regulation mechanism. Other studies have indeed described that CXCL1 and -2 are associated with tumor progression but left the underlying regulation mechanisms untouched. CXCL1 is known to promote the migration of breast cancer cells in vitro (42) and tumor growth, metastasis, as well as angiogenesis in mouse squamous cell carcinoma (41). Moreover Massaguè et al. have shown that highly metastatic subclones derived from MDA-MB-231 cells by dilution cloning overexpress CXCL1 and -2 (12,13). Li et al. (43) show a direct effect of CXCL1 and -2 overexpression on the invasive behavior of MDA-MB-231 and MCF-7 cells that can be abolished through the silencing of these genes.

Although originally identified on leukocytes, chemokines and their functional receptors are expressed by malignantly transformed cells (28,44). While many human cancers have a complex chemokine network that influences the extent of tumor cell growth, invasion, survival, migration and angiogenesis (45,46), we do not have a clear picture of the overall chemokine repertoire of an individual human cancer type by now. There exists evidence that CXCL1 expression in primary tumors is associated with decreased survival and the correlation between this chemokine and malignant melanoma is already well known (47,48), whereas the role of CXCL1 and -2 in mammary carcinoma and its progression is still poorly investigated. Up to now, there is only one report regarding the role of CXCL1 in breast cancer progression (11). We have recently described strong effects of Curcumin on hematogenous metastases in a murine model (9). Yet it is difficult to establish the role of CXCL1 and -2 in this type of experiment since the effect would become ‘evident’ in the metastases that do not form and not in those that do. As a matter of fact, our own analysis revealed that the few metastases formed in Curcumin-treated animals are of identical morphology and dimension as those formed in untreated controls. Curcumin thus affects the formation but not the growth of metastases (9).

Downregulation of CXCL1 and -2 has a clear effect on the metastatic potential of MDA-MB-231 cells since silencing of each of the two cytokines reduces the expression of several prometastatic genes that are contained in the lung metastasis signature developed using the same cellular model (12).

Very recently it has been shown that the expression of these two chemokines relies on the inactivation of the tumor suppressor SYK, a tyrosine kinase that apparently acts upstream of NF{kappa}B (43) and which we found not to be expressed in MDA-MB-231 cells (data not shown).

We have recently reported that Curcumin induces apoptosis and represses invasion of human breast cancer cells by downregulating the activation of NF{kappa}B (9). Knockdown of this transcription factor by gene silencing resulted in a reduced expression of the two proinflammatory chemokines in human breast cancer cells. We and others elucidated that Curcumin prevents NF{kappa}B activation by inhibiting phosphorylation and degradation of I{kappa}B{alpha}, which acts as inhibitor of cellular translocation and activation of p65 (9,10). We performed silencing experiments in order to make clear whether I{kappa}B{alpha}, which acts upstream of NF{kappa}B, influences chemokine expression. In silencing experiments using siRNA oligos directed against I{kappa}B{alpha}, its gene expression could be halved leading to a 5-fold increase of CXCL1 and -2 syntheses, which can be attributed to the increased activation of p65 caused by the lack of its inhibitor. We are the first to outline the causal relationship of NF{kappa}B in the molecular regulation of the two chemotactic cytokines CXCL1 and -2 that can be repressed by inactivation of this inflammatory-related transcription factor in human breast cancer cells. Moreover, we provide evidence that this mechanism requires intact I{kappa}B{alpha} expression.

Additionally, we found a higher metastatic potential in breast cancer cells in which I{kappa}B{alpha} was knocked down by gene silencing. The expression of various metastasis-related genes was significantly upregulated in I{kappa}B{alpha}-silenced cells indicating the involvement of NF{kappa}B. The increased activation of p65, which is caused by the lack of its inhibitor I{kappa}B{alpha}, leads to elevated metastatic potential of the breast cancer cells.

We found that some of the metastasis-related genes we investigated are affected in opposite direction by I{kappa}B{alpha} silencing and CXCL1/2 silencing. It is therefore probable that CXCL1 and -2 induce a positive feedback on NF{kappa}B activation that in turn leads to an increased metastatic potential. Thereby, we provide evidence that Curcumin reduces breast cancer metastasis through the inhibition of NF{kappa}B activation, which in turn impairs the expression of two prometastatic cytokines, CXCL1 and -2, which regulate the expression of a series of metastasis-promoting genes among which we found CXCR4, the receptor for SDF1/CXCL12. CXCL12 is released from metastasis target tissues and attracts CXCR4-expressing cells by means of chemotaxis (28). This appears to be interrupted in cells that loose CXCL1 or -2 expressions. In conclusion, we suggest that Curcumin reduces breast cancer metastasis through NF{kappa}B-mediated expression of the two prometastatic cytokines, CXCL1 and -2, which in turn reduces expression of the chemotactic receptor CXCR4 along with other metastasis-promoting genes.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Table can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Deutsche Akademischer Austauschdienst/Conferenza dei Rettori delle Università Italiane, programma Vigoni to B.E.B.; the Ministero della Salute, the Ministero dell'Istruzione, Università e Ricerca (FIRB—LITBIO), the Istituto Superiore Sanità (programma Italia-USA), the Compagnia San Paolo di Torino and the Comitato Interministeriale per la Programmazione Economica—Regione Liguria to U.P.


   Acknowledgments
 
We thank Adriana Albini for support in the initial phase of the work and Michele Di Candia for microarray hybridizations. Furthermore, we would like to acknowledge Dean Brenner for the high-performance liquid chromatography analysis of the Curcumin preparation.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received July 19, 2007; revised October 31, 2007; accepted November 4, 2007.


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