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Curcumin Inhibits Prostate Cancer Metastasis in vivo by Targeting the Inflammatory Cytokines CXCL1 and -2.

Authors:

Abstract

In America and Western Europe, prostate cancer is the second leading cause of death in men. Emerging evidence suggests that chronic inflammation is a major risk factor for the development and metastatic progression of prostate cancer. We previously reported that the chemopreventive polyphenol curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast cancer metastases. In this study, we analyze the effects of curcumin on prostate carcinoma growth, apoptosis and metastasis. We show that curcumin inhibits translocation of NFκB to the nucleus through the inhibition of the IκB-kinase (IKKβ, leading to stabilization of the inhibitor of NFκB, IκBα, in PC-3 prostate carcinoma cells. Inhibition of NFκB activity reduces expression of CXCL1 and -2 and abolishes the autocrine/paracrine loop that links the two chemokines to NFκB. The combination of curcumin with the synthetic IKKβ inhibitor, SC-541, shows no additive or synergistic effects indicating that the two compounds share the target. Treatment of the cells with curcumin and siRNA-based knockdown of CXCL1 and -2 induce apoptosis, inhibit proliferation and downregulate several important metastasis-promoting factors like COX2, SPARC and EFEMP. In an orthotopic mouse model of hematogenous metastasis, treatment with curcumin inhibits statistically significantly formation of lung metastases. In conclusion, chronic inflammation can induce a metastasis prone phenotype in prostate cancer cells by maintaining a positive proinflammatory and prometastatic feedback loop between NFκB and CXCL1/-2. Curcumin disrupts this feedback loop by the inhibition of NFκB signaling leading to reduced metastasis formation in vivo.
For Peer Review
Curcumin Inhibits Prostate Cancer Metastasis in vivo by
Targeting the Inflammatory Cytokines CXCL1 and -2
Journal:
Carcinogenesis
Manuscript ID:
CARCIN-2012-00565.R1
Manuscript Type:
Original Manuscript
Date Submitted by the Author:
21-Aug-2012
Complete List of Authors:
Killian, Peter; LMU-Munich, Dept. of Clinical Chemistry and Clinical
Biochemistry
Kronski, Emanuel; LMU-Munich, Dept. of Clinical Chemistry and Clinical
Biochemistry
Michalik, Katharina; LMU-Munich, Dept. of Clinical Chemistry and Clinical
Biochemistry
Barbieri, Ottavia; AOU San Martino-IST National Cancer Research Institute,
IRCCS
Astigiano, Simonetta; AOU San Martino-IST National Cancer Research
Institute, IRCCS
Sommerhoff, Christian; LMU-Munich, Dept. of Clinical Chemistry and
Clinical Biochemistry
Pfeffer, Ulrich; University Hospital San Martino - National Cancer Research
Institute, Integrated Molecular Pathology
Nerlich, Andreas; Academic Hospital Munich-Bogenhausen, Institute of
Pathology
Bachmeier, Beatrice; LMU-Munich, Dept. of Clinical Chemistry and Clinical
Biochemistry
Keywords:
chemoprevention, metastases in vivo, prostate cancer, inflammatory
cytokines, Curcumin
Carcinogenesis
Carcinogenesis Advance Access published October 5, 2012
at Azienda Sanitaria Liguria on October 9, 2012http://carcin.oxfordjournals.org/Downloaded from
For Peer Review
Curcumin Inhibits Prostate Cancer Metastasis in vivo by Targeting
the Inflammatory Cytokines CXCL1 and -2
Peter H. Killian
1
, Emanuel Kronski
1
, Katharina Michalik
1
, Ottavia Barbieri
2,3
, Simonetta Astigiano
2
,
Christian P. Sommerhoff
1
, Ulrich Pfeffer
4
, Andreas G. Nerlich
5
, Beatrice E. Bachmeier
1,4,*
1
Department of Clinical Chemistry and Clinical Biochemistry, Surgical Hospital, Ludwig-Maximilians-
University Munich, Munich, Germany;
2
IRCCS AOU San Martino-IST National Cancer Research Institute, Genoa, Italy.
3
Department of Experimental Medicine, University of Genoa, Genoa, Italy.
4
Integrated Molecular Pathology, University Hospital San Martino - National Cancer Research
Institute, Genoa, Italy.
5
Institute of Pathology, Academic Hospital Munich-Bogenhausen, Munich, Germany
* corresponding author:
Running title: Curcumin Inhibits Prostate Cancer Metastasis in vivo
Keywords: chemoprevention / metastasis in vivo / prostate cancer / inflammatory cytokines /
curcumin
Direct all correspondence to:
PD Dr. Beatrice E. Bachmeier,
Dept. of Clinical Chemistry and Clinical Biochemistry
Ludwig-Maximilians-University
Nussbaumstr. 20
D-80336 Munich, Germany
Phone: +49-89-50073926, Fax: +49-89-5160-4740
e-mail: bachmeier.beatrice@gmail.com
bachmeier@med.uni-muenchen.de
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Abstract
In America and Western Europe prostate cancer is the second leading cause of death in men.
Emerging evidence suggests that chronic inflammation is a major risk factor for the development and
metastatic progression of prostate cancer.
We previously reported that the chemopreventive polyphenol Curcumin inhibits the expression of the
pro-inflammatory cytokines CXCL1 and -2 leading to diminished formation of breast cancer
metastases. Here we analyse the effects of Curcumin on prostate carcinoma growth, apoptosis and
metastasis. We show that Curcumin inhibits translocation of NFκB to the nucleus through the inhibition
of the IκB-kinase, IKKβ, leading to stabilization of the inhibitor of NFκB, IκBα, in PC3 prostate
carcinoma cells. Inhibition of NFκB activity reduces expression of CXCL1 and -2 and abolishes the
autocrine/paracrine loop that links the two chemokines to NFκB. The combination of Curcumin with the
synthetic IKKβ inhibitor, SC-541, shows no additive or synergistic effects indicating that the two
compounds share the target. Treatment of the cells with Curcumin as wells as siRNA based knock-
down of CXCL1 and -2 induce apoptosis, inhibit proliferation, and down-regulate several important
metastasis-promoting factors like COX2, SPARC, and EFEMP. In an orthotopic mouse model of
haematogenous metastasis, treatment with Curcumin inhibits statistically significantly formation of lung
metastases.
In conclusion, chronic inflammation can induce a metastasis prone phenotype in prostate cancer cells
by maintaining a positive pro-inflammatory and pro-metastatic feed-back loop between NFκB and
CXCL1/-2. Curcumin disrupts this feed-back loop by the inhibition of NFκB signalling leading to
reduced metastasis formation in vivo.
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Introduction
Curcumin (diferuloylmethane; (1E,6E)-1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)
is a polyphenol that naturally occurs in the zingiberaceae Curcuma longa and is used as turmeric, the
main ingredient of curry. It is also used as a food additive, E100, for its strong yellow color.
In the last twenty years, many studies have addressed pharmacological activities of curcumin and
especially its anti-cancer activities are now well established [1]. Its anti-tumoral activities have been
observed for many cancers including colon [2], breast [3], head and neck [4], lung [5] and prostate [6]
cancer. However, pro-tumoral activities have also been observed [7].
The anti-cancer activities are apparently mainly mediated by its inhibitory effect on the transcription
factor complex, nuclear factor kappa B, NFκB, [8] that plays a central role in inflammation and (anti-)
apoptosis and radio- and chemoresistance. Inhibition of NFκB is achieved by curcumin through the
inhibition of IKK (inhibitor of kappaB kinase) [9,10] either directly [10] or through the action on up-
stream activators of IKK [9]. Inflammation is considered as a major factor for tumor progression [11]
and inhibition of NFκB activation and translocation is a common theme in cancer chemoprevention
[12]. Anti-apoptosis appears to be an important mechanism of resistance to chemotherapy that might
be controlled by the inhibition of NFκB [13] and, indeed, curcumin shows such activity [14,15].
Curcumin has also been reported to affect other signaling pathways such as src/Akt [16], c-jun/AP1
[17], protein kinase C [18], sonic hedgehog [19] and others [1]. Its activity as a low affinity ligand of the
estrogen receptor has also been proposed [20].
Curcumin is a lipophilic compound and intestinal resorption is low. Free curcumin is detected in
plasma at low levels [21]. Curcumin is reduced to di- and tetrahydrocurcumin by intestinal E.coli [22].
Major metabolites are glucuronide conjugates [23]. Exposure to the polyphenol is direct for cancers of
the gastrointestinal tract but despite its reduced bioavailability, the effects of curcumin on the
progression of other solid tumors are well documented. We have recently reported a significant effect
on the formation of breast cancer metastases in a murine model of hematogenous metastasis where
curcumin reduced the number of lung metastases [3] through the down-regulation of NFκB-dependent
expression of chemokines [24].
These data point at an application of curcumin for the prevention of cancer progression. Given its anti-
cancer and anti-inflammatory activities, curcumin becomes a natural candidate for the prevention of
inflammation related cancers. Moreover, prevention is particularly interesting for clinical situations in
which a low risk tumor is not necessarily excised immediately upon diagnosis. These two features
apply to prostate cancer since inflammation plays an important role in its etiology and progression [25]
and “watchful waiting” is an option for selected groups of elder patients [26]. In addition, benign
prostate hyperplasia, a condition that can develop into cancer, is also linked to inflammation [27] with
cyclooxygenase-2, PTGS2/COX2, being a central mediator [28]. COX2 is among the NFκB targets
that are down-regulated by curcumin in breast cancer cells [24].
We therefore wished to establish whether curcumin can inhibit prostate cancer progression to
metastasis. We analyzed the effects of the polyphenol on the highly aggressive PC-3 prostate cancer
cells in a murine model of hematogenous metastasis and identified the molecular mediators of the
significant anti-metastatic effect observed.
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Material and Methods
Cell Types and Culture Conditions
The human prostate cancer cell line PC-3 used in this study was obtained from the American Type
Tissue Culture Collection (Rockville, MD, USA). The cell line is androgen receptor insensitive and was
initiated from a bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old male
Caucasian [29].
The cell line is commonly used for prostate cancer studies and is well defined in its growth, invasive,
and metastatic characteristics.
The cell line was cultured at 37°C in a humidified atmosphere of 5% CO
2
. The cells were grown in
RPMI medium (PAN, Aidenbach, Germany) supplemented with 5% heat inactivated fetal calf serum
(PAA, Pasching, Austria), 1% L-glutamine solution (200mM) (PAN, Aidenbach, Germany), 1% sodium
pyruvate solution (100mM) (PAN, Aidenbach, Germany), non-essential amino acids and vitamins
(PAN, Aidenbach, Germany). Medium was changed every 2d.
Curcumin Treatment of Cells
Curcumin with a purity of 95% was purchased from Fluka (Buchs, Switzerland), dissolved in 0.5 M
NaOH as a 25mM stock solution and stored at -20 °C. For the use in cell culture a 2.5 mM solution in
sterile PBS was prepared. According to the IC
50
value of PC-3 cells determined by us previously
Curcumin was applied at an end concentration of 15 µM for all assays. For controls the carrier (0.5 M
NaOH) diluted accordingly to the Curcumin working solutions in PBS was applied to the cells.
Gene Silencing
RNA interference was used to generate specific knockdowns of p65 (NFκB), IκBα, CXCL1 and
CXCL2 mRNA transcripts in the human prostate cancer cell line PC-3. siRNAs
[r(GAUCAAUGGCUACACAGGA) d(TT) and r(UCCUGUGUAGCCAUUGAUC) d(TT)] targeted to
NFκB were synthesized and annealed (Qiagen, Hilden, Germany). Predesigned annealed double-
stranded siRNAs targeted to IκBα, CXCL1, CXCL2, and CXCR2 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 PC-3 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 NFκB, IκBα, CXCL1,
CXCL2, and CXCR2 or to an irrelevant control 30 min prior to the application to the cells.
Preparation of conditioned media
Cell culture supernatants of Curcumin and mock-treated PC-3 cells were collected and centrifuged 15
min at 4000g in order to remove dead cells. The supernatants were used for western blots.
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Preparation of cellular extracts
Cells were washed three times with phosphate-buffered saline and collected by scraping and
centrifuged. Lysis buffer (10 mM Na
3
PO
4
, 0.4 M NaCl and 0.2% Triton X-100) supplemented with 1mM
PMSF (Fluka, Buchs, Switzerland) and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, USA)
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
-80°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 and cDNA
Total RNAs were isolated from cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according
to the manufacturer’s instructions. Thereafter, oligo dT primed cDNAs were synthesized using the First
Strand cDNA Synthesis Kit (GE Healthcare, Buckinghamshire, UK) following the manufacturer’s
instructions.
Quantitative RT-PCR
qRT-PCR was performed on a Light-Cycler (Roche, Mannheim, Germany) using the QuantiTectTM
SYBR® Green PCR kit (Qiagen, Hilden, Germany). Primers for the CXCL1, CXCL2, p65 (NFκB),
IκBα, CXCR2, COX2, SPARC, ALDH3A1, and EFEMP1 target genes as well as for the housekeeping
gene HPRT were designed using the Primer3 program and purchased from TibMolbiol (Berlin,
Germany). All sequences are listed in supplementary table 1. PCR conditions were set according to
the manufacturer’s instructions provided with the QuantiTectTM SYBR® Green PCR kit. All
experiments were carried out in duplicates as recommended for the use of the LightCycler. The
specificity of the RT-PCR products was proven by the appropriate melting curves (specific melting
temperature) and by the expected size of the PCR products (data not shown). Expression data were
normalized on the house keeping gene as indicated in the results section.
Western blots
Conditioned media from Curcumin-treated (24 h) and respective non-treated control cells as well as
conditioned media from p65-, IκBα-, 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-, IκBα-, CXCL1- or CXCL2-silenced and non-silenced control cells were analyzed
using a p65, p-p65, IκBα, p- IκBα, COX2, SPARC and EFEMP antibodies (Cell Signalling, MA, USA).
For stimulation of IκBα phosphorylation cells were incubated with 10µM TNFα (Sigma, Deisenhofen,
Germany) prior to cell lysis. Likewise for inhibition of IκBα phosphorylation 10µM of a kinase β inhibitor
(SC-514, EMD, Calbiochem, Gibbstown, USA) was used in cell culture. Equal amounts of protein were
subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the intracellular amount
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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 (Dianova, 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 (Invitrogen, Carlsbad, CA, USA). Blots were performed as previously described by us in
detail [24]. 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/).
Apoptosis Assay
Apoptotic cell death was determined by an enzyme-linked immunoassay (Cell Death Detection ELISA
PLUS
, Roche Applied Science, Mannheim, Germany) to detect fragmented DNA and histones
(mononucleosomes and oligonucleosomes). Human prostate cancer cells PC-3 were seeded on 24-
well plates and either transfected with small interfering RNAs directed against CXCL1 and -2 and
corresponding control oligos or treated with Curcumin and corresponding carrier. All experiments were
performed in triplicates. After an appropriate incubation period depending on the experiment cells were
washed with PBS, supernatants were collected for analysis of necrosis and cells were lysed and
processed following the instructions of the manufacturer.
Immunfluoresence staining for NF
κ
B p65 Location
Cells were plated on SuperFrost glass slides for adherence and treated the next day with Curcumin or
transfected with small interfering RNAs directed against CXCL1 and -2 and corresponding control
oligos in triplicates. Slides were airdried for 1 h at room temperature and fixed with ice-cold
acetone/methanol (1:1). After brief washing in PBS, slides were blocked with a blocking solution
(Biogenex, San Ramon, CA) for 1 h and then incubated with a 1:100 dilution of rabbit polyclonal anti-
human p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After overnight incubation, the
slides were washed and then incubated with goat antirabbit IgG-Alexa 594 (Invitrogen - Molecular
Probes, Carlsbad, CA) for 1 h and counter-stained for nuclei with 1 µg/ml 4',6-diamidino-2-
phenylindole (DAPI) for 5 min. Stained slides were mounted with mounting medium (Vector Labs,
Burlingame, CA) and analyzed under a fluorescence microscope with digital image capture (Leica,
Bensheim, Germany).
Cell doubling
PC-3 cells were trypsinized and equal cell numbers were plated in 25ccm flasks. The next day cells
were either treated with Curcumin or transfected with small non-coding RNA oligos (siRNAs)
specifically directed against CXCL1 and -2 or corresponding control oligos. After another incubation
period of 1-5 days, cells were washed with PBS, trypsinized and the cell number was determined
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using a cell counter (Casy1TTC cell counter, Roche, Germany). All experiments were performed in
triplicates.
Hematogenous Metastases in Immunodeficient Mice
Animal studies and research protocols were reviewed and approved by the institutional ethics
committee with and were conducted in accordance with the national current regulations and guidelines
for the care and use of laboratory animals (D.L. 27/01/1992, n. 116). Five-week-old CD-1 Foxn1
nu
male mice were obtained from Charles River Laboratories and were maintained under specific
pathogen-free conditions and given sterile food and water ad libitum. Before intracardiac tumor
inoculation, mice were anaesthetized with an i.p. mixture of ketamine (50mg/kg) and xylazine
(5mg/kg). Animal health and survival rate was observed until their euthanasia due to one of the
following medical reasons: severe weights loss (exceeding 20% of original body mass),
hyperventilation, paralysis, or bone fracture. PC-3 cells were collected by trypsinization and washed
and resuspended in PBS. 5 x 105 cells were injected into the heart of the mice. Mice were fed with
LASCRdiet™ CRD55131 (LASvendi, Soest, Germany) containing 1% Casein (controls) or 1%
Curcumin (Fluka, Buchs, Switzerland).
On day 35 after inoculation, mice were humanely sacrificed. Following sacrifice of the animals all
internal organs, including the brain, the vertebral column, humeri, and femora were removed and
immersed for 24-36h in buffered formaldehyde, pH 7.4. Subsequently, all material was dissected and
completely embedded into paraffin as routinely performed. Samples containing bone were first
decalcified in 0.1M EDTA until complete decalcification. Sections from all resulting tissue blocks were
stained for H&E. In addition, particular attention was paid to the dissection of the lungs resulting in all
samples in a cross-section through the largest diameter of the organs. Thereby, the pulmonary work
up was standardized so that comparably sized cross-sections through all lung samples were achieved.
Following embedding into paraffin, serial sections were prepared from the lungs which were stained by
immunohistochemistry in addition to the H&E stain. The immunostainings comprised the localization of
(human) cytokeratin (pan-keratin), p53 protein and the proliferation marker Ki-67 (all antibodies DAKO,
Hamburg, Germany). All organ sections were analyzed by light microscopy and the presence and
number of tumor cells/tumor cell aggregates was recorded. In the lung samples, all tumor cells/tumor
cell aggregates present within the pulmonary parenchyma (intrapulmonary) were distinguished from
those present at the pleural surface and/or seen in mediastinal soft tissue (peripulmonary). Metastases
were identified by typical morphology, positive reaction for (human type) cytokeratin and enhanced
proliferative capacity (Ki-67) or enhanced positivity of tumor cell nuclei for p53 protein. All metastases
were counted irrespective of the metastasis size or the number of tumor cells present. The resulting
metastasis frequencies were statistically evaluated.
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 ANOVA was used to test for
statistical significance (p<0.05), and when significance was determined, Bonferroni’s Multiple
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Comparison Test was performed post hoc, as indicated in the figure legends. Statistical analysis was
performed using the Prism software (GraphPad, San Diego, CA, USA).
Promoter Analysis
The sequences 5000bp upstream and 1000bp downstream of the transcription start site of the genes
CXCL1 (NM_001511.2), CXCL2 (NM_002089.3), and CXCR2 (NM_001557.3) were extracted using
the human, mouse and rat promoter extraction service, PromoSer
(http://biowulf.bu.edu/zlab/PromoSer/; [30] and analyzed using the transcription factor binding search
tool TFSEARCH 1.3 (http://www.cbrc.jp/research/db/TFSEARCH.html) that identifies sequence
elements that match to known transcription factor bindings sites stored in the Transfac database [31]
and calculates a score ranging from 0 to 100 for sequences that divert from the consensus based on
the degree of conservation of each single nucleotide. Matches with a score above 85 were collected.
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Results
Curcumin down-regulates the inflammatory cytokines CXCL1 and -2 in metastatic prostate
cancer by targeting NF
κ
B signaling
We previously reported that Curcumin inhibits the expression of the pro-inflammatory cytokines
CXCL1 and -2 leading to diminished formation of breast cancer metastases [24]. Here we show in a
model of metastatic prostate cancer, that Curcumin downregulates the two cytokines by targeting the
NFκB pathway and acting as kinase inhibitor.
The analysis of 281 human prostate cancers by microarray gene expression profiling [32] reveals that
CXCL1 and 2 as well as CXCR2 are expressed in many human prostate cancers. The highly variable
levels of expression (CXCL1: mean 982.6 range 270.6 – 8192.0; CXCL2: mean 2166.0 range 340.1
9607.9; CXCR2: mean 913.3 range 464 – 15825.9) probably reflecting differential inflammation status
of these tissues (see supplementary figure 1). We therefore analyzed expression of these cytokines
and their receptor in human prostate cancer cell lines and found that among the commonly used
prostate cancer cell lines LNCaP, DU154 and PC3 only the latter expressed CXCL1 and -2 as well as
CXCR2 (see supplementary figure 1). Therefore we selected PC3 cells as in vitro model for metastatic
prostate cancer in our study.
In a first step we investigated the effect of Curcumin on the expression of CXCL1 and -2 mRNA (qRT-
PCR) in metastatic prostate cancer cells PC-3. A 24h treatment with the polyphenol (“Cur”) reduces
statistically significantly CXCL1 transcription about 40% (Fig 1a, left upper panel) and CXCL2
transcription about 25 % (Fig 1a, right upper panel) in comparison to the expression rate of PC-3 cells
treated with the carrier alone (Fig. 1a indicated with ctrl). Well in accordance with the transcription
data, the corresponding protein levels (Western Blot) of CXCL1 and -2 were diminished in conditioned
media of Curcumin treated PC-3 cells (Fig. 1a, upper middle panel). The decrease of CXCL1
concentration in cell culture supernatants of Curcumin treated PC-3 cells was about 60% (Fig. 1a, left
side) and that of CXCL2 was about 50% 24 h after Curcumin treatment. Densitometric analysis of the
Western Blot data followed by statistical evaluation by student’s t-test revealed that the differences in
CXCL1 and -2 amounts secreted into the cell culture supernatants between Curcumin treated and
carrier treated PC-3 cells were statistically highly significant. 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 (Fig. 1a, “loading control”).
In order to unravel the molecular mechanism behind the inhibitory effect of Curcumin on CXCL1 and -
2, we analyzed in depth the role of NFκB signalling in this context. Our previous studies using a model
of metastatic breast cancer already exposed that p65 and IκBα are involved [24]. Here we extend our
studies on metastatic prostate cancer and moreover we investigate the underlying regulation
mechanism more in detail by going further upstream in the NFκB signalling pathway.
We analyzed the promoter regions of CXCL1, -2 and CXCR2 from 5000bp upstream to 1000bp
downstream of the transcription start site. The promoters of CXCL1 and 2 share an almost identical
proximal promoter region (92% identity in the region -137 to transcription start site) and no significant
homology in distal promoter elements. This region contains a perfect NFκB binding site
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(GGGAATTTCC, -73 for CXCL1 and -75 for CXCL2). An additional, imperfect binding site is located
close to the transcription start site in position -22 and -23. Further imperfect binding sites are located in
the 5’ upstream untranslated region for both genes. The CXCR2 promoter contains bona fide NFκB
binding sites 1071 and 1034bp upstream and 85bp downstream of the transcription start site (see
supplementary table 2).
In this context we modulated the NFκB pathway in PC-3 cells by specifically silencing the gene
transcription of p65 and IκBα by RNA interference. As determined by qRT-PCR 24h, 48h and 72h
after transfection of the cells with small non-coding RNA oligos, we achieved knock-down efficiencies
of up to 95 % (after 72h) for p65 and of up to 82 % (after 24h) for IκBα as compared to control cells
transfected with non-target directed siRNAs (Fig. 1b and 1d, columns indicated with p65 or IκBα
respectively).
To verify the efficiency of gene silencing on protein level, we performed W estern blotting analysis of
lysates. Consistent with our mRNA data, cellular extracts of p65 or IκBα silenced PC-3 cells revealed
an almost complete knock-down (~92%) of p65 protein (Fig. 1c, left panel) and an about 60 % knock
down of IκBα protein (Fig. 1e, left panel) 72 h after transfection with specific siRNAs as compared to
cells transfected with a non-target directed siRNA.
As consequence of p65 silencing, expression of CXCL1 and -2 mRNAs was statistically significantly
diminished to a minimum of 60 % and 55 % respectively after 72h (Fig. 1b, columns indicated with
“CXCL1” and “CXCL2”).
As expected, silencing of IκBα in PC-3 cells led to a result opposed to that observed for p65 silencing
and accordingly CXCL1 and -2 mRNA levels increased about 2 fold each 72h after transfection as
compared to control cells transfected with non-target directed siRNAs (Fig. 1d, columns indicated with
“CXCL1” and “CXCL2”).
Well in line with the results from qRT-PCR, protein levels of CXCL1 and -2 secreted into the
conditioned media of PC-3 cells decreased when p65 was silenced (Fig. 1c, middle and right panels)
and augmented when IκBα was transiently knocked down (Fig. 1e, right panels). The efficiency of
transient gene silencing of p65 and IκBα by specific siRNAs was ~ 90% for both after 24h on mRNA
level (Fig. 1b and d) and ~90% and 60% respectively after 72h on protein level (Fig. 1c and e, left
panels). Amounts of secreted CXCL1 and -2 proteins diminished 45% and 40% respectively when p65
was silenced for 72h as compared to control cells transfected with a nonsense oligo (Fig. 1c middle
and right panels). On the contrary, CXCL1 and -2 levels increased 3 fold and 6 fold respectively when
IκBα was transiently knocked down for 72h (Fig. 1e middle and right panels). All differences found
were statistically highly significant ranging from p<0.01 up to p<0.001 as indicated in the appropriate
figures.
We furthermore wished to know whether Curcumin inhibits phosphorylation and thereby functions as a
kinase inhibitor in metastatic prostate cancer cells (Fig. 2a). IκBα is phosphorylated by the inhibitor of
the IκB kinase complex, mainly IKKβ. The effect of Curcumin could therefore be due to inhibition of
these kinases. We therefore compared the effects of Curcumin with those obtained using the IκB
kinase β inhibitor SC-514.
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We stimulated phosphorylation of p65 and IκBα in PC-3 cells for 10 min with TNFα, resulting in
statistically significant 1.3 fold and 2.5 fold inductions of p-p65 and p-IκBα respectively.
Phosphorylation of p65 could be diminished 1.7 fold after 2h treatment with SC-514. A similar effect
could be achieved by treating PC-3 cells 2h with Curcumin. Combined application of Curcumin and
SC-514 also led to inhibition of phosphorylation of p65 but the two drugs did not yield an additive or
synergistic effect making a common inhibitory mechanism most likely. SC-514 diminished
phosphorylation of IκBα 3 fold after 2h. Curcumin led to a similar yet weaker reduction of IκBα
phosphorylation. These results indicate that the polyphenol directly acts as an inhibitor of IκB
kinase β .
In order to investigate whether the inhibitory effect of Curcumin on phosphorylation of IκBα and p65
has a functional consequence on p65 translocation, we monitored the localization of this NFκB subunit
in different cellular compartments by immunofluorescence using specific antibodies (Fig. 2b). In PC-3
cells treated with the carrier alone, p65 was evenly distributed between cytoplasm and nucleus of the
cells (A-C). Already 1h after Curcumin treatment, p65 translocation into the nuclei was impaired and
the NFκB subunit remained in the cytoplasm of the prostate cancer cells (E-G). After 2h treatment with
Curcumin this effect became even more evident and most of the nuclei were free of p65 (I-L). DNA in
the nuclei was stained with DAPI (C, G, and L). Photographs of the cells using bright light show the
morphology of the cells (D, H, and M)
Taken together, these results show that Curcumin acts as kinase inhibitor within the NFκB signaling
cascade in metastatic prostate cancer cells. By blocking the phosphorylation of IκBα, Curcumin has
an effect further down-stream on phosphorylation of p65, which in consequence impairs its
translocation into the nucleus of the tumor cells (Fig. 2). As a result inhibition of NFκB leads to
diminished expression rates of the pro-inflammatory cytokines CXCL1 and -2 (shown in Fig. 1).
The pro-inflammatory cytokines CXCL1/-2 act in a feedback loop reversely on the NF
κ
B
pathway
Besides the regulatory effect of NFκB on CXCL1 and -2 expressions we demonstrate here that the two
inflammatory cytokines themselves act on single factors of the NFκB pathway (Fig. 3). In detail, we
first analyzed the silencing efficiency after transfection of the prostate cancer cells with siRNA oligos
targeted specifically against CXCL1 and -2 (Fig. 3a).
Compared to a nonsilencing control (normalized expression level was set to 1 in qRT-PCR
experiments, Fig. 3a upper panel) CXCL1 expression was downregulated about 60% after 24 and 48h
and about 50% after 72h, whereas CXCL2 expression was inhibited about 85% after 24h, 70% after
48h and 60% after 72h. On the corresponding protein level CXCL1 and -2 expressions were silenced
about 50% and 40% respectively as evidenced by Western Blots followed by densitometric analysis
(Fig. 3a lower panels). All results were statistically highly significant with p values from **p<0.01 to
***p<0.001 (student’s t-test).
We then show that silencing of CXCL1 and -2 down-regulates p65 expression (Fig. 3b left upper
panel), phosphorylation (Fig. 3b, left lower panel) and translocation (Fig. 3c) while it induces as
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expected the expression (Fig. 3b, right upper panel) and block phosphorylation (Fig. 3b, right lower
panel) of the inhibitor of kappaB (IκBα).
Already 24h after CXCL1 and -2 silencing, p65 mRNA expression was impaired about 25% with
further highly statistically significant decrease (p<0.001) down to about 80-90% after 72h of silencing
of the pro-inflammatory cytokines (Fig. 3b, left upper panel). On the contrary, the expression of the
inhibitor of p65 (IκBα) was 50% induced upon specific CXCL1 and -2 knock-down for 24h. Prolonged
silencing of CXCL1 for 48h resulted in almost 2 fold increase of IκBα expression, while silencing of
CXCL2 was most efficient after 72h and led to an 2.5 fold increase of with a statistical significance of
p<0.001 (Fig. 3b, right upper panel).
Densitometric analysis of the Western blot data revealed that phosphorylation of p65 was diminished
about 40% by silencing of CXCL1 and 55% by knocking down CXCL2. Phosphorylation of IκBα was
only diminished about 60% by gene knock-down of CXCL1, whereas the effect of silencing of CXCL2
was not significant.
In order to investigate the involvement of the receptor of CXCL1 and -2, in feeding back on NFκB, we
silenced CXCR2 by transient siRNA knock-down, and analyzed the effect on p65 and IκBα after 48h
by qRT-PCR (Fig. 3c). Silencing efficiency on CXCR2 was about 40% (left panel) 48h after
transfection with specific siRNA oligos. Interestingly 24h Curcumin treatment of the prostate cancer
cells led to diminished CXCR2 expression (***p<0.001) and the combination of CXCR2 silencing
together with Curcumin treatment had a synergistic effect on CXCR2 down-regulation which was most
efficient after 48h with about 75% (***p<0.001). Well in line with our hypothesis, p65 expression was
diminished about 40% (middle panel) and IκBα expression was induced about 3-fold (right panel)
As a consequence of diminished phosphorylation, p65 should not translocate into the nucleus and
therefore we monitored the localization of this NFκB subunit in different cellular compartments after
CXCL1 and -2 silencing (Fig. 3d). In PC-3 cells transfected with a non-silencing control oligo (nonsi),
p65 was evenly distributed between cytoplasm and nucleus of the cells (A-C). After CXCL1 silencing
(CXCL1-si, E-G) as well as after CXCL2 silencing (CXCL2-si, I-L), p65 translocation into the nuclei is
impaired and the NFκB subunit remains dephosphorylated in the cytoplasm of the prostate cancer
cells. DNA in the nuclei was stained with DAPI (C, G, and L). Photographs of the cells using bright
light show the morphology of the cells (D, H, and M)
CXCL1 and -2 inhibit growth and induce apoptosis in metastatic prostate cancer
We wished to know whether the effects of CXCL1 and -2 expressions on the tumor progression
associated pathway NFκB translates directly into functional changes of tumor cell biology and
therefore analyzed possible consequences of CXCL1 and -2 silencing on proliferation and apoptosis.
Cell doubling rates were remarkably diminished after Curcumin treatment as well as after CXCL1 and -
2 silencing within an observation period of up to 5 days (Fig 4a). Growth rates of cells were mostly
diminished by Curcumin treatment (-○-) with a rate of about 50% as compared to controls (-◊-).
Likewise, silencing of CXCL1 (-▼-) and CXCL2 (-♦-) inhibited growth of prostate cancer cells about
45% and 35% respectively. Statistical significance was ***p<0.001 (Bonferroni)
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Next, we examined the effect of CXCL1 and -2 expressions on their ability to induce apoptosis, since
this function is critical for suppression of tumor formation and metastasis.
Using quantitative RT-PCR technique, we analyzed CXCL1 and 2-silenced human metastatic prostate
cancer cells PC-3 for expression of the apoptosis related factors bcl2 and surviving/birc5 and
compared the effect of CXCL1 and -2 expression with that of Curcumin treatment in an experiment
performed in parallel (Fig. 4b). Statistically significant downregulation (*p<0.05; **p<0.01, ***p<0.01;
ANOVA with Bonferroni’s post test) of the two survival related factors bcl2 and surviving/birc5 could be
achieved 72 h after transfection with small non-coding RNAs directed against CXCL1 and -2, yet to a
different extent. While CXCL1 silencing led to a downregulation of 55% and 65% for bcl2 and birc5
respectively, CXCL2 inhibited bcl2 expression by about 60% and that of birc 5 only by 10%. Curcumin
treatment for 24h inhibited bcl2 expression by about 50% and birc5 expression by about 60%, when
compared to PC-3 cells treated with only the carrier.
Functional apoptosis/necrosis assays revealed that CXCL1 and -2 silencing leads to enhanced
apoptosis (“Apo”) as well as necrosis (“Nec”) (Fig. 4c) in a highly statistically significant manner
(*p<0.05, **p<0.01 and ***p<0.001; ANOVA with Bonferroni’s post test). By silencing of CXCL1 and -2
for 72h in human metastatic prostate cancer cells PC-3, apoptosis rate could be almost doubled as
compared to PC-3 cells transfected with an appropriate control oligo (Fig. 4c, left side “Apo”). Similarly
necrosis rate was significantly increased statistically in CXCL1 and -2 silenced PC-3 cells as
compared to an appropriate control (Fig. 4c, right side “Nec”). Comparing the effect of CXCL1 and -2
silencing to Curcumin treatment after 72h, we found that the polyphenol acts more weakly on
apoptosis with a induction rate of 50%, but more strongly on necrosis with a induction rate of 60%.
Curcumin down-regulates metastatic factors via CXCL1 and -2
In order to investigate whether Curcumin reduces the metastatic potential of prostate cancer cells, we
analyzed the expression of a series of metastasis-related genes after treatment of metastatic PC-3
cells with the polyphenol (Fig. 5a). We chose genes whose functional involvement has been shown
through the analysis of highly metastatic tumor cells with pulmonary tropism [33]. After 24h Curcumin
treatment, mRNA expression of the metastasis-related genes SPARC (osteonectin), COX2 (PTGS2,
prostaglandin-endoperoxide-synthase 2), ALDH3A1 (aldehyde-dehydrogenase 3 family member A1),
and EFEMP (EGF containing fibulin like extracellular matrix protein 1) was statistically significantly
(**p<0.01; ***p<0.001; student’s t-test) downregulated, whereby down-regulation was most efficient for
COX2 with a reduced expression level down to about 45% in Curcumin treated cells as compared to
carrier treated cells (Fig. 5a, left graph “qRT-PCR”). On the corresponding protein level, Curcumin
down-regulated COX 2 about 50% (**p<0.01), SPARC about 25% (***p<0.001) and EFEMP1 about
40% as evidenced by Western Blots followed by densitometry (Fig. 5a, middle and right panels).
We furthermore asked whether downregulation of the two pro-inflammatory cytokines can affect the
metastatic potential of the cells. To answer this question, we analyzed the expression of metastasis-
related genes in CXCL1- and CXCL2-silenced PC-3 cells. We demonstrate that 72h silencing of the
two cytokines is followed by downregulation of SPARC, COX2, ALDH3A1, and EFEMP as evidenced
by quantitative qRT–PCR (Figure 5b, left panel). Transcription of COX2 and SPARC that were already
among the most down-regulated factors by Curcumin, was reduced about 75% and 50-80%
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respectively by transiently knocking down the expressions of the two cytokines. Interestingly ALDH3A1
mRNA synthesis, which was inhibited by Curcumin only by 25%, was almost abolished by CXCL1 and
-2 silencing.
Impaired expression of COX2, SPARC and EFEMP1 could also be seen on the level of the
corresponding proteins. Our Western Blot results after 72h of of CXCL1- or CXCL2-silencing in PC-3
cells (Figure 5b, middle panel; lanes indicated with CXCL1-si and CXCL2-si, respectively) followed by
densitometric analysis (Fig. 5b, right panel) demonstrate that COX2 and SPARC expressions were
statistically significantly abrogated by about 50% and 60% respectively in cell lysates 72h after
transfection with the specific oligo directed against CXCL1 when compared to cells transfected with a
non-silencing oligo (lanes indicated with nonsi). The same effect could be observed in CXCL2 silenced
PC-3 cells but to a much weaker extend. Here COX2 and SPARC expressions were diminished only
by about 10% and 30% when compared to cells transfected with a non-silencing oligo (lanes indicated
with nonsi), however with statistical significances of **p<0.01 and ***p<0.001 respectively (student’s t-
test). A weak downregulation of EFEMP1 protein of about 15% could only be achieved by CXCL2-
silencing (Fig. 5b, middle and right panel). Intracellular β-actin levels were monitored as a loading
control.
Metastatic process in prostate cancer is driven by the CXCL1/-2 – NF
κ
B axis
In order to further investigate the involvement of NFκB in regulating the metastatic potential of prostate
cancer cells through CXCL1 and -2, we complemented the list of genes altered after IκBα silencing
(Figure 1d and e) with the metastasis-related genes COX2 and SPARC (Fig. 5c). In accordance with
the knockdown of IκBα, mRNA expression levels of both genes significantly increased (**p<0.01;
**p<0.001). While the expression of COX2 was induced more than 40 fold, 72h after IκBα silencing,
SPARC expression was up-regulated about 2.5 fold when compared with control cells that were
transfected with a non-target-directed siRNA (nonsi). Similar to CXCL1 and CXCL2, the rise in the
expression of the metastasis-related genes COX2 and SPARC can be attributed to the increased
activation of NFκB, which is caused by the lack of its inhibitor IκBα, leading to an elevated metastatic
potential of the prostate cancer cells.
Mouse Metastasis of Prostate Cancer Xenografts is reduced by Curcumin
5 x 10
5
subconfluent PC-3 cells were injected into the heart of nude mice that were subsequently
divided into two groups that received standard or Curcumin diets. This route of administration, though
leading to suboptimal absorption of the polyphenol that is mainly excreted with the faeces, was chosen
in consideration of the probable route of administration for dietary chemopreventive agents. Mice were
observed for 5 weeks within which the two groups did not show any differences, especially, Curcumin
treated mice did not reveal any side effects of the treatment, and the weights of the animals of the two
groups were comparable. After five weeks, mice were sacrificed and all internal organs, the vertebral
column, and both humeri and femora were collected and examined.
Lung metastases were counted after preparation of eosin-hematoxylin stained paraffin sections. In
both study groups, tumor cells/ cell aggregates were seen in the intrapulmonary and the
peripulmonary compartment, though to different numbers. Peripulmonary metastases are more likely
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to be derived from direct dissemination during the intercardiac injection. We therefore limited our
analysis to intrapulmonary metastases that are of hematogenous origin. The tumor cell morphology
showed characteristic atypia.
Significant expression of human p53 protein was observed only in tumor cells. Vitality of the tumor
cells was confirmed by a high number (80-90%) of proliferating Ki-67 positive cells thus excluding
tumor cell dormancy (Fig. 6a).
Tumors of treated and untreated animals were similar in dimension, morphology and histology. Figure
6b demonstrates that Curcumin prevented the formation of lung metastases in a highly significant
manner (Mann-Whitney test, p=0.0241). The effect of Curcumin on lung metastasis becomes also
evident from supplementary table 3 that shows the mice subdivided in ranks with no, few (< 10), and
many (>10) metastases. The number of animals with few metastases (< 10) was increased in treated
(11 of 15 animals; 74 %) as compared to untreated animals (9 of 16 animals; 55%). Five Curcumin
treated and none of the control animals remained metastases free. 7 controls and only 4 treated
animals had high total metastases counts (>10).
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Discussion
Many studies have shown anti-cancer effects of Curcumin [1,34]. Given the widespread use of
Curcumin as a spice and in consideration of the absence of toxicity even of high dosages, the use of
the polyphenol for chemoprevention has been proposed. Cancer patients who have obtained adjuvant
therapy and face a residual risk of relapse might wish to continue a non toxic preventive therapy after
adjuvant therapy. Finally, the introduction of more sophisticated prognostic procedures might induce
low risk cancer patients to opt against chemotherapy leaving room for non toxic preventive treatments
[35].
Here we address the specific situation of prostate cancer. Benign prostate hyperplasia (BPH) and
prostate in situ neoplasia (PIN) are potential precursor lesions of prostate cancer although it is not
clear whether there is a continuity between these situations [36,37]. In addition, many elder men have
prostate cancer but do not know and the recent recommendation against prostate cancer (PSA)
screenings [38] in men over 65 years old that is going to be extended to younger men [39], suggests
the introduction of dietary integrators that lower the risk of prostate cancer or its progression to
clinically overt and eventually metastatic disease. These situations justify preventive treatments that
target the potential of prostate (cancer) cells to grow and to metastasize.
Curcumin is a plant derived compound that is particularly suited for prevention of prostate cancer
formation and progression since it is known to act on the central activator of inflammation, NFκB, and
prostate hyperplasia and cancer are known to be driven by inflammation [25]. Yet the inflammatory
forms of prostate cancer are far from being as aggressive as for example inflammatory breast cancer
that will hardly respond to bland chemopreventive drugs.
Several studies have shown effects of Curcumin on prostate cancer cell survival through the down-
regulation of anti-apoptotic survival genes in vitro [40,41] and in vivo [42,43] as well radiosensitizing
effects [6,44]. The effects on the highly aggressive androgen independent prostate cancer cell line
PC-3 have also been analyzed [6,43] (see also [45]). We have chosen this model assuming that if
Curcumin can reduce formation of metastases by PC-3 cells, similar effects in less aggressive forms
of cancer become even more likely.
We show that Curcumin acts on the activation of NFκB through the stabilization of IκB in prostate
cancer cells just like it has be shown for other cells (for a review see [46]). Inhibition of IκK appears to
be the true mechanism since Curcumin shows similar effects as the synthetic inhibitor SC-514 but
administration of both, SC-514 and Curcumin, does not lead to additive or synergistic effects.
Similar to what we have previously observed in breast cancer cells, Curcumin down-regulates the
inflammatory cytokines CXCL1 and -2. We show that both cytokines are under the control of NFκB
since transcriptional silencing of the NFκB subunit p65 leads to reduced expression whereas silencing
of IκB leads to increased expression of the cytokines. None of the two cytokines is listed as a known
target of NFκB in man, CXCL2 is listed as a target in mouse (http://bioinfo.lifl.fr/NF-KB/;
http://www.bu.edu/nf-kb/gene-resources/target-genes/; see also [47]). We also show for the first time
that the two cytokines establish a positive feedback loop inasmuch as phosphorylation of p65 and of
IκBα was reduced in cells where the expression of the cytokines was abolished by siRNA silencing,
phosphorylation of p65 and of IκBα was reduced. Diminished phosphorylation of IκBα stabilizes the
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inhibitor which in turn reduces phosphorylation of p65 and blocks the NFκB complex in the cytoplasm
as evident from immunofluorescence analyses. The feedback appears to be mediated by CXCR2, the
receptor for the two cytokines, since its silencing reduces p65 transcription and phosphorylation,
similar to what we observed after treatment with Curcumin. CXCR2 has been shown to mediate
resistance to chemotherapy that induces an autocrine NFκB/cytokine feedback loop [48] that, as we
show here, is interrupted by Curcumin. Knock-down of CXCR2 has been described to reduce the
invasive potential and the number of metastases formed by xenografts of breast cancer cells [49].
CXCR2 itself is regulated by NFκB in prostate cells [50] and, as expected, responds to the treatment
with Curcumin as we show here. It is therefore most likely that the observed effect of Curcumin on the
formation of metastases is at least in part due to the interruption of the feedback loop that involves
CXCR2.
Treatment of PC-3 cells with Curcumin as well as silencing of CXCL1 and -2 reduce cell growth. The
anti-apoptotic molecules BCL2 and BIRC5/survivin are down-regulated by Curcumin as well as by
CXCL1 and -2 silencing. This explains the induction of apoptosis that we observed in our experiments.
Anti-apoptotic effects of Curcumin also determine its radio- and chemosensitizing effects [51].
In addition Curcumin down-regulates COX2 (PTGS2) whose overexpression has also been linked to
prostate hyperplasia [28,52]. COX2 is a known NFκB dependent mediator of inflammation and has
already been identified as a target of other chemopreventive compounds [12,53].
Taken together, these data delineate the molecular mechanisms by which Curcumin reduces survival
and growth of prostate cancer cells on the one hand and the ability of the cells to form metastases on
the other hand. Curcumin interrupts an important positive feedback loop between the cytokines and
NFκB that is responsible for the activation of several mediators of metastasis. This is particularly
important in prostate cancer since inflammation plays a crucial role in its progression towards more
aggressive growth and metastasis.
Indeed, our in vivo experiment demonstrates that Curcumin treated animals show a significant
reduction in the number of lung metastases formed from circulating prostate cancer cells after
intracardial injection into the left ventricle. In the group of Curcumin treated animals but not in the
control group, several animals remain metastasis free despite the large number of cells injected. The
hematogeneous metastasis assay specifically addresses the capacity of the cells to extravasate and
to colonize a target tissue and the number of metastases formed is a direct measure of the metastatic
potential.
Curcumin therefore appears particularly suited for prostate cancer prevention in healthy elder men, in
patients with benign prostate hyperplasia and eventually low grade prostate cancers in elder patients
where “watchful waiting” might remain an option.
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Supplementary Figure 1
Microarray gene expression analysis of 281 human prostate cancers
CXCL1, CXCL2, and CXCR2 are expressed at highly variable levels by many prostate cancers.
Dashed lines indicate maximum, mean and minimum expression levels of all genes present on the
microarray. Data were obtained from Gene Expression Omnibus microarray data repository, accession
number GSE16560 [54]
Supplementary Table 1
Primer Sequences
Supplementary Table 2
The promoter regions 5000bp upstream and 1000bp downstream of the transcription start site of
CXCL1 and -2 and CXCR2 were analyzed for the presence of sequence motifs corresponding to the
canonical NFκB binding site using TFSearch. The positions are indicated in relation to the transcription
start site, nucleotides that divert from the consensus sequence are underscored, the score indicated
considers number of substitutions and degree of conservation of the single nucleotides.
Supplementary Table 3
Curcumin Inhibits Lung Metastases.
Curcumin treated animals had less lung metastases. Seven animals in the control group and only four
treated animals had high total metastases counts (>10); the number of animals with few metastases (<
10) was higher in treated (11 of 15 animals; 74 %) than in untreated animals (9 of 16 animals; 55 %).
Five Curcumin treated and none of the control animals remained metastases free.
Acknowledgements:
This work was made possible through grants from Compagnia San Paolo and Regione Liguria to UP.
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TABLE AND FIGURES LEGENDS
Figure 1.
Curcumin impairs the expression of CXCL1 and CXCL2 via the NF
κ
B signalling pathway.
(a) Curcumin treatment of PC-3 cells (15µM for 24h) down-regulates mRNA expression of CXCL1 by
about 40% and that of CXCL2 by about 25% (upper panel indicated as “qRT-PCR”). Statistical
evaluation of the qRT-PCR results by student’s t-test resulted in p-values of *p=0.0101 for CXCL1 and
***p=0.0002 for CXCL2. Western Blot analysis reveals that CXCL1 protein secretion is diminished by
more than 50% and that of CXCL2 by about 45% (panel indicated as “Western Blot”) compared to
carrier treated cells as evidenced by densitometric evaluation of the band intensities (CXCL1:
**p=0.0011, CXCL2: ***p=0.0003; student’s t-test).
(b) The NFκB subunit p65 was transiently knocked down for 24, 48 and 72h in PC-3 cells and
quantitative RT-PCR analysis was performed to monitor the effects on CXCL1 and -2 expressions. All
data are shown as fraction of values in cells transfected with siRNAs. (*p<0.05, **p<0.01 and
***p<0.001; analysis of variance with Bonferromi`s multiple comparison test). 72h after transfection the
efficiency of p65 knockdown was ~ 90%, which resulted in decreased CXCL1 and -2 mRNA
expressions rates down to ~ 50%.
(c) Western blot analyses of cellular extracts from p65-silenced PC-3 cells (left panel) showed a ~ 10
fold reduction of p65 protein 72h after transfection with siRNA targeted against p65 (lanes indicated
with p65-si) in comparison to controls (lanes indicated with nonsi), which was statistically highly
significant (p<0.0001). To assure that equal amounts of total protein were loaded onto the gels β-actin
was used as a loading control (left lower panel). Western blot analyses of conditioned media of PC-3
cells harbouring the knockdown of p65 (middle and right panel) revealed a 45% reduction of secreted
CXCL1 and a 40% reduction of CXCL2 72h after silencing (lanes indicated with p65-si) compared to
controls (lanes indicated with nonsi).
(d) The inhibitor of p65 - IκBα - was silenced for 24, 48 and 72h in PC-3 cells to investigate whether
CXCL1 and 2 expression was induced CXCL1 and -2 mRNA expression data are shown as fraction of
values in cells transfected with siRNAs (*p<0.05, **p<0.01 and ***p<0.001; analysis of variance with
Bonferromi`s multiple comparison test). The knockdown of IκBα had an efficiency of ~ 70% after 24h
and faded out to ~50% after 72h. mRNA levels for CXCL1 and CXCL2 increased after IκBα
knockdown with a maximum of about 2 fold expression after 72h as compared to controls.
(e) Western blot analyses of cellular extracts from IκBα-silenced PC-3 cells showed a 60% reduction
of IκBα protein 72h after transfection with specific siRNAs (lanes indicated with IκBα-si) in comparison
to controls (lanes indicated with nonsi). The knock-down of IκBα had a statistical significance of
p=0.0004. To assure equal amounts of total protein were subjected to the gels, β-actin was used as a
loading control. Western blot analyses of conditioned media of PC-3 cells harbouring the knockdown
of IκBα (lanes indicated with IκBα-si) revealed a 3 fold increase of secreted CXCL1 and a 6 fold
increase of CXCL2 after 72h compared to controls (lanes indicated with nonsi).
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All experiments were performed at least in triplicates. Western Blot results were quantified by
densitometry followed by statistical analysis using student’s t-test. Statistical significances were
*p<0.05, **p<0.01 and ***p<0.001.
Figure 2
Curcumin acts as kinase inhibitor in metastatic prostate cancer cells.
Fig 2a left panel: Phosphorylation of p65 and IκBα was stimulated in PC-3 cells for 10 min with TNFα,
resulting in statistically significant 1.3 fold and 2.5 fold inductions of p-p65 and p-IκBα respectively.
Phosphorylation of p65 could be diminished 1.7 fold after 2h treatment with the commercial IκB kinase
β inhibitor SC-514. A similar effect could be achieved by treating PC-3 cells for 2h with Cur. Combined
application of Curcumin and SC-514 also led to inhibition of phosphorylation of p65 but the two drugs
did not yield an additive or synergistic effect making a common inhibitory mechanism most likely. SC-
514 diminished phosphorylation of IκBα 3 fold after 2h. Curcumin led to a similar yet weaker reduction
of IκBα phosphorylation. These results indicate that the polyphenol directly acts as an inhibitor of IκB
kinase β .
Fig. 2b: Localization of the NFκB subunit p65 in different cellular compartments of PC3 cells was
monitored by immunofluorescence using specific antibodies. In cells treated with the carrier alone, p65
was evenly distributed between cytoplasm and nucleus (A-C). 1h after Curcumin treatment, p65
translocation into the nuclei was impaired and p65 remained unphosphorylated in the cytoplasm (E-G).
After 2h treatment with Curcumin this effect became even more evident and almost all nuclei were free
of p65 (I-L). DNA in the nuclei was stained with DAPI (C, G, and L). Photographs using bright light
show the morphology of the cells (D, H, and M). Magnification: x20
Figure 3
CXCL1/-2 act in a feedback loop reversely on the NF
κ
B pathway
Fig. 3a: Silencing efficiency after transfection of the prostate cancer cells with siRNA oligos targeted
specifically against CXCL1 and -2 compared to a nonsilencing control was about 60% after 24 and
48h for CXCL1 and about 85% after 24h, 70% after 48h and 60% after 72h for CXCL2 mRNA
expression (upper panels “qRT-PCR”). On the protein level CXCL1 and -2 expressions were silenced
by about 50% and 40% respectively as evidenced by Western Blots followed by densitometric analysis
(Fig. 3a lower panels). All results were statistically highly significant with p values from **p<0.01 to
***p<0.001 (student’s t-test).
Fig. 3b: 24h after CXCL1 and -2 silencing, p65 mRNA expression was impaired down to a minimum of
80-90% after 72h of silencing of the pro-inflammatory cytokines (left upper panel). In contrary, the
expression of the inhibitor of p65 (IκBα) was induced upon specific CXCL1 and -2 knock-down up to a
maximum of 2- and 2.5-fold after 48h and 72h respectively (right upper panel). Densitometric analysis
of the Western blot data revealed that phosphorylation of p65 was diminished by about 40% by
silencing of CXCL1 and by 55% by knocking down CXCL2. Phosphorylation of IκBα was only
diminished by about 60% by gene knock-down of CXCL1, whereas the effect of silencing of CXCL2
was not significant.
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Results were statistically significant with p values of *p<0.05, **p<0.01 and ***p<0.001 (student’s t-
test).
Fig 3c: Left panel: CXCR2 expression is down-regulated about 20% upon 24h of Curcumin treatment
and about 30% upon 48h of CXCR2 silencing; combination of CXCR2 silencing together with
Curcumin treatment for 48h resulted in a 70% diminished CXCR2 expression. Middle panel:
Expression of the NFκB subunit p65 was impaired upon 48h of CXCR2 silencing in PC3 cells. Right
panel: Well in line, IκBα expression was induced 3-fold upon 48h CXCR2 silencing. All results were
statistically highly significant (One-way ANOVA, with Bonferroni’s Multiple Comparison Test post hoc
***p<0.001). All experiments were performed in triplicates.
Fig. 3d: Localization of p65 was monitored in different cellular compartments after 48h of CXCL1 and -
2 silencing. In PC-3 cells transfected with a non-silencing control oligo (nonsi), p65 was evenly
distributed between cytoplasm and nucleus of the cells (“nonsi”, A-C). After CXCL1 silencing (“CXCL1-
si”, E-G) as well as after CXCL2 silencing (“CXCL2-si”, I-L), p65 translocation into the nuclei was
impaired and the NFκB subunit remained in the cytoplasm of the prostate cancer cells. DNA in the
nuclei was stained with DAPI (C, G, and L). Photographs using bright light show the morphology of the
cells (D, H, and M). Magnification: x20
Figure 4
CXCL1 and -2 inhibit growth and induce apoptosis in metastatic prostate cancer
Fig. 4a: After 5 days, growth rates of cells were most strongly reduced by Curcumin treatment (-○-)
with a rate of about 50% as compared to controls (-◊-). Likewise, silencing of CXCL1 (-▼-) and CXCL2
(-♦-) inhibited growth of prostate cancer cells by about 45% and 35% respectively. Statistical
significance was ***p<0.001 (student’s t-test).
Fig. 4b: Statistically significant downregulation (*p<0.05; **p<0.01, ***p<0.01; ANOVA with
Bonferroni’s post test) of the two survival related factors bcl2 and surviving/birc5 could be achieved 72
h after transfection with small non-coding RNAs directed against CXCL1 and -2. CXCL1 silencing led
to a downregulation of 55% and 65% for bcl2 and birc5 respectively, while CXCL2 inhibited bcl2
expression by about 60% and that of birc 5 by only 10%. 24h Curcumin treatment inhibited bcl2
expression about 50% and birc5 expression by about 60%, when compared to PC-3 cells treated with
only the carrier.
Fig. 4c: CXCL1 and -2 silencing leads to enhanced apoptosis (“Apo”) as well as necrosis (“Nec”) in a
highly statistically significant manner (*p<0.05, **p<0.01 and ***p<0.001; ANOVA with Bonferroni’s
post test). By silencing of CXCL1 and -2 for 72h in human metastatic prostate cancer cells PC-3,
apoptosis rate could be almost doubled as compared to PC-3 cells transfected with an appropriate
control oligo (left side “Apo”). Similarly necrosis rate was significantly increased in CXCL1 and -2
silenced PC-3 cells as compared to an appropriate control (right side “Nec”). Comparing the effect of
CXCL1 and -2 silencing to Curcumin treatment for 72h, we found that the polyphenol acts more
weakly on apoptosis with a induction rate of 50%, but more strongly on necrosis with a induction rate
of 60%.
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Figure 5
Curcumin down-regulates metastatic factors via CXCL1 and -2
Fig. 5a left panel (“qRT-PCR”): After 24h Curcumin treatment, mRNA expression of SPARC, COX2,
ALDH3A1 and EFEMP was statistically significantly (**p<0.01; ***p<0.001; student’s t-test)
downregulated in Curcumin treated cells as compared to carrier treated cells
Fig. 5a middle and right panel: On the corresponding protein level, 24h Curcumin treatment down-
regulated COX 2 50% (**p<0.01), SPARC by about 25% (***p<0.001) and EFEMP1 by about 40%
(Western Blots followed by densitometry).
Fig. 5b left panel (“qRT-PCR”): Silencing of the CXCL1 and -2 leads to downregulation of SPARC,
COX2, ALDH3A1, and EFEMP. Inhibition of metastatis correlated factors in prostate cancer cells was
more pronounced in CXCL2- than in CXCL1-silenced cells, at least for SPARC, ALDH3A1 and
EFEMP. ALDH3A1 mRNA synthesis, which was reduced by Curcumin only 25%, was almost
abolished by CXCL1 and -2 silencing.
Fig. 5b middle and right panel: Impaired expression of COX2, SPARC and EFEMP1 could also be
seen on the level of the corresponding proteins. COX2 and SPARC expressions were statistically
significantly abrogated by about 50% and 60% respectively in cell lysates 72h after transfection with
the specific oligo directed against CXCL1 (lanes indicated with CXCL1-si). Likewise COX2 and
SPARC expressions were diminished only by about 10% and 30% after CXCL2 silencing (lanes
indicated with CXCL2-si) as compared to cells transfected with a non-silencing oligo (lanes indicated
with nonsi). A weak downregulation of EFEMP1 protein of about 15% could only be achieved by
CXCL2-silencing.
All results were statistically significant with *p<0.05, **p<0.01 and ***p<0.001 (student’s t-test).
Intracellular β-actin levels were monitored as a loading control.
Metastatic process in prostate cancer is driven by the CXCL1/-2 – NFκ
κκ
κB axis
Fig. 5c: mRNA expression levels of the metastasis-related genes COX2 and SPARC increased
statistically highly significantly (**p<0.01; **p<0.001) after knockdown of IκBα by RNAi technique.
While the expression of COX2 was induced more than 40 fold, 72h after IκBα silencing, SPARC
expression was up-regulated about 2.5 fold when compared with control cells that were transfected
with a non-target-directed siRNA (nonsi).
Figure 6
Mouse Metastasis of Prostate Cancer Xenografts is reduced by Curcumin
5 x 10
5
PC3 cells were injected into the heart of immunodeficient mice. Animals were untreated
(control) or treated with Curcumin in the diet and sacrificed after 5 weeks. Lungs were removed fixed
and four sections for each animal were analyzed for metastases.
Fig. 5a: Histology of metastases formed. The immunolocalization of (human) cytokeratin in the
periphery of a mouse lung specimen reveals multiple selectively and positively stained tumor cells and
small tumor cell aggregates (brown color) (A, anti-human cytokeratin).
Immunohistochemical staining for p53 protein of a mouse lung specimen also shows multiple
positively labeled cell nuclei (brown color) indicating implanted prostate cancer metastases (B, anti-
human p53 protein).
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Localization of the cell proliferation antigen Ki-67 with positive labeling of isolated tumor cell groups
(brown color) shows cell proliferation of the transplanted tumor cells (C, anti-human Ki-67 antigen
(MIB-1)).
The overview of lung tissue 5 weeks after intracardiac tumor cell injection shows small groups of cells
with typical morphological features of tumor cells (polymorphism, atypia, mitotic activity) (D, H&E).
Original magnification: x 400
b) Number of metastases formed. The scattergrams report the numbers of metastases found in each
of the untreated (control, n=16) and Curcumin treated (Curcumin; n=15) animals (mean of 4 sections
analyzed). Mean number of metastases per animal are reduced in Curcumin treated animals. Note the
high number of animals with no or very few metastases in treated animals (Mann-Whitney p=0.0241).
Page 26 of 36Carcinogenesis
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288x409mm (300 x 300 DPI)
Page 27 of 36 Carcinogenesis
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Curcumin acts as kinase inhibitor in metastatic prostate cancer cells.
Fig 2a left panel: Phosphorylation of p65 and IkBa was stimulated in PC-
3 cells for 1h with TNFa, resulting in
statistically significant 1.3 fold and 2.5 fold inductions of p-p65 and p-IkBa respectively. Phosphorylation of
p65 could be diminished 1.7 fold after 2h treatment with the commercial IkB kinase b inhibitor SC-514. A
similar effect could be achieved by treating PC-3 cells for 2h with Cur. Combined application of Curcumin
and SC-514 also led to inhibition of phosphorylation of p65 but the two drugs did not yield an additive or
synergistic effect making a common inhibitory mechanism most likely. SC-514 diminished phosphorylation
of IkBa 3 fold after 2h. Curcumin led to a similar yet weaker reduction of IκBα phosphorylation. These
results indicate that the polyphenol directly acts as an inhibitor of IkB kinase b .
Fig. 2b: Localization of the NFkB subunit p65 in different cellular compartments of PC3 cells was monitored
by immunofluorescence using specific antibodies. In cells treated with the carrier alone, p65 was present
predominantly in the nuclear compartments (A-C). 1h after Curcumin treatment, p65 translocation into the
nuclei was impaired and p65 remained unphosphorylated in the cytoplasm (E-G). After 2h treatment with
Page 28 of 36Carcinogenesis
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Curcumin this effect became even more evident and all nuclei were free of p65 (I-L). DNA in the nuclei was
stained with DAPI (C, G, and L). Photographs using bright lig
ht show the morphology of the cells (D, H, and
M). Magnification: x20
281x369mm (300 x 300 DPI)
Page 29 of 36 Carcinogenesis
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CXCL1/-2 act in a feedback loop reversely on the NFκB pathway
Fig. 3a: Silencing efficiency after transfection of the prostate cancer cells with siRNA oligos targeted
specifically against CXCL1 and -2 compared to a nonsilencing control was about 60% after 24 and 48h for
CXCL1 and about 85% after 24h, 70% after 48h and 60% after 72h for CXCL2 mRNA expression (upper
panels “qRT-PCR”). On the protein level CXCL1 and -2 expressions were silenced by about 50% and 40%
respectively as evidenced by Western Blots followed by densitometric analysis (Fig. 3a lower panels). All
results were statistically highly significant with p values from **p<0.01 to ***p<0.001 (student’s t-test).
Fig. 3b: 24h after CXCL1 and -2 silencing, p65 mRNA expression was impaired down to a minimum of 80-
90% after 72h of silencing of the pro-
inflammatory cytokines (left upper panel). In contrary, the expression
of the inhibitor of p65 (IκBα) was induced upon specific CXCL1 and -2 knock-down up to a maximum of 2-
and 2.5-fold after 48h and 72h respectively (right upper panel). Densitometric analysis of the Western blot
data revealed that phosphorylation of p65 was diminished by about 40% by silencing of CXCL1 and by 55%
by knocking down CXCL2. Phosphorylation of IκBα was only diminished by about 60% by gene knock-down
Page 30 of 36Carcinogenesis
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of CXCL1, whereas the effect of silencing of CXCL2 was not significant.
Results were statistically significant with p values of *p<0.05, **p<0.01 and ***p<0.001 (student’s t-
test).
Fig 3c: Left panel: CXCR2 expression is down-regulated about 20% upon 24h of Curcumin treatment and
about 30% upon 48h of CXCR2 silencing; combination of CXCR2 silencing together with Curcumin treatment
for 48h resulted in a 70% diminished CXCR2 expression. Middle panel: Expression o
f the NFκB subunit p65
was impaired upon 48h of CXCR2 silencing in PC3 cells. Right panel: Well in line, IκBα expression was
induced 3-fold upon 48h CXCR2 silencing. All results were statistically highly significant (One-way ANOVA,
with Bonferroni’s Multiple Comparison Test post hoc ***p<0.001). All experiments were performed in
triplicates.
Fig. 3d: Localization of p65 was monitored in different cellular compartments after 48h of CXCL1 and -2
silencing. In PC-3 cells transfected with a non-silencing control oligo (nonsi), p65 was evenly distributed
between cytoplasm and nucleus of the cells (“nonsi”, A-C). After CXCL1 silencing (“CXCL1-si”, E-G) as well
as after CXCL2 silencing (“CXCL2-si”, I-L), p65 translocation into the nuclei was impaired and the NFκB
subunit remained in the cytoplasm of the prostate cancer cells. DNA in the nuclei was stained with DAPI (C,
G, and L). Photographs using bright light show the morphology of the cells (D, H, and M). Magnification:
x20
204x294mm (300 x 300 DPI)
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CXCL1 and -2 inhibit growth and induce apoptosis in metastatic prostate cancer
Fig. 4a: After 6 days, growth rates of cells were most strongly reduced by Curcumin treatment (--) with a
rate of about 50% as compared to controls (-◊-). Likewise, silencing of CXCL1 (--) and CXCL2 (--)
inhibited growth of prostate cancer cells by about 45% and 35% respectively. Statistical significance was
***p<0.001 (student’s t-test).
Fig. 4b: Statistically significant downregulation (*p<0.05; **p<0.01, ***p<0.01; ANOVA with Bonferroni’s
post test) of the two survival related factors bcl2 and surviving/birc5 could be achieved 72 h after
transfection with small non-coding RNAs directed against CXCL1 and -2. CXCL1 silencing led to a
downregulation of 55% and 65% for bcl2 and birc5 respectively, while CXCL2 inhibited bcl2 expression by
about 60% and that of birc 5 by only 10%. Curcumin treatment inhibited bcl2 expression about 50% and
birc5 expression by about 60%, when compared to PC-3 cells treated with only the carrier.
Fig. 4c: CXCL1 and -2 silencing leads to enhanced apoptosis (“Apo”) as well as necrosis (“Nec”) in a highly
statistically significant manner (*p<0.05, **p<0.01 and ***p<0.001; ANOVA with Bonferroni’s post test).
Page 32 of 36Carcinogenesis
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By silencing of CXCL1 and -2 for 72h in human metastatic prostate cancer cells PC-3, apoptosis rate could
be almost doubled as compared to PC-3 cells transfected with an appropriate control oligo (left side “Apo”).
Similarly necrosis rate was significantly increased in CXCL1 and -2 silenced PC-3 cells as compared to an
appropriate control (right side “Nec”). Comparing the effect of CXCL1 and -2 silencing to Curcumin
treatment, we found that the polyphenol acts more weakly on apoptosis with a induction rate of 50%, but
more strongly on necrosis with a induction rate of 60%.
276x495mm (300 x 300 DPI)
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Curcumin down-regulates metastatic factors via CXCL1 and -2
Fig. 5a left panel (“qRT-
PCR”): After 24h Curcumin treatment, mRNA expression of SPARC, COX2, ALDH3A1
and EFEMP was statistically significantly (**p<0.01; ***p<0.001; student’s t-test) downregulated in
Curcumin treated cells as compared to carrier treated cells
Fig. 5a middle and right panel: On the corresponding protein level, Curcumin down-regulated COX 2 50%
(**p<0.01), SPARC by about 25% (***p<0.001) and EFEMP1 by about 40% (Western Blots followed by
densitometry).
Fig. 5b left panel (“qRT-PCR”): Silencing of the CXCL1 and -2 leads to downregulation of SPARC, COX2,
ALDH3A1, and EFEMP. Inhibition of metastatis correlated factors in prostate cancer cells was more
pronounced in CXCL2- than in CXCL1-silenced cells, at least for SPARC, ALDH3A1 and EFEMP. ALDH3A1
mRNA synthesis, which was reduced by Curcumin only 25%, was almost abolished by CXCL1 and -2
silencing.
Fig. 5b middle and right panel: Impaired ex
pression of COX2, SPARC and EFEMP1 could also be seen on the
Page 34 of 36Carcinogenesis
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level of the corresponding proteins. COX2 and SPARC expressions were statistically significantly abrogated
by about 50% and 60% respectively in cell lysates 24 h after transfection with the specific oligo directed
against CXCL1 (lanes indicated with CXCL1-si). Likewise COX2 and SPARC
expressions were diminished only
by about 10% and 30% after CXCL2 silencing (lanes indicated with CXCL2-si) as compared to cells
transfected with a non-silencing oli
go (lanes indicated with nonsi). A weak downregulation of EFEMP1 protein
of about 15% could only be achieved by CXCL2-silencing.
All results were statistically significant with *p<0.05, **p<0.01 and ***p<0.001 (student’s t-test).
Intracellular β-actin levels were monitored as a loading control.
Metastatic process in prostate cancer is driven by the CXCL1/-2 – NFκB axis
Fig. 5c: mRNA expression levels of the metastasis-related genes COX2 and SPARC increased statistically
highly significantly (**p<0.01; **p<0.001) after knockdown of IκBα by RNAi technique. While the
expression of COX2 was induced more than 40 fold, 72h after IκBα silencing, SPARC expression was up-
regulated about 2.5 fold when compared with control cells that were transfected with a non-target-directed
siRNA (nonsi).
275x370mm (300 x 300 DPI)
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Mouse Metastasis of Prostate Cancer Xenografts is reduced by Curcumin
5 x 105 PC3 cells were injected into the heart of immunodeficient mice. Animals were untreated (control) or
treated with Curcumin in the diet and sacrificed after 5 weeks. Lungs were removed fixed and four sections
for each animal were analyzed for metastases.
Fig. 5a: Histology of metastases formed. The immunolocalization of (human) cytokeratin in the periphery of
a mouse lung specimen reveals multiple selectively and positively stained tumor cells and small tumor cell
aggregates (brown color) (A, anti-human cytokeratin).
Immunohistochemical staining for p53 protein of a mouse lung specimen also shows multiple positively
labeled cell nuclei (brown color) indicating implanted prostate cancer metastases (B, anti-human p53
protein).
Localization of the cell proliferation antigen Ki-67 with positive labeling of isolated tumor cell groups (brown
color) shows cell proliferation of the transplanted tumor cells (C, anti-human Ki-67 antigen (MIB-1)).
The overview of lung tissue 5 weeks after intracardiac tumor cell injection shows small groups of cells with
Page 36 of 36Carcinogenesis
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typical morphological features of tumor cells (polymorphism, atypia, mitotic activity) (D, H&E).
Original magnification: x 400
b) Number of metastases formed. The scattergrams report the numbers of metastases found in each of the
untreated (control, n=16) and Curcumin treated (Curcumin; n=15) animals (mean of 4 sections analyzed).
Mean number of metastases per animal are reduced in Curcumin treated animals. Note the high number of
animals with no or very few metastases in treated animals (Mann-Whitney p=0.0241).
262x384mm (300 x 300 DPI)
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Microarray gene expression analysis of 281 human prostate cancers
CXCL1, CXCL2, and CXCR2 are expressed at highly variable levels by many prostate cancers. Dashed lines
indicate maximum, mean and minimum expression levels of all genes pre
sent on the microarray. Data were
obtained from Gene Expression Omnibus microarray data repository, accession number GSE16560 [54]
338x254mm (72 x 72 DPI)
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Supplementary Table 1
HPRT
sense 5’-CTCAACTTTAACTGGAAAGAATGTC-3’
antisense
5’-TCCTTTTCACCAGCAAGCT-3’
CXCL1
sense 5’-CTTGCCTCAATCCTGCATC-3’
antisense
5’-CCTTCTGGTCAGTTGGATTTG-3’
CXCL2
sense 5’-CGAAGTCATAGCCACACTCAAG-3’
antisense
5’-CTTCTGGTCAGTTGGATTTGC-3’
COX2
sense 5’-GGTGATGAGCAGTTGTTCCAG-3’
antisense
5’-TGCACATAATCTTCAATCACAATC-3’
SPARC
sense 5’-GATGAGACAGAGGTGGTGGAAG-3’
antisense
5’-CTGCACCATCATCAAATTCTCC-3’
ALDH3A1
sense 5’-CCCTGACTACATCCTCTGTGACC-3’
antisense
5’-CCGGGATTTCTTAGCATCTTC-3’
EFEMP1
sense 5’-AGTCACAGGACACCGAAGAAAC-3’
antisense
5’-TCATCAATATCTTTGCATTGCTG-3’
p65
sense 5’-ACGAGCTTGTAGGAAAGGACTG-3’
antisense
5’-ATAGGAACTTGGAAGGGGTTGT-3’
Iκ
κκ
κBα
αα
α
sense 5’-CCAGGGCTATTCTCCCTACC-3’
antisense
5’-GCTCGTCCTCTGTGAACTCC-3’
Page 39 of 36 Carcinogenesis
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Supplementary Table 2
NFκB binding sites in CXCL1,
-
2 and CXCR2 promoters
position
sequence
score
orientation
consensus
GGGRNNYYCC
100
+/
-
CXCL1
-
73
GGGAATTTCC
100
+
-
22
GGGGTTCGCG
88.7
+
+327
GGGAATTCAC
89.1
+
+361
GTGAAGTCCC
86.6
+
+774
GGATATTCCC
89.4
+
CXCL2
-
75
GGGAATTTCC
100
+
-
23
GGGGTTCGCC
88.7
+
+357
GTGAAGTCCC
86.6
+
+651
GCGTCTGCCC
88.1
+
CXCR2
-
1074
GGGTATTCCC
91.0
+
-
1031
GGGACTTCCT
85.7
+
+85
GTGAAAATCC
85.4
+
Page 40 of 36Carcinogenesis
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Supplementary Table 3
Effects of Curcumin on lung metastases
# metastases # (%) control animals
(n=16)
# (%) Curcumin treated
animals
(n=15)
0 0 (0) 5 (33)
< 10 9 (55) 11 (74)
> 10 7 (44) 4 (27)
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... CUR has the potential to reduce inflammation associated with prostate cancer. Killian et al. investigated the effects of the chemokines CXCL-1 and CXCL-2 in prostate cancer cells and showed that CUR treatment significantly reduces CXCL-1 and CXCL-2 transcription [146]. CUR also inhibits NF-κB by blocking the phosphorylation of NF-κB inhibitor-α and inhibiting the downstream phosphorylation of P65, leading to decreased CXCL-1 and CXCL-2 expression [146]. ...
... Killian et al. investigated the effects of the chemokines CXCL-1 and CXCL-2 in prostate cancer cells and showed that CUR treatment significantly reduces CXCL-1 and CXCL-2 transcription [146]. CUR also inhibits NF-κB by blocking the phosphorylation of NF-κB inhibitor-α and inhibiting the downstream phosphorylation of P65, leading to decreased CXCL-1 and CXCL-2 expression [146]. One study in humans with prostate cancer combined pomegranate, green tea, broccoli, and turmeric into a capsule and monitored PSA levels [147]. ...
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Prostate cancer is one of the leading causes of death for men worldwide. The development of resistance, toxicity, and side effects of conventional therapies have made prostate cancer treatment become more intensive and aggressive. Many phytochemicals isolated from plants have shown to be tumor cytotoxic. In vitro laboratory studies have revealed that natural compounds can affect cancer cell proliferation by modulating many crucial cellular signaling pathways frequently dysregulated in prostate cancer. A multitude of natural compounds have been found to induce cell cycle arrest, promote apoptosis, inhibit cancer cell growth, and suppress angiogenesis. In addition, combinatorial use of natural compounds with hormone and/or chemotherapeutic drugs seems to be a promising strategy to enhance the therapeutic effect in a less toxic manner, as suggested by pre-clinical studies. In this context, we systematically reviewed the currently available literature of naturally occurring compounds isolated from vegetables, fruits, teas, and herbs, with their relevant mechanisms of action in prostate cancer. As there is increasing data on how phytochemicals interfere with diverse molecular pathways in prostate cancer, this review discusses and emphasizes the implicated molecular pathways of cell proliferation, cell cycle control, apoptosis, and autophagy as important processes that control tumor angiogenesis, invasion, and metastasis. In conclusion, the elucidation of the natural compounds’ chemical structure-based anti-cancer mechanisms will facilitate drug development and the optimization of drug combinations. Phytochemicals, as anti-cancer agents in the treatment of prostate cancer, can have significant health benefits for humans.
... In fact, it is not fully understood how Curcumin is transported to the target sites in order to exert its therapeutic effects. Still, in vivo studies have already documented the anti-metastatic effects of orally administered Curcumin [1,2]. ...
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Curcumin is one of the most interesting plant-derived polyphenols with a high potential for therapeutic, and even diagnostic, application in various diseases [...]
... Curcumin has been extensively investigated for its cellular and molecular modes of action against cancer [17,18], diabetes [19], neurological ailments [20], and osteoarthritis [21], and even entered several clinical trials [22,23]. Curcumin inhibits cancer cell proliferation [24,25], DNA repair along the p53-p21/GADD45A-cyclin/CDK-Rb/E2F-DNMT1 axis [26,27], metastasis by the NF-κB/c-JUN/MMP pathway [28], and the CXC-chemokine/NF-κB signaling pathway [29][30][31] as well as angiogenesis by the protein kinase C/NF-κB/AP-1 pathway [32,33]. EGFR is upregulated in several tumor types, including lung and colorectal tumors making EGFR an exquisite therapeutic target. ...
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