Cancer Therapy: Preclinical
High-Throughput Cell-Based Screening of 4910 Known Drugs and
Drug-like Small Molecules Identifies Disulfiram as an
Inhibitor of Prostate Cancer Cell Growth
Kristiina Iljin,1,2Kirsi Ketola,2Paula Vainio,2Pasi Halonen,1Pekka Kohonen,2Vidal Fey,1
Roland C. Grafström,1,3Merja Perälä,1and Olli Kallioniemi1,2,4
Purpose: To identify novel therapeutic opportunities for patients with prostate cancer, we
applied high-throughput screening to systematically explore most currently marketed
drugs and drug-like molecules for their efficacy against a panel of prostate cancer cells.
Experimental Design: We carried out a high-throughput cell-based screening with pro-
liferation as a primary end-point using a library of 4,910 drug-like small molecule com-
pounds in four prostate cancer (VCaP, LNCaP, DU 145, and PC-3) and two nonmalignant
prostate epithelial cell lines (RWPE-1 and EP156T). The EC50values were determined for
each cell type to identify cancer selective compounds. The in vivo effect of disulfiram
(DSF) was studied in VCaP cell xenografts, and gene microarray and combinatorial
studies with copper or zinc were done in vitro for mechanistic exploration.
Results: Most of the effective compounds, including antineoplastic agents, were non-
selective and found to inhibit both cancer and control cells in equal amounts. In con-
trast, histone deacetylase inhibitor trichostatin A, thiram, DSF, and monensin were
identified as selective antineoplastic agents that inhibited VCaP and LNCaP cell prolif-
eration at nanomolar concentrations. DSF reduced tumor growth in vivo, induced me-
tallothionein expression, and reduced DNA replication by downregulating MCM mRNA
expression. The effect of DSF was potentiated by copper in vitro.
Conclusions: We identified three novel cancer-selective growth inhibitory compounds
for human prostate cancer cells among marketed drugs. We then validated DSF as a
potential prostate cancer therapeutic agent. These kinds of pharmacologically well-
known molecules can be readily translated to in vivo preclinical studies and clinical
trials. (Clin Cancer Res 2009;15(19):6070–8)
Besides surgery and radiation therapy, androgen deprivation
remains the main first-line therapeutic option for prostate can-
cer patients, and the predominant therapy for patients with ad-
vanced and metastatic disease. However, hormonal therapy is
not curative, and often, androgen-independent, drug-resistant
disease develops. Such tumors remain virtually impossible to
treat with current medications. The median survival time for
men with androgen-independent cancer is around 2 years un-
derlining the need to develop better therapies (1).
The majority of prostate tumors from patients with an andro-
gen-independent disease overexpress androgen receptor (AR),
thereby sensitizing prostate cancer cells to low levels of andro-
gens. Also other mechanisms behind androgen independency
have been described, such as AR mutations activating the recep-
tor in response to nonandrogenic ligands (2). Because AR plays
an essential role also in androgen-independent prostate cancers,
AR and its coregulators are potential drug targets for the disease.
Recently, a frequent gene fusion between the androgen-regulat-
ed prostate-specific protease TMPRSS2 and the ERG transcrip-
tion factor was discovered (3). In addition to TMPRSS2-ERG,
other driver genes, as well as oncogenic ETS factors (e.g.,
ETV1, ETV4, and ETV5) have been identified as gene fusions
in prostate tumors. It has been shown that ERG fusion gene
may be bypassed at later stages of cancer progression, indicating
Authors' Affiliations:1Medical Biotechnology, VTT Technical Research
Centre of Finland, Turku;2Turku Centre for Biotechnology, University of
Turku, Turku, Finland;3Institute of Environmental Medicine, Karolinska
Institutet, Stockholm, Sweden; and4Institute for Molecular Medicine,
Finland (FIMM), University of Helsinki, Helsinki, Finland
Received 4/23/09; revised 6/17/09; accepted 6/17/09; published OnlineFirst
Grant support: Marie Curie Canceromics (MEXT-CT-2003-2728), EU-PRIMA
project (contract # LSHC-CT-204-504587), Academy of Finland, Cancer Or-
ganizations of Finland, and Sigrid Juselius Foundation.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Requests for reprints: Olli Kallioniemi, Institute for Molecular Medi-
cine, University of Helsinki and Medical Biotechnology, VTT Tech-
nical Research Centre of Finland, Itäinen Pitkäkatu 4, Turku,
Finland. Phone: 358-40-5698192; Fax: 358-20-722-2840. E-mail: Olli.
F 2009 American Association for Cancer Research.
6070Clin Cancer Res 2009;15(19) October 1, 2009 www.aacrjournals.org
that a more efficient treatment for these cancers would be of
great clinical interest (4, 5).
The AR and ETS status of the available prostate cancer model
cell lines are relatively well known. The VCaP cell line is an es-
tablished model of TMPRSS2-ERG gene fusion–positive pros-
tate cancers (3, 6). LNCaP cells overexpress ETV1 oncogene
(7). Both VCaP and LNCaP cells express AR, which is lost in
in LNCaP cells, AR has a point mutation (T877A) enabling
progestagens, estradiol, and antiandrogens to activate androgen
signaling(9). TheAR-T877Amutation hasalso been found from
prostatic tissues derived from patients with a metastatic prostate
cancer, indicating potential biological relevance (10).
In this study, we performed a cell-based screen with a library
of 4,910 drug-like small molecule compounds, including most
currently marketed drugs in the VCaP, LNCaP, PC-3, and DU
145 prostate cancer cells as well as in the RWPE-1 and
EP156T nonmalignant prostate epithelial cells. Due to this se-
lection of compounds analyzed, any interesting drugs that show
efficacy would be possible to rapidly test in vivo in preclinical
models and also in clinical trials. We focused our analysis to-
ward identifying antiproliferative compounds that act in pros-
tate cancer cells but lack impact on normal or transformed
prostate epithelial cells. Of the several growth inhibitory com-
pounds identified, we chose disulfiram (tetraethylthiuram di-
sulfide; DSF) for further analysis in the VCaP cells.
Materials and Methods
received from Drs. Adrie van Bokhoven (University of Colorado Health
Sciences Center, Denver, Colorado) and Kenneth Pienta (University of
Michigan, Michigan) and were grown in RPMI 1640. LNCaP and LNCaP
The prostate carcinoma cell lines VCaP and DuCaP were
C4-2 cells were received from Dr. Marco Cecchini (University of Bern,
Bern, Switzerland) and grown in T-Medium (Invitrogen). The prostate
cell lines RWPE-1 (11), PC-3, and DU 145 were purchased from Ameri-
can Type Culture Collection (LGC Promochem AB), and the nonmalig-
nantEP156Tprostateepithelial cells werereceived fromDr. Varda Rotter
recommended by the distributor (12). Primary prostate epithelial cells
were ordered from Lonza.
Five compound libraries were used in high-through-
put (HT) screenings (HTS). The libraries, summary of the compounds,
and the final concentrations used in the screening plates were the fol-
lowing: Biomol (80 known kinase and phosphatase inhibitors; 10, 1,
0.1, and 0.01 μmol/L), LOPAC (1,280 existing Food and Drug Admin-
istration–approved drugs and other compounds with pharmacological-
ly relevant structures; 1 and 0.1 μmol/L), IBIS (1,473 compounds
derived from natural sources; 1 and 0.1 μmol/L), Microsource Spectrum
(2,000 compounds including most of the known drugs and other bio-
active compounds and natural products; 1 and 0.1 μmol/L), and an in-
house library (77 experimental compounds; 10, 1, and 0.1 μmol/L).
For the validation of primary screens, DSF, thiram, and monensin
were purchased from Sigma-Aldrich. DSF and thiram were dissolved
in DMSO, whereas monensin was dissolved in methanol. CuCl2and
ZnCl2were ordered from Sigma.
CellTiter-Blue cell viability assay (Promega, Inc.) was
done in 384-well plates (Falcon). Before screening, cell number was ti-
trated for each cell line separately to ensure that cell proliferation re-
mained in a linear-exponential phase throughout the experiment.
Plates containing 50 nL of compound stock solutions or DMSO as con-
trols were diluted with 15 μL of cell culture media, and appropriate
amount of cells (1,000-2,000 per well) were plated in 35 μL of media.
After 72-h incubation, fluorometric CellTiter-Blue assay was done ac-
cording to the manufacturer's instructions. The Envision Multilabel
Plate Reader (Perkin-Elmer) was used for signal quantification.
Statistical methods applied for hit identification in HTS experiments
included plate normalization using B-score (13). Compounds reducing
cell viability by at least three SDs from the median of the controls were
considered as putative hits. The SD of the null distribution was esti-
mated using the statistically robust Huber's Estimator, winsorizing at
1.5 SDs. Classic multidimensional scaling of a data matrix, also known
as principal coordinates analysis (14), was used to visualize the dis-
tances between the screens. The Pearson correlation distance measure
was used for the screening data.
To obtain the absolute differences, the raw data were normalized us-
ing a loess method similar to the method implemented in the cellHTS2
R-package (15). The statistical outliers were down-weighted when a
polynomial surface was fitted to the intensities within each assay plate
using local regression. This ensured a robust fit, although some plates
had a much higher hit-rate than others (16). The fit, representing a sys-
tematic background signal, was then subtracted from the raw signal va-
lues. The data were then log2-transformed and centered on the plate
median. The following formula was used to calculate the absolute per-
centage-wise response: -100% + 2^loess_log_value%.
Determination of EC50values.
EC50assays were done on 384-well
plates with 2,000 cells per well plated in their respective growth media
and left to attach overnight. A 10-fold dilution series (10 pmol/
L-10 μmol/L) of selected compounds were added to the cells, and the
plates were incubated for 48 h. Cell viability was determined with Cell-
Titer-Blue assay described above. The EC50data were analyzed with
GraphPadPrism 4 software (GraphPad Software, Inc.).
Quantitative reverse transcriptase PCR.
∼70% confluence before treatment with 1 μmol/L DSF for 6 and
24 h. Cells were harvested and total RNA was extracted using RNeasy
(Qiagen) according to the manufacturer's protocol. Reverse transcrip-
tion using 1 μg of total RNA was done with Applied Biosystem's
cDNA syntesis kit. TaqMan gene expression probes and primers from
the Universal Probe Library (Roche Diagnostics) were used to study
ERG, AR, minichromosome maintenance complex genes (MCM2
VCaP cells were grown into
Therapeutic options for advanced and hormone-
refractory prostate cancer are limited and treatment
responses often unsatisfactory. We explored here
most currently marketed drugs and drug-like mole-
cules for their efficacy against prostate cancer cells.
Histone deacetylase (HDAC) inhibitor trichostatin A
(TSA), thiram, disulfiram (DSF), and monensin were
identified as the only agents that had antitumor effi-
cacy at nanomolar concentrations without affecting
nonmalignant control cells. Because DSF has a fa-
vorable safety profile, it was selected for further
studies. Gene expression analyses indicated that
DSF did not influence androgen receptor expression,
but induced metallothionein genes and downregu-
lated DNA replication. DSF also reduced VCaP pros-
tate cancer xenograft growth in vivo but was not able
to block it. Copper potentiated the antiproliferative
effects of DSF in cultured prostate cancer cells, sug-
gesting potential for increasing efficacy by combina-
torial approaches. In summary, we have identified
novel indications for known drugs that could be
readily translated to in vivo preclinical studies and
6071 Clin Cancer Res 2009;15(19) October 1, 2009www.aacrjournals.org
HTS for Selective Inhibitors against Prostate Cancer
and MCM5), metallothioneins (MT1A, MT1B, MT1F, MT1G, MT1X,
and MT2A), and β-actin mRNA expression. Real-time quantitative
PCR was done using ABI Prism 7900 (Applied Biosystems). Quantita-
tion was carried out using theΔΔCT method with RQ manager 1.2 soft-
ware (Applied Biosystems). Average expression of the untreated control
samples was considered for the calculation of the fold changes. mRNA
expression of two to four replicate samples was studied.
siRNA transfection, RNA isolation, and real-time qPCR.
lecules targeting metallotioneins and MCMs were transfected into VCaP
and LNCaP cells by using reverse transfection with siLentFect transfec-
tion (Bio-Rad) reagent in Opti-MEM (Invitrogen). Four siRNA mole-
cules were ordered from Qiagen (HP GenomeWide) against MCM5,
MT1A, MT1B, MT1F, MT1G, MT1X, and MT2A and one validated siRNA
molecule for MCM2. AllStars Negative Control and siRNA against PLK1
were used as controls in experiments. The final siRNA concentration
was 13 nmol/L.
Western blot analysis.
For protein extraction and Western blot anal-
ysis, cells were plated at 70% confluency and left to attach overnight.
VCaP cells were treated with the indicated compounds for the indicated
time. The cell extracts were separated by SDS-PAGE and transferred to a
nitrocellulose membrane. Western blot analysis was done using specific
antibodies against AR (1:1,000 dilution, mouse monoclonal, Labvi-
sion), cleaved poly ADP ribose polymerase (1:4,000 dilution, rabbit
Becton Dickinson). Signal was detected with 1: 4,000 dilutions of
appropriate horseradish peroxidase–conjugated secondary antibodies
(all from Invitrogen Molecular Probes), followed by visualization with
the enhanced chemiluminescence reagent (Amersham Biosciences).
Gene expression analysis using BeadArrays.
to ∼70% confluency before treatments with 1 μmol/L DSF for 3, 6,
and 24 h before harvesting. Total RNA was extracted using RNeasy
(Qiagen) according to the manufacturer's protocol. Integrity of the
RNA before hybridization was monitored using a Bioanalyzer 2100
(Agilent) according to manufacturer's instructions. Purified total RNA
(500 ng) was amplified with the TotalPrep kit (Ambion) and the bio-
tin-labeled cRNA was hybridized to Sentrix HumanRef-8 Expression
VCaP cells were grown
Fig. 1. Cell viability analysis of high-throughput compound screens. A, number of hits obtained per HT screen. HTS assay was done twice in
TMPRSS2-ERG–positive VCaP cells and once in LNCaP, PC-3, and DU 145 prostate cancer cells as well as in RWPE-1 and EP156T nontumorigenic
prostate epithelial cells. B, overview of the B-score–normalized data from the high-throughput compound screening in VCaP cells. C, pair-wise
correlations calculated based on B-score–normalized data. D, multidimensional scaling with principal coordinates analysis visualizes the distribution of
prostate cell lines based on their compound responses.
6072Clin Cancer Res 2009;15(19) October 1, 2009 www.aacrjournals.org
Cancer Therapy: Preclinical
BeadChips (Illumina). The arrays were scanned with the BeadArray
Statistical analysis of gene expression data.
data were quantile normalized and analyzed with the R/Bioconductor
software (17). Statistical analysis of differential gene expression after
the compound treatments was done using the empirical Bayes statistics
implemented in the eBayes function of the limma package (18). Gene
expression profiles of the compound-treated samples were compared
with the DMSO-treated negative control samples. The threshold for dif-
ferential expression was q < 0.05 after the Benjamini-Hochberg multi-
ple testing correction. The functional Gene Ontology and pathway
annotations were analyzed for the sets of differentially expressed genes
using DAVID (19, 20). To identify drugs with similar or opposite effects
on gene expression, Connectivity Map 02 was used (21).
Athymic nude (Athymic Nude-Foxn 1nu,
Harlan Winkelman) male mice, 5 wk of age, were injected s.c. in the
flank with 1 × 106VCaP cells in 200 μL of Matrigel without growth
factors (BD Biosciences, Inc.). Tumor volumes were calculated by cali-
per measurements done weekly to monitor tumor growth (tumor
volume = LW2× 0.56). When tumor size reached 200 mm3, the mice
were divided into two groups: (a) vehicle (olive oil) only and (b) DSF
(200 mg/kg/d). Mice were treated daily for 3 wk.
The hit criteria in HT compound screening
(B-score lower than -3 SD from the median) correspond to a P value
of <0.01. In siRNA screening, hit criteria was mean -2 SD,
corresponding to a P value of <0.05. Statistical analyses of xenograft
growth as well as quantitative reverse transcription PCR results were
The raw gene expression
done by using the Student's t test. These results are presented as the
mean ± SD. The following P values were used to show statistical signif-
icance: *, P < 0.05; **, P < 0.01; and ***, P < 0.005.
pounds, we carried out HTS in AR and TMPRSS2-ERG
fusion–positive VCaP cells (two replicate screens), in LNCaP
(AR positive, ERG negative), and in PC-3 and DU 145 (both
AR and ERG negative), prostate cancer cells as well as in non-
malignant prostate epithelial cells RWPE-1 and EP156T (both
AR positive, ERG negative). Because the growth rate of these
different cell lines varied, cell amounts were carefully titrated
to be on a linear range before HTS. Compound libraries com-
prising 4,910 small molecule compounds, including most
currently marketed drugs, were screened with at least two dif-
ferent concentrations. The cell viability was determined after
3-day incubation with compounds using a fluorescent assay.
The compounds that qualified as hits inhibited cell viability
(B-score) by at least three SDs from the median of the controls.
The number of hits per screen is presented in Fig. 1A. The HTS
results showed that the nonmalignant RWPE-1 and EP156T
cells were more sensitive to growth inhibition than the prostate
cancer cell lines (P = 0.04). B-score–normalized data from HT
compound screen with VCaP cells is shown in Fig. 1B and, for
the other screens, in Supplementary Fig. S1. The pair-wise
correlations between the cell viability results (B-scores)
obtained from different cell lines were 0.61 to 0.86, reflecting
the fact that the majority of compounds had similar effects
cipal coordinates analysis was used to visualize the distribution
of prostate cell lines based on their compound responses
(Fig. 1D), with normal epithelium-derived cell lines forming
one group and the different prostate cancer cell lines forming
Because VCaP cells originate from a hormone-refractory
prostate cancer patient, are representative of the clinical pros-
tate cancers in terms of the AR and ERG status, and were most
distant from nonmalignant prostate epithelial cells in terms of
their compound responses, these cells were selected for further
exploration. Replicate compound screens in the VCaP cells car-
ried out on different days were highly correlated (r = 0.859)
and yielded to a reproducible hit rate of 4.7% with 230
hits (listed in Supplementary Table S1). Several compounds,
To identify selective antineoplastic com-
Fig. 2. The absolute cell viability results from the initial HTS for thiram,
DSF, and monensin (all at 1 μmol/L concentration) indicate prostate cancer
cell selectivity in their growth inhibitory potential. Right, compound and
the library (MS, Microsource Spectrum; LOPAC).
Table 1. EC50values (nmol/L) for thiram, DSF and monensin in various prostate epithelial cells
95 ± 14
72 ± 47
222 ± 104
243 ± 224
94 ± 19
60 ± 18
170 ± 36
97 ± 22
39 ± 19
9 ± 2
90 ± 37
42 ± 16
Abbreviation: PrEC, primary prostate epithelial cell.
6073 Clin Cancer Res 2009;15(19) October 1, 2009www.aacrjournals.org
HTS for Selective Inhibitors against Prostate Cancer
such as Taxol, vinblastine sulfate, staurosporine, DSF, mitoxan-
trone, colcichine, celastrol, and actimomycin D, were identified
as hits from multiple libraries included in our HT screen,
supporting the overall reliability and functionality of the
assayandapproach. TheHDACinhibitor TSAwastheonlyselec-
tive VCaP inhibitor, which confirms our previous observation
where we specifically tested only HDAC inhibitors in prostate
Identification of selective antineoplastic compounds.
B-scores of VCaP, LNCaP, DU 145, and PC-3 prostate cancer
cell screens were compared with those seen in the nonmalig-
nant RWPE-1 and EP156T prostate epithelial cells (Supplemen-
tary Table S1). In addition to TSA, only three compounds,
thiram (tetramethylthiuram disulfide), tetraethylthiuram disul-
fide (DSF; identified as a hit from two libraries), and monensin
sodium, showed selective antineoplastic effects and were there-
fore selected for further analysis (Supplementary Fig. S2; Fig. 2).
Validation of the HTS results.
DSF, and monensin were determined in VCaP, DuCaP,
LNCaP, LNCaP C4-2, DU 145, PC-3, EP156T, RWPE-1 and
PrEC cells with a 10-fold dilution series and cell viability
measurements. All these compounds inhibit the TMPRSS2-
ERG gene fusion–positive VCaP and DuCaP cell viability at
nanomolar concentrations (Table 1). LNCaP cells were
∼2-fold less sensitive than VCaP cells, and the other prostate
cancer cells studied were >10-fold less sensitive. Interestingly,
the androgen-independent LNCaP variant C4-2 was as sensi-
tive to DSF and monensin as VCaP cells. The EC50values for
thiram and DSF in VCaP cells were >100-fold lower than in
nontumorigenic RWPE-1, EP156T, or primary prostate epithe-
lial cell cells (EC50values, >10,000 nmol/L), confirming the
selective antineoplastic action of these compounds. Also,
monensin was >25-fold more potent in reducing VCaP cell
viability than either one of the nonmalignant prostate epithe-
lial cells studied.
Thiram and DSF are structurally homologous members of di-
thiocarbamate family. In addition to acting as an aldehyde de-
hydrogenase inhibitor, DSF has been shown to inhibit, e.g.,
DNA topoisomerases, matrix metalloproteinases, and ABC drug
transport proteins, and thereby to have antitumor and chemo-
sensitizing activities (23, 24). Monensin is a Na+/H+antiporter
with antibiotic and antimalarial activities. It has been widely
used as a biochemical tool to block intracellular protein trans-
port and to improve growth rates in cattle (25). Because DSF is
a relatively safe drug used for decades as a Food and Drug Ad-
ministration–approved alcohol abuse deterrent, it was chosen
for more detailed mechanistic studies.
DSF induces the expression of metallothioneins and reduces the
expression of ERG and minichromosome maintenance complex
To study the growth inhibitory mechanism of DSF in
prostate cancer cells, VCaP cells were exposed to 0 (DMSO con-
trol) or 1 μmol/L DSF for 3, 6, 24, or 48 hours. Samples were
collected and prepared for quantitative PCR and genome-wide
gene expression studies. First, ERG and AR mRNA expression
was studied in response to DSF by quantitative PCR. The results
indicate that ERG mRNA expression is reduced, whereas AR ex-
pression in not consistently affected in response to DSF treat-
ment alone (Fig. 3A). Second, for genome-wide expression
analysis, early time points were chosen (3, 6, and 24 hours)
to gain insights into direct mechanisms rather than secondary
alterations caused by DSF. The gene ontology analysis of differ-
The EC50values for thiram,
entially expressed genes at 3- and 6-hour DSF exposure were
enriched in metal-binding activities, whereas the most signifi-
cantly altered biological process in response to 24-hour
exposure was decreased DNA replication (Supplementary
Table S2). The metal binding group consisted of zinc transpor-
ters and metallothioneins, a group of low molecular weight
proteins that regulate the availability of essential metals and
protecting cells against DNA damage and oxidative stress. The
quantitative PCR results confirmed induction of MT1B, MT1G,
MT1F, MT1X, and MT2A mRNA expression in response to
6-hour DSF exposure, whereas at 24-hour metallothionein
expression had declined nearly back to the baseline (Fig. 3B).
The DNA replication cluster with downregulated mRNA expres-
sion was validated by studying minichromosome maintenance
complex component (MCM) mRNA expression in response to
DSF (Fig. 3C). MCM complexes unwind the double stranded
DNA, recruit DNA polymerases, and initiate DNA synthesis.
The MCM proteins are highly expressed in malignant human
cancer cells and precancerous cells undergoing malignant trans-
To get additional clues about the biological processes al-
tered in response to DSF, the differentially expressed genes
in DSF exposed VCaP cells after 6 and 24 hours were com-
pared with the >7,000 expression profiles representing drug
responses to >1,309 compounds using Connectivity Map.
Fig. 3. DSF induced changes in gene expression in VCaP cells. A,
quantitative reverse transcription-PCR analysis of ERG and AR mRNA
expression in VCaP cells in response to 6, 24, and 48 h of exposure to DSF
compared with DMSO controls. B, quantitative reverse transcription-PCR
analysis of metallothionein (MT1B, MT1F, MT1G, MT1X, and MT2A)
mRNAs and (C) minichromosome MCM (MCM2 and MCM5) mRNAs in
VCaP cells exposed to 6 or 24 h DSF compared with DMSO controls.
6074Clin Cancer Res 2009;15(19) October 1, 2009www.aacrjournals.org
Cancer Therapy: Preclinical
The results indicate the highest enrichment with 6-hour DSF
treatment and 12,13-EODE (Supplementary Table S3). This
compound, 12,13-cis epoxide of linoleic acid is generated by
neutrophils during the oxidative burst (27). Also irinotecan, a
topoisomerase 1 inhibitor was among the most enriched
drugs when compared with 6-hour DSF treatment. These
correlations support the gene ontology results indicating
oxidative stress and inhibition of DNA replication as the
DSF-induced biological processes. Interestingly, HDAC inhibi-
tors MS-275 and TSA were among drugs altering gene expres-
sion in an opposite direction than DSF (data not shown),
indicating that although both HDACi and DSF are potent
growth inhibitory compounds in VCaP cells, their mechan-
isms of action may be completely different.
Metallothioneins and minichromosome maintenance complex
genes regulate prostate cancer cell viability.
metallotioneins and MCM genes affect prostate cancer prolifer-
ation, siRNA transfections were done in VCaP and LNCaP pros-
tate cancer cells and cell viability was measured 48 or 72 hours
after transfection. The results indicate that silencing of any one
of the three genes MCM5, MT1F, or MT1G, is alone sufficient to
reduce the proliferation of both VCaP and LNCaP cells (Fig. 4),
suggesting that these genes may be among the critical mediators
of the molecular mechanisms by which DSF exerts its growth
inhibitory effects. For these siRNAs, the silencing of the
corresponding gene was confirmed by quantitative PCR in
VCaP cells (Supplementary Fig. S3).
DSF inhibits VCaP cell xenograft growth.
itory potential of DSF in vivo on prostate cancer growth, VCaP
cell xenograft experiments were done in immunocompromized
mice. Male nude mice were injected s.c. in the flank with VCaP
cells. When tumor size was ∼200 mm3, mice were divided into
two groups and daily treatments with 200 mg/kg/day DSF in
olive oil or olive oil only were administered p.o for 3 weeks.
Tumor growth was monitored by measuring tumor size with
calipers and calculating the approximate tumor volume. The re-
To find out whether
To study the inhib-
sults indicate that DSF reduced tumor growth up to 40% but
was not able to block it, indicating the need for a combinatorial
treatment (Fig. 5A).
Copper sensitized VCaP cells to DSF-induced cell death.
vious studies with melanoma and breast cancer cells have
shown that the growth inhibitory potential of DSF was poten-
tiated with copper or zinc cotreatment (28, 29). Combinatorial
effects of DSF and copper or zinc were studied in VCaP cells.
VCaP cells were treated with 20 μmol/L CuCl2alone, DSF
alone, or with the DSF-CuCl2combination and compared with
cells treated with DMSO. Similar experiments were done with
20 μmol/L ZnCl2. Apoptotic changes with spherical and de-
tached cells were clearly visible only in the DSF-CuCl2
complex–treated samples after 6-hour treatment. Cell viability
assay results indicated a significant reduction in cell viability
only in response to DSF-CuCl2complex treatment (Fig. 5B).
CuCl2and DSF cotreatment reduced AR protein levels and in-
duced poly ADP ribose polymerase cleavage, whereas neither of
the agents had an effect alone (Supplementary Fig. S4). Because
metallothioneins regulate the intracellular zinc and copper le-
vels and DSF induces metallothionein mRNA expression, we
studied metallothionein expression in VCaP cells in response
to DSF, CuCl2, and DSF-CuCl2cotreatment. The results indicate
that in response to DSF-CuCl2cotreatment, the expression of all
metallothioneins analyzed is more highly induced, whereas
MCM2 and MCM5 expression is even more repressed, than in
response to either one of the agents alone (Fig. 5C and D).
In this study, we used an unbiased HTS approach to identify
clinically compatible drugs to inhibit human prostate cancer
growth. Four prostate cancer cell lines and two nontumorigenic
prostate epithelial cells were screened with a library of 4,910
drug-like small molecule compounds including most of the
currently marketed drugs. We wanted to focus on drugs that
are preferentially inhibiting cancer cells. It turned out that the
vast majority of anticancer drugs are equally effective in cancer
and control cells. For example, docetaxel, currently used in the
clinic to treat patients with hormone-refractory prostate cancer,
was identified as a nonselective hit in our screen. The other HTS
hit compounds already in ongoing clinical trials5in prostate
cancer included doxorubicin, Taxol/paclitaxel, mitoxantrone,
suramin, camptothecin, staurosporine, ixabepilone, 17-AAG,
epothilone B, MS-275, and vinblastine sulfate salt. Also, cardiac
glycosides, previously identified as hits inhibiting KLK expres-
sion in a recent HTS study with breast cancer cells, and reported
to have antiproliferative and apoptotic effects also in PC-3,
LNCaP, and DU145 cells (30, 31), were identified among anti-
proliferative compounds in our screen, supporting the overall
functionality of our assay. Only a few cancer cell selective
growth inhibitory compounds were identified in the HTS exper-
iment. In agreement with our previous data, TSA was among
the most selective antiproliferative compounds for VCaP cells
(22, 32). The identification of HDAC inhibitors among the se-
lective compounds in an unbiased HTS approach supports our
previous conclusions that epigenetic reprogramming is charac-
teristic to ERG-positive prostate cancers. In addition, three nov-
el cancer selective agents, thiram, DSF, and monensin, were
Fig. 4. Metallothioneins and minichromosome MCM regulate prostate
cancer cell viability. Cell viability analysis in response to metallothionein
(MT1A, MT1B, MT1F, MT1G, MT1X, and MT2A) and minichromosome
MCM gene 5 (MCM5) silencing in VCaP and LNCaP prostate cancer cells.
Cell viability is presented as percentage of PLK1 siRNA–induced growth
inhibition. Only results exceeding the hit limit are shown. AllStars Negative
control siRNA and lipid only were used (SiLentFect without siRNAs) as
6075 Clin Cancer Res 2009;15(19) October 1, 2009www.aacrjournals.org
HTS for Selective Inhibitors against Prostate Cancer
identified. These agents inhibit VCaP, LNCaP, and LNCaP C4-2
prostate cancer cell proliferation at nanomolar concentrations,
but had little effect on control cells.
Previous studies already indicate that thiram, DSF, and mon-
ensin have antitumorigenic effects in culture and in various tu-
mor xenograft models, but their potential in prostate cancer
treatment has not been previously explored. Thiram has been
shown to block tumor angiogenesis in C6 glioma xenograft
model, and to reduce metastases and spontaneous leukemia
in rodents (33, 34). However, large doses of thiram have also
been reported to cause toxic effects such as neurotoxicity and
muscle dysfunction in rats (35). Studies in cultured cells in-
dicate that DSF inhibits myeloma, leukemia, lymphoma,
small cell lung cancer, cervical adenocarcinoma, melanoma,
neuroblastoma, and colorectal cancer cell survival as well as
osteosarcoma invasion (36, 37). Monensin in turn has been
reported to inhibit myeloma, renal cell carcinoma, colon can-
cer, lymphoma, and leukemia cell growth in vitro (38-41).
Comparison of prostate and breast cancer cell responses to
thiram, DSF, or monensin indicated that prostate cancer cells
are in general more sensitive to these drugs in vitro than
breast cancer cells (data not shown).
Due to its excellent safety profile and long-term use as an
alcohol deterrent in the clinic, DSF was selected for more de-
tailed mechanistic studies. Genome-wide expression profiling
results indicated that DSF induced metallothionein expres-
sion and downregulated DNA replication in VCaP cells. Me-
tallothioneins are intracellular proteins that regulate zinc and
copper availability, detoxify toxic metals, and protect cells
against oxidative stress. Decrease of VCaP cell growth was
linked to inhibition of DNA replication by reduced expres-
sion of minichromosome maintenance complex genes. In vivo
studies using VCaP cell xenografts showed reduced tumor
growth in response to DSF exposure. However, DSF alone
was not able to completely block tumor growth, indicating
need for combinatorial approaches.
Previous studies with melanoma and breast cancer cells indi-
cate that DSF given in combination with zinc or copper re-
duced proliferation in vitro at lower concentrations than DSF
alone (28, 29). Zinc is known to play an important role in
Fig. 5. DSF reduces VCaP xenograft growth and is potentiated by copper in culture. A, DSF reduces VCaP cell xenograft growth in vivo. Points,
mean of tumor volume in each experimental group; bars, SD. Tumor size was significantly decreased in DSF-treated mice when compared with vehicle
controls on measurement days 5, 9, and 16. B, VCaP cells were exposed to DMSO, DSF, copper or zinc chloride, or a combination of copper or zinc chloride
together with DSF for 6 h. Cell viability was measured by a fluorometric cell viability assay and results are presented relative to the DMSO control. C,
quantitative reverse transcription PCR analysis of metallothionein mRNA expression in VCaP cells in response DMSO, 1 μmol/L DSF, 20 μmol/L CuCl2, or
a combination of 20 μmol/L CuCl2and 1 μmol/L DSF exposure for 6 h. Normalized results are presented relative to CuCl2+DSF exposure. D, quantitative
reverse transcription PCR analysis of MCM2 and MCM5 expression in same samples as in C. Results are presented relative to DMSO control.
6076Clin Cancer Res 2009;15(19) October 1, 2009www.aacrjournals.org
Cancer Therapy: Preclinical
various cellular processes such as differentiation by regulating,
e.g., transcription factor activities, defense against free radicals,
and maintaining genomic stability. In prostate, zinc levels have
been reported to be lower in cancer than in normal tissue (42),
and increase in dietary zinc has been associated with a decrease
in the incidence of prostate cancer (43). A recent study also re-
ported that zinc did not induce DNA damage in normal cells
but only in cancer cells (44). However, Zn-DSF cotreatment did
not affect VCaP cell viability, whereas copper potentiated the
DSF effect and induced cell death. High serum and tissue levels
of copper have been found in many types of human cancers
including prostate, and hence, copper could act as a tumor-
specific sensitizer by, e.g., creating free radicals and thereby
DSF response (45). However, physiologic copper levels within
VCaP prostate cancer cells are not sufficient to block tumor
growth and that additional copper supply could be needed to
enhance the DSF response also in vivo.
Taken together, our systematic unbiased screen of known
drugs and drug-like molecules in prostate models provides
the first evidence that prostate cancer cell growth can be effi-
ciently and selectively inhibited by thiram, DSF, and monensin.
Thus far, there are clinical trials ongoing only with DSF in non–
small cell lung cancer, stage IV melanoma, and metastatic mel-
anoma.5Published results of a case report on DSF-treated
metastatic melanoma have been very promising (28). Our re-
sults indicate that DSF may have potential also in prostate can-
cer treatment, most likely in combination with other drugs or
with specific sensitizers, such as copper. In summary, results
from this study provide several novel starting points for pre-
clinical and eventually clinical efforts to treat prostate cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
We thank Finnish DNA Microarray Centre for performing the Illumina
experiments and Niko Sahlberg, Arttu Heinonen, and Jouni Latoniitty for
excellent technical assistance with HTS.
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