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Indexed and abstracted in Science Citation Index Expanded and in Journal Citation Reports/Science Edition
Bratisl Med J 2017; 118 (8)
460–466
DOI: 10.4149/BLL_2017_089
EXPERIMENTAL STUDY
Phthalate induced toxicity in prostate cancer cell lines and
effects of alpha lipoic acid
Kismali G1, Yurdakok-Dikmen B2, Kuzukiran O3, Arslan P4, Filazi A2
Ankara University Faculty of Veterinary Medicine, Department of Biochemistry, Ankara, Turkey.
gorkemkismali@yahoo.com
ABSTRACT
OBJECTIVE: The effects of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n-butyl phthalate, ben-
zylbutyl phthalate, di-2-ethylhexyl phthalate were investigated on human prostate cancer cell lines DU145 and
PC3 in vitro.
MATERIALS AND METHODS: Standards of dimethyl phthalate, diethyl phthalate, di-isobutyl phthalate, dibutyl
phthalate, benzyl butyl phthalate, and di-ethyl hexyl phthalate were used. Alpha lipoic acid was used as an-
tioxidant compound. DU145 and PC3 human prostate carcinoma cells were used. MTT assay were used for
cytotoxicity assay.
RESULTS: A low dose proliferative effect of phthalates in vitro was observed. With the hypothesis of the inhibi-
tion of aerobic glycolysis activity in cancer treatment, α-lipoic acid was applied to cells; where as a contrary to
previous studies, no change in the cell proliferation was observed. In combination with ALA, at IC50 and lower
doses, an increase of the cytotoxic effect was found for DIBP, DBP and BBP; while for DMP, DEP and DEHP,
a decrease was observed for DU145 cells. In PC3 cells, a decrease was observed for DMP, DEP and DBPs;
while no signifi cant difference were observed for DEHP, DIBP and BBP.
CONCLUSSION: The present study demonstrates preliminary information regarding the low dose proliferative
effects of phthalates in prostate cancer in vitro (Tab. 2, Fig. 2, Ref. 65). Text in PDF www.elis.sk.
KEY WORDS: phthalate, prostate cancer, DU145, PC3, alpha lipoic acid, in vitro.
1Ankara University Faculty of Veterinary Medicine, Department of Bio-
chemistry, Ankara, Turkey, 2Ankara University Faculty of Veterinary Medi-
cine, Department of Pharmacology and Toxicology, Ankara, Turkey, 3Etlik
Veterinary Control Central Research Institute, Ankara, Turkey, and 4Ankara
University Faculty of Biology, Ankara, Turkey
Address for correspondence: G. Kismali, PhD, Ankara University, Fac-
ulty of Veterinary Medicine, Department of Biochemistry, 06110 Ankara,
Turkey.
Phone: +90.3123170315/4424, Fax: +903123176073
Introduction
Prostate cancer, as among the most commonly diagnosed can-
cer in men; has no effective treatment; while steroid hormones
such as androgen were found to infl uence their growth and sur-
vival. Prostate cancer was found to be an ideal candidate for exog-
enous preventive measures, such as dietary and pharmacological
prevention, due to the high prevalence, long latency, endocrine
dependency, availability of serum markers (e.g. prostate-specifi c
antigen) and the histological precursor lesions. However, there is
currently no strong evidence to suggest that dietary interventions
can reduce/induce the risk of prostate cancer (1).
Alpha lipoic acid (ALA), a naturally occurring cofactor, is
important in the assortment of enzyme complexes controlling
metabolism, including the conversion of pyruvate to energy in
the mitochondrion. This compound is shown to be included in a
variety of biological process associated with oxidative stress, in-
cluding cancer (2, 3). ALA was found to generate reactive oxygen
species (ROS), triggering the mitochondrial pathway of apopto-
sis in cancer cells, which contributes ALA-dependent cell death
in various types of cancer cells in experimental studies, includ-
ing lung (4, 5) colon, (6) breast (7, 8), leukemia (9, 10) and liver
(11). Due to its powerful antioxidant capacity and importance
in glycose metabolism by supporting pyruvate dehydrogenase
reaction and oxidation of glycose, ALA has turned into a promis-
ing complementary therapeutic agent in the eradication of tumor
cells. The mechanism of action of ALA is complex and differs
according to the cancerous cell type (12). ALA was found to in-
hibit the second messenger NF-κB (nuclear factor kB), leading to
decreased proliferation, metastasis, invasion, chemo/radio resis-
tance and infl ammation of cancer cells (13–15). ALA was found
to induce the hyperacetylation of histones related to the prolifera-
tion of many types of cancer cells, which would eventually lead
to apoptosis (16). As mentioned previously, the increased uptake
of oxidizable substrates into the mitochondrion of cancerous cells
also stimulate apoptosis. Antimutagenic and anticlastogenic ef-
fects of this compound has also been studied (17, 18). Due to its
anti-infl ammatory property, protective effects on nerve damage
and neuropathy from chemo drugs like the platinum, related to
its anti-infl ammatory property were also described previously
(19, 20). Epidemiology and experimental research indicate dis-
cordance for the relationship between ALA and prostate cancer.
Increased risk has been associated previously (21–23). Azrad et
al (23) defi ned the genetic variation related to ALA metabolism,
Kismali G et al. Phthalate induced toxicity in prostate cancer cell lines…
xx
461
where the prostatic ALA, independent of diet, was found to be
signifi cantly and positively associated with biomarkers of aggres-
sive disease affecting the tumor proliferation rates. Meanwhile,
these studies show drawbacks such as collection of the accurate
dietary data, inter-individual differences in the metabolism of
ALA or the sampling sizes (24).
Phthalates, known as the plasticizers (making plastics more
fl exible or soft), have a variety of commercial uses, including
personal-care products (e.g. perfumes, lotions, cosmetics), paints,
food, construction industry, and certain medical devices and phar-
maceuticals (25). These ubiquitous environmental, endocrine dis-
rupting contaminants, were found to have adverse effects on male
reproductive health (26, 27). Irreversible changes in the male re-
productive tract due to phthalate exposure, even in the prenatal
period, is shown to interfere with the androgen signaling pathway,
causing permanent adverse effects on reproductive development
corresponding a decline in male fertility due to changes in sperm
concentration and semen quality. These compounds are also as-
sociated with an impaired development and alter the regular func-
tion of prostate (26). Exposure to DEHP (di-2-ethylhexyl phthal-
ate), DEHA (di(2-ethylhexyl)adipate), (28) and DIBP (diisobutyl
phthalate) (29) in the diet, were found to result in decreased weight
of the prostate.
The effects of phthalates on prostate cancer cells were studied
extensively especially in LNCaP cells; since this cell line was
found to express estrogen receptor-α, estrogen receptor-β and
androgen receptors (ARs), which were linked to the endocrine
disrupting property of phthalates. DBP was found to promote LN-
CaP prostate cancer proliferation through the crosstalk between
TGF-β and ER signaling pathway (30). Meanwhile, Hruba et al
(31) showed that, at lower concentrations, DEHP (50 μM) and
DBP (50 μM) were found to suppress cell cycle proliferation in
a dose-dependent manner through induction of accumulation of
cells within G1 phase of the cell cycle. Previously, DEHP (3 mM)
and its main metabolite MEHP (mono(2-ethylhexyl)phthalate-3
μM) caused production of reactive oxygen species, activation
of p53 tumor suppressor and induction of p21WAF/Cip1cyclin-
dependent kinase inhibitor; where this effect was inhibited by
selenium (32). DBP was also shown to promote LNCaP cell
proliferation by upregulating the gene expression of c-myc and
cyclin D1 and by downregulating the expression of p21 (15).
DEHP was also found to weakly reduce AR protein levels after
long-term exposure (8 days), while only DBP partially inhibited
expression of the prostate-specifi c antigen (KLK3) gene, a model
AR transcriptional target. Overall, it was stated that DEHP and
DBP may have negative effects on the proliferation of LNCaP
cells, independent of AR modulations. Possible involvement of
AR or phenotypic changes such as modulation of neuroendo-
crine trans differentiation (NED) due to phthalate exposure are
still unknown (31). The relationship between phthalate/alpha
lipoic acid and male reproduction has recently been studied in
an in vivo model. Bi-n-butyl phthalate (BNBBP) was found to
cause testicular toxicity through testosterone, follicule stimulating
hormones (FSH) and antioxidant enzymes in Wistar rats; where
ALA was found to mitigate BNBP-induced testicular toxicity
through antioxidant mechanism and by direct free radical scav-
enging activity (33).
While the majority of the prostatic cancers are adenocarci-
nomas characterized by the expression of luminal differentiation
markers AR and prostate-specifi c antigen (PSA), where LNCaP
cells are used as the main in vitro model; androgen independent
models DU-145 and PC3 (as a model for small cell neuroendocrine
carcinoma) are used in studies for the evaluation of the effects of
chemicals independent of AR and more aggressive phenotypes
(34, 35). Therefore, the aim of the current study was to evalu-
ate the effects of phthalates on androgen independent cell lines
DU-145 and PC3 and to assess the possible interaction with the
antioxidant ALA.
Materials and method
Chemicals
Standards of dimethyl phthalate (DMP), diethyl phthalate
(DEP), di-isobutyl phthalate (DIBP), dibutyl phthalate (DBP),
benzyl butyl phthalate (BBP), and di-ethyl hexyl phthalate (DEHP)
were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Al-
pha lipoic acid (DL-Thioctic acid, 98+ %) was purchased from
Acros Organics (New Jersey, USA).
Cell culture conditions
DU145 (HTB-81) human prostate carcinoma cells derived
from the brain metastatic site and PC3 (CRL-1435), grade IV hu-
man prostate adenocarcinoma cells derived from the bone meta-
static site used in the study were acquired from the American
Type Culture Collection (ATCC™). All cell culture procedures
were performed under strict sterile conditions and kept inside a
5 % CO2 incubator at 37 °C. Cells were cultivated using RPMI
1640 medium (Gibco®) supplemented with 10 % fetal bovine
serum (Thermo Fisher Scientifi c) and penicillin-streptomycin
(100 U/ml, Thermo Fisher Scientifi c). Cell culture medium was
replaced every other day. Cell growth was checked using phase-
contrast microscopy.
Cytotoxicity
Sub-culturing and/or cell cultivation was carried out when a
confl uent monolayer of cells was observed over the majority of
growth surface via Juli FL software (Seoul, Korea). For the cy-
totoxicity assays, cells were seeded in 96-well microplates at a
density of 3 x 104 cells/mL in 100 μL. The microplates were in-
cubated for 24 h to allow for cell attachment and growth in the
plates while the following day 20 μL phthalate was added to the
media for another 24 h incubation for cytotoxicity assays of MTT
((3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide).
Based on the preliminary assays, half dilutions between 0.0061–
12.5 μg/ml for DMP, DIBP and BBP; 0.0002–0.5 for DEP and
DBP; 0.0006–1.25 μg/ml were applied. Concentration of alpha
lipoic acid was decided according to preliminary cytotoxicity
studies and our previous study (10). Treatments at each dose were
conducted at three replicates in the same plate and all the experi-
ments were repeated four times. Medium only and 0.1 % Triton
Bratisl Med J 2017; 118 (8)
460–466
462
X-100 served as negative and positive controls, respectively. MTT
assays were performed soon after the incubation. Cell viability
was quantifi ed using SpectraMax i3/i3x Multi-Mode Detection
Platform (Molecular Devices, Sunnyvale, CA) at 540 nm for MTT.
Statistical analysis
Percent cytotoxicity were calculated for each concentration
using Microsoft Excel computer program. Regression analysis
was done using the plotted values against the corresponding doses
by SPSS 17.0 where the highest correlation coeffi cient (R2) is se-
lected for the fi t and IC50 values are calculated. Results for ALA
and phthalate combination were presented as the means ± SDs.
Statistical analysis was done using one way analysis of variance
(ANOVA) for multiple samples and Student’s t-test for compar-
ing paired sample sets. p values less than 0.05 were considered
statistically signifi cant.
Results
Ic50 Values for the Tested Phthalates: Among the tested phthal-
ates, DEHP induced the highest cytoxocity on DU 145 cells; where
the least cytotoxic compound was DMP in the same cell line. PC3
cells were more susceptible to DMP, DEP and BBP than DU 145
cells (p > 0.05). Among average IC50 values for both cells, the
most cytotoxic compound was DEHP followed by DBP. PC3 cells
were found to be more susceptible to the tested phthalates com-
pared to DU 145 cells (Tab. 1).
Phthalate Combination with Alpha Lipoic Acid: Following
the co-administration of ALA with IC50 doses of pyrethroids, an
increase of the cytotoxic effect were found for DIBP, DBP and
BBP (13.09, 25.22, 5.36 %, respectively); while for DMP, DEP
and DEHP, a decrease (9.27, 8.12, 7.76 %, respectively) was ob-
served for DU145 cells (Fig. 1 a, Tab. 2). In PC3 cells, a decrease
was observed for DMP, DEP and DBPs (26.58, 17.01 and 16.02
%, respectively); while no signifi cant difference were observed
for DEHP, DIBP and BBP (p > 0.05) (Fig. 2, Tab. 2).
Discussion
Endocrine disrupting compounds were found to infl uence the
development and progression of prostate cancer mainly through
estrogen reprogramming of the prostate gland resulting perma-
nent alterations and gene expression for prostatic lesions with ag-
ing (36, 37). Epidemiologic evidence linked prostate cancer and
environmental contaminants with endocrine disrupting potential
such as pesticides (chlorpyrifos, fonofos, coumaphos, phorate,
permethrin) (38–40) bisphenol A (41), PCBs (42), dioxin (43), cad-
mium (44), and arsenic (45) which are known to have estrogenic
activities. Estrogens have been implicated as potential agents in
the development and progression of prostate cancer through hor-
monal dysregulation, hyperprolactinemia, infl ammation, which
would lead mutations and DNA damage and epigenotoxigenic
pathways (46, 47).
Fig. 1. Phthalate combination with alpha lipoic acid for DU145 cell line.
Fig. 2. Phthalate combination with alpha lipoic acid for PC3 cell line.
DU 145 PC 3
ALA 50.77±3.23 -22.13±-2.00
DMP 81.25±0.62 38.15±2.24
DMP+ALA 56.66±0.19 11.57±0.95
DEP 84.34±0.45 34.81±3.44
DEP+ALA 65.22±4.62 17.80±1.97
DEHP 90.00±16.30 47.97±0.14
DEHP+ALA 58.11±0.87 48.39±3.97
DIBP 60.66±6.72 17.88±3.46
DIBP+ALA 63.27±3.15 17.64±2.84
DBP 95.84±0.45 32.74±2.83
DBP+ALA 54.67±1.00 16.72±1.41
BBP 78.23±0.18 30.35±3.26
BBP+ALA 58.20±0.10 28.00±2.44
Tab. 2. Phthalate combination with alpha lipoic acid for DU145 and
PC3 cell lines.
DU 145 PC 3
Dimethyl phthalate (DMP) 2390.48 1301.78
Diethyl phthalate (DEP) 1905.53 477.13
Diisobutyl phthalate (DIBP) 449.74 785.80
Di-n-butyl phthalate (DBP) 27.32 77.21
Benzylbutyl phthalate (BBP) 93.15 44.25
Di-2-ethylhexyl phthalate (DEHP) 22.82 78.69
Tab. 1. IC50 values (ppb) of tested phthalates on prostate cancer cells.
Kismali G et al. Phthalate induced toxicity in prostate cancer cell lines…
xx
463
In order to study the genetic and molecular changes of pros-
tate cancer development and progression, in vitro culture models
such as LNCaP, DU145, PC3 and TSU-Pvl were developed. In the
nuclear compartment of PC3 cells, the presence of high affi nity
estrogen binding sites were reported; indicating the evidence for a
specifi c estrogen receptor; where signifi cant proliferative activity
was inhibited (48). This was supported by Matsumura et al (49)
where phytoestrogengenistein was found to inhibit the prolifera-
tive activity and induced the expression of p21, a regulator of cell
cycle progression and ERβ in the PC-3 cells. Lau et al (50) tested
the receptor-mediated estrogenic and antiestrogenic action of nor-
mal and malignant prostatic epithelial cells; where LNCaP cells
(androgen-sensitive human prostate adenocarcinoma cells) were
found to express Erβ, and estrogen responsive genes (progester-
one receptor and pS2), DU145 expressed ER-β and PR, and PC-3
cells exhibited ER-α, ER-β, and pS2 mRNA. Relative potencies
of their estrogenic activities of the phthalate compounds tested in
the current study descended in the order BBP > DiBP > DBP >
DEHP > DEP > DMP; where BBP showed its estrogenic activity
mainly through Erβ. DMP and DEP did not induce Erα-β ago-
nism or Erβ/AR antagonism (51). In the current study, the least
toxic compounds on both cells were DEP and DMP, this would
suggest a possible estrogen receptor dependency for the toxic ef-
fects of these two compounds. Also in the current study, DMP,
DEP and BBP induced less cytotoxic effects on DU145 cells
than PC-3 and vice versa for DIBP, DBP and DEHP cells. Even
though DU145 and PC3 were reported to be AR negative (52),
both cells were found to express detectable levels of AR mRNA
and protein, where levels of AR protein were found to increase
after the androgen ligand (dihydrotestosterone) treatment (53).
The expression of AR in PC3 and DU145 cell line were found
to inhibit the cell proliferation; through upregulation of p21 by
androgen signaling through AR (53, 54). From this point, andro-
gen antagonist phthalates such as DiBP, DBP and BBP would
expected to have lower cytotoxic activity; meanwhile DMP and
DEP (not AR antagonism) were found to have the least cytotox-
icity. For DiBP, DBP and BBP; relative inhibitory concentration
(RIC20) for AR antagonistic activity were found as 6.2x10–6,
4.8x10–6, 2.9x10–6 M (51) respectively; while IC50 values in the
current study for the same compounds were 1.44X10–6, 1.23x10–7
and 3.3x10–7 M for DU-145 cells; 2.51x10–6, 3.47x10–7, 1.59x10–7
for PC-3 cells. Since the concentration for the cytotoxic effects of
DBP and BBP are lower than the levels causing possible antian-
drogenic effects, AR pathway could not be attributed directly. AR
are linked to different phosphorylation sites, which are expected
to induce different functions and phosphorylation process is cell
type specifi c,(51) the differences in the cytotoxic effects between
DU-145 and PC-3 along with the different types of phthalates,
might be related to the AR, ER receptor affi nity and phosphory-
lation of these receptors.
Erβas, a mediator of epithelial differentiation and as an anti-
proliferative molecule, regulating many molecular pathways in-
cluding upregulation of apoptotic genes (55) is expressed in both
DU145 and PC3 cells (50). Among phthalates, BBP, which effects
directly as an agonist for Erβ (51), is expected to have higher tu-
mor-suppressing function (55). This was confi rmed in the current
study for BBP, being the most cytotoxic compound in PC-3 cells,
which express both Erβ and Erα. Interestingly, BBP was found to
be the third cytotoxic compound in DU145 cells, which express
Erβ only. Recently, the opposing roles of ERα and ERβ in prostate
cancer are under discussion; (56) since the tumor-promoting roles
of ERβ2 and ERβ5 isoforms were identifi ed. Since these isoforms
play an important role in tumor progression and currently, no in-
formation is available for phthalates, further studies are required
to understand estrogen receptor mediated effects of phthalates in
prostate cancer.
Neuroendocrine differentiation (NED) as a structural and
functional feature of prostate cancer, appears during advanced
stages, and found to be responsible for treatment resistance and
poor prognosis.(57) Androgen depletion is also correlated to the
induction of NED in prostate cancer cells in vitro (54, 58). Mean-
while, androgen-deprivation conditions did not induce NED in
PC3 and DU145 cells (59). Therefore, the results of the cur-
rent study could not be discussed within NED perspective; while
neuron-specifi c enolase and chromogenin A expression could be
studied in future.
Contradictory results in the previous studies with LNCaP
cell lines and phthalates, raise concerns over more complicated
molecular mechanisms behind the mechanism of action of these
compounds. DBP at 1 μM treatment induced cell proliferation;
(30) while at 50 μM decreased cell proliferation independent from
AR expression and activity (31). DEHP induced cytoxicity at 3
mM concentration through induction of reactive oxygen species
(ROS) and activation of nuclear p53 and p21 proteins; (60) while
this effect was found at much lower concentrations (50 μM) in
the study by Hruba et al (31) Experimental and epidemiological
evidence for the non-monotonic dose response relationship of en-
docrine disrupting compounds reveal a need for different strate-
gic methods for the risk assessment of these substance in human
health (61). Among these compounds, phthalates were found to
induce adverse effects at low concentrations (62). Low dose ex-
posure to DEHP (100 μg DEHP/kg/day) was found to alter sperm
morphology and chromatin DNA damage leading sperm toxicity
in rats (63), and increase susceptibility to testicular autoimmunity
(increase in IFN-γ positive cells) and damage to blood testis bar-
rier in mice (64).
The use of the powerful antioxidant, ALA; which is involved
in many important biological and biochemical cellular processes,
is used in the ancillary treatment of many diseases, such as dia-
betes, cardiovascular, neurodegenerative, autoimmune diseases,
cancer and AIDS (3, 11, 12). Meanwhile, their use as a poten-
tial anti-cancer agent is discussed for prostatic cancer patients
where epidemiologic and experimental researches indicate dis-
cordance (24). Recently, prostatic ALA, was signifi cantly and
positively associated with biomarkers of aggressive prostatic
cancer progression and tumor proliferation rates (21–23). Choi
et al (65) studied the effects of ALA on the antioxidant system
in prostatic cancer cells PC-3, LNCaP, and RWPE-2 cell lines
where the expression of Ref-1 protein was increased with 125,
250, and 500 μM of ALA in PC-3 signifi cantly. Treatment of LN-
Bratisl Med J 2017; 118 (8)
460–466
464
CaP cells with increasing concentrations of ALA (0, 0.125, 0.5,
1, 10, 125, 250, 500 μM, 1 mM, and 2 mM) resulted in a dose-
dependent decrease in cell viability, where signifi cant induction
of cell loss was observed at 250 μM; whereas no information is
available for PC-3 cells. In our study, we used a similar dose 200
μM for ALA, a slightly lower dose than the IC50. The mRNA
expressions of SOD-1, SOD-2, catalase, and GSH-Px were also
found to be decreased by ALA in PC-3 with 125, 50 and 500
μM treatment along with an increase of Ref-1 protein, which has
multifunctional roles involved in oxidative DNA damage repair
(65). In the current study, ALA were found to increase the cy-
totoxicity of the estrogen receptor agonist phthalates (51), BBP,
DIBP and DBP signifi cantly. According to current literature,
information regarding ALA and estrogen receptor is missing.
Therefore, current study might provide a preliminary information
for the mechanism of action of ALA through estrogen receptor
(especially Erα).
Conclusion
Future directions on the development of effective therapeutic
strategy for the prostate cancer would be linked to the effective
control on the hormonal and neuroendocrine transdifferentiation
pathways. Meanwhile, various molecular differences of the tumor
type and epigenetic factors including endocrine disrupting com-
pounds, like phthalates, makes the accurate treatment diffi cult
and the progression more aggressive. Combination therapies to
reduce the resistance of chemotherapeutics, such as antioxidants
would be directed. In the current study, the responses of two
different cell lines DU-145 and PC3 on exposure to phthalates
were found to be different and the cytotoxic effects of estrogen
receptor agonist phthalates (DIBP, DBP and BBP) were found to
increase the cytotoxic effects in PC3 cells, which are known to
be a more aggressive tumor type than DU145 cells. Even though
the current study has the limitation of providing in vitro data that
might not carry over to in vivo conditions, it could be suggested
that the combination ALA upon exposure to estrogenic environ-
mental contaminants might be benefi cial for the progression of
the prostatic tumor.
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Received March 13, 2017.
Accepted April 19, 2017.
