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Objective: The effects of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n-butyl phthalate, benzylbutyl 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 antioxidant 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 inhibition 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 significant 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).
Indexed and abstracted in Science Citation Index Expanded and in Journal Citation Reports/Science Edition
Bratisl Med J 2017; 118 (8)
DOI: 10.4149/BLL_2017_089
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.
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 signi 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
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,
Phone: +90.3123170315/4424, Fax: +903123176073
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 in 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-speci 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 in 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-in ammatory property, protective effects on nerve damage
and neuropathy from chemo drugs like the platinum, related to
its anti-in 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) de ned the genetic variation related to ALA metabolism,
Kismali G et al. Phthalate induced toxicity in prostate cancer cell lines…
where the prostatic ALA, independent of diet, was found to be
signi 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
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-speci 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-speci 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
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 Scienti c) and penicillin-streptomycin
(100 U/ml, Thermo Fisher Scienti c). Cell culture medium was
replaced every other day. Cell growth was checked using phase-
contrast microscopy.
Sub-culturing and/or cell cultivation was carried out when a
con 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)
X-100 served as negative and positive controls, respectively. MTT
assays were performed soon after the incubation. Cell viability
was quanti 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 coef cient (R2) is se-
lected for the 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 signi cant.
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 signi cant difference were observed
for DEHP, DIBP and BBP (p > 0.05) (Fig. 2, Tab. 2).
Endocrine disrupting compounds were found to in 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, in 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…
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 af nity
estrogen binding sites were reported; indicating the evidence for a
speci c estrogen receptor; where signi 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 speci 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 af 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 con 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 identi 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-speci 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 signi 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 signi cantly. Treatment of LN-
Bratisl Med J 2017; 118 (8)
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 signi 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 signi 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).
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 dif 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 bene cial for the progression of
the prostatic tumor.
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Received March 13, 2017.
Accepted April 19, 2017.
... In the study by Pérez-Albaladejo et al. (2017), in which human placenta JEG-3 cells were exposed for 24 h, dibutyl phthalate (DBPh) was very weakly cytotoxic (IC 50 = 466 ± 63 μM) and no significant cytotoxic effect was observed for benzyl butyl phthalate (BBPh), di(2-ethylhexyl)phthalate (DEHPh) and dimethyl phthalate. Surprisingly, phthalates were found strongly cytotoxic in PC3 and DU145 human prostate cancer cells at IC50 concentrations differing from 2.4 μg/mL (DMPh) to 0.023 μg/mL (DEHPh) (Kismali et al., 2017). ...
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This study was undertaken to assess cytotoxic effects of selected aluminium compounds, parabens and phthalates in combination with silver nanoparticles (AgNP, 15 and 45 nm by STEM, Ag15 and Ag45, respectively) on cell lines of the human breast epithelium, normal (MCF-10A) and transformed (MDA-MB-231 and MCF-7). Combination indices were the most spectacular at effective concentrations (ED) inducing 25% decrease in viability for the combinations of Ag15 with AlCl3 for MDA-MB-231 cells or aluminium zirconium tetrachlorohydrex Gly (AlZr) for MCF-10A and MCF-7 cells, where rather strong antagonism was revealed. As the ED values increased, those effects were enhanced (e.g. Ag15+AlCl3 for MDA-MB-231) or reversed into synergism (e.g. Ag15+AlZr for MCF-7). Another strong effect was observed for aluminium chloride hydroxide, which increasing ED, induced synergistic effect with both Ag15 and Ag45 on MCF-10A cells. Another interesting synergistic effect was observed for DBPh, but only in combination with Ag45 on MCF-10A and MCF-7. The results on cytotoxicity, cell cycle and oxidative stress induction indicate complex response of the cell lines to combined treatment with silver nanoparticles and the chemicals, which were influenced by diverse factors, such as physico-chemical characteristics of AgNP, method of their synthesis, concentrations used, and finally cell type.
... No upper limit of LA administration has been defined in clinical use, and no adverse effects have been noticed over time at higher doses [33,34]. In regard to cancer treatment, recent studies, among many others, have confirmed that LA can inhibit cell proliferation in cervical, breast, colon, prostate, and lung cancers [24,[35][36][37][38]. Most importantly, the combination of LA and PTX has shown an enhanced inhibitory effect on breast cancer cell proliferation [25]. ...
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Paclitaxel-lipoate (IDD-1040) is a conjugate formed by the chemical joining of the two compounds, by condensing a lipoic acid moiety to the C2 of paclitaxel. IDD-1040 was evaluated for its anti-tumor activity and potential druggability, using an in vivo non-small-cell, lung cancer (NSCLC) xenograft mouse model. In the in vivo studies, IDD-1040 showed a maximum tolerated dose (MTD) of 250 mg/kg compared to paclitaxel (PTX), with an MTD of 20 mg/kg. Most interesting, IDD-1040 demonstrated higher anti-tumor activity, and its inhibitory activity on tumor volume (cell growth) was dose-dependent. That anti-tumor activity persisted for two weeks after cessation of IDD-1040 treatment, as opposed to PTX cessation, after which the tumor relapsed, confirming that IDD-1040 exhibits superior tumor inhibition. Similar to PTX treatment, no marked body weight decrease was observed during IDD-1040 treatment, indicating a low toxicity profile. The increase in animal body weight noted over time was due to the increasing weight of tumors, recorded in all the mouse test groups. The results also showed that mortality rate of mice was reduced by treatment with IDD-1040, more so than with PTX. Furthermore, in a preliminary study on the ex vivo distribution of IDD-1040, neutropenia was primarily concentrated in the liver 1 h after injection, and most of the drug was metabolized by the liver in 24 h. All of these results demonstrate IDD-1040's great potential as a candidate drug for cancer treatment.
... Blood bags are an important source of exposure to DEHP, with exposure levels of 0.7 mg/kg/time in transfused persons. Previous studies documented that DEHP is cytotoxic in different cell lines (Kismali et al. 2017;Ma et al. 2018) and showed, also, that this phthalate is deleterious in various organs, like the reproductive tract, liver, lungs, and heart (Tickner et al. 2001). ...
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Because of the extensive use of phthalates for domestic, medical, and industrial applications, the evaluation of their toxic effects is of major concern to public health. The aim of the present study was to assess the propensity of di (2-ethylhexyl) phthalate (DEHP), one of the most used phthalates, to cause oxidative cardiac damage in mice. DEHP was administered intraperitoneally at doses of 5, 50, and 200 mg/kg body weight for 30 consecutive days in BALB/c mice. We assessed the effect of DEHP on cardiac injury using biochemical profile (such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatinine phosphokinase (CPK), total cholesterol (T-CHOL), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C)), parameters related to myocardiac oxidative stress, such as malondialdehyde (MDA) level, protein carbonyl (PC) concentration, and DNA fragmentation. In addition, we evaluated antioxidant status; enzymatic (catalase (CAT) and superoxide dismutase (SOD) activities) and non-enzymatic (protein-bound sulfhydryl concentration (PSH)) antioxidants. Acetylcholinesterase (AChE) activity and histopathological changes were also assessed in heart mice treated with DEHP. Our results showed that DEHP induced an elevation of serum marker enzymes and perturbated the lipid profile. In addition, this phthalate increased lipid peroxidation, protein carbonyl levels, and DNA fragmentation in the heart in a dose-dependent manner. Antioxidant status was also perturbated by the increase of the CAT and SOD activities and the decrease of the protein-bound sulfhydryl concentration. AChE activity was also inhibited in the heart following the treatment with DEHP. These biochemical alterations were also confirmed by histopathological changes. Increased free radical production at various doses of DEHP would result in impairment of the redox status leading to an enhanced dose-dependent cardiotoxicity.
... It suggested that the chemical possessed the potential to inhibit the progression of the prostatic tumor. [78] Dioctyl phthalate Dioctyl phthalate (DOP) released from PVC products can be taken up by pharmaceuticals, foods, and then transported into the body fluid of animals and human beings, apples regarded as a model found apples from different places showed a various concentration of DBP because of polluted in varying degrees. [79] DOP was found to exhibit antiandrogenic function on endocrine disruptors on the developmental reproductive system in male rates. ...
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Phthalates are widely used in polymer materials as a plasticizer. These compounds possess potent toxic variations depending on their chemical structures. However, a growing body of evidence indicates that phthalate compounds are undoubtedly discovered in secondary metabolites of organisms, including plants, animals and microorganisms. This review firstly summarizes biological sources of various phthalates and their bioactivities reported during the past few decades as well as their environmental toxicities and public health risks. It suggests that these organisms are one of important sources of natural phthalates with diverse profiles of bioactivity and toxicity.
... 61,62 Despite the many beneficial effects of exchange transfusion highlighted above, plasticizers leaking from the storage bags were found to significantly accumulate in the plasma of transfused recipients with SCD. It is interesting to note that chronic exposure to plasticizers has been previously associated with prostate cancer and fertility issues in males 63 and reproductive outcomes in females in laboratory studies. 64,65 Because phthalate plasticizers progressively accumulate in storage units as a function of storage time up to mM levels, 14 it is interesting to note that exchange transfusion would theoretically result in hundred-μM to low-mM levels of phthalate plasticizers in plasma and, in a population exposed chronically to transfusion such as patients with SCD, could promote untoward consequences beyond factors such as mortality, as tested in recent randomized clinical trials on the age of blood. ...
BACKGROUND Exchange transfusion is a mainstay in the treatment of sickle cell anemia. Transfusion recipients with sickle cell disease (SCD) can be transfused over 10 units per therapy, an intervention that replaces circulating sickle red blood cells (RBCs) with donor RBCs. Storage of RBCs makes the intervention logistically feasible. The average storage duration for units transfused at the Duke University Medical Center is approximately 2 weeks, a time window that should anticipate the accumulation of irreversible storage lesion to the RBCs. However, no metabolomics study has been performed to date to investigate the impact of exchange transfusion on recipients' plasma and RBC phenotypes. STUDY DESIGN AND METHODS Plasma and RBCs were collected from patients with sickle cell anemia before transfusion and within 5 hours after exchange transfusion with up to 11 units, prior to metabolomics analyses. RESULTS Exchange transfusion significantly decreased plasma levels of markers of systemic hypoxemia like lactate, succinate, sphingosine 1‐phosphate, and 2‐hydroxyglutarate. These metabolites accumulated in transfused RBCs, suggesting that RBCs may act as scavenger/reservoirs. Transfused RBCs displayed higher glycolysis, total adenylate pools, and 2,3‐diphosphoglycerate, consistent with increased capacity to deliver oxygen. Plasma levels of acyl‐carnitines and amino acids decreased, while fatty acids and potentially harmful phthalates increased upon exchange transfusion. CONCLUSION Metabolic phenotypes confirm the benefits of the transfusion therapy in transfusion recipients with SCD and the reversibility of some of the metabolic storage lesion upon transfusion in vivo in 2‐week‐old RBCs. However, results also suggest that potentially harmful plasticizers are transfused.
... The analysis was performed by flow cytometry (C). mentioned conditions is associated with the phthalate and phthalate metabolites-mediated haemolysis and eryptosis (James-Todd et al., 2016;Harley et al., 2017;Piecha et al., 2016;Kismali et al., 2017). ...
Phthalates have been extensively used as plasticizers in various fields, including food, cosmetic, and pharmaceutical industry. Those compounds do not form covalent bonds to substances they are being added to, and thus they may migrate easily and penetrate various products used every day. They may reach organisms with air, food, or by a direct skin contact. Significant levels of phthalates and their metabolites are found in urine, breast milk, blood serum, venous blood, and cord blood. The purpose of this study was to assess the simple toxicity (haemolysis) and programmed death (eryptosis) caused by following phthalates: di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP) and their metabolites: mono-n-butyl phthalate (MBP) and mono-benzyl phthalate (MBzP) in vitro in human RBCs. RBCs were incubated with the above mentioned compounds at concentrations ranging between 0.5 and 500 μg/mL for 24 h. Obtained results demonstrated that DBP and BBP possess higher haemolytic properties compared to their metabolites. The lethal concentration (LC50) was determined. The value was 126.37 ± 5.94 μg/mL for DBP, and 103.65 ± 4.03 μg/mL for BBP, and for metabolites the LC50value was over 500 μg/mL. All compounds induced eryptosis causing translocation of phosphatidylserine, increased cytosolic calcium ions level, increased caspase-3 and calpain activation in human erythrocytes. BBP caused translocation of phosphatidylserine at a lower concentration compared to DBP. In case of other parameters, more pronounced changes were evoked by DBP at lower concentrations. Metabolites showed a significantly lower toxicity compared to parent compounds.
Despite several modifiable and non-modifiable risk factors of hormone-associated cancers have been established, less heed has been paid to chemicals, those having the potential to thwart the body’s normal detox system and affect the endocrine-hormonal pathways. Phthalates are endocrine-disrupting chemicals, most widely manufactured and used indiscriminately in several industries, including processed, ultra-processed and packaged food, single-use plastics, household and personal care/cosmetic products including diapers and electronics. The general population is regularly being exposed to phthalates on contact with these products, especially women and children are most vulnerable. It is therefore highly crucial to monitor and evaluate the biological burden of plasticizing phthalates in humans and understand the potential mechanisms of etiological link between pervasive exposure to phthalates and development of chronic diseases such as cancer through epigenetic and/or genetic alterations. It is also important to identify sustainable and scalable interventions for increasing public awareness, and restricting chronic phthalate exposure to individual and the population at large through relevant policy legislations, particularly in low-income and middle-income countries, such as India.
Exploring new bioactive compounds from endophytic fungi with the anticipation of novel alpha-glucosidase inhibitors (AGIs) has led to the identification of few interesting small compounds. In the present study, the major chunk of purified fraction (‘DFR8’: Diaporthe sp. Fraction number 8) had a compound 3-hydroxybenzoic acid (compound 4), reported for the first time in an endophytic fungus Diaporthe sp. isolated from a medicinal plant, Simarouba glauca DC. The fraction (‘DFR8’) was purified from ethyl acetate extract of Diaporthe sp. using the activity-based column purification method, and the fraction showed the highest AG inhibition (IC50 = 22.85 μg/mL). The fraction also showed significant antioxidant properties (DPPH: IC50=206.60 µg/mL and ABTS: IC50=59.65 µg/mL) and contained phenolic compounds. Apart from compound 4, the GC–MS analysis of ‘DFR8’ revealed the presence of 16 other compounds at lesser concentrations, and some of them are already known for their antioxidant and alpha-glucosidase inhibition (AGI) activities. Interestingly, compound 4 (3-hydroxybenzoic acid) had the highest binding energy against both yeast and human AG enzymes when compared to the remaining compounds. Additionally, 3-hydroxybenzoic acid obeyed the crucial rules of drug-likeness properties during ADME/Toxicity evaluations, indicating that the compound could be developed into an effective AG inhibitor, which needs further detailed investigations.
Elevated demand and extensive exploitation of cosmetics in day-to-day life have hiked up its industrial productions worldwide. Organic and inorganic chemicals like parabens, phthalates, sulfates, and so forth are being applied as constituents towards the formulations, which tend to be the mainspring ecological complication due to their enduring nature and accumulation properties in various sections of the ecosystem. These cosmetic chemicals get accrued into the terrestrial and aquatic systems on account of various anthropogenic activities involving agricultural runoff, industrial discharge, and domestic effluents. Recently, the use of microbes for remediating persistent cosmetic chemicals has gained immense interest. Among different forms of the microbial community being applied as an environmental beneficiary, algae play a vital role in both terrestrial and aquatic ecosystems by their biologically beneficial metabolites and molecules, resulting in the biobenign and efficacious consequences. The use of various bacterial, fungal, and higher plant species has been studied intensely for their bioremediation elements. The bioremediating property of the algal cells through biosorption, bioassimilation, biotransformation, and biodegradation has made it favorable for the removal of persistent and toxic pollutants from the environment. However, the research investigation concerned with the bioremediation potential of the algal kingdom is limited. This review summarizes and provides updated and comprehensive insights into the potential remediation capabilities of algal species against ecologically hazardous pollutants concerning cosmetic chemicals.
Background: Red blood cell exchange (RCE) transfusions are a mainstay in the treatment of sickle cell anemia (SCA), and allow a temporary restoration of physiological parameters with respect to erythrocyte oxygen carrying capacity and systems metabolism. Recently, we noted that 1) RCE significantly impacts recipients' metabolism in SCA; 2) fresh and end-of-storage red blood cell (RBC) units differently impact systems of metabolism in healthy autologous recipients; and 3) phosphate/inosine/pyruvate/adenine (PIPA) solution reverses the metabolic age of stored RBCs. Therefore, we hypothesized that RCE with PIPA-treated RBC units could further increase the metabolic benefits of RCE in SCA patients. Study design and methods: Circulating plasma and erythrocytes were collected from patients with SCA before and after RCE, with either conventional or PIPA-treated RBC units, prior to metabolomics analyses. Results: Consistent with prior work, RCE significantly decreased circulating levels of markers of systemic hypoxemia (lactate, succinate) and decreased plasma levels of acyl-carnitines and amino acids. However, PIPA-treated exchanges were superior to untreated RCEs, with a higher energy state and an increased capacity to activate the pentose phosphate pathway and glutamine metabolism. In addition, RBCs and plasma from recipients of PIPA-treated RBC units resulted in significantly decreased levels of post-transfusion plasticizers, though at the expense of higher circulating levels of oxidized purines (hypoxanthine, xanthine, and the antioxidant urate). Conclusion: Transfusion of PIPA-treated RBCs further increases the metabolic benefits of RCE to patients with SCA, significantly reducing the levels of post-transfusion plasticizers.
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Chemotherapeutic failure and metastasis are the main causes of high mortality rate in lung cancer. Alteration of cellular redox status in response to endogenous stimuli or exogenous compounds has a significant impact on cell signaling and behavior. Herein we divulge for the first time that lung cancer cells exposed to α-lipoic acid (LA) resulted in a higher level of cellular superoxide anion (O2·-) and hydrogen peroxide (H2O2), and such an increase of the specific reactive oxygen species (ROS) downregulated integrin β1 and β3, the integrins known for potentiating aggressive behavior and metastasis. The LA-treated cells exhibited significant decrease in their abilities to survive in detached condition and grow in anchorage-independent soft agar assay. Furthermore, LA sensitized the cells to cisplatin, etoposide and paclitaxel-induced apoptosis. For underlying mechanism, we found that the treatment of the cells with LA significantly decreased integrin β1 and β3, while had no effect on integrin α5 and αv. Interestingly, survival protein p-AKT and anti-apoptotic protein Bcl-2 were reduced in an association to such integrin modulations. Using ROS probes and selective anti-oxidants, we have shown that H2O2 and O2·- induced by LA are key players for the decrease of β1 and β3 integrins, respectively. These findings indicate a novel effect of LA as well as specific ROS, O2·- and H2O2 in integrin regulation, anoikis and chemotherapeutic sensitizations.
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The role of androgens in prostate cancer is obvious due to the fact that androgen signalling is the main regulator of prostate growth and function. Androgen deprivation therapy is a mainstay treatment for advanced prostate cancer. However, prostate cancer often becomes androgen-independent, which in consequence leads to lethal and incurable disease. In addition, oestrogens play a crucial role in prostate cancer, especially in elder men in whom the overall ratio of oestrogens to androgens is increasing. This review summarizes the current knowledge on molecular mechanisms through which oestrogens are involved in prostate cancer development. We focused on commonly alternated molecular signalling pathways contributing to tumourgenesis in prostate cancer.
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This review deals with alpha-lipoic acid (LA) from the point of its chemical and biological characteristics affecting enzymatic activities that are part of cellular biochemical processes in normal and cancer cells. This includes attributes of LA that are related to its ability to act as a free-radicals scavenger and also as a radical generator. LA is discussed in the light of its physico-chemical features, toxicity, biochemical bases of LA biological activities, and mechanisms of action. Additionally, it is discussed how these properties of LA are reflected by results of in vivo experiments with cancer cells and in experimental cancer chemotherapy. Finally, the results of LA Use in human Cancer chemotherapy and as chemopreventive agent are discussed in the light of LA future inclusion into chemotherapeutic protocols.
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Experimental studies investigating the effects of endocrine disruptors frequently identify potential unconventional dose-response relationships called non-monotonic dose-response (NMDR) relationships. Standardized approaches for investigating NMDR relationships in a risk assessment context are missing. The aim of this work was to develop criteria for assessing the strength of NMDR relationships. A literature search was conducted to identify published studies that report NMDR relationships with endocrine disruptors. Fifty-one experimental studies that investigated various effects associated with endocrine disruption elicited by many substances were selected. Scoring criteria were applied by adaptation of an approach previously used for identification of hormesis-type dose-response relationships. Out of the 148 NMDR relationships analyzed, 82 were categorized with this method as having a “moderate” to “high” level of plausibility for various effects. Numerous modes of action described in the literature can explain such phenomena. NMDR can arise from numerous molecular mechanisms such as opposing effects induced by multiple receptors differing by their affinity, receptor desensitization, negative feedback with increasing dose, or dose-dependent metabolism modulation. A stepwise decision tree was developed as a tool to standardize the analysis of NMDR relationships observed in the literature with the final aim to use these results in a Risk Assessment purpose. This decision tree was finally applied to studies focused on the effects of bisphenol A.
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Among endocrine disrupting chemicals, phthalates are of important concern due to widespread human exposure and environmental contamination. Eventhough, the use of some phthalates has been restricted for toys, some plastic and food contact materials, mix exposure to these contaminants at very low concentrations in various matrices are still been reported. In the current research, the effects of the mixture of some phthalates were studied. Di‐n‐butyl phthalate (DBP), n-butyl benzyl phthalate (BBP), di-2-ethylhexyl phthalate (DEHP), diisononyl phthalate (DiNP), di-n-octyl phthalate (DNOP) and diisodecyl phthalate (DiDP) on two different colorectal adenocarcinoma cell lines DLD-1 and HT29 were studied as described before. Cells were treated with increasing log concentrations (0.33 ppt to 33.33 ppb) of the phthalate mixture and following 24 h exposure cell viability/proliferation were measured by MTT, neutral red and crystal violet along with the LDH activity. Cell viability/proliferation was found to have increased by phthalate treatment at concentrations <33.33 ppt. Phthalate mixture induced an increase in HT29 proliferation as 10.94% at 33.33 ppt and 60.87% at 3.33 ppt. Whereas this proliferation relation at lower concentrations was not found for DLD1 cells. The present study demonstrates a preliminary information regarding the low dose induction of proliferation by phthalate mixtures in vitro. Further studies are needed on the nonmonotonic dose-response effect of phthalates to reevaluate the reference doses defined by governments.
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Neuroendocrine differentiation (NED) marks a structural and functional feature of certain cancers, including prostate cancer (PCa), whereby the malignant tissue contains a significant proportion of cells displaying neuronal, endocrine, or mixed features. NED cells produce, and can secrete, a cocktail of mediators commonly encountered in the nervous system, which may stimulate and coordinate cancer growth. In PCa, NED appears during advanced stages, subsequent to treatment, and accompanies treatment resistance and poor prognosis. However, the term " neuroendocrine " in this context is intrinsically vague. This article seeks to provide a framework on which a unified view of NED might emerge. First, we review the mutually beneficial interplay between PCa and neural structures, mainly supported by cell biology experiments and neurological conditions. Next, we address the correlations between PCa and neural functions, as described in the literature. Based upon the integration of clinical and basic observations, we suggest that it is legitimate to seek for true neural differentiation, or neuromimicry, in cancer progression, most notably in PCa cells exhibiting what is commonly described as NED.
Exposure to di-(2-ethylhexyl) phthalate (DEHP) induces spermatogenic disturbance (SD) through oxidative stress, and affects the immune system by acting as an adjuvant. Recently, we reported that in mice, a low dose of DEHP, which did not affect spermatogenesis, was able to alter the testicular immune microenvironment. Experimental autoimmune orchitis (EAO) can be induced by repeated immunization with testicular antigens, and its pathology is characterized by production of autoantibodies and SD. In the present study, we investigated the effect of a low-dose DEHP on the susceptibility of mice to EAO. The exposure to DEHP-containing feed (0.01%) caused a modest functional damage to the blood-testis barrier (BTB) with an increase in testicular number of interferon gamma (IFN-γ)-positive cells and resulted in the production of autoantibodies targeting haploid cells, but did not affect spermatogenesis. While only single immunization with testicular antigens caused very mild EAO, the concurrent DEHP exposure induced severe EAO with significant increases in number of interferon gamma-positive cells and macrophages, as well as lymphocytic infiltration and serum autoantibody titer accompanied by severe SD. To summarize, the exposure of mice to the low-dose DEHP does not induce significant SD, but it may cause an increase in IFN-γ positive cells and modest functional damage to the BTB in the testis. These changes lead to an autoimmune response against haploid cell autoantigens, resulting in increased susceptibility to EAO.
Aim of Study: This research indicated to evaluate the effects of piperlongumine (PL), a biologically active alkaloid, and alpha lipoic acid (ALA), a naturally occurring cofactor existed in multienzyme complexes regulating metabolism on leukemia cells. Excessive production of reactive oxygen species (ROS) can lead to oxidative stress, a state that has been observed in several hematopoietic malignancies, including acute and chronic myeloid leukemias. The importance of the association between oxidative stress and malignancy is not currently clear; however, there is evidence that tumor-derived ROS may promote cell survival, migration and metastasis, proliferation and even drug-resistance depending on the origin of the cancer. Increased oxidative stress in leukemic cells may represent a potential therapeutic target, although there are differing opinions on whether therapeutic strategies should aim to antagonize or further promote oxidative stress in leukemic cells. Materials and Methods: The effects of PL alone (5, 15, 30 μM) and in combination (30 μM) with ALA (200 μM) on Jurkat, NB4 and MEC1 leukemia cell lines were investigated through MTT, caspase-3 and cyclooxygenase-2 (COX-2) activities. Results: Inhibition of COX-2 and the induction of caspase-3 cleavage in Nb4 (acute promyelocytic leukemia) cells were found to be significant following PL application and synergistic effects with combination of ALA (inhibition of COX-2 as 23.74% and 3.55-fold increase of caspase-3). Conclusion: PL and ALA may have a potential value as a therapeutic agent for patients with acute promyelocytic leukemia. Keywords: Alpha lipoic acid, COX-2, leukemic cell lines, oxidative stress, piperlongumine