Induction of Cyp450 enzymes by 4-thiazolidinone-based derivatives in 3T3-L1 cells in vitro

Article abstract of DOI:10.1007/s00210-020-02025-7

4-Thiazolidinones and related derivatives are regarded as privileged structures in medicinal chemistry and a source of new drug-like compounds. To date it is known that thiazolidinones are able to induce CYP1A1 activity in 3T3-L1 cells. Therefore, to extend the knowledge of the mechanism of thiazolidinones in the cell, four chemically synthesized heterocycles were tested on 3T3-L1 cells. The 3T3-L1 cells were exposed to Les-2194, Les-3640, Les-5935, and Les-6166. Our study showed that 1 μM βNF, Les-2194, and Les-6166 decreased the expression of Ahr mRNA. In turn, βNF, Les-2194, and Les-3640 increased the Cyp1a1 mRNA expression at the same time interval. On the other hand, Les-5935 was found to decrease the Cyp1a1 mRNA expression. Interestingly, the expression of Cyp1a2 mRNA was activated only by βNF and Les-2194. The expression of Cyp1b1 mRNA in the 3T3 cell line increased after the βNF and Les-2194 treatment but declined after the exposure to Les-5935 and Les-6166. Moreover, the Les-2194 and Les-5935 compounds were shown to increase the activity of EROD, MROD, and PROD. Les-3640 increased the activity of EROD and decreased the activity of PROD. In turn, the treatment with Les-6166 resulted in an increase in the activity of EROD and a decrease in the activity of MROD and PROD in the 3T3-L1 cells.

Full text of DOI:10.1007/s00210-020-02025-7

Naunyn-Schmiedeberg's Archives of Pharmacology
https://doi.org/10.1007/s00210-020-02025-7
ORIGINAL ARTICLE
Induction of Cyp450 enzymes by 4-thiazolidinone-based derivatives
in 3T3-L1 cells in vitro
Konrad A. Szychowski1 & Bartosz Skóra1 & Anna Kryshchyshyn-Dylevych2 & Danylo Kaminskyy2
Kamila Rybczyńska-Tkaczyk3 & Roman Lesyk1,2 & Jan Gmiński1
&
Received: 18 September 2020 /Accepted: 15 November 2020
#
The Author(s) 2020
Abstract
4-Thiazolidinones and related derivatives are regarded as privileged structures in medicinal chemistry and a source of new drug-
like compounds. To date it is known that thiazolidinones are able to induce CYP1A1 activity in 3T3-L1 cells. Therefore, to
extend the knowledge of the mechanism of thiazolidinones in the cell, four chemically synthesized heterocycles were tested on
3T3-L1 cells. The 3T3-L1 cells were exposed to Les-2194, Les-3640, Les-5935, and Les-6166. Our study showed that 1 μM
βNF, Les-2194, and Les-6166 decreased the expression of Ahr mRNA. In turn, βNF, Les-2194, and Les-3640 increased the
Cyp1a1 mRNA expression at the same time interval. On the other hand, Les-5935 was found to decrease the Cyp1a1 mRNA
expression. Interestingly, the expression of Cyp1a2 mRNA was activated only by βNF and Les-2194. The expression of Cyp1b1
mRNA in the 3T3 cell line increased after the βNF and Les-2194 treatment but declined after the exposure to Les-5935 and Les-
6166. Moreover, the Les-2194 and Les-5935 compounds were shown to increase the activity of EROD, MROD, and PROD. Les-
3640 increased the activity of EROD and decreased the activity of PROD. In turn, the treatment with Les-6166 resulted in an
increase in the activity of EROD and a decrease in the activity of MROD and PROD in the 3T3-L1 cells.
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Keywords CYP450 4-Thiazolidinone Cyp1a1 3T3-L1 cells Cyp1b1 AhR
Highlights
• Les-2194, Les-3640, Les-5935, and Les-6166 do not affect cell viability
in the range from 100 nM to 1 μM.
• Compounds Les-3640 and Les-6166 at the 50 and 100 μM concentra-
tions activate caspase-3 in 3T3-L1 cells.
• Les-2194 increases the activity of EROD, MROD, and PROD and the
Cyp1a2 mRNA expression in 3T3-L1 cells.
• Les-2194 and Les-3640 increase the Cyp1a1 mRNA expression in 3T3-
L1 cells.
• Les-6166 increases the activity of EROD and decreases the activity of
MROD and PROD in 3T3-L1 cells.
* Konrad A. Szychowski
Jan Gmiński
kszychowski@wsiz.edu.pl
jgminski@wsiz.edu.pl
Bartosz Skóra
bskora@wsiz.edu.pl
1
2
3
Department of Lifestyle Disorders and Regenerative Medicine,
University of Information Technology and Management in Rzeszow,
Sucharskiego 2, 35-225 Rzeszow, Poland
Anna Kryshchyshyn-Dylevych
kryshchyshyn.a@gmail.com
Danylo Kaminskyy
dankaminskyy@gmail.com
Department of Pharmaceutical, Organic and Bioorganic Chemistry,
Danylo Halytsky Lviv National Medical University, Pekarska 69,
Lviv 79010, Ukraine
Kamila Rybczyńska-Tkaczyk
kamila.rybczynska-tkaczyk@up.lublin.pl
Department of Environmental Microbiology, University of Life
Sciences, Leszczyńskiego 7, 20-069 Lublin, Poland
Roman Lesyk
rlesyk@wsiz.edu.pl

Naunyn-Schmiedeberg's Arch Pharmacol
Abbreviations
After activation, PPARs bind with the retinoid X receptor
(RXR) and form PPARs/RXR heterodimers, which regulate
transcription of genes involved in different metabolic path-
ways (Bardot et al. 1993). Similarly, the aryl hydrocarbon
receptor (AhR) requires participation of RXR for activation
of gene expression (Hessel-Pras et al. 2018). Moreover,
Wójtowicz et al. (2017) described crosstalk between AhR
and PPARγ receptors in mouse neurons. Similarly, as report-
ed by Kim et al. (2008), troglitazone (PPARγ agonist) affects
induction of Cyp1a1, whose expression is AhR dependent, in
murine hepatoma Hepa-1c1c7 cells. It has also been described
that alpha-naphthoflavone (αNF), which is an AhR antago-
nist, inhibits differentiation of 3T3-L1 pre-adipocytes, which
is a well-documented PPARγ-dependent process (He et al.
2013). To date, PPARγ activators have been reported to act
as carcinogens as well, as described in some papers (Scatena
et al. 2008).
AhR
Aryl hydrocarbon receptor
DMSO Dimethyl sulfoxide
PBS
Phosphate-buffered saline
PPARs Peroxisome proliferator-activated receptors
ROS
XO
Reactive oxygen species
Xanthine oxidize
αNF
βNF
Alpha-naphthoflavone
Beta-naphthoflavone
EROD Ethoxyresorufin-O-deethylase
MROD 7-Methoxyresorufin O-demethylation
PROD
Pentoxyresorufm O-dealkylase
DMEM Dulbecco’s Modified Eagle’s Medium
Introduction
4-Thiazolidinones and related derivatives are regarded as
privileged structures in medicinal chemistry and are a source
of new drug-like compounds (Tripathi et al. 2014; Kaminskyy
et al. 2017a). In terms of modern medicinal chemistry, 4-
thiazolidinones possess a variety of biological activities (both
in screening campaigns and directed experiments). They are
also regarded as a tool for synthesis of related heterocycles,
simplified analogs, and diverse complex heterocycles in vari-
ous approaches. Moreover, among the variety of
thiazolidinone subtypes, 5-ene-4-thiazolidinones are of spe-
cial interest as hit and lead compounds with anticancer, anti-
microbial, antiviral, and antitrypanosomal activities
(Kaminskyy et al. 2017a; Lesyk 2020a, b). However, a new
trend in medicinal chemistry refers to these compounds as
frequent hitters or pan-assay interference compounds, which
are useless due to their possible low selectivity. This is chal-
lenged by the Michael acceptor property of 5-ene-4-
thiazolidinones (Kaminskyy et al. 2017b). Such a thesis is
actively discussed in the literature and requires further inves-
tigation. Nevertheless, we established earlier that annealing of
the thiazolidine core into fused analogs (thiopyrano[2,3-d-
]thiazoles, chromeno[2,3-d]thiazoles, thiazolo[4,5-b]pyridine,
etc.) is one of the prominent optimization directions that helps
to conserve the activity pattern of synthetic precursors (5-ene-
4-thiazolidinones), decrease the toxicity, and avoid the
Michael acceptor properties as well (Kryshchyshyn et al.
2018).
It is well known that many xenobiotics can accumulate in
adipose tissue (Barouki 2013). Unfortunately, the conse-
quences of this accumulation are still poorly understood
(Jackson et al. 2017). Moreover, adipose tissue can also me-
tabolize xenobiotics to a small extent with the involvement of
cytochrome P450 enzymes (Ellero et al. 2010), e.g., CYP1A1,
which has been examined extensively for its capacity to acti-
vate compounds with carcinogenic properties
(Androutsopoulos et al. 2009). Exposure to chemicals and
environmental carcinogens is thought to increase the level of
CYP1A1 expression in extrahepatic tissues through the AhR
receptor (Nebert et al. 2000; Di Bello et al. 2007).
Dysregulation of these pathways may be implicated in tumor
progression (Androutsopoulos et al. 2009). Similarly
CYP1B1, which is expressed in liver and extrahepatic tissues,
carries out the metabolism of numerous xenobiotics, including
the metabolic activation of polycyclic aromatic hydrocarbons
(Spink et al. 1998). Moreover, CYP1B1 has also been shown
to be important in regulating endogenous metabolic pathways,
including the metabolism of steroid hormones, fatty acids,
melatonin, and vitamins (Li et al. 2017). CYP1B1 and its
interaction with PPARs, estrogen receptors (ERs), and retinoic
acid receptors (RARs) contribute to the maintenance of the
homeostasis of these endogenous compounds (Angus et al.
1999; Nebert and Dalton 2006). Accumulating data indicate
that modulation of CYP1B1 can decrease adipogenesis and
tumorigenesis and prevent obesity, hypertension, atheroscle-
rosis, and cancer (Malik et al. 2012; Song et al. 2016; Li et al.
2017). Therefore, it may be feasible to consider CYP1B1 as a
therapeutic target for the treatment of metabolic diseases (Li
et al. 2017; Alzahrani and Rajendran 2020).
To date, it has been described that 4-thiazolidinones deri-
vates can be peroxisome proliferator-activated receptor
(PPAR) agonists (Wright et al. 2014; Zhou et al. 2015;
Szychowski et al. 2017a). PPARs are members of the nuclear
receptor superfamily that can be activated by various natural
fatty acids and xenobiotics (Berger et al. 2005; Scatena et al.
2008; Wojtowicz et al. 2014). Three subtypes of PPARs, i.e.,
PPARα, PPARβ, and PPARγ, have been identified (Lea et al.
2004; Scatena et al. 2008; Tachibana et al. 2008).
The molecular mode of the anticancer action of the
abovementioned compounds is still unclear, but it is believed
to be related with PPAR pathways (Lu et al. 2011; Asati et al.
2014). Our previous study has demonstrated that 4-
thiazolidinone-based derivatives (thiopyrano[2,3-d]thiazole

Naunyn-Schmiedeberg's Arch Pharmacol
derivative Les-2194 and thiazolidinone-pyrazoline hybrid
molecule Les-3640) cause apoptotic cell death in human squa-
mous carcinoma cells (SCC-15) with key involvement of
PPARγ (Szychowski et al. 2017a, b). Moreover, another com-
pound studied by our team 5Z-(4-fluorobenzylidene)-2-(4-
hydroxyphenylamino)-thiazol-4-one (Les-236) has been
found to exhibit broad cytotoxic and proapoptotic action in
SCC-15, lung carcinoma (A549), colon adenocarcinoma
(CACO-2), neuroblastoma (SH-SY5Y), and skin fibroblast
(BJ) cell lines (Szychowski et al. 2019).
At present, there are very limited data on the influence of
anticancer 4-thiazolidinone-based derivatives on Cyp1a1,
Cyp1b1, Cyp1a2, and Cyp2b10 expression or activity.
Moreover, the impact of Les-2194, Les-3640, Les-5935
(chromeno[2,3-d]thiazole derivative), or Les-6166
(thiazolidinone-indole hybrid) on induction of Cyp1a1,
Cyp1b1, Cyp1a2, and Cyp2b10 expression or activity has
never been studied. These compounds represent the most
promising classes of 4-thiazolidinone-based derivatives as po-
tential anticancer agents. In particular, Les-2194 (Havrylyuk
et al. 2012), Les-3640 (Kryshchyshyn et al. 2012), and Les-
5935 (Table S1, ESI) inhibited the growth of sixty cancer cell
lines representing nine neoplastic diseases at sub-micromolar
and micromolar concentrations according to US NCI proto-
cols (Shoemaker 2006).
(Mm99999915_g1), Ahr (Mm01291777_m1), Cyp1a1
(Mm00487218_m1), Cyp1b1 (Mm00487229_m1), Cyp1a2
(Mm00487224_m1), and Cyp2b10 (Mm01972453_s1) were
purchased from Thermo Fisher Scientific (Grand Island, NY,
USA).
Synthesis and physicochemical data of the tested com-
pounds were described previously: Les-2194, rel-N-(2,4-
dichlorophenyl)-2-[(5aR,11bR)-2-oxo-5a,11b-dihydro-
2H,5H-chromeno[4',3':4,5]thiopyrano[2,3-d][1,3]thiazol-
3(6H)-yl]acetamide (Kryshchyshyn et al. 2012), and Les-
3640, 3-{2-[5-(4-dimethylaminophenyl)-3-phenyl-4,5-
dihydropyrazol-1-yl]-4-oxo-4,5-dihydro-1,3-thiazol-5-yli-
dene}-2,3-dihydro-1H-indol-2-one (Havrylyuk et al. 2012).
Spectral and analytical data for Les-5935 (9H-
benzo[5,6]chromeno[2,3-d][1,3]thiazol-9-one) and Les-6166
(methyl 5-fluoro-3-[2-(4-hydroxyanilino)-4-oxo-4,5-dihydro-
1,3-thiazol-5-ylidenmethyl]-1H-2-indolecarboxylate) are de-
scribed in the Supplementary information.
All stock solutions were prepared by dissolving the com-
pounds in DMSO. The final concentration of DMSO in the
culture medium was always 0.1%.
Synthesis of anticancer 4-thiazolidinone-based
derivatives
Due to the lipophilic nature of the studied compounds, the
mouse non-cancerous preadipocyte 3T3-L1 cell line was se-
lected for metabolism studies. In addition, it is possible that
they accumulate in adipose tissue. Therefore, the aim of our
study was to determine whether the 4-thiazolidinone-based
derivatives induce the expression and/or activity of Cyp1a1,
Cyp1b1, Cyp1a2, and Cyp2b10 in the 3T3-L1 cell line.
The synthesis of Les-2194 and Les-3166 was performed ac-
cording to the method described by Kryshchyshyn et al.
(2012) and Havrylyuk et al. (2012), respectively.
General procedure for the synthesis of 9H-benzo[5,6]
chromeno[2,3-d][1,3]thiazol-9-one (Les-5935)
Mixtures of 4-aminothiazol-2(5H)-one (5 mmol), 2-
hydroxynaphthalene-1-carbaldehyde (5 mmol), and sodium
acetate (5 mmol) were heated at reflux for 2 h in a mixture
of AcOH and Ac2O (10 mL, 1:1). After cooling, the precipi-
tate formed was filtered off, washed with AcOH, H2O, and
EtOH, and recrystallized from a mixture of DMF/EtOH (2:1).
Spectral and analytical data for compound Les-5935 is pro-
vided in supplementary material 1.
Materials and methods
Reagents
Dulbecco’s Modified Eagle’s Medium (DMEM) without phe-
nol red (10-013CV) and phosphate-buffered saline without
calcium and magnesium (PBS) (21-040-CVR) were pur-
chased from Corning (Manassas, VA, USA). Trypsin, peni-
cillin, streptomycin, amphotericin B, β-naphthoflavone
(βNF) (N3633), and dimethyl sulfoxide (DMSO) were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA). Substrates
for P450 cytochrome ethoxyresorufin-O-deethylase (EROD)
(5725-91-7), 7-methoxyresorufin O-demethylation (MROD)
(5725-89-3), and pentoxyresorufm O-dealkylase (PROD)
(87687-03-04) were supplied by Tocris Bioscience. Fetal bo-
vine serum (FBS) (E5050-02), RNA isolation kits (E0309),
and fast probe qPCR master mix (Rox) (E0423) were pur-
chased from EURx (Gdańsk, Poland). TaqMan probes corre-
sponding to specific genes encoding G ap d h
General procedure for the synthesis of methyl
5-fluoro-3-[2-(4-hydroxyanilino)
-4-oxo-4,5-dihydro-1,3-thiazol-5-ylidenmethyl]
-1H-2-indolecarboxylate (Les-6166)
A mixture of 0.1 mole of 4-hydroxyphenylthiourea (0.1 mol),
methyl 5-fluoro-3-formylindole-2-carboxylate (0.11 mol),
chloroacetic acid (0.1 mol), and fused sodium acetate (0.1
mol) in 100 mL of acetic acid was heated under reflux for 3
h. The crystalline precipitate was filtered off, washed with
acetic acid, water, ethanol, and diethyl ether, and then recrys-
tallized from a mixture DMF-acetic acid (1:2). Spectral and

Naunyn-Schmiedeberg's Arch Pharmacol
analytical data for compound Les-6166 is provided in supple-
mentary material 1.
Ac-DEVD-pNA at 37 °C. Cells treated with 1 μM
staurosporine were used as a positive control (data not
shown). After 30 min, the absorbance of the lysates at
405 nm was measured using a microplate reader (FilterMax
F5). The amount of the colorimetric product was continuously
monitored for 120 min. The data were analyzed using Multi-
Mode Analysis software and normalized to the absorbance in
the vehicle-treated cells.
3T3-L1 cell culture
Mouse embryonic fibroblast cell line 3T3-L1 was obtained
from the American Type Culture Collection (ATCC, distrib-
utor: LGC Standards, Łomianki, Poland). The 3T3-L1 cell
line was maintained in phenol red-free DMEM supplemented
with 10% FBS, 100 U/mL penicillin, 0.10 mg/mL streptomy-
cin, and 250 ng/mL amphotericin B. The cells were main-
tained at 37 °C in a humidified atmosphere with 5% CO2.
They were seeded in 96-well culture plates at a density of 6
× 103 (for the 24-h treatment), 5 × 103 (for the 48-h treatment),
and 4 × 103 (for the 72-h treatment) per well and initially
cultured before the experiment for 24 h. Subsequently, the
medium was replaced with a fresh one by raising the concen-
trations (100 nM, 1 μM, 10 μM, 50 μM, and 100 μM) of Les-
2194, Les-3640, Les-5935, and Les-6166 (resazurin reduction
and caspase-3 activity).
qPCR analysis of mRNAs specific to genes encoding
Ahr, Cyp1a1, Cyp1b1, Cyp1a2, and Cyp2b10
The quantitative polymerase chain reaction (qPCR) method
was chosen to determine whether the tested compounds in-
crease gene expression. The experiment was conducted with a
procedure described previously (Szychowski et al. 2017a).
For the qPCR assay, 3T3-L1 cells were seeded onto 12-well
plates and initially cultured for 24 h. After 6-h exposure to
1 μM βNF, Les-2194, Les-3640, Les-5935, or Les-6166, the
samples were collected, and total RNA was extracted from the
3T3-L1 cells using a RNA isolation kit (EURx, Gdańsk,
Polska) according to the manufacturer’s instructions. Both
the quality and quantity of the RNA were determined spectro-
photometrically at 260 and 280 nm, respectively (ND/1000
UV/Vis; Thermo Fisher NanoDrop, USA). Two-step real-
time reverse transcription (RT)-PCR was conducted with both
the RT reaction and the quantitative PCR (qPCR) run using
the CFX real-time system (Bio-Rad, USA). The RT reaction
was carried out at a final volume of 20 μL with 800 ng RNA
(as a cDNA template) using the cDNA reverse transcription
kit in accordance with the manufacturer’s instructions.
Products of the RT reaction were amplified using the fast
probe qPCR master mix (EURx) with TaqMan probes as
primers for specific genes encoding Gapdh, Ahr, Cyp1a1,
Cyp1b1, Cyp1a2, and Cyp2b10. The amplification was car-
ried out in a total volume of 20 μL containing 1× fast probe
qPCR master mix (EURs) and 1.0 μL of the RT product,
which was used as the PCR template. The standard qPCR
procedures were carried out as follows: 2 min at 50 °C and
10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and
1 min at 60 °C. The threshold value (Ct) for each sample was
set during the exponential phase, and the ΔΔCt method was
used for data analysis. Gapdh was used as the reference gene.
Resazurin reduction assay
The resazurin reduction assay was used to determine the rate
of cell metabolism and, based on this, cell viability
(Rampersad 2012). Resazurin is a redox dye showing colori-
metric and fluorometric changes depending on the cell meta-
bolic activity. The resazurin reduction assay is based on the
ability of metabolically active cells to convert blue resazurin
(non-fluorescent form) to red resorufin (fluorescent form). To
determine viability, the cells were seeded in 96-well plates.
They were incubated in different series of the Les-2194, Les-
3640, Les-5935, and Les-6166 compounds (100 nM, 1 μM,
10 μM, 50 μM, and 100 μM) for 24 h, 48 h, and 72 h at 37 °C.
Next, the dilutions of the compounds were removed, and the
DMEM medium containing 1% FBS and 10% of resazurin
was added. Then, after 30 and 60 min of incubation, fluores-
cence measurements were made using a microplate reader
(FilterMax F5). The maximum excitation spectrum was 530
nm, and the emission was 590 nm. The data were analyzed
using Multi-Mode Analysis software and normalized to the
fluorescence in the vehicle-treated control (% of control).
Caspase-3 activity
CYP450 activities (EROD, MROD, and PROD assays)
Caspase-3 activity was used as a marker of cell apoptosis and
was assessed according to Nicholson et al. (1995). To measure
caspase-3 activity, the cells were seeded on 96-well plates and
exposed to Les-2194, Les-3640, Les-5935, and Les-6166.
After thawing (− 80 °C), the cells were lysed using lysis buffer
(50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS,
1 mM EDTA, 10% glycerol, and 10 mM DTT) at 10 °C for
10 min. The lysates were incubated in the caspase-3 substrate
To confirm the results of the qPCR reaction and their transla-
tion into functional protein, the activity of the studied cyto-
chromes was tested. We estimated the activity of the Cyp1a1/
Cyp1b1, Cyp1a2, and Cyp2b1 enzymes using the fluoromet-
ric ethoxyresorufin-O-deethylase (EROD), 7-
methoxyresorufin O-demethylation (MROD), and
pentoxyresorufm O-dealkylase (PROD) substrates,

Naunyn-Schmiedeberg's Arch Pharmacol
respectively. The fluorescence EROD, MROD, and PROD
assays were performed according to the method proposed by
Kennedy et al. (1993). For the assays, the cells were seeded on
12-well plates and initially cultured for 24 h. Measurement of
the EROD, MROD, and PROD activity was performed after 24
or 48 h of exposure to 1 μM βNF, Les-2194, Les-3640, Les-
5935, and Les-6166. To perform the EROD, MROD, and
PROD assays, lysed cells were transferred into multiwell plates,
and the fluorescent product resorufin was quantified within the
wells with a fluorescence plate reader (FilterMax F5) at an
excitation wavelength of 530 nm and an emission wavelength
of 590 nm. The protein concentration was determined spectro-
photometrically in triplicate for each sample at 280 nm using
the ND/1000 UV/Vis Thermo Fisher NanoDrop device.
performed to measure the effects of Les-2194, Les-3640, Les-
5935, and Les-6166 on the viability of 3T3-L1 preadipocytes.
After the 24-h exposure to 100 nM, 1 μM, 10 μM, 50 μM,
and 100 μM of Les-2194, Les-3640, Les-5935, or Les-6166,
only 100 μM of Les-2194 and Les-3640 induced a decrease in
resazurin reduction in the 3T3-L1 cells (a decrease by 16.43
and 15.77%, respectively, compared to the control) (Fig. 2a).
Similarly, after the 48-h exposure to 100 nM, 1 μM, 10 μM,
50 μM, and 100 μM of Les-2194, Les-3640, Les-5935, or Les-
6166, 100 μM of Les-2194 and Les-3640 caused a decrease in
resazurin reduction in the 3T3-L1 cells (a decrease by 17.07 and
24.83% respectively, compared to the control). Moreover, Les-
6166 decreased resazurin reduction at the concentrations of
50 μM and 100 μM (a decrease by 16.44 and 28.19%, respec-
tively, compared to the control). In turn, compound Les-5935 at
the concentration of 100 μM increased resazurin reduction by
14.19%, compared to the control (Fig. 2b).
Statistical analysis
The data are presented as means SD of three independent
experiments. Each treatment was repeated six times in each
independent experiment (n = 18). The data were analyzed with
one-way analysis of variance (ANOVA) followed by Tukey’s
multiple comparison procedure ***P < 0.001, **P < 0.01,
and *P < 0.05 vs. the control.
After the 72-h exposure to the studied compounds, or Les-
6166, similar as in the previous variant, 100 μM of Les-2194
and Les-3640 contributed to a decrease in resazurin reduction
in the 3T3-L1 cells (a decrease by 17.88 and 13.20%, respec-
tively, compared to the control). Les-6166 decreased resazurin
reduction at the concentrations of 10 μM, 50 μM, and 100 μM
(a decrease by 16.15, 28.77, and 26.65%, respectively, com-
pared to the control). In turn, compound Les-5935 at the con-
centration of 10 μM, 50 μM, and 100 μM increased resazurin
reduction by 13.12, 11.68, and 11.75%, respectively, com-
pared to the control (Fig. 2c).
Results
Synthesis of 4-thiazolidinone-based derivatives
The general methods for the synthesis of the target
thiazolidinone-based compounds Les-2194, Les-3640, Les-
5935, and Les-6166 are presented in Fig. 1. Les-2194 was
obtained via N-alkylation of rel-(5aR,11bR)-3,5a,6,11b-
tetrahydro-2H,5H-chromeno[4',3':4,5]thiopyrano[2,3-
d]thiazol-2-one by 2-chloro-N-(3,4-dichlorophenyl)-acetamide.
Les-3640 and Les-6166 were synthesized in a one-pot three-
component reaction involving [2+3]-cyclocondensation of ap-
propriate S,N-binucleophile and chloroacetic acid followed by
the Knoevenagel reaction with an appropriate oxo compound.
In the case of Les-3640, 3-phenyl-5-(dimethylaminophenyl)-1-
thiocarbamoyl-2-pyrazoline was used as the S,N-binucleophile,
and isatin was used as the oxo compound; for the Les-6166
synthesis, 4-hydroxyphenylthiourea and methyl 5-fluoro-3-
formylindole-2-carboxylate were used, respectively. The
chromeno[2,3-d]thiazole Les-5935 was obtained via one-pot
condensation of 4-aminothiazol-2(5H)-one and 2-
hydroxynaphthalene-1-carbaldehyde.
Measurement of caspase-3 activity
To date, thiazolidinone-based compounds have been shown to
initiate the apoptotic process in various cancerous cell lines
(Szychowski et al. 2017a,b, 2019). Caspase-3 activity assay
was performed to measure the effects of Les-2194, Les-3640,
Les-5935, and Les-6166 on the apoptotic process in the 3T3-
L1 cell line.
After the 24-h exposure to 100 nM, 1 μM, 10 μM, 50 μM,
and 100 μM of Les-2194, Les-3640, Les-5935, or Les-6166,
compounds Les-2194 and Les-5935 did not affect caspase-3
activity in the 3T3-L1 cells. Compounds Les-3640 and Les-
6166 at the concentrations of 50 μM and 100 μM caused an
increase in caspase-3 activity (an increase by 44.27 and
79.75%, respectively, for Les-3640 and an increase by 95.09
and 147.44%, respectively, for Les-6166) (Fig. 3a).
After the 48-h exposure to the studied compounds, as in the
case of the 24-h exposure, compounds Les-2194 and Les-
5935 did not affect caspase-3 activity in the 3T3-L1 cells.
Moreover, compounds Les-3640 and Les-6166 at the concen-
trations of 50 μM and 100 μM induced an increase in caspase-
3 activity (an increase by 40.33 and 82.33%, respectively, for
Les-3640 and an increase by 98.26 and 151.47%, respective-
ly, for Les-6166) (Fig. 3b).
Measurement of resazurin reduction
Several thiazolidinone-based compounds have been previous-
ly shown to impact the viability of tumor cells (Szychowski
et al. 2017a,b, 2019). Resazurin reduction experiments were

Naunyn-Schmiedeberg's Arch Pharmacol
Cl
Cl
CHO
Fig. 1 Synthesis of target
thiazolidinone-based compounds.
Les-2194 is described in
Kryshchyshyn et al. (2012), and
Les-3640 is described in
H
O
S
H
S
S
N
H
H
N
Cl
N
H
O
N
Cl
NH
O
O
N
O
O
S
O
H
S
NEt3, AcOH
S
O
Cl
KOH, EtOH
H
Havrylyuk et al. (2012)
Les-2194
H3C
O
H3C
N
O
CH3
N
O
ClCH2COOH
+
Ph
N
H
H3C
HN
N
N
S
O
S
AcONa, AcOH
N
N
NH2
Les-3640
Les-5935
CHO
OH
NH2
Ph
S
S
NH4OH
N
N
NH
O
S
S
S
AcONa, Ac2O, AcOH
O
O
NH2
O
CHO
COOMe
MeOOC
HN
HN
S
F
N
ClCH COOH
+
2
S
OH
N
H
N
H
AcONa, AcOH
Les-6166
F
OH
Similarly, after the 72-h exposure to the studied com-
pounds, Les-2194 and Les-5935 did not affect caspase-3 ac-
tivity in the 3T3-L1 cells. Compound Les-6166 at the concen-
trations of 50 μM and 100 μM increased caspase-3 activity
(an increase by 90.85 and 163.21%, respectively, compared to
the control). Compound Les-3640 caused an increase in
caspase-3 activity at the concentrations of 10 μM, 50 μM,
and 100 μM (an increase by 16.18; 47.36 and 85.06%, respec-
tively, compared to the control) (Fig. 3c).
βNF, Les-2194, and Les-3640 increased the Cyp1a1 mRNA
expression by 1240051.71, 1325.58, and 106.18%, respec-
tively, compared to the control. In turn, Les-5935 decreased
the Cyp1a1 mRNA expression by 56.16%, compared to the
control (Fig. 4b). Interestingly, the expression of Cyp1a2
mRNA was activated only by βNF and Les-2194 (41.22%
compared to the expression induced by βNF); therefore,
βNF was regarded to be the control in this analysis (Fig.
4c). After the βNF and Les-2194 treatment, the expression
of Cyp1b1 mRNA in the 3T3 cell line increased by 105.11
and 33.04%, respectively, compared to the control. In turn,
Les-5935 and Les-6166 decreased the expression of Cyp1b1
mRNA by 23.66 and 27.02%, respectively, compared to the
control (Fig. 4d). No expression of Cyp2b10 mRNA was de-
tected in the 3T3 cell line (Fig. 4e).
Expression of Ahr, Cyp1a1, Cyp1b1, Cyp1a2, and
Cyp2b10 mRNA
AhR and AhR-related genes are involved in the metabolism
and detoxification system of different ligands, such as drugs,
tetrachlorodibenzo-p-dioxin (TCDD), or βNF (Tian et al.
2015). To date, the Les-2194 and Les-3640 compounds have
been shown to exhibit properties of PPARγ receptor agonists
(Szychowski et al. 2017a, b). Therefore, due to crosstalk be-
tween AhR and PPARγ receptors, investigations of the
mRNA expression of Ahr and Ahr-related genes were
performed.
The expression of Ahr, Cyp1a1, Cyp1b1, Cyp1a2, and
Cyp2b10 mRNA was measured after 6 h of exposure to
βNF, Les-2194, Les-3640, Les-5935, or Les-6166. βNF,
Les-2194, and Les-6166 were found to decrease the expres-
sion of Ahr mRNA by 57.97, 17.00, and 25.69% respectively,
compared to the control (Fig. 4a). At the same time interval,
CYP450 activities
Currently, it is well known that mRNA expression does not
fully translate into its functions (Perl et al. 2017). Therefore,
enzymatic activity assays EROD, MROD, and PROD were
carried out to confirm or deny that changes in mRNA expres-
sion affect protein activity.
EROD assay
After 24 and 48 h of exposure of the 3T3-L1 cells to 1 μM
Les-2194, Les-5935, or Les-6166, we observed an increase in

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 2 Effect of 100 nM, 1 μM,
10 μM, 50 μM, or 100 μM of
Les-2194, Les-3640, Les-5935,
or Les-6166 on resazurin reduc-
tion after 24 h (a), 48 h (b), and
72 h (c) of exposure of 3T3-L1
fibroblasts. Control vehicle only
and 1 μM β-naphthoflavone
(βNF) were included in the ex-
periments. Data are expressed as
means SD of three independent
experiments, each of which
consisted of six replicates per
treatment group
EROD activity. An increase by 76.45 and 102.90% compared
to the control was observed in the case of Les-2194. Les-5935
increased the activity by 84.31 and 83.90%, compared to the
control, whereas an 81.29 and 74.30% increase was induced
by Les-6166, compared to control. In turn, Les-3640 in-
creased the EROD activity only after 48 h (by 35.40% com-
pared to the control) (Fig. 5a).
PROD activity, compared to the control. After 48 h, Les-5935
increased the PROD activity by 61.95%, compared to the
control. Les-6266 induced a decrease in the PROD activity
at both time intervals (a decrease by 36.00 and 38.43%, com-
pared to the control) (Fig. 5b).
MROD assay
PROD assay
βNF reduced the MROD activity only after 48 h of exposure
(a decrease by 24.89%, compared to the control). Likewise
previously, Les-2194 increased the MROD activity in the
3T3-L1 cells after 24 and 48 h (an increase by 22.76 and
37.98%, respectively, compared to the control). After 24-h
exposure, Les-5935 increased the MROD activity by
After 24 and 48 h of exposure of the 3T3-L1 cells to 1 μM
Les-2194, the PROD activity was increased by 61.70 and
67.67%, respectively, compared to the control. After 24 h of
exposure to Les-3640, we observed an 18.70% decrease in the

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 3 Effect of 100 nM, 1 μM,
10 μM, 50 μM, or 100 μM of
Les-2194, Les-3640, Les-5935,
or Les-6166 on caspase-3 activity
after 24 h (a), 48 h (b), and 72 h
(c) of exposure of 3T3-L1 fibro-
blasts (a). Control vehicle only
and 1 μM β-naphthoflavone
(βNF) were included in the ex-
periments. Data are expressed as
means SD of three independent
experiments, each of which
consisted of six replicates per
treatment group
34.64%, compared to the control. In turn, Les-3640 and Les-
6166 contributed to a decrease in the MROD activity at both
time intervals, i.e., by 34.10 and 23.55% in the case of Les-
3640 and by 36.02 and 32.83% in the case of Les-6166, com-
pared to the control (Fig. 5c).
highest micromolar concentrations in the 3T3-L1 cells.
Interestingly, compound Les-5935 after 72 h of exposure in
the concentration range from 10 to 100 μM induced an in-
crease in the resazurin reduction assay. Moreover, at all the
time intervals studied, compounds Les-2194 and Les-5935
did not affect caspase-3 activity in the 3T3-L1 cells.
However, compounds Les-3640 and Les-6166 at the concen-
trations of 50 μM and 100 μM contributed to an increase in
caspase-3 activity. Therefore, it can be assumed that the com-
pounds applied in the range 100 nM to 1 μM neither are toxic
nor initiate apoptosis in the normal mouse fibroblast 3T3-L1
cell line. Our previous studies show that Les-2194 and Les-
3640 increased reactive oxygen species (ROS) production in
Discussion
The present experiments showed that, after 24, 48, and 72 h of
exposure to 100 nM, 1 μM, 10 μM, 50 μM, and 100 μM, the
Les-2194, Les-3640, and Les-6166 compounds caused a sig-
nificant decrease in the resazurin reduction assay only at the

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 4 Effect of 1 μM Les-2194, Les-3640, Les-5935, or Les-6166 and
1 μM β-naphthoflavone (βNF) on the mRNA expression of Ahr (a),
Cyp1a1 (b), Cyp1a2 (c), Cyp1b1 (d), and Cyp2b10 (e) genes after 6 h
of exposure of 3T3-L1 fibroblasts. The lack of expression of Cyp2b10
mRNA was confirmed by the use of mouse astrocyte cells as a positive
control; see supplementary material 2 for details. Data are expressed as
means SD of three independent experiments, each of which consisted of
six replicates per treatment group. **P < 0.01 and ***P < 0.001 versus
the control cells
the SCC-15 cell line. Furthermore, both Les-2194 and Les-
3640 decrease cell viability (measured by the resazurin reduc-
tion assay) and increase cell apoptosis (measured as caspase-3
activity) (Szychowski et al. 2017b). Moreover, our study
shows that both Les-2194 and Les-3640 act in a PPARγ-
dependent way (Szychowski et al. 2017a). Resveratrol, which
is known as a PPARγ agonist, has been described to inhibit
AhR-induced cytochrome CYP1A1 enzyme activity and ex-
pression (Ciolino and Yeh 1999). Based on this, the next stage
of our study was to check the impact of 1 μM Les-2194, Les-
3640, Les-5935, or Les-6166 on expression of Cyp1a1,
Cyp1b1, Cyp1a2, and Cyp2b10 mRNA in the 3T3-L1 cell
line. In the experiments, 1 μM βNF, i.e., a known agonist of
the AhR receptor, was used as a positive control. The results
showed that βNF decreased the expression of AhR receptor
mRNA but increased the expression of the AhR-dependent
genes Cyp1a1, Cyp1b1, and Cyp1a2. Unfortunately, no
expression of Cyp2b10 was detected in the experiments. As
shown by literature data, the cyp2b10 expression in the 3T3
cell line is very low and decreases during cell differentiation
(Roth et al. 2008; Mesnier et al. 2015; Kong et al. 2018).
Therefore, this may be the reason why we did not observe
Cyp2b10 mRNA expression in the 3T3-L1 cell line.
They demonstrated that Les-2194 affected the expression
of AhR, Cyp1a1, Cyp1b1, and Cyp1a2 mRNA in a similar
way to βNF. However, Les-3640 increased only the Cyp1a1
mRNA expression, while Les-5935 decreased the mRNA ex-
pression of this enzyme. The Les-6166 compound decreased
only the expression of AhR receptor mRNA. It is important to
underline that only βNF and Les-2194 induced the expression
of Cyp1a2 mRNA in the 3T3-L1 cell line.
Our previous study demonstrated that Les-2194 and Les-
3640 act in a PPARγ-dependent way in SCC-15 cells and
similar to known PPARγ agonists (rosiglitazone and

Naunyn-Schmiedeberg's Arch Pharmacol
CYP1A1, CYP1A2, and CYP1B1 (Beischlag et al. 2008), it
can be assumed that PPARγ agonists should affect the expres-
sion of these genes as well.
On the other hand, it has been reported that the PPARα ago-
nist ciprofibrate can inhibit the expression of Cyp1a1 and Cyp1a2
mRNA and protein by interfering with AhR-dependent signaling
in rat liver (Gallagher et al. 1995; Shaban et al. 2004). Such data
suggest that Les-5935 and Les-6166 may be at least weak
PPARα agonists. The final part of our study was to evaluate
the effect of Les-2194, Les-3640, Les-5935, or Les-6166 on the
activity of Cyp1a1, Cyp1b1, Cyp1a2, and Cyp2b10 in the 3T3-
L1 cell line. It has been shown in various studies that EROD
activities can be related to both CYP1A1 and CYP1A2 isoen-
zymes (Zamaratskaia and Zlabek 2009). However, some authors
have suggested that the EROD assay measures CYP1B1 activity
as well (Deeni et al. 2013). The MROD assay has been applied
for measurement of the CYP1A2 activity (Conaway et al. 1996),
whereas the PROD assay has been used to measure the CYP2B
activity (Séïde et al. 2016). Our data showed an increase in the
EROD activity after 24 and 48 h in the 3T3-L1 cells exposed to
1 μM Les-2194, Les-5935, or Les-6166; however, Les-3640
increased the EROD activity only after 48 h. In the case of Les-
2194, the data on the Cyp1a1, Cyp1a1, and Cyp1b1 mRNA
expression and activity measured by the EROD assay are consis-
tent. The increase in the EROD activity induced by Les-6166
may be explained by the increase in the Cyp1a1 mRNA expres-
sion. Unfortunately, in the case Les-5935 and Les-3640, the in-
creased EROD activity is not supported by the expression. This
phenomenon may be a feedback effect where high enzyme ac-
tivity inhibits its expression (Zanger and Schwab 2013).
Our data showed that only Les-2194 and Les-5935 in-
creased the MROD activity, while βNF, Les-3640, and Les-
6166 decreased the activity in the 3T3-L1 cells. To date,
MROD activity has been correlated with CYP1A2 expression
(Conaway et al. 1996). However, in our experiments, we ob-
served increased expression of Cyp1a2 mRNA only after the
treatment of the cells with Les-2194. It cannot be excluded
that Les-5935 affects only the activity of Cyp1a2 without
significant induction of mRNA expression.
Our data showed that Les-2194 and Les-5935 contributed
to an increase in the PROD activity, while the Les-6266 com-
pound decreased it at both time intervals. Interestingly, the
experiments showed no expression of Cyp2b10 in 3T3-L1.
A single CYP2B gene, i.e., CYP2B6, has been described in
humans. In turn, mice have five Cyp2b genes: Cyp2b9,
Cyp2b10, Cyp2b13, Cyp2b19, and Cyp2b23 (Nelson et al.
2004). In mice, the Cyp2b9, Cyp2b10, and Cyp2b13 forms
are primarily expressed in the liver (Finger et al.
2011). The most important is Cyp2b10, and its expres-
sion is regulated by CAR (Honkakoski et al. 1998;
Wang et al. 2003; Kretschmer and Baldwin 2005).
Therefore, it cannot be excluded that the Les- com-
pounds affect other than Cyp2b10 mRNA expression.
Fig. 5 Effect of 1 μM Les-2194, Les-3640, Les-5935, or Les-6166 and
1 μM β-naphthoflavone (βNF) on EROD (a), MROD (b), and PROD (c)
activities after 24 and 48 h of exposure of 3T3-L1 fibroblasts. Data are
expressed as means SD of three independent experiments, each of
which consisted of six replicates per treatment group. **P < 0.01 and
***P < 0.001 versus the control cells
pioglitazone) (Szychowski et al. 2017a). Moreover, it was
described that PPARγ agonists could activate the expression
of CYP1A1 in mouse and human cells in vitro (Kim et al.
2008; Wójtowicz et al. 2017), which is probably possible
due to the crosstalk of the PPARγ and AhR receptor pathways
(Wójtowicz et al. 2017). Unfortunately, the data on the role of
PPARγ agonists in the expression of CYP1A1, CYP1B1, or
CYP2B1 mRNA (in mouse Cyp2b10) are very limited.
However, since AhR control the expression of, e.g.,

Naunyn-Schmiedeberg's Arch Pharmacol
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Recently, it has been reported that different thiazolidinone
derivatives exerted CYP inhibition activity in several isoforms
(CYP1A2, CYP3A4, and CYP2C19) at 10 μM, but there was
no CYP inhibition activity of CYP2C9 and CYP2D6 in hu-
man and rat liver microsomes (Ashraf et al. 2020).
Unfortunately, the data on the CYP expression and/or activity
after thiazolidinone treatment are limited.
It is well described that CYP1A1, CYP1A2, and CYP1B1 play
an important role in the detoxication of environmental carcinogens
and in the metabolic activation of dietary compounds with cancer
preventive activity (Zanger and Schwab 2013; Alzahrani and
Rajendran 2020). Moreover, the contribution of CYPs to cancer
progression or prevention may depend on the balance of
procarcinogen activation/detoxication and extrahepatic drug me-
tabolism (Androutsopoulos et al. 2009). Similarly, CYP2B6 is
involved in drug clearance (Zanger and Schwab 2013).
References
Alzahrani AM, Rajendran P (2020) The multifarious link between cyto-
chrome P450s and cancer. Oxidative Med Cell Longev 2020:1–18.
https://doi.org/10.1155/2020/3028387
Androutsopoulos VP, Tsatsakis AM, Spandidos DA (2009) Cytochrome
P450 CYP1A1: wider roles in cancer progression and prevention.
BMC Cancer 9:187. https://doi.org/10.1186/1471-2407-9-187
Angus WG, Larsen MC, Jefcoate CR (1999) Expression of CYP1A1 and
CYP1B1 depends on cell-specific factors in human breast cancer
cell lines: role of estrogen receptor status. Carcinogenesis 20:947–
955
Asati V, Mahapatra DK, Bharti SK (2014) Thiazolidine-2,4-diones as
multi-targeted scaffold in medicinal chemistry: potential anticancer
agents. Eur J Med Chem 87:814–833. https://doi.org/10.1016/j.
ejmech.2014.10.025
Ashraf S, Saeed A, Moon SH, Flörke U, Kim SH, Ashraf Z, Yaseen M,
Latif M (2020) Design, synthesis and biological evaluation of 2-
(naphthoyl) iminothiazolidin-4-ones as potential anticancer agents.
ChemistrySelect 5:3965–3970. https://doi.org/10.1002/slct.
202000579
Bardot O, Aldridge TC, Latruffe N, Green S (1993) PPAR-RXR hetero-
dimer activates a peroxisome proliferator response element up-
stream of the bifunctional enzyme gene. Biochem Biophys Res
Commun 192:37–45. https://doi.org/10.1006/bbrc.1993.1378
Barouki R (2013) Les xénobiotiques accumulés dans le tissu adipeux
peuvent-ils contribuer à un risque carcinogène ? Ann Endocrinol
(Paris) 74:154–155. https://doi.org/10.1016/j.ando.2013.03.022
Beischlag TV, Luis Morales J, Hollingshead BD, Perdew GH (2008) The
aryl hydrocarbon receptor complex and the control of gene expres-
sion. Crit Rev Eukaryot Gene Expr 18:207–250. https://doi.org/10.
2964/jsik.kuni0223
Conclusions
Our study is the first to explore the role of the Les-2194, Les-
3640, Les-5935, and Les-6166 compounds in the 3T3-L1 cell
line. The experiments show that compounds Les-3640 and Les-
6166 at the 50 and 100 μM concentrations activate caspase-3 in
3T3-L1 cells and can be toxic in these concentrations.
Moreover, the study has shown that only Les-2194 acts simi-
larly to our positive control βNF by affecting the mRNA ex-
pression of AhR, Cyp1a1, Cyp1a2, and Cyp1b1 in the 3T3-L1
cell line. Interestingly, both Les-2194 and Les-5935 increased
the activity of EROD, MROD, and PROD. Given the important
role of the AhR receptor in the detoxification system and its
crosstalk with the PPARγ receptor, which is crucial in adipo-
cyte differentiation, more research in this field is needed to
explain the mechanism of the Les-2194, Les-3640, Les-5935,
or Les-6166 action on drug metabolism or the detoxification
system and adipocyte differentiation.
Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s00210-020-02025-7.
Berger JP, Akiyama TE, Meinke PT (2005) PPARs: therapeutic targets
for metabolic disease. Trends Pharmacol Sci 26:244–251. https://
doi.org/10.1016/j.tips.2005.03.003
Ciolino HP, Yeh GC (1999) Inhibition of aryl hydrocarbon-induced cy-
tochrome P-450 1A1 enzyme activity and CYP1A1 expression by
resveratrol. Mol Pharmacol 56:760–767
Authors’ contributions K.S. designed the research. A.K-L, D.K., and R.L.
synthesized the compound used in the experiments. K.S., B.S., and J.G.
performed the cell culture research. K.S. and R.L. analyzed the data and
wrote the manuscript. J.G. obtained financial support for the project. All
authors read and approved the manuscript. The authors declare that all data
were generated in-house and that no paper mill was used.
Conaway CC, Jiao D, Chung FL (1996) Inhibition of rat liver cytochrome
P450 isozymes by isothiocyanates and their conjugates: a structure-
activity relationship study. Carcinogenesis 17:2423–2427. https://
doi.org/10.1093/carcin/17.11.2423
Funding This work was supported by statutory funds from the University
of Information Technology and Management in Rzeszow, Poland (DS
503-07-01-21).
Deeni YY, Ibbotson SH, Woods JA, Wolf CR, Smith G (2013)
Cytochrome P450 CYP1B1 interacts with 8-methoxypsoralen (8-
MOP) and influences psoralen-ultraviolet A (PUVA) sensitivity.
PLoS One 8:2–8. https://doi.org/10.1371/journal.pone.0075494
Di Bello D, Vaccaro E, Longo V et al (2007) Presence and inducibility by
β-naphthoflavone of CYP1A1, CYP1B1 and phase II enzymes in
Compliance with ethical standards
Conflict of interests The authors declare that they have no conflict of
interests.

Naunyn-Schmiedeberg's Arch Pharmacol
Trematomus bernacchii, an Antarctic fish. Aquat Toxicol 84:19–26.
https://doi.org/10.1016/j.aquatox.2007.05.010
Lea MA, Sura M, Desbordes C (2004) Inhibition of cell proliferation by
potential peroxisome proliferator-activated receptor (PPAR) gamma
agonists and antagonists. Anticancer Res 24:2765–2771
Lesyk R (2020a) Drug design: 4-thiazolidinones applications. Part 1.
Synthetic routes to the drug-like molecules. J Med Sci 89:e406.
doi: 10.20883/medical.406
Lesyk R (2020b) Drug design: 4-thiazolidinones applications. Part 2.
Pharmacological profiles. J Med Sci e407.
Li F, Zhu W, Gonzalez FJ (2017) Potential role of CYP1B1 in the devel-
opment and treatment of metabolic diseases. Pharmacol Ther 178:
18–30. https://doi.org/10.1016/j.pharmthera.2017.03.007
Ellero S, Chakhtoura G, Barreau C, Langouët S, Benelli C, Penicaud L,
Beaune P, de Waziers I (2010) Xenobiotic-metabolizing cyto-
chromes P450 in human white adipose tissue: expression and induc-
tion. Drug Metab Dispos 38:679–686. https://doi.org/10.1124/dmd.
109.029249
Finger JH, Smith CM, Hayamizu TF, McCright IJ, Eppig JT, Kadin JA,
Richardson JE, Ringwald M (2011) The mouse gene expression
database (GXD): 2011 update. Nucleic Acids Res 39:835–841.
https://doi.org/10.1093/nar/gkq1132
Gallagher EP, Buetler TM, Stapleton PL, Wang CH, Stahl DL, Eaton DL
(1995) The effects of diquat and ciprofibrate on mRNA expression
and catalytic activities of hepatic xenobiotic metabolizing and anti-
oxidant enzymes in rat liver. Toxicol Appl Pharmacol 134:81–91.
https://doi.org/10.1006/taap.1995.1171
Havrylyuk D, Zimenkovsky B, Vasylenko O, Gzella A, Lesyk R (2012)
Synthesis of new 4-thiazolidinone-, pyrazoline-, and isatin-based
conjugates with promising antitumor activity. J Med Chem 55:
8630–8641. https://doi.org/10.1021/jm300789g
Lu W, Che P, Zhang Y, Li H, Zou S, Zhu J, Deng J, Shen X, Jiang H, Li J,
Huang J (2011) HL005 - a new selective PPARγ antagonist specif-
ically inhibits the proliferation of MCF-7. J Steroid Biochem Mol
Biol 124:112–120. https://doi.org/10.1016/j.jsbmb.2011.01.019
Malik KU, Jennings BL, Yaghini FA, Sahan-Firat S, Song CY, Estes
AM, Fang XR (2012) Contribution of cytochrome P450 1B1 to
hypertension and associated pathophysiology: a novel target for an-
tihypertensive agents. Prostaglandins Other Lipid Mediat 98:69–74.
https://doi.org/10.1016/j.prostaglandins.2011.12.003
He Q, Huang C, Zhao L, Feng J, Shi Q, Wang D, Wang S (2013) α-
Naphthoflavone inhibits 3T3-L1 pre-adipocytes differentiation via
modulating p38MAPK signaling. Int J Clin Exp Pathol 6:168–178
Hessel-Pras S, Ehlers A, Braeuning A, Lampen A (2018) The aryl hy-
drocarbon receptor and retinoid receptors cross-talk at the Cyp1A1
promoter in vitro. EXCLI J 17:246–256. https://doi.org/10.17179/
excli2018-1147
Honkakoski P, Zelko I, Sueyoshi T, Negishi M (1998) The nuclear or-
phan receptor CAR-retinoid X receptor heterodimer activates the
phenobarbital-responsive enhancer module of the CYP2B gene.
Mol Cell Biol 18:5652–5658. https://doi.org/10.1128/mcb.18.10.
5652
Mesnier A, Champion S, Louis L, Sauzet C, May P, Portugal H,
Benbrahim K, Abraldes J, Alessi MC, Amiot-Carlin MJ, Peiretti
F, Piccerelle P, Nalbone G, Villard PH (2015) The transcriptional
effects of PCB118 and PCB153 on the liver, adipose tissue, muscle
and colon of mice: highlighting of Glut4 and Lipin1 as main target
genes for PCB induced metabolic disorders. PLoS One 10:0–17.
doi: https://doi.org/10.1371/journal.pone.0128847, e0128847
Nebert DW, Dalton TP (2006) The role of cytochrome P450 enzymes in
endogenous signalling pathways and environmental carcinogenesis.
Nat Rev Cancer 6:947–960. https://doi.org/10.1038/nrc2015
Nebert DW, Roe AL, Dieter MZ, Solis WA, Yang Y, Dalton TP (2000)
Role of the aromatic hydrocarbon receptor and [Ah] gene battery in
the oxidative stress response, cell cycle control, and apoptosis.
Biochem Pharmacol 59:65–85. https://doi.org/10.1016/S0006-
2952(99)00310-X
Jackson E, Shoemaker R, Larian N, Cassis L (2017) Adipose tissue as a
site of toxin accumulation. Compr Physiol 7:1085–1135. https://doi.
org/10.1002/cphy.c160038
Kaminskyy D, Kryshchyshyn A, Lesyk R (2017a) 5-Ene-4-
thiazolidinones – an efficient tool in medicinal chemistry. Eur J
Med Chem 140:542–594. https://doi.org/10.1016/j.ejmech.2017.
09.031
Kaminskyy D, Kryshchyshyn A, Lesyk R (2017b) Recent developments
with rhodanine as a scaffold for drug discovery. Expert Opin Drug
Discovery 12:1233–1252. https://doi.org/10.1080/17460441.2017.
1388370
Kennedy SW, Lorenzen A, James CA, Collins BT (1993)
Ethoxyresorufin-O-deethylase and porphyrin analysis in chicken
embryo hepatocyte cultures with a fluorescence multiwell plate
reader. Anal Biochem 211:102–112. https://doi.org/10.1006/abio.
1993.1239
Kim HG, Han EH, Jeong HG (2008) Effect of troglitazone on CYP1A1
induction. Toxicology 246:166–171. https://doi.org/10.1016/j.tox.
2008.01.003
Kong X, Yao T, Zhou P, et al (2018) Brown adipose tissue controls
skeletal muscle function via the secretion of myostatin. Cell Metab
28:631-643.e3. doi: https://doi.org/10.1016/j.cmet.2018.07.004
Kretschmer XC, Baldwin WS (2005) CAR and PXR: Xenosensors of
endocrine disrupters? Chem Biol Interact 155:111–128. https://doi.
org/10.1016/j.cbi.2005.06.003
Kryshchyshyn A, Atamanyuk D, Lesyk R (2012) Fused thiopyrano[2,3-
d]thiazole derivatives as potential anticancer agents. Sci Pharm 80:
509–529. https://doi.org/10.3797/scipharm.1204-02
Kryshchyshyn A, Roman O, Lozynskyi A, Lesyk R (2018)
Thiopyrano[2,3-d]thiazoles as new efficient scaffolds in medicinal
chemistry. Sci Pharm 86:26. https://doi.org/10.3390/
scipharm86020026
Nelson DR, Zeldin DC, Hoffman SMG, Maltais LJ, Wain HM, Nebert
DW (2004) Comparison of cytochrome P450 (CYP) genes from the
mouse and human genomes, including nomenclature recommenda-
tions for genes, pseudogenes and alternative-splice variants.
Pharmacogenetics 14:1–18. https://doi.org/10.1097/01.fpc.
0000054151.92680.31
Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK,
Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA,
Munday NA, Raju SM, Smulson ME, Yamin TT, Yu VL, Miller
DK (1995) Identification and inhibition of the ICE/CED-3 protease
necessary for mammalian apoptosis. Nature 376:37–43. https://doi.
org/10.1038/376037a0
Perl K, Ushakov K, Pozniak Y, Yizhar-Barnea O, Bhonker Y,
Shivatzki S, Geiger T, Avraham KB, Shamir R (2017)
Reduced changes in protein compared to mRNA levels across
non-proliferating tissues. BMC Genomics 18:305. https://doi.
org/10.1186/s12864-017-3683-9
Rampersad SN (2012) Multiple applications of Alamar Blue as an indi-
cator of metabolic function and cellular health in cell viability bio-
assays. Sensors 12:12347–12360. https://doi.org/10.3390/
s120912347
Roth A, Looser R, Kaufmann M, Blättler SM, Rencurel F, Huang W,
Moore DD, Meyer UA (2008) Regulatory cross-talk between drug
metabolism and lipid homeostasis: constitutive androstane receptor
and pregnane X receptor increase Insig-1 expression. Mol
Pharmacol 73:1282–1289. https://doi.org/10.1124/mol.107.041012
Scatena R, Bottoni P, Giardina B (2008) Mitochondria, PPARs, and
cancer: is receptor-independent action of PPAR agonists a key?
PPAR Res 2008:1–10. https://doi.org/10.1155/2008/256251

Naunyn-Schmiedeberg's Arch Pharmacol
Séïde M, Marion M, Mateescu MA, Averill-Bates DA (2016) The fungi-
cide thiabendazole causes apoptosis in rat hepatocytes. Toxicol in
Vitro 32:232–239. https://doi.org/10.1016/j.tiv.2015.12.018
Shaban Z, El-Shazly S, Ishizuka M et al (2004) PPARgamma-dependent
modulation of hepatic CYP1A by clofibric acid in rats. Arch Toxicol
78. https://doi.org/10.1007/s00204-004-0569-9
Shoemaker RH (2006) The NCI60 human tumour cell line anticancer
drug screen. Nat Rev Cancer 6:813–823. https://doi.org/10.1038/
nrc1951
Song CY, Ghafoor K, Ghafoor HU, Khan NS, Thirunavukkarasu S,
Jennings BL, Estes AM, Zaidi S, Bridges D, Tso P, Gonzalez FJ,
Malik KU (2016) Cytochrome P450 1B1 contributes to the devel-
opment of atherosclerosis and hypertension in apolipoprotein E–
deficient mice. Hypertension 67:206–213. https://doi.org/10.1161/
HYPERTENSIONAHA.115.06427
Spink DC, Spink BC, Cao JQ et al (1998) Differential expression of
CYP1A1 and CYP1B1 in human breast epithelial cells and breast
tumor cells. Carcinogenesis 19:291–298. https://doi.org/10.1093/
carcin/19.2.291
Tripathi AC, Gupta SJ, Fatima GN, et al (2014) 4-Thiazolidinones: the
advances continue…. Eur J Med Chem 72:52–77. https://doi.org/10.
1016/j.ejmech.2013.11.017
Wang H, Faucette S, Sueyoshi T, Moore R, Ferguson S, Negishi M,
LeCluyse EL (2003) A novel distal enhancer module regulated by
pregnane X receptor/constitutive androstane receptor is essential for
the maximal induction of CYP2B6 gene expression. J Biol Chem
278:14146–14152. https://doi.org/10.1074/jbc.M212482200
Wojtowicz AK, Szychowski KA, Kajta M (2014) PPAR-γ agonist
GW1929 but not antagonist GW9662 reduces TBBPA-induced neu-
rotoxicity in primary neocortical cells. Neurotox Res 25:311–322.
https://doi.org/10.1007/s12640-013-9434-z
Wójtowicz AK, Szychowski KA, Wnuk A, Kajta M (2017) Dibutyl
phthalate (DBP)-induced apoptosis and neurotoxicity are mediated
via the aryl hydrocarbon receptor (AhR) but not by estrogen receptor
alpha (ERα), estrogen receptor beta (ERβ), or peroxisome
proliferator-activated receptor gamma (PPARγ) in mouse C.
Neurotox Res 31:77–89. https://doi.org/10.1007/s12640-016-9665-
x
Szychowski KA, Leja ML, Kaminskyy DV, Kryshchyshyn AP, Binduga
UE, Pinyazhko OR, Lesyk RB, Tobiasz J, Gmiński J (2017a)
Anticancer properties of 4-thiazolidinone derivatives depend on per-
oxisome proliferator-activated receptor gamma (PPARγ). Eur J
Med Chem 141:162–168. https://doi.org/10.1016/j.ejmech.2017.
09.071
Szychowski KA, Leja ML, Kaminskyy DV, Binduga UE, Pinyazhko
OR, Lesyk RB, Gmiński J (2017b) Study of novel anticancer 4-
thiazolidinone derivatives. Chem Biol Interact 262:46–56. https://
doi.org/10.1016/j.cbi.2016.12.008
Szychowski KA, Kaminskyy DV, Leja ML, Kryshchyshyn AP, Lesyk
RB, Tobiasz J, Wnuk M, Pomianek T, Gmiński J (2019) Anticancer
properties of 5Z-(4-fluorobenzylidene)-2-(4-hydroxyphenylamino)-
thiazol-4-one. Sci Rep 9:10609. https://doi.org/10.1038/s41598-
019-47177-6
Tachibana K, Yamasaki D, Ishimoto K, Doi T (2008) The role of PPARs
in cancer. PPAR Res 2008:1–15. https://doi.org/10.1155/2008/
102737
Wright MB, Bortolini M, Tadayyon M, Bopst M (2014) Minireview:
challenges and opportunities in development of PPAR agonists.
Mol Endocrinol 28:1756–1768. https://doi.org/10.1210/me.2013-
1427
Zamaratskaia G, Zlabek V (2009) EROD and MROD as markers of
cytochrome P450 1A activities in hepatic microsomes from entire
and castrated male pigs. Sensors 9:2134–2147. https://doi.org/10.
3390/s90302134
Zanger UM, Schwab M (2013) Cytochrome P450 enzymes in drug me-
tabolism: regulation of gene expression, enzyme activities, and im-
pact of genetic variation. Pharmacol Ther 138:103–141. https://doi.
org/10.1016/j.pharmthera.2012.12.007
Zhou L, Zhong Y, Xue MZ, Kuang D, Cao XW, Zhao ZJ, Li HL, Xu YF,
Wang R (2015) Design, synthesis and evaluation of PPAR gamma
binding activity of 2-thioxo-4-thiazolidinone derivatives. Chin
Chem Lett 26:63–68. https://doi.org/10.1016/j.cclet.2014.10.008
Tian J, Feng Y, Fu H, Xie HQ, Jiang JX, Zhao B (2015) The aryl hydro-
carbon receptor: a key bridging molecule of external and internal
chemical signals. Environ Sci Technol 49:9518–9531. https://doi.
org/10.1021/acs.est.5b00385
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