Selective cytotoxic and genotoxic activities of 5-(2-bromo-5-methoxybenzylidene)-thiazolidine-2,4-dione against NCI-H292 human lung carcinoma cells

Article abstract of DOI:10.1016/j.pharep.2017.11.008

Background: Thiazolidine-2,4-dione ring system is used as a pharmacophore to build various heterocyclic compounds aimed to interact with biological targets. In the present study, benzylidene-2,4-thiazolidinedione derivatives (compounds 2–5) were synthesized and screened against cancer cell lines and the genotoxicity and cytotoxicity of the most active compound (5) was investigated on normal and lung cancer cell line. Methods: For in vitro cytotoxic screening, the MTT assay was used for HL60 and K562 (leukemia), MCF-7 (breast adenocarcinoma), HT29 (colon adenocarcinoma), HEp-2 (cervix carcinoma) and NCI-H292 (lung carcinoma) tumor cell lines and Alamar-blue assay was used for non-tumor cells (PBMC, human peripheral blood mononuclear cells) were used. Cell morphology was visualized after Giemsa-May-Grunwald staining. DNA content, phosphatidylserine externalization and mitochondrial depolarization were measured by flow cytometry. Genotoxicity was assessed by Comet assay. Results: 5-(2-Bromo-5-methoxybenzylidene)-thiazolidine-2,4-dione (5) presented the most potent cytotoxicity, especially against NCI-H292 lung cancer cell line, with IC₅₀ value of 1.26 μg/mL after 72 h incubation. None of the compounds were cytotoxic to PBMC. After 48 h incubation, externalization of phosphatidylserine, mitochondrial depolarization, internucleosomal DNA fragmentation and morphological alterations consistent with apoptosis were observed in NCI-H292 cells treated with compound (5). In addition, compound (5) also induced genotoxicity in NCI-H292 cells (2.8-fold increase in damage index compared to the negative control), but not in PBMC. Conclusion: Compound 5 presented selective cytotoxic and genotoxic activity against pulmonary carcinoma (NCI-H292 cells).

Full text of DOI:10.1016/j.pharep.2017.11.008

Accepted Manuscript
Title: Selective cytotoxic and genotoxic activities of
5
-(2-Bromo-5-methoxybenzylidene)-thiazolidine-2,4-dione
against NCI-H292 human lung carcinoma cells
Authors: Maria do D. Rodrigues, Priscila B.G.S. Santiago,
Karla M.R. Marques, Val e´ ria R.A. Pereira, Maria C.A.B. de
Castro, Jeanne C.L.L. Cantalice, Teresinha G. da Silva,
M oˆ nica L. Adam, Silene C. do Nascimento, Julianna F.C. de
Albuquerque, Gardenia C.G. Militao
PII:
S1734-1140(17)30061-0
DOI:
Reference:
https://doi.org/10.1016/j.pharep.2017.11.008
PHAREP 818
To appear in:
Received date:
Revised date:
Accepted date:
26-1-2017
29-9-2017
20-11-2017
Please cite this article as: Maria do D.Rodrigues, Priscila B.G.S.Santiago, Karla
M.R.Marques, Val e´ ria R.A.Pereira, Maria C.A.B.de Castro, Jeanne C.L.L.Cantalice,
Teresinha G.da Silva, M oˆ nica L.Adam, Silene C.do Nascimento, Julianna F.C.de
Albuquerque, Gardenia C.G.Militao, Selective cytotoxic and genotoxic activities of
5-(2-Bromo-5-methoxybenzylidene)-thiazolidine-2,4-dione against NCI-H292 human
lung carcinoma cells (2010), https://doi.org/10.1016/j.pharep.2017.11.008
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Selective cytotoxic and genotoxic activities of 5-(2-Bromo-5-methoxybenzylidene)-
thiazolidine-2,4-dione against NCI-H292 human lung carcinoma cells
a
a
a
Maria do D. Rodrigues , Priscila B. G. S. Santiago , Karla M. R. Marques , Valéria R.
b
bc
a
A. Pereira , Maria C. A. B. de Castro , Jeanne C. L. L. Cantalice , Teresinha G. da
a
d
a
a
Silva , Mônica L. Adam , Silene C. do Nascimento , Julianna F. C. de Albuquerque ,
Gardenia C. G. Militaoe*
aDepartment of Antibiotics, Federal University of Pernambuco, 50670-901, Recife, PE,
Brasil
b
Department of Immunology, Oswaldo Cruz Foundation, Aggeu Magalhães Research
Center, Campus da UFPE, 50670-420 - Recife, PE – Brasil
c
Parasitology Laboratory, Federal University of Pernambuco; Academic Center of
Vitoria, Pernambuco, Brazil
d
Department of Biological Sciences, Federal University of Pernambuco; Academic
Center of Vitoria, Pernambuco, Brazil
e
Department of Physiology and Pharmacology, Federal University of Pernambuco,
5
0670-901, Recife, PE, Brasil
*Corresponding author: Gardenia Carmen Gadelha Militao, Physiology and
Pharmacology Department. Federal University of Pernambuco, Rua Prof. Moraes Rego
S/N. Cidade Universitária CEP:50.670-901, Recife, PE, Brazil. Phone +55 31 81 2126-
8
531, E-mail address: gcgadelha@yahoo.com.br
ABSTRACT
Background: Thiazolidine-2,4-dione ring system is used as a pharmacophore to build
various heterocyclic compounds aimed to interact with biological targets. In the present
study, benzylidene-2,4-thiazolidinedione derivatives (compounds 2 to 5) were
synthesized and screened against cancer cell lines and the genotoxicity and cytotoxicity
of the most active compound (5) was investigated on normal and lung cancer cell line.
Methods: For in vitro cytotoxic screening, the MTT assay was used for HL60 and K562
(
(
leukemia), MCF-7 (breast adenocarcinoma), HT29 (colon adenocarcinoma), HEp-2
cervix carcinoma) and NCI-H292 (lung carcinoma) tumor cell lines and Alamar-blue
assay was used for non-tumor cells (PBMC, human peripheral blood mononuclear cells)
were used. Cell morphology was visualized after Giemsa-May-Grunwald staining. DNA
content, phosphatidylserine externalization and mitochondrial depolarization were
measured by flow cytometry. Genotoxicity was assessed by Comet assay.
Results: 5-(2-Bromo-5-methoxybenzylidene)-thiazolidine-2,4-dione (5) presented the
most potent cytotoxicity, especially against NCI-H292 lung cancer cell line, with IC50
value of 1.26 µg/mL after 72 h incubation. None of the compounds were cytotoxic to

PBMC. After 48 h incubation, externalization of phosphatidylserine, mitochondrial
depolarization, internucleosomal DNA fragmentation and morphological alterations
consistent with apoptosis were observed in NCI-H292 cells treated with compound (5).
In addition, compound (5) also induced genotoxicity in NCI-H292 cells (2.8-fold
increase in damage index compared to the negative control), but not in PBMC.
Conclusion: Compound 5 presented selective cytotoxic and genotoxic activity against
pulmonary carcinoma (NCI-H292 cells)
Keywords: Cytotoxicity, genotoxicity, 5-benzylidene-2,4-thiazolidinedione, lung
cancer
Introduction
Cancer is one of the leading causes of mortality worldwide characterized by
sustained chronic proliferation of immortal cells, evasion of growth suppressors’
programs, resistance to cell death, development of angiogenesis, tissue invasion and
metastasis [1]. The most common sites of cancers diagnosed among men are lung,
prostate, colorectal, stomach and liver. Meanwhile, most common sites of cancer in
women are breast, colorectal, lung, cervix and stomach. The total number of cancer
related deaths worldwide raised from 8.2 million in 2012 to 8.8 million in 2015 and
lung cancers caused approximately 1.69 million deaths in 2015 [2–4].
Thiazolidine-2,4-dione ring (Figure 1) system exhibits a broad spectrum of
biological activities, such as hypoglycemic [5,6], anti-mycobacterial [7], anti-
inflammatory [8], antileishmanial [9], antibiotics [7,10] and antitumor [11]. Much
attention has been given to the construction of new derivatives of thiazolidinediones
with anticancer activity [12–14].
Heterocyclic molecules belonging to the thiazolidinedione class represents a new
alternative in the search for drugs with antineoplastic potential. Thiazolidinediones
exhibit antitumor effects through both PPARγ-dependent and -independent mechanisms
[15,16]. The role of PPARγ (peroxisome proliferator-activated receptors) in tumor cells
has been extensively investigated and treatment with PPARγ agonists exert biological
effects such as cell growth control, motility, differentiation and apoptosis [16,17]
PPARγ-independent mechanism of tumor cell death induced by thiazolidinediones
treatments includes proteasome-dependent degradation of cyclins (D1 and D3) with
consequent cell cycle blockage at G -S transition, induction of cellular acidosis through
1
+
+
inhibition of the Na /H exchanger and release of apoptotic factors from the
mitochondria due to the production of reactive oxygen species (ROS) [15].
Among many thiazolidinediones derivatives with distinct biological activities, 5-
benzylidene-2,4-thiazolidinedione and its analogues presented cytotoxic activity against
a variety of different cell lines: compound 2-[4-[(2,4-Dioxothiazolidin-5-ylidene)
methyl] phenoxy]-N- [3-(trifluromethyl) phenyl] acetamide was active against K562
leukemia, MCF-7 (breast cancer), PC3 (Prostate cancer), KB (Nasopharyngeal cancer)
and GURAV (Oral cancer) [18]; 5-(4-(2-(piperidin-1-
yl)ethoxy)benzylidene)thiazolidine-2,4-dione was active against a panel of 60 human
tumor cell [19]. In fact, a wide variety of substituents in the thiazolidine-2,4-dione

(TZD) nucleus lead to a huge diversity of compounds targeting different signaling
pathways related to cell proliferation or cell death [11].
One of the most important characteristic for a new molecule with action against
cancer is its selectivity toward tumor cells, since many anticancer drugs cause diverse
cytotoxic and genotoxic damages also in normal cells. Although there are many reports
indicating a wide range of biological activities for compounds with a thiazolidine-2,4-
dione nucleus, genotoxicity studies in normal cells are still insufficient. In this context,
this study aimed to evaluate in vitro antitumor effects of 5-benzylidene-2,4-
thiazolidinedione derivatives, as well as to investigate the possible mechanisms of
action, and genotoxic outcomes of these compounds on human normal and tumor cells.
Material and Methods
Synthesis of thiazolidine-2,4-dione derivatives compounds
The synthesis methodology of compounds 1 to 5 was already described [9].
Briefly, the thiazolidine-2,4-dione (1) was obtained by the method already described
[20,21]. This reaction occurs by condensation of monochloroacetic acid and thiourea in
an aqueous medium under reflux for 24 h. The series of thiazolidine-2,4-dione
derivatives were originated by changes in the 5 position of the heterocyclic ring (Fig. 1).
The compounds5-(2-bromo-6-fluorobenzylidene)-thiazolidine-2,4-dione (2), 5-(2-
Hydroxy-3-bromo-5-chlorobenzylidene)-thiazolidine-2,4-dione (3), 5-(2-Hydroxy-5-
clorobenzylidene)-thiazolidine-2,4-dione (4) e 5-(2-bromo-5-methoxybenzylidene)-
thiazolidine-2,4-dione (5) were synthesized according to protocol described previously
-
3
[
9] (Fig. 2). A solution of thiazolidine-2,4-dione (0.2 g, 1.70 x 10 mol) in ethanol, (7.0
-3
mL) containing piperidine (2 drops) and aromatic aldehyde (0.184 g, 2.25 x 10 mol)
was heated (70 C), under stirring, for 5-9 h. Afterward, the product was cooled in an ice
o
bath, filtered and recrystallized with an appropriate solvent. The resulting precipitate
was filtered off and recrystallized from acetic acid to give the compounds (2-5). All
compounds were identified by IR spectroscopy method, NMR and HRMS
(
Supplementary material). The yields obtained ranged from 73 to 79%.
Cell lines
The cell lines NCI-H292 (Mucoepidermoid pulmonary carcinoma), HL60
Promyelocytic leukemia), HT-29 (Colon adenocarcinoma), K562 (Chronic
(
myelogenous leukemia), HEp-2 (cervix carcinoma) and MCF-7 (Breast
adenocarcinoma) were obtained from The Rio de Janeiro cell bank, Brazil. The cells
were maintained in DMEM or RPMI 1640 medium. All cell lines were supplemented
with 10% fetal bovine serum (GIBCO), 1% antibiotic solution (penicillin 5000
Units/mL + streptomycin 5000 µg/mL) and 1% L-glutamine 200 mM.
Cytotoxic activity in tumor cells

The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
Sigma Aldrich Co., St. Louis, MO/USA) reduction assay was used for cytotoxicity.
Tumor cells were plated in 96-well plates (10 cells/mL for adherent cells or 3×10
(
5
5
cells/mL for leukemia). Tested compounds (0.39–25 µg/mL) dissolved in DMEM with
DMSO 0.007% for the lowest concentration (0.39 µg/mL) to 0.5 % for the highest
concentration (25 µg/mL) of compounds were added to each well. Subsequently, cells
were incubated for 72 h. The compound 5 was also evaluated after 24 and 48 h of
incubation in NCI-H292 cells. Control groups received the same amount of DMSO.
After 69 h of treatment 25 µL of MTT (5 mg/mL) was added, three hours later the
supernatant was removed and the MTT-formazan product was dissolved in 100 µL of
DMSO, and absorbance was measured at 570 nm in plate spectrophotometer.
Doxorubicin (0.01–5 µg/mL) was used as positive control. Data are presented as IC50
(concentration that cause 50% of cell growth inhibition) values with their 95%
confidence intervals (CI 95%) obtained by non linear regression after normalize the
absorbance results against untreated control samples [22].
Peripheral blood mononuclear cells isolation and cytotoxic activity (PBMC)
Mononuclear cells (lymphocytes and monocytes) were obtained from the
peripheral blood of healthy volunteers collected in sterile tubes with heparin solution
(
BD Vacutainer ™) as an anticoagulant. The protocol was approved by The Human
o
Research Ethics Committee (N CAAE 48809515700005208). The total blood (6 ml)
was diluted in 3 ml PBS and layered onto 2 ml Ficoll®-Hystopaque (Sigma). For phase
separation of the solution, the tube was centrifugated at 1500 rpm for 30 minutes. After
centrifugation the mononuclear cells were concentrated in the middle layer located
between the plasma (light phase) and erythrocytes (dark phase). Afterwards the PBMC
were transferred to another tube with PBS to a final volume of 11 ml. The cells were
pelleted by centrifugation and resuspended in RPMI 1640 medium supplemented with
2
0% fetal bovine serum and 1% antibiotics 1% antibiotic solution (penicillin 5000
Units/mL + streptomycin 5000 µg/mL). Phytohemagglutinin 2% (Sigma) was added to
the medium to stimulate the proliferation of lymphocytes. Cells were seeded at 106
cells/mL in 96 well plates. After 24 hours of incubation, compounds were dissolved in
DMEM with DMSO and added to each well. Doxorubicin (0.078 to 10 μM) was used as
a positive control. The negative control received the same amount of DMSO. Twenty-
four hours before the end of the incubation period (total time of 72h), 10 μL of Alamar
Blue stock solution (0.312 mg/mL) was added to each well. The absorbances were
measured at 570 nm (oxidized state) and 595 nm (reduced state) in a plate
spectrophotometer. Cell proliferation that was calculated by the following formula: %
proliferating = ALW – (AHW x R0) x 100, where ALW is the absorbance at the lowest
wavelength (570 nm) e AHW is the absorbance at highest wavelength (595 nm),
respectively. The R0 was calculated according to the formula: R0 = (absorbance of
medium with Alamar Blue minus absorbance of medium without Alamar blue at 570
nm)/( absorbance of medium with Alamar Blue minus absorbance of medium without
Alamar blue at 595 nm) [23].

Evaluation of hemolytic potential in human erythrocytes
Blood was collected from healthy volunteers. Erythrocytes were pelleted by
centrifugation (3000rpm/5min) and resuspended in saline (0.85% NaCl + 10 mM
CaCl ) to obtain 2% erythrocytes solution (SE) 2%. The 96-well plates were prepared
2
according to the following: 100 μl of saline (negative control); 50 μl of saline solution
and 50 μl of vehicle with 1% DMSO (Blank); 80 μL saline + 20 μL Triton X - 100 1%
(positive control); 50 μl of saline solution and 50 μl of compounds (1.95 to 250 µg/ mL)
diluted in 1% DMSO. Then 100μl of the solution of erythrocytes was plated in each
well. After incubation for 1 h under constant agitation at room temperature, the
supernatant was analyzed in automatic plate reader (450nm). The 50% effective
concentration (EC50) and their 95% confidence intervals were determined from
nonlinear regression.
Morphological changes induced compound 5 (5-(2-bromo-5-methoxybenzylidene)-
thiazolidine-2,4-dione) on lung adenocarcinoma (NCI-H292)
Compound 5, the most active compound, was selected for further investigation
5
of cytotoxicity on lung carcinoma NCI-H292. Cells cells (10 cells/mL) were treated
for 48 h with Compound 5 at 8.0 and 16.0 µg/mL, the IC50 and 2x IC50 for 48h,
respectively. Afterward, slides were prepared by cytospin centrifugation. To evaluate
the morphology, the cells were fixed with methanol for 1 minute and stained with May-
Grunwald-Giemsa. Doxorubicin (0.5 µg/mL) was used as a positive control.
Analysis by Flow Cytometry
Externalization of phosphatidylserine in NCI-H292 after treatment with compound 5
The externalization of phosphatidylserine was evaluated by annexin/propidium
iodite staining. NCI-H292 cells were treated with compound 5 at 8 and 16 µg/mL for
4
8 hours, the IC50 and 2x IC50 for 48 h, respectively. Doxorubicin (0.5 µg/mL) was used
as positive control. Cells were harvested and incubated with AnnexinFitV-FITC kit
according to the manufacturer (Sigma). Cell fluorescence was then determined by flow
cytometry on a BD cytometry - FACS Calibur - CBA. Twenty thousand events per
experiment were acquired and cell debris were omitted from the analysis. The
percentages of viable cells, early apoptotic, late apoptotic and necrotic cells were
graphed using Prism 5.0 (GraphPad Software Inc.)
Measurement of mitochondria transmembrane potential in NCI-H292 after treatment
with compound 5
NCI-H292 cells treated with compound 5 at 8 and 16 µg/mL for 48 hours, the
IC50 and 2 x IC50 for 48 h, respectively and doxorubicin (0.5 µg/mL) were centrifuged at
2
000 rpm for 5 minutes, washed with PBS and stained with 200 μL of rhodamine 123 (1
µg/ml) for 15 min. Subsequently, cells were pelleted again and incubated with PBS for
0 minutes. Cell fluorescence was then determined by flow cytometry. Twenty
3

thousand events per experiment were acquired and cell debris were omitted from the
analysis [24].
DNA content of NCI-H292 cells (DNA fragmentation and cell cycle) after treatment
with compound 5
Cells were collected, pelleted and fixed with cold ethanol (70%) for 1h. After
washing with phosphate buffer saline to remove ethanol, treated and untreated cells
were incubated with 100 µL of lysis solution (0.1% sodium citrate, 0.1% triton X - 100
and 2 µg/ml of propidium iodide). After 30 minutes cell fluorescence of the samples
were analyzed on a BD cytometry - FACS Calibur – CBA. FlowJo was used to
calculate the cell cycle parameters, based on the cells PI emission (analyzed in the
FACSCalibur FL2 channel that captures 564-606 nm emissions). Twenty thousand
events per experiment were acquired and cell debris were omitted from the analysis
[25].
Genotoxic effect of compound 5 in tumor and normal cell lines - Comet assay
We evaluated the genotoxic effect of compound 5 using the comet assay [26].
5
Approximately 2 x 10 cells/mL of the NCIH-292 were treated with compound 5 for
4
8h. To investigate the initial effects on DNA damage induced by compound 5 we used
the 72 h IC50 and 2 x IC50, 1.26 and 2.5 μg/mL, respectively. For normal cells, we
choose 2 x IC50 and 4 x IC50 found in cancer cells. PBMC were treated with compound
5
with 2.5 and 5.0 µg/mL for 48 h. Doxorubicin (0.5 µg / mL) was used as a positive
control. The negative control received the same amount of DMSO as treated samples
0.5%). After treatment, cells were harvested and 15 μL of the cell suspension was
(
embedded in low melting point agarose at 37°C. The homogenate was then casted on
o
agarose-coated glass slide. Slides were stored in the dark at 4 C for 20 min before
adding electrophoresis buffer. Gel electrophoresis was performed at 40 V for 20 minute
and 300 mA). The slides were neutralized in 0.2 M Tris buffer (pH 7.5) for 15 minutes
and fixed with 98% ethanol for 5 minutes. After drying, the slides were stored in a
refrigerator until staining. Staining was performed through 1:1000 Gel Red
(BiotargetGelRed®) addition onto each slide. We analyzed the slides using a
fluorescence microscope (Olympus – BX series). Approximately 100 cells per treatment
were analyzed and scored from 0 to 4 points depending on the degree of damage in the
nucleoid as the following criteria: (a) class 0: undamaged, with no tail; (b) class 1: with
tail shorter than the diameter of the head (nucleus); (c) class 2: with tail length between
one and two times the diameter of the head; (d) class 3: with tail longer than two times
the diameter of the head; and (e) class 4: comets with no heads. The damage index (DI)
can vary from 0 (0x100) to 400 (4x100) [27]. Examples of comets from our experiment
are provided for better understanding of the classification used (Fig. 3).
Statistical analysis
For cytotoxicity assays, IC50 values and their 95% confidence intervals were
obtained by non-linear regression. In order to determine the differences, the data were

compared by analysis of variance (ANOVA) followed by Dunnett`s test (p <0.05). The
GraphPad Prism version 5.00 was used for statistical analysis.
Results
Cytotoxic activity of thiazolidine-2,4-dione derivatives in tumor cells
Four compounds were tested in six human tumor cell lines HEp-2, HT-29, HL-
6
7
0, MCF-7, K562, and NCI-H 292 and cytotoxicity was determined by MTT assay after
2 hours of incubation. Compounds 2, 3 and 4 showed low cytotoxic activity with IC50
values ranging from 9.0 μg/ml to 25.0 μg/ml (Table 1). Compound 5 was the most
active compound with lowest IC50 value (1.2 μg/mL) for lung cancer (NCI-H292). All
cell lines were inhibited by the positive control doxorubicin, being HEp-2 and HT-29
the most resistant cell lines. In order to investigate the time dependency of compound 5
cytotoxic activity, NCI-H292 cells were treated for 24 h and 48 h. Compound 5
presented IC50> 25 μg/mL after 24 hours of treatment and IC50 of 7.9 μg/mL after 48
hours of treatment (Fig 4)
Cytotoxic activity of thiazolidine-2,4-dione derivatives on peripheral blood
mononuclear cells (PBMC) and hemolytic activity
Compounds that cause non-specific cell death targeting the cell plasmatic
membrane are very toxic. A positive hemolytic assay excludes the compound from
being used as therapeutic drug. None of the compounds tested were toxic to human
erythrocytes (EC50> 125 μg/mL). In addition, the compounds were tested in a model of
human normal cells: peripheral blood mononuclear cells. After isolation from human
blood, PBMC cultivated under mitogenic stimulus works as a model of normal cells.
Compounds 2 to 5 were not cytotoxic to PBMC after 72 h of treatment (IC50> 25
μg/mL). The results indicated that the compound 5 showed selectivity for tumor cell
lines since it causes tumor cell growth inhibition but it does not cause hemolysis of
human erythrocytes neither cytotoxicity toward PBMC.
Analysis of cell morphology -May - Grünwald – Giemsa staining
The cell morphology was analyzed by optical microscopy after 48 h of incubation.
NCI-H292 untreated cells (negative control - CN) showed typical cellular morphology of
adherent cells with intact nuclear and plasmatic membrane and the presence of mitotic
cells (Fig. 5). Cells treated with compound 5 at 8.0 µg/mL and 16.0 µg/mL exhibit
morphological changes consistent with apoptosis including the cell volume reduction and
nuclear fragmentation (Fig. 5). Doxorubicin (0.5 µg/mL) reduced the number of cells,
induced reduction of cell volume, chromatin condensation and nuclear fragmentation in
NCI- H292 cells.
Phosphatidylserine externalization analysis and mitochondrial transmembrane
potential by flow citometry

The percentage of NCI-H292 apoptotic cells was significantly higher (p<0.05)
after 48h treatment with compound 5 at 16 µg/mL (64%) compared to the negative
control (15%). Doxorubicin 0.5 µg/mL also induced apoptosis (51%) compared to the
negative control (p<0.05). None of the treatments induced increase on necrotic cells
(
Fig. 6 and Fig. 7). The mitochondrial membrane potential evaluation was performed
by flow cytometry. Rhodamine 123, a green-fluorescent dye, is sequestered by active
mitochondria. When mitochondria depolarizes due to membrane damage, the dye is lost
and the average fluorescence decrease. After exposure of NCI-H292 to compound 5
(
16.0 μg/mL) for 48 hours a significant increase (p<0.05) in mitochondrial
depolarization was observed (Fig. 6 and Fig. 7). Doxorubicin (0.5 μg/ml) was also able
to promote significant mitochondrial depolarization at the concentration tested (p<0.05).
Analysis of cell cycle and DNA fragmentation
NCI-H292 cells were treated with compound 5 for 48 h (Fig. 6) and 72 h (Fig.
9
) at IC50 and 2 x IC50 and DNA content was quantified by flow cytometry. After 48 h
of treatment, an increase on subdiploid DNA was observed when cells were treated with
compound 5 at 16 µg/mL (16.12% after 48h) compared to negative control (2.4% after
4
8h) (p<0.05). After 48 h there were no significant differences at cells cycle phases
(G0/G1, S and G2/M) between treated and control cells (Fig. 7). After 72 h of treatment
compound 5 at 1.26 µg/mL caused an increase of cells on G0 / G1 (59.8 % after 72 h)
vs. negative control (41.3 % after 72 h) (p<0.05). When cells were treated with 2.52
µg/mL of compound 5 during 72 h, an increase on subdiploid DNA (27.4 % after 72 h)
was observed (negative control 3.79 % after 72 h) (p<0.05).
Genotoxicity assessment in PBMC and NCI-H292
The in vitro genotoxic activity on both PBMC and lung cancer cell line NCI-
H292 was assessed after 48 h by the Comet assay. To analyze the extent of damage to
DNA molecules, data were expressed as damage index (ID) ranging from 0 (no damage)
to 400 (maximum damage) (Fig. 10). No DNA damage was detected on PBMC after 48
h of treatment at any tested concentration of Compound 5 (2.52 µg/mL e 5.0 µg/mL)
compared to the negative control; Conversely, Doxorubicin (0.5 µg/mL) caused
significant DNA damage. For lung cancer cell line NCI-H292, compound 5 (1.26
µg/mL e 2.52 µg/mL) caused an increase on damage index of DNA at both tested
concentrations (1.26 µg/mL e 2.52 µg/mL). Doxorubicin also increased DNA damage
on NCI-H292.
Discussion
Recently, compounds 2, 3, 4 and 5 were synthesized in satisfactory yields and
tested against pteridine reductase 1 (PTR1), a promising enzyme as target for the
development of antileishmanial drugs. Compound 3, the first non-competitive inhibitor
of PTR1, presented a two-digit micromolar potency (less than 50 uM) in comparison
with compounds 2, 4 and 5 [9]. Nonetheless, cytotoxic and genotoxic potentialities on
neoplastic or normal cells have not been described yet.

Compounds 2 to 5 were screened against six tumor cell lines (HEP-2, HL-60,
HT-29, K562, MCF-7 and NCI-H292) and 5 presented lowest IC50 values. The most
sensitive cell line treated with compound 5 was NCI-H292 (IC50 value of 1.26 μg/mL).
The presence of an electron releasing substituent methoxy group increased the activity
of compound 5, while in compound 2 the presence of two strong electron withdrawing
atoms (bromine and fluorine) reduced the cytotoxic activity. Previously, some studies
have reported in vitro cytotoxic activities of thiazolidine-2,4-dione derivatives upon
tumor lines of different histological types [18,28,29]. Amongst ten compounds
synthesized, (2-[4-[(2,4-Dioxothiazolidin-5-ylidene) methyl] phenoxy]-N-[3-
(trifluromethyl) phenyl] acetamide) showed potent cytotoxicity against five of the seven
cell lines tested (breast cancer MCF-7, prostate cancer PC3, nasopharyngeal KB, cancer
oral cancer GURAV and leukemia K562) [18]. Another group of derivatives where the
substitution reaction occurs at the third position of the 2,4- thiazolidinedione ring
presented two active compounds against MCF-7 cell line [28]. Recently, two series of
thiazolidinediones were synthesized and their cytotoxicity evaluated in prostate
adenocarcinoma PC-3, breast adenocarcinoma MDA-MB-231, and fibrosarcoma HT-
1
080 cancer cell lines. Most of the compounds synthesized were active against at least
one cell line, being 5-{4-[(3-(4-Chlorophenyl)-3,4-dihydro-4-oxoquinazolin-2-
yl)methoxy]benzylidene}thiazolidine-2,4-dione the most active compound. Moreover,
mechanistic investigation revealed pro-apoptotic activity of the most cytotoxic
compound [29] .
In order to verify whether the cytotoxicity is related to direct cell membrane
damage, the compounds were incubated with human erythrocytes. None of the
compounds induced lysis of human erythrocytes (EC50> 125 μg/mL). Probably, the
cytotoxicity of compound 5 toward tumor cells occurs by a more specific mechanism
other than a immediate and direct disruption of cell membrane. In fact, treatment of
NCI-H292 cells for a brief period such as 24 h with compound 5 did not reduce cell
growth. Only after 48 hours of treatment, a significant cytotoxicity on NCI-H292 was
observed with maximum effect achieved after 72 h. Lung cancer is currently the leading
cause of cancer death worldwide and for most patients with this disease current
treatments do not promote cure [3,30]. Additionally, cytotoxic action on normal cells
(
2
peripheral blood mononuclear cells) was not observed with compounds 2 to 5 (IC50
5 μg/ml). Compound 5 demonstrated antiproliferative selectivity to lung cancer cells,
since the tumor cells were affected by the treatment but not normal cells.
>
Morphological and biochemical patterns in NCI-H292 human lung cancer cells
incubated with compound 5 were investigated. NCI-H292 treated cells exhibited cell
volume reduction and DNA fragmentation (8 and 16 μg/mL), suggesting activation of
apoptotic events. The process of cell death by apoptosis has some characteristics such as
cell volume reduction, chromatin condensation, and nuclear fragmentation without
extravasation of the cellular content [31]. So, unlike what happens in necrosis, no
inflammatory process occurs [32]. Mitochondrial membrane depolarization and loss of
plasma membrane phospholipid asymmetry also occur during the apoptosis process
[33].
To confirm if morphological alterations in NCI-H292 treated cells were consistent with
apoptosis, phosphatidylserine (PS) externalization was evaluated. An increase on NCI-

H292 apoptosis was observed after 48 h of treatment with compound 5 and such
findings were accompanied by mitochondrial depolarization and DNA fragmentation
(16 μg/mL). After 72 h of incubation, compound 5 caused DNA fragmentation in NCI-
H292 cells at 6-fold lower concentration (2.5 μg/mL) than that used for 48 h analyses
and cell cycle arrest at G0/G1 at 1.26 μg/mL. A report has shown that hybrid molecules
containing 5-benzilidene thiazolidine-2, 4-dione induced apoptosis on leukemia cells by
activation of the extrinsic and the intrinsic pathways of cell death [14]. Pioglitazone
(PGZ), a thiazolidinedione compound, induced growth inhibition and apoptosis of
human B lymphocytic leukemia (SD1 cells). Treatment of these cells with the PPARγ
ligand pioglitazone resulted in growth inhibition in a dose-dependent manner which was
associated with a G1 to S cell cycle arrest after 3 days of treatment. After 4 days of
treatment PGZ caused significant apoptosis in lymphocytic leukemic cell lines [34].
TZD18, Another PPARα and PPARγ agonist structurally related to the
thiazolidinedione, induced G1 cell cycle arrest in Ph+ lymphocytic leukemia cell line at
1
0 uM and at higher concentration (20 µM) induced apoptosis in a time-dependent
fashion [35]. Thus, G1 arrest may represent one of the underlying mechanisms for
subsequent cell apoptosis.
Thiazolidine-2,4-dione (TZDs) were evaluated regarding the expression of
proteins that control the transition from the G1 to S cell cycle´s phase in human prostate
cancer cell lines [36]. Inhibitory concentrations of TZDs rosiglitazone and ciglitazone
induced expression of p21 (Inhibitor of cyclin-dependent kinase) and decrease of cyclin
D1 levels in PC-3 cells. Cell cycle arrest occurred due to the decrease in the level of
cyclin proteins that regulate cell cycle progression [36].
The genotoxicity of compound 5 was accessed by alkaline comet assay. This
technique allows quantitative evaluation of recent damage to DNA by single and double
breaks [37] and has advantages over others DNA damage methods such as the
micronucleus test because of its high sensitivity and early detection of DNA injury.
Compound 5 did not cause genotoxicity in PBMC even for a prolonged period of
treatment with 5 µg/mL (48 h), but NCI-H292 cells presented significant DNA damage
at 4-fold lower concentrations than that used for normal cells (1.25 µg/mL).
Doxorubicin (0.5 μg mL) also produced DNA damage in normal cells. DNA damage
induced by compound 5 in NCI-H292 initiated with low concentration as detected by
the comet assay and became more extensive with higher concentrations of compound 5,
as detailed by flow cytometry assay. Although 5-benzylidene-thiazolidine-2,4-dione
derivatives have been reported in a large number of biological activities, there are still a
few studies that investigate possible genotoxic effects in normal lines. Recently, some
thiazacridine derivatives (ATZD), a new class of cytotoxic agents combining one
acridine nucleus with thiazolidine group, were described as non-genotoxic upon human
lymphocytes [38]. Progress in the treatment of locally stage III Non–Small Cell Lung
Cancer has been achieved with two chemotherapy doublets in combination with
thoracic radiotherapy: 1) full-dose cisplatin plus etoposide (PE) for two cycles; and 2)
weekly low-dose paclitaxel plus carboplatin (PC). However, advanced lung cancer has
been resistant to traditional chemotherapy [39]. This data reinforce the need for
encouragement of smoking cessation as a preventive approach and the development of
new chemotherapeutic agents to treat lung cancer. Hence, we have identified compound
5
- (2-bromo-5-methoxybenzylidene) thiazolidine-2,4-dione as a selective cytotoxic and

pro-apoptotic agent against lung carcinoma cells. Furthermore, compound 5 did not
show genotoxicity to normal cells.
Conflict of interest
None
Funding
The authors thank Brazilian National Research Council (CNPq) and Research
Foundation of Pernambuco State (FACEPE) for financial support.
Acknowledgements
We thank Maria Cicalese for English editing.
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H
N
4
O
O
3
2
S
5
1
Fig. 1.Thiazolidine-2,4-dione ring
O
H
H
O
O
O
N
R1
4
N
H O
2
RCHO
+
Cl
reflux
O
O
H N
NH
2
OH
S
2
S
R1
1
2
-5
R1
R1
R1
F
Br
HO
HO
Br
Br
Cl
Cl
O
CH3
2
3
5
Fig. 2. Synthetic routes and structure of compounds 2 to 5

Fig. 3. Photomicrograph of comets classified by visual inspection into five categories:
score zero representing undamaged cells and score 1 to 4 representing increasing
damage. 1000 x magnification.
1
00
24h
48h
72h
8
0
60
40
20
0
0
.39 0.78 1.56 3.12 6.25 12.5 25.0 (g/mL)
Compound 5
Fig. 4. Time-dependent growth inhibition effect of compound 5 in NCI-H292 cell line.
Tumor cells were treated with 7 different concentrations at 24, 48 and 72 h.

Fig. 5. Cell Morphology of NCI-H292 cells after 48 h of treatment. (NC) negative
control treated with the vehicle alone DMEM 0.5% DMSO. Doxorubicin 0.5 μg/mL
was used as positive control (Dox). Cells were treated with compound 5 at the
concentrations 8.0 µg/mL and 16.0 µg/mL. Arrows indicate the aspects observed:
volume reduction (short arrows) and cellular debris (curved arrows). Cells were stained
with May - Grunwald - Giemsa and visualized by light microscopy with 400X
magnification.
A
1
B
VIABLE
APOPTOSIS
NECROSIS
1500
00
8
0
*
1000
*
60
*
*
*
40
*
500
20
*
0
0
NC
Dox
8
16 g/mL
NC
Dox
8
16
g/mL
Compound 5
Compound 5
Fig. 6. A- Externalization of phosphatidylserine on lung cancer cell line NCI-H292
analyzed by flow cytometry after 48 hours incubation with compound 5. B-Effect of
compound 5 in NCI-H 292 transmembrane mitochondrial potential determined by flow
cytometry after 48 hour of treatment. The negative control (NC) was treated only with
DMEM 0.5% DMSO. Doxorubicin (0.5 µg/mL) was used as positive control (Dox).

Values are mean ± standard deviation of 3 independent experiments (n = 6). *p<0.05
compared to negative control by ANOVA followed by Dunnett's test.
Negative control
Dox (0.5 µg/mL)
Compound 5 (8 µg/mL) Compound 5 (16 µg/mL)
A
B
Intrinsic cell
fluorescence - not
stained
Rhodamine
stained cells
Green fluorescence
C
Red fluorescence
Fig. 7. Effect of Compound 5 in NCI-H292 cell line apoptosis parameters and DNA
content after 48 h treatment. A) Representative flow cytometric dot plots showing the
percentage of cells in viable, apoptotic and necrotic stages. B) Representative flow
cytometric histograms of cells with mithochondrial despolarization. C) Cell cycle
histograms. Dox: doxorubicin.

80
Sub G1
G0/G1
S
G2/M
a
60
40
a
*
20
*
0

g/mL
NC
Dox
8
16
Compound 5
Fig. 8. The effect of compound 5 at IC50 and 2 x IC50 concentrations on the cell cycle of
NCI -H292 was determined by flow cytometry after 48 h of incubation. The negative
control (NC) was treated only with DMEM 0.5% DMSO. Doxorubicin (0.5 µg/mL) was
used as positive control (Dox). Values are mean ± standard deviation of 3 independent
a
experiments (n = 6). *p<0.05 when Sub G1 was compared to negative and p<0.05
when G2/M was compared to negative control by ANOVA followed by Dunnett's test.
Sub G1
G0/G1
a
S
G2/M
1
00
c
8
0
60
40
*
20
a
b
b
0

g/mL
NC
Dox
1.26
2.52
Compound 5
Fig. 9. The effect of compound 5 at IC50 and 2 x IC50 concentrations on the cell cycle of
NCI-H292 determined by flow cytometry after 72 h of incubation. The negative control
(NC) was treated only with DMEM 0.5% DMSO. Doxorubicin (0.5 µg/mL) was used as
positive control (Dox). Values are mean ± standard deviation of 3 independent
a
b
experiments (n = 6). p<0.05 when G0/G1 was compared to negative control. p<0.05
c
when S phase was compared to negative control. p<0.05 when G2/M was compared to
negative control. *p<0.05 when Sub G1 was compared to negative control. All of them
were compared by ANOVA followed by Dunnett's test.

B
A
400
400
300
300
*
*
*
*
2
00
200
100
0
100
0
NC
0.5
2.5
5.0 g/mL
NC
Dox
1.26
2.52 g/mL
Compound 5
Compound 5
Fig. 10. Damage Index (ID) obtained by Comet assay. A) Peripheral blood mononuclear
cells (PBMC) isolated from healthy human volunteers. B) NCI-H292 lung cancer cells.
Both normal (PBMC) and cancer cells (NCI-H292) were treated with DMEM 0.5%
DMSO (NC); Doxorubicin 0.5 μg/mL as positive control (Dox) and compound 5 for 48
hours. Values (damage index of 100 cells at 2 slides; n = 2) corresponds to the mean ±
SD of two independent experiments.* p˂0.05 compared to the negative control (NC)
and analyzed by ANOVA followed by Dunnett´s post test.
Table 1 Cytotoxicity of thiazolidine-2,4-dione derivatives after 72 hours of incubation.
The compound concentration that causes 50% inhibition of cell growth and their 95%
confidence interval (CI95%) are expressed in µg/mL. Doxorubicin was used as positive
control (DOX).
Tumor cell line IC50 (CI95%) µg/mL
Compound HEp-2
HL-60
HT-29
>25
K-562
MCF-7
NCI-H292
9.0
20.2
15.4
2
>25
>25
>25
>25
>25
(6.8-11.9)
(16.4-24.7) (12.9-18.4)
16.1
11.6
(8.7-15.4)
17.9 10.9
(15.6-20.6) (8.8-13.5)
3
>25
(13.9-18.9)
10.2
17.0
(8.2-35.1)
16.9
>25
4
>25
(7.7-13.3)
(12.8-22.4)
2.0
6.8
(0.2-14.5)
3.5
(1.8-6.3)
1.26
(0.9-1.7)
5
>25
(1.5-2.7)
1
.2
0.03
(0.02-0.03)
0.7
(0.3-1.0)
0.24
0.16-0.39
0.5
0.36-0.74
0.3
0.17-0.9
DOX
(0.5-2.4)
IC50: Concentration that causes 50% inhibition of cell growth, and (CI95%) confidence interval. Dox:
doxorubicin