Anticancer effects of synthetic hexahydrobenzo [g]chromen-4-one derivatives on human breast cancer cell lines

Article abstract of DOI:10.1007/s12282-016-0704-5

Background: Cancer results from a series of molecular changes that alter the normal function of cells. Breast cancer is the second leading cause of cancer death in women. To develop novel anticancer agents, new series of chromen derivatives were synthesized and evaluated for their cytotoxic activity against human breast cancer cell lines. Method: The growth inhibitory activities of synthesized hexahydrobenzo chromen-4-one were screened against six human cancer cell lines using an in vitro cell culture system (MTT assay). Fluorochrome staining (acridine orange/ethidium bromide double staining) and DNA fragmentation by the diphenylamine method were used to investigate the effects of most potent compounds on the process of apoptosis in breast cancer cell lines. To determine the mechanism of apoptosis, ROS and NOX production in treated breast cancer cells with compounds was evaluated. Results: The cytotoxicity data of tested compounds demonstrate these compounds had varying degree of toxicity. Compound 7h was the most potent compound with IC₅₀?=?1.8?±?0.6?μg/mL against T-47D cell line. Analyses of the compounds treated (MCF-7, MDA-MB-231, and T-47D) cells by acridine orange/ethidium bromide double staining and DNA fragmentation by the diphenylamine method showed that the synthetic compounds induce apoptosis in the cells. A significant increase in ROS production was observed in T-47D cells treated with IC₅₀ value of compound 7g. Incubation with IC₅₀ value of synthetic compounds increased the NOX production in cell lines, especially T-47D cells. Conclusion: Our results show that most compounds have a significant anti-proliferative activity against six human cancer cell lines. The observations confirm that chromen derivatives have induced the cell death through apoptosis.

Full text of DOI:10.1007/s12282-016-0704-5

Breast Cancer
DOI 10.1007/s12282-016-0704-5
ORIGINAL ARTICLE
Anticancer effects of synthetic hexahydrobenzo [g]chromen-4-one
derivatives on human breast cancer cell lines
2
3
•
Mahboobeh Pordeli1 Maryam Nakhjiri Maliheh Safavi
•
•
Sussan Kabudanian Ardestani1 Alireza Foroumadi
2
•
Received: 18 February 2016 / Accepted: 13 May 2016
Ó The Japanese Breast Cancer Society 2016
Abstract
T-47D) cells by acridine orange/ethidium bromide double
staining and DNA fragmentation by the diphenylamine
method showed that the synthetic compounds induce
apoptosis in the cells. A significant increase in ROS pro-
duction was observed in T-47D cells treated with IC50
value of compound 7g. Incubation with IC50 value of
synthetic compounds increased the NOX production in cell
lines, especially T-47D cells.
Background Cancer results from a series of molecular
changes that alter the normal function of cells. Breast
cancer is the second leading cause of cancer death in
women. To develop novel anticancer agents, new series of
chromen derivatives were synthesized and evaluated for
their cytotoxic activity against human breast cancer cell
lines.
Method The growth inhibitory activities of synthesized
hexahydrobenzo chromen-4-one were screened against six
human cancer cell lines using an in vitro cell culture sys-
tem (MTT assay). Fluorochrome staining (acridine orange/
ethidium bromide double staining) and DNA fragmentation
by the diphenylamine method were used to investigate the
effects of most potent compounds on the process of
apoptosis in breast cancer cell lines. To determine the
mechanism of apoptosis, ROS and NOX production in
treated breast cancer cells with compounds was evaluated.
Results The cytotoxicity data of tested compounds
demonstrate these compounds had varying degree of toxi-
city. Compound 7h was the most potent compound with
IC50 = 1.8 0.6 lg/mL against T-47D cell line. Analyses
of the compounds treated (MCF-7, MDA-MB-231, and
Conclusion Our results show that most compounds have a
significant anti-proliferative activity against six human
cancer cell lines. The observations confirm that chromen
derivatives have induced the cell death through apoptosis.
Keywords Apoptosis Á Chromen Á Anticancer
Introduction
Cancer is an extremely heterogeneous disease which is
leading to uncontrolled cell growth and leading cause of
death worldwide. Due to cancer progression, drug resis-
tance and side effects of current drugs, researchers are
seeking new synthetic drugs with fewer side effects for the
treatment of cancer. During the past years, many studies
have been done on the biological activity of chromen
derivatives. Recently, the antibacterial, anti-fungal, and
antitumor properties of chromen have been studied [1, 2].
Previously, Liu et al. have reported cytotoxicity of
phenyl-4H-chromen-4-one as derivatives of chromens [3].
In addition, a series of chromen derivatives previously
were synthesized and were considered for their activity
against MCF-7 and MDA-MB-468 breast cancer cell lines
[4].
& Maliheh Safavi
safavi_maliheh@yahoo.com; m.safavi@irost.ir
1
Department of Biochemistry, Institute of Biochemistry and
Biophysics, University of Tehran, Tehran, Iran
2
Department of Medicinal Chemistry, Faculty of Pharmacy
and Drug Design and Development Research Center, Tehran
University of Medical Sciences, Tehran, Iran
3
Department of Biotechnology, Iranian Research Organization
for Science and Technology, Tehran, P. O. Box 3353-5111,
Iran
Whereas apoptosis or programmed cell death con-
tributes to the antitumor activity of many chemotherapeutic
123

Breast Cancer
drugs; therefore, evaluation of induction of apoptosis by
the compounds is a key factor. Apoptosis is a normal
process and one kind of cell death which plays a crucial
role to maintain the integrity and tissue homeostasis of
multi-cellular organisms. It has been known that inade-
quate or abnormal inhibition of apoptotic pathways are
intimately involved in the development of cancer [5].
In recent years, a number of studies have shown that
oxidative stress plays a pivotal role in apoptosis [6, 7].
Reactive oxygen species (ROS) and nitric oxide (NO)
generation are important mediators that can induce DNA
damage. DNA damage activates p53, a transcription factor,
which is transported to the nucleus and bound to DNA and
mediated transcriptional activation of many genes whose
products trigger cell-cycle arrest, apoptosis, or DNA repair.
Actually, ROS is a collective term that is used to
describe a number of reactive molecules and free radicals
derived from the incomplete reduction of oxygen. These
molecules are generated as by-products of normal cellular
metabolism primarily in the mitochondria. In addition,
ROS are generated by xenobiotic exposure and as essential
mediators of metal catalyzed oxidation reactions. ROS can
act as essential biomolecules in the regulation of cellular
functions and as toxic by-products of metabolism in
according to differences in the concentrations of ROS
produced. ROS is useful to the cell, because they play roles
in cell signaling, including; apoptosis; gene expression; and
the activation of cell-signaling cascades [8].
cytotoxic effect on a panel of six human cancer cell lines:
breast cancer cells (MCF-7, MDA-MB231, and T-47D),
hepatocarcinoma (HepG2), neuroblastoma cell line (SK-N-
MC), and mouth Epidermal Carcinoma Cells (KB). Then,
we assessed apoptosis as pathway for cell-death induction
by the compounds on breast cancer cell lines. Increased
production of ROS and NOX has been recognized as a
critical factor in cancer [13]. In addition, a key feature of
apoptosis in many cell types is the induction of genomic
DNA fragmentation [14]. Thus, the ROS and NOX pro-
duction and DNA fragmentation on three breast cancer cell
lines were investigated.
Materials and methods
Materials
The cell-culture medium (RPMI 1640), fetal bovine serum
(FBS), streptomycin, and penicillin were purchased from
Gibco BRL (life technologies, Paisley, Scotland). MTT [3-
(4, 5-dimethyl tiazol-2, 5-diphenyl tetrazolium bromide],
ethidium bromide, acridine orange, VCL3, Griess reagent,
2, 7 dichlorofluorescein diacetate (DCFH-DA), trypsin–
EDTA solution, and dimethyl sulfoxide (DMSO) were
obtained from Sigma Chemical Company (Germany). The
culture plates were purchased from Nunc (Brand products,
Denmark).
NO, an important mediator of both physiological and
pathophysiological processes, has wide-ranging and varied
effects on cellular physiology, signal transduction, and cell
survival [9]. The results from many studies have demon-
strated that NO is a Doubled-Edged Sword in cancer and
the role of NO in the carcinogenic process depends on its
concentration. Low levels of NO can be pro-angiogenic
and pro-tumor formations, whereas its high concentration
acts cytotoxically on tumor cells [10, 11].
General procedure for the preparation
of compounds 7a–k
Dry hydrogen chloride gas was passed through an ice-cold
solution of 2, 3, 6, 7, 8, 9-Hexahydro-6, 6, 9, 9-tetram-
ethylbenzo[g]chromen-4-one (5) (0.03 mol) and substi-
tuted benzaldehyde (0. 03 mol) in ethanol (10 mL) for
5 min. The reaction mixture was allowed to stand at room
temperature for 24 h. The precipitate was filtered, dried,
and crystallized from ethanol (scheme 1).
NO producers has been reported to inhibit HIF-1a
accumulation and activation in hypoxic malignant cells and
considered to develop a novel cancer therapy. NO pro-
ducers were also reported to cause DNA damage via the
generation of peroxynitrite (ONOO–) and N2O3. Perox-
ynitrite can attack on the sugar-phosphate backbone on the
DNA and lead to single-strand DNA breaks. N2O3 can
alkylate DNA through nitrosation of amines and formation
of N-nitrosamines ions and subsequent DNA-crosslinks
[11, 12]. While NO-releasing drugs are under development,
NOS-encoding cDNA sequence could be transfer into
cancer cells for the gene therapy purposes [11].To find
novel potent derivatives with improved cytotoxicity than
current drugs on cancer cells, a series of chromen deriva-
tives (Table 1) prepared and were examined for their
Selected spectroscopic data for the most potent
synthetic compounds (7e, 7g, 7h)
(E)-3-(3-hydroxy-4-methoxybenzylidene)-6,6,9,9-tetram-
ethyl-2,3,6,7,8,9-hexahydrobenzo[g]chromen-4-one (7e).
Yield 35 %; mp 205–208 °C; 1HNMR (CDCl3,
500 MHz) d: 7.98 (s, 1H, H-ethylene), 7.77 (s, 1H, H5-
Chroman), 6.94–6.86 (m, 4H, H20, H50, H60-phenyl and
H10-Chroman), 5.71 (s, 1H, OH), 5.33 (s, 2H, H2-chro-
man), 3.96 (s, 3H, OMe), 1.69 (s, 4H, H7 and H8-chro-
man), 1.32 (s, 6H, Me), and 1.29 (s, 6H, Me).
IR (KBr) m: 3339, 1657.
123

Breast Cancer
Table 1 Structure, molecular
mass, and molecular formula of
the synthesized benzylidene
hexahydrobenzo[g]chromen-4-
one derivatives
Cmpds Name
Name
Molecu Formula
Structure
lar
weight
O
O
7a
(E)-3-(3,5-
374.52 C26H30O2
dimethylbenzylidene)
6,6,9,9-tetramethyl-
2,3,6,7,8,9-hexahydrobenzo
[g]chromen-4-one
-
O
7b
(E)-3-(3-bromo-4,5-
dimethoxybenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
485.41 C26H29BrO
4
OMe
OMe
O
Br
hexahydrobenzo[g]chrom
en-4-one
O
7c
(E)-3-(4-hydroxy-3-
methoxybenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
392.49 C25H28O4
OMe
OH
O
hexahydrobenzo[g]chrom
en-4-one
O
O
7d
(E)-3-(3-bromo-4-
hydroxy-5-
471.38 C25H27BrO
4
OMe
OH
methoxybenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
Br
hexahydrobenzo[g]chrom
en-4-one
(E)-3-(3-chloro-4,5-dimethoxybenzylidene)-6,6,9,9-te-
tramethyl-2,3,6,7,8,9-hexahydrobenzo[g]chromen-4-one
(7g).
(E)-3-(3-chlorobenzylidene)-6,6,9,9-tetramethyl-
2,3,6,7,8,9-hexahydrobenzo[g]chromen-4-one (7h).
Yield 40 %; mp: 138–139 °C; 1HNMR (CDCl3,
500 MHz) d: 7.98 (s, 1H, H-ethylene), 7.77 (s, 1H, H5-
Chroman), 7.4–7.38 (m, 2H, H50 and H60-phenyl),
7.31–7.29 (m, 1H, H40-phenyl), 7.21–7.16 (m, 1H, H20-
phenyl), 6.91 (s, 1H, H10-Chroman), 5.27 (s, 2H, H2-
chroman), 1.70 (s, 4H, H7 and H8-chroman), 1.33 (s, 6H,
Me), and 1.30 (s, 6H, Me).
Yield 12 %;mp 112–114 °C; 1HNMR (CDCl3,
400 MHz) d: 7.96 (s, 1H, H-ethylene), 7.70 (s, 1H, H5-
Chroman), 6.98–6.82 (m, 2H, H20-phenyl and H10-Chro-
man), 6.77 (s, 1H, H60-phenyl), 5.28 (s, 2H, H2-chroman),
3.92 (s, 3H, OMe), 3.91 (s, 3H, OMe), 1.69 (s, 4H, H7 and
H8-chroman), 1.31 (s, 6H, Me), and 1.29 (s, 6H, Me).
IR (KBr) m: 1676.
IR (KBr) m:1660.
123

Breast Cancer
Table 1 continued
O
O
7e
(E)-3-(3-hydroxy-4-
methoxybenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
392.49 C25H28O4
OH
OMe
hexahydrobenzo[g]chrom
en-4-one
O
O
(E)-3-(3-chloro-4-hydroxy- 426.93 C25H27ClO
OMe
5-methoxybenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
4
OH
Cl
7f
hexahydrobenzo[g]chrom
en-4-one
O
O
(E)-3-(3-chloro-4,5-
dimethoxybenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
440.96 C26H29ClO
4
OMe
OMe
Cl
7g
7h
hexahydrobenzo[g]chrom
en-4-one
O
O
(E)-3-(3-
380.91 C24H25ClO
2
Cl
chlorobenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
hexahydrobenzo[g]chrom
en-4-one
7i
O
O
(E)-3-(3-
376.49 C25H28O3
OMe
methoxybenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
hexahydrobenzo[g]chrom
en-4-one
Cell lines and culture
purchased from the National Cell Bank of Iran (Pastor
Institute, Tehran, Iran). The cells were grown at 37 °C in
humidified air with 5 % CO2 in RPMI 1640 supplemented
with penicillin (100 U/ml), streptomycin (100 mg/ml),
1 %L-Glutamine, and 10 % heat-inactivated fetal calf
serum.
The six cell lines, including breast cancer cells (MCF-7,
MDA-MB231 and T-47D), hepatocarcinoma cell line
(HepG2), Neuroblastoma cell line (SK-N-MC), and
mouth epidermal carcinoma cell line (KB) were
123

Breast Cancer
Table 1 continued
O
O
OMe
7j
(E)-3-(2-
376.49 C25H28O3
methoxybenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
hexahydrobenzo[g]chrom
en-4-one
7k
O
O
Cl
(E)-3-(2,3-
415.35 C24H24Cl2
O2
Cl
dichlorobenzylidene)-
6,6,9,9-tetramethyl-
2,3,6,7,8,9-
hexahydrobenzo[g]chrom
en-4-one
Scheme 1 Synthesis of
compounds 7a–k. a HCl,
b phenol, dichloromethane,
aluminum chloride, reflux,
45 min then HCl 25 %,
a
b
Cl
Cl
OH
OH
OH
extracted with CHCl3 and dried
over Na2SO4.
3
2
1
c Trifluoromethanesulfonic
acid, 30 min \ 5 °C, reflux,
40 min, 65°C, extracted with
CHCl3 and dried over Na2SO4.
d NaOH, 4 h, rt, HCl 10 %,
extracted with ethyl acetate and
dried Na2SO4. e Dry HCl gas,
substituted benzaldehyde,
5 min, 24 h, rt
O
O
d
c
OH
Cl
O
5
4
O
R1
O
O
R1
R4
R2
R3
R2
R3
e
H
+
R4
6
7a-7k
MTT cytotoxicity assay
purple formozan crystals by mitochondrial dehydrogenases
in viable cells. The cells (1 9 104) were treated with the
chromen-4-one compounds in a 96-well plate and incu-
bated overnight. Negative control wells contained the same
number of cells with DMSO %0.1 and without any test
compounds, while positive control wells contained
A colorimetric assay using the tetrazolium salt MTT was
used to assess the cytotoxicity of synthesized compounds
and etoposide and doxorubicin [15]. MTT assay is based on
the cleavage of the yellow tetrazolium salt which turned to
123

Breast Cancer
etoposide and doxorubicin. After overnight incubation at
37 °C, the growth medium was removed and 200 lL of
medium supplemented with 4 different final concentrations
(1, 5, 10, 20 lg/mL) of synthetic compounds were added in
triplicate. The test compounds were all first dissolved in
DMSO and then diluted in the growth medium. To perform
the MTT assay, after 24 h treatment, the culture medium
was removed and 200-lL phenol red-free medium con-
taining MTT (0.5 mg/mL) was added to wells. After 4-h
incubation under standard conditions, the MTT solution
was removed and 100 lL of DMSO were added into each
well to solubilize the formazan crystals. The absorbance
was measured at 492 nm with a microplate reader (Gen5,
Powerwave xs2, BioTek, America). The IC50 value was
defined as the drug concentration that produced a 50 %
reduction of absorbance at 492 nm in drug-treated cells as
compared with untreated controls containing DMSO
0.1 %.
defense against the harmful effects of ROS, catalyzes the
conversion of two superoxide anions into a molecule of
hydrogen peroxide (H2O2) and oxygen (O2). Therefore,
the oxidation of DCFH to DCF has been used quite
extensively for the quantitation of H2O2 [17, 18]. In this
study, breast cancer cells were seeded in 6-well plates at a
density of, 5 9 105 cells/well and cultured at 37 °C
overnight. Cells were treated with IC50 dose of the
compounds (7e, 7g and 7h) for 24 h. After that, cells
were incubated with DCFH-DA (10 mM) for 1 h and then
they were washed with PBS and suspended in 500-lL
PBS, and the fluorescence intensity was measured using a
spectrofluorometer at excitation and emission wavelength
of 495 and 525 nm, respectively. DCF standard curve was
prepared by diluting the 1-mM DCF stock in cell culture
media, and the amount of ROS was subsequently esti-
mated from DCF production.
Detection of nitric oxide
Fluorescence microscopic analysis of cell death
Measurement NO in biological systems needs careful
considerations. NO can rapidly oxidize to Nitrite (NO2–)
and nitrate (NO3–) by oxygen. NO has a very short half-life
in biological matrix (t1/2 \ 5 s) [19, 20]; therefore, a direct
measurement of its production is difficult and the deter-
mination of total nitrate and nitrite concentration (NOx) is
routinely used as an index of NO production in biological
fluids and cell-culture medium [21]. In this study, the
nitrite concentration was measured in the culture medium
as an indicator of NO production using the Griess reaction
method. Briefly, 5 9 105 cells/well of three breast cancer
cell lines were grown in 6-well plates and after overnight
incubation, the cells treated with IC50 concentration of
compounds (7e, 7g and 7h) for 4 h. After this time, 100 lL
of each supernatant was mixed with 50-lL VCl3 as a
reductant for the reduction of nitrate-to-nitrite.
Morphological changes in cells were studied by acridine
orange/ethidium bromide (AO/EB) double staining assay
[16]. Acridine orange is taken up by both viable and nonviable
cells and would fluoresce green if intercalated into double
stranded DNA or fluoresce red when bound to single-stranded
nucleic acids RNA and DNA which single-stranded DNA
dominatesindeadcells. Ethidiumbromidewasexcludedfrom
living cells, but is taken up only by cells with ruptured
membrane that allows the entrance of ethidium bromide to
intercalate into DNA and emits red fluorescence. MCF-7,
T-47D, and MDA-MB-231 cell lines were grown in 6-well
plates (5 9 105 cells/well) and after overnight incubation
treated with IC50 concentration of selected compounds (7e, 7g
and 7h) for 24 h. After treatment, cells were harvested and
washed twice with phosphate buffer saline (PBS). 1 lL of dye
mixture (100-lg/mL AO and 100-lg/mL EB) in PBS was
mixed with 25 lL of cell suspension (5 9 105 cells/well) on a
clean microscope slide. The suspension was immediately
examinedbyaAxoscope2plusfluorescencemicroscopefrom
Zeiss (Germany) at 409 magnification.
The same volume of Griess reagent which contains 1 %
sulfanilamide in 5 % phosphoric acid and 0.1 % naph-
thylethylenediamine dihydrochloride in water was added.
The Griess Reagent System is based on the chemical dia-
zotization reaction that was originally described by Griess
in 1879 [22], which uses sulfanilamide under acidic con-
ditions to form diazonium salt which readily couples with
N-(1-naphthalenediamine) to develop a highly colored azo
dye that can be detected at 543 nm [23].
Detection of reactive oxygen species
Intracellular ROS generation was assessed using cell
permeable fluorogenic probe 20, 70-dichlorofluorescin
diacetate (DCFH-DA). This nonpolar compound is
deacetylated to the membrane-impermeant polar deriva-
tive 20, 70-Dichlorodihydrofluorescin (DCFH) by cellular
esterases when it is taken up by the cell. DCFH is non-
fluorescent, but is rapidly oxidized to the highly fluores-
cent DCF by intracellular H2O2 and other peroxides. In
the cells, superoxide dismutase (SOD), as a mechanism of
Evaluation of DNA fragmentation
A simple spectrophotometric method for measuring DNA
in proliferating cells is described. The method is an adap-
tation of the widely used diphenylamine (DPA) reaction to
examine DNA in cells grown in a 96-well plate. This assay
was capable of detecting as little as 10-ng DNA and could
123

Breast Cancer
be used to measure DNA in stored as well as viable tissue
samples [24]. Cells treated with test compounds for 24 h
were harvested and centrifuged at 2000 RPM, for 10 min
(4 °C) to obtain a cell pellet; then, 0.5-mL Tris Triton
EDTA (TTE) buffer was added to the pellet and cen-
trifuged at 13000g for 10 min. Then, 0.5 mL of 25 % tri-
chloro acetic acid (TCA) was added to both tubes and
vortexed vigorously. Both tubes were kept overnight at
4 °C followed by centrifugation at 13000g. The super-
natant was removed and the pallet obtained was hydrolyzed
by the addition of 80 lL of 5 % TCA and heating at 90 °C
for 15 min. This was followed by addition of 160 lL of
freshly prepared DPA and incubation at 37 °C for 24 h.
Finally, optical density of the solution was read at 620 nm
in an ELISA reader, and the percentage of DNA frag-
mentation was expressed by the formula:
MB-231,T-47D, HEPG-2, SK-NMC, and KB cells were
treated with (1, 5, 10, and 20 lg/mL) of synthetic com-
pounds for 24 h. Cell viability and cell growth were
evaluated by MTT reduction assay. The IC50 values of
compounds 7a–7k in comparison to etoposide and dox-
orubicin as positive control are showed in Table 2. The
dose-dependent cytotoxicity effects of most potent com-
pounds (7e, 7g, and 7h) and etoposide against three breast
cancer cell lines are presented in Fig. 1. Results revealed
that most compounds showed more potent cytotoxic
activity than etoposide. The most potent compound is 7h
with the IC50 value of 1.8 0.6 lg/mL which is five-fold
more active than etoposide against T-47D. In addition, this
compound showed desirable growth inhibitory activity
against MDA-MB-231 (IC50 = 2.4 0.6 lg/mL). After
that, compound 7e with IC50 = 2.5 0.8 lg/mL against
MDA-MB-231 and compound 7g with IC50
=
% DNA fragmentation
O:D:of supernatant
2.9 0.9 lg/mL against the T-47D were the potent com-
pounds. Since breast cancer is the most common cancer
and second leading cause of cancer death in women, the
effects and mechanisms of action of the most potent
compounds on breast cancer cells were investigated.
¼
 100:
O:D:of supernatant þ O:D:of pellet
Statistical analysis
Acridine orange/ethidium bromide doubles staining
The experimental results are presented as mean values plus
or minus the standard deviation (SD) of three independent
experiments. The test value of p \ 0.05 was considered
highly significant by Student’s t-test.
Acridine orange/ethidium bromide double staining was
applied to observe the morphological changes in cell death.
Three potent compounds, such as 7e, 7g, and 7h, which
showed a better cytotoxic activity against all three cancer
cell lines, were nominated for the next stages. As shown in
Fig. 2 the non-apoptotic control cells were stained green
and the apoptotic cells had orange particles in their nuclei
due to nuclear DNA fragmentation. According to the
results, the synthetic compounds induced apoptosis in three
cancer cell lines. According to Fig. 3, the highest
Results
Growth inhibition and cell viability
To measure the cytotoxic activity of the synthesized
compounds in human cancer cells, such as MCF-7, MDA-
Table 2 Cytotoxic activity (IC50s, lg/ml) of compounds 7a-k against human cancer cell lines after 24 h treatment
Compounds
R1
R2
R3
R4
SK-NMC
HEPG-2
KB
MCF-7
MDA-MB-231 T-47D
7a
H
Me
H
Me [100
[100
[100
[100
[100
[100
7b
H
OMe OMe Br
6.4 1.6
4.6 1.1
8.2 2.2
3.1 1.3
[100
6.08 2.6
5.5 2.2
[100
12.8 2.2
5.8 1.6
[100
4.9 1.6
4.1 0.9
18.5 0.6
3.1 0.8
15.4 2.4
3.3 0.1
6.1 2.02
5.06 2.2
[100
4.9 2.2
3.7 1.4
5.9 1.5
2.5 0.8
16.7 2.3
3.4 0.7
2.4 0.6
6.09 0.4
5.3 0.6
[100
7c
H
OMe OH
OMe OH
H
7d
H
Br
H
7e
H
OH
OMe
3.5 0.7
[100
4
1.3
3.6 0.3
12.8 0.9
2.9 0.9
1.8 0.6
4.6 0.8
[100
7f
H
OMe OH
Cl
[100
7g
H
OMe OMe Cl
3.6 0.8
5.6 0.9
6.6 3.3
[100
4.2 0.9
4.1 1.1
5.2 1.8
[100
6.6 2.4
6.3 2.05
10.1 1.5
[100
7h
H
Cl
H
H
H
H
H
H
H
H
7i
H
OMe
H
2.9
1
7j
OMe
Cl
[100
[100
7 k
Cl
[100
8.3 3.9
[100
13.7 1.9
[100
16.6 6.5
[100
7.2 0.9
[100
8.5 1.4
Etoposid
Doxorubicin
9.25 1.008
0.007 0.003 0.5 0.001 0.3 0.002 0.02 0.002 0.006 0.001 0.03 0.002
123

Breast Cancer
Fig. 1 Cytotoxic effects of
compounds on three breast
cancer cell lines. Cell viability
assays using the cells treated
with increasing doses of
etoposide (10, 15, 20, and
30 lg/mL) and synthesized
compounds (1, 5, 10, and 20 lg/
mL)
percentage of apoptotic cells in this assay was 52 %,
resulting from treatment T-47D cell lines with the com-
pound 7h. In addition, Compounds 7e and 7g induced
apoptosis in 33 and 48 % of T-47D cell lines. Compound
7e, 7g, and 7h could cause 37, 35.5, and 30 % apoptosis
against MCF-7 cells, respectively. Exposure of MDA-MB-
231 cells to IC50 concentration of tested compounds could
induce apoptosis in 39, 33, and 40 % of the cells by 7e, 7g,
and 7h, respectively.
in the regulation of various physiological processes. NO is
used in various types of disorders like AIDS, cancer,
Alzheimer’s, and arthritis by cytotoxic effects. As shown
in Fig. 5, all compounds could induce NO generation in
three cancer cell lines. The most percentage of NO pro-
duction was related to compound 7e in T-47D cells. In
addition, the highest amount of NO induction was
demonstrated against T-47D cells.
DNA fragmentation
The compounds treatment causes ROS production
in human breast cancer cells
DNA fragmentation is a marker of apoptosis, and cells
exhibiting the morphological characteristics associated
with DNA fragmentation are referred to as ‘‘apoptotic
cells’’ [26]. Figure 6 demonstrates the mean percentage of
DNA fragmentation induced in breast cancer cell lines by
synthesized compounds. According to the results, com-
pound 7h was the most potent in DNA fragmentation
against T-47D and MDA-MB-231.
Because ROS are implicated in apoptosis induction by a
variety of natural anticancer agents [25], we questioned if
pro-apoptotic response to compounds was mediated by
ROS generation. The ROS generation was measured as one
of the indicators of apoptosis in breast cancer cells. As
shown in Fig. 4, our data demonstrate that all compounds
have induced considerably production of ROS in three
breast cancer cell lines. The generation of ROS in T-47D
cells had the highest percentage among the other breast
cancer cell lines, and 7g was the powerful compound in
production of ROS.
Discussion
In the present study, the cytotoxic activities of hexahy-
drobenzo [g]chromen-4-one derivatives 7a–k are reported
against a panel of human cancer cell lines, including MCF-
7, MDA-MB-231, T-47D, HepG2, SK-N-MC, and KB. Our
results show that most compounds have a significant anti-
proliferative activity against six human cancer cell lines.
Many chemotherapeutic drugs have been shown to induce
The compounds treatment causes NOX production
in human breast cancer cells
NO is an important chemical mediator generated by
endothelial cells, macrophages, neurons, etc., and involved
123

Breast Cancer
Fig. 2 Morphological changes
of apoptosis cell in three cell
lines. White arrow indicates live
cells, dashed arrow shows
apoptosis. a DMSO 0.1 % as
control, b cells after exposure to
Etoposide for 24 h, c cells
treated with compound 7e for
24 h, d cells treated with
compound 7g for 24 h, and
e cells treated with compound
7h for 24 h
apoptosis in various cancer types. It is, therefore, important
to understand how each drug acts to induce apoptosis on
certain cancers [27]. Global statistics show that the annual
incidence of breast cancer is increasing, and there is an
essential need to develop potent cytotoxic drugs for inva-
sive breast cancer; Therefore, in this study, we decided to
123

Breast Cancer
Fig. 3 Percent of apoptotic cell
treated with selected
compounds for 24 h
Fig. 4 Effect of compounds on
ROS generation in breast cancer
cell lines. Cells were cultured in
RPMI 1640 medium in the
present of IC50 concentration of
selected compounds and
compared with untreated cells
(control). The bars are
mean SD for three
experiments. *Significantly
different from control cells
(P \ 0.05)
Fig. 5 Effect of compounds on NOX production in breast cancer cell
lines. Cells were cultured in RPMI 1640 medium in the present of
IC50 concentration of selected compounds and compared with
untreated cells (control). The bars are mean SD for three times
study which is significantly different from control. *Significantly
different from control cells (P \ 0.05)
investigate the effects and mechanisms of action of the
most potent compounds on breast cancer cells.
DNA fragmentation is an endpoint characteristic of
apoptosis occurring after caspase activation. Recent studies
have suggested that caspase-3 and caspase-7 are essential
for DNA fragmentation [28]. According to the results,
DNA fragmentation showed in three cell lines. However,
MCF-7 cell line used in this study did not express caspase-
3. Therefore, we suggested that is another mechanism for
DNA fragmentation in MCF-7 cell line that is a caspase-3
independent pathway [27]. Our results show that the
The apoptotic effects of the compounds (7a–k) were
investigated on breast cancer cell lines using acridine
orange in combination with ethidium bromide staining, and
the percentage of apoptotic cells in the early stages of cell
death was investigated. According to the results, Com-
pounds 7g and 7h induced 48 and 52 % apoptosis in T-47D
cells, respectively.
123

Breast Cancer
Fig. 6 Percentage of DNA
fragmentation induced in breast
cancer cell lines with selected
compounds for 24 h
compounds 7g and 7h caused 38.5 and 45 % DNA frag-
mentation in T-47D cells, respectively, which was much
higher than DNA fragmentation in other cells treated with
compounds.
of the intrinsic pathway, which results in cytochrome c
release from the mitochondria and activation of Caspase-9.
In addition, NO increases superoxide generation and per-
oxynitrite formation through mitochondrial electron trans-
port chain inhibition [38].
Chemotherapic agents have been shown to produce
oxidative stress in cells, because during apoptosis, cyto-
chrome c is released from mitochondria to the cytosol
[29, 30]. ROS are essential for life, because low levels of
ROS regulate cellular signaling and play an important role
in normal cell proliferation [31]. Many cellular processes
result in the generation of ROS and excess ROS induce
oxidative DNA damage, genomic instability, and activation
of the mitochondrially mediated intrinsic apoptotic path-
way [32]. According to the results of this study, synthetic
compounds produced oxidative stress in cancer cells which
treated with these compounds. Because ROS attack DNA,
resulting in structural changes and DNA dysfunction, we
hypothesized that our compounds may induce DNA dam-
age. DNA fragmentation assay confirmed that compounds
caused DNA damage in three breast cancer cell lines.
One critical factor involved in apoptosis is Bcl-2, which
is highly expressed in 40–80 % of breast cancer patients,
and is also common in other tumors [33]. MCF-7 cell line
normally expresses higher level of Bcl-2 in comparison
with two other breast cancer cell lines [34]. Our results
showed that compounds increase ROS production in three
breast cancer cell lines compare with control, especially in
T-47D. These results suggest that as regards MCF-7, cell
line showed lower level of ROS, it can be related to Bcl-2.
It has been proposed that Bcl-2 protects against apoptosis
by inhibiting the generation or action of ROS [35, 36].
Indeed, p53 effectively regulate the basal levels of ROS
and there is a crosstalk between the basal levels of cellular
ROS and p53 [37]. Our results showed that ROS formation
in the untreated and treated MCF-7 cells with wild-type
P53 is lower than two other untreated and treated breast
cancer cell lines with mutant-type p53.
Our result indicates that MCF-7 cells (wild-type p53)
produced lower amount of NO than cell lines T-47D
(mutant-type p53) and MDA-MB-231 (mutant-type p53).
In addition, it has been reported previously that there may
be a negative feedback loop existing between NO pro-
duction and wild-type p53 tumor suppressor gene [37]. NO
induces wild-type p53, which is itself a negative regulator
of iNOS that binds to its promoter, resulting in the down-
regulation of iNOS expression [39, 40]. The possible
mechanisms associated with the reduction in NO produc-
tion in MCF-7 cells may be the presence of wild-type p53
in the feedback loop which suppresses the expression of
NO in comparison with two other cancer cell lines with
mutant-type p53. P53 controls overproduction of NO, and,
therefore, the potential for NO-induced DNA damage,
through repression of iNOS gene promoter [37].
Recent results have shown that NO can stimulate p53
expression and apoptosis in breast cancer cell lines
[39, 40]. NO from NOS or NO donors have been demon-
strated to trigger p53 in cancer cells via DNA damage by
peroxynitrite (ONOO-), which can induce apoptosis
[11, 12]. This is a possible process by which NO may
induce death of tumor MCF-7 cells.
Results from the present study suggested that ROS and
NO might act as the signal molecules for inducing cell
death, but it might be through different mechanisms.
T-47D cell line treated with compounds showed higher
levels of ROS and NO production in comparison to other
two breast cancer cell lines. In addition, DNA fragmenta-
tion and apoptosis percent in T-47D cells treated with 7g
and 7h were higher than the other cells, which confirmed
the involvement of oxidative stress in inducing apoptosis
T-47D. All of these observations confirm that the cell death
caused by chromen derivatives is through apoptosis.
NO induces the intrinsic apoptotic pathway to induce
cell death. NO activates BAX and BAK, integral members
123

Breast Cancer
Acknowledgments This research has been supported by a grant
from Iran National Science Foundation (INSF).
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