Synthesis, spectroscopy, thermal analysis, magnetic properties and biological activity studies of Cu(II) and Co(II) complexes with Schiff base dye ligands

Article abstract of DOI:10.3390/molecules17066449

Three azo group-containing Schiff base ligands, namely 1-{3-[(3- hydroxypropylimino) methyl]-4-hydroxyphenylazo}-4-nitrobenzene (2a), 1-{3-[(3-hydroxypropylimino) methyl]-4-hydroxyphenylazo}-2-chloro-4-nitrobenzene (2b) and 1-{3-[(3-hydroxypropylimino) methyl]-4-hydroxyphenylazo}-4-chloro-3- nitrobenzene (2c) were prepared. The ligands were characterized by elemental analysis, FTIR spectroscopy, UV-Vis spectroscopy, 13C- and 1H-NMR spectroscopy and thermogravimetric analysis. Next the corresponding copper(II) and cobalt(II) metal complexes were synthesized and characterized by the physicochemical and spectroscopic methods of elemental analysis, FTIR spectroscopy, UV-Vis spectroscopy, magnetic moment measurements, and thermogravimetric analysis (TGA) and (DSC). The room temperature effective magnetic moments of complexes are 1.45, 1.56, 1.62, 2.16, 2.26 and 2.80 B.M. for complexes 3a, 3b, 3c, 4a 4b, and 4c, respectively, indicating that the complexes are paramagnetic with considerable electronic communication between the two metal centers.

Full text of DOI:10.3390/molecules17066449

Molecules 2012, 17, 6449-6464; doi:10.3390/molecules17066449
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
The Coumarin Psoralidin Enhances Anticancer Effect of Tumor
Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)
Joanna Bronikowska †, Ewelina Szliszka †, Dagmara Jaworska, Zenon P. Czuba and
Wojciech Krol *
Chair and Department of Microbiology and Immunology, Medical University of Silesia in Katowice,
Jordana 19, 41-808 Zabrze, Poland; E-Mails: jbronikowska@sum.edu.pl (J.B.);
eszliszka@sum.edu.pl (E.S.); djaworska@sum.edu.pl (D.J.); zczuba@sum.edu.pl (Z.P.C.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: wkrol@sum.edu.pl;
Tel./Fax: +480-322-722-554.
Received: 29 March 2012; in revised form: 15 May 2012 / Accepted: 21 May 2012 /
Published: 29 May 2012
Abstract: Coumarins are a very common type of secondary plant metabolites with a broad
spectrum of biological activities. Psoralidin is a naturally occurring furanocoumarin
isolated from Psoralea corylifolia possessing anticancer and chemopreventive properties.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in
cancer cells with no toxicity toward normal tissues. Endogenous TRAIL plays an important
role in immune surveillance and defence against cancer cells. Coumarins can modulate
TRAIL-mediated apoptosis in cancer cells. We examined the cytotoxic and apoptotic
activities of psoralidin in combination with TRAIL on HeLa cancer cells. The cytotoxicity
was measured by MTT and LDH assays. The apoptosis was detected using annexin
V-FITC staining and mitochondrial membrane potential was evaluated using DePsipher
staining by fluorescence microscopy. Death receptor (TRAIL-R1/DR4 and TRAIL-
R2/DR5) expression was analyzed using flow cytometry. Psoralidin enhanced TRAIL-
induced apoptosis in HeLa cells through increased expression of TRAIL-R2 death receptor
and depolarization of mitochondrial membrane potential. Our study indicated that
psoralidin augmented the anticancer effects of TRAIL and confirmed a potential use of
coumarins in cancer chemoprevention.

Molecules 2012, 17
Keywords: coumarin; psoralidin; apoptosis; TRAIL; cancer cells; chemoprevention
6450
1. Introduction
Bioactive compounds from fruits, vegetables, spices and herbs are widely considered to be valuable
for human health [1–3]. Coumarins (1,2-benzopyrones) constitute an important and large class of
oxygen heterocycles, often found as secondary plant metabolites. They are categorized and subdivided
as follows: simple coumarins, furanocoumarins (linear and angular types), pyranocoumarins (linear
and angular types), dicoumarins, and phenylcoumarins. Coumarins naturally present in many plants
(primarily in angiosperms) possess anticoagulant, antimicrobial, antioxidant, anti-inflammatory,
anti-allergic and anticancer properties [4,5].
Psoralidin (Figure 1) is one of the major (angular type) furanocoumarins isolated from the seeds of
Psoralea corylifolia (Leguminosae), a medicinal plant widely distributed in Southeastern Asian
countries [6,7]. Psoralidin has been shown to induce cytotoxicity against gastric (SNU-1, SNU-16),
colon (HT-29) and breast (MCF-7) cancer cells. Psoralidin also caused apoptosis in androgen-dependent
(LNCaP, C4-2B) and androgen-independent (DU-145, PC-3) prostate cancer cells and inhibited
growth of PC-3 xenograft tumor in nude mice [8–12].
Figure 1. Chemical structure of psoralidin.
HO
O
O
O
H3C
CH3
OH
Chemoprevention is a method of tumor control in which malignancy is prevented or reversed by
nutritional or pharmacological intervention using natural or synthetic substances [13–18]. The role of
bioactive compounds in cancer prevention has been confirmed in numerous laboratory and epidemiological
studies [18–22]. Coumarins or their synthetic derivatives have been found to exhibit a variety of
biological activities and have raised considerable interest because of their potential beneficial effects
of human health and chemoprevention [4,5]. Therefore, the development of natural or synthetic
coumarins is becoming increasingly recognized as an effective strategy in cancer prevention.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent stimulator of apoptosis
in cancer cells, is an important immune effector molecule in the surveillance and defence against
developing tumors. Endogenous TRAIL is expressed on the surface of T lymphocytes, natural killer
cells, dendritic cells, neutrophils, monocytes or macrophages and can be cleaved into a soluble,
secreted form [18,23,24]. TRAIL triggers apoptosis in cancer cells through its interaction with specific
death receptors. There are two receptors, TRAIL-R1/DR4 and TRAIL-R2/DR5, that by extracellular
domains recognize and bind ligand. The death receptors contain complete and functional intracellular
death domains responsible for the activation of apoptosis pathway in cancer cells [25,26]. However,

Molecules 2012, 17
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some cancer cells are resistant to TRAIL-mediated death. The expression of the death receptors in
cancer cells could be involved in TRAIL-resistance [23–26].
Our previous findings demonstrated that psoralidin augmented TRAIL-mediated apoptosis and
overcame TRAIL-resistance in LNCaP prostate cancer cells [27]. In the present study we report for the
first time the molecular mechanism by which psoralidin enhances TRAIL-induced apoptosis in cancer
cells on HeLa cell line model. The TRAIL-induced apoptotic signaling pathway is a potential target for
the coumarins in the tumor cells. Overcoming of TRAIL-resistance by psoralidin may be one of the
mechanisms responsible for the cancer chemopreventive properties of coumarins.
2. Results and Discussion
2.1. Cytotoxic and Apoptotic Activities of Psoralidin in HeLa Cancer Cells
Psoralidin is one of the most active ingredients identified in P. corylifolia. The extracts from seeds
of P. corylifolia, as well as psoralidin, possess antimicrobial, antioxidant, anti-inflammatory,
antimutagenic and anticancer activities [6,7,27–33]. Previous experimental data from in vitro and
in vivo studies showed that psoralidin induced cytotoxicity and apoptosis in cancer cells [9–12,27]. We
tested cytotoxic and apoptotic activities of psoralidin against HeLa cells after 48 incubation time.
Psoralidin at concentrations of 20–50 μM induced 2.4 ± 0.5%–11.4 ± 0.8% cytotoxicity in HeLa cells
in a concentration-dependent manner (Figure 2). Our results indicate that cytotoxic effect of the
compound against HeLa cells was mediated through apoptosis. The percentage of necrotic cells
examined by lactate dehydrogenase assay and fluorescence microscopy with Ethidium Homodimer III
was near 0%. At the concentration of 50 μM psoralidin induced 13.5 ± 1.2% apoptosis in HeLa cells.
Figure 2. Cytotoxic activity of psoralidin in HeLa cancer cells. The cancer cells were
incubated for 48 h with psoralidin at the concentrations of 20 μM and 50 μM. The
percentage of cell death was measured by MTT cytotoxicity assay. The values represent
mean ± SD of three independent experiments performed in quadruplicate (n = 3)
(***p < 0.001 compared with control).
14
***
12
10
8
6
4
***
2
0
0
20
50
Psoralidin[µM]

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2.2. Cytotoxic and Apoptotic Activities of TRAIL in HeLa Cancer Cells
TRAIL is a member of the tumor necrosis factor (TNF) superfamily, which includes potent inducers
of apoptosis in a wide variety of cancer cells [23,24]. Recombinant human TRAIL used in our study is
a soluble protein based on a natural endogenous ligand. We and others have reported that HeLa cells
are resistant to TRAIL-mediated death [25,34–36]. TRAIL induced 2.7 ± 0.4%–10.8 ± 0.7%
cytotoxicity in HeLa cells in a concentration-dependent manner (Figure 3). TRAIL induced the
cytotoxic effect in cancer cells via the apoptotic route. The necrotic cell death percentage of HeLa cells
examined by lactate dehydrogenase assay and fluorescence microscopy with Ethidium Homodimer III
was near 0%. The apoptotic effect of TRAIL at a concentration of 100 ng/mL was 10.8 ± 1.0%.
Concentrations of TRAIL of 200 ng/mL or higher did not significantly increase the cytotoxic and
apoptotic effects on HeLa cells (our unpublished observations).
Figure 3. Cytotoxic activity of TRAIL in HeLa cancer cells. The cancer cells were
incubated for 48 h with TRAIL at the concentrations of 50–200 ng/mL. The percentage of
cell death was measured by MTT cytotoxicity assay. The values represent mean ± SD
of three independent experiments performed in quadruplicate (n = 3) (*** p < 0.001
compared with control).
14
12
***
***
10
8
6
4
***
2
0
0
50
100
200
TRAIL [ng/mL]
2.3. Cytotoxic and Apoptotic Activities of TRAIL in Combination with Psoralidin in HeLa Cancer Cells
Several cellular mechanisms contribute to the overall anti-cancer activity of coumarins. TRAIL is
considered to be a tumor-selective, apoptosis-inducing cytokine and TRAIL-mediated apoptotic
pathway is a potential target for bioactive coumarins. TRAIL-resistant cancer cells can be sensitized to
TRAIL-mediated cytotoxicity by various compounds [25,34–40].
We investigated the cytotoxic and apoptotic activity of TRAIL in combination with psoralidin on
HeLa cancer cells. The cytotoxicity of TRAIL at the concentration of 100 ng/mL in combination with
psoralidin at the concentrations of 20 μM and 50 μM in HeLa cells is demonstrated in Figure 4. As
shown in Figures 2 and 3, little cytotoxicity was observed with psoralidin or TRAIL alone. HeLa cell
cotreatment with TRAIL and psoralidin increased the percentage of cell deaths to 28.7 ± 0.8%–66.9 ± 0.8%.

Molecules 2012, 17
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Using Webb’s method [41] it was calculated that for TRAIL at the concentration of 100 ng/mL in
combination with psoralidin at the concentrations of 20 μM and 50 μM, the synergistic effects were
13% and 21%, respectively. The obtained experimental values are higher than calculated and confirm
this synergistic effect.
Figure 4. Cytotoxic activity of TRAIL in combination with psoralidin in HeLa cancer
cells. The cancer cells were incubated for 48 h with TRAIL at the concentration of
100 ng/mL and psoralidin at the concentrations of 20 μM and 50 μM. The percentage of
cell deaths was measured by MTT cytotoxicity assay. The values represent mean ± SD of
three independent experiments performed in quadruplicate (n = 3) (*** p < 0.001
###
+++
compared with control,
with TRAIL).
p < 0.001 compared with psoralidin and
p < 0.001 compared
80
70
60
50
40
30
20
10
+++
###
***
+++
###
***
***
***
***
20
0
Psoralidin
[µM]
-
-
-
50
-
20
50
TRAIL
100
-
100
100
[ng/mL]
Psoralidin in combination with TRAIL induced cytotoxic effects in cancer cells via the apoptotic
route. The necrotic cell death percentage of HeLa cells examined by lactate dehydrogenase assay and
fluorescence microscopy with Ethidium Homodimer III was near 0%. Psoralidin significantly
enhanced TRAIL-induced apoptosis in HeLa cancer cells (Figure 5).
The percentage of apoptotic cells stained with annexin V-FITC detected by fluorescence
microscopy after a 48-hour exposure to TRAIL at a concentration of 100 ng/mL and psoralidin at
a concentration of 50 μM was elevated to 67.1 ± 3.3%. Like in the case of the cytotoxicity the
calculated synergistic effect was 23%. The obtained experimental values are higher than calculated and
confirm this synergistic effect.
TRAIL plays a significant role in immune surveillance and defense mechanisms against tumor
cells [18,23]. The results suggest that psoralidin can sensitize cancer cells to immune effector
mechanisms such as TRAIL-mediated apoptosis.

Molecules 2012, 17
Figure 5. TRAIL induced apoptosis in combination with psoralidin in HeLa cancer cells.
6454
The cancer cells were incubated for 48 h with TRAIL at the concentrations of
100 ng/mL and psoralidin at the concentration of 50 μM. Detection of apoptotic cell deaths
by fluorescence microscopic using annexin V-FITC, Ethidium Homodimer III and Hoechst
33342 staining: (a) control cells, (b) cells incubated with TRAIL, (c) cells incubated with
psoralidin, (d) cells incubated with TRAIL and psoralidin. The values represent mean ± SD
of three independent experiments performed in quadruplicate (n = 3) (*** p < 0.001
compared with control, ### p < 0.001 compared with psoralidin and +++ p < 0.001 compared
with TRAIL). The healthy cells (stained with Hoechst 33342) emitted blue fluorescence
and apoptotic cells (stained with Annexin V-FITC and Hoechst 33342) emitted green and
blue fluorescence. Cells undergoing apoptosis showed nuclei shrinkage, chromatin
condensation and nuclei fragmentation, indicated by arrows.
80
+++
###
***
70
60
50
40
30
20
10
0
***
***
Psoralidin [µM]
TRAIL [ng/mL]
-
-
-
50
-
50
100
100
a
b
c
d

Molecules 2012, 17
6455
Our previous findings demonstrated that psoralidin reversed TRAIL-resistance and significantly
augmented the apoptotic activity of TRAIL in LNCaP prostate cancer cells [27]. Srinivasan et al.
reported the induction of apoptosis by psoralidin in DU-145 and PC-3 prostate cancer cells. The cancer
cells were co-treated with psoralidin and recombinant TNF-alpha, that belongs also like TRAIL to the
TNF superfamily. The authors confirmed that psoralidin overcame TNF-alpha-mediated resistance in
prostate cancer cell lines [11].
Other coumarins also sensitize cancer cells to TRAIL-mediated death. Esculetin (6,7-dihydroxy-
coumarin) is a coumarin with chemopreventive and anticancer properties found in Artemisia capillaries,
Citrus limonia and Euphorbia lathyris. Kok et al. demonstrated the enhancement of TRAIL-induced
apoptosis in SAS oral cancer cells by esculetin [42].
There are many factors contributing to the resistance to TRAIL-induced apoptosis [18,23].
Therefore, further investigations will be required to explain the molecular mechanisms by which
psoralidin sensitize cancer cells to TRAIL-mediated death.
2.4. The Mechanism by Which Psoralidin Sensitize HeLa Cancer Cells to TRAIL-Induced Apoptosis
Coumarin and its natural or synthetic derivatives exert anticancer potential against HeLa cells.
Numerous findings confirmed that coumarins induced programmed death in cancer cells targeting
several molecules associated with multiple signaling pathways [43–46].
2.4.1. Effects of Psoralidin on Death Receptor Expression in HeLa Cancer Cells
TRAIL triggers cell death in various cancers through its interaction with death receptor
TRAIL-R1/DR4 and/or TRAIL-R2/DR5, which contain a cytoplasmic death-domains capable of
recruiting apoptosis signaling molecules and inducing apoptosis [18,23,25]. However, many tumor
cells are resistant to TRAIL-mediated apoptosis [16–18,24–27,34–40]. Various natural agents have
been demonstrated to augment TRAIL-induced apoptosis through induction of TRAIL-R2 expression,
indicating that death receptor expression levels might be involved in resistance to
TRAIL [16,18,25,26,47]. Thus, induction of TRAIL-R2 expression could enhance cytotoxicity and
apoptosis mediated by TRAIL.
We analyzed the expression of TRAIL-R1 and TRAIL-R2 in HeLa cells after a 48-hour treatment
with 50 μM psoralidin by flow cytometry (Figure 6). Psoralidin significantly increased TRAIL-R2 and
slightly TRAIL-R1 expression in HeLa cell surface. The compound sensitized cancer cells to TRAIL
through the extrinsic (receptor) apoptotic pathway via up-regulation of TRAIL-R2.
Our results corroborate those of previous studies showing that treatment of DU-145 and PC-3
prostate cancer cells with psoralidin markedly increase the expression of death receptor TRAIL-R1 and
TRAIL-R2 [11]. Esculetin augmented TRAIL-induced apoptosis in SAS oral cancer cells by
up-regulation of TRAIL-R2 [42].

Molecules 2012, 17
Figure 6. Effects of psoralidin on death receptor expression in HeLa cancer cells. Cells
6456
were incubated for 48 h with psoralidin at the concentration of 50 μM. The surface
expression of (a) TRAIL-R1 and (b) TRAIL-R2 on cancer cell was measured by flow
cytometry. The values represent mean ± SD of three independent experiments performed in
duplicate (n = 3).
500
TRAIL - R1 expression
a
p < 0.001 Psoralidin v. Control
and Isotype control
400
300
200
100
0
Control
Isotype control Psoralidin
500
400
300
200
100
0
TRAIL - R2 expression
p < 0.001 Psoralidin v. Control
and Isotype control
b
Control
Isotype control Psoralidin
2.4.2. Effects of TRAIL and Psoralidin on Mitochondrial Membrane Potential (ΔΨm) in HeLa
Cancer Cells
Mitochondrial membrane depolarization is one of the first intracellular changes following the onset
of apoptosis [16,17]. We evaluated whether psoralidin sensitizes cancer cells to TRAIL-induced
mitochondrial dysfunction.
The treatment of HeLa cells with 100 ng/mL TRAIL or 50 μM psoralidin alone caused little effect
on ΔΨm (7.25 ± 0.71% and 10.63 ± 0.74%, respectively). The combination of TRAIL with psoralidin
enhanced ΔΨm loss in a large percentage of cancer cells (58.38 ± 1.41%) and induced a significant
disruption of the ΔΨm (Figure 7). These results demonstrated the engagement also of the intrinsic
(mitochondrial) apoptotic pathway in HeLa cells co-treated with TRAIL and psoralidin.

Molecules 2012, 17
Figure 7. Effects of TRAIL in combination with psoralidin on the mitochondrial
6457
membrane potential (ΔΨm) in HeLa cancer cells. Cells were incubated for 48 h with
TRAIL at the concentration of 100 ng/mL and/or psoralidin at the concentration of 50 μM.
The values represent the mean ± SD of three independent experiments performed in
###
duplicate n = 3 (*** p < 0.001 compared with control,
p < 0.001 compared with
+++
Psoralidin and
p < 0.001 compared with TRAIL). TRAIL in combination with
psoralidin induced loss of ΔΨm in HeLa cells. Disruption of ΔΨm in cancer cells was
assessed by fluorescent microscopic analysis of DePsipher staining: (a) control cells; (b)
cells incubated with TRAIL; (c) cells incubated with psoralidin; (d) cells incubated with
TRAIL and psoralidin. Red fluorescence is emitted from the red aggregates of DePsipher,
which are formed within mitochondria in healthy cells. Green fluorescence reveals the
monomeric form of the DePsipher molecule, which appears in the cytosol after
mitochondrial membrane depolarisation (indicated by arrows).
70
+++
###
60
50
40
30
20
10
0
***
***
***
Psoralidin [μM]
-
-
-
50
-
50
TRAIL [ng/mL]
100
100
a
b
c
d

Molecules 2012, 17
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Srinivasan et al. described a significant increase in the expression of cytochrome c in DU-145 and
PC-3 cells after exposure of psoralidin, indicating the involvement of mitochondrial membrane
potential. Additionally, psoralidin treatment causes a decrease of total Bid, that suggest the crosstalk
between both apoptotic pathways, the extrinsic (receptor) and intrinsic (mitochondrial). Psoralidin
alters in cancer cells, both receptor-mediated and mitochondrial, signals lead to apoptosis [11]. In
contrast, Kok et al. showed that esculetin does not influence the mitochondrial-dependent pathway in
SAS cells [42].
3. Experimental
3.1. Coumarin
Psoralidin was obtained from Alexis Biochemicals (San Diego, CA, USA). The compound was
dissolved in DMSO (50 mM) to a final concentration of 0.1% (v/v) in the culture media.
3.2. TRAIL
Soluble recombinant human TRAIL (rhsTRAIL) was purchased from PeproTech Inc. (Rocky Hill,
NJ, USA).
3.3. Cancer Cell Culture
The experiments were performed on a HeLa human cervical cancer cell line (DSMZ (Deutsche
Sammlung von Mikroorganismen und Zellkulturen) GmbH-German Collection of Microorganism and
Cell Cultures, Braunschweig, Germany). The HeLa cells were grown in monolayer cultures in RPMI
1640 containing 10% fetal bovine serum (FBS) with 4 mM L-glutamine, 100 U/mL penicillin and
100 μg/mL streptomycin. The cancer cells were grown at 37 °C in atmosphere with 5% CO2 [34–36].
All reagents for cell culture were obtained from PAA Laboratories (Pasching, Austria).
3.4. Cytotoxicity Assay
The cytotoxicity was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay as described previously [48,49]. The HeLa cells (2.5 × 105/mL) were seeded
24 h before the experiments in a 96-well plate. Psoralidin (20–50 μM) and/or TRAIL (50–200 ng/mL)
were added to the cells, and 48 h later the medium was removed and 20 μL MTT solutions prepared at
5 mg/mL (Sigma Chemical Company, St. Louis, MO, USA) were added to each well for 4 h. The
resulting crystals were dissolved in DMSO. Controls included native cells and medium alone. The
spectrophotometric absorbance of each well was measured using a microplate reader (ELx 800,
Bio-Tek Instruments Inc., Winooski, VT, USA) at 550 nm. The percent cytotoxicity was calculated by
the formula: percent cytotoxicity (cell death) = [1 − (absorbance of experimental wells/absorbance of
control wells)] × 100%.

Molecules 2012, 17
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3.5. Lactate Dehydrogenase Release Assay
Lactate dehydrogenase (LDH) is a stable cytosolic enzyme that is released upon membrane damage
in necrotic cells. LDH activity was measured using a commercial cytotoxicity assay kit (Roche
Diagnostics GmbH, Mannheim, Germany), in which LDH released in culture supernatants is measured
with a coupled enzymatic assay, resulting in conversion of a tetrazolium salt into red formazan
product. The HeLa cells were treated with various concentrations of psoralidin (20–50 μM) alone and
in combination with TRAIL (50–200 ng/mL) for the indicated period of time. The sample solution
(supernatant) was removed and LDH released from cells was measured in culture medium. The
maximal release was obtained after treating control cells with 1% Triton X-100 (Sigma Chemical
Company) for 10 min at room temperature [39,40,50,51]. The necrotic percentage was expressed using
the formula: (sample value/maximal release) × 100%.
3.6. Determination of Apoptotic Cell Death by Fluorescence Microscopy with Annexin V-FITC Staining
Apoptotic cells were quantified by the fluorescence microscopy method using the Apoptotic &
Necrotic & Healthy Cells Quantification Kit from Biotium, Inc. (Hayward, CA, USA) according to the
manufacturer’s instruction [17,27,34,36]. The HeLa cells (2.5 × 105/mL) were seeded 24 h before the
experiments in a 24-well plate. Psoralidin (20–50 μM) and/or TRAIL (50–200 ng/mL) were added to
the cancer cells, and 48 h later the cells were washed with PBS and detached from cell culture wells by
trypsin. Next, the HeLa cells were centrifuged to discard supernatant, washed with PBS and
resuspended in Binding Buffer (100 μL/sample). To each tube were added: 5 μL of Annexin V-FITC,
5 μL of Ethidium Homodimer III and 5 μL of Hoechst 33342 solutions. The samples were incubated at
room temperature for 15 min in the dark. After staining the cancer cells were washed with Binding
Buffer and placed on a glass slide and covered with a glass coverslip. The total number of 200 cells per
sample was taken for analysis. The stained cells were observed under a fluorescence inverted
microscope IX51 (Olympus, Tokyo, Japan) using filter set for FITC, TRITC and DAPI. The healthy
cells (stained with Hoechst 33342) emitted blue fluorescence, apoptotic cells (stained with Annexin
V-FITC and Hoechst 33342) emitted green and blue fluorescence and necrotic cells (stained with
Ethidium Homodimer III and Hoechst 33342) emitted red and blue fluorescence. Cancer cells stained
with triple colours blue, red and green, were dead cells progressing from apoptotic cell population. The
cells were counted each time from the representative area containing 100 cells and apoptotic cells were
expressed as percentage of total cells.
3.7. Flow Cytometric Analysis of Death Receptor Expression on the Cancer Cell Surface
The cell surface expression of death receptors TRAIL-R1 and TRAIL-R2 was determined by flow
cytometry (LSR II, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). HeLa cells
(2.5 × 105/mL) were seeded in 24-well plates for 24 h and exposed to psoralidin (50 μM) for
48 h. Cells were then harvested using solution of trypsin and ethylenediaminetetraacetic acid (EDTA),
washed twice in PBS and resuspended in PBS containing 0.5% bovine serum albumin (BSA). HeLa
cells were incubated with 10 μL phycoerythrin-conjugated anti-TRAIL-R1 or anti-TRAIL-R2
monoclonal antibody (R&D Systems) at 4 °C for 45 min. After staining, the cells were washed with

Molecules 2012, 17
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PBS and analysed using flow cytometry [26,47]. The control sample consisted of cells in a separate
tube treated with phycoerythrin-labelled mouse IgG1 or mouse IgG2B (R&D Systems).
3.8. Evaluation of Mitochondrial Membrane Potential by DePsipher
The DePsipher Kit (R&D Systems) was used to measure the mitochondrial membrane potential
using fluorescence microscopy [17,24,47,51]. HeLa cells (2.5 × 105/mL) were seeded in a 24-well
plate 24 h prior to the experiments. TRAIL (100 ng/mL) and/or psoralidin (50 μM) were added, and
48 h later, the cells were washed with PBS and harvested by trypsinisation. The cells were incubated in
the dark with DePsipher (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanin iodide)
solution at a concentration of 5 μg/mL for 30 min at 37 °C, washed with reaction buffer with stabiliser,
placed on a glass slide and covered with a glass cover slip. The stained cells were observed with a
fluorescence inverted microscope using filter sets for FITC and TRITC. DePsipher undergoes
potential-dependent accumulation in the mitochondria, which is indicated by a fluorescence emission
shift from red (590 nm) to green (530 nm).
3.9. Statistical Analysis
The results are expressed as means ± S.D. obtained from three separate experiments performed in
duplicate or quadruplicate. Statistical significance was evaluated using ANOVA or t Student’s test.
P-values < 0.05 were considered significant.
4. Conclusions
Psoralidin enhanced TRAIL-induced apoptosis in HeLa cells and overcame TRAIL-resistance by
engaging both intrinsic and extrinsic apoptotic pathway via increase expression of TRAIL-R2 and
induction of loss of ΔΨm. Together, our results suggest that psoralidin exerts their anticancer and
chemopreventive effects through the modulation of TRAIL-mediated death in tumor cells.
Acknowledgments
This project was supported by research grant KNW-1-017/P/1/0 from the Medical University of
Silesia in Katowice (Poland).
References and Notes
1. Almeida, I.M.; Barreira, J.C.; Oliveira, M.B.; Ferreira, I.C. Dietary antioxidant supplements:
Benefits of their combined use. Food Chem. Toxicol. 2011, 49, 3232–3237.
2. Barros, L.; Duenas, M.; Carvalho, A.M.; Ferreira, I.C.; Santos-Buelga, C. Characterization of
phenolic compounds in flowers of wild medicinal plants from Northeastern Portugal.
Food Chem. Toxicol. 2012, 50, 1576–1582.
3. Pinela, J.; Barros, L.; Carvalho, A.M.; Ferreira, I.C. Nutritional composition and antioxidant
activity of four tomato (Lycopersicon esculentum L.) farmer’ varieties in Northeastern Portugal
home gardens. Food Chem. Toxicol. 2012, 50, 829–834.

Molecules 2012, 17
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4. Wu, L.; Wang, X.; Xu, W.; Farzaneh, F.; Xu, R. The structure and pharmacological functions of
coumarins and their derivatives. Curr. Med. Chem. 2009, 16, 4236–4260.
5. Riveiro, M.E.; De Kimpe, N.; Moglioni, A.; Vázquez, R.; Monczor, F.; Shayo, C.; Davio, C.
Coumarins: Old compounds with novel promising therapeutic perspectives. Curr. Med. Chem.
2010, 17, 1325–1338.
6. Zhao, L.H.; Huang, C.Y.; Shan, Z.; Xiang, B.G.; Mei, L.H. Fingerprint analysis of
Psoralea corylifolia by HLPC and LC-MS. J. Chromatogr. B 2005, 821, 67–74.
7. Xiao, D.; Li, G.; Chen, L.; Zhang, Z.; Yin, J.; Wu, T.; Cheng, Z.; Wei, X.; Wang, Z. Isolation of
antioxidants from Psoralea corylifolia fruits using high-speed counter-current chromatography
guided by thin layer chromatography-antioxidant autographic assay. J. Chromatogr. A 2010,
1217, 5470–5476.
8. Yang, Y.M.; Hyun, J.W.; Sung, M.S.; Chung, H.S., Kim, B.K.; Paik, W.H.; Kang, S.S.; Park, J.G.
The cytotoxicity of psoralidin from Psoralea corylifolia. Planta Med. 1996, 62, 353–354.
9. Mar, W.; Je, K.H.; Seo, E.K. Cytotoxic constituents of Psoralea corylifolia. Arch. Pharm. Res.
2001, 24, 211–213.
10. Pahari, P.; Rohr, J. Total synthesis of psoralidin, an anticancer natural product J. Org. Chem.
2009, 74, 2750–2754.
11. Srinivasan, S.; Kumar, R.; Koduru, S.; Chandramouli, A.; Damodaran, C. Inhibiting TNF-mediated
signaling: A novel therapeutic paradigm for androgen independent prostate cancer. Apoptosis
2010, 15, 153–161.
12. Kumar, R.; Srinivasan, S.; Pahari, P.; Rohr, J.; Damodaran, C. Activating stress-activated protein
kinase-mediated cell death and inhibiting epidermal growth factor receptor signaling: A promising
therapeutic strategy for prostate cancer. Mol. Cancer Ther. 2010, 9, 2488–2496.
13. Krenn, L.; Paper, D.H. Inhibition of angiogenesis and inflammation by an extract of red clover
(Trifolium pratense L.). Phytomedicine 2009, 16, 1083–1088.
14. Ozmen, A.; Bauer, S.; Gridling, M.; Singhuber, J.; Krasteva, S.; Madlener, S.; Vo, T.P.; Stark, N.;
Saiko, P.; Fritzer-Szekeres, M.; et al. In vitro anti-neoplastic activity of the ethno-pharmaceutical
plant Hypericum adenotrichum Spach endemic to Western Turkey. Oncol. Rep. 2009, 22, 845–52.
15. Ozmen, A.; Madlener, S.; Bauer, S.; Krasteva, S.; Vonach, C.; Giessrigl, B.; Gridling, M.; Viola, K.;
Stark, N.; Saiko, P.; et al. In vitro anti-leukemic activity of the ethno-pharmacological plant
Scutellaria orientalis ssp. carica endemic to western Turkey. Phytomedicine 2010, 17, 55–62.
16. Szliszka, E.; Zydowicz, G.; Janoszka, B.; Dobosz, C.; Kowalczyk-Ziomek, G.; Krol, W. Ethanolic
extract of Brazilian green propolis sensitizes prostate cancer cells to TRAIL-induced apoptosis.
Int. J. Oncol. 2011, 38, 941–953.
17. Szliszka, E.; Krol, W. Soy isoflavones augment the effect of TRAIL-mediated apoptotic death in
prostate cancer cells. Oncol. Rep. 2011, 26, 533–541.
18. Szliszka, E.; Krol, W. The role of dietary polyphenols in tumor necrosis factor-related apoptosis
inducing ligand (TRAIL)-induced apoptosis for cancer chemoprevention. Eur. J. Cancer Prev.
2011, 20, 63–69.
19. Vanamala, J.; Leonardi, T.; Patil, B.S.; Taddeo, S.S.; Murphy, M.E.; Pike, L.M.; Chapkin, RS.;
Lupton, J.R.; Turner, N.D. Suppression of colon carcinogenesis by bioactive compounds in
grapefruit. Carcinogenesis 2006, 27, 1257–1265.

Molecules 2012, 17
6462
20. Turner, N.D.; Paulhill, K.J.; Warren, C.A.; Davidson, L.A.; Chapkin, R.S.; Lupton, J.R.; Carroll, R.J.;
Wang, N. Quercetin suppresses early colon carcinogenesis partly through inhibition of
inflammatory mediators. Acta Hortic. 2009, 841, 237–242.
21. Warren, C.A.; Paulhill, K.J.; Davidson, L.A.; Lupton, J.R.; Taddeo, S.S.; Hong, M.Y.; Carroll, R.J.;
Chapkin, R.S.; Turner, N.D. Quercetin may suppress rat aberrant crypt foci formation by
suppressing inflammatory mediators that influence proliferation and apoptosis. J. Nutr. 2009, 139,
101–105.
22. Leonardi, T.; Vanamala, J.; Taddeo, S.S.; Davidson, L.A.; Murphy, M.E.; Patil, B.S.; Wang, N.;
Carroll, R.J.; Chapkin, R.S.; Lupton, J.R.; et al. Apigenin and naringenin suppress colon
carcinogenesis through the aberrant crypt stage in azoxymethane-treated rats. Exp. Biol. Med.
2010, 235, 710–717.
23. Lee, J.Y.; Huerta-Yepez, S.; Vega, M.; Baritaki, S.; Spandidos, D.A.; Bonavida, B. The NO
TRAIL to YES TRAIL in cancer therapy (review). Int. J. Oncol. 2007, 31, 685–691.
24. Szliszka, E.; Helewski, K.J.; Mizgala, E.; Krol, W. The dietary flavonol fisetin enhances the
apoptosis-inducing potential of TRAIL in prostate cancer cells. Int. J. Oncol. 2011, 39, 771–779.
25. Horinanka, M.; Yoshida, T.; Shiraishi, T.; Nakata, S.; Wakada, M.; Nakanishi, R.; Nishino, H.;
Sakai, T. The combination of TRAIL and luteolin enhances apoptosis in human cervival cancer
HeLa cells. Biochem. Biophys. Res. Comun. 2005, 333, 833–838.
26. Szliszka, E.; Mazur, B.; Zydowicz, G.; Czuba, Z.P.; Krol, W. TRAIL-induced apoptosis
and expression of death receptor TRAIL-R1 and TRAIL-R2 in bladder cancer cells.
Folia Histochem. Cytobiol. 2009, 47, 579–585.
27. Szliszka, E.; Czuba, Z.P.; Sedek, L.; Paradysz, A.; Krol, W. Enhanced TRAIL-mediated apoptosis
in prostate cancer cells by the bioactive compounds neobavaisoflavone and psoralidin isolated
from Psoralea corylifolia. Pharmacol. Rep. 2011, 63, 139–148.
28. Latha, P.G.; Evans, D.A.; Panikkar, K.R.; Jayavardhanan, K.K. Immunomodulatory and
antitumour properties of Psoralea corylifolia seeds. Fitoterapia 2000, 71, 223–231.
29. Khatune, N.A.; Islam, M.E.; Haque, M.E.; Khondkar, P.; Rahman, M.M. Antibacterial
compounds from seed of Psoralea corylifolia. Fitoterapia 2004, 75, 228–230.
30. Ruan, B.; Kong, L.Y.; Takaya, Y.; Niwa, M. Studies on the chemical constituents of
Psoralea corylifolia L. J. Asian Nat. Prod. Res. 2007, 9, 41–44.
31. Lee, S.; Nam, K.; Mar, W. Induction of quinone reductase activity by psoralidin isolated from
Psoralea corylifolia in mouse Hepa 1c1c7 cells. Arch. Pharm. Res. 2009, 32, 1061–1065.
32. Szliszka, E.; Skaba, D.; Czuba, Z.P.; Krol. W. Inhibition of inflammatory mediators by
neobavaisoflavone in activated RAW264.7 macrophages. Molecules 2011, 16, 3701–3712.
33. Chiou, W.; Don, M.; Liao, J., Wei, B. Psoralidin inhibits LPS-induced iNOS expression via
repressing Syk-mediated activation of P13K-IKK-Iκβ signaling pathways. Eur. J. Pharmacol.
2011, 650, 102–109.
34. Szliszka, E.; Czuba, Z.P.; Jernas, K.; Krol, W. Dietary flavonoids sensitize HeLa cells to tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL). Int. J. Mol. Sci. 2008, 9, 56–64.
35. Szliszka, E.; Czuba, Z.P.; Domino, M.; Mazur, B.; Zydowicz, G.; Krol, W. Ethanolic extract of
propolis (EEP) enhances the apoptosis-inducing potential of TRAIL in cancer cells. Molecules
2009, 14, 738–754.

Molecules 2012, 17
6463
36. Bronikowska, J.; Szliszka, E.; Czuba, Z.P.; Zwolinski, D.; Szmydki, B.; Krol, W. The
combination of TRAIL and isoflavones enhances apoptosis in cancer cells. Molecules 2010, 15,
2000–2015.
37. Szliszka, E.; Majcher, A.; Domino, M.; Pietsz, G.; Krol, W. Cytotoxic activity of tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) against bladder cancer cells after using
chemotherapeutic drugs. Urol. Pol. 2007, 60, 138–142.
38. Szliszka, E.; Bronikowska, J.; Majcher, A.; Miszkiewicz, J.; Krol, W. Enhanced sensitivity of
hormone-refractory prostate cancer cells to tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL) mediated cytotoxicity by taxanes. CEJ Urol. 2009, 62, 29–34.
39. Szliszka, E.; Gebka, J.; Bronikowska, J.; Krol, W. Dietary flavones enhance the effect of tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL) on bladder cancer cells. CEJ Urol.
2010, 63, 138–143.
40. Szliszka, E.; Czuba, Z.P.; Kawczyk-Krupka, A.; Sieron-Stoltny, A.; Sieron, A.; Krol, W. Chlorin-
based photodynamic therapy enhances the effect of tumor necrosis factor-related apoptosis-
inducing ligand (TRAIL) in bladder cancer cells. Med. Sci. Monit. 2012, 18, BR47–BR53.
41. Webb, J.L. Effect of more than One Inhibitor. In Enzyme and Metabolic Inhibitors, 1st ed.;
Academic Press: New York, NY, USA, 1963; Volume 1, pp. 487–512.
42. Kok, S.H.; Yeh, C.C.; Chen, M.L.; Kuo, M.Y. Esculetin enhances TRAIL-induced apoptosis
through DR5 upregulation in human oral cancer SAS cells. Oral Oncol. 2009, 45, 1067–1072.
43. Chuang, J.; Huang, Y.; Lu H.; Ho, H.; Yang, J.; Li, T.; Chang, N.; Chung J. Coumarin induces
cell cycle arrest and apoptosis in human cervical cancer HeLa cells through a mitochondria- and
caspase-3 dependent mechanism and NF-κB down-regulation. In Vivo 2007, 21, 1003–1010.
44. Goel, A.; Prasad, A.K.; Parmar, V.S.; Ghosh, B.; Saini, N. Apoptogenic effect of 7,8-diacetoxy-4-
methylcoumarin and 7,8-diacetoxy-4-methylthiocoumarin in human lung adenocarcinoma cell
line: Role of NF-κB, Akt, ROS and MAP kinase pathway. Chem. Biol. Interact. 2009, 179, 363–374.
45. Yang, J.; Xiao, Y.; He, X.; Qiu, G.; Hu, X. Aesculetin-induced apoptosis through a ROS-mediated
mitochondrial dysfunction pathway in human cervical cancer cells. J. Asian Nat. Prod. Res. 2010,
12, 185–193.
46. Bhattacharyya, S.S.; Paul, S.; De, A.; Das, D.; Samadder, A.; Boujedaini, N.; Khuda-Bukhsh, A.R.
Poly (lactide-co-glycolide) acid nanoencapsulation of synthetic coumarin: Cytotoxicity and
bio-distribution in mice, in cancer cell line and interaction with calf thymus DNA as target.
Toxicol. Appl. Pharmacol. 2011, 253, 270–281.
47. Szliszka, E.; Czuba, Z.P.; Mertas, A.; Paradysz, A.; Krol, W. The dietary isoflavone biochanin-A
sensitizes prostate cancer cells to TRAIL-induced apoptosis. Urol. Oncol. 2011,
doi:10.1016/j.urolonc.2011.01.019.
48. Stankovic, M.S.; Curcic, M.G.; Zizic, J.B.; Topuzovic, M.D.; Solujic, S.R.; Markovic, S.D.
Teucrium plant species as natural sources of novel anticancer compounds: Antiproliferative,
proapoptotic and antioxidant properties. Int. J. Mol. Sci. 2011, 12, 4190–4205.
49. Cheah, S.; Appleton, D.R.; Lee, S.; Lam, M.; Hamid, A.; Hadi, A.; Mustafa, M.R. Panduratin A
inhibits the growth of A549 cells through induction of apoptosis and inhibiotion of NF-KappaB
translocation. Molecules 2011, 16, 2583–2598.

Molecules 2012, 17
6464
50. Szliszka, E.; Czuba, Z.P.; Mazur, B.; Sedek, L.; Paradysz, A.; Krol, W. Chalcones enhance
TRAIL-induced apoptosis in prostate cancer cells. Int. J. Mol. Sci. 2010, 11, 1–13.
51. Szliszka, E.; Czuba, Z.P.; Mazur, B.; Paradysz, A.; Krol. W. Chalcones and dihydrochalcones
augment TRAIL-mediated apoptosis in prostate cancer cells. Molecules 2010, 15, 5336–5353.
Sample Availability: Samples of the compound psoralidin are available from the Alexis Biochemicals
(San Diego, CA, USA).
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