Capsaicin-induced genotoxic stress does not promote apoptosis in A549 human lung and DU145 prostate cancer cells

Abstract

Capsaicin is the major pungent component of the hot chili peppers of the genus Capsicum, which are consumed worldwide as a food additive. More recently, the selective action of capsaicin against cancer cells has been reported. Capsaicin was found to induce apoptosis and inhibit proliferation of a wide range of cancer cells in vitro, whereas being inactive against normal cells. As data on capsaicin-induced genotoxicity are limited and the effects of capsaicin against human lung A549 and DU145 prostate cancer cells were not explored in detail, we were interested in determining whether capsaicin-associated genotoxicity may also provoke A549 and DU145 cell death. Capsaicin-induced decrease in metabolic activity and cell proliferation, and changes in the cell cycle were limited to high concentrations used (≥100μM), whereas, at lower concentrations, capsaicin stimulated both DNA double strand breaks and micronuclei production. Capsaicin was unable to provoke apoptotic cell death when used up to 250μM concentrations. Capsaicin induced oxidative stress, but was ineffective in provoking the dissipation of the mitochondrial inner transmembrane potential. A different magnitude of p53 binding protein 1 (53BP1) recruitment contributed to diverse capsaicin-induced genotoxic effects in DU145 and A549 cells. Capsaicin was also found to be a DNA hypermethylating agent in A549 cells. In summary, we have shown that genotoxic effects of capsaicin may contribute to limited susceptibility of DU145 and A549 cancer cells to apoptosis in vitro, which may question the usefulness of capsaicin-based anticancer therapy, at least in a case of lung and prostate cancer.
Copyright © 2015 Elsevier B.V. All rights reserved.

Full text

Mutation Research 779 (2015) 23–34
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and
Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox
Community address: www.elsevier.com/locate/mutres
Capsaicin-induced genotoxic stress does not promote apoptosis in
A549 human lung and DU145 prostate cancer cells
Anna Lewinskaa,∗, Paulina Jaroszb, Joanna Czechb, Iwona Rzeszutekb,
Anna Bielak-Zmijewskac, Wioleta Grabowskac, Maciej Wnukb
aDepartment of Biochemistry and Cell Biology, University of Rzeszow, Zelwerowicza 4, 35-601 Rzeszow, Poland
bDepartment of Genetics, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa, Poland
cLaboratory of Molecular Bases of Aging, Nencki Institute of Experimental Biology, PAS, Pasteura 3, 02-093 Warsaw, Poland
article info
Article history:
Received 29 November 2014
Received in revised form 8 February 2015
Accepted 10 February 2015
Available online 16 February 2015
Keywords:
Capsaicin
Cancer cells
DNA damage
Micronuclei
Oxidative stress
DNA methylation
abstract
Capsaicin is the major pungent component of the hot chili peppers of the genus Capsicum, which are
consumed worldwide as a food additive. More recently, the selective action of capsaicin against can-
cer cells has been reported. Capsaicin was found to induce apoptosis and inhibit proliferation of a wide
range of cancer cells in vitro, whereas being inactive against normal cells. As data on capsaicin-induced
genotoxicity are limited and the effects of capsaicin against human lung A549 and DU145 prostate can-
cer cells were not explored in detail, we were interested in determining whether capsaicin-associated
genotoxicity may also provoke A549 and DU145 cell death. Capsaicin-induced decrease in metabolic
activity and cell proliferation, and changes in the cell cycle were limited to high concentrations used
(≥100 ␮M), whereas, at lower concentrations, capsaicin stimulated both DNA double strand breaks and
micronuclei production. Capsaicin was unable to provoke apoptotic cell death when used up to 250 ␮M
concentrations. Capsaicin induced oxidative stress, but was ineffective in provoking the dissipation of the
mitochondrial inner transmembrane potential. A different magnitude of p53 binding protein 1 (53BP1)
recruitment contributed to diverse capsaicin-induced genotoxic effects in DU145 and A549 cells. Cap-
saicin was also found to be a DNA hypermethylating agent in A549 cells. In summary, we have shown
that genotoxic effects of capsaicin may contribute to limited susceptibility of DU145 and A549 cancer
cells to apoptosis in vitro, which may question the usefulness of capsaicin-based anticancer therapy, at
least in a case of lung and prostate cancer.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Capsaicin, a homovanillic acid derivate (8-methyl-N-vanillyl-6-
nonenamide), is the most abundant pungent ingredient found in
pepper plants (genus Capsicum, family Solanaceae) that is widely
used as a spice [1]. Capsaicin has been previously used to treat pain
and inflammation associated with neuropathic pain conditions,
such as rheumatoid arthritis, cluster headaches, herpes zoster and
vasomotor rhinitis [2–5]. More recently, anticancer activity of cap-
saicin has been demonstrated [6]. Capsaicin-mediated apoptosis
and/or antiproliferative potential has been reported for numer-
ous cancer cell lines, e.g., in leukemia cells [7], multiple myeloma
cells [8], cutaneous cell carcinoma [9], glioma cells [10], tongue
cancer cells [11], nasopharyngeal carcinoma cells [12], esophageal
∗Corresponding author. Tel.: +48 17 785 54 03; fax: +48 17 872 12 65.
E-mail address: alewinska@o2.pl (A. Lewinska).
carcinoma cells [13], gastric cancer cells [14], pancreatic cancer
cells [15], hepatocarcinoma cells [16], colon carcinoma cells [17],
small cell lung cancer [18], breast cancer cells [19] and prostate
cancer cells [20]. It is widely accepted that capsaicin-associated
anticancer effects may be executed by reactive oxygen species
(ROS) production, cell cycle arrest, regulation of transcription fac-
tor expression and changes in growth/survival signal transduction
pathways, such as EGFR/HER-2 pathway or NF-␬B inactivation
[9,15,19,21–24]. In contrast, capsaicin-induced autophagy may
result in the suppression of endoplasmic reticulum (ER) stress-
mediated apoptosis, which, in turn, may cause an attenuation of
cancer cell death response, e.g., in malignant breast cells [25].
Because capsaicin carcinogenic and tumorigenic properties have
also been suggested [26], the mechanisms underlying capsaicin
cytotoxicity and chemotherapeutic activity require clarification.
It has been postulated and rebutted that capsaicin may stim-
ulate genotoxic effects [27–30]. These discrepancies may rely on
different protocols and diverse biological materials used to address
http://dx.doi.org/10.1016/j.mrgentox.2015.02.003
1383-5718/© 2015 Elsevier B.V. All rights reserved.

24 A. Lewinska et al. / Mutation Research 779 (2015) 23–34
capsaicin-induced genotoxicity [27–30]. Of course, in a case of anti-
cancer therapy it would be beneficial if capsaicin-induced excessive
DNA damage would provoke apoptotic cell death and cancer cell
elimination.
In the present study, a link between capsaicin-induced geno-
toxicity and cytotoxicity has been evaluated. To address capsaicin
genotoxic potential, we used two distinct cancer cell lines, namely,
A549 lung adenocarcinoma and DU145 prostate adenocarci-
noma cells, because data on capsaicin-induced cytotoxicity and
genotoxicity against these cancer cells are limited and lacking,
respectively. Surprisingly, capsaicin-associated genotoxic effects
were not accompanied by apoptosis, which may have implications
for capsaicin-based anticancer therapy.
2. Materials and methods
2.1. Reagents
Capsaicin (12084, analytical standard grade, ≥99%) was pur-
chased from Sigma (Poznan, Poland), phosphate-buffered saline
(PBS) was obtained from Gibco, Invitrogen Corporation (Grand
Island, NY, USA), rhodamine G6 and dihydroethidium were pur-
chased from Molecular Probes (Leiden, Netherlands) and dimethyl
sulfoxide (DMSO) was purchased from BioShop (LabEmpire, Rzes-
zow, Poland). Capsaicin was dissolved in DMSO and added to the
medium to a given final concentration. The DMSO concentration
in the cell culture did not exceed 0.1%, which did not influence
the cell survival. All other reagents, if not mentioned otherwise,
were purchased from Sigma (Poznan, Poland) and were of analytical
grade.
2.2. Cell culture
Human prostate carcinoma cells (DU145) and human lung car-
cinoma cells (A549) were obtained from ATCC (LGC Standards,
Lomianki, Poland). Cells (3000 cells/cm2) were cultured at 37 ◦Cin
Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with
10% fetal calf serum (FCS) and antibiotic and antimycotic mix solu-
tion (100 U/ml penicillin, 0.1 mg/ml streptomycin and 0.25 ␮g/ml
amphotericin B) in a humidified atmosphere in the presence of 5%
CO2until they reached confluence. Typically, cells were passaged
by trypsinization and maintained in DMEM.
2.3. MTT assay
Capsaicin-mediated metabolic activity was estimated as a
function of redox potential as an ability of live cells to
metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) to formazan [31]. The ability of cells to reduce
MTT provides an indication of the mitochondrial integrity and
activity, which, in turn, may be interpreted as a measure of cell
number/proliferation/viability/survival/toxicity. Cells were seeded
onto 96-well plates at a density of 2 ×103cells per well, grown
for 24 h and then the medium was discarded and replaced with
fresh medium with a range of concentrations of capsaicin (from 5 to
250 ␮M) and incubated for another 24 h or 48h. Then, the medium
was removed and cells were supplemented with MTT-containing
medium (500 ␮g/ml working concentration). After 4-h incubation
at 37 ◦C, the medium was removed and formazan crystals were
dissolved in DMSO (30 min, room temperature). Absorbance was
read at 570 nm (measurement wavelength) and at 630 nm (refer-
ence wavelength) using a Tecan Infinite®M200 absorbance mode
microplate reader. Metabolic activity (activity of mitochondrial
dehydrogenases) was calculated as a A(A570 −A630). Metabolic
activity at standard growth conditions is considered as 100%.
2.4. BrdU incorporation test
Capsaicin-mediated changes in cell proliferation were esti-
mated as the ability of cells to synthesize DNA, namely,
5-bromo-2-deoxyuridine (BrdU) incorporation using Cell Prolif-
eration ELISA, BrdU (colorimetric) test (Roche) according to the
manufacturer’s instructions. Briefly, cancer cells were seeded onto
96-well plates at a density of 2 ×103cells per well, grown for
24 h and then the medium was discarded and replaced with fresh
medium with a range of concentrations of capsaicin (from 5 to
150 ␮M) and incubated for another 24 h. Then, the medium was
removed and cells were incubated with 10 ␮M BrdU for 6 h. After
cell fixation, BrdU-labeled DNA was denatured and detected with
anti-BrdU antibody and substrate solution. Photometric detection
was performed with a Tecan Infinite®M200 absorbance mode
microplate reader (measurement wavelength: 370 nm, reference
wavelength: 492 nm). The amount of BrdU-labeled DNA reflecting
cell proliferation was calculated as a A(A370 –A492). Cell prolifer-
ation at standard growth conditions is considered as 100%.
2.5. Annexin V staining and multi-caspase assay
After 24-h treatment with capsaicin (0–250 ␮M), apoptotic
markers, namely, phosphatidylserine externalization and pan
caspase activity were evaluated. The live, early apoptotic, late apop-
totic and dead cells were assessed using the MuseTM Cell Analyzer
and both the MuseTM Annexin V and Dead Cell Assay Kit and the
MuseTM Multi-caspase Kit according to manufacturer’s instructions
(Merck Millipore, Warsaw, Poland). Briefly, Annexin V was used
to detect phosphatidylserine on the external membrane of apop-
totic cells and a derivatized VAD-peptide (Val-Ala-Asp tripeptide)
was used to detect the activity of multiple caspases (caspase-1,
3, 4, 5, 6, 7, 8, and 9) [32]. The VAD peptide (fluorescent-labeled
inhibitor of caspases, FLICA), derivatized with a fluorescent group
and a fluoromethyl ketone (FMK) moiety, is membrane permeable
and non-cytotoxic and binds to the activated caspases, resulting
in a fluorescent signal proportional to the number of active cas-
pases in the cell [32]. A dead cell marker, 7-aminoactinomycin D
(7-AAD) was also used as an indicator of cell membrane integrity.
Four populations of cells were detected:
•non-apoptotic cells (live cells): Annexin V (−) and 7-AAD (−); and
caspase (−) and 7-AAD (−),
•early apoptotic cells: Annexin V (+) and 7-AAD (−); and caspase
(+) cells exhibiting pan caspase activity: caspase (+) and 7-AAD
(−),
•late stage apoptotic and dead cells: Annexin V (+) and 7-AAD (+);
and late stage of caspase activity cells: caspase (+) and 7-AAD (+),
•mostly nuclear debris (necrotic cells): Annexin V (−) and 7-AAD
(+); and caspase (−) and 7-AAD (+).
The calculations were performed automatically, and the
Annexin V and multi-caspase profiles (dot plots) were displayed
using MuseTM Annexin V and dead cell software module and the
MuseTM multi-caspase software module, respectively. As positive
controls, 0.5-h and 3-h treatments with 10 mM hydrogen peroxide
and 10 mM tert-butyl hydroperoxide (tBOOH) were used [33].
2.6. Cell cycle analysis (DNA content)
After 24-h treatment with capsaicin, DNA content analysis was
performed using flow cytometry method as described by Korwek at
al. [34]. The analysis of 104cells was performed using FACSCalibur
and CellQuestPro software.

A. Lewinska et al. / Mutation Research 779 (2015) 23–34 25
2.7. Oxidative stress parameters
After 24-h treatment with capsaicin, steady-state level of
reactive oxygen species (ROS) in the cell culture medium, total
intracellular ROS production and superoxide production were
measured with 2,7-dichlorodihydrofluorescein diacetate (H2DCF-
DA) and dihydroethidium, respectively. Briefly, 5 ␮MH
2DCF-DA
was added to the medium (supernatant obtained after cell
centrifugation) and fluorescence of the 2,7-dichlorofluorescein
(DCF) formed was monitored in a Tecan Infinite®M200 flu-
orescence mode microplate reader. Measurement conditions
were: ex = 495 nm and em =525 nm. Steady-state level of ROS
is presented as RFU (relative fluorescence unit). For ROS and
superoxide kinetics, cells (2.5 ×105cells/ml) were washed and
suspended in PBS containing 0.1% glucose, 0.5 mM EDTA and
5␮MH
2DCF-DA or 5 ␮M dihydroethidium, respectively. After
15-min incubation in the dark at 37 ◦C, fluorescence intensity
due to oxidation of 2,7-dichlorodihydrofluorescein diacetate
(H2DCF-DA) to 2,7-dichlorofluorescein (DCF) and dihydroethid-
ium to ethidium was monitored in a Tecan Infinite®M200
fluorescence mode microplate reader. Measurement conditions
were: ex = 495 nm and em = 525 nm; temperature 37 ◦C and
ex = 518 nm and em = 605 nm; temperature 37 ◦C, respectively.
Data are presented as RFU (relative fluorescence unit) per minute.
2.8. Mitochondrial membrane potential (␺m)
After 24-h treatment with capsaicin, cancer cells (2.5 ×105
cells/ml) were incubated with 5 ␮M rhodamine G6 in PBS con-
taining 0.1% glucose, 0.5 mM EDTA at 37 ◦C for 15 min, washed
and fluorescence intensity reflecting mitochondrial membrane
potential (MMP) was monitored in a Tecan Infinite®M200 fluo-
rescence mode microplate reader. Measurement conditions were:
ex = 528 nm and em = 551 nm; temperature 37 ◦C. Mitochondrial
membrane potential is presented as RFU (relative fluorescence
unit).
2.9. Cytokinesis-block micronucleus (CBMN) assay
To evaluate capsaicin-mediated micronuclei generation, after
24-h treatment with capsaicin, a CBMN assay was performed using
BDTM Gentest Micronucleus Assay Kit using the standard proto-
col according to the manufacturer’s instructions. Briefly, the cells
were cultured for 24 h, then capsaicin (5–250 ␮M) was added for
another 24 h and after that the cells were subjected to cytokinesis
blocking solution for another 24 h. A total of 500 binucleated cells
per well [35,36] were scored using an In Cell Analyzer 2000 (GE
Healthcare, UK) equipped with a high performance CCD camera.
Three replicates per concentration were used (a total of 1500 cells
per concentration). For a positive control, 24-h treatment with
100 ng/ml mitomycin C was used [37].
2.10. Comet assay
DNA double strand breaks (DSBs) were assessed using neutral
single-cell microgel electrophoresis (comet assay). After 24-h treat-
ment with capsaicin, cells were suspended in PBS, mixed with low
melting (LM) agarose (0.7%), added to agarose (LM) slides, lyzed
with proteinase K (0.5 mg/ml) and reduced glutathione (2 mg/ml)
in lysis solution (1.25 M NaCl, 50 mM EDTA, 100 mM Tris–HCl, 0.01%
N-lauroylsarcosine sodium salt, pH 10) at 37 ◦C for 2 h and pro-
ceeded with electrophoresis (neutral comet assay buffer: 100 mM
Tris–HCl, 0.5 M NaCl, 1 mM EDTA, 0.2% DMSO, pH 10). Subse-
quently, slides were stained with 0.25 ␮M YOYO-1 (Invitrogen
Corporation, Grand Island, NY, USA) in 2.5% DMSO and 0.5% sucrose,
mounted with a coverslip and digital comet images were imme-
diately captured with an Olympus BX61 fluorescence microscope
equipped with a DP72 CCD camera and Olympus CellF software. The
CCD capture conditions were: exposure time 81 ms, magnification
400×. Images were saved as TIFF files. At least 150 comets were
measured per each sample triplicate using AutoComet Software
http://autocomet.com/index.php (TriTek Corp.). The % tail DNA was
used as a parameter of DNA damage. For a positive control, 4-h
treatment with 10 mM hydrogen peroxide was used.
2.11. 53BP1 immunostaining
For 53BP1 immunostaining, interphase nuclei were used. After
24-h treatment with capsaicin in the 96-well plate, cancer cells
were fixed with 3.7% formaldehyde containing 0.1% Triton X-100
in PBS for 20 min. Subsequently, the cells were incubated with 1%
bovine serum albumin (BSA) in PBST (phosphate buffered saline
containing 0.25% Triton X-100) at room temperature for 30 min.
After washing with PBST, the cells were incubated with a rab-
bit polyclonal antibody against 53BP1 (Novus Biologicals, Poland)
(diluted 1:200 in PBST–BSA (PBST containing 1% BSA)) overnight
at 4 ◦C, and with, a FITC-conjugated, secondary polyclonal anti-
body against rabbit IgG (BD Biosciences, Germany) (diluted 1:200
in PBST–BSA) at room temperature for 1 h. Nuclei were visualized
with Hoechst 33,342. Digital cell images were captured with an In
Cell Analyzer 2000 (GE Healthcare, UK) equipped with a high per-
formance CCD camera. Cells with 0, 1, 2–5 and more than 5 53BP1
foci were scored [%].
2.12. Global DNA methylation
DNA methylation was estimated as a 5-methyl-2-
deoxycytidine (5mdC) level using both high performance liquid
chromatography (HPLC) and enzyme-linked immunosorbent assay
(ELISA).
For HPLC analysis, DNA from control and capsaicin-treated cells
was purified by phenol chloroform extraction and then digested
by a nuclease P1/alkaline phosphatase DNA degradation method
[38]. Typically, 50 ␮g of good quality DNA was used for diges-
tion. Using HPLC, in DNA hydrolysates from cancer cells treated
for 24 h with capsaicin (5–100 ␮M), 5-methyl-2-deoxycytidine
(5mdC) and 2-deoxycytidine (dC) were determined using UV
detector. Separation of the 2-deoxyribonucleosides was performed
using 250 ×4.6 mm 5 ␮m Hypersil GOLD Column equipped with
10 ×4mm 5␮m Hypersil GOLD drop in guards precolumn. The
nucleosides were eluted with 5% methanol using a HPLC grade
mixture: methanol:water (20%:80%) (v/v) with sodium phosphate
monobasic, pH 3.2–3.4 (34,899 Sigma, Poland) and a flow rate of
1 ml/min. The injection volume of sample was 20 ␮l. Typically,
three replicate injections were performed for each biological sam-
ple. The eluate was monitored with UV detector at 279 nm (5mdC
detection) and 271 nm (dC detection). Linear calibration curves
were obtained in the concentrations ranging from 2.5 nmol/20 ␮lto
20 nmol/20 ␮l for dC and from 0.25 nmol/20 ␮l to 2nmol/20 ␮l for
5mdC. Calibration curves were constructed from triplicate injec-
tions for each concentration. The 5mdC content in genomic DNA
was expressed as a ratio of 5mdC/(dC + 5mdC) [%]. Acquisition
and quantitative analysis of the chromatograms were carried out
using Chromeleon 4.3 software (Dionex Corporation). For a neg-
ative control, 24-h treatment with a DNA methylation inhibitor
5-aza-2-deoxycytidine (5-aza-dC) (5 ␮M) was used [37]. For a pos-
itive control, 24-h treatment with a DNA hypermethylating agent
hydroxyurea (HU) (100 ␮M) was used [37].
For ELISA, MethylFlashTM Methylated DNA Quantification Kit
(Epigentek, Farmingdale, NY, USA) was used according to the man-
ufacturer’s instructions. Briefly, after 24-h treatment with 10 or

26 A. Lewinska et al. / Mutation Research 779 (2015) 23–34
Fig. 1. Capsaicin-mediated changes in metabolic activity (A) and proliferation (B). (A) MTT assay. Metabolic activity at standard growth conditions is considered as 100%.
The bars indicate the SD, n=5, ***p< 0.001 compared with 24-h control, ###p< 0.001 compared with 48-h control (ANOVA and Dunnett’s a posteriori test). DU145 cells (left),
A549 cells (right). (B) Cell proliferation was estimated as the ability of cells to synthesize DNA: BrdU incorporation assay using Cell Proliferation ELISA. Cell proliferation at
standard growth conditions is considered as 100%. The bars indicate the SD, n=5, *** p< 0.001 compared with 24-h control (ANOVA and Dunnett’s a posteriori test). DU145
cells (left), A549 cells (right).
100 ␮M capsaicin, DNA from cancer cells (100 ng) was subjected
to 5mdC content analysis. The positive and negative controls were
always performed. The positive control is a methylated polynu-
cleotide containing 50% of 5mdC, whereas the negative control
is an unmethylated polynucleotide containing 50% of cytosine.
Absorbance was read at 450 nm using a Tecan Infinite®M200
absorbance mode microplate reader. The calculation was made on
the basis of a standard curve obtained for positive control solutions
and 5mdC content is presented as ng 5mdC per 100 ng DNA.
2.13. Statistical analysis
The results represent the mean ±SD from at least three inde-
pendent experiments. The obtained data conform the ANOVA
assumptions as evaluated using Shapiro–Wilk normality test and
Levene test for the equality of variances. Differences between
capsaicin-treated cells versus control cells were assessed using the
one-way analysis of variance (ANOVA) with post hoc testing using
Dunnett’s multiple comparison test. A p-value <0.05 was consid-
ered significant.
The statistical analyses were performed using StatSoft, Inc.
(2005), STATISTICA, version 7.0 (www.statsoft.com).
3. Results
3.1. Capsaicin-mediated cytotoxicity, apoptosis and changes in
the cell cycle
Firstly, cytotoxic/cytostatic potential of capsaicin was inves-
tigated. Because capsaicin was dissolved initially in DMSO, the
possibility of solvent interference was evaluated and we were
unable to observe any DMSO-mediated effects (data not shown).
We used a wide range of capsaicin concentrations from 5 ␮Mto
250 ␮M and two different cancer cell lines: DU145 prostate cancer
cells and A549 lung cancer cells. Both cell lines were found to be
insensitive to 24-h treatment with nutraceutical, when capsaicin
was used at concentrations up to 50 ␮M(Fig. 1A).
For both cell lines, IC50 value was established to be approx-
imately 200 ␮M(Fig. 1A). At the highest concentration used,
250 ␮M, and 24-h incubation time, capsaicin caused a reduction
of metabolic activity of about 80% and 65% in DU145 and A549
cells compared with untreated control, respectively (p< 0.001)
(Fig. 1A). After 48-h treatment, capsaicin was slightly more effec-
tive, especially when higher concentrations were used (Fig. 1A).
Capsaicin also caused a moderate inhibition of cancer cell prolif-
eration (Fig. 1B). At concentration of 150 ␮M, capsaicin lowered
BrdU incorporation of about 30% and 50% in DU145 and A549
cells compared with untreated control, respectively (p< 0.001)
(Fig. 1B).
The level of dead cells was unaffected after capsaicin treatment
using trypan blue exclusion assay (data not shown). Capsaicin,
when used up to 250 ␮M, was also unable to provoke apoptotic
cell death in cancer cells as estimated using Annexin V stain-
ing and multi-caspase assay (Fig. 2). Capsaicin treatment did not
result in either phosphatidylserine externalization or pan caspase
activity, which are widely accepted apoptotic markers (Fig. 2). In
contrast, treatment with the well-known oxidants: hydrogen per-
oxide and tert-butyl hydroperoxide induced apoptosis (Fig. 2). It
is possible that, at the highest concentrations used, the capsaicin-
mediated decrease in metabolic activity estimated using the MTT
assay reflects the antiproliferative and cytostatic action of capsaicin
rather than its ability to stimulate apoptotic cell death.
Capsaicin-associated cell cycle analysis revealed some minor
changes in the particular phases of the cell cycle of DU145 cells,
when capsaicin was used up to 200 ␮M(Fig. 3). The changes in
the G1, S and G2/M phases were statistically insignificant (Fig. 3).
However, at the highest concentration examined (250 ␮M), the
percentage of cells in the S phase dropped from 26% (control con-
ditions) to 17% (250 ␮M capsaicin) and the percentage of cells
in the G1 phase increased from 46% (control conditions) to 58%
(250 ␮M capsaicin) (Fig. 3). These effects were statistically sig-
nificant (p< 0.05) (Fig. 3). The level of polyploidy events was
comparable between control conditions and capsaicin treatment
(Fig. 3). Similar observations were noticed for A549 cells (data not
shown).

A. Lewinska et al. / Mutation Research 779 (2015) 23–34 27
Fig. 2. Capsaicin-mediated apoptosis. After 24-h treatment with capsaicin (0–250 ␮M), apoptosis was assessed using the MuseTM Cell Analyzer and the MuseTM Annexin V
and Dead Cell Assay Kit (panel I) and the MuseTM Multi-caspase Kit (panel II). Panel I: representative Annexin V profiles (dot plots) of DU145 cells are presented. (A) control,
(B) 50 ␮M capsaicin, (C) 100 ␮M capsaicin, (D) 150 ␮M capsaicin, (E) 200␮M capsaicin, (F) 250 ␮M capsaicin. Panel II: representative multi-caspase profiles (dot plots) of
A549 cells (top) and DU145 cells (bottom) are presented. (A and F) control, (B and G) 150 ␮M capsaicin, (C and H) 250␮M capsaicin, (D) 10 mM H2O2, 3 h, (E) 10 mM tBOOH,
3 h, (I) 10 mM H2O2, 0.5 h, (J) 10 mM tBOOH, 0.5 h.

28 A. Lewinska et al. / Mutation Research 779 (2015) 23–34
Fig. 3. Cell cycle analysis after capsaicin treatment. After 24-h treatment with capsaicin (0–250 ␮M), cell cycle analysis was performed using flow cytometry. (A) Repre-
sentative histograms of DU145 cells are shown. M1: G1, M2: sub-G1, M3: G2/M, M4: S, M5: polyploidy. (B) Graph showing % of cells in a particular phase of cell cycle and
polyploidy events. The bars indicate the SD, n=3, *p< 0.05 compared with 24-h control (ANOVA and Dunnett’s a posteriori test).
3.2. Capsaicin-induced oxidative stress
Capsaicin acted as a prooxidant, because it stimulated both
extracellular and intracellular redox imbalance (Fig. 4). The effects
were concentration-dependent and similar for two cancer cell lines
examined (Fig. 4).
After capsaicin treatment, culture medium was found more
oxidative compared with untreated control (Fig. 4A). The level
of reactive oxygen species (ROS) was increased about 15% after
treatment with 150 ␮M capsaicin compared with the control con-
ditions (p< 0.001) (Fig 4A). After treatment with 150 ␮M capsaicin,
intracellular ROS and superoxide production was augmented
approximately 2-fold and 4-fold, respectively (p< 0.001) (Fig. 4B
and C). As cells were suffered from oxidative stress, we were inter-
ested in determining whether the disequilibrium in the redox
homeostasis may affect mitochondria, namely, the mitochondrial
membrane potential (MMP). Surprisingly, no changes in rhodamine
G6 fluorescence reflecting MMP were observed (Fig. 4D). The effect
was comparable for two cancer cell lines used (Fig. 4D).
3.3. Capsaicin-mediated genotoxicity
Capsaicin, at a very mild inhibitory concentration of 100 ␮M
(15% reduction of metabolic activity, Fig. 1A), was found an inducer
of DNA double strand breaks (DSBs) (Fig. 5A).
Capsaicin, in a concentration-dependent manner, caused
increase in DNA double strand breaks (Fig. 5A). After treatment with
100 ␮M capsaicin, the level of DNA DSBs was elevated 2.5- and 3-
fold compared with untreated control in A549 cells and DU145 cells,
respectively (p< 0.05) (Fig. 5A). Capsaicin also caused micronuclei
(MN) formation (p< 0.05) (Fig. 5B). However, the effect was not
concentration-dependent, especially for DU145 cells (Fig. 5B). Cap-
saicin, at concentrations up to 100 ␮M, increased MN production up
to 4-fold (p< 0.05), whereas at the highest concentration (250 ␮M),
a decrease in MN production to control level was observed in DU145
cells (Fig. 5B). In contrast, treatment with capsaicin, at concentra-
tions up to 250 ␮M, stimulated MN production from 2- to 3-fold
compared with untreated control in A549 cells (p< 0.05). We also
investigated capsaicin-mediated formation of p53 binding protein
(53BP1) foci, which are considered to be accumulated at site of DSBs
being a part of DNA repair process. Surprisingly, an inverse correla-
tion between 53BP1 foci and MN generation was shown in DU145
cells (Fig. 5C). In contrast, in A549 cells, in which the level of MN pro-
duction was relatively high after treatment with capsaicin at a wide
range of concentrations, and did not decrease at higher concentra-
tions, the formation of 53BP1 foci was also high and comparable
between concentrations used (Fig. 5C).
3.4. Capsaicin-mediated epigenetic changes
Capsaicin-mediated changes in the global DNA methylation
were also evaluated. After capsaicin treatment, the level of
5-methyl-2-deoxycytidine (5mdC) was unchanged in DU145,
whereas capsaicin was found to be a potent DNA hypermethylating
agent in A549 cells (Fig. 6).

A. Lewinska et al. / Mutation Research 779 (2015) 23–34 29
Fig. 4. Capsaicin-mediated oxidative stress (A–C) and changes in mitochondrial membrane potential (D). (A) Steady-state level of reactive oxygen species (ROS) in the cell
culture medium. (B) Intracellular ROS production. (C) Intracellular superoxide production. Fluorescence intensity was monitored in a Tecan Infinite®M200 fluorescence
mode microplate reader. The bars indicate the SD, n=5, ***p< 0.001 compared with 24-h control (ANOVA and Dunnett’s a posteriori test). DU145 cells (left), A549 cells (right).
(D) Fluorescence intensity of rhodamine G6 reflecting mitochondrial membrane potential (MMP) was monitored in a Tecan Infinite®M200 fluorescence mode microplate
reader. The bars indicate the SD, n= 5. DU145 cells (left), A549 cells (right).

30 A. Lewinska et al. / Mutation Research 779 (2015) 23–34
Fig. 5. Capsaicin-mediated genotoxicity. After 24-h treatment with capsaicin, DNA double strand breaks (DSBs) (A), micronuclei production (B) and 53BP1 foci (C) were
analyzed. (A) DSBs were assessed using neutral comet assay. As a DNA damage marker, the % tail DNA was used. A positive control (4-h treatment with 10 mM hydrogen
peroxide) is also shown. The bars indicate the SD, n= 150, ** p< 0.01, *p< 0.05 compared with 24-h control (ANOVA and Dunnett’s a posteriori test). DU145 cells (left), A549
cells (right). (B) CBMN assay. A positive control (24-h treatment with 100 ng/ml mitomycin C) is also shown. The bars indicate the SD, n=1500, *p< 0.05 compared with 24-h
control (ANOVA and Dunnett’s a posteriori test). DU145 cells (left), A549 cells (right). (C) 53BP1 foci were revealed using 53BP1 immunostaining. Cells with 0, 1, 2–5 and
more than 5 53BP1 foci were scored [%]. DU145 cells (left), A549 cells (right).
HPLC analysis revealed that a concentration of 10 ␮M of cap-
saicin caused an increase in 5mdC level of approximately 20%
compared with untreated control in A549 cells (p< 0.001), whereas
ELISA-based 5mdC quantification showed a 40% increase in 5mdC
level compared with control conditions (p< 0.05) (Fig. 6).
4. Discussion
In the present study, capsaicin genotoxic potential in two can-
cer cell lines DU145 and A549 has been demonstrated. Capsaicin
induced DNA double strand breaks (DSBs) and chromosomal dam-
age, and capsaicin-associated genomic instability affected capsaicin
pro-apoptotic activity and cytotoxicity, which, in turn, may lead to
limited effectiveness of capsaicin-based anticancer therapy. DNA
damaging agents may affect DNA yielding modified bases, intra-
and inter-strand cross-links, cyclobutane pyrimidine dimers, 6-
4 photoproducts, single- and double-stranded DNA breaks. Upon
sensing DNA damage, a well co-ordinated network of signaling cas-
cade, termed the DNA damage response (DDR), is induced [39].
Depending on the magnitude of DNA damage, different cellular
processes may be activated, such as cell cycle checkpoint control,
transcription, DNA repair machinery, senescence and/or apoptotic
cell death [39]. Thus, there can be two outcomes of genotoxic injury:
survival or apoptosis.
The concentration- and time-dependent anticancer potential
of capsaicin in vitro has been well-documented [1]. Capsaicin is
believed to be toxic to cancer cells when added at the micromo-
lar range [1]. The maximal antiproliferative activity of capsaicin

A. Lewinska et al. / Mutation Research 779 (2015) 23–34 31
Fig. 6. Capsaicin-mediated changes in the global DNA methylation. After 24-h treatment with capsaicin, DNA methylation was estimated as a 5-methyl-2-deoxycytidine
(5mdC) level using HPLC (A) and ELISA (B). The 5mdC content in genomic DNA was expressed as a ratio of 5mdC/(dC + 5mdC) [%] (A) and as ng 5mdC/100ng DNA (B). (A)
The cells treated with a DNA methylation inhibitor 5-aza-2-deoxycytidine (5-aza-dC) (5 ␮M, 24-h treatment) served as a negative control, whereas cells treated with a DNA
hypermethylating agent hydroxyurea (HU) (100 ␮M, 24-h treatment) served as a positive control. The bars indicate the SD, n=5, *p< 0.05, *** p< 0.001 compared with 24-h
control (ANOVA and Dunnett’s a posteriori test). DU145 cells (left), A549 cells (right).
has been observed at about 200–300 ␮M[1]. The response to cap-
saicin may also be cancer cell type-dependent [1]. The sensitivity
of A549 and DU145 cells to capsaicin was comparable as estimated
using MTT test. We have established IC50 value to be approximately
200 ␮M for both cell lines examined. Capsaicin was not able to
inhibit completely the proliferation of cancer cells. The maximum
antiproliferative effect of approximately 30% and 50% for DU145
and A549 cells compared with untreated control was observed,
respectively. Moreover, capsaicin did not stimulate apoptosis or
massive changes in the cell cycle. We were unable to observe any
significant changes in sub-G1 population or changes in Annexin V
staining or pan caspase activity after capsaicin treatment. Data on
capsaicin effects on DU145 and A549 cells are scarce and contradic-
tory. Cytotoxicity of dihydrocapsaicin (DHC), a saturated structural
analog of capsaicin, against A549 cells was found moderate [40].
A549 cells did not respond to DHC treatment when DHC was used
up to 200 ␮M, whereas 400 ␮M DHC treatment resulted in decrease
in metabolic activity of about 30% compared with untreated con-
trol [40]. After 200 ␮M DHC treatment, the level of apoptotic cells
increased to about 10% [40]. In contrast, 100 ␮M capsaicin was
established to be IC50 value in A549 cells, whilst 200 ␮M capsaicin
accounted for 90% inhibition of cell metabolic activity [41]. After
100 ␮M capsaicin treatment, clonal proliferation of DU145 cells
was reduced to 20% of control level [20].10␮M capsaicin was
established to be IC50 value in DU145 cells [20]. Beside cancer cell
type-specific response to capsaicin [33], capsaicin-based toxicolog-
ical data may be also affected by some other factors, e.g., capsaicin
stability during particular experimental conditions, which, in turn,
may yield contradictory results, e.g., discrepancies between IC50
values across studies may occur [1].
It is widely accepted that cancer cells are characterized by
higher levels of reactive oxygen species (ROS) compared with
non-cancerous cells due to oncogenic stimulation and increased
metabolic activities [42], which promotes their proliferation and
cell growth [43]. The role of ROS in cancer biology is rather complex,
because both cancer-suppressing and cancer-promoting actions of
ROS have been documented, which depends on ROS levels [43].
In general, cancer cells upregulate glycolysis even when oxygen
is abundant, a phenomenon called the Warburg effect (or aero-
bic glycolysis) [44,45]. The upregulation of glycolysis may serve
for the synthesis of ATP, ribonucleotides and amino acids, but
also for reduced nicotinamide adenine dinucleotide phosphate
(NADPH) production [46], which can remove ROS [46]. Pyruvate
kinase M2 (PKM2), an essential regulator of aerobic glycolysis
in cancer cells, was also found a participant in a negative feed-
back loop controlled by cellular oxidative stress [46]. Oxidation of
PKM2 on Cys358 caused its inactivation, thereby inducing NAPDH
production by the pentose phosphate pathway, and increasing
cellular redox buffering capacity [46]. This strategy allows can-
cer cells to control a proper redox status and may be considered
a survival mechanism for cancer cells, especially under hypoxic
conditions [45,46]. Thus, a combination of targeting the Warburg
effect and the manipulations in cancer cell redox homeostasis may
have anticancer effects [45]. Indeed, redox-based cancer therapy
involving nutraceutical-mediated modulation in ROS levels and sig-
naling molecules/pathways associated with drug resistance seems
promising [43].
Because anticancer effects of capsaicin are accompanied by
oxidative stress [15,17,47], we were interested in determin-
ing whether cellular redox homeostasis is affected in A549 and
DU145 cells after capsaicin treatment. Indeed, after capsaicin
treatment, ROS level in the culture medium was increased,
and ROS and superoxide production was elevated in A549 and
DU145 cells. Nevertheless, capsaicin-mediated disequilibrium in
the intracellular redox state did not provoke the dissipation of
the mitochondrial inner transmembrane potential (␺m)-based

32 A. Lewinska et al. / Mutation Research 779 (2015) 23–34
apoptotic cell death in cancer cells, which is inconsistent with avail-
able data on capsaicin-induced ROS-based apoptosis. Capsaicin
was reported to inhibit cancer cell-specific isoform of the outside
cell surface-localized NADH oxidase (ENOX2), which may result in
ROS elevation triggering mitochondrial-dependent apoptosis [6].
Capsaicin-induced ROS may also inhibit antioxidant protein thiore-
doxin, a negative regulator of apoptosis signal-regulating kinase
1 (ASK1), leading to activation of the ASK1 cascade and apopto-
sis in pancreatic cancer cells [48]. Moreover, capsaicin may affect
the activities of mitochondrial complexes: I and III, which may
cause ROS accumulation, ATP level depletion and downregulation
of antioxidant enzymes, which, in turn, may contribute to apoptosis
in pancreatic cancer cells [15].
In contrast to limited cytotoxicity, capsaicin was found an
inducer of genotoxic events in A549 and DU145 cells. Capsaicin
promoted DNA double strand breaks (DSBs) and micronuclei for-
mation. It has been previously reported that capsaicin may induce
DNA damage both in nonmalignant cells and malignant cells
[27,30]. Capsaicin caused sister chromatid exchanges and micronu-
clei production in human lymphocytes [30] and stimulated DNA
strand breaks in human neuroblastoma cells SHSY-5Y and MCF-7
breast cancer cells [27,49]. However, genotoxic potential of cap-
saicin has been also questioned [29]. Using four genotoxicity assays,
namely, the Ames test, mouse lymphoma cell mutation test, mouse
in vivo bone marrow micronucleus test and chromosomal aberra-
tion in human peripheral blood lymphocytes (HPBL) assay, weak
genotoxic activity of capsaicin has been observed [29]. The authors
suggested that inadequate purity of capsaicin and the use of pepper
plant extracts instead of pure, analytical standard grade capsaicin
may yield false positive results on capsaicin genotoxic potential
[29]. Nevertheless, we used high purity capsaicin, so genotoxic
activity of capsaicin against A549 and DU145 cells was not due to
impurities.
When DSBs occur, a complex cellular response is induced to
promote DNA repair and maintain genome integrity [50]. During
cellular response to DNA damage, p53 binding protein (53BP1)-
dependent pathway is activated: 53BP1 is recruited to sites of
DNA damage due to methylation state-specific recognition of his-
tone H4-K20 by 53BP1 [51]. The biological role of 53BP1 is far
from being understood. Nevertheless, 53BP1 has been shown to be
involved in the regulation of activation of the G2/M phase check-
point [52,53] and the intra-S phase checkpoint [54], and repair
of DNA DSBs via non-homologous end-joining (NHEJ) [55].We
have monitored 53BP1 foci during capsaicin-mediated genotoxicity
and found a negative correlation between micronuclei production
and 53BP1 foci, especially in DU145 cells. A link between recruit-
ment of 53BP1 and resolution of DNA damage has been already
reported [56]. In 53BP1-depleted WI38 human fibroblasts sub-
jected to DNA damage-promoting conditions, the percentage of
micronuclei-positive cells was increased [56]. Moreover, a more
robust activation or upregulation of 53BP1 in response to DNA
damage resulted in lower level of chromosomal damage [56]. Thus,
53BP1 was suggested to contribute to genomic stability. Perhaps,
a more potent recruitment of 53BP1 may account for diminished
MN generation after DU145 cell treatment with capsaicin at higher
concentrations.
The introduction of DNA damage in a cancer cell by geno-
toxic agents/chemicals would be a beneficial approach if DNA
damage-associated cell death would occur. However, unresolved
DNA damage may also promote gene mutation and/or genomic
instability, which, in turn, may affect cell susceptibility to cell
cycle checkpoint control and apoptosis. Thus, nutraceutical-based
genotoxic agents with the ability to eliminate rapidly cancer
cells without causing potentially detrimental genomic instabili-
ties and/or mutagenic side effects should be screened. Perhaps,
capsaicin is not a promising candidate for anticancer therapy,
because capsaicin-induced genotoxicity is accompanied by lim-
ited cytotoxicity in A549 and DU145 cells. Capsaicin-mediated
genomic instability may be a part of an adaptive response of can-
cer cells that allow them to survive after capsaicin treatment. More
recently, capsaicin-induced autophagy was found to protect human
breast cancer cells against apoptosis [25]. Autophagy is a lysosome-
dependent and energy supplying degradation process that is a part
of cellular stress response promoting cell survival [57]. Capsaicin-
induced DNA damage provoked autophagy through AMPK␣–mTOR
signaling pathway, which, in turn, regulated activation of ATM,
DNA–PKcs and PARP-1, and prolonged breast cancer cell survival
[49]. Capsaicin-mediated induction of DNA repair signaling and
autophagy may interrupt the treatment of human breast cancer
[49]. However, autophagy genes may be considered another target
of anticancer therapy.
Epigenetic changes in cancer cells may involve global DNA
hypomethylation resulting in the expression of quiescent proto-
oncogenes and prometastatic genes and promoting tumor
progression, and promoter-localized hypermethylation causing
transcriptional silencing of tumor suppressor genes and affecting
the control of tumorigenesis [58]. Nutraceuticals may modulate
cancer cell epigenome, e.g., dietary phytochemicals have been
shown to affect DNA methylation, histone modifications and
miRNA expression, which may promote cancer cell chemosensiti-
zation, being a particularly attractive anticancer strategy [59,60].
Because data on capsaicin-mediated epigenetic changes in can-
cer cells are lacking, we decided to evaluate capsaicin-associated
global DNA methylation. The level of 5-methyl-2-deoxycytidine
(5mdC) was unaffected in DU145 cells after capsaicin treatment,
whereas capsaicin was found to be a potent DNA hypermethy-
lating agent in A549 cells. Thus, capsaicin epigenetic action may
be considered cancer cell-specific and at least in A549 cells cap-
saicin may promote DNA hypermethylation-mediated changes in
gene expression. More recently, capsaicin has been documented a
potent modulator of microRNAs in cancer cells, which may affect
gene expression profiles [61,62]. Capsaicin restored the p53/miR-
34a regulatory axis and decreased survival of non-small cell lung
carcinoma cells [61]. Capsaicin also downregulated miR-520a-5p in
human chronic myeloid leukemia cells (K562 cells), which resulted
in cell growth retardation and apoptosis via targeting STAT3 [62].
In conclusion, we showed that capsaicin may induce DNA and
chromosomal damage in A549 and DU145 cancer cells, which may
contribute to limited susceptibility of these cells to apoptotic cell
death and may challenge the use of capsaicin in anticancer thera-
pies, at least in lung and prostate cancer treatment.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This study was supported by Grant from the National Science
Center, 2013/11/D/NZ7/00939. Experiments using flow cytometry
were performed in the Laboratory of Cytometry at the Nencki Insti-
tute of Experimental Biology.
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