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Boldo prevents UV light and nitric oxide-mediated plasmid
DNA damage and reduces the expression of Hsp70 protein
in melanoma cancer cellsjphp_1320 1219..1229
Alessandra Russoa, Venera Cardileb, Silvia Caggiab,
Germán Guntherc, Nicolas Troncosodand Juan Garbarinoe
Departments of aBiological Chemistry, Medical Chemistry and Molecular Biology, bPhysiological Sciences,
University of Catania, Catania, Italy, cLaboratorio de Cinética y Fotoquímica, Facultad de Ciencias
Químicas y Farmacéuticas, Universidad de Chile, dLo Vicuña & Cia., Santiago and eDepartment of
Chemistry, University T.F. Santa Maria, Valparaiso, Chile
Abstract
Objectives This study was designed to investigate the potential protective effect of a
methanolic extract of Peumus boldus leaves on UV light and nitric oxide (NO)-mediated
DNA damage. In addition, we investigated the growth inhibitory activity of this natural
product against human melanoma cells (M14).
Methods Boldine, catechin, quercetin and rutin were identified using a HPLC method.
The extract was incubated with plasmid DNA and, before irradiating the samples with UV-R,
H2O2was added. For analysis of DNA single-strand breaks induced by NO, the experiments
were performed by incubating the extract with Angeli’s salt. In the study on M14 cell line,
cell viability was measured using MTT assay. Release of lactate dehydrogenase, a marker of
membrane breakdown, was also measured. For the detection of apoptosis, the evaluation
of DNA fragmentation (COMET assay) and caspase-3 activity assay were employed.
The expression of heat shock protein 70 (Hsp70) was detected by Western blot analysis.
Generation of reactive oxygen species was measured by using a fluorescent probe.
Key findings The extract (demonstrating the synergistic effect of the constituents boldine
and flavonoids), showed a protective effect on plasmid DNA and selectively inhibited the
growth of melanoma cells. But a novel finding was that apoptosis evoked by this natural
product in M14 cells, appears to be mediated, at least in part, via the inhibition of Hsp70
expression, which may be correlated with a modulation of redox-sensitive mechanisms.
Conclusions These results confirm the promising biological properties of Peumus boldus
and encourage in-vivo investigations into its potential anti-cancer activity.
Keywords apoptosis; heat shock protein; melanoma cells; Peumus boldus; ultraviolet
radiation
Introduction
Boldo consists of the dried leaf of Peumus boldus Molina (Monimiaceae), an evergreen
shrub or a small tree growing in central and southern Chile. It has been used for its medicinal
properties by diverse indigenous groups, including the Mapuche ethnia who lived in Chile
before the arrival of the Spanish in the fifteenth century.[1] Actually, boldo is widely used in
Chilean folk medicine and is recognised as a medicinal herb in Pharmacopoeia.[1] Introduced
in France in about 1870 by Bourgoin and Verne, this species is described in the French
Pharmacopoeia. It is also included in the Pharmacopoeias of Switzerland, Germany, Brazil,
Chile, Portugal, Rumania and Spain and it is employed in the form of infusions, tinctures and
extracts.[1,2] Its choleretic and cholagogue properties are often reported. Besides these main
indications, boldo is also used as a diuretic, urinary tract anti-inflammatory agent, sto-
machic, sedative and emetic, and in the treatment of headache, earache, toothache and
rheumatism.[2] Boldo leaves contain 0.4–0.5% of at least 17 different alkaloids belonging to
the large benzylisoquinoline-derived family. Boldine is the major alkaloid and its content in
boldo leaves is 0.12%.[3] Leaves of P. boldus also contain essential oils of complex and
variable composition, tannins and flavonoids, such as catechin and flavonol aglycons,
kaempferol and quercetin, and their glycosides (i.e. rutin).[1,4] Catechin is the flavonoid most
abundant and, with the alkaloid boldine, is the main contributor to the antioxidant activity of
Research Paper
JPP 2011, 63: 1219–1229
© 2011 The Authors
JPP © 2011 Royal
Pharmaceutical Society
Received February 15, 2011
Accepted May 24, 2011
DOI
10.1111/j.2042-7158.2011.01320.x
ISSN 0022-3573
Correspondence: Alessandra
Russo, Department of Biological
Chemistry, Medical Chemistry
and Molecular Biology,
University of Catania, V.le A.
Doria 6, 95125 Catania, Italy.
E-mail: alrusso@unict.it;
ales0303@libero.it
1219
boldo leaf extracts.[1,4] For this high catechin content of boldo
leaves and its bioactivity, it has been suggested that quality
control of boldo leaf has to combine the analysis of catechin
as well as the characteristic aporphine alkaloids.[4]
In addition to its antioxidant properties, the boldine mol-
ecule has two major absorption peaks, at 280 and 302 nm.[2,5]
The latter would confer boldine a UV light-filtering property
relevant to a photo-protective action. Hidalgo et al.[6] showed
that it displays a photo-protector effect against UV-B both
in vitro and in vivo in mice. More recently, Rancan et al.[7]
investigated the photo-filtering properties of boldine in
humans. These authors observed that the topical application
of boldine protected the skin against erythema formation.
Also catechin and rutin, in our previous study, showed a
protective effect on DNA damage induced by hydroxyl radi-
cals (.OH) generated from UV photolysis of H2O2.[8] Exposure
to ultraviolet radiation (UV-R) induces genotoxic effects that
contribute not only to skin photoaging but also to skin car-
cinogenesis.[9] Ninety percent of skin cancer cases have been
attributed to the solar UV radiation, particularly its UV-B
component which is greatly absorbed by cellular DNA. Also,
UV-B indirectly damages DNA through reactive oxygen
species (ROS) formation, which facilitate the oxidation of
DNA and premature skin ageing possibly resulting in skin
cancer.[10] In view of these considerations, we analysed, using
an HPLC method, a methanolic extract from leaves of
P. boldus for boldine, catechin, quercetin and rutin content,
and we examined its effect on pBR322 DNA cleavage induced
by .OH generated from UV-photolysis of hydrogen peroxide
(H2O2) and by nitric oxide (NO).
Boldine and flavonoids quercetin and catechin have been
shown to exhibit anti-cancer activity in preclinical studies.[11–13]
Our previous studies found that this methanolic extract exhib-
ited comparable degrees of anti-growth effect on human cancer
epithelial cell lines, probably by the induction of apoptosis.[14]
Therefore, we also investigated the activity of this natural
product against the human melanoma cell line, M14. Several
biochemical parameters were tested, such as cell viabi-
lity (3(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium
bromide, MTT, assay) and cell membrane integrity (lactate
dehydrogenase, LDH, release).[15] For the detection of apopto-
sis, the evaluation of DNAfragmentation (COMET assay) and
caspase-3 activity assay were employed.[15]
The molecular chaperone Hsp70 acts at multiple steps in a
protein’s life cycle, including the processes of folding, traf-
ficking, remodelling and degradation. The protective presence
of Hsp70 can be beneficial for the whole organism, if Hsp70
is expressed in normal cells; however, in cancer cells, Hsp70
is a negative prognostic marker.[16] In cancer cells, the expres-
sion of Hsp70 is abnormally high, and Hsp70 may participate
in oncogenesis and in resistance to chemotherapy. Its tumo-
rigenic potential seems to correlate with its anti-apoptotic
ability.[17] However, only a small number of pharmacological
Hsp70 inhibitors have been identified; these include the fla-
vonol quercetin.[18] Therefore, in this study, a possible rela-
tionship between Hsp70 expression and cell death elicited by
extract from leaves of P. boldus in M14 cells, was investi-
gated. The possible induction of oxidative stress was also
evaluated by performing a fluorescent analysis of intracellular
ROS production.[15]
Taken together, our results demonstrate, for the first time,
that a methanolic extract of the leaves of P. boldus, for the
synergistic activity of its components (boldine and flavonoids,
catechin, quercetin and rutin, evidenced by HPLC analysis), is
able to exhibit a protective effect on NO and UV radiation-
induced DNA cleavage and to inhibit the growth of melanoma
cells. In addition, our data seem to indicate that the apoptosis
evoked by this natural product in M14 cells, at least in part,
appears to be induced by a reduction of Hsp70 expression,
associated with an increase of ROS production. The results
obtained in our experimental conditions have more value,
considering that, in all the parameters examined, the metha-
nolic extract from leaves of Peumus boldus exhibited no effect
on normal human cells.
Materials and Methods
Chemicals
All reagents were of commercial quality and were used
as received. pBR322 plasmid DNA, diethylenetriamine-
pentaacetic acid (DTPA), 3(4,5-dimethylthiazol-2-yl)2,5-
diphenyl-tetrazolium bromide (MTT) and b-nicotinamide-
adenine dinucleotide (NADH) were obtained from Sigma
Aldrich Co (St Louis, USA). All other chemicals were pur-
chased from Sigma Aldrich Co (St Louis, USA) and Gibco
BRL Life Technologies (Grand Island, USA).
Plant material
The leaves of P. boldo were collected at Quintay (Valparaiso)
in January 2006. A voucher specimen (voucher specimen
No. 12–07) was deposited in the Department of Chemistry,
Universidad Santa Maria, Valparaiso, Chile. The leaves of
P. boldo were exhaustively extracted with methanol and con-
centrated under vacuum to a residue (yield 165 g (14.34%)).
Boldine, catechin, quercetin and rutin concentration was
evaluated in the methanolic extract from leaves of P. boldus
by chromatography, using an HPLC Waters system equipped
with detection by diode arrangements. These compounds
were identified by comparing their retention times (Rt)
and spectra obtained with those of standards. For boldine
the following conditions were used: dilution in methanol
(33.6 mg in 20 ml), isocratic elution with methyl alcohol and
phosphate buffer 0.025 m, at 1 ml/min. Detection was done at
the maximum of the boldine spectra, 310 nm, Rt 2.1 min. For
catechin a dilution of 50 mg in 10 ml of solvent (methanol :
water 80 : 20), a system coupled in a series of two monolithic
columns with precolumn of 10 mm and elution with solvent
mixture (methanol : water 80 : 20) were used. Detection was
done at 280 nm, Rt 11.5 min. For quercetin and rutin the
following conditions were used: dilution in methanol (26 mg
in 20 ml), isocratic elution with acetonitrile and phosphate
buffer 0.025 m, at 1 ml/min (acetonitrile : phosphate buffer
80 : 20); detection was done at 370 nm. The retention times
were: quercetin =Rt 18 min; rutin =Rt 4 min.
Activity in cell-free systems
DNA cleavage induced by hydrogen peroxide
UV photolysis
The experiments were performed, as previously reported,[15] in
a volume of 20 ml containing 33 mmin bp of pBR322 plasmid
1220 Journal of Pharmacy and Pharmacology 2011; 63: 1219–1229
DNA in 5 mmphosphate saline buffer (pH 7.4), and the
methanolic extract from P. boldus leaves at different concen-
trations. Immediately before irradiating the samples with UV
light, H2O2was added to a final concentration of 2.5 mm.
Untreated pBR322 plasmid was included as a control in each
run of gel electrophoresis, conducted at 1.5 V/cm for 15 h.
Gel was stained in ethidium bromide (1 mg/ml; 30 min) and
photographed on Polaroid-Type 667 positive land film. The
intensity of each scDNA band was quantified by means of
densitometry. Parallel experiments, were also carried out in
the presence of boldine and quercetin.
Analysis of DNA single-strand breaks induced by
Angeli’s salt
The experiments were performed, as previously reported,[15]
by incubating pBR322 plasmid DNA in 100 mmsodium
phosphate buffer, pH 7.4, containing 0.1 mmDTPA, 0.15 mm
Angeli’s salt (prepared in 0.01 nNaOH), an appropriate
amount of HCl to neutralise the NaOH present in the solution
of Angeli’s salt, and the methanolic extract from P. boldus
leaves, at different concentrations, at 37°C for 1 h (final
volume 10 ml, final pH 7.5). Untreated pBR322 plasmid was
included as a control in each run of gel electrophoresis, con-
ducted at 1.5 V/cm for 15 h. Gel was stained in ethidium
bromide (1 mg/ml; 30 min) and photographed on Polaroid-
Type 667 positive land film. The intensity of each scDNA
band was quantified by means of densitometry. Parallel
experiments, were also carried out in the presence of boldine,
catechin, quercetin and rutin.
Study in cell culture
Cell culture and treatments
M14 human melanoma cells were grown in RPMI containing
10% fetal calf serum (FCS), 100 U/ml penicillin, 100 mg/ml
streptomycin and 25 mg/ml fungizone. Normal human
non-immortalised buccal fibroblast cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% FCS, 100 U/ml penicillin, 100 mg/ml strep-
tomycin, and 25 mg/ml fungizone. The cells were plated at a
constant density to obtain identical experimental conditions
in the different tests, thus to achieve a high accuracy of the
measurements.
In the MTT assay the cells were plated at 6 ¥103cells per
well for human cancer cells, and at 2 ¥104cells per well for
normal human non-immortalised buccal fibroblast cells,
in a 96-well flat-bottomed 200 ml microplate. In other tests,
the cells were plated at 8 ¥105cells (2 ml) for human cancer
cells, and 2 ¥106cells (2 ml) for normal human non-
immortalised buccal fibroblast cells, per 35 mm culture dish.
After 24 h incubation at 37°C under humidified 5% carbon
dioxide to allow cell attachment, the cells were treated with
different concentrations of methanolic extract from P. boldus
leaves and incubated for 72 h under the same conditions. This
time of treatment was chosen since no effect of methanolic
extract from P. boldus leaves was observed before 72 h of
treatment, at least for parameters examined by us. In the MTT
assay the cells were also exposed to pure compounds boldine,
catechin, quercetin and rutin. Stock solutions of the extract
and pure compounds were prepared in ethanol and the final
concentration of this solvent was kept constant at 0.25%.
Control cells received ethanol alone.
MTT bioassay
MTT assay was performed as described previously.[15] The
optical density of each well sample was measured with a
microplate spectrophotometer reader (Digital and Analog
Systems, Rome, Italy) at 550 nm.
Lactate dehydrogenase release
LDH release was spectrophotometrically measured in the
culture medium and in the cellular lysates at 340 nm by anal-
ysing NADH reduction during the pyruvate-lactate trans-
formation, as previously reported.[15] The percentage of LDH
released was calculated as percentage of the total amount,
considered as the sum of the enzymatic activity present in the
cellular lysate and that in the culture medium. A Hitachi
U-2000 spectrophotometer (Hitachi, Tokyo, Japan) was used.
DNA analysis by COMET assay
The presence of DNA fragmentation was examined by
single-cell gel electrophoresis (COMET assay), as previously
reported.[15] Software (Leica-QWIN) allowed us to assess the
quantitative and qualitative extent of DNA damage by mea-
suring: (a) tail length (TL), intensity (TI) and area (TA); (b)
head length (HL), intensity (HI) and area (HA). Finally,
the program using these parameters calculates the level of
DNA damage as: (a) the percentage of the fragmented DNA
(TDNA) and (b) tail moment (TMOM). The tail moment is
defined as the product of the percentage of DNA in the tail of
the comet and TD value, which is obtained by calculating the
distance between the centre of mass of the comet head and the
centre of mass of the tail. The percentage of DNA in the comet
tail was calculated as the rate of the fluorescence intensity in
the comet tail relative to the total fluorescence; 100 randomly
selected cells were analysed per sample.
Activity of caspase-3
The activity of caspase-3 was determined by using the
Caspase colorimetric assay Kit (Sigma RBI, St Louis, USA).
This assay measures the cleavage of a specific colorimetric
caspase substrate, acetyl-Asp-Glu-Val-Asp p-nitroanilide
(Ac-DEVD-pNA). pNA (p-nitroaniline) is released from the
substrate upon cleavage by caspase. Free pNA produces a
yellow colour that is monitored by a Hitachi U-2000 spec-
trophotometer (Hitachi, Tokyo, Japan) at l=405 nm. The
caspase-3 activity was measured in cell lysates according to
the manufacture’s protocol. The total protein content, used to
reflect cell number and measured as previously described,[15]
was evaluated for each sample, and the results are reported as
OD405 nm/mg protein and compared with control.
Western blot analysis
The expression of heat shock protein 70 (Hsp70) was evaluated
by Western blot analysis. Briefly, the untreated and treated M14
cells were washed twice with ice-cold phosphate-buffered
saline (PBS) and collected with lysing buffer (10 mmTris-HCl
plus 10 mmKCl, 2 mmMgCl2, 0.6 mmPhenylmethanesul-
fonyl fluoride and 1% SDS, pH 7.4). After cooling for 30 min
at 0°C, cells were sonicated. Twenty micrograms of total
Boldo prevents melanoma Alessandra Russo et al.1221
protein, present in the supernatant, were loaded on each lane
and separated by 4–12% Novex Bis-Tris gel electrophoresis
(NuPAGE; Invitrogen, Milan, Italy). Proteins were then trans-
ferred to nitrocellulose membranes (Invitrogen, Italy) in a wet
system. The transfer of proteins was verified by staining the
nitrocellulose membranes with Ponceau S and the Novex
Bis-Tris gel with Brillant blue R. Membranes were blocked
in Tris-buffered saline containing 0.01% Tween-20 (TBST)
and 5% non-fat dry milk at 4°C overnight. Mouse monoclonal
anti-Hsp70 (1 : 200 dilution) (Santa Cruz Biotechnology,
Santa Cruz, CA) and mouse monoclonal anti-a–tubulin
(1 : 5000 dilution) (Sigma, Milan, Italy) antibodies were
diluted in TBST and membranes incubated for 24 h at
room temperature. Antibodies were detected with horseradish
peroxidase-conjugated secondary antibody using the enhanced
chemiluminescence detection Supersignal West Pico Chemi-
luminescent Substrate (Pierce Chemical Co., Rockford, USA).
Bands were measured densitometrically by ImageJ software
and their relative density calculated based on the density of
the a-tubulin bands in each sample. Values were expressed as
arbitrary densitometric units corresponding to signal intensity.
Reactive oxygen species assay
ROS determination was performed by using a fluorescent
probe (DCFH-DA) as previously described.[15] The fluores-
cence (corresponding to the radical species-oxidised DCF)
was monitored spectrofluorometrically using a Hitachi
F-2000 spectrofluorimeter (Hitachi, Tokyo, Japan): excitation
488 nm, emission 525 nm. The total protein content,
measured as previously described,[15] was evaluated for
each sample, and the results are reported as fluorescence
intensity/mg protein and compared with control.
Statistical analysis
Each value represents the mean ⫾SD of three experiments
performed in quadruplicate. Results were analysed using one-
way analysis of variance followed by Dunnett’s post-hoc
test for multiple comparisons with control. All statistical
analyses were performed using the statistical software
package SYSTAT, version 9 (Systat Inc., Evanston, USA).
Differences were considered significant at P<0.05.
Results
Analysis of extract
The methanolic extract was subjected to HPLC analysis
to determine boldine, catechin, quercetin and rutin
(Figures 1–3). The identification of these compounds was
evaluated by comparing their retention times (Rt) and spectra
obtained with those of standards. The concentrations obtained
for these compounds were: boldine 1.05%, Rt. 2.1 min; cat-
echin 1.44%, Rt. 11.5 min; quercetin 0.1%, Rt. 18 min; rutin
0.14%, Rt. 4.0 min.
Biological activity in cell-free systems
The methanolic extract from P. boldus leaves was tested for its
potential DNA-protective activity on pBR322 DNA cleavage
induced by hydroxyl radicals (.OH), generated from UV pho-
tolysis of H2O2, and by NO. DNA derived from pBR322
plasmid showed two bands on agarose gel electrophoresis; the
faster moving band corresponded to the native form of super-
coiled circular DNA (scDNA) and the slower moving band
was the open circular form (ocDNA). The UV irradiation of
DNA in the presence of H2O2suppresses the formation of
scDNA, producing ocDNA and linear form (linDNA), indi-
cating that .OH generated from UV photolysis of H2O2pro-
duces DNA strand scission.[8,15] The addition of the extract,
at 200–800 mg/ml concentrations, to the reaction mixture
suppressed the formation of linDNA and induced a partial
recovery of scDNA. In fact, the intensity of scDNA bands for
plasmid DNA, treated with H2O2in the presence of 800 mg/ml
methanolic extract, was 94% (Table 1). The treatment of
plasmid DNA with extract alone did not change the migration
pattern (data not shown). To examine the role of the pure
compounds, their effect was also investigated using the same
experimental conditions. The results showed that boldine and
quercetin, tested at a concentration (50 mm) greater than that
present in the methanol extract from leaves of P. boldus, had
no effect (Table 1). On the other hand, our previous study
0.25
0.20
0.15
0.10
Boldine
(a)
(b)
0.05
0.00
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.0 0.5 1.0 1.5
Time (min)
Absorbance (au)Absorbance (au)
2.0 2.5 3.0
01234
Time (min)
Figure 1 HPLC chromatograms at 310 nm of methanolic extract from
P. boldus leaves (a) and standard solution of boldine (b).
1222 Journal of Pharmacy and Pharmacology 2011; 63: 1219–1229
reported that catechin and rutin exhibited a protective effect
on plasmid DNA damage induced by UV photolysis of H2O2
only at doses higher than 200 mm.
Angeli’s salt, a NO donor,[19] as previously reported,[15]
induced in our experimental condition, a significant decrease
in the scDNA band intensity (Table 2). The methanolic
extract, at 200–800 mg/ml concentration, was able to reduce
the NO-induced DNA damage, acting as a nitric oxide donor
agent (Table 2). The treatment of plasmid DNA with extract
alone did not change the migration pattern (data not shown).
Also in this assay, pure compounds boldine, catechin, quer-
cetin and rutin, tested at a concentration (50 mm) greater than
that present in the methanol extract from leaves of P. boldus,
exhibited no protective activity on NO-induced DNA damage
(Table 2).
Study in cell culture
The methanolic extract from P. boldus leaves was tested
in vitro for its potential activity against cell growth in human
melanoma cells, M14, using MTT assay. The results, sum-
marised in Figure 4, show that the methanolic extract at
5–40 mg/ml concentrations, exhibited a significant inhibitory
effect (P<0.001) on M14 cell growth. In particular, the vital-
ity was 4% in cells exposed to 40 mg/ml concentration. Inter-
estingly, the natural product examined, in our experimental
conditions (5–40 mg/ml), revealed no cytotoxic effect against
normal human buccal fibroblast cells (Figure 4), considered a
useful model to evaluate the cytotoxic effects of carcino-
gens[20] and the tumour-specific cytotoxicity of potential anti-
neoplastic agents.[21]
In an attempt to elucidate the role of boldine and flavonoids,
catechin, quercetin and rutin, in the potential cancer cell anti-
growth activity of the extract from leaves of P. boldus, we also
evaluated the effect of these pure compounds. The results
obtained revealed that all compounds examined, also at con-
centrations greater than those present in the extract, were
unable to affect the vitality of cancer cells (Figure 5). Only
catechin, at 25 and 50 mmconcentrations, showed a significant
inhibitory effect (P<0.001) on M14 cells.
LDH is a soluble enzyme located in the cytosol, which is
released into the surrounding culture medium upon cell
damage and lysis. Measuring LDH in the culture medium can
therefore be used as an indicator of membrane integrity, and
thus a measurement of cytotoxicity.[15] No statistically signifi-
cant increase in LDH release was observed in M14 cells
treated with the methanolic extract from P. boldus leavesat5
and 10 mg/ml concentrations (Table 3). Conversely, a signifi-
cant increase in LDH was observed at higher concentrations
(20 and 40 mg/ml) (Table 3).
Nuclear DNA was analysed using single-cell gel electro-
phoresis (SCGE), known as COMET assay, a sensitive
method for the visualisation of DNA damage measured at the
level of individual cells. The COMET assay also allows us to
distinguish apoptotic from necrotic cells based on the DNA
fragmentation pattern.[22] The COMET pattern significantly
differs between apoptotic and control cultures as well as
between apoptotic and necrotic cultures. Quantification of the
COMET data, in our experimental condition, is reported as
TDNA and TMOM in Table 4. The results clearly show an
increase in both TDNA and TMOM at 5 and 10 mg/ml con-
centrations. These findings seem to suggest that the extract
from P. boldus leaves induced, also at lower concentration of
5mg/ml, cell death by apoptosis, because data in the literature
indicate that only COMETS with high values of TMOM can
be related to apoptosis.[23]
Active caspases cleave several important intracellular pro-
teins, leading to the morphological and biochemical changes
associated with apoptosis, such as oligonucleosomal fragmen-
tation of chromosomal DNA.[24] Caspase-3 is the major execu-
tioner caspase in the caspase cascade, therefore experiments
were performed to characterise the role of activation of this
protein in cell growth inhibition mediated by tested extract.As
shown in Figure 6, the activity of caspase-3, measured by
pNA (p-nitroaniline), released from the specific caspase sub-
strate, and reported as OD405 nm/mg protein, was significantly
increased in M14 cells treated with the methanolic extract at 5
and 10 mg/ml concentrations, supporting the hypothesis that
the cell growth inhibition, demonstrated in these experimental
conditions, was correlated to an early signal of apoptosis.
Conversely, at 20 and 40 mg/ml concentrations, the activity of
this protease returned to control values.
We also tested the effect of this natural product on normal
human non-immortalised fibroblast cells. At 5–10 mg/ml, the
extract was ineffective in inducing apoptosis in these cells, as
demonstrated by the results presented in Table 4 (COMET
assay) and Figure 6 (caspase-3 activity). Also at 20 and
40 mg/ml concentrations, this natural product did not exhibit
cytotoxic activity; in fact the LDH release was unmodified
with respect to the values observed in control untreated cells
(Table 3).
0,10 Catechin
a
b
Absorbance (au)Absorbance (au)
0,08
0,06
0,04
0,02
0,00
0,35
0,30
0,25
0,20
0,15
0,10
0,05
0,00
0 5 10 15
Time (min)
20 25 30
0 5 10 15
Time (min)
20 25 30
Figure 2 HPLC chromatograms at 280 nm of methanolic extract from
P. boldus leaves (a) and standard solution of catechin (b).
Boldo prevents melanoma Alessandra Russo et al.1223
Hsp70, because of its chaperone function, has been shown
to affect the apoptotic process and thereby increases the sur-
vival of cells exposed to a wide range of lethal stimuli. Hsp70
has also been shown to act as an inhibitor of apoptosis in
cancer cells, increasing the tumorigenicity of these cells.[16,17]
Therefore, a possible relationship between Hsp70 expression
and cell death elicited by extract from leaves of P. boldus in
M14 cells, was investigated. In Figure 7 the results of Hsp70
immunoblotting are reported. The data show a reduction of
levels of this protein in M14 cells treated with the methanolic
extract from P. boldus leaves at 5–20 mg/ml concentrations,
compared with the values of control untreated cells. Hsp70
expression was undetectable in cancer cells exposed to extract
at 40 mg/ml concentration. (data not shown). In normal human
fibroblast cells, the level of Hsp70 expression was unmodified
at all concentrations tested (5–20 mg/ml) (Figure 7).
ROS have been reported to be involved in cell death
induced by a variety of stimuli and different antitumoral
agents. We therefore examined whether tested extract-induced
cell death may be correlated at an elevation of ROS. To
assess changes in intracellular ROS levels, we employed an
oxidation-sensitive fluorescent probe DCFH-DA. DCFH-DA
can be taken up into cells, and then oxidised by ROS to its
Quercetin
Rutin
0.010
Absorbance (au)
Absorbance (au)
0.008
0.006
0.004
0.002
0.000
–0.002
–0.004
0 5 10 15
Time (min)
Time (min)
20
02468
25 30
0.10
Absorbance (au)
0.08
0.06
0.04
0.02
0.00
0.10
0.12
0.14
Absorbance (au)
0.08
0.06
0.04
0.02
0.00
0 5 10 15
Time (min)
20
02134
Time (min)
5
(a)
(b)
(c)
Figure 3 HPLC chromatograms at 370 nm of methanolic extract from P. boldus leaves (a) and standard solutions of quercetin (b) and rutin (c).
Table 1 Effect of methanolic extract from P. boldus leaves (BOE) and
pure compounds, boldine and quercetin on DNA cleavage induced by the
photolysis of H2O2
Treatment Densitometric units of supercoiled DNA
(% of native DNA)
scDNA 100
BOE
200 mg/ml 44 ⫾4.6*
400 mg/ml 83 ⫾7.1
800 mg/ml 94 ⫾6.3
Boldine
50 mm_
Quercetin
50 mm_
The hydroxyl radicals generated by the photolysis of H2O2suppressed the
supercoiled DNA (scDNA). The values are expressed as densitometric
units obtained by scanning the agarose gel electrophoresis photos. Each
value represents the mean ⫾SD of three experiments performed in
quadruplicate. *P<0.001 vs supercoiled DNA.
1224 Journal of Pharmacy and Pharmacology 2011; 63: 1219–1229
fluorescent derivative DCF. We found that the DCF fluores-
cence increased in a concentration-dependent manner in M14
cells treated with the extract from P. boldus leaves (Figure 8).
Alternatively, also in this assay the extract exhibited insignifi-
cant effect on fibroblast cells, also at higher concentration of
40 mg/ml (Figure 8).
Discussion
The skin is the largest organ of the human body and a primary
target for an array of environmental insults. As ozone is
becoming thinner, our skin becomes more in contact with a
variety of harmful agents such as induction of oxidative stress
and exposure to UV-R. UV-R has been identified as a cause of
several hazardous cutaneous effects, including immune sup-
pression, dermatitis, premature aging and skin cancer. UV-R
can cause direct biological damage, or indirect damage via the
production of ROS. These cause oxidative damage to DNA,
proteins and lipids.[25] The epidermis contains antioxidant
defences including the enzymes, superoxide dismutase, glu-
tathione peroxidase and catalase, which remove ROS from the
skin. Increased production of ROS following exposure to
UV-R can deplete these antioxidant defences, leaving the skin
vulnerable to attack from ROS.[25] In addition, it has become
clear that perturbations or defects in the signaling cascade of
NO and reactive nitrogen intermediates have been shown to be
associated with common forms of skin diseases. It has been
reported that NO liberated following UV-R irradiation plays a
significant role in initiating erythema and inflammation.[26]
NO can combine with UV-induced superoxide to form perox-
ynitrite which exists in equilibrium with peroxynitrous acid.
These reactive nitrogen species are very toxic, and can cause
DNA damage, nitrosylation of tyrosine residues in proteins,
and initiate lipid peroxidation, all of which interfere with
cellular function.[26] Various compounds in foods as well as in
medicinal plants have been widely used for wound-healing,
anti-aging, and disease treatments in the skin. Their possible
use in the prevention of skin cancer, including melanoma, has
been suggested. The biological activity of these natural com-
pounds, has been correlated in part to their capacity to contrast
the oxidative and nitrosative stress.[26] Our results suggest that
also the methanolic extract from leaves of P. boldus, contain-
ing boldine 1.05%, catechin 1.44%, quercetin 0.1% and rutin
0.14%, which were detected using a HPLC Waters system
(Figures 1–3), could act in this way in skin protection. In
fact, it exhibited protection against DNA damage induced
by.OH radicals, generated by UV-photolysis of H2O2
(Table 1), and like carboxy-PTIO (2-(4-Carboxyphenyl)-
4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium
salt), a nitric oxide scavenger,[27] it was able to reduce
NO-induced DNA single-strand breakage (Table 2). Under
the same experimental conditions, boldine, catechin, querce-
tin and rutin, also at concentrations greater than those present
in the extract, as previously reported[8] and shown in Tables 1
and 2, exhibited no protective activity on plasmid DNA
damage, indicating that the extract capacity to prevent DNA
damage is most likely attributed to the synergistic effect of
different constituents. In the present study, we also show that
only the extract from leaves of P. boldus inhibited the growth
of M14 human cancer cell line (Figure 4). In fact, boldine,
catechin, quercetin and rutin also at the concentrations greater
than those present in the extract, were unable to affect the
vitality of cancer cells (Figure 5). Only catechin at 25 and
50 mM concentrations showed a significant inhibitory effect
(P<0.001). On the other hand, the recent studies of Gerhardt
et al.[11] reported that boldine, after 72 h of treatment,
decreased the cell number of different glioma cell lines at
doses higher than 80 mM, and in cell-type specific manner.
Interestingly, the extract from leaves of P. boldus exhibited
no effect on the viability of normal fibroblast cells (Figure 4).
These findings encouraged us to explore the mechanisms that
may be involved in the selective growth inhibitory activity of
the extract on M14 cells.
Table 2 Effect of methanolic extract from P. boldus leaves (BOE) and
pure compounds boldine, catechin, quercetin and rutin on Angeli’s salt-
mediated DNA damage
Treatment Densitometric units of supercoiled DNA
(% of native DNA)
scDNA 100
Angeli’s salt
0.2 mm8.0 ⫾0.7*
BOE
200 mg/ml 37 ⫾4.6*,**
400 mg/ml 56 ⫾6.6*,**
800 mg/ml 83 ⫾5.5**
Boldine
50 mm_
Catechin
50 mm_
Quercetin
50 mm_
Rutin
50 mm_
The values are expressed as densitometric units obtained by scanning the
agarose gel electrophoresis photos. Each value represents the mean ⫾SD
of three experiments performed in quadruplicate. *P<0.001 vs super-
coiled DNA; **P<0.001 vs Angeli’s salt-treated DNA.
120
100
80
60
40
20
0C5 10
Fibroblast cells
M14 cells
**
*
*
20 40
Cellular vitality (% control)
Concn (μ/ml)
μg/ml
Figure 4 Cell growth, assayed using MTT test, of fibroblast and M14
cells untreated and treated with the methanolic extract from P. boldus
leaves at different concentrations for 72 h. Stock solution of extract was
prepared in ethanol and the final concentration of this solvent was kept
constant at 0.25%. Control cultures received ethanol alone. Each value
represents the mean ⫾SD of three experiments, performed in quadrupli-
cate. *P<0.001 vs control untreated cells.
Boldo prevents melanoma Alessandra Russo et al.1225
120
100
80
60
40
C 6.25 12.5 25
Boldine
50 6.25 12.5 25
Catechin
50 6.25 12.5 25
Quercetin
50 6.25 12.5 25
Rutin
50 μM
20
0
Cellular vitality (% control)
*
*
Figure 5 Cell growth, assayed using MTT test, of M14 cells untreated and treated with pure compounds boldine, catechin, quercetin and rutin at
different concentrations for 72 h. Stock solution of pure compounds was prepared in ethanol and the final concentration of this solvent was kept
constant at 0.25%. Control cultures received ethanol alone. Each value represents the mean ⫾SD of three experiments, performed in quadruplicate.
*P<0.001 vs control untreated cells.
Table 3 Lactate dehydrogenase (LDH) release, expressed as percent-
age of LDH released into the cell medium with respect to total LDH, in
fibroblast and M14 cells treated with different concentrations of metha-
nolic extract from P. boldus leaves (BOE)
Treatment % LDH released
Fibroblast cells
Control 5.0 ⫾0.5
BOE
5mg/ml 7.2 ⫾0.7
10 mg/ml 6.6 ⫾0.5
20 mg/ml 8.3 ⫾0.3
40 mg/ml 5.1 ⫾0.4
M14 cells
Control 8.0 ⫾0.9
BOE
5mg/ml 9.7 ⫾0.7
10 mg/ml 9.5 ⫾1.2
20 mg/ml 40.1 ⫾3.7*
40 mg/ml 86.3 ⫾2.8*
The values are the mean ⫾SD of three experiments performed in qua-
druplicate. *P<0.001 vs control untreated cells.
Table 4 COMET assay of genomic DNA of fibroblast and M14 cells
untreated and treated with methanolic extract from P. boldus leaves
(BOE)
Treatment TDNA TMOM
Fibroblast cells
Control 26.5 ⫾2.0 89 ⫾4.2
BOE
5mg/ml 34 ⫾3.0 75 ⫾10
10 mg/ml 29 ⫾6.0 96 ⫾11
20 mg/ml 24 ⫾2.1 82 ⫾23
40 mg/ml 34 ⫾2.7 98 ⫾9
M14 cells
Control 19.3 ⫾3.0 91 ⫾4.5
BOE
5mg/ml 105 ⫾3.0* 1075 ⫾15*
10 mg/ml 149 ⫾6.0* 1135 ⫾12*
20 mg/ml 49 ⫾2.1* 323 ⫾21*
40 mg/ml 45 ⫾2.5* 327 ⫾9*
TDNA, % of the fragmented DNA; TMOM, tail moment expressed as the
product of TD (distance between head and tail) and TDNA. The values
are the mean ⫾SD of three experiments performed in quadruplicate.
*P<0.001vs control untreated cells.
1226 Journal of Pharmacy and Pharmacology 2011; 63: 1219–1229
In cutaneous cells, there is a homeostatic relationship
between cell proliferation and apoptosis. Alterations in either
cell proliferation or cell death can lead to a loss of growth
control, and thus play a major role in the process of tumori-
genesis. Defects of apoptotic pathways influence also drug
resistance, and because of these defects chemotherapy often
fails. Recent studies have suggested that the resistance of
human melanoma to apoptosis is an important mechanism
underlying this cancer’s aggressiveness and its poor response
to chemotherapeutic agents. The induction of apoptosis in
tumor cells is considered very useful in the management and
therapy of cancer, including melanoma.[28] It is thus consid-
ered important to screen apoptotic inducers from plants, either
in the form of extracts or as components isolated from them.
Consistent with this approach, our data suggest that the
methanolic extract from leaves of P. boldus is able to induce
apoptosis in melanoma cancer cells. In fact, a high DNA
fragmentation (Comet assay), occurred in M14 cells exposed
to this extract at 5–10 mg/ml concentrations. One pathway of
caspase activation is the intrinsic or mitochondrial pathway,
from which cytochrome c is released into the cytosol, which
interacts with cytosolic apoptosis protease-activating factor-1
μg/ml
Fibroblast cells
M14 cells
*
*
p-nitroaniline release
(OD405 nm/mg protein)
400
350
300
250
200
150
100
50
0
C 5 10 20 40
Concn (μ/ml)
Figure 6 Caspase-3 activity, determined by using the Caspase colori-
metric assay Kit (Sigma RBI St Louis, USA), in fibroblast and M14 cells
untreated and treated with the methanolic extract from P. boldus leaves at
different concentrations for 72 h. Stock solution of extract was prepared
in ethanol and the final concentration of this solvent was kept constant at
0.25%. Control cultures received ethanol alone. Each value represents the
mean ⫾SD of three experiments, performed in quadruplicate. *P<0.001
vs control untreated cells.
*
*
*
μg/ml
C
Hsp70
a-tubulin
510
Fibroblast cells
20
μg/ml
C
Hsp70
a-tubulin
510
2
1,5
1
0,5
0
M14 cells
20
Fibroblast cells
M14 cells
Hsp70 level (ADU)
C 5 10 20
Concn (μ/ml)
μg/ml
Figure 7 Levels of Hsp70 protein in fibroblast and M14 cells untreated and treated with the methanolic extract from P. boldus leaves at different
concentrations for 72 h. Stock solution of extract was prepared in ethanol and the final concentration of this solvent was kept constant at 0.25%. Control
cultures received ethanol alone. Values are expressed as arbitrary densitometric units (ADU) corresponding to signal intensity present on the
autoradiography of Western blots. Each value represents the mean ⫾SD of three experiments, performed in quadruplicate. *P<0.001 vs control
untreated cells.
*
*
*
Fibroblast cells
M14 cells
Fluorescence intensity/mg protein
(% of control)
1000
900
800
700
600
500
400
300
200
100
0C 5 10 20 40
Concn (μg/ml)
μg/ml
Figure 8 Reactive oxygen species (ROS) determination, performed by
using a fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA),
in fibroblast and M14 cells untreated and treated with the methanolic
extract from P. boldus leaves at different concentrations for 72 h. Stock
solution of extract was prepared in ethanol and the final concentration of
this solvent was kept constant at 0.25%. Control cultures received ethanol
alone. Each value represents the mean ⫾SD of three experiments, per-
formed in quadruplicate. °P<0.05, P<0.001 vs control untreated cells.
Boldo prevents melanoma Alessandra Russo et al.1227
(Apaf-1) and procaspase-9 to form the apoptosome, the
caspase-3 activation complex.[17] Our results show that, fol-
lowing incubation with Boldo extract, caspase-3 activity was
significantly (P<0.001)increased in M14 cancer cell line
(Figure 6), thus strongly suggesting that the mitochondrial
apoptotic pathway is involved in Boldo extract-induced apo-
ptosis. Alternatively, it was ineffective in inducing apoptosis
in human non-immortalised fibroblast cells, as shown in
Table 4 (COMET assay) and Figure 6 (caspase-3 activity).
The molecular chaperone Hsp70 acts at multiple steps in a
protein’s life cycle, including during the processes of folding,
trafficking, remodeling and degradation. The protective pres-
ence of Hsp70 can be beneficial for the whole organism, if
Hsp70 is expressed in normal cells, however in cancer cells,
Hsp70 is negative prognostic marker.[16] In cancer cells, the
expression of Hsp70 is abnormally high, and Hsp70 may
participate in oncogenesis and in resistance to chemotherapy.
Its tumorigenic potential seems to correlate with its ability to
disable apoptosis. Antisense constructs of Hsp70 have been
shown to sensitise cancer cells to apoptosis and to eradicate
tumors in several models.[17] Elevated Hsp70 levels block
the apoptotic pathway at different levels. Some studies have
suggested that Hsp70 may inhibit apoptosis by acting down-
stream of mitochondria and cytochrome c release.[17] This
anti-apoptotic effect was explained by the Hsp70-mediated
modulation of the apoptosome.[17] Indeed, Hsp70 has been
demonstrated to directly bind to the cytosolic apoptosis
protease-activating factor-1 (Apaf-1), thereby preventing the
recruitment of procaspase-9 to the apoptosome. Our data rein-
force the well documented existence of a linkage between
Hsp70 expression and cancer cell demise, and permit to
hypothesise that the reduction of Hsp70 levels induced in M14
by methanolic extract from leaves of P. boldus could allow
induction of apoptosis. In fact, the results obtained clearly
demonstrate that P. boldus leaf extract, at 5 and 10 mg/ml,
induced a reduction of Hsp70 expression (Figure 7)
correlated with a high DNA fragmentation (Table 4) and a
significant increase of the caspase-3 enzyme activity
(Figure 6). Alternatively, at higher concentrations, the extract
induced extreme damage, associated with a lower levels of
Hsp70 expression (Figure 7), and evidenced by a different
pattern of DNA damage (COMET assay) (Table 4), a high
LDH release (Table 3) and a reduction in the caspase-3 activ-
ity (Figure 6). The release of cytochrome c from mitochondria
and the inhibition of the mitochondrial respiratory chain was
assumed to result in the overproduction of ROS, which would
act as mediators of the death signaling pathway.[29] Studies
have shown that the addition of ROS or the depletion of
endogenous antioxidants can induce programmed cell
death.[30] Our data suggest that the significant increase of ROS
production, at 5–10 mg/ml concentrations, probably induced
by Hsp70 down-modulation, could amplify the apoptosis cas-
cades. Alternatively, at higher concentrations (20–40 mg/ml),
when the capacity of the cells to sustain Hsp70 synthesis is
reduced, our results seem to indicate that necrosis cell death,
associated with a high LDH release (Table 3), and demon-
strated by COMET assay values (Table 4), was induced by a
further increase in ROS production, generating intolerable
oxidative stress in cancer cells that are already near a thresh-
old for tolerating ROS.[31] Our hypothesis is further confirmed
by caspase-3 activity results, demonstrating a reduction in
the activity of this protease at higher concentration, 20 and
40 mg/ml (Figure 6). Our data obtained with normal fibroblast
cells also seem to support the existence of a correlation
between the Hsp70 down-modulation and a modification of
intracellular redox state. In fact, according to previous studies
showing that the cytotoxic effect of Hsp70 down-modulation
is particularly strong in transformed cells and undetectable in
normal cells,[17] boldo extract treatment exhibited no activity
on human normal fibroblast cells. This natural product had no
effect on ROS production at 5 and 10 mg/ml concentrations,
neither did it induce a stress response at higher concentrations
(20 and 40 mg/ml), as demonstrated by LDH release evalua-
tion (Table 3), COMET assay (Table 4) and Hsp70 levels
(Figure 7). On the other hand, while it has been reported
that phenolic phytochemicals have antioxidant/protective
properties in normal tissues and cells, they can, paradoxically,
induce the formation of ROS to achieve an intolerable level of
high oxidative stress in some cancer cells.[31]
Conclusions
Our data provided the first evidence that boldo, known from
ancient times for its health-beneficial characteristics, for the
synergistic effect of different constituents boldine and fla-
vonoids, is able to contrast UV light and NO-mediated DNA
damage. Therefore, they suggest its possible use for the pre-
vention of afflictions correlated to UV-R, such as skin cancer,
including melanoma.
The extract from leaves of P. boldus has also shown inter-
esting potential anti-tumour activity. Our results, in fact dem-
onstrate the capacity of this natural product, but not of pure
compounds boldine and flavonoids, catechin, quercetin and
rutin, to selectively attenuate the growth of M14 cells. The
central and novel finding in this pre-clinical study is that
apoptosis induced by this natural product in M14 cells appears
to be mediated, at least in part, via the inhibition of Hsp70
expression, which may be correlated with a modulation of
redox-sensitive mechanisms. In addition, we have provided
further support that Hsp70 confers resistance to apoptosis in
melanoma cancer cells. Therefore, the combination of boldo
with other anti-melanoma therapies could be considered a
promising strategy that warrants further in-vivo evaluation.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest
to disclose.
Acknowledgements
The authors would like to thank Professor Peter Fiedler for
proofreading the manuscript.
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Boldo prevents melanoma Alessandra Russo et al.1229
