DOI:http://dx.doi.org/10.7314/APJCP.2013.14.9.

5317
Antioxidant and Apoptotic Effects of an Extract of Urtica dioica on the MCF-7 Human Breast Cancer Cell Line

RESEARCH ARTICLE

Antioxidant and Apoptotic Effects of an Aqueous Extract of

Urtica dioica on the MCF-7 Human Breast Cancer Cell Line
Sadegh Fattahi1, Ali Motevalizadeh Ardekani2, Ebrahim Zabihi1, Zeinab Abedian1,
Amrollah Mostafazadeh1, Roghayeh Pourbagher1, Haleh Akhavan-Niaki1*
Abstract
Breast cancer is the most prevalent cancer and one of the leading causes of death among women in the world.
Plants and herbs may play an important role in complementary or alternative treatment. The aim of this study
was to evaluate the antioxidant and anti-proliferative potential of Urtica dioica. The anti oxidant activity of an
aqueous extract of Urtica dioica leaf was measured by MTT assay and the FRAP method while its anti-proliferative
activity on the human breast cancer cell line (MCF-7) and fibroblasts isolated from foreskin tissue was evaluated
using MTT assay. Mechanisms leading to apoptosis were also investigated at the molecular level by measuring
the amount of anti and pro-apoptotic proteins and at the cellular level by studying DNA fragmentation and
annexin V staining by flow cytometry. The aqueous extract of Urtica dioica showed antioxidant effects with a
correlation coefficient of r2=0.997. Dose-dependent and anti-proliferative effects of the extract were observed
only on MCF-7 cells after 72 hrs with an IC50 value of 2 mg/ml. This anti proliferative activity was associated
with an increase of apoptosis as demonstrated by DNA fragmentation, the appearance of apoptotic cells in
flow cytometry analysis and an increase of the amount of calpain 1, calpastatin, caspase 3, caspase 9, Bax and
Bcl-2, all proteins involved in the apoptotic pathway. This is the first time such in vitro antiproliferative effect
of aqueous extract of Urtica dioica leaf has been described for a breast cancer cell line. Our findings warrant
further research on Urtica dioica as a potential chemotherapeutic agent for breast cancer.
Keywords: Breast cancer - Urtica dioica - MCF-7 - apoptosis - anti oxidant

Asian Pac J Cancer Prev, 14 (9), 5317-5323

Introduction made to use herbs for cancer cure (Olaku and White,
2011; Huang et al., 2013; Suzuki et al., 2013). Due to
Breast cancer is the most prevalent cancer and one the presence of flavonoids, aromatic compounds with
of the leading causes of death among women in the antioxidant properties, plants can be effective in reducing
world. New statistics showed that in the US one out of the high level of oxidative stress present in cancerous
every eight women will suffer from breast cancer and cells. An association between consumption of fruits and
annually more than 200,000 new cases of breast cancer are vegetables with lower risk of cancer is widely accepted
diagnosed (DeSantis et al., 2011). According to a recent (Terry et al., 2001; Campbell et al., 2009). Urtica dioica
assessment based on varying levels of human development (stinging nettle) is an herbaceous perennial flowering plant
it was predicted that the incidence of all-cancer cases which grows wild in Iran. Urtica dioica has a variety of
including breast cancer as the most common cancer in uses as a medicine in disease treatment such as diabetes,
women will almost double by 2030 (Bray et al., 2012). atherosclerosis, cardiovascular disease, prostate cancer
Therefore, providing effective treatment strategies will (Konrad et al., 2000; Testai et al., 2002; El Haouari et al.,
play an important role in promoting community health, 2006; Bnouham et al., 2010). In this study, we investigated
particularly women’s health. Surgery, chemotherapy and the antioxidant and antiproliferative activity of the
radiation therapy are currently used in the treatment of aqueous extract of Urtica dioica on breast cancer MCF-7
breast cancer, but have serious side effects. Plants and cell line with various cellular and molecular methods.
herbs due to some advantages such as being natural
sources, having low side effect and low cost and also being Materials and Methods
easily accessible in comparison to common treatment
methods, may play an important role in the treatment Plant extracts’ preparation
of the cancer. In recent years, many attempts have been Plant materials used in this research were leaves of

Cellular and Molecular Biology Research Center, Babol University of Medical Sciences, Babol, 2Reproductive Biotechnology
1

Research Center, Avicenna Research Institute, ACECR, Tehran, Iran *For correspondence: halehakhavan@yahoo.com
Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 5317
Sadegh Fattahi et al
Urtica dioica harvested in September, 2011 from Babol MTT assay
(Iran). The leaves were cleaned with water and then dried Anti proliferative activities of the Urtica dioica
at room temperature away from direct sunlight and finely against MCF-7 breast cancer cell line and Fibroblasts
grounded with a grinder. Aqueous extraction procedures was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2,
were performed by boiling 15 gr leaves in 300 ml of 5% 5-diphenyltetrazolium bromide (MTT) assay. For this
ethanol by stirring for 15 min and then filtering through #1 purpose MCF-7 cells were plated at a density of 5×103
filter paper (Whatman Inc., Hillsboro, OR, USA), followed cells/well in a 96-well plate with 100 µl of culture medium
by centrifugation at 5000 rpm for 15 min. The supernatant in each well. Fibroblast cells were plated at a density 5×105
was evaporated at room temperature until dryness and cells/well in a 24-well plate with 1 ml of culture medium
the crude extracts were dissolved in deionized water and in each well. After 24 hrs incubation at 37°C in 5%CO2,
then filtered through a 0.2 μm filter and stored at -20oC culture media were removed and replaced by new media
for further analysis. containing different concentrations (0.375, 0.75, 1.5 and 3
mg/ml) of the extract and cells were incubated for 24, 48
Antioxidant assay by MTT and 72 hrs. After period time, the medium was discarded
Antioxidant activity was evaluated according and the adherent cells were washed with phosphate buffer
to Muraina et al. (Muraina et al., 2009) with slight solution (PBS), then MTT stock solution (5 mg/ml) was
modification. Briefly, two-fold dilution of 12 mg/ml of added to each well and the plates were further incubated
extract was prepared with 100 ml of distilled water in a for 4 hrs at 37°C. Following which resulted crystals were
96-well plate. Then, 10 µl of MTT (5 mg/ml) was added dissolved in acidic isopropanol. After complete dissolving
to every well and OD was measured at 590 nm after 0 and of formazan blue, absorbance of formazan dye at 570 nm
8 hrs incubation at 37oC. Also, for extract stability and was measured using a microplate reader (Rayto, China).
blank, different concentrations of extract without MTT
were prepared. 250 µM Ascorbic acid was used as the Apoptosis assay with ethidium bromide and acridine
positive control. All assays were repeated three times. orange staining
To determine if the aqueous extract of Urtica dioica
Ferric reducing antioxidant power assay induced apoptosis in MCF-7 cells, cells were stained
The ability to reduce ferric ions was measured using with acridine orange (AO) and ethidium bromide (EB) as
a modified version of the method described by Benzie fluorescent probes. The fluorescent cells were examined
and Strain (1996). An aliquot (50 µl) of the extract was by fluorescence microscope. AO is a vital dye which
added to 1.5 ml of FRAP reagent containing 10 parts of stains cells with intact (normal cell) or fragmented nuclei
0.3M sodium acetate buffer at pH 3.6, 1 part of 10 mM 2, (apoptotic) in green, while Ethidium bromide is taken
4, 6- tris (2-pyridyl)-1, 3, 5-triazine (TPTZ) solution and up only by dead cells (necrotic cells) and stains them
1 part of 20 mM FeCl3. 6H2O solution and the reaction orange-red. Briefly, cells were treated with 2 mg/ml (IC50)
mixture was incubated at 37°C for 15 min. After which the aqueous extract of Urtica dioica for 72 hrs in a 6-well
absorbance was measured at 593 nm. A calibration curve plate. Floating and attached cells were then collected
was prepared, using an aqueous solution of ascorbic acid and washed with PBS, the pellet was dissolved in 250 µl
[62.5-1000 µM (serial dilution), r2=0.999]. FRAP values PBS. Eight µl of a mixture of fluorescent dyes containing
were expressed on a fresh weight basis as micromoles of 100 pg/ml AO (Sigma Chemical, St. Louis, MO) and 100
ascorbic acid equivalent per gram of sample. pg/ml EB (Sigma, USA) was added to the 100 µl cells
(1×106) and mixed gently at room temperature. Ten μl of
Cell lines the mixture was loaded on a neobar and examined under
The estrogen-receptor MCF-7 cell line was purchased a fluorescence microscope.
from Pasteur institute, Tehran, Iran. The cells were cultured
in RPMI-1640 medium (PAA, Austria) supplemented with Flow cytometry with annexin V-propidium iodide (PI) staining
10% fetal bovine serum and 1% antibiotics (penicillin/ Apoptotic cells were quantified by Annexin V-FITC
streptomycin (Invitrogen) in a humidified atmosphere and Propidium iodide (PI) double staining, using an
containing 5%CO2 and 95% air, at 37°C. Primary human Annexin V-FITC apoptosis detection kit (BioVision
Fibroblasts were isolated from fresh foreskin isolated from Research Products, Mountain View, CA, USA) according
children aged between 1.5-30 months who underwent to manufacturer’s protocols. MCF-7 Cells were incubated
routine circumcision as previously described (Pandamooz with 0, 0.375, 0.75, 1.5 and 3 mg/ml aqueous extract of
et al., 2012). Briefly 1-2 mm3 pieces of foreskin were Urtica dioica for 72 hrs, the floating and trypsinized-
incubated at 37°C for 2 hrs in a 15 ml falcon tube in the adherent cells were collected and washed twice with
presence of 0.5% dispase II (Sigma-Aldrich) then digested PBS, adjusted to 500 µl of the binding buffer (5×105
with 0.1% crude collagenase (Sigma-Aldrich, C2674). cells). Then, 5 µl of Annexin V–FITC and 5 µl of PI were
Cells were collected upon release every 20 minutes from added and cells were gently vortexed. Cells were then
the aqueous phase of the tube, passed through 70 μm incubated for 15 min at room temperature (25°C) in the
cell strainer (BD Bioscience) and centrifuged before dark. Finally, cells were analyzed by a flow cytometer
resuspending in high-glucose DMEM (PAA cat : E15- (Becton Dickinson).
883), supplemented with 10% fetal bovine serum (FBS)
(PAA, cat: A 15-15) and 1% penicillin/streptomycin (PAA DNA fragmentation assay
cat: P11-010). DNA preparation and agarose gel electrophoresis was
5318 Asian Pacific Journal of Cancer Prevention, Vol 14, 2013
 DOI:http://dx.doi.org/10.7314/APJCP.2013.14.9.5317
Antioxidant and Apoptotic Effects of an Extract of Urtica dioica on the MCF-7 Human Breast Cancer Cell Line
carried out according to a method previously reported Table 1. Antioxidant Activities of the Aqueous Extract
by Herrmann et al. (Herrmann et al., 1994) with slight of Urtica Dioica and Ascorbic Acid Evaluated by MTT
modification. Briefly, cells were cultured in 25 cm3 flask Assay
and treated with 2 mg/ml of extract for 72 hrs. After Concentration 0 h After 8 hrs
harvesting the cells samples were washed with phosphate mg/ml OD Mean±SD OD Mean±SD
buffer saline and pelleted by centrifugation. The cell
pellets were suspended in 100 µl lysis buffer (1% NP- 12 0.352667±0.023965* 0.793±0.077582*
6 0.241667±0.021032* 0.450667±0.02318*
40 in 20 mM EDTA, 50 mM Tris-HCl, and pH 7.5) and
3 0.199333±0.033081* 0.302667±0.032624*
after incubation at room temperature for 15 min, treated 1.5 0.173667±0.026083 0.212±0.032924
for 10 min with SDS (final concentration 1%) and RNase 0.75 0.168±0.032078 0.192667±0.030989
A (final concentration 5 mg/ml) at 37°C followed by 0 0 0 100.0
digestion with proteinase K (final concentration 1 mg/ml) 250µM VC† 0.112±0.012583 0.246±0.020339 6.
for at least 5 hrs at 56°C. After addition of 1/4 vol. 10 M *The p value <0.05; †Vitamin C (Ascorbic Acid)
ammonium acetate the DNA was precipitated with 1 vol.
75.0
ice-cold isopropanol, mixed thoroughly and kept for 1 h
at-20°C after which the solution was centrifuged for 20
56
min at 12,000 rpm. The nucleic acids pellet was washed
with 200 µl 75% ice-cold ethanol and air-dried for 10 min 50.0
at room temperature and finally dissolved in gel loading
buffer for further separation by electrophoresis in 1.2%
agarose gels. 25.0
Immunoassay (Enzyme-linked immunosorbent assay) Figure 1. A) Antioxidant Activity of the Aqueous 31

ELISA Extract of Urtica Dioica, 0 h (Dark Blue) and After 8

To evaluate the apoptotic pathway in MCF-7 cells hrs (Light Blue) by MTT; and B) Regression Factor 0
after treatment with Urtica dioica extract, the amounts Squared for the Linear Regression, r2=0.997
of calpain1, calpestatin, caspase 3, caspase 9, Bax and
Table 2 Antioxidant Activity of Urtica dioica Extract
Bcl-2 were measured by ELISA kit (Glory Science Co.,
by FRAP Assay
Ltd, USA). After treating cells with Urtica dioica extract
for 72hrs, floated and adherent cells were pelleted and Ascorbic acid Urtica diocia
washed in cold PBS, and then cells were dissolved in PBS Concentration 250µM 0.75 mg/ml
and lysed with one freeze/thaw cycle at -80oC. Lysed cells OD Mean±SD 0.508±0.0132 0.113±0.0176
were centrifuged at 2000 rpm for 10 min to remove cell-
debris. Protein concentration was determined according to
the method proposed by Bradford (1976), using BSA as a
standard protein. Equal amounts of protein were subjected
to ELISA kit according to kit instructions.

Results
The antioxidant activity of Urtica dioica leave extract
The 15 gr of the powdered leave of Urtica dioica
were observed to yield 2.32±0.288 gr of the aqueous
extract corresponding to 15.46±1.92%. The antioxidant
activity of the aqueous extract of Urtica dioica leave was
evaluated by two methods, MTT and Ferric Reducing
assays, Ascorbic acid, was considered as positive control.
Due to the absorption of the aqueous extract at 590 nm, we
measured the absorption of different concentrations of the
extract (12-0.75 mg/ml) in the beginning and after eight
hrs (Table1 and Figure 1A). As shown in Figure 1B, the Figure 2. Dose-dependent Antiproliferative Effects of
aqueous extract of Urtica dioica have antioxidant effect Aqueous Extracts of Urtica dioica Against MCF-7 cell
with correlation r2=0.997. line (A) and Fibroblast (C) after 72 hrs Incubation. B)
In Ferric reducing assay, because of extract absorption IC50 Value of Aqueous Extract of Urtica dioica towards
in 593 nm wavelength and rapid enzymatic reactions, we MCF-7 Cell Line. Each value represents mean of three
only made the measurement for 0.75 mg/ml of the extract independent experiments±SD
which showed no absorption at 593 nm (Table2).
the human breast cancer MCF-7 cell line and fibroblast are
Effects of aqueous extracts on cell viability represented in Figure 2. Cell proliferation was analyzed
Anti proliferative activities of different concentrations after 24, 48 and 72 hrs. Data analysis showed that there
of the aqueous extract of Urtica dioica on the growth of was no regular difference in cell viability after 24 and
Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 5319
Sadegh Fattahi et al
48 hrs (data not show) but after 72 hrs a dose-dependent aqueous extract of Urtica dioica, cells were stained with
manner anti proliferative activity was observed with acridine orange and ethidium bromide fluorescence dyes.
correlation r2=0.936 for MCF-7 but not fibroblast (Figure Annexin-V and PI were used to detect apoptotic cells. The
2B and 2C). Statistical analysis showed that there was no morphological changes of the MCF-7 cells-treated with 2
significant difference in cell viability for MCF-7 cell line mg/ml Urtica dioica extract for 72 hrs were compared to
between 0.75 mg/ml and 1.5mg/ml and also between 1.5 untreated control after acridine orange/ethidium bromide
and 3 mg/ml concentrations (p>0.05). But the difference (AO/EB) fluorescence staining. As shown in Figure
between 0.375 mg/ml and 0.75 mg/ml was significant 3A, the apoptotic nuclei clearly had highly condensed
(p<0.05). Also fibroblasts treated with 0.375-2 mg/ml chromatin that was uniformly fluorescent. In the control
extract showed no significant difference in viability the cells nuclei were bigger than those of the cells treated
compared to non treated control, but in the presence of with Urtica dioica extracts. Quantitative assessment of
3 mg/ml extract, a significant viability difference with apoptosis was conducted using an Annexin-V assay kit.
non treated control was observed. Concentrations of the As shown in Figure 3C and 3D, treatment with different
extracts which exhibited 50% growth inhibition (IC50 concentrations of Urtica dioica resulted in an increase
value) for MCF-7 cells were 2 mg/ml as shown in Figure in the percentage of early and late apoptosis (lower and
2B. upper right quadrant, respectively) in a concentration
dependent manner. We also assessed the effects of Urtica
Urtica dioica induced apoptosis in MCF-7 cells. dioica on induction of apoptosis in MCF-7 cells by DNA
Most anti-cancer drugs induce apoptosis, as a primary fragmentation assay. DNA extracted after 72 hrs treatment
mechanism for inhibition of cell proliferation. To further with 2 mg/ml of aqueous extract of Urtica dioica revealed
investigate the cause of the decrease of cell viability by the a ladder corresponding to DNA fragmentation in agarose
gel electrophoresis (Figure 3B) which gave an indication
of apoptotic cell death induction by Urtica dioica extract.
Furthermore, ELISA results emphasized that Urtica dioica
extract caused cell death in MCF-7 cells via apoptosis
proteins increase (Table 3). As shown in Table 4, the ratio
of different apoptotic proteins is approximately equal to 1
for each concentration of aqueous extract of Urtica dioica
analyzed and therefore the amounts of those proteins are
similar with no significant difference between them.

Discussion
Recent analyses raise concerns about the increase of
cancer prevalence in the near future (Bray et al., 2012).
Therefore, looking for natural and low cost drugs that
prevent cancer development is becoming an important
purpose for researchers. In this study, we investigated
Figure 3. A) Morphological Observation with Acridine antioxidant, antiprolifrative and apoptotic effects of
Orange and Ethidium Bromide (AO/EB) Stain. MCF- aqueous leaf extract of Urtica dioica on MCF-7 cell line.
7 cells were treated without and with Urtica dioica aqueous The antioxidant activity of the aqueous extract of Urtica
extract, 2 mg/ml for 72 hrs. Magnification: 400×. B) DNA dioica leave was evaluated by MTT and FRAP assay. Both
Ladder Formation in MCF-7 Cells Treated with 2 mg/ methods confirmed that the aqueous extract of Urtica
ml Urtica dioica for 72 hrs. Lane 1, standard DNA marker
(100bp ladder), lane 2, control; lane 3, 2mg/ml treated cells. C) Table 4. Increase Rate of Different Apoptotic Proteins
Flow Cytometric Analysis of Apoptosis/Necrotic Cell After Treatment with Urtica diocia Extract
Death using Annexin-V-FITC/PI Staining of MCF-7 Different Calpain 1 Caplestatin Caspase Bax Bcl-2
Cells Treated with Urtica diocia. Quadrant 2 represents late Concentration/
apoptotic cells; Quadrant 3 represents live cells and Quadrant 4 control 3 9
represents early apoptotic cells. D) Percentage of Apoptotic 0.75/Control 9.12 11.37 13.3 10.44 9.5 8.94
(Early and Late) MCF-7 Cells Induced by Different 1.5/Control 2.76 3.18 5.1 4.05 3 2.76
3/Control 2.7 3.5 4.7 3.19 3.11 3.26
Concentrations of Urtica dioica after 72 hrs Treatment

Table 3. Amount of Different Apoptotic Proteins After Treatment with Urtica dioica Extract
Concentration Total protein Calpain 1 Calpestatin Caspase 3 Caspase 9 Bax Bcl-2
mg/ml loaded µg concentration IU/L concentration IU/L ng/ml IU/L ng/ml ng/ml
Control 20 139.4 267.2 2.2 318.9 19.05 2.77
0.75 20 1273 3039 29.4 3331 181 24.76
1.5 20 385 850 11.41 1294 57.2 7.03
3 20 377.4 938 10.47 1019 59.3 8.3

5320 Asian Pacific Journal of Cancer Prevention, Vol 14, 2013

 DOI:http://dx.doi.org/10.7314/APJCP.2013.14.9.5317
Antioxidant and Apoptotic Effects of an Extract of Urtica dioica on the MCF-7 Human Breast Cancer Cell Line
dioica contains antioxidant activity. Although while Urtica dioica extract for 72 hrs, apoptotic nuclei clearly
comparing to ascorbic acid, 15 gr powdered leave of had highly condensed chromatin that was uniformly
Urtica dioica had an antioxidant activity approximately fluorescent in comparison with control. This suggests that
equivalent to 26.192 mg and 30.311 mg of pure ascorbic the cell death observed was due to apoptosis, but not
acid in MTT and FRAP assays respectively, but this necrosis. For further analysis of apoptosis, quantitative
difference in the amount of antioxidant activity may be assessment was conducted using flow cytometry with
due to different substrates used in these two methods. Annexin-V FITC kit. Our findings indicated that treatment
Moreover, Flavonoids such as Quercetin, Kaempferol with different concentrations of Urtica dioica extract
(Apak et al., 2007) and so which may be present in Urtica resulted in an increase in the percentage of early and late
dioica may have different abilities to convert substrate to apoptotic cells in a concentration dependent manner with
product. As drugs used to treat cancer are cytotoxic and/ r2=0.984 similar to the antiproliferation assay. This data
or cytostatic, the potential cytotoxic effect of Urtica dioica emphasizes that Urtica dioica affects cell proliferation by
was investigated by determining its effect on the viability activating the apoptosis pathway. A family of cytosolic
of MCF-7 cell line and fibroblast cells with MTT assay. proteases, the caspases, plays an essential role in apoptosis.
The results showed that Urtica dioica aqueous extract They can be divided into two major subgroups, initiator
caused significant growth inhibition of MCF-7 cells in a (caspases-2, -8, -9, and -10), which activate the effector
dose-dependent manner with correlation r2=0.936. In a caspases (caspases-3, -6 and -7). Initiator caspases are
related study, the cytotoxic activity of methanolic extract activated by apoptotic signals, resulting in the activation
of Urtica dioica on prostate cancer cells determined by of the effector caspases (Devarajan et al., 2002; Mc Geea
MTT assay showed a 30% maximum growth reduction at et al., 2002). One of the major pathways for activation of
concentrations between 1-6 mg/ml after day 5 (Konrad et caspase-9 (initiator caspase) involves calcium overload
al., 2000). In another study, the percentage of cell survival of mitochondria to trigger cytochrome c release (Yu et al.,
of MCF-7 cells following 72 hrs exposure to 50 µg/ml 2007), leading to the activation of caspase-9, and the
ethanol extracts fractions from Urtica dioca leaves and subsequent activation of caspase-3. Calpain1 and its
stems were demonstrated to be 93.12±8.88 (Abu-Dahab endogenous specific inhibitor, calpastatin, which is also
and Afifi, 2007). Recently, the cytotoxicity effect of Urtica activated in some apoptotic systems, like caspase-3, is a
dioica on MCF-7 and MDA 231 cell lines was determined cytosolic cysteine protease, but requires Ca2+ for its
using the XTT assay (Guler, 2013). The survival activity (Cui et al., 2007; Salehin et al., 2010). Caspase
differences observed could be due to differences in cell 3, the most commonly activated caspase that plays a
types investigated (as there are different MCF-7 cell line critical role in apoptotic pathway by cleaving some
types) and the extraction procedures. The potent proteins, exhibits some of the typical morphological and
cytotoxicity activity of the extract of Urtica dioica could biochemical features such as membrane blebbing,
be due to its polyphenols which have a vast range of induction of phagocytes migration to the site of apoptotic
actions such as antioxidant, antibacterial, antimutagenic, cells and DNA fragmentation (Jänicke, 2009). The Bcl-2
and antiproliferative properties (Peterson and Dwyer. proteins family also have important role in apoptosis and
1998; Nijveldt et al., 2001; Apak et al., 2007). Interestingly, are divided into anti-apoptotic (Bcl-2, Bcl-xL and Bcl-W)
Bosetti et al. reported an inverse association between and pro-apoptotic proteins (Bax, Bak and Bid). By
flavanoids and breast cancer risk (Bosetti et al., 2005). modifying mitochondrial membrane permeability, these
The observed cytotoxic effect of 3 mg/ml Urtica dioica proteins cause the release of cytochrome c and subsequently
extract on foreskin fibroblasts might be due to the fact that caspase cascade activation and consequently initiate
foreskin fibroblasts are in some characters similar to foetal apoptosis (Harris and Thompson, 2000). Bax/Bcl-2 ratio
fibroblasts (Chen et al., 1989). In fact, previous studies may be used to determine cell survival or death after
indicated that tumor-derived fibroblasts, skin fibroblasts treatment by an apoptotic drug (Oltvai et al., 1993). Our
of breast cancer patients and foetal fiboblasts secrete a data indicate that following the addition of Urtica dioica
migration stimulating factor (MSF) which is not secreted aqueous extract, MCF-7 cells displayed significant
by normal fibroblasts (Schor et al., 1988a; 1988b). Schor increases in calpain 1, calpastatin, caspase 3, caspase 9,
et al. (1987) showed that cancer development susceptibility Bax and Bcl-2 activity in an inversely dose-dependent
in adults increased in the presence of foetal-like fibroblasts. manner with the ratio of these different proteins remaining
Therefore, foreskin fibroblasts growth inhibition caused approximately equal to 1. The lower activity of apoptotic
by the high concentration of Urtica dioica extract, can be proteins observed with high concentrations of Urtica dioca
interpreted as an anticancer effect. Since, most drugs used extract, could be due to the fact that apopotosis takes place
in cancer treatment also induce apoptosis, a type of sooner with those concentrations compared to low
programmed cell death characterized by specific concentrations (as confirmed by our flow cytometry
morphological changes such as nuclear fragmentation, analyses (Figure 3C, D), leading to subsequent proteins
condensation of chromatin, blebbing of the plasma degradation. Although molecular studies concerning the
membrane, and the presence of apoptotic bodies (Janicke. mechanism of apoptosis induction in MCF-7 cell lines are
2009), to further investigate the cause of the decrease of contradictory, indicating for some caspase 9 activation
cell viability by the aqueous extract of Urtica diocia, we (Cui et al., 2007; Liang et al., 2001), others demonstrating
used different methods to evaluate qualitatively and both caspase 9 and caspase 3 indepent or independent
quantitatively apoptotic cells. Quality assessment with mechanisms (Ofir et al., 2002; Shim et al., 2007) or
AO/EB staining showed that after treating cells with caspase 3 dependent activation under some circumstances
Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 5321
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