cytotoxic effect of an aqueous extract of lepidium sativum...

Indian Journal of Traditional Knowledge

Vol. 12 (4), October 2013, pp. 605-614

Cytotoxic effect of an aqueous extract of Lepidium sativum L. seeds on human

breast cancer cells

Sawsan Hassan Mahassni* & Roaa Mahdi Al-Reemi

Biochemistry Department, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

E-mail: *[email protected]

Received 18.02.13, revised 16.07.13

Easily available alternative and natural remedies for breast cancer are sought to reduce or eliminate the disadvantages

of conventional treatments. Garden cress seeds (L. sativum L.) are known to contain several chemopreventive and

chemotherapeutic constituents. The effect of L. sativum plant extract on cancers is generally attributed to the constituent

isothiocyanates, specifically benzyl isothiocyanate, which are also present in the seeds. The current work examines, for the

first time, the cytotoxic effect of the aqueous extract of L. sativum seeds on MCF-7 breast cancer cells, using the trypan blue

dye exclusion and sulforhodamine B assays, compared to its effect on normal human HFS cells. The MCF-7 cell culture

morphology, after exposure to the extract, was studied using light microscopy. Finally, HPLC was used to ascertain whether

benzyl isothiocyanate is contained in the seeds extract. The results showed that the L. sativum seeds aqueous extract had a

significant cytotoxic effect on MCF-7 cells, by causing significant time- and dose-dependent decreases in their viability. In

contrast, the HFS cells were significantly more resistant to the cytotoxic effect of the extract. Therefore, the current study

provides strong evidence of the L. sativum seeds extract’s ability to inhibit growth of breast cancer cells.

Keywords: Cancer chemotherapy, Chemoprevention, Breast cancer, Lepidium sativum, Garden cress

IPC Int. Cl.8: A61K 36/00, A01D 4/04, A01D 12/15, A01D 4/34

Breast cancer is the most commonly diagnosed cancer

in women worldwide, and the second leading cause of

cancer-related deaths among women1. Conventional

treatments of breast cancer are costly, have serious

side effects, and may cause new cancers to occur.

Thus, it is of the utmost importance to find effective

chemopreventive or chemotherapeutic agents that

have minor or no side effects and that are easily and

economically available. It is well known that some

herbs have strong and beneficial effects when

consumed on a regular basis. Some are used as easily

available natural remedies to prevent or treat cancers2-5

,

especially in poor developing countries, but without

sufficient scientific proof.

Cruciferous vegetables (Family: Brassicaceae) and

their seeds are used in alternative and traditional

systems of medicine and healing in many countries.

An important member of this group of vegetables is

the garden cress (L. sativum L.) plant and its seeds,

which are used as nutritional constituents and

common ingredients in folk remedies used mainly in

Arabian and Asian countries. L. sativum seeds are

recommended for the treatment of many ailments and

they have many therapeutic effects6, 7

.

The seeds of the L. sativum plant contain many

phytochemicals and their secondary metabolites8,9

,

such as alkaloids, tannins, flavonoids, and

isothiocyanates, which have a wide range of

biological activities and known healing effects.

Phenolic compounds are present in L. sativum seeds10

,

and they have been shown11-13

to have diverse

functions, such as anti-inflammatory activities that

protect the human body from oxidative stress that

may lead to cancer.

The oil of the L. sativum seeds is rich in alpha

linolenic acid, a ω-3 fatty acid, which has been shown

to have chemopreventive14-16

and chemotherapeutic

effects15-17

on different types of cancers, including

breast, in both animals and human cell line models.

The major secondary compounds of L. sativum

seeds are the glucosinolates18-20

. Glucotropaeolin, the

main glucosinolate in L. sativum, is degraded to

produce benzyl thiocyanate and benzyl

isothiocyanate21,22

. Benzyl isothiocyanate (BITC) is

the most abundant and important isothiocyanate found

in the seeds2,23

. Isothiocyanates have been shown2, 24-29

________

*Corresponding author

INDIAN J TRADITIONAL KNOWLEDGE, VOL. 12, No. 4, OCTOBER 2013

606

to be capable of inhibiting both the formation

(chemopreventive) and development (anti-cancer) of

cancer through multiple pathways. Xiao et al.23

showed that BITC induces significant apoptosis and

cell cycle arrest of cancer cells, but this effect may be

cell line specific, significantly suppressing the growth

of cells such as MDA-MB-231 (estrogen-

independent) and MCF-7 (estrogen-responsive)

human breast cancer cell lines, although the

mechanism of cell death is not fully understood.

Many studies, as detailed above, have shown that

certain constituents of the L. sativum plant, and its

extracts have chemopreventive and chemotherapeutic

effects, but no studies have been done on the effects

of any extracts of L. sativum seeds on the viability and

growth of cancer cells. In fact, and after extensive

literature search, we are the only researchers to study

the effects of the aqueous extract of L. sativum seeds

on human breast cancer cells30

. Therefore, in this

work the potential of the aqueous extract of

L. sativum seeds to induce death of breast cancer cells

in tissue culture was investigated in the hope of

finding a natural, cheap, and easily available

treatment for human breast cancer. Thus, this is the

first study of the cytotoxic effects of the L. sativum

seeds extract on cancer cells, and specifically breast

cancer.

Materials and methods Cell lines and culturing of cells

The human breast cancer cell line MCF-7

(Michigan Cancer Foundation–7) was obtained from

the National Cancer Institute, Cairo University, Cairo,

Egypt. This cell line is an epithelial invasive breast

ducal carcinoma cell line that is hormone-sensitive.

Normal human skin fibroblasts (HFS) were acquired

from foreskin circumcision operations, King

Abdulaziz University Hospital, Jeddah, Saudi Arabia.

Cells were cultivated in Dulbecco's minimal essential

medium (DMEM) supplemented with 10% fetal

bovine serum (FBS) (both from GIBCO, Grand

Island, NY, USA), 1% L-glutamine, and 1%

penicillin/streptomycin and incubated in 5% CO2, at

37 °C, and 96% relative humidity. All tissue culture

work was done at the Tissue Culture Unit, at King

Fahd Medical Research Center, Jeddah, Saudi Arabia.

Culture flasks, 6-well cell culture plates, and Petri

dishes were purchased from Falcon (Becton

Dickinson, Milton Keynes, UK). Polystyrene 24-well

and 96-well cell culture plates were purchased from

Corning (Corning, NY, USA).

Aqueous extraction of L. sativum seeds

Dry L. sativum seeds were obtained from a local

herbalist shop in Jeddah. The type obtained was

grown in Al-Qaseem area in Saudi Arabia as

authenticated by the herbalist and confirmed by a

taxonomist.

Three methods based on traditional ways of

extraction and consumption of L. sativum seeds were

used. The dry seeds were ground, in an electric

grinder, to a coarse powder. Using the traditional

Moroccan method, 1 gm of powdered seeds was

mixed with 100 ml de-ionized water and boiled for

10 minutes, after which the extract was left for

15 minutes at room temperature31-33

. For both the

second and third methods, 1 gm of powdered seeds

was stirred with 99.8 ml of de-ionized water and 0.2

ml of DMSO. For the second method, the mixture was

stirred overnight, while for the third method the

mixture was boiled for 10 minutes. The extracts were

then left at room temperature for 15 minutes. DMSO

(0.2%) was added to insure that all ingredients of the

seeds were dissolved. The amount of DMSO used was

at the allowable tolerable concentration (< 0.2%) for

cells26-27

.

Each of the three extracts was filtrated first by sterile

gauze or any size filter paper, and then by a 9.0 cm

filter paper (Whatman Company, NY, USA) to

remove the gel-like substances. These resultant

filtrates were secondly filtered by a 0.22 µm pore size

sterile filter system (Corning Incorporated, Corning,

NY, USA). These final stock solutions were frozen

at −80 °C and used up to six weeks only.

Cytotoxic assay of the three extracts on viability of MCF-7 cells

MCF-7 cells (1×105 cell/ml) suspended in DMEM

containing 10% FBS were plated onto 24-well plates

at 1 ml per well. After 24 hrs of incubation, the

original media was replaced by serum-free media

(SFM) that contains one of several concentrations of

the extracts (extract:media (vol/vol), 1:1, 1:10, 1:100,

1:1000, or 1:10000). As for the controls for each

extract, only SFM was used for the first extract, while

for the second and third extracts 0.1% DMSO was

added in addition to SFM. The cells were harvested

by trypsinization and counted by a Vi-CELL XR cell

viability analyzer (Beckman Dickinson, Miami,

Florida, USA), after 24, 48, and 72 hrs of exposure to

the extract.

Cytotoxic assay of the extract on MCF-7 and HFS cells

Using the third extract here, and for all subsequent

experiments, its cytotoxic effects on the viability of

MAHASSNI & AL-REEMI: CYTOTOXICITY AQUE EXT L. SATIVUM SEEDS HUMAN BREAST CANCER

607

MCF-7 and HFS cells were determined as described

above. Different concentrations of the extract

[extract:SFM (vol/vol); 1:3 (25% extract), 1:1 (50%),

and 3:1 (75%)] were used. The control was SFM

containing 0.1% DMSO. The number of living cells

was determined following 24, 48, 72, 96 and 120 hrs

incubation with the extract.

Proliferative assay

The anti-proliferative effects of the extract on

MCF-7 and HFS cells were determined using the

sulforhodamine B (SRB) assay34

.

MCF-7 and HFS cells were separately cultivated in

96-well microtiter plates, at 1×104 cell/well in DMEM

containing 10% FBS. After 24 hrs, 100 µl of SFM that

contains a concentration (20%, 30%, 40%, 50%, 60%,

70%, or 80%) of extract was added. The treated cells

were allowed to incubate for 48 hrs. Adherent cells

were then fixed by the addition of 50 µl of cold 40%

(w/v) trichloroacetic acid in distilled water, and

subsequently incubated for 60 min at 4°C. Using a

plate washer, the plates were then washed 5 times

with deionized water. Next, the cells were stained

with 100 µl SRB stain (Biomedicals, Illkirch, France)

(0.4% w/v in 1% acetic acid) and subsequently

incubated for 10 min at room temperature. Unbound

stain was removed and the plate was washed with

200 µl of 1% acetic acid, and then incubated for 5 min

at room temperature. Subsequently, the plates were

air-dried and 200 µl of Tris base (10 mM, pH 10.5)

were added to each well. Finally, the plates were

shaken gently for 5 minutes, after which the

absorbance (OD) of each well was read on an ELx800

ELISA reader (BioTek Instruments Inc., Winooski,

Vermont, USA) at 490 nm.

Evaluation of cell culture morphology

The effects of the extract on cell culture

morphology of both MCF-7 and HFS cells were

determined by light microscopy, after staining the

cells with Coomassie blue. MCF-7 (1×105cell/ml)

and HFS cells (1.5×105cell/ml) were each suspended

in DMEM containing 10% FBS and then cultivated, at

5 ml per well, onto 6-well plates. After the formation

of one layer of cells, the original DMEM was

replaced with 5 ml of SFM containing one of the

different concentrations of the extract (25%, 50%, or

75%). After 48 hrs, adherent cells were washed twice

with 3 ml of PBS (Sigma-Aldrich, St. Louis, MO,

USA). Subsequently, 2 ml of 4 % formaldehyde were

added for 5 min to fix the cells. After discarding the

formaldehyde, 2 ml of Coomassie blue were added for

5-10 min to stain the fixed (non-living) cells. The

plates were then washed several times with tap water,

left to dry, and finally observed and photographed

under an Eclipse TS100 inverted microscope (Nikon,

Tokyo, Japan) equipped with DS-Fil digital camera

(Nikon, Tokyo, Japan).

Detection of benzyl isothiocyanate by paired-ion HPLC

To determine whether benzyl isothiocyanate is a

constituent of the seeds extract, it was subjected to

paired-ion HPLC as described previously

35. BITC was

monitored at 250 nm. The two solvents used in the

gradient were 6 mM tetraethylammonium bromide in

water and acetonitrile. Using the above conditions,

Zhang35

reported a retention time of 14.9 minutes for

BITC.

Statistical analysis

The data are represented as mean ± standard

deviation (SD). Statistical comparisons were

performed using the three-way ANOVA followed by

the Dunnett’s multiple comparisons test to determine

the statistical significances of the effects of different

incubation times and extract concentrations on the

viability of cells. A statistically significant difference

was 0.01 ≤ P < 0.05, 0.001 ≤ P < 0.01 was a

statistically high significant difference, while 0.05 ≤ P

< 0.10 was considered tending toward being

statistically significant.

Results Cytotoxic effects of the three extracts on the viability of

MCF-7 cells

Using the three-way ANOVA test (Fig. 1), the

number of living cells treated with each extract was not

significantly affected with increasing concentrations of

the extracts compared with the respective controls,

except for the highest concentration (1:1) for each

extract where the number of living cells decreased

highly significantly (P = 0.001) compared to the

controls. The comparison between the three extracts

for each incubation time gave highly significant P

values equal to 0.00, 0,001, and 0.00 after 24, 48, and

72 hrs of exposure, respectively. This means that

there were highly significant differences between the

effects of the extracts at each incubation period.

The number of living MCF-7 cells that were treated

with the extracts increased highly significantly after

72 hrs of exposure to the first (P = 0.001), second

(P = 0.002), and third (P = 0.008) extracts, and

significantly after 48 hrs (P = 0.042) of exposure to


608

the second extract compared with the number of cells

after 24 hrs of exposure for each extract (Fig. 1).

As a whole, the effects of the three extracts on

MCF-7 viability were close to each other. The third

extract was the extract of choice for all subsequent

experiments because of the slightly, but not

significantly, more depressing effect of the highest

concentration on the cell viability compared to the

other two extracts. The number of living MCF-7 cells

treated with the highest concentration (1:1) of the

third extract compared to the control showed a trend

toward a significant decrease (P = 0.053), while cells

treated with the same concentration of the first and

second extracts (P = 0.257 and 0.104, respectively)

did not show any differences from the controls.

Additionally, the third extract contains DMSO, which

is important to ensure that the active components of

the seeds were extracted. Thus, the highest

concentration (1:1; 50%) of the third extract with two

new additional concentrations (1:3; 25% and 3:1; 75%

extract) were used to treat both MCF-7 and HFS cells

in all subsequent experiments.

Cytotoxic effects of the extract on the viability of MCF-7 and

HFS cells

Using the three-way ANOVA test (Fig. 2), the

viability of MCF-7 cells decreased with increasing

concentrations of the extract and exposure time

(Fig. 2 A), thus showing time- and dose-dependency.

The viability of MCF-7 cells was decreased by the

three concentrations (25%, 50%, and 75%) of the

extract, compared with the respective controls, after

24, 48, 72, 96, and 120 hrs. The decrease in the

viability was highly significant for all concentrations

and incubation times, except for the 25% extract

concentration at 24 hrs of incubation, which showed a

significant decrease.

The Dunnett’s two-sided test was used to compare

the number of living cells for each extract

concentration and incubation time with the cells’

viability at its respective 24 hrs incubation (Table 1).

The number of living control MCF-7 cells was highly

significantly increased after incubating with the

extract for 48, 72, and 120 hrs, while it was

significantly increased after 96 hrs of incubation.

When MCF-7 cells were incubated with 25% extract,

there were no significant changes in the number of

living cells with increasing incubation time. On the

other hand, cells treated with 50% extract led to a

highly significant decrease in the number of living

cells after 72, 96, and 120 hrs, but no difference

Fig. 1 Cytotoxic effects of the different concentrations of the

three L. sativum seeds extracts on the viability of MCF-7 cells

after different incubation times. (A) Cytotoxic effects of the first

extract, (B) Cytotoxic effects of the second extract, and (C)

Cytotoxic effects of the third extract. Each bar represents one

experiment.

Fig. 2 Cytotoxic effects of L. sativum seeds extract on the five

day viability for (A) MCF-7 and (B) HFS cells, using the time-

dose manner. Each bar represents the mean ± S.D. of, at least,

triplicate experiments. Each concentration of extract (25%, 50%,

and 75%) is compared to its respective control. One star (*)

indicates that P < 0.05, a significant difference, and two stars (**)

indicate that P < 0.01, a highly significant difference, using the

three-way ANOVA test followed by the Dunnett’s multiple

comparisons test.


609

resulted after 48 hrs. Finally, at 75% extract, there

was a significant decrease in the number of living

cells after 96 hrs and a highly significant decrease

after 120 hrs, while the 48 and 72 hrs of incubation

showed no differences. Each of these experiments

was performed in triplicates.

As for the viability of HFS cells, the Dunnett’s

two-sided test was used for the analysis (Fig. 2 B) of

the differences between each respective control and

the different concentrations of extract and incubation

times, as done for MCF-7 cells above. After 24 hrs of

incubation, the three concentrations (25%, 50%, and

75%) of the extract caused a highly significant

decrease in the number of living HFS cells compared

to the control. Highly significant decreases were also

observed for the 50% and 75% concentrations after

72 hrs and for the 75% after 96 and 120 hrs,

comparing each incubation time to its control.

Significant decreases were observed for the 50% and

75% concentrations after 48 hrs of incubation, for the

25% after 72 hrs, and for the 25% and 50%

concentrations after 120 hrs of incubation, comparing

each to its respective control.

To compare each extract concentration at each

incubation time with the 24 hrs incubation, the

Dunnett’s two-sided test was used (Table 2), as done

above. The numbers of living HFS cells that were

treated with the 25% and 50% concentrations of

L. sativum seeds extract, in addition to the control,

were not significantly different from the number of

cells after 24 hrs. The number of living HFS cells

treated with 75% of extract did not show any

significant differences, except for the 96 and 120 hrs

of incubation where the viable HFS cells decreased

significantly. Thus, the effects of the extracts on HFS

cells were not time-or dose-dependent.

Antiproliferative effects of the extract

Using the SRB assay, the three-way ANOVA

demonstrated (Fig. 3) that the normal HFS cells were

found to be relatively insensitive to growth inhibition

by the extract, compared with MCF-7 cells, since the

number of HFS cells continued increasing with the

increased concentration of the extract. After 48 hrs of

exposure, the proliferation of MCF-7 cells was highly

significantly decreased with increasing concentrations

of the extract compared with the control. On the other

hand, the HFS cells proliferation increased

significantly with increasing extract concentrations

for all concentrations, except for 80%.

The effects of the extract on cell culture morphology

The sheet like growth of cells (cell density) of both

MCF-7 and HFS cells (Fig. 4) was lost with the

appearance of signs of apoptosis. However, the effect

Table 1 Effects of incubation times for extract concentrations on

MCF-7 cells viability. Results are compared with viability after

24 hrs (control). P values were determined using the Dunnett’s two

sided test

Incubation Control 25% 50% 75%

48 hrs 0.000HS 0.469 NS 0.052 NS 0.927 NS

72 hrs 0.000HS 0.239 NS 0.006 HS 1 NS

96 hrs 0.013S 0.234 NS 0.000HS 0.002 S

120 hrs 0.009 HS 0.397 NS 0.000HS 0.001 HS

HS: Highly Significant (0.001 ≤ P < 0.01)

S: Significant (0.01 ≤ P < 0.05)

NS: Not Significant

Table 2 Effects of incubation times with extract on HFS cells

viability. Results are compared with viability after 24 hrs (control).

P values were determined using the Dunnett’s two-sided test

Incubation Control 25% 50% 75%

48 hrs 0.53 1 0.774 0.998

72 hrs 0.736 0.742 0.822 0.829

96 hrs 0.218 0.348 0.97 0.039 S

120 hrs 0.874 0.869 0.979 0.049 S

S: Significant (0.01 ≤ P < 0.05)

Fig. 3 Antiproliferative effects of different extract

concentrations on MCF-7 and HFS cells after 48 hours of

treatment. Each point is the mean ± S.D. of 12 independent

experiments. One star (*) indicates that P < 0.05 and two stars

(**) indicate that P < 0.01, both a significant difference compared

to the control by the three-way ANOVA followed by the

Dunnett’s multiple comparisons test..


610

was more marked on MCF-7 cells compared with

HFS. It was also observed that the effect was more

evident with increasing concentrations of the extract

as the amount of apoptotic cells increased. There was

a more significant effect of the 75% concentration on

both cells, but the effect was more pronounced on

MCF-7 cells where more apoptotic MCF-7 cells were

observed compared to apoptotic HFS cells.

Fig. 4 Effects of L. sativum seeds extract on cell density after 48 hours of treatment. The cells were examined under TS100-F inverted

microscope using a 10× objective lens. (A1-A4) represents the effects of extract on MCF-7 cells (control, extract at 25%, 50%, and 75%

respectively). (B1-B4) represents the effects of the extract on HFS cells (control, extract at 25%, 50%, and 75% respectively).


611

Paired-ion HPLC separation of the extract

Benzyl isothiocyanate was not detected in the

extract by using HPLC, as shown in Fig. 5, since there

were no peaks at a retention time of 14.9 minutes.

Discussion Despite its great medicinal value and many

beneficial effects, garden cress has not received the

attention it deserves. To our knowledge, other than

the current study here and our previous study30

, there

are no other in-vitro or in-vivo studies on the anti-

cancer effects of garden cress seeds extract. A number

of studies2, 23, 24, 27, 29

have been done on benzyl

isothiocyanate, which is contained in garden cress

seeds, and on extracts of other cruciferous

vegetables36-37

.

In this study, three traditional extraction methods

of L. sativum seeds were used. An aqueous extract

was chosen rather than an alcoholic one to better

emulate the natural route of use of the seeds for

therapeutic treatments. It is well known that an

alcoholic extract is superior to an aqueous one since

more of the active substances would be extracted and

solubilized in the extract. The three extracts were

tested for their growth depression, or cytotoxic effect,

on MCF-7 breast cancer cells. The number of MCF-7

cells treated with the three extracts at different

concentrations (1:10, 1:100, 1:1000, or 1:10000) and

for different incubation times (24, 48, and 72 hrs)

showed no significant differences compared to the

control. It was expected that the viability of MCF-7

cells would decrease with increasing concentration of

extract. This lack of decreased viability may be due to

the presence of nutritional constituents in the extract

or insufficient amounts of cytotoxic components at

these low concentrations. Only the highest

concentration of all three extracts, at all incubation

periods, showed significant decreases (P = 0.001) in

the number of living MCF-7 cells compared with the

respective controls. The effects for the third extract

were similar to the other two extracts, but it had a

higher cytotoxic effect tending toward significance

(P = 0.053) at the highest concentration, and thus it

was used to carry out the rest of experimental work.

An added component of this extract was DMSO,

which was used to ensure the extraction of most

ingredients of the seeds.

Using the trypan blue dye exclusion assay, the

extract had a non-selective cytotoxic effect on the

viability of MCF-7 and HFS cells in a time and dose-

dependent manner. The extract showed strong

cytotoxic effects on the viability of MCF-7 cells with

an IC50 ~25% compared with HFS that had an

IC50 ~50% after 24 hrs of extract exposure. The

higher concentration of extract (75%) decreased the

viability by ~72% and ~87% for MCF-7 cells and

~60% and ~50% for HFS cells, compared with the

control, after 24 and 48 hrs of extract exposure,

respectively. Therefore, the HFS cell line was

relatively less sensitive to the cytotoxicity of the

extract compared with MCF-7 cells.

The antiproliferative effects of the extract on both

MCF-7 and HFS cells were also determined by using

the SRB assay. The extract had a significant

antiproliferative effect on MCF-7 cells after treatment

for 48 hrs with an IC50 ~40%-50% while the extract

had a proliferative effect on HFS that significantly

increased with increasing concentrations, except for

the 80% concentration which tended toward a

significant increase compared with the control.

The differences in the IC50 between the trypan

blue dye exclusion assay and the SRB assay may be

due to the fact that in the SRB assay the experimental

media that contains the different concentrations of the

extract (20%, 30%, 40%, 50%, 60%, 70%, or 80%)

was added without first discarding the original media

(as instructed in the method). This led to the dilution

of each concentration to the half, making the actual

used concentrations to become 10%, 15%, 20%, 25%,

30%, 35%, and 40%. Thus, the IC50~25% obtained

for MCF-7 cells using the trypan blue dye exclusion

assay is nearly the same as the IC50 ~40%-50%

obtained by using the SRB assay, which should be

adjusted to IC50 ~20-25%.

From this experiment it may be concluded that the

70% concentration is the desired therapeutic dose,

since it inhibited the growth of MCF-7 by 64.03% but

Fig. 5 Paired-ion HPLC separation of L. sativum seeds extract.


612

increased the growth of HFS by 133.33%. The

number of MCF-7 cells was higher in the SRB assay

compared with the trypan blue dye exclusion assay at

the same dose and time, which may be due to the

presence of dead cells that were not detached, yet the

ODs of their adhesion proteins were read as living

cells, and due to the added dilution of the extract as

indicated above.

It has been shown that38

the ethanolic L. sativum

leaves extract had no anti-proliferative activity on

MCF-7. Other researchers have shown5 that the

hydroalcoholic extract of L. sativum leaves inhibits

division and growth of MCF-7 cells by 2.68% and

amelanotic melanoma cell line C32 by 4.32%, but it

does not have antiproliferative activity on a prostate

cancer or renal adenocarcinoma cell lines. The leaves

extracts are similar to the seeds extracts in

composition, but they differ in the amounts of

different components8.

In the current study, morphological changes

occurred in MCF-7 and HFS cells, compared to the

control, upon exposure to 25%, 50%, and 75%

L. sativum seeds extract for 48 hrs. Marked decreases

in the densities of Coomassie blue stained cells were

observed with increasing concentration of the extract.

Loss of cell-to-cell contact was observed markedly in

cultures of both types of cells at 75% extract

concentration.

HPLC of the extract showed that BITC was not

present. This finding was expected since BITC is

polar and thus would be extracted more easily using

alcohols. Therefore, it is evident that the cytotoxic

effects of the extract are not attributed to the presence

of BITC, which is known to possess anticancer

activities. Thus, other ingredients present in the

extract must be responsible for the observed activities.

The aqueous extract was equally, and in some

experiments more, effective against MCF-7 cells

compared to HFS cells. In general, the highest (75%)

dose of extract was cytotoxic for both MCF-7 and

HFS cells in most assays. The resulting effect of

garden cress seeds extract on the cancer cells is

especially important since the MCF-7 cells are known

to be strongly resistant to chemotherapeutic drugs.

Significance of the study and some constructive recommendations

Many herbs used as nonconventional treatments are

easily prepared and administered without the need of

medical personnel with the goal that they provide a

way of self treatment or cure. The low or no toxic

effects of these treatments make them more amenable

for use. Thus, the main advantages of such treatments

are that they may be prepared and administered at

home by the patient herself or a family member. This

leads to such treatments being easily used, not require

a specially trained health practitioner or hospitals, and

possibly increase patient compliance. This is

especially important and essential in developing

countries where a majority of the populations are

poor, have limited resources, and live in remote rural

areas that are far from hospitals making expensive

treatments at hospitals or health centers inaccessible,

impractical, and of low priority. These factors make

herbal medicines even more important to developing

countries.

Further study of the individual components of the

aqueous L. sativum seeds extract is recommended to

try to determine the ingredient(s) that may be

responsible for its antineoplastic effects, which may

be used as natural and low cost drugs to fight cancer

with minimal or no side effects. It is also

recommended to test the cytotoxic effects of the

extract on other types of cancers in general and breast

cancers in particular. In addition, it is recommended

to test higher and lower concentrations of the extract

and to use it in combination with other promising

nonconventional treatments or chemical

chemotherapy to enhance its effects.

Conclusion

The findings of this study highlight the usefulness

of using local and easily available plant products and

constituents in treating or preventing diseases. The

findings are encouraging and warrant further work on

the aqueous extract of L. sativum seeds and its effects

on breast cancer and possibly even on other types of

cancers.

Funding: This study was funded by King

Abdulaziz City for Science and Technology.

References 1 American Cancer Society, Cancer Facts & Figures 2010,

(GA: American Cancer Society, Inc.), 2010.

2 Kassie F, Laky B, Gminski R, Mersch-Sundermann V,

Scharf G, Lhoste E & Kansmüller S, Effects of garden and

water cress juices and their constituents, benzyl and

phenethyl isothiocyanates, towards benzo(a)pyrene-induced

DNA damage: a model study with the single cell gel

electrophoresis/Hep G2 assay, Chemico-Biological

Interactions, 142 (2002) 285-296.

3 Shoieb A M, Elgayyar M, Dudrick P S, Bell J L & Tithof P K,

In-vitro inhibition of growth and induction of apoptosis in

cancer cell lines by thymoquinone, Int J Onccol, 22(2003)

107-113.


613

4 Campbell C T, Prince M, Landry G M, Kha V & Kleiner

H E, Pro-apoptotic effects of 1'-acetoxychavicol acetate in

human breast carcinoma cells, Toxicol Lett, 173 (2007)

151-160.

5 Conforti F, Ioele G, Statti G A, Marrelli M, Ragno G &

Menichini F, Antiproliferative activity against human tumor

cell lines and toxicity test on Mediterranean dietary plants,

Food Chem Toxicol, 46 (2008) 3325-3332.

6 Kasabe P J, Patil P N, Kamble D D & Dandge P B,

Nutritional, elemental analysis and antioxidant activity of

garden cress (Lepidium sativum L.) seeds, Int J Pharm

Pharmaceut Sci, 4 (2012) 392-395.

7 Sharma S & Agarwal N, Nourishing and healing prowess of

garden cress (Lepidium sativum L.), Indian J Nat Prod

Resour, 2(2011) 292-297.

8 Maier U H, Gundlach H & Zenk M H, Seven imidazole

alkaloids from Lepidium sativum, Phytochemistry, 49 (1998)

1791-1795.

9 Mali R G, Mahajan S G & Mehta A A, Studies on

bronchodilatory effect of Lepidium sativum against allergen

induced bronchospasm in guinea pigs, Pharmacog Mag, 4

(2008) 189-192.

10 Orlovskaya T V & Chelombit'ko V A, Phenolic compounds

from Lepidium sativum, Chem Nat Comp, 43(2007) 323.

11 Güvenç A, Okada Y, Akkol E K, Duman H, Okuyama T &

Çalış İ, Investigations of anti-inflammatory, antinociceptive,

antioxidant and aldose reductase inhibitory activities of

phenolic compounds from Sideritis brevibracteata,

J Ethnopharmacol, 61(1998) 161-166.

12 Wojdyło A, Oszmiański J & Czemerys R, Antioxidant

activity and phenolic compounds in 32 selected herbs, Food

Chem, 105(2007) 940–949.

13 Conforti F, Sosa S, Marrelli M, Menichini F, Statti G A,

Uzunov D, Tubaro A & Menichini F, The protective ability

of Mediterranean dietary plants against the oxidative

damage: The role of radical oxygen species in inflammation

and the polyphenol, flavonoid and sterol contents, Food

Chem, 112(2009) 587-594.

14 Simopoulos A P, Omega-3 fatty acids in health and disease

and in growth and development, The Am J Clin Nutr, 1(1991)

438-63.

15 Berquin I M, Edwards I J & Chen Y Q, Multi-targeted

therapy of cancer by omega-3 fatty acids, Cancer Letters,

269 (2008) 363–377.

16 Diwakara B T, Duttaa P K, Lokeshb B R & Naidu K A,

Bio-availability and metabolism of n-3 fatty acid rich garden

cress (Lepidium sativum) seed oil in albino rats,

Prostaglandins, Leukotrienes and Essential Fatty Acids, 78

(2008) 123–130.

17 Tisdale M J & Dhesi J K, Inhibition of Weight Loss by u-3

Fatty Acids in an Experimental Cachexia Model, Cancer

Res, 50(1990) 5022-5026.

18 Gokavi S S, Malleshi N G & Guo M, Chemical Composition

of Garden Cress (Lepidium sativum) Seeds and Its Fractions

and Use of Bran as a Functional Ingredient, Plant Foods

Hum Nutr, 59 (2005) 105-111.

19 Matthäus B & Angelini L G, Anti-nutritive constituents in

oilseed crops from Italy, Ind Crops Prod, 21(2005) 89-99.

20 Karazhiyan H, Razavi S M A, Phillips G O, Fang Y,

Al-Assaf S, Nishinari K & Farhoosh R, Rheological

properties of Lepidium sativum seed extract as a function of

concentration, temperature and time, Food Hydrocolloids, 23

(2009) 2062-2068.

21 Hasapis X & Macleod A J, Benzylglucosinolate

degradation in heat-treated Lepidium sativum seeds and

detection of a thiocyanate-forming factor, Phytochemistry,

21(1982) 1009-1013.

22 Tisdale M J & Dhesi J K, Successful herbivore attack due to

metabolic diversion of a plant chemical defense, The Nat

Acad Sci, 101 (2004) 4859–4864.

23 Xiao D, Vogel V & Singh S V, Benzyl isothiocyanate–

induced apoptosis in human breast cancer cells is initiated by

reactive oxygen species and regulated by Bax and Bak,

Molecular Cancer Therapeutics, 5 (2006) 2931-2945.

24 Kassie F, Pool-Zobel B, Parzefall W & Knasmüller S,

Genotoxic effects of benzyl isothiocyanate, a natural

chemopreventive agent, Mutagenesis, 14 (1999) 595-604.

25 Goosen T C, Mills D E & Hollenberg P F, Effects of Benzyl

Isothiocyanate on Rat and Human Cytochromes P450:

Identification of Metabolites Formed by P450 2B1, The J

Pharmacl Exp Therap, 296 (2001) 198-206.

26 Tseng E, Scott-Ramsay E A & Morris M E, Dietary Organic

Isothiocyanates Are Cytotoxic in Human Breast Cancer

MCF-7 and Mammary Epithelial MCF-12A Cell Lines, Exp

Biol Med, 229(2004) 835-842.

27 Zhang R, Loganathan S, Humphreys I & Srivastava S K,

Benzyl Isothiocyanate-Induced DNA Damage Causes

G2/M Cell Cycle Arrest and Apoptosis in Human

Pancreatic Cancer Cells, The Journal of Nutrition, 136

(2006) 2728-2734.

28 Pledgie-Tracy A, Sobolewski M D & Davidson N E,

Sulforaphane induces cell type–specific apoptosis in human

breast cancer cell lines, Molecular Cancer Therapeutics, 6

(2007) 1013-1021.

29 Kassie F, Rabot S, Uhl M, Huber W, Qin H M, Helma C,

Schulte-Hermann R & Knasmüller S, Chemoprotective

effects of garden cress (Lepidium sativum) and its

constituents towards 2-amino-3-methyl-imidazo[4,5-

f]quinoline (IQ)-induced genotoxic effects and colonic

preneoplastic lesions, Carcinogenesis, 23(2002) 1155-1161.

30 Mahassni S H & Al-Reemi R M Apoptosis and necrosis of

human breast cancer cells by an aqueous extract of garden

cress (Lepidium sativum) seeds, Saudi Journal of Biological

Sciences, 20 (2013) 131–139.

31 Eddouks M, Maghrani M, Zeggwagh N A & Michel J B,

Study of the hypoglycaemic activity of Lepidium sativum L.

aqueous extract in normal and diabetic rats,

J Ethnopharmacol, 97 (2005) 391-395.

32 Maghrani M, Zeggwagh N-A, Michel J-B & Eddouks M,

Antihypertensive effect of Lepidium sativum L. in

spontaneously hypertensive rats, J Ethnopharmacol,

100(2005) 193-197.

33 Patel U, Kulkarni M, Undale V & Bhosale A, Evaluation of

Diuretic Activity of Aqueous and Methanol Extracts of

Lepidium sativum Garden Cress (Cruciferae) in Rats, Trop J

Pharmaceut Res, 8 (2009) 215-219.

34 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J,

Vistica D, Warren J T, Bokesch H, Kenney S & Boyd M R,

New colorimetric cytotoxicity assay for cancer-drug

screening, J Nat Cancer Inst, 82 (1990) 1107-1112.

35 Zhang Y, Role of glutathione in the accumulation of

anticarcinogenic isothiocyanates and their glutathione


614

conjugates by murine hepatoma cells, Carcinogenesis,

21(2000) 1175-1182.

36 Kassie F, Uhl M, Rabot S, Grasl-Kraupp B, Verkerk R,

Kundi M, Chabicovsky M, Schulte-Hermann R &

Knasmüller1 S, Chemoprevention of 2-amino-3-

methylimidazo[4,5-f]quinoline (IQ)-induced colonic and

hepatic preneoplastic lesions in the F344 rat by cruciferous

vegetables administered simultaneously with the carcinogen,

Carcinogenesis, 24 (2003) 255-261.

37 Brandi G, Schiavano G F, Zaffaroni N, Marco C D, Paiardini M,

Cervasi B & Magnani M, Mechanisms of Action and

Antiproliferative Properties of Brassica oleracea Juice in

Human Breast Cancer Cell Lines, The Am Soc Nutr Sci,

135(2005) 1503-1509.

38 Abu-Dahab R & Afifi F, Antiproliferative activity of

selected medicinal plants of Jordan against a breast

adenocarcinoma cell line (MCF-7), Sci Pharmaceut, 75

(2007) 121-136.

cytotoxic effect of an aqueous extract of lepidium sativum...

Documents