Cytotoxic Assay Compounds Genestein Against Breast Cancer Cells MCF-7, In Vitro

SUMMARY
Breast cancer is a malignant tumor that invades breast tissue and disease is frightening
for women. The cause of breast cancer is not known explicitly, there is no cure for the disease
that has been in an advanced stage, and there is no proper way to prevention.
Phytoestrogens are reported can prevent cancer caused by hormonal disorders such as
breast cancer, prostate cancer and colon cancer. Phytoestrogens are secondary metabolite
who syntheses by plants, among others in the form of plant compounds genestein that have
estrogenic activity. Several studies report that giving genestein can inhibit proliferation of
cancerous cells, because it can inhibit tyrosin kinase. In addition genestein can also inhibit
invasion tumor cells and angiogenesis so that it can serve as a antimetastasis compound.
Genestein exposure on prepubertas cause a loss of the target for the transformation of
malignan so supposedly can reduce risk of breast cancer. Soy and soy products are products
that contain genestein can be instrumental in preventing risk of developing breast cancer.
Soybeans containing phytoestrogens include genestein is a part of daily meal
consumed society. Therefore it is necessary to research the study of utilization
phytoestrogens in soy beans for prevention of breast cancer.
In vitro research aims to know genestein dose that causes cell death MCF-7 (LC50)
need to be performed as a second stage of research on in vivo.
In vitro research conducted using MCF-7 cell lines, with negative control is DMSO
and positive control is Doxorubicin. Genestein concentration tested was 100 g/ml, 200
g/ml,300 g/ml and 400 g/ml is used as a comparison, normal cells (Vero cells). Results of
in vitro studies indicate that treatment genestein on 208,31 g/ml concentration can cause
death of breast cancer MCF-7 cell lines of 50 (LC50). Genestein treatment LC50
concentration on MCF-7 cell lines were only a small percentage of the death cause Vero cell
lines on 200 g/ml concentration only lead to cell death 11,14% so it safe and not harmful to
normal cells.
Based on LC50 Genestein obtained in this research, need to do further testing in vivo,
using animal testing mice strain C3H has high sensitivity to induction of breast cancer cells,
with a test dose of soy beans in the formula genestein (milk from soybean seeds and flour of
soybean tempeh), so formula can be obtained from soy beans that are better and effective way
to prevent breast cancer.
keywords: genestein, breast cancer, MCF-7 cell lines, vero cell lines, LC50.
Introduction
Breast cancer is a malignant tumor that invades breast tissue and disease is frightening
for women (purwoastuti, 2008). Nina bin Hatim (2012) mentions that in 2008 Global Cancer
Statistics data shows number of cancer patients reached 24.6 million. The American Cancer
Society estimates that in 2011, in United States the number of women who died of breast
cancer, a mentioned person 39.520 reaches that deaths from breast cancer are higher than
other cancer deaths. Meanwhile, in Indonesia there is no statistical data that describes number
of breast cancer patients, but an extrapolation of the calculation shows the prevalence of
breast cancer sufferers in Indonesia amounted to 876.665 people (Kusminarto, 2008).
A cause of the occurrence of breast cancer is not known explicitly, there is no way to
cure disease has reached an advanced stage, and there is no proper way to prevention
(Anonim, 2009). It is said that the efforts of healing through surgery, chemotherapy and radio
therapy has been effective but haven't done much and just enough help for the treatment of
cancer detected at early stage (Nina bin Hatim, 2012). It further mentioned that the way
treatment is an expensive cost.
Kusminarto (2008) mentioned that the world of medicine has not found a way to
prevent breast cancer. It is said that if there were an effective means of early detection of
breast cancer, it will be a lot of lives can be saved, due to the presence of known early breast
cancer, more likely it can be cured. Therefore appropriate prevention efforts and effectively
needs to be constantly reviewed and researched.
Many factors can cause occure of breast cancer include age, socio-economic status,
reproductive status, exogenus hormone therapy, lifestyle, and family history. Most cases of
breast cancer occur in women post-menopause and depend on estrogen (Young et al., 2006)
Phytoestrogens are reported can prevent cancer due to hormonal disturbances, such as
breast cancer, prostate cancer, and colon cancer, because estrgogenic activity are considered
to be influential in cancer prevention. Phytoestrogens also has antioxidant activity, can
influence intracellular enzyme, cell proliferation and protein synthesis (Davies et al., 1999).
Giving genistein and estrogens can reduce size of glandular epithelium mamae area
and increase density of structure of alveolar lobulo. Mentioned also that exposure to genistein
at the age of prepubertas leads to loss of target for malignant transformation, thus reducing
risk of breast cancer (Cabanes, 2004).
Giving genistein through injections in mice and being exposed to carcinogens in
adulthood, suggesting risk of breast cancer growth and declining,. Giving genistein during
pre-puberty also can reduce risk of breast cancer during adulthood (Thomsen et al., 2006).
 Soy and soy products have an important role in lowering risk exposed to a wide range
of degenerative diseases, among other due to the presence of Phytoestrogens in soy beans.
Phytoestrogens in soy beans, such as genistein has oestrogenic activity (Koswara, 2008).
Genistein can suppress chromosome and DNA changes in liver and glands mamae on mice
with exposure DMBA (Jin and McDonald, 2002).
Soy beans are foods consumed by the public. Soy beans contains Phytoestrogens
genestein that can prevent breast cancer, so it has the potential to be utilized as a breast
cancer prevention is cheap and easily available. Therefore, need to review formula of soy
beans and effective dose to prevent risk of breast cancer, so that research needs to be done
utilization of Phytoestrogens from soy beans for prevention of breast cancer.

TINJAUAN PUSTAKA
Breast cancer is a malignant tumor that invades breast tissue and disease is frightening
for women (Purwoastuti, 2008). Cancer is characterized by abnormal growth of cells which
can spread and can destroy organs and tissues of the body. Cancers are classified according to
the tissue affected, e.g. breast cancer, uterine cancer, prostate cancer, colon cancer (Koswara,
2008). Most cases of breast cancer occur in women post-menopause and depends on the
activity of estrogen (Young et al., 2006).
Endogenus estrogen naturally plays an important role in the development of a variety
of networks, in particular the reproductive organs including the glands, while estrogen
mamae eksogenus can play a role in increasing the risk of breast cancer and cancer-related
hormonal (Thomsen et al., 2006).
Phytoestrogens are reported can prevent cancer caused by hormonal disturbances,
such as breast cancer, prostate cancer, and colon cancer because of the nature of anti-
estrogennya. Phytoestrogens also showed antioxidant activity and influence on the breeding
of cells, intracellular enzymes and protein synthesis (Davies et al., 1999). Phytoestrogens can
work through a hormonal mechanism to lower risk of cancer with estrogen receptor binding
or interaction with enzymes involved in the biosynthesis of steroids and metabolism
(Cotterchio, 2006).
Koswara (2008) mentions that in seeds of soybean contained Phytoestrogens or
isoflavones such as compounds genistein can produce estrogen activity. Also mentioned by
Duffy et al. (2007) that soy beans contained a major isoflavones is genistein.
Genestein (4,5,7-trihydroxyisoflavone), is a heterocyclic compound difenol that
structurally like as 17-estradiol so that genestein can be bound to estrogen receptors. In
addition to having oestrogenic effects which is able to inhibit proliferation of cancer cells,
genestein are also capable inhibit tyrosine kinase, stabilize topoisomerase II and was
instrumental in setting up transcription on the growth factor-. With these mechanisms,
genestein could disrupt cell cycle, triggering apoptosis and induces differentiation of different
types of cancer cells. In addition genestein is also capable of inhibit tumor cell invasion and
angiogenesis activity so genestein allegedly serves as a antimetastatis compound (Vantyghem
et al., 2005).
Jin and McDonald (2002) reported that giving of isoflavones containing 250 g/g
genestein can inhibit breast tumors in mice. It further said that genestein have affinity with
estrogen receptors and ability to inhibit tyrosine kinase. An increase activity of tyrosine
kinase interferes coordination cell and increase occurrence of cell changes towards cancer so
that inhibition of tyrosine kinase is thought to be able to prevent occurre of cancer.
In addition genestein can also stimulate differentiation of glandular mamae became
resistant to occurrence of cancer (Rowlands et al., 2002). It is said that the effect of giving
genestein in breast cancer depends on the time of giving, due to changes in development of
breasts in women throughout his life is related to hormonal changes during puberty,
pregnancy and menopause. Lamartiniere (2002), reported that giving genestein of prepubertas
period may inhibit breast cancer induced by chemical compound.
Genestein in subcutaneous in mice after birth until 21st day with a dose of 1-10 mg/kg
per day is apparently causing mice became resistant to breast cancer induced by DMBA when
mature. Experiment on in vitro fertilization, genestein and other isoflavones in low
concentrations (<10 M) worked as agonist estrogen, but at high concentrations (> 10 M) in
addition genestein have oestrogenic activity also has a biological effect on cells that
eventually suppress cell growth (Murril et al., 1996). Other studies using genestein high doses
(500 g/kg weight) on day 16, 18, and 20 after birth, reducing formation of breast tumors as
an adult at the time of DMBA induction (Padilla-Banks et al., 2006).
Also reported by Thomsen et al. (2006) that giving of genistein through injections in
mice and after being exposed to carcinogens, showed a decrease in risk of breast cancer. It
further said that giving of genistein during pre-puberty also reduces risk of breast tumors
when mature.
Cabanes et al. (2004) stated that exposure genestein during pre-puberty leads to loss
of target for malignant transformation, thus reducing risk of breast cancer. Decrease of risk
can occur when genistein given via injection (Lamartiniere et al., 1995: Murrill et al., 1996)
or given away via food in doses of 250 g/g (Fritz et al., 1998). On the other hand, it was
reported that are given genestein of the mice as much as 750 g/g indicate that tumor growth
is not unlike real mice compared to controls (Santell et al., 2000). Therefore the proper dose
of giving genestein and can prevent breast cancer needs to be examined further.
Soy beans are foods consumed by the public. Soybean seeds are contained inside a
phytoestrogens genestein (Koswara, 2008). Fermented soy beans have a higher content of
genistein, necessitating soy beans formulation of precise and can be useful to prevent breast
cancer.

RESEARCH METHODS
The research was carried out in the Laboratorium Penelitian dan Pengujian Terpadu
(LPPT) at Gadjah Mada University, Yogyakarta. The research aims to know dose of
genestein that causes MCF-7 cell death (LC50).
Preparation of MCF-7 cells was done together as shown by Puspitasari and Ulfa
(2009) as follows: Ampules containing MCF-7 cells were taken from a storage in a tank of
liquid nitrogen, soon thawing in water bath with temperature 370 C until melted. Ampules are
opened and moved into the new conical tube containing RPMI 1640 medium 6-10 ml. RPMI
1640 added 10% FBS and 2% streptomicin and Penicilin 0.5% fungizon was prepared, then
sentrifuge at 1500 rpm for 5 minutes. Supernatan thrown, pellets plus 6-10 ml MK, slowly
resuspation until homogeneous, then the cell input tissue culture flask and incubated at a
temperature of 370 C with CO2 flow 5%. After 24 hours the medium changed and grown
cells to konfluen. Cell culture checked to see density his cell. Cell culture is calculated using
haemacytometer on 4 chamber that each contains 16 boxes and taken his average, then
multiplied by a dilution factor and correction factors for each major area (volume 104 ml),
with formula Djajanegara and Wahyudi (2009) as follows:
n/4 x P x 104 sel/ml

where:
n : number of cells counted
4 : total calculated haemocytometer booth
104 : haemocytometer capacity per ml
P : dilution factor
The number of ml of the transferred cells to produce = the total number of cells required
number of cells counted

Then added medium growers a number of ml is required. For treatment of MCF-7 cells
required 5 x 103 cells/well.
The toxicity assay of genestein against cancer cells MCF-7 needs 5x103 cell/well.
Some 100 l suspension of cancer cells input into each well plate. Then, incubated the cell
with serial concentrations of genestein treatment done i.e: 0 g/ml or control (G0), 100 g/ml
(G1), 200 g/ml (G2), 300 g/ml (G3), 400 g/ml (G4), in medium culture (370 C, CO2 5%),
during 24-48 hours, with three replicates. At the end of incubation, cell culture media is
removed and washed with PBS 1 time, then added MTT 100 l each well, and incubated for
24 hours, until formazan crystal formed. After crystal formazan formed, in cell culture added
stopper SDS 10 in 0.1 N HCl, incubated in the dark last night. Then read with elisa reader
with a wavelength of 550 nm. The living cell will be reacted to with MTT to form purple
(Turalely et al., 2012). As positive controls are used giving of Doxorubicin and for
comparator, testing was also performed against normal cells (Vero cells).
The percentage of death cells is calculated with: the number of living cells control
reduced the number of living cells treatment, divided the number of living cells control
multiplied 100%. LC50 value calculated by Probit analysis.

RESULTS
As negative controls, have made cytotoxic assay with DMSO treatment, which is a
solvent of Genestein to be tested. The solvent DMSO treatment tested on the highest
concentration of 400 g/ml. The observations and calculations show that DMSO does not
cause the death of breast cancer cells MCF-7 with values -7,47%. This indicates that DMSO
directly is not toxic and can be used as a solvent in cytotoxic assay Genestein against breast
cancer cells MCF-7.
As positive controls used Doxorubicin treatment which is a substance used in
chemotherapy, to see the sensitivity of MCF-7 cells. Results of the observation and
calculation of the percentage of cell death in MCF7 breast cancer Doxorubicin treatment as
shown in table 2 :
Table 2. A percentage Death Cells on Doxorubicin treatment as positif control against
breast cancer cell MCF-7

No Concentration (g/ml) Percentage of death cells (%)

1 0,25 36,19
2 0,50 46,90
3 1,00 51,65
4 2,00 62,48
5 4,00 66,83
6 8,00 73,82

Based on the above table, it is known that Doxorubicin treatment at low

concentrations has caused death of MCF-7, and at high concentrations 1 g/ml has caused the
death of MCF-7 cells more than 50%. A treatment with the higher concentration of
Doxorubicin that causes death of breast cancer cells MCF-7 higher anyway. Influence of
concentration of Doxorubicin to the death cells MCF-7 can be described as Figure 1:
100
80 Series1
DEATH CELLS

60 Linear (Series1)
40 y = 4.0956x + 45.561
R = 0.76
20
0
0 2 4 6 8 10
DOXORUBICIN CONCENTRATION

Figure 1. Graphics Percentage of death cells on doxorubicin treatment as positif control

against breast cancer cells MCF-7

Based on the results of observation and calculation above, then it can be calculated
that the concentration of Doxorubicin can cause death of MCF-7 50% (LC50) was on the
concentration 1.08 g/ml, that means cells MCF-7 that will be used are sensitive to substance
of chemotherapy and can be used for further testing.
Doksorubisin is a commonly used drug for treatment of breast cancer. Mechanism of
doksorubisin in cancer cells is associated with the process of inhibition synthesis DNA and
RNA, DNA repair processes and increase mutates. Doksorubisin also suggests inhibit
topoisomerase II enzyme activity that results in a mutation of DNA with a served DNA
strand then associated. These drugs can affect every phase on cell division cycle. However,
doksorubisin has a more powerful effectiveness on cell cycle in S phase (Semeniuk et al.,
2004).
 A treatment Genestein on MCF-7 cells, result of observation in microscope it appears
that influence Genestein can cause death of breast cancer MCF-7 cells. A figure MCF-7 cells
of who died and who lived before giving MTT as seen in Figure 2 and Figure 3:

Furthermore, the result observations and calculations of percentage cell death during
treatment Genestein against breast cancer MCF-7 cells, as seen in Table 3:

Table 3. A Percentage death cell on treatment Genestein against Breast Cancer MCF-7 cells
No Konsentrasi (g/ml) Persentase Kematian Sel (%)
1 100 33,16
2 200 49,11
3 300 56,14
4 400 67,55

Based on the table above, known as that the treatment Genestein can cause death
breast cancer MCF-7 cells 50% (LC50) is located at concentrations between 200 g/ml-300
g/ml. The influence of concentration Genestein of cell death MCF-7 can be described as
Figure 4:
Figure 4. A percentage of graphics death cell on treatment genestein against brest
cancer MCF-7 cells

Based on the results of observation and calculation above, then it can be calculated
that a treatment Genestein can cause death breast cancer cells 50% (LC50) in concentration
208,31 g/ml. Genistein can suppress cancer proliferation and induce apoptosis (cell death),
this is because genestein can act as triggers cell cycle arrest of cancer cells in mechanisms of
checkpoint.
Checkpoint serves to detect damage or mutations in DNA. If there is any damage or
mutations in DNA, checkpoint will spur cell cycle arrest for repair DNA. When a mechanism
of cell cycle arrest is not enough guarantee repair of damaged DNA, the cell will undergo
apoptosis (Ren et al., 2003). Inhibition of cancer cell growth caused by cell cycle arrest
eventually results of inhibit proliferation cells. Mentioning that genestein can induce G2/M
phase on cell cycle in gastric cancer cell melanoma (Cassagrande and Darbon, 2000).
Genestein is difenol heterocyclic compounds, it has the structural resemble with 17-
estradiol so that it can be bound to estrogen receptors. In addition genestein has estrogenic
effects which is able to inhibit proliferation of cancerous cells, genestein also capable of
inhibiting tyrosine kinase due to compete in ATP (Adlercreutz et al., 1995), as well as
stabilize topoisomerase II and was instrumental in setting up transcription on growth factor-.
With these mechanisms, genestein could disrupt cell cycle and trigger apoptosis of cancer
cells (Vantyghem et al., 2005). It further mentioned by Ren et al. (2003) that genistein
inhibiting topoisomerase I and II. The enzyme topoisomerase I play a role in cutting and
splitting a single strand of DNA and a type II topoisomerase enzim play role cut and break
down double-stranded DNA. Inhibition of enzyme topoisomerase will stabilize a complex
topoisomerase and causing truncated and mutates of DNA. DNA mutations can cause
expression of Bax and Bak protein that is proapoptosis protein.
Genestein is also capable of inhibiting tumor cell invasion and angiogenesis activity
so that it can function as compounds antimetastatis (Vantyghem et al., 2005). Angiogenesis is
the process of formation new blood vessels, so it necessary for proliferation, invasion and
metastasis of cancer cells. Genestein can inhibit vascular endothelial cell proliferation and
growth of cancer cells (Fotsis et al., 1995). It further said that TGF- is the main factor that
regulates cell proliferation and genistein may inhibit TGF-, so as inhibit angiogenesis..
Disruption of angiogenesis process cause cancer cells die as results of decreased nutrients and
oxygen (Ren et al., 2003).
As a comparison, a treatment Genestein also made against to Vero cells (normal
cells). Seems that microscope observations, influence of Genestein may also cause death of
Vero cells. Vero cells picture of who died and who lived before and after giving of MTT, as
shown in Figure 5 and Figure 6:

Figure 6. Vero cells on treatment Genestein after giving MTT, on that obsevation with
microscope inverted with maginification 10x10

Further observations and calculations of percentage death cell on Genestein treatment

against in Vero cells, as seen in Table 4:
 Table 4. A Percentage of death cells on treatment Genestein against vero cells
No Konsentrasi (g/ml) Persentase Kematian Sel (%)
1 100 5,84
2 200 11,14
3 300 17,67
4 400 34,78

Based on the table above, known as that treatment of Genestein up to 400 g/ml
concentrations can not achieve a 50% Vero cells death, it does mean that a treatments
Genestein up to 400 g/ml concentration are still far from under LC50. Likewise, when
compared with LC50 Genestein on MCF-7 cells i.e. 208,31 g/ml, then the concentration of
Genestein 200 g/ml against vero cells can causes death cell 11,14%, so harmless to normal
cells.

CONCLUSION DAN SUGGESTION:

Based on the results of in vitro assay, influence Genestein of death breast cancer
MCF-7 cells, some conclusions is:
- Genestein treatment that can be lead to death of breast cancer MCF-7 cells on 50%
(LC50) is at concentration 208,31 g/ml
- A treatment of Genestein on LC50 concentration against MCF-7 cells were only a
small percentage of death cells on normal cells, i.e., at concentrations Genestein: 200
g/ml causing death 11,14% on vero cells (normal cell)
- Based on a Genestein concentration of LC50 is 208,31 g/ml, need to do further
testing in vivo, with a test dose of soy beans in the formula genestein (milk from
soybean seeds and flour soybean tempeh), so can be obtained formula from soy beans
that are better and effective way to prevent breast cancer