sulforaphane (s-antioxidant) from broccoli (brassica oleracea) as antiproliferation and inducer of...

7/28/2019 SULFORAPHANE (S-ANTIOXIDANT) FROM BROCCOLI (Brassica oleracea) AS ANTIPROLIFERATION AND INDUCER OF A

1/30

SPECIAL STUDY I

SULFORAPHANE (S-ANTIOXIDANT)

FROM BROCCOLI (Brassica oleracea)

AS ANTIPROLIFERATION AND INDUCER OF APOPTOSIS

IN BREAST CANCER CELLS

BY:

FARADILLA NOVITA ANGGREINI

NIM.0802005008

2nd

SEMESTER

SUPERVISOR:

DR. WAYAN SUGIRITAMA, M.KES

FACULTY OF MEDICINE UDAYANA UNIVERSITY

DENPASAR

2009


2/30

ii

PREFACE

I would like to say thanks to the Lord for His charity, because of Him, I can finish

this scientific writing as my final report on the time that have been given to me.

Scientific writing based on the literatur titled Sulforaphane (S-Antioxidant) from

Broccoli (Brassica Oleracea) as Antiproliferation and Inducer of Apoptosis in

Breast Cancer Cells was made in order to complete and pass final report of

Special Study I in 2nd semester. Wishes that I can be able and applicate my ability

to compile scientific writing systematically which comes from valid literatures.

In this chance, I would thank to:

1. dr. Wayan Sugiritama, M.Kes as my supervisor for the guidances incompiling this scientific writing,

2. dr. Ida Ayu Ika Wahyuniari, M.Kes / dr. I Gusti Ayu Dewi Ratnayanti,S.Ked as evaluator, and

3. All parties that have given supports for me in compiling this scientificwriting neither morally or materially.

I recognize that this writing still far away from perfection. Accordingly, I wish

more suggestions and critics for making this writing better. Finally, I also hope

this scientific writing can give positive contribution for the development of

knowledge, especially in medical field.

Denpasar, 23r of July 2009

Writer


3/30

iii

CONTENTS

Content Page

REPORT COVER ........................................................................................ i

PREFACE ..................................................................................................... ii

CONTENTS .................................................................................................. iii

TABLE LISTS ............................................................................................... iv

FIGURE LISTS ............................................................................................ v

ABBREVIATIONS ...................................................................................... vi

SECTION I INTRODUCTION1.1 Background ............................................................................... 11.2 Problems .................................................................................... 31.3 Aims .......................................................................................... 31.4 Benefits ..................................................................................... 3

SECTION II LITERATURE REVIEW

2.1 Cell Cycle and Apoptosis .......................................................... 42.2

Sulforaphane ............................................................................. 7

2.2.1 Molecular and Chemical Structure of Sulforaphane 82.2.2 Source of Sulforaphane ............................................ 82.2.3 Mechanism of Sulforaphane in Breast Cancer Treatmet . 9

2.2.3.1Sulforaphane Inhibit Cell Proliferation in BreastCancer Cells .................................................. 9

2.2.3.2Sulforaphane Induce Apoptosis of Breast CancerCells ............................................................... 14

SECTION III CONCLUSION

3.1 Conclusion ................................................................................. 213.2 Suggestions ............................................................................... 21

REFERENCES ............................................................................................. 23

APPENDIX

Original Articles


4/30

iv

TABLE LISTS

Table 1. Taxonomy of Broccoli and Cauliflower .......................................... 9


5/30

v

FIGURE LISTS

Figure 1. Cell Cycle .................................................................................. 4

Figure 2. Mechanism of Apoptosis ........................................................... 6

Figure 3. Pathway of Apoptosis ............................................................... 7

Figure 4. Extrinsic and Instrinsic Pathway of Apoptosis ......................... 7

Figure 5. Chemical Structure of Sulforaphane ........................................ 8

Figure 6. Broccoli and Cauliflower ......................................................... 8

Figure 7. Sulforaphane Inhibits Cell Growth in Several Human Breast Cancer

Cell Lines .................................................................................. 10Figure 8. Sulforaphane Down-regulates ER-, EGFR, and HER-2 Protein in

Multiple Breast Cancer Cell Lines ........................................... 11

Figure 9. Sulforaphane Induced a G2-M Block and Increased Cyclin B1 Protein

Expression in Breast Cancer Cells ........................................... 11

Figure 10. Reduction ofDNA Synthesis in MCF-7 cell (A) and MDA-MB-231

cell (B) ...................................................................................... 12

Figure 11. Cell Growth Inhibitor Induced by Cauliflower Juice ................. 13

Figure 12. Expression of the G1 CDKandPhosphorylationpRb Level in MCF-7

Cell ........................................................................................... 13

Figure 13. The Analysis Result from Effect of Sulforaphane on Apoptotic

Proteins byElectrophoresis ..................................................... 14

Figure 14. Sulforaphane Induced Apoptosis Associated with Induction of Bax

and Bak Protein ........................................................................ 17

Figure 15. Analysis Result the Release ofCytochrome c from Mitochondria to

the Cytosol by Immunoblotting ................................................ 18


the Cytosol byImmunohistochemistry ..................................... 19

Figure 17. Increasing ofApaf-1 Protein Levels, Inhibition ofXIAPProtein, and

Mitochondrial Translocation of Bax ........................................ 20


6/30

vi

ABBREVIATIONS

GLS = Glucosinolates

ITCs =Isothiocyanates

ROS =Reactive Oxygen Species

CDKs = Cyclin-Dependent Kinases

pRb =Retinoblastoma protein

DNA =Deoxyribonucleic Acid

mRNA = messenger-Ribonucleic Acid

ER =Estrogen ReceptorTRADD = Tumor-necrosis factor Receptor Associated Death Domain

TRAF 2 = Tumor-necrosis factor Receptor Associated Factor 2

FAAD =Fas-associated Death Domain

DKO =Double Knockout

PARP =Poly (ADP-Ribose) Polymerase

XIAP =X-linked Inhibitor of Apoptosis

HO-1 =Heme Oxygenase-1

NQO1 =NAD(P)H:Quinone Oxidoreductase-1

NRF2 =Nuclear-factor erythroid 2-Related Factor 2

EGFR =Epidermal Growth Factor Receptor

HER-2 =Human EGFR-2

MEFs =Mouse Embrionic Fibroblasts

RPE =Retinal Pigment Epithelial


7/30

1

SECTION I

INTRODUCTION

1.1BackgroundCancer is the abnormal cell growth in a tissue that escape from the normal

control, can become invasive and metastases to the other tissues or organs by

blood and lymph circulation. Cancer is caused by alteration in oncogenes,

tumor suppressor genes, and microRNA genes (Croce, 2008). In cancer cells,

protooncogenes (a gene typically involved in cell growth or proliferation)

mutated to oncogen, this incidence caused overproliferation in cancer cells.

Tumor suppressor genes are genes to stop or inhibit cell proliferation in

normal condition, e.g. pRb and p53. If there was DNA damage in cell, tumor

suppressor gene would stop the proliferation in order to repairDNA or cell

apoptosis. However in cancer cells, tumor suppressor genes is inactivated and

no one can stop the proliferation ofDNA damaged cells (Dyke, 2007).

In women, breast cancer is the common cancer and is leading cause of cancer

death. More than one million women were diagnosed (22% of all female

cancer diagnoses) and 373 000 women died (14% of all cancer deaths among

women) of breast cancer in 2000 (Althuis, 2005). Complication of metastatic

breast cancer cells are very dangerous, because this cells can metastate to the

other organs or tissue like bone marrow, lungs, liver and brain, develop more

colonies of cancer cells in that place (Chiang and Massague, 2008). Until thistime, no medication which can cure cancer totally 100%. Only hormonal

theraphy with SERMs, such as tamoxifen and aromatase inhibitor become

standard treatment of women with ER+ breast cancer. This medication only

can prevent the development of invasive breast cancer (Anonim, 2006).

Consumers of higher levels antioxidant in vegetables have attracted particular

attention of some people to prevent the disease, especially cancer which


8/30

2

associated with free radical attack. Free radical is any chemical species that

has one or more unpaired electrons, unstable, and highly reactive. Many foods

contain free radicals, e.g.junk foodand fried food with waste cooking oilin its

cooking process. Some people want to keep their body healthy by consume

nature food which processed by good cooking process. Many plant-derived

substances, collectively termed phytonutrients or phytochemicals are

becoming increasingly known for their antioxidant activity. For example,

sulforaphane, the sulfur containing phytochemicals can be found in the

cruciferous vegetables from genus Brassica (broccoli and cauliflower). Data

from several epidemiologic studies have suggested that diets rich in

cruciferous vegetables, such as broccoli and cauliflower, reduce the risk of

developing many common cancer including breast cancer (Pledgie-Tracy, et

al., 2007). Broccoli and cauliflower contain high concentrations of

glucosinolates that can be hydrolyzed by the plant enzyme, myrosinase, or

intestinal microflora to isothiocyanates (sulforaphane), potent inducers of

cytoprotective enzymes and inhibitors of carcinogenesis (Cornblatt, et al.,

2007). As antioxidant, sulforaphane known as inducer of cytoprotective

enzymes, such as NQO1 (NAD(P)H:Quinone Oxidoreductase-1) and HO-1

(Heme Oxygenase-1), which NQO1 protects cells from oxidative damage by

two-electrons reduction of quinones, suppressing oxidative cycling and ROS

(Reactive Oxygen Species) generation, while HO-1 which is an important

enzyme in heme catabolism, lead to production of biliverdin, which upon

reduction forms the reactive oxygen scavenger bilirubin (Cornblatt, et al.,

2007). In this scientific paper, it would be explained about cell cycle and

mechanism of apoptosis, mechanism of sulforaphane in breast cancertreatment which focused on inhibition of cell proliferation and induction of

apoptosis in breast cancer cells.


9/30

3

1.2Problems1. How do the process of cell cycle and apoptosis?2. How can sulforaphane inhibit the proliferation of the breast cancer cells?3. How can sulforaphane induce the apoptosis of the breast cancer cells?

1.3Aims1. To understand the process of cell cycle and apoptosis.2. To understand the mechanism of sulforaphane in inhibiting the

proliferation of breast cancer cells.3. To understand the mechanism of sulforaphane in inducing the apoptosis of

breast cancer cells.

1.4BenefitsFor the writer:

1. The writer can understand the process of cell cycle and apoptosis,2. The writer can understand the mechanism of sulforaphane as antioxidant

in inhibiting the cell proliferation and inducing the apoptosis in the breast

cancer cells, and

3. The writer can increase the knowledge and experience in compiling thescientific writing based on the literature.

For the reader:

1. The reader can understand the process of cell cycle and apoptosis, and2. The reader can understand the mechanism of sulforaphane as antioxidant

in inhibiting the cell proliferation and inducing the apoptosis in the

prevention and treatment of breast cancer.


10/30

4

SECTION II

LITERATURE REVIEW

2.1 Cell Cycle and Apoptosis

The cell cycle is a series of events within the cell that prepare the cell for

dividing into two daughter cells. Cell cycle is divided into two major events:

interphase and mitosis. Mitosis is a shoter perior of cell cycle (only 5% of cell

cycle), it consist of five phases: prophase, prometaphase, metaphase,

anaphase, and telophase. Cell will divide its nucleus and cytoplasm giving rise

to two daughter cells. Interphase is the longer period of cell cycle (95% of cell

cycle), it consist of three phases: G1 phase, S phase, and G2 phase. In this

phase, cell will increase its size and content and replicates its genetic material.

Cells that have left the cell cycle are said to be in a resting stage, in G 0 phase

or stable phase (Gartner and Hiatt, 2007).

Figure 1. Cell Cycle (Gartner and Hiatt, 2007).


11/30

5

Daughter cells formed during mitosis enter the G1 phase, synthesize their

RNA, regulatory proteins essential to DNA replication, and enzymes necessary

to carry out these synthesize activities. In S phase, the cells synthesize their

DNA. During G2 phase, RNA and proteinsessential to cell division are

synthesize, the energy for mitosis is stored, tubulin is synthesize into

microtubules required for mitosis, and DNA replication is analyzed for

possible error. Mechanical force (e.g. stretching of smooth muscle), injury to

the tissue (e.g. ischemia), and cell death inducing the cells to enter the cell

cycle which is caused by release of ligands (growth factor). These ligands

induce the expression of protooncogene (a gene that control the cell

proliferation). Mutation on this gene as known as oncogene will cause over

cell proliferation, leads to cancer. Capability of the cell to begin and advance

through the cell cylce is govern by the presence and interaction of a group of

related proteins known as cyclin, with specific cyclin-dependent kinases

(CDKs), they are (Gartner and Hiatt, 2007):

- Cyclin D synthesized during early G1 phase binds to CDK4 and CDK6.Cyclin Esynthesize during late G1 phase binds to CDK2, permit the cell to

enter and proggress through S phase.

- Cyclin A binds to CDK2 and CDK1, permit the cell to leave S phase andenter G2 phase.

- Cyclin B binds to CDK1, allow cell to leave the G2 phase and enter Mphase.

Apoptosis is proggrammed cell death. Normally, the cell would stop to growth

and finally did the apoptosis. Apoptosis differ from necrosis. Necrosis is deathof cell in living tissue by patholigical condition, as a result from toxicity or

hypoxia (oxygen deficiency). If necrosis occurred, the cell which get injurious

stimulus will breakdown (lisis), while in apoptosis, the cell will develop its

apoptotic body (Mitchell and Cotran, 2003).


12/30

6

Figure 2. Mechanism of Apoptosis (Mitchell and Cotran, 2003).

Apoptosis can occur through two of pathways, they are instrinsic pathway

(mitochondria-mediated caspase cascade / stress pathway) and extrinsic pathway

(death-receptor pathway). Intrinsic pathway is triggered by proteins that contain

theBCL2 homology 3 domain, this domain inactivatesBCL2 andBCL-XL (which

normally inhibit apoptosis) and thereby activates the caspases that induce

apoptosis. Whereas the death-receptor pathway is activated by the binding ofFas

ligand, TRAIL, and tumor necrosis factor , to their corresponding (death)

receptors on the cell surface. Activation of death receptors activates caspases that

cause cell death (Croce, 2007).


13/30

7

Figure 3. Pathway of Apoptosis (Croce, 2007)

Figure 4. Extrinsic and Intrinsic Pathway of Apoptosis

(Mitchell dan Cotran, 2003).

2.2SulforaphaneSulforaphane is a naturally occuring member of the isothiocyanates family of

cancer chemopreventive agents that has attracted particular attention due to its

potent anticancer effects. As indirect antioxidant, sulforaphane can provide

potent and sustained protection against oxidant injury by inducing the broad

phase 2 enzyme response in cells. Upregulation of the phase 2 response


14/30

8

protectsRPEand retina from the damaging effects of photo-oxidant ROSand

electrophiles. A study from Cano et. al., indicated that sulforaphane protected

RPE cells from chemical oxidant stress, as evidenced by the diminished

intracellular redox shifts and the increasedRPEcell viability.

2.2.1 Molecular and Chemical Structure of SulforaphaneMolecular structure of sulforaphane is 1-isothiocyanato-4-(methylsulfinyl)-

butane. Its chemical structure is CH3-SO-(CH2)4-N=C=S (Choi and Singh,

2005).

Figure 5. Chemical Structure of Sulforaphane (Ebert, et. al., 2007).

2.2.2 Source of SulforaphaneSulforaphane is widely available in cruciferous vegetable genus Brassica, e.g.

broccoli and cauliflower. Broccoli and cauliflower is a member of cabbage

family. Broccoli resembled with cauliflower, its difference is only come from

its flowers colour. Broccoli was green, while cauliflower was white. Usually,

broccoli and cauliflower were eaten by boiling them, or directly eat them.

Figure 6. Broccoli and Cauliflower


15/30

9

Broccoli and cauliflower contain high concentration ofglucosinolates that can

be hydrolyzed by the plant enzyme (myrosinase) or intestinal microflora to

isothiocyanates (sulforaphane), known as potent inducer of cytoprotective

enzymes and inhibitor of carcinogenesis (Cornblatt, et. al., 2007). This is the

taxonomy of broccoli and cauliflower:

Table 1. Taxonomy of Broccoli and Cauliflower (ITIS, 2009).

Regnum Plantae (Plant)

Subregnum Tracheobionta (Vascular Plant)

Divisio Magnoliophyta (Angiospermae)

Class Magnoliopsida (Dicotyledone)

Subclass Diileniidae

Ordo Capparales

Familia Brassicaceae

Genus Brassica

Species Brassica olerace (Cabbage)

Variety Brassica oleracea var. botrytis(Broccoli, Cauliflower)

2.2.3 Mechanism of Sulforaphane in Breast Cancer TreatmentCancer cell is the cell that its gene has been mutated and hyperactively growth

by over proliferation. To cover that problems, sulforaphane as indirect

antioxidant provide inhibition of cell proliferation and apoptosis induction in

breast cancer cells.

2.2.3.1Sulforaphane Inhibit Cell Proliferation in Breast Cancer CellsSulforaphane inhibit breast cancer cell growth by decreased expression of

critical proteins involved in cancer cell growth: estrogen receptor (ER-),

epidermal growth factor receptor (EGFR), and human EGFR-2 (HER-2);

induced a G2-M phase block and increased expression of cyclin B1. Pledgie-


16/30

10

Tracy, et. al., have done their research which gave sulforaphane treatment 0-

25 mol/L in four breast cancer cell lines: MDA-MB-231, MDA-MB-468,

MCF-7, and T47D. From this research, it was found that:

- ER expressed in both MCF-7 and T47D cells, whereas MDA-MB-231 andMDA-MB-468 cells lack expression of ER,

- EGFR is overexpressed in MDA-MB-468 cell, highly exprexxed in T47Dcell, and expressed at lower level in MDA-MB-231 and MCF-7 cells,

- HER-2 is expressed at lower level in ER- MDA-MB-231 and MDA-MB-468 cells, expressed at higher level in ER+ MCF-7 and T47D cells.

Figure 7. Sulforaphane Inhibits Cell Growth in Several Human Breast Cancer

Cell Lines (Pledgie-Tracy, et. al., 2007).

15 and 25 mol/L of sulforaphane treatment down-regulatedEGFR andHER-

2 in all cell lines, was accompanied by a decrease in the mRNA expression of

both EGFR and HER-2. ER mRNA was also down-regulated in MCF-7 and

T47D cells, but no alteration ofER protein expression in MDA-MB-231 and

MDA-MB-468 cells.


17/30

11

Figure 8. Sulforaphane Down-regulatesER-,EGFR, andHER-2 Protein in

Multiple Breast Cancer Cell Lines (Pledgie-Tracy, et. al., 2007).

From Pledgie-Tracy et. al.s research, treatment with sulforaphane in 15

mol/L concentration increased the percentage of cells in G2-M phase,

decreased the percentage of cells in G1 and S phase within 24 hours of

treatment, and increased in cells in sub-G1 phase after 72 hours of

sulforaphane treatment. G2-M block was also accompanied by an increase in

cyclin B1, but not cyclin D1 protein expression in both MDA-MB-231 and

MCF-7 cells.

Figure 9. Sulforaphane Induced a G2-M Block and Increased Cyclin B1

Protein Expression in Breast Cancer Cells (Pledgie-Tracy, et. al., 2007).


18/30

12

Similar research by Brandi et. al. has been conducted to understand the

mechanism of antiproliferation properties ofBrassica oleracea (cauliflower)

juice. From the treatment can be indicated that cauliflower juice is a potent

inhibitor ofDNA synthesis in human breast cancer cell. It proved by reducing

DNA synthesis in MCF-7 sel with treatment of 3.5 mL/L cauliflower juice and

inhibiting ER- cells growth during 72 hours of treatment.

Figure 10. Reduction ofDNA Synthesis in MCF-7 cell (A) and MDA-MB-

231 cell (B) (Brandi, et. al., 2005).

Cauliflower juice at lower concentration also affect the cell cycle, the juice

suppressed cell growth without inducing a specific block of the cell cycle,

paralleled by a decrease in cell number in G0/G1 phase and increase in the

percentage of debris. Moreover, cauliflower juice is evaluated to know its

cytostatic and cytotoxic effect. As the result, growth arrest in juice-treated

cells is associated with a cytostatic mechanism and with necrotic cell death

that occurs at the higher concentrations utilized up to 25% in 20 mL/L of juice

as shown by loss of viability and increase ofdebris.


19/30

13

Figure 11. Cell Growth Inhibitor Induced by Cauliflower Juice

(Brandi, et. al., 2005).

Increased level ofp27 CDKinhibitor and decreased level ofCDK6protein in

MCF-7 cell have occurred after treatment of 10 mol juice during 22-72

hours. Decreased amount of total and hyperphosphorylation ofpRb (key

substrate for the G1CDKs) shown at lower concentration of juice.

Figure 12. Expression of the G1 CDKandPhosphorylationpRb Level in

MCF-7 cell (Brandi, et. al., 2005).


20/30

14

2.2.3.2Sulforaphane Induce Apoptosis of Breast Cancer CellsSulforaphane induce apoptosis in breast cancer cells through two pathways:

extrinsic and intrinsic pathway. Pledgie-Tracy, et. al. have proved it

succesfully that sulforaphane activated two apoptosic patways in breast cancer

cells. This is the result of electrophoresis which describe the effect of

sulforaphane on apoptotic proteins in four breast cancer cell lines:

Figure 13. The Analysis Result from Effect of Sulforaphane on Apoptotic

Proteins byElectrophoresis (Pledgie-Tracy, et. al., 2007).


21/30

15

From this electrophoresis result, it can be concluded that treatment with 15 or

25 mol/L sulforaphane in MDA-MB-231 cell increased the expression ofFas

ligand and the cleavage of caspase-3, caspase-8, an also PARP cleavage

without altered expression ofBcl-2 or cytochrome c localization (extrinsic

pathway). Whereas treatment with sulforaphane in MDA-MB-468 and T47D

cells activated cleavage ofPARP, caspase-3, dan caspase-9; decreased Bcl-2

expression; and activated the release ofcytochrome c from the mitochondria to

the cytosol (intrinsic pathway). From Brandi, et. al. research, it was found

increasing ofTRADD (180%), TRAF-2 (120%),BID (170%), and decreasing

FADD (50%) expression in MCF-7 cell which has been treated with

cauliflower juice.

A result from research which has been done by Choi and Singh, it was also

shown that treatment with sulforaphane induced the expression of Bax and

Bak proteins that have a critical role in the process of apoptosis, and also

caused mitochondria translocation of Bax to induce the release of apoptogenic

molecules from the mitochondria to the cytosol which result on the activation

of caspase (caused by induction ofApaf-1 protein and disfunction ofXIAP

protein) and cell death. Choi and Singh treatedMouse Embrionic Fibroblasts

(MEFs) from four types of different mice: Wild-type, Bax knockout(Bax -/-),

Bak knockout (Bak -/-), and Bax-Bak double knockout (DKO) which have

been immortalized by transfection with a plasmid containing SV40 genomic

DNA with sulforaphane (>99% pure).To evaluate the effect of sulforaphane

treatment on cytoplasmic histone-associated DNA fragmentation, similartreatment has been done in BEAS2B (normal human broncial epithelial cell

line), PrEC (normal prostate epithelial cell line), H1299 (human lung cancer

cell line), and LNCaP (human prostate cancer cell line). As the result from

that treatment: both cancer cells (H1299 and LNCaP) were significantly more

sensitive to sulforaphane-induced cytoplasmic histone-associated DNA

fragmentation, this result indicated that normal epithelial cells (BEAS2B and

PrEC) were significantly more resistant to apoptosis induction by


22/30

16

sulforaphane compared with cancer cells. Treatment with sulforaphane also

reduced the viability of MEFs Wild-type which was determined by trypan blue

dye assay, statistically significant increased in cytoplasmic histone-associated

DNA fragmentation (apoptosis induction has been detected), increased the

level of Bax and Bak protein expression. This result indicated that reduced

viability of MEFs Wild-type in the presence of sulforaphane was indeed due to

apoptosis induction by Bax and Bak proteins.


23/30

17

Figure 14. Sulforaphane Induced Apoptosis Associated with Induction of Bax

and Bak Protein (Choi and Singh, 2005).

The release of apoptogenic molecules including cytochrome c and

Smac/DIABLO from mitochondria to the cytosol, is a critical event in

apoptosis induction by a variety of stimuli. There are the result from


24/30

18

immunoblotting and immunohistochemistry which describe the release of

cytochrome c from mitochondria to the cytosol in MEFs:


the Cytosol by Immunoblotting(Choi and Singh, 2005).


25/30

19


the Cytosol byImmunohistochemistry (Choi and Singh, 2005).

From this result, it has been shown tnat treatment with sulforaphane in MEFs

Wild-type caused the release ofcytochrome c from mitochondria to the cytosol

after a hour of treatment (evidenced by a yellow-orange staining of the

mitochondria due to merge of green and red fluorescence) and also caused

cytosolic release ofSmac/DIABLO after 24 hours, this effect was regulated by

both Bax and Bak proteins, as shown in the absence of cytosolic release of

cytochrome c by immunoblottinganalysis in the control of MEFs Wild-type

(less Bax and Bak).Sulforaphane also causedproteolytic cleavage ofcaspase-

9 and caspase-3 in MEFs Wild-type, but less in MEFs DKO. In MEFs Wild-

type, Bax and Baksingle knockoutwere found the reduction ofXIAP protein

expression level (inhibitor of caspase activity) in higher concentration of

sulforaphane, but absence in MEFs DKO and increased the level ofApaf-1

protein (inducer of caspase-9 activation) after 8 hours of treatment in four

types of cells. These result suggested that sulforaphane-mediated induction of

Apaf-1 protein might be regulated by Bax and Bak proteins. In normal cells,


26/30

20

the Bax protein exist in an inactive form in the cytosol but can be induced to

change conformation and translocate to thye mitochondria in response to

certain apoptotis stimuli. In this research, Bax protein in MEFs Wild-type

translocated to the mitochondria after 12 hours of treatment (yellow-orange

staining).

Figure 17. Increasing ofApaf-1 Protein Levels, Inhibition ofXIAPProtein,

and Mitochondrial Translocation of Bax (Choi and Singh, 2005).


27/30

21

SECTION III

CONCLUSION

3.1 Conclusion

Sulforaphane as phytochemical antioxidant can inhibit cell proliferation in

breast cancer cells by suppressing or reducing the action ofER-,EGFR, and

HER-2 proteins which involved in breast cancer cell growth, inducing G2-M

block by increasing the cyclin B1 levels, as potent inhibitor of DNA synthesis,

increasing p27 CDK inhibitor, reducing CDK6 levels in G0/G1 phase, and

decreasing pRb levels and hyperphosphorylation ofpRb. Sulforaphane which

can be found in cruciferous vegetables e.g. broccoli and cauliflower (Brassica

oleracea) also can induce the apoptosis of breast cancer cells through two

pathways: extrinsic pathway by up-regulating Fas ligand which results in

activation of caspase-3, caspase-8, and proteolytic cleavage ofPARP; and

intrinsic pathway (mitochondria-mediated caspase cascade) by activating

caspase-3 and caspase-9 (as a result caused by induction ofApaf-1 protein

and disfuntion ofXIAPprotein), decreasing Bcl-2 expression, and activating

the release of apoptogenic molecules e.g. cytochrome c and Smac/DIABLO

from mitochondria to the cytosol. Sulforaphane also up-regulating TRADD

and TRAF-2 expression, and inducing Bax and Bak proteins as inducer of

apoptosis.

3.2 Suggestions

1. It is necessary to observe continuously and research about mechanism ofsulforaphane in breast cancer treatment in contributing medical knowledge

especially oncology.

2. The community should take more attention and awareness to the diseaseespecially breast cancer, because until this time, theres no medication

which can cure cancer totally 100%.


28/30

22

3. Broccoli and cauliflower should be introduced to the wide community asvegetables rich in phytochemicalantioxidant (sulforaphane) which useful

in preventing and management of cancer, especially breast cancer.


29/30

23

REFERENCES

Althuis, D. Michelle, Jaclyn M. Dozier, William F. Anderson, Susan S. Devesa,

Louise A. Brinton. 2004. Global Trends in Breast Cancer Incidence and

Mortality 1973-1997. International Journal of Epidemiology 34:405-412

(Access on 13Juli 2009 at http://www.highwire.org)

Anonim. 2006.Fact Sheet: Breast Cancer. National Institutes of Health:USA

Brandi, Giorgio, Giuditta F. Schiavano, Nadia Zaffaroni, Cinzia De Marco, Mirko

Paiardini, Barbara Cervasi, et. al. 2005. Mechanisms of Action and

Antiproliferative Properties of Brassica oleracea Juice in Human BreastCancer Cell Lines. The Journal of Nutrition 22:1503-1509 (Access on 7

Juli 2009 at http://www.jn.nutrition.org)

Cano, Marisol del V., Johann M. reyes, Choul Y. Park, Xiangqun Gao, Keisuka

Mori, Roy S. Chuck, et. al. 2008. Demonstration by redox Fluorometry

that Sulforaphane Protects Retinal Pigment Epithelial Cells Againt

Oxidative Stress. Investigative Ophthalmology and Visual Science

49(6):2606-2612 (Access on 21 Juli 2009 at http://www.highwire.org)

Chiang, Anne C. dan Joan massague. 2008. Molecular Basis of Metastasis. The

New England Journal of Medicine 359:2814-2823 (Access on 18 Juli 2009

at http://www.nejm.org)

Choi, Sunga dan Shivendra V. Singh. 2005. Bax and Bak Are Required for

Apoptosis Induction by Sulforaphane, a Cruciferous Vegetable-Derived

Cancer Chemopreventive Agent. Cancer Research 65:2035-2043 (Access

on 9Juli 2009 at http://www.aacrjournals.org)

Cornblatt, Brian S., Lingxiang Ye, Albena T. Dinkova-Kostova, Melanie Erb, Jed

W. Fahey, Navin K. Singh, et. al. 2007. Preclinical and Clinical

Evaluation of Sulforaphane for Chemoprevention in the Breast.

Carcinogenesis 28:1485-1490 (Access on 9

Juli 2009 at

http://www.oxfordjournals.org)

Croce, Carlo M. 2008.Molecular Origins of Cancer: Oncogenes and Cancer. The

New England Journal of Medicine 358:502-511 (Access on 24 Juni 2009

at http://www.nejm.org)


30/30

24

Dyke, Terry V. 2007. p53 and Tumor Suppression. The New England Journal of

Medicine 356:79-81 (Access on 18 Juli 2009 at http://www.nejm.org)

Ebert, Bettina, Albercht Seidel dan Alfonso Lampen. 2007. Phytochemicals

Induce Breast Cancer Resistance Protein in Caco-2 Cells and Enhance the

Transport of Benzo[a]pyrene-3-sulfate. Toxicological Sciences 96:227-

236 (Access on 9 Juli 2009 at http://www.highwire.org)

Gartner, Leslie P. dan James L. Hiatt. 2007. Color Textbook of Histology Third

Edition. Saunders Elsevier:China

ITIS. 2009. Brassica oleracea var. botrytis Taxonomic Serial No.: 530957.

http://www.itis.gov (Access on 13Juli 2009)

Mitchell, Richard N. dan Ramzi S. Cotran. 2003. Robbins Basic Pathology

Seventh Edition. Saunders Company:London

Pledgie-Tracy, Allison, Michele D. Sobolewski, dan Nancy E. Davidson. 2007.

Sulforaphane Induces Cell Type-Specific Apoptosis in Human Breast

Cancer Cell Lines. Molecular Cancer Therapeutics 6(3):1013-1021

(Access on 11July 2009 at http://www.highwire.org)

sulforaphane (s-antioxidant) from broccoli (brassica oleracea) as antiproliferation and inducer of...

Documents