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

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 in compiling 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 scientific writing 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, 23rd of July 2009

Writer

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CONTENTS
Content REPORT COVER PREFACE CONTENTS TABLE LISTS FIGURE LISTS Page ........................................................................................ i ii

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.................................................................................................. iii ............................................................................................... iv ............................................................................................ v

ABBREVIATIONS

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SECTION I INTRODUCTION 1.1 1.2 1.3 1.4 Background Problems Aims Benefits ............................................................................... .................................................................................... 1 3 3 3

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SECTION II LITERATURE REVIEW 2.1 2.2 Cell Cycle and Apoptosis Sulforaphane 2.2.1 2.2.2 2.2.3 .......................................................... 4 7 8 8

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Molecular and Chemical Structure of Sulforaphane Source of Sulforaphane ............................................

Mechanism of Sulforaphane in Breast Cancer Treatmet . 9 2.2.3.1 Sulforaphane Inhibit Cell Proliferation in Breast Cancer Cells .................................................. 9

2.2.3.2 Sulforaphane Induce Apoptosis of Breast Cancer Cells SECTION III CONCLUSION 3.1 3.2 Conclusion Suggestions ................................................................................. 21 ............................................................................... 21 ............................................................... 14

REFERENCES APPENDIX

............................................................................................. 23

Original Articles

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

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FIGURE LISTS
Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Cell Cycle .................................................................................. ........................................................... ............................................................... ......................... 4 6 7 7 8 8

Mechanism of Apoptosis Pathway of Apoptosis

Extrinsic and Instrinsic Pathway of Apoptosis Chemical Structure of Sulforaphane Broccoli and Cauliflower

........................................

.........................................................

Sulforaphane Inhibits Cell Growth in Several Human Breast Cancer Cell Lines .................................................................................. 10 Sulforaphane Down-regulates ER-, EGFR, and HER-2 Protein in Multiple Breast Cancer Cell Lines ........................................... 11

Figure 8.

Figure 9.

Sulforaphane Induced a G2-M Block and Increased Cyclin B1 Protein Expression in Breast Cancer Cells ........................................... 11

Figure 10. Reduction of DNA Synthesis in MCF-7 cell (A) and MDA-MB-231 cell (B) ...................................................................................... 12 ................. 13

Figure 11. Cell Growth Inhibitor Induced by Cauliflower Juice

Figure 12. Expression of the G1 CDK and Phosphorylation pRb Level in MCF-7 Cell ........................................................................................... 13 Figure 13. The Analysis Result from Effect of Sulforaphane on Apoptotic Proteins by Electrophoresis ..................................................... 14

Figure 14. Sulforaphane Induced Apoptosis Associated with Induction of Bax and Bak Protein ........................................................................ 17

Figure 15. Analysis Result the Release of Cytochrome c from Mitochondria to the Cytosol by Immunoblotting ................................................ 18

Figure 16. Analysis Result the Release of Cytochrome c from Mitochondria to the Cytosol by Immunohistochemistry ..................................... 19

Figure 17. Increasing of Apaf-1 Protein Levels, Inhibition of XIAP Protein, and Mitochondrial Translocation of Bax ........................................ 20

ABBREVIATIONS
GLS ITCs ROS CDKs pRb DNA mRNA ER TRADD TRAF 2 FAAD DKO PARP XIAP HO-1 NQO1 NRF2 EGFR HER-2 MEFs RPE = Glucosinolates = Isothiocyanates = Reactive Oxygen Species = Cyclin-Dependent Kinases = Retinoblastoma protein = Deoxyribonucleic Acid = messenger-Ribonucleic Acid = Estrogen Receptor = Tumor-necrosis factor Receptor Associated Death Domain = Tumor-necrosis factor Receptor Associated Factor 2 = Fas-associated Death Domain = Double Knockout = Poly (ADP-Ribose) Polymerase = X-linked Inhibitor of Apoptosis = Heme Oxygenase-1 = NAD(P)H:Quinone Oxidoreductase-1 = Nuclear-factor erythroid 2-Related Factor 2 = Epidermal Growth Factor Receptor = Human EGFR-2 = Mouse Embrionic Fibroblasts = Retinal Pigment Epithelial

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SECTION I INTRODUCTION
1.1 Background Cancer 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 repair DNA or cell apoptosis. However in cancer cells, tumor suppressor genes is inactivated and no one can stop the proliferation of DNA 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 this time, 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

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 food and fried food with waste cooking oil in 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 cancer treatment which focused on inhibition of cell proliferation and induction of apoptosis in breast cancer cells.

1.2 Problems 1. 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?

1.3 Aims 1. 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.

1.4 Benefits For 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 the scientific writing based on the literature. For the reader: 1. The reader can understand the process of cell cycle and apoptosis, and 2. 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.

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 G0 phase or stable phase (Gartner and Hiatt, 2007).

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

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 E synthesize 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 and enter G2 phase. Cyclin B binds to CDK1, allow cell to leave the G2 phase and enter M phase.

Apoptosis is proggrammed cell death. Normally, the cell would stop to growth and finally did the apoptosis. Apoptosis differ from necrosis. Necrosis is death of 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).

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 the BCL2 homology 3 domain, this domain inactivates BCL2 and BCL-XL (which normally inhibit apoptosis) and thereby activates the caspases that induce apoptosis. Whereas the death-receptor pathway is activated by the binding of Fas 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).

Figure 3. Pathway of Apoptosis (Croce, 2007)

Figure 4. Extrinsic and Intrinsic Pathway of Apoptosis (Mitchell dan Cotran, 2003).

2.2 Sulforaphane Sulforaphane 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

protects RPE and retina from the damaging effects of photo-oxidant ROS and 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 increased RPE cell viability.

2.2.1

Molecular and Chemical Structure of Sulforaphane

Molecular 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 Sulforaphane

Sulforaphane 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

Broccoli and cauliflower contain high concentration of glucosinolates 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 Subregnum Divisio Class Subclass Ordo Familia Genus Species Variety Plantae (Plant) Tracheobionta (Vascular Plant) Magnoliophyta (Angiospermae) Magnoliopsida (Dicotyledone) Diileniidae Capparales Brassicaceae Brassica Brassica olerace (Cabbage) Brassica oleracea var. botrytis (Broccoli, Cauliflower)

2.2.3

Mechanism of Sulforaphane in Breast Cancer Treatment

Cancer 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.

2.2.3.1 Sulforaphane Inhibit Cell Proliferation in Breast Cancer Cells Sulforaphane 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-

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Tracy, et. al., have done their research which gave sulforaphane treatment 025 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 and MDA-MB-468 cells lack expression of ER, EGFR is overexpressed in MDA-MB-468 cell, highly exprexxed in T47D cell, 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-MB468 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-regulated EGFR and HER2 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 of ER protein expression in MDA-MB-231 and MDA-MB-468 cells.

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Figure 8. Sulforaphane Down-regulates ER-, EGFR, and HER-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).

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Similar research by Brandi et. al. has been conducted to understand the mechanism of antiproliferation properties of Brassica oleracea (cauliflower) juice. From the treatment can be indicated that cauliflower juice is a potent inhibitor of DNA 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 of DNA Synthesis in MCF-7 cell (A) and MDA-MB231 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 of debris.

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Figure 11. Cell Growth Inhibitor Induced by Cauliflower Juice (Brandi, et. al., 2005).

Increased level of p27 CDK inhibitor and decreased level of CDK6 protein in MCF-7 cell have occurred after treatment of 10 mol juice during 22-72 hours. Decreased amount of total and hyperphosphorylation of pRb (key substrate for the G1 CDKs) shown at lower concentration of juice.

Figure 12. Expression of the G1 CDK and Phosphorylation pRb Level in MCF-7 cell (Brandi, et. al., 2005).

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2.2.3.2 Sulforaphane Induce Apoptosis of Breast Cancer Cells Sulforaphane 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 by Electrophoresis (Pledgie-Tracy, et. al., 2007).

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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 of Fas ligand and the cleavage of caspase-3, caspase-8, an also PARP cleavage without altered expression of Bcl-2 or cytochrome c localization (extrinsic pathway). Whereas treatment with sulforaphane in MDA-MB-468 and T47D cells activated cleavage of PARP, caspase-3, dan caspase-9; decreased Bcl-2 expression; and activated the release of cytochrome c from the mitochondria to the cytosol (intrinsic pathway). From Brandi, et. al. research, it was found increasing of TRADD (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 of Apaf-1 protein and disfunction of XIAP protein) and cell death. Choi and Singh treated Mouse 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, similar treatment 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

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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.

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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

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immunoblotting and immunohistochemistry which describe the release of cytochrome c from mitochondria to the cytosol in MEFs:

Figure 15. Analysis Result the Release of Cytochrome c from Mitochondria to the Cytosol by Immunoblotting (Choi and Singh, 2005).

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Figure 16. Analysis Result the Release of Cytochrome c from Mitochondria to the Cytosol by Immunohistochemistry (Choi and Singh, 2005).

From this result, it has been shown tnat treatment with sulforaphane in MEFs Wild-type caused the release of cytochrome 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 of Smac/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 immunoblotting analysis in the control of MEFs Wild-type (less Bax and Bak). Sulforaphane also caused proteolytic cleavage of caspase9 and caspase-3 in MEFs Wild-type, but less in MEFs DKO. In MEFs Wildtype, Bax and Bak single knockout were found the reduction of XIAP protein expression level (inhibitor of caspase activity) in higher concentration of sulforaphane, but absence in MEFs DKO and increased the level of Apaf-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,

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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 of Apaf-1 Protein Levels, Inhibition of XIAP Protein, and Mitochondrial Translocation of Bax (Choi and Singh, 2005).

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SECTION III CONCLUSION

3.1 Conclusion Sulforaphane as phytochemical antioxidant can inhibit cell proliferation in breast cancer cells by suppressing or reducing the action of ER-, 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 of pRb. 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 of PARP; and intrinsic pathway (mitochondria-mediated caspase cascade) by activating caspase-3 and caspase-9 (as a result caused by induction of Apaf-1 protein and disfuntion of XIAP protein), 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 of sulforaphane in breast cancer treatment in contributing medical knowledge especially oncology. 2. The community should take more attention and awareness to the disease especially breast cancer, because until this time, theres no medication which can cure cancer totally 100%.

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3. Broccoli and cauliflower should be introduced to the wide community as vegetables rich in phytochemical antioxidant (sulforaphane) which useful in preventing and management of cancer, especially breast cancer.

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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 13 Juli 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 Breast Cancer 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 9 Juli 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)

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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 96:227236 (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 13 Juli 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 11 July 2009 at http://www.highwire.org)