沙漠研究 19-1, 367- 370 (2009 )Journal of Arid Land Studies“Desert Technology IX ”
Refereed Paper

Anti-Proliferative and Apoptotic Effects of Oleuropein and Hydroxytyrosol on
Human Breast Cancer MCF-7 Cells
Junkyu HAN1, Terence P.N. TALORETE1, Parida YAMADA1, Hiroko ISODA1
Abstract: Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation,and induced cell apoptosis in MCF-7 cells. MTT assay guided 200 μg/ml of oleuropein or 50μg/ml of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein orhydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cellproliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited
statistically significant block of G1 to S phase transition manifested by the increase of cellnumber in G0/G1 phase.
Key words: Apoptosis, Hydroxytyrosol, MCF-7 cells, Oleuropein

1. Introduction
The phenolic compounds of olive oil and leaf are a complex mixture of compounds that include 3, 4dihydroxyphenylethanol (hydroxytyrosol), 4-hydroxyphenylethanol (tyrosol), 4-hydroxyphenylacetic acid,protocatechuic acid, caffeic acid and p-coumaric acid, among others (Caponio et al., 1999). The
concentration of the phenolic fraction is several times more in olive leaf than in oil and varies dependingon the cultivar, climate (Abaza et al., 2005). Even though anticancer properties of oleuropein andhydroxytyrosol were confirmed in vitro with different cell lines, studies of their protective effect frombreast cancer have not been demonstrated. Since many epidemiological studies suggest the possible
correlation between olive products consumption and incidence of breast cancer here we tried to elucidate
the possible effect of the main phenolic compounds hydroxytyrosol and oleuropein of olive leaf on breast
cancer using human breast cancer cell line MCF-7.
2. Materials and methods
2.1. Cell culture
MCF-7 cells were obtained from American Type Culture Collection (ATCC), and maintained inDMEM, supplemented with 10% FBS (Sigma), 1% 5000 units/ml penicillin, 5000 μg/ml streptomycin(Sigma). The cells were incubated in an atmosphere of 5% CO2 at 37 ˚C. All cells used in this study werebetween passages 5 and 15.
2.2. Growth inhibition assay
MCF-7 cells were seeded in 96-well plates at 2×103 cells/well in 100 μl of DMEM with 10% FBS.
After 24 h the cells were treated with olive leaf extract (0.001, 0.01, 0.02, 0.1% (w/v)) for 12 h. Also
MCF-7 cells were seeded in 96-well plates at 2×103, 2×104 , 2×105 cells/well in 100 μl of DMEM with
10% FBS. After 24 h the cells were treated with oleuropein (25, 50, 100, 200 μg/ml) or hydroxytyrosol(6.25, 12.5, 25, 50 μg/ml) for 12 h. The treated cells were washed with PBS and 10 μl of MTT solution (5mg/ml MTT in PBS) was added to each well. Cells were incubated for 12 h at 37 ˚C at the end of which
100 μl 10% SDS was added and incubated further for 6 h at 37 ˚C. Optical densities at 570 nm were
measured using a plate reader (Powerscan HT; Dainippon Pharmaceutical, Osaka, Japan). The relative cell
viability in percentage was calculated as (A570 of treated samples/A570 of untreated samples) ×100.
2.3. Cell proliferation assay
Cells were regularly seeded into 6-well plates at 2×104 cells/well and incubated at 37 ˚C for 24 h.
Cells were treated in fresh medium with Cells were incubated with oleuropein (200 μg/ml) orhydroxytyrosol (50 μg/ml) for 3, 6, and 12 h. At the end of each time period, the cells were trypsinized to1

Alliance for Research on North Africa (ARENA), Graduate School of Life and Environmental Sciences, Universityof Tsukuba, 1-1-1 Tennodai, Tsukuba City, Ibaraki 305-8572, Japan;

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produce a singlecell suspension, and the viable cell number in each well was counted using the ViaCountAssay (Guava Technologies, Hayward, CA).
2.4. Multicaspase assay by flow cytometry
Apoptosis was assessed using a flow cytometry based multicaspase assay kits (Guava Technologies,Burlingame, CA). The multicaspase assay is based on measurement of caspase enzymes activated during
apoptosis. MCF-7 cells (2×105 cells/plate) were plated on tissue culture dishes (100 mm) in 10% FBS
medium. Cells were incubated with oleuropein (200 μg/ml) or hydroxytyrosol (50 μg/ml) for 3, 6 and 12 h.
Cells were trypsinized, washed in PBS and stained with sulforhodamine-valyl-alanyl-aspartylfluoromethyl-ketone (SR-VAD-FMK), and 7-amino-actinomycin D (7-AAD) according to themanufactures instructions. Cell populations were quantified using Guava personal cytometer (GuavaTechnologies, Hayward, CA).
2.5. Cell cycle analysis
MCF-7 cells (2×105 cells/plate) were exposed to concentrations of oleuropein (100 μg/ml) orhydroxytyrosol (25 μg/ml) for 24 h and the treated cells were collected by trypsinisation. The cells werefixed with 70% ethanol and stored at -20 ˚C. On the day of analysis, the cells were washed with PBS andsuspended in 250 μl of PBS. To the cell suspension 1.0 ml of phosphate-citrate buffer was added and thesuspension was incubated at room temperature for 5 min to facilitate the extraction of low molecularweight DNA. Following centrifugation the cells were resuspended in 500 μl DNA staining solution (20μg/ml propidium iodide, 200 μg/ml DNase free RNase and 0.1% Triton X-100) and incubated in the dark atroom temperature for 30 min. Cell cycle distribution was determined by fluorescence-activated cell sorting
analysis of propidium iodide-stained ethanol-fixed cells using a Guava EasyCyte (Guava Technologies,Hayward, CA).
3. Results and discussion
For the screening of anticancer effect of olive leaf we used olive leaf extract to treat breast cancer
MCF-7 cells. MTT assay results have shown that after 48 h treatment with the extract at the dosage of 1%
cancer cell proliferation was inhibited compared to the vehicle treated control cells (Fig. 1). The rest oftreatment concentrations did not show cytotoxic effect. Since the major phenolic compounds of olive leaf
are oleuropein and hydroxytyrosol, in subsequent anticancer assays we assumed that this cytotoxic effectcould be mainly due to presence of these compounds and tried to elucidate their tumor suppressive effects(Fig. 2).
This data may suggest that phenolic compounds of olive leaf have health protective effect rather thanhealing effect. Constant consumption of olive products such as leaf tea and oil may enhance this protective
effect thus, suggesting their daily consumption is more effective than the infrequent intake.
Since caspase activation is the classic feature of apoptosis we performed multicaspase assay using
fluorochrome conjugated caspase inhibitor sulforhodamine-valyl-alanyl-aspartyl-fluoromethylketone (SRVAD-FMK). This inhibitor is cell permeable and non-cytotoxic and binds to multiple caspases activatedduring apoptosis. Also the cell impermanent dye 7-aminoactinomycin D (7-ADD) was included in theFig. 1. Cytotoxic effect of olive
leaf extract.

Fig. 2. Influence of initial cell number on the concentration-dependent
decrease in viability of human breast cancer MCF-7 cells incubatedwith oleuropein (A) and hydroxytyrosol (B).

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Fig. 3. Effect of hydroxytyrosol and oleuropein on MCF-7 cell cycle.

assay to detect dead cells from apoptotic ones. From the multicaspase assay we could observe thatcaspases were activated soon after treatment with both compounds, besides, induction of the enzymes’
activation was greater in oleuropein treated cells compare to the hydroxytyrosol treated cells in thebeginning of the treatment.
Because it has been reported that the cells undergoing apoptosis are arrested in G1/G0 phase of cell
division phase we could verify the induction of apoptosis of breast cancer cells by hydroxytyrosol andoleuropein using cell cycle analysis. In this assay we treated MCF-7 cells with 100 µg/ml of oleuropein
and 25 µg/ml of hydroxytyrosol. When cells were treated for 24 h with changed concentration ranges, thecell cycle arrest at G1 phase was evident, accompanying a decrease in G2/M phase when compared withthe untreated control cells. From this result we suggest that apoptotic cell death of MCF-7 cells afterhydroxytyrosol and oleuropein treatment was accompanied with the block of cell cycle at G1 phase (Fig. 3).
Our data confirmed that the cells treated with hydroxytyrosol and oleuropein exhibited statisticallysignificant block of G1 to S phase transition manifested by the increase of cell number in G0/G1 phase.
Although the oleuropein notably induced less cell cycle arrest than hydroxytyrosol it was still capable tosignificantly promote breast cancer apoptotic cell death.
4. Conclusion
Our results have shown that main phenolic compounds of olive leaf hydroxytyrosol and oleuropein
induced apoptotic cell death of human breast cancer MCF-7 cells. Our findings may support the breast370

cancer protective results of epidemiological studies of Mediterranean diet with correlation of olive
products’ consumption. Even though the cancer protective effect of the main olive oil component oleic
acid in gene regulation was demonstrated further studies are needed to explore molecular mechanisms oftumor suppressive effect of other olive tree products such as olive leaf because they also could be thepotent inducers of such effect if consumed frequently.
Aknowledgement
This study was supported in part by funds from the Grant-in-Aid for Scientific Research (Grant No.
17255011)
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