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University оf Kragujevac, Faculty of Science
CENTRE FOR PRE-CLINICAL TESTING OF ACTIVE SUBSTANCES
LABORATORY FOR CELL AND MOLECULAR BIOLOGY
Number
Pl/06
Radoja Domanovića 12, 34000 Kragujevac, Serbia
http://cpctas.pmf.kg.ac.rs
 e-mail: cpctas@kg.ac.rs
User request
Material reception /
Responsible person
Start of testing
Milan Stanković,
Department for Biology and
October, 2010.
Milena Ćurčić
Ecology, Faculty of Science
University of Kragujevac
Active substance
Methanolic extracts of leaves
and seed cones from Taxus
baccata
Title
Objective
Material,
methods,
patients
Analysis
MTT cell viability assay,
HCT-116 and MDA-MB-231
Acridine orange/Ethidium
cell lines
bromide double staining
UM.01, UM.02, UM.03, UM.04,
UM.05, UM.21
ANTIPROLIFERATIVE AND PROAPOPTOTIC ACTIVITIES OF
METHANOLIC EXTRACTS OF LEAVES AND SEED CONES FROM T.
baccata L. ON HCT-116 AND MDA-MB-231 CELL LINES
The aim of this study was to determinate the antprolifertive effects of methanol
extracts of methanolic extracts of leaves and seed cones from T. baccata on colon
cancer adenocarcinoma cell line HCT-116, as well analysis of cell death.
Drugs
The HCT-116 cells were treated with various concentrations of methanolic
extracts of leaves and seed cones from T. baccata ranging from 50 to 1000 μg/ml
for 24 and 72 h.
Cell preparation and culturing (UM.01, UM.02, UM.03, UM.04)
HCT-116 cell line, human colon cancer was obtained from American Type
Culture Collection. Cells were maintained in DMEM supplemented by 10% FBS,
with 100 units/ml penicillin and 100 µg/ml streptomycin. Cells were cultured in a
humidified atmosphere of 5% CO2 at 37 °C. Cells were growth in 75 cm2 culture
bottles supplied with 15 ml DMEM, and after a few passages cells were seeded
in 96-well plate. All studies were done with cells at 70 to 80% confluence.
Cell viability assay (MTT assay)(UМ.05)
HCT-116 cells were seeded in a 96-well plate (10 000 cells per well). After 24 h
of cells incubation, the medium was replaced with 100 μl medium containing
various doses of methanolic extracts at different concentrations (50, 100, 250,
500, 750 and 1000 μg/ml) for 24 and 72 h. Untreated cells served as the control.
After 24 and 72 h of treatment the cell viability was determined by MTT assay
(Mosman, 1983=. The proliferation test is based on the color reaction of
mitochondrial dehydrogenase in living cells by MTT. At the end of the treatment
period, MTT (final concentration 5 mg/ml PBS) was added to each well, which
was then incubated at 37 °C in 5% CO2 for 2-4 h. The colored crystals of
produced formazan were dissolved in 150 μl DMSO. The absorbance was
measured at 570 nm on Microplate Reader. Cell proliferation was calculated as
the ratio of absorbance of treated group divided by the absorbance of control
group, multiplied by 100 to give a percentage proliferation.
Milena Ćurčić, MSci
Model system
Snežana Marković, Ph.D.
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Fluorescence microscopic analysis of cell death (UM.21)
We used acridine orange/ethidium bromide (AO/EB) double staining assay
(Baskić et al., 2006). Acridine orange is taken up by both viable and nonviable
cells and emits green fluorescence if interrelated into double stranded nucleic
acid (DNA) or red fluorescence if bound to single stranded nucleic acid (RNA).
Ethidium bromide is taken up only by nonviable cells and emits red fluorescence
by intercalation into DNA. We distinguished four types of cells according to the
fluorescence emission and the morphological aspect of chromatin condensation in
the stained nuclei. Viable cells have uniform bright green nuclei with organized
structure. Early apoptotic cells (which still have intact membranes but have
started to undergo DNA cleavage) have green nuclei, but perinuclear chromatin
condensation is visible as bright green patches or fragments. Late apoptotic cells
have orange to red nuclei with condensed or fragmented chromatin. Necrotic cell
have a uniformly orange to red nuclei with condensed structure. The amount of
200 µl of dye mixture (100 µl/mg AO and 100 µl/mg EB in distilled water) was
mixed with 2 ml cell suspension (30 000 cells/ml) in 6-well plate. The suspension
was immediately examined and viewed under Nikon inverted fluorescence
microscope (Ti-Eclipse) at 400x magnification. We observed untreated cells as
controls and cells treated with methanolic extract of different Teucrium species in
250 µg/ml concentrations for 24 h of exposure. A minimum of 300 cells were
counted in each sample.
Statistical analysis
Results
The data are expressed as the means ± standard errors (SE). Biological activity is
result of three individual experiments, performed in triplicate for each dose.
Statistical significance was determined using the Student’s t-test. A p value <
0.05 was considered as significant. The effect of each extract were expressed by
IC50 (inhibitory dose which inhibit 50% growth cells) and by the magnitude of
maximal effect in exposed cells. The IC50 values were calculated from the dose
curves by a computer program (CalcuSyn).
Antiproliferative activity
A dose-dependent MTT reduction (or color change from yellow to purple) was
observed in extracts-treated HCT-116 cells. The shape of dose response curves
indicates a inhibition of cell growth in dose-dependent manner in 24 h treatment.
It means that in higher concentration better activity was observed. Cells growth
were significant lower (p<0.05) when we compared the cell growth of extractstreated cells with control cells.
On 24 and 72 h exposure to extracts of leaves in different concentrations,
cytotoxic effects were over 50% for each concentration. Effects were higher after
72 h exposure (proliferation of HCT-116 human colon carcinoma cells treated
with this extract was inhibited in a time-dependent manner).
After 24 and 72 h exposure of HCT-116 cells to extracts of seed cones, cytotoxic
effects were over 50% for all concentrations, except the concentration of 50
μg/ml. Cytotoxic effects of lower concentrations were not higher after 72 h
exposure. The results indicate that cytotoxic effects of lower concentrations
didn’t strengthen with the time of exposure, already extract has acute cytotoxic
effect on HCT-116 human colon cancer cells and then after longer time of
exposure cells were recovered.
Both extract inhibited MDA-MB-231 cell growth in dose-dependent manner in
24 and 72 h treatment. Cells growth were significant lower (p<0.05), except in
the lowest concentrations (50 and 100 μg/ml) when we compared cell growth of
extracts-treated cells with control cells.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
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Table 4 presents in vitro cytotoxic activity of the two investigated methanolic
extracts. The effect of both extracts were expressed by IC50 (inhibitory dose wich
inhibit 50% growth cells). Methanolic extract of leaves from T. baccata on HCT116 cells has noteworthy effect after 24 h exposure, and pronounced effect after
72 h. Both extracts from T. baccata hasn’t significant cytotoxic effects on MDAMB-231 cell line.
Fluorescence microscopic analysis of cell death
The results obtained with AO/EB double staining are presented in Figure 3 and
Figure 4. Compared with spontaneous apoptosis observed in HCT-116 control
cells (early apoptotic 3.19%, 0% late apoptotic and 0% necrotic cells) treated
cells by 250 µg/ml methanolic extract of leaves resulted in increased percentages
of early apoptotic (66.55%), late apopoptotic cells (21.84%), increase in total
number of apoptotic cells (88.90%) and appear necrotic cells (2.39%).
Methanolic extract of seed cones showed increased percentages of early apoptotic
(53.85%) and slightly increased percentage of late apoptotic (0.43%) cells, and
increase in total number of apoptotic cells (54.27%).
Compared with spontaneous apoptosis observed in MDA-MB-231 control cells
(early apoptotic 1.47%, 0.37% late apoptotic and 0% necrotic cells) treated cells
by 250 µg/ml methanolic extract of leaves resulted in increased percentages of
early apoptotic (43.59%). Methanolic extract of seed cones showed increased
percentages of early apoptotic (35.8%), slightly increased percentage of late
apoptotic (6.05%) cells, increase in total number of apoptotic cells (41.85%) and
appear necrotic cells (2.42%).
Discussion
Conclusion
According to the results of investigations, our in vitro data indicated that
methanolic extracts of leaves and seed cones from T.baccata have the
antiproliferative and proapoptotic effects and could be considered as suitable
candidates for further studies to find the effective anticancer components.
Derived data showed that methanolic extract of leaves has a better
cytotoxic effect than methanolic extract of seed cones and that HCT-166
cell line showed quite higher sensibility on plant extracts. Finding data
indicate that treatment with methanolic extracts of leaves and seed cones
from T. baccata were induced apoptosis in HCT-116 and MDA-MB-231
cell lines.
References
Notes
Sign
Mosmann T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Meth.1983, 65,
55-63.
Baskić D, Popović S, Ristić P, Arsenijević NN. Analysis of cyclohexamideinduced apoptosis in human leukocytes: Fluorescence microscopy using
annexin V/propidium iodide versus acridin orange/ethidium bromide. Cell
Biol. Int. 2006, 30, 924-932.
This report applies only to the tested substances. Responsible for the report,
(accuracy and technical explanations of results) are researcher and manager and
are considered the report's authors, which they have confirmed with their
signature. The report should not be used or reproduced partially, except in its
entirety in form of the publication of results as an integral part of the report.
Publication of results based on this report must be approved by the authors.
Responsible person for testing
Milena Ćurčić, MSci
Responsible person for Laboratory
Snežana Marković, Ph.D.
4
Milena Ćurčić
Date
Dr Snežana Marković
21.12.2010.
Figure and table legends
Figure 1. The dose response curves of effects of leaves and seed cones from T. baccata on cell
growth on HCT-116 cells, after 24 and 72 h exposure. The cells were treated with methanolic
extracts in concentration range from 50 to 1000 µg/ml. The antiproliferative effects were
measured by MTT assay. Results were expressed as the means ± SE from three independent
determinations.
Figure 2. The dose response curves of effects of leaves and seed cones from T. baccata on cell growth on
MDA-MB-231 cells, after 24 and 72 h exposure. The cells were treated with methanolic extracts in
concentration range from 50 to 1000 µg/ml. The antiproliferative effects were measured by MTT assay.
Results were expressed as the means ± SE from three independent determinations.
Figure 3. Different values of viable, apoptotic and necrotic HCT-116 cells as percentages of all cells
measured by AO/EB fluorescence staining, after treatment by 250 µg/ml methanolic extracts. Percentages
of cells were measured 24 hours after treatment.
Figure 4. Different values of viable, apoptotic and necrotic MDA-MB-231 cells, as percentages
of all cells measured by AO/EB fluorescence staining, after treatment by 250 µg/ml methanolic
extracts. Percentages of cells were measured 24 hours after treatment.
Table 1. Growth inhibitory effects - IC50 values (μg/ml) of methanolic extracts on HCT-116 and MDAMB-231 cell line after 24 and 72 h exposure.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
5
Figure 1.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
6
Figure 2.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
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Figure 3.
Figure 4.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
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Table 1.
IC50 µg/ml
tested extracts
after 24 h
HCT-116
MDA-MB-231
T. baccata – leaves
14.4±4.5
356.47±5.7
T. baccata – seed cones
49.69±7.6
>1000
Milena Ćurčić, MSci
after 72 h
HCT-116
MDA-MB-231
4.59±1.5
246.87±1.68
133.53±0.35
604.25±8.41
Snežana Marković, Ph.D.