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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
ChPl/01
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
June, 2011.
Milena Ćurčić
Ecology, Faculty of Science
University of Kragujevac
Active substance
Methanolic extracts of:
leaves and fruits from Ligustrum
vulgare L. ;
Pd(apox) complex
Title
Objective
Material,
methods,
patients
Model system
HCT-116 cell line
Analysis
MTT cell viability assay,
Acridine orange/Ethidium
bromide double staining
UM.01, UM.02, UM.03, UM.04,
UM.05, UM.21
Antiproliferative and proapoptotic activity of Ligustrum vulgare L. extracts in
cotreatment with Pd(apox) complex
The aim of this study was to determinate the growth inhibitory effects of
methanolic extracts of leaves and fruits from L. vulgare on human colon cancer
cell line (HCT-116 cells) and their synergistic effect with chemotherapeutic drug
- Pd(apox) complex.
Drugs
The HCT-116 cells were treated with various concentrations of methanolic
extracts of leaves and fruits from L. vulgare ranging from 1 to 250 μg/ml in
combination with Pd(apox) complex (100 and 250 μM).
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.
For investigation of combination effect of plant extracts and Pd(apox) complex
on cell proliferation 10000 cells/well were seeded in 96-well plates. After 24 h
the cells were exposed to four concentrations of methanolic extracts of leaves and
fruits from L. vulgare (1, 50, 100 and 250 μg/ml). Pd(apox) complex (100 and
250 μM) was added after 3, 6 and 24 h of cell incubation with plant extracts.
Absorbencies were measured and cell were counting after 24 h (for cells where
Pd(apox) was added after 3 and 6 hours of cell exposure to plant extracts) and 72
h (for cells where Pd(apox) was added after 24 hours of cell exposure to plant
extracts) from initial treatments with plants. AO/EB assay for analysis of cell
death also was used in same time intervals and same combinations of
concentrations. Cells were also treated with Pd(apox) complex (100 and 250 μM)
alone.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
2
Results
Cell viability assay (MTT assay)(UМ.05)
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.
Fluorescence microscopic analysis of cell death (UM.21)
We used acridine orange/ethidium bromide (AO/EB) double staining assay
(Baskic, 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
20 µl of dye mixture (10 µl/mg AO and 10 µl/mg EB in distilled water) was
mixed with 100 µl cell suspension (10 000 cells/ml) in 96-well plate. After
incubation times with drugs the suspension was immediately examined and
viewed under Nikon inverted fluorescence microscope (Ti-Eclipse) at 400x
magnification. We observed untreated cells as controls. A minimum of 300 cells
were counted in each sample. Results were expressed as means ± SE for three
independent determinations.
Statistical analysis
The data are expressed as the means ± standard errors (SE). Biological activity is
result of three individual experiments, performed in triplicate for each dose. 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).
In this study, the antiproliferative and proapoptotic activity of the methanolic
extracts from L. vulgare and the chemotherapeutic drug - Pd(apox) complex were
investigated in HCT-116 cells by MTT cell viability assay and AO/EB assay.
Different combinations of Pd(apox) and plant extracts were used to identify those
combinations had the highest potential to decrease viability of HCT-116 cells.
Combination assays were performed using appropriate concentrations of
methanolic extracts from L. vulgare (1, 50, 100 and 250 μg/ml) with appropriate
concentrations of Pd(apox) complex (100 and 250 μM). Pd(apox) complex was
added after 3, 6 and 24 hours of cell incubation with plant extract, but
absorbencies were measured after 24 h (for cells where Pd(apox) was added after
3 and 6 hours of cell exposure to plant extracts) and 72 h (for cells where
Pd(apox) was added after 24 hours of cell exposure to plant extracts) from initial
treatments with plants. Cells treated with the same final concentrations of the
extracts or chemotherapeutic drugs alone were also examined.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
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Our results indicate that Pd(apox) complex has the very weak antiproliferative
activity (96.9% and 94.9% of viability cells in treatment by 100 μM and 250 μM
Pd(apox) for 24 h; 96.73% and 92.67% of viability cells in treatment by 100 μM
and 250 μM Pd(apox) for 72 h).
Our study showed that L. vulgare extracts were selectively toxic against HCT116 cell line in different time periods and that, in combination with Pd(apox)
complex, produced an increased growth inhibitory effect with lower IC50 values
(Table 1). IC50 values were calculated in relation to the concentration of plant
extract. The addition of Pd(apox) complex significantly reduces the IC50 values,
although Pd (in individual treatment) didn’t show good antiproliferative activity.
It is probably because Pd(apox) complex and plant extracts have synergistic
effects on inhibition of cell proliferation.
Table 2 summarizes the results obtained with AO/EB double staining of different
drug combinations, in different time periods. A time dependent increase in
induction of apoptosis also was observed. HCT-116 treated with different drug
combinations showed increased percentages of early apoptotic cells (the higest
increase of early apoptotic cells showed combination of methanolic extract of
leaves from L. vulgare and 250 μM Pd(apox) complex – 45.21%, after 6 h), and
increased percentage of late apoptotic cells (the highest increase showed
combination of methanolic extract of leaves from L. vulgare and 250 μM
Pd(apox) complex, after 72 h).
Discussion
Conclusion
References
Notes
Sign
Date
In summary, our results demonstrate that combinations of leaves and fruits
methanolic extracts from L.vulgare with Pd(apox) complex in HCT-116 cell lines
have a better effect than either agent alone. Further studies are needed to assess
the underlying mechanism(s), signal transduction pathways, leading to growth
inhibition induced by single agents and combinations both in vitro.
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
Responsible person for Laboratory
Milena Ćurčić
Dr Snežana Marković
12.08.2011.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
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Figure and table legends
Figure 1. Antiproliferative activity of methanolic extract of leaves from L. vulgare alone and in
combinations with Pd(apox) complex on HCT-116 cell line. Cells were treated with plant extracts and
after 3, 6 and 24 h of incubation Pd(apox) complex was added. The antiproliferative activity were
measured by MTT assay after 24 (1 and 2) and 72 h (3) of initial treatment with plant extracts. Results
were expressed as means ± SE for three independent determinations.
Figure 2. Antiproliferative activity of methanolic extract of fruits from L. vulgare alone and in
combinations with Pd(apox) complex on HCT-116 cell line. Cells were treated with plant extracts and
after 3, 6 and 24 h of incubation Pd(apox) complex was added. The antiproliferative activity were
measured by MTT assay after 24 (1 and 2) and 72 h (3) of initial treatment with plant extracts. Results
were expressed as means ± SE for three independent determinations.
Table 1. Growth inhibitory effects – IC50 values (μg/ml) of methanolic extracts of leaves and fruits from
L. vulgare in combination with Pd(apox) complex on HCT-116 cell line. Cells were treated with plant
extracts and after 3, 6 and 24 h of incubation Pd(apox) complex was added. The antiproliferative activities
were measured by MTT assay after 24 and 72 h of initial treatment with plant extracts. Results were
expressed as means ± SE for three independent determinations.
Table 2. Different values of viable, apoptotic and necrotic cells as percentage of all cells measured by
AO/EB fluorescence staining. Cells were treated by 100 µg/ml plant extracts and after 3, 6 and 24 h of
incubation Pd(apox) complex was added. The cell percentages were measured after 24 and 72 h of initial
treatment with plant extracts. Results were expressed as means ± SE for three independent
determinations.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
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Figure 1.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
6
Figure 2.
Milena Ćurčić, MSci
Snežana Marković, Ph.D.
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Table 1.
combination of drugs
Extract of leaves
and 250 μM Pd
Extract of leaves
And 100 μM Pd
Extract of fruits
and 250 μM Pd
Extract of fruits
and 100 μM Pd
Measured after 24 h
Pd(apox) complex added after Pd(apox) complex added after
3 h of cell incubation with
6 h of cell incubation with
plant extract
plant extract
Measured after 72 h
Pd(apox) complex added after
24 h of cell incubation with
plant extract
121.10±3.25
5.46±0.75
4.79±0.65
269.54±2.89
13.79±1.21
27.16±1.47
422.17±5.41
8.07±1.01
6.61±0.25
347.98±4.63
33.36±1.56
145.44±1.26
Table 2.
Time of cell
counting
Pd(apox)
complex
was added after
an appropriate
time of
incubation with
plant extracts
after 3 h
24 h after
initial
treatment
with plant
extracts
after 6 h
72 h after
initial
treatment
with plant
extracts
after 24 h
24 h after treatment
Milena Ćurčić, MSci
combination of drugs
VC
EA
LA
N
leaves extract and 100 μM Pd
74.13±0.93
25.70±0.47
0.17±0.29
-
leaves extract and 250 μM Pd
62.35±6.35
36.52±4.39
1.13±0.39
-
fruits extract and 100 μM Pd
79.13±0.77
20.66±1.24
0.21±0.74
-
fruits extract and 250 μM Pd
68.92±4.05
31.08±1.87
-
-
leaves extract and 100 μM Pd
44.98±4.76
41.13±0.79
13.69±1.25
0.20±2.56
leaves extract and 250 μM Pd
32.15±2.68
45.21±5.05
20.83±1.74
1.81±3.21
fruits extract and 100 μM Pd
59.13±1.57
32.69±0.41
8.18±1.13
-
fruits extract and 250 μM Pd
leaves extract and 100 μM Pd
50.14±2.91
37.13±1.58
39.74±2.31
26.71±1.87
10.12±2.01
34.34±1.36
1.81±0.97
leaves extract and 250 μM Pd
30.48±2.02
29.52±2.14
37.86±1.91
2.14±1.67
fruits extract and 100 μM Pd
40.81±2.14
30.61±1.57
25.95±1.36
2.63±1.07
fruits extract and 250 μM Pd
42.93±1.64
31.28±1.42
24.78±1.42
1.01±1.34
100 μM Pd(apox) complex
250 μM Pd(apox) complex
98.14±0.89
96.62±3.81
1.86±0.28
3.38±1.09
-
-
Snežana Marković, Ph.D.