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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/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
Marijana Kosaric
October, 2010.
Dragana Đačić
Faculty of Science, University
of Kragujevac
Active substance
methanol lichenes extracts
L8 – Umbilicaria
crustulosa (Ach.) Frey
L18 – Parmeliopsis
ambigua (Wulfen) Nyl.
L15 – Umbilicaria
polyphylla (L.) Baumg.
L19 – Lecanora
muralis (Schreber) Rabenh.
L21 – Parmelia saxatilis (L.)
Ach.
Title
Objective
Material,
methods, patients
Model system
HCT-116
Analysis
Cell viability assay (MTT assay)
Acridine orange/Ethidium
bromide double staining
UM.01, UM.02, UM.03,
UM.04, UM.05, UM.21
CYTOTOXIC ACTIVITIES OF LICHENES METHANOL EXTRACTS
The aim of this study was to determinate the antprolifertive effects of methanol
extracts of lichenes on colon cancer adenocarcinoma cell line HCT-116, as well
analysis of cell death.
Cell preparation and culturing(UM.01, UM.02, UM.03, UM.04)
The colon cancer adenocarcinoma cell line HCT-116 was obtained from the
American Tissue Culture Collection (Manassas, VA, USA). These cells were
propagated and maintained in DMEM (Dulbecco’s Modified Eagle Medium),
(Gibco, USA) and supplemented with 10% fetal bovine serum (PAA), antibiotics
100 IU/mL penicillin and 100 μg/mL streptomycin. 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. Cells were cultured in a humidified atmosphere of 5%
CO2 at 37 °C.
Cell viability assay (MTT assay) (UM.05)
After 24 and 72 h of treatment the cell viability was determined by MTT assay.
The proliferation test is based on the color reaction of mitochondrial
dehydrogenase from living cells with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide, Sigma, USA). HCT-116 cells was seeded in a 96well plate (104 cells per well). After 24 h incubation cells were treated with 100
µL of each concentrations of licnenes methanol extracts (in concentration of 501000 µg/ml) for 24 and 72 h. Untreated cells served as a control. At the end of
the tretmant period, MTT (final concentration 5 mg/ml PBS) was added to each
well, wich was then incubated at 37 °C in 5% CO2 for 2 h. The colored crystals
of produced formazan were dissolved in DMSO (dimethyl sulfoxide, Sigma,
USA). The absorbance was measured at 550 nm. Cell proliferation was calculated
Dragana Đačić, MSci
Snežana Marković, Ph.D.
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as a ratio of the apsorbance of the treated group divided by the apsorbance of
control group, multiplied by 100 to give percentage proliferation (Mosmann,
1983).
Fluorescence microscopic analysis of cell death (AO/EB) double staining
(UM.21)
For analysis of cell death, we used fluorescent assays Acridine orange/ethidium
bromide (AO/EB) double staining. Acridine orange is taken up by both viable
and nonviable cells and emits green fluorescence if intercalated 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. (1) Viable cells have uniform bright
green nuclei with organized structure, also have orange cytoplasm. (2) 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. (3) Late apoptotic cells have orange
to red nuclei with condensed or fragmented chromatin. (4) Necrotic cells have a
uniformly orange to red nuclei with organized structure (Baskic et al. 2006).
HCT-116 cells was seeded in a 6-well plate (3x104 cells per well). After 24 h
incubation cells were treated with 2 ml (250 μg/ml) of each licnenes methanol
extracts for 24 and 72 h. Untreatd cells served as a control. Incubation was
performed at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity.
After 24 and 72 h of treatment, we were added 200 μl of dye mixture (100 μl of
100 mg/ml AO and 100 μl of 100 mg/ml EB in distilled water) in each well. The
suspension was immediately (fast uptake) examined by fluorescence microscopy
(NICON Eclipse Ti) at 400x magnification. A minimum of 300 cells was counted
in every sample.
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
magnitude of correlation between variables was done using a SPSS (Chicago, IL)
statistical software package (SPSS for Windows, ver. 17, 2008). 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 EC50 values
were calculated from the dose curves by a computer program (CalcuSyn).
Results
1. Antiproliferative activity
After cell seeding in standard DMEM medium cells were exposed to different
drugs concentrations for 24 and 72 h at 37 °C. Percent cell survival, evaluated by
MTT assay (see Materials and methods), was calculated as the ratio between
absorbance at each dose of the drugs and absorbance of untreated control x 100.
The results obtained with antiproliferative assays are represented in Fig. 1-5.
According to the American National Cancer Institute (NCI), the criteria of
cytotoxic activity for the crude extracts is an IC50 < 30 μg/ml (Itharat et al. 2004).
Cell viability was reduced in dose-dependent manner for L8 and L18 methanol
extracts. The longer time exposure induced higer cell sensitivity, except in lower
concentrations (Figure 1 and Figure 2). When compared percent of viable cells
after 24 and after 72 h, cell viability was reduced in time-dependent manner for
Dragana Đačić, MSci
Snežana Marković, Ph.D.
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higer concentration.
The shape of dose response curves indicates a significant inhibition of cell
growth in dose-dependent and time-dependent manner for L15 – Umbilicaria
polyphylla (L.) Baumg. and L19 – Lecanora muralis (Schreber) Rabenh (Figure
3 and Figure 4). Our results show better activity in higer concentration. The
longer time exposure induced higer cell sensitivity (Figure 3 and Figure 4).
According to the American National Cancer Institute (NCI), the criteria of
cytotoxic activity for the active substances extracted from L8, L18, L15 and L19
were completely unable to inhibit HCT-116 cell growth at the used
concentrations (IC50 >30 μg/ml) (Table 1) and this extracts demonstrated an
intermediate activity after 24 and 72 h.
The shape of dose response curves indicates a significant inhibition of cell
growth in dose-dependent manner for L21 (Figure 5). Maximal inhibition after
24-hours exposure was observed for concentration of 1000 μg/ml and after 72hours exposure that concentration was 250 μg/ml for L21 extracts.
The longer time exposure induced higher cell sensitivity (Figure 5). When
compared percent of viable cells after 24 and after 72 h, cell viability was
reduced in time-dependent manner for all concentration.
According to the American National Cancer Institute (NCI), the criteria (Itharat
et al. 2004) of cytotoxic activity for the active substances extracted from L21 was
completely inhibited HCT-116 cell growth at the used concentrations (IC50 =14.7
μg/ml) (Table 1) and this extracts demonstrated higher activity after 72 h.
The data resulting from three different experiments are summarized in Table 1.
2. The results obtained with AO/EB double staining
The ability of the lichens extracts to induce apoptosis was initially screened by
using acridine orange/ ethidium bromide staining. Control cells showed bright
green nucleus with uniform intensity and had not taken up ethidium bromide,
where the apoptotic cells appeared orange in color (Figure 6 and Figure 7). The
all lichens extract treated cells showed obvious nuclear condensation after 24 h
treatment. Our results in AO/EB apoptotic test showed condensation of chromatin
and nuclear fragmentation and cell shrinkage which were clerly observed on
fluorescence microscopy (Figure 6 and Figure 7).
Florescence microscopic images clearly showed nuclear disintegration of cells
which were treated by methanolic extracts of all lichenes species compared with
that of the untreated control cells when stained with acridine orange and ethidium
bromide. Compared with spontaneus apoptosis observed in control cells (early
apoptotic 3.2%, 0% late apoptotic and 0% necrotic cells) HCT-116 was treated
by 250 µg/ml methanolic extract of all lichenes showed increased percentages of
early apoptotic for 24 h. The methanolic extract of L21 wich exhibited the
highest antiproliferative potential with IC50 of 96.85 μg/ml (Table 1) showed
increased percentages of early apoptotic (90.15%), increased percentage of late
apoptotic (0.98%) cells after 24 h. Acridine orange/ethidium bromide staining of
HCT 116 cells to detect apoptosis induced by 250 μg/ml dose of lichens extracts:
8, 18, 15, 19 and 21. Compared with the spontaneous apoptosis observed in
control cells (early apoptotic 28.8% after 72 h), HCT 116 treated with 250 μg/ml
dose of different lichens extract showed slightly increased percentages of late
apoptotic (Figure 7). The all lichens extract treated cells showed obvious nuclear
condensation after 72 h treatment. Our results showed increase of late apoptosis
(29.25%) and significant increase of necrosis (70.75%) cells treated with
methanol extract of L21 after 72 h (IC=14.7 μg/ml).
Dragana Đačić, MSci
Snežana Marković, Ph.D.
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Discussion
Conclusion
References
Notes
All of tested drugs show significant inhibition of cell proliferation. The maximal
effects to inhibit proliferated had extract from L21 for 72 h. The methanol
fraction of L21 which inhibited the highest antiproliferative potential among the
five active lichens with IC50 values of 14.7 μg/ml fall within the NCI criteria, thus
are considered as of promising anticancer potential. The active substances
extracted from other lichens was completely unable to inhibit HCT-116 cell
growth at the used concentrations (IC50>30 μg/ml), and this extracts
demonstrated an intermediate activity after 24 and 72 h.
Based on the above cytomorphological changes and cell death the effect of
methanol extract in these cells were indicative of early apoptosis after 24 h and
late apoptosis after 72 h.
To further determine the activity and mechanism of action of the methanol
extract, or adders different extracts, we would like to isolate and identify the
active principle and investigate the effects in more detail.
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 cycloheximideinduced apoptosis in human leukocytes: Fluorescence microscopy using annexin
V/propidium iodide versus acridin orange/ethidium bromide. Cell Biol Int 2006;
30(11): 924-932.
Itharat A, Houghton PJ, Eno-Amooquaye E, Burke PJ, Sampson JH, Raman A. In
vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer.
J Ethnopharmacol. 2004; 90: 33–38.
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.
Sign
Responsible person for testing
Dragana Đačić
Date
04.02.2011.
Dragana Đačić, MSci
Responsible person for Laboratory
Dr Snežana Marković
Snežana Marković, Ph.D.
5
Figure 1. The dose responses curve of effects of methanol extract L8 on cell growth in HCT-116 cells
after 24 and 72 h exposure. The cells were treated with methanol extract L8 in concentration range
from 50-1000 µg/ml. The antiproliferative effects was measured by MTT assay after 24 and 72 h
exposure. Results were expressed as the means ± SE from two independent determinations.
Figure 2. The dose responses curve of effects of methanol extract L18 on cell growth in HCT-116 cells
after 24 and 72 h exposure. The cells were treated with methanol extract L18 in concentration range
from 50-1000 µg/ml.. The antiproliferative effects was measured by MTT assay. Result were expressed as
the means ± SE from two independent determinations.
Figure 3. The dose responses curve of effects of methanol extracts L15 on cell growth in HCT-116 cells
after 24 and 72 h exposure. The cells were treated with methanol extract L15 in concentration range
from 50-1000 µg/ml.The antiproliferative effects was measured by MTT assay. Result were expressed as
the means ± SE from two independent determinations.
Figure 4. The dose responses curve of effects of methanol extracts L19 on cell growth in HCT-116 cells
after 24 and 72 h exposure. The cells were treated with methanol extract L19 in concentration range
from 50-1000 µg/ml. The antiproliferative effects was measured by MTT assay. Result were expressed as
the means ± SE from two independent determinations.
Figure 5. The dose responses curve of effects of methanol extracts L21 on cell growth in HCT-116 cells
after 24 and 72 h exposure. The cells were treated with methanol extract L21 in concentration range
from 50-1000 µg/ml. The antiproliferative effects was measured by MTT assay. Result were expressed as
the means ± SE from two independent determinations.
Figure 6. Effect of lichens extract on apoptosis HCT 116 cells were stained with AO/EB and analyzed
under a fluorescence microscope after 24 h.
Figure 7. Effect of lichens extract on apoptosis HCT 116 cells were stained with AO/EB and analyzed
under a fluorescence microscope after 72 h.
Figure 8. AO/EB staining of HCT-116 cells to detect apoptosis and necrosis induced by methanolic
extracts of L8 (2), L18 (3), L15 (4), L19 (5), L21 (6) in concentration of 250 µg/ml, after 24 hours of
exposure. Untreated cells were observed as a control cells (1). Magnification on fluorescent microscope
was 400×.
Figure 9. AO/EB staining of HCT-116 cells to detect apoptosis and necrosis induced by methanolic
extracts of L8 (2), L18 (3), L15 (4), L19 (5), L21 (6) in concentration of 250 µg/ml, after 72 hours of
exposure. Untreated cells were observed as a control cells (1). Magnification on fluorescent microscope
was 400×.
Table 1. Growth inhibitory effects - IC50 values (μg/ml) of methanolic extracts on HCT-116 cell line after
24 and 72 h exposure.
Dragana Đačić, MSci
Snežana Marković, Ph.D.
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Figure 1.
Figure 2.
Figure 3.
Figure 4.
Dragana Đačić, MSci
Snežana Marković, Ph.D.
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Figure 5.
Dragana Đačić, MSci
Snežana Marković, Ph.D.
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Figure 6.
Fluorescence microscope analysis of cell death AO/EB
double staning 250 µg/ml 24 h
120
100
80
Viable cells (%)
Early apoptotic cells (%)
60
Late apoptotic cells (%)
40
Necrosis cells (%)
20
0
Control
24 h
Control
8
18
15
19
21
8
18
Viable cells
(%)
96.8
53.51
23.74
50.49
34.07
Dragana Đačić, MSci
15
19
21
Early apoptotic
Cells (%)
3.2
44.69
58.99
42.24
62.92
90.15
Late apoptotic
cells (%)
Necrosis cells
(%)
1.8
12.94
2.6
1.48
9.8
4.3
4.6
1.48
Snežana Marković, Ph.D.
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Figure 7.
Fluorescence microscope analysis of cell death AO/EB double
staining 250 µg/ml 72 h
100
90
80
70
60
Viable cells (%)
50
Early apoptotic cells (%)
40
Late apoptotic cells (%)
30
Necrosis cells (%)
20
10
0
Control
8
18
72 h
Viable cells (%)
Control
8
18
15
19
21
71.12
32.78
13.23
4.7
Dragana Đačić, MSci
15
19
Early apoptotic
Cells (%)
28.88
67.21
33.03
85.81
91.18
21
Late apoptotic
cells (%)
Necrosis cells
(%)
46.69
0.96
4.06
29.25
20.26
70.75
Snežana Marković, Ph.D.
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Figure 8.
Figure 9.
Dragana Đačić, MSci
Snežana Marković, Ph.D.
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Table 1.
Tested extracts
lichens
8
18
15
19
21
Dragana Đačić, MSci
IC 50 (µg/ml)
24 h
72 h
401.444±20.05
299.579±18.87
194.41±8.16
331.108±11.58
96.857±3.29
497.656±33.84
280.434±11.21
153.956±5.85
286.808±17.49
14.775±0.57
Snežana Marković, Ph.D.