University оf Kragujevac, Faculty of Science
CENTRE FOR PRE-CLINICAL TESTING OF ACTIVE SUBSTANCES
LABORATORY FOR CELL AND MOLECULAR BIOLOGY
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
Milena Ćurčić
July, 2011.
Dragana Đačić
Faculy of Science
Kragujevac
Number
Pl-Ch/06
Active substance
methanol mushroom extracts
Phellinus linteus
Model system
HCT-116
Analysis
Acridine orange/Ethidium
bromide double staining
[Pt(dpe-(S,S)-eddp)Cl4]
Title
Objective
Material,
methods, patients
UM.01, UM.02, UM.03,
UM.04, UM.21
SYNERGISTIC EFFECTS OF MUSHROOM METHANOL EXTRACTS
AND PLATINUM COMPLEX [Pt(dpe-(S,S)-eddp)Cl4] ON THE TYPE OF
CELL DEATH OF HCT-116 CELL LINE
The aim of this study was to determinate type of cell death in single treament
with methanol extracts of mushroom Phellinus linteus, platinum complex
[Pt(dpe-(S,S)-eddp)Cl4] and synergistic effects of coteratment with methanol
extracts of mushroom Phellinus linteus and platinum complex, [Pt(dpe-(S,S)eddp)Cl4] on colon cancer adenocarcinoma cell line HCT-116.
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.
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
Dragana Đačić, MSci
Snežana Marković, Ph.D.
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 96-well plate (3x104 cells per well).
Treatment with platinum complex ([Pt(dp-(S,S)-eddp)Cl4]) - after 24 h of
incubation cells were treated with 100 µl of cisplatin complex ([Pt(dp-(S,S)eddp)Cl4]) in concentration of 10 µM.
Treatment with methanol extract of Phellinus linteus - after 24 h cells were
treated with 100 µl of methanol extract of Phellinus lintheus in concentration
range (5-250 µg/ml).
Cotreatment with methanol extract of Phellinus linteus and cisplatin complex
([Pt(dpe-(S,S)-eddp)Cl4]). After 24 h of incubation cells were treated with 50 µl
of Phellinus liteus methanol extract in concentration range (10-250 µg/ml). After
6 h of incubation with methanol extract cells were treated with 50 µl of 20 µM
[Pt(dpe-(S,S)-eddp)Cl4] complex. Final concentration of cisplatin complex was
10 µM in each well. The effects in all treatments were observed after 24 and 72 h.
Untreated 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 20 μl of dye mixture (10 μl of 100 mg/ml AO and 10 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.
Results
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 IC50 values
were calculated from the dose curves by a computer program (CalcuSyn).
1. The results obtained with AO/EB double staining
The ability of methanol extract of Phellinus linteus, platinum complex and
cotreatment with methanol mushroom extracts and [Pt(dp-(S,S)-eddp)Cl4] 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 2 (a), 3(a)). The treated cells showed obvious
nuclear condensation after 24 h treatment in all tested concentration. 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 2-6). Results of AO/EB double staining present on Figure 1.
Florescence microscopic images clearly showed nuclear disintegration of cells
which were treated by methanolic extracts in all concetration compared with
untreated control cells. Compared with spontaneus apoptosis observed in control
cells (early apoptotic 4.58%, 0% late apoptotic and 0% necrotic cells) HCT-116
was treated by 100 µl of methanolic extract of Phelinus liteus in concetration
range (10-250 µg/ml) showed increased percentages of early apoptotic and late
apoptosis for 24 h. The highest effect on induced late apoptosis (68%) show
Dragana Đačić, MSci
Snežana Marković, Ph.D.
treatment with exstract Phellinus linteus in highest concentration (250 µg/ml) for
24 h and significant increase necrosis (50%) after 72 h of exsposure, until the
treatment of concentration 10 µg/ml show increase of early apoptotic cells
(31.73%) for 24 h and late apoptotic (48.66%) cells for 72 h (Figure2-3).
The effect of treatment with platinum complex [Pt(dpe-(S,S)-eddp)Cl4] show,
increased percentages of early apoptosis (11.04%) and late apoptosis (10.12%)
after 24 h of exsposure, when compared with control cells (Figure 4). After 72 h
of treatment our results showed increased percentages of early apoptosis
(43.19%) and late apoptosis (6.56%) cells (Figure 5).
The synergistic effect of methanolic extract of Phelinus linteus and [Pt(dpe-(S,S)eddp)Cl4] wich exhibited the highest antiproliferative potential with IC50 of
111.81±7.19 μg/ml (Table 1) showed increased percentages of early apoptotic
and late apoptosis in lower concentration, and significant increased percentages
of late apoptosis (35.15%) and necrotic cell (64.85%) in highest concentrations
after 24 h of exsposure. Our results showed significant increase of necrosis
(99.4%) cells treated with 250 μg/ml methanolic extract of Phelinus linteus and
10 µM [Pt (dpe-(S,S)-eddp)Cl4] after 72 h (IC=13.16±2.04μg/ml) (Figure 6,
Table 1).
Discussion
Conclusion
References
Notes
All of tested drugs show significant increase of cells death.
Based on the above cytomorphological changes and cell death the effect of
methanol extract in HCT116 cell line was showed significant increased early
apoptosis and late apoptosis after 24 h and late apoptosis and necrosis after 72 h
compared with untreated (control) cells.
Acridine orange/ethidium bromide staining of HCT 116 cells, treated by 10 µM
dose of [Pt (dpe-(S,S)-eddp)Cl4], was detected apoptosis after 24 h and increase
early and late apoptosis after 72 h of exposure compared with control cells.
The methanol fraction of Phellinus linteus with 10 µM [Pt(dpe-(S,S)-eddp)Cl4] in
cotreatment show the highest antiproliferative potential, had highest effect to
induced apoptosis and after 24 h and necrosis after 72 h.
The maximal effects to induced cell death had methanol extract from Phellinus
linteus in cotreatment with 10 µM [Pt(dpe-(S,S)-eddp)Cl4] for 72 h.
Our results showed to cotreatment had the highest effect to induced condensation
of chromatin and nuclear fragmentation and cell shrinkage compared with control
cells or sigles treatments of methanolic extract of Phelinus liteus and platinum
complex [Pt(dpe-(S,S)-eddp)Cl4].
In this study, we have found that Phellinus linteus extract, when combined with
antitumor agents, showed strong effects to induced apoptosis and necrosis on
HCT 116 colon cancer cell lines (in vitro).
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.
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.
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
Dragana Đačić, MSci
Snežana Marković, Ph.D.
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
05.09.2011.
Responsible person for Laboratory
Dr Snežana Marković
Figure 1. Effect of [Pt(dpe-(S,S)-eddp)Cl4], methanolic extract of Phellinus linteus and cotreatment of
methanolic extract of Phellinus linteus and [Pt(dpe-(S,S)-eddp)Cl4] on cell death of HCT-116 cell lines
were stained with AO/EB and analyzed under a fluorescence microscope after 24 h and 72 h.
Figure 2. AO/EB staining of HCT-116 cells to detect apoptosis and necrosis induced by methanolic
extracts of mushrom Phellinus linteus in concentrations of 250 (b), 50 (c), 25 (d), 10 (e) µM, after 24
hours of exposure. Untreated cells were observed as a control cells (a). Magnification on fluorescent
microscope was 400×.
VC-Viable cells; EA-Early apoptotic cells; LA-Late apoptotic cells; NC-Necrotic cells
Figure 3. AO/EB staining of HCT-116 cells to detect apoptosis and necrosis induced by methanolic
extracts of mushroom Phellinus linteus in concentrations of 250 (b), 50 (c), 25 (d), 10 (e) µM, after 72
hours of exposure. Untreated cells were observed as a control cells (a). Magnification on fluorescent
microscope was 400×.
VC-Viable cells; EA-Early apoptotic cells; LA-Late apoptotic cells; NC-Necrotic cells
Figure 4. AO/EB staining of HCT-116 cells to detect apoptosis and necrosis induced by [Pt(dpe-(S,S)eddp)Cl4] in concentration of 10 µM, after 24 and 72 hours of exposure. Untreated cells were observed as
a control cells (1). Magnification on fluorescent microscope was 400×.
VC-Viable cells; EA-Early apoptotic cells; LA-Late apoptotic cells; NC-Necrotic cells
Figure 5. AO/EB staining of HCT-116 cells to detect apoptosis and necrosis induced by cotretment
methanolic extracts of mushroom Phellinus linteus and [Pt(dpe-(S,S)-eddp)Cl4]. Concentrations of
methanol extract were be 250 (a), 50 (b), 25 (c), 10 (d) µM and final concentration of cisplatin was be 10
µM. Changes were observated after 24 hours of exposure. Magnification on fluorescent microscope was
400×.
VC-Viable cells; EA-Early apoptotic cells; LA-Late apoptotic cells; NC-Necrotic cells
Figure 6. AO/EB staining of HCT-116 cells to detect apoptosis and necrosis induced by cotretment
methanolic extracts of mushroom Phellinus linteus and [Pt(dpe-(S,S)-eddp)Cl4]. Concentrations of
methanol extract were be 250(a), 50 (b), 25 (c), 10 (d) µM and final concentration of cisplatin was be 10
µM. Changes were observated after 72 hours of exposure. Magnification on fluorescent microscope was
400×.
VC-Viable cells; EA-Early apoptotic cells; LA-Late apoptotic cells; NC-Necrotic cells
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.
Figure 1.
Figure 2.
Dragana Đačić, MSci
Snežana Marković, Ph.D.
Figure 3
Figure 4.
Figure 5.
Dragana Đačić, MSci
Snežana Marković, Ph.D.
Figure 6.
Table 1.
Tested extract
mushrom+10 µM
[Pt(dpe-(S,S)-eddp)Cl4]
Phellinus liteus
Dragana Đačić, MSci
IC 50 (µg/ml)
24 h
72 h
111.81±7.19
13.16±2.04
Snežana Marković, Ph.D.