University оf Kragujevac, Faculty of Science
CENTRE FOR PRE-CLINICAL TESTING OF ACTIVE SUBSTANCES
LABORATORY FOR CELL AND MOLECULAR BIOLOGY
Number
Pl-Ch/02
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
Dejan Poslon, Medical
June 2011.
Dragana Đačić
herbs, Novi Sad /
Prof. dr Srećko Trifunović,
Department of Chemistry,
Faculty of Science,
University of Kragujevac.
Active substance
methanol mushroom extracts
Ganoderma lucidum
Letinus edodes
Phellinus linteus
Coprinus comatus
Cordiceps sinensis
Model system
HCT-116
Analysis
Cell viability assay (MTT assay)
UM.01, UM.02, UM.03,
UM.04, UM.05,
cisplatin,
[[Pt(dbu-(S,S)-eddp)Cl4],
[Pt(dpe-(S,S)-eddp)Cl4]
Title
ANTIPROLIFERATIVE EFFECTS OF MUSHROOM METHANOL
EXTRACTS IN COTREATMENT WITH PLATINUM COMPLEXES ON
HCT-116 CELL LINE
The aim of this study was to determinate the antiprolifeative effects of methanol
Objective
extracts of mushroom in cotreatment with cisplatin or platinum complexes [Pt(dbu(S,S)-eddp)Cl4], [Pt(dpe-(S,S)-eddp)Cl4] on colon cancer adenocarcinoma cell line
HCT-116.
Material,
Cell preparation and culturing(UM.01, UM.02, UM.03, UM.04)
methods, patients 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]-2,5-diphenyltetrazolium
bromide, Sigma, USA). HCT-116 cells was seeded in a 96-well plate (104 cells per
well). After 24 h incubation cells were treated with 50 µL of 100 µg/ml of
mushroom methanol extracts and with 50 µL, 20 µM concentration of cisplatin or
platinum complexes ([Pt(dbu-(S,S)-eddp)Cl4], [Pt(dpe-(S,S)-eddp)Cl4]) for 24 and
Dragana Đačić, MSci
Snežana Marković, Ph.D.
72 h. Final concentration of cisplatin or platinum complexes in each well was 10
µM. Untreated cells served as a control. 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 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 as a ratio of the absorbance
of the treated group divided by the absorbance of control group, multiplied by 100
to give percentage proliferation (Mosmann, 1983).
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. 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. 1-3.
The results of antiproliferative effects of mushrooms methanolic extracts + 10
µM cisplatin show inhibition of cell growth in tested concentration (Figure 1).
Maximal inhibition of cells proliferation was observed in cotreatment with
methanol extarct of Phellinus linteus and after 24-hours of exposure.
Compared antiproliferative effect of five mushrooms in coteratments with
[Pt(dbu-(S,S)-eddp)Cl4] our results showed that the highest effect of inhibition of
cell growth had methanol extract of Phellinus linteus (Figure 2).
MTT cell viability assay showed that cotreatment with methanolic extract of
Phelinus linteus and [Pt(dpe-(S,S)-eddp)Cl4] induced significant inhibition of cell
growth in dose- and time-dependent manner on HCT-116 (Figure 3). This
cotreatment had the higherst effect on inhibition of cell growth.
The results show the antiproliferative effects of cotreatment with Phelinus linteus
and [Pt(dpe-(S,S)-eddp-)Cl4] complex is highest on HCT-116 for 72 h of
exposure (Figure 3).
Discussion
Conclusion
The maximal effects to inhibit cell proliferation had methanol extract of Phellinus
linteus in cotreatment with 10 µM [Pt(dpe-(S,S)-eddp)Cl4] for 72 h. The methanol
fraction of Phellinus linteus in cotreatment with 10 µM [Pt(dpe-(S,S)-eddp)Cl4]
had the highest antiproliferative potential among the five active mushroom. The
active substances extracted from other mushroom in cotreatment with 10 µM
cisplatin, [Pt(dbu-(S,S)-eddp)Cl4], [Pt(dpe-(S,S)-eddp)Cl4] was completely unable
to inhibit HCT-116 cell growth at the used concentrations, and this extracts could
be described as low cytotoxic. The active substances extracted from mushroom
Phellinus linteus in cotreatment with 10 µM [Pt(dpe-(S,S)-eddp)Cl4] was
completely capable to inhibit HCT-116 cell growth at the used concentrations,
and this extracts could be described as cytotoxic after 24 and 72 h .
Dragana Đačić, MSci
Snežana Marković, Ph.D.
References
Notes
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
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
31.08.2011.
Responsible person for Laboratory
Dr Snežana Marković
Figure 1. The dose responses curve of antiproliferative effects of methanol extracts Ganoderma
lucidum, Letinus edodes, Phellinus linteus, Coprinus comatus, Cordiceps sinensis and cisplatin,
administrated in cotreatment, on HCT-116 cells after 24 and 72 h exposure. The cells were treated with
methanol extracts mushrooms in concentration of 100 µg/ml and 10µM cisplatin. The antiproliferative
effects were measured by MTT assay after 24 and 72 h exposure. Results were expressed as the means ±
SE from three independent determinations.
Figure 2. The dose responses curve of antiproliferative effects of methanol extracts Ganoderma
lucidum, Letinus edodes, Phellinus linteus, Coprinus comatus, Cordiceps sinensis and [Pt(dbu(S,S)-eddp)Cl4], administrated in cotreatment on HCT-116 cells after 24 and 72 h exposure. The cells
were treated with methanol extract mushrooms in concentration of 100 µg/ml and 10µM [Pt(dbu-(S,S)eddp)Cl4]. The antiproliferative effects were measured by MTT assay. Results were expressed as the
means ± SE from three independent determinations.
Figure 3. The dose responses curve of antiproliferative effects of methanol extracts Ganoderma
lucidum, Letinus edodes, Phellinus linteus, Coprinus comatus, Cordiceps sinensis and [Pt(dpe(S,S)-eddp)Cl4], administrated in cotreatment on HCT-116 cells after 24 and 72 h exposure. The cells
were treated with methanol extract mushrooms in concentration 100 µg/ml and 10µM [Pt(dpe-(S,S)eddp)Cl4].The antiproliferative effects was measured by MTT assay. Results were expressed as the means
± SE from three independent determinations.
Dragana Đačić, MSci
Snežana Marković, Ph.D.
Figure 1.
Figure 2.
Dragana Đačić, MSci
Snežana Marković, Ph.D.
Figure 3.
Dragana Đačić, MSci
Snežana Marković, Ph.D.