University оf Kragujevac, Faculty of Science
CENTRE FOR PRE-CLINICAL TESTING OF ACTIVE SUBSTANCES
LABORATORY FOR CELL AND MOLECULAR BIOLOGY
Number
Pl-Ch/04
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
July, 2011.
Dragana Đačić
herbs, Novi Sad
Prof. dr Srećko Trifunović,
Department of Chemistry,
Faculty of Science,
University of Kragujevac.
Active substance
Methanol mushroom extracts
Model system
HCT-116
Analysis
Cell viability assay (MTT assay)
Phellinus linteus
[Pt(dpe-(S,S)-eddp)Cl4]
Title
Objective
Material,
methods, patients
UM.01, UM.02, UM.03,
UM.04, UM.05,
ANTIPROLIFERATIVE EFFECTS OF MUSHROOM METHANOL
EXTRACTS IN COTREATMENT WITH [Pt(dpe-(S,S)-eddp)Cl4] ON
HCT-116 CELL LINE
The aim of this study was to determinate the antiprolifeative effects of methanol
extracts of mushroom in cotreatment with 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.
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 each concentrations of
mushrom methanol extract (in concentration of 10-500 µg/ml). After 6 h of
incubation cells treated with 50 µL 20 µM concentration of [Pt(dpe-(S,S)-eddp)Cl4]
and effects were observated afer 24 and 72 h. Final concentration of cisplatin in
each well was 10 µM. 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 as a
Dragana Đačić, MSci
Snežana Marković, Ph.D.
ratio of the apsorbance of the treated group divided by the apsorbance 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.
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 1). This
cotreatment had the high effect on inhibition of cell growth. IC50 values for
cotreatment with methanolic extract of Phelinus linteus and [Pt(dpe-(S,S)eddp)Cl4] were 111.81±7.19 and 13.16±2.04 µg/ml (Table 1), for 24- and 72-h of
exposure for HCT-116. The longer time exposure induced higer cell sensitivity.
When compared percent of viable cells after 24 and after 72 h, our results show
better activity after 72 h of exsposure. Maximal inhibition was observed for
concentration of 250 µg/ml and after 72-hours of exposure for all cotereatmens.
Discussion
Conclusion
References
Notes
The methanol fraction of Phellinus linteus in cotreatment with 10 µM [Pt(dpe(S,S)-eddp)Cl4] had the highest antiproliferative potential with IC50 values of
13.16±2.02 μg/ml. 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.
Dragana Đačić, MSci
Responsible person for Laboratory
Dr Snežana Marković
Snežana Marković, Ph.D.
Figure 1. The dose responses curve of antiproliferative effects of methanol extracts Phellinus linteus and
[Pt(dpe-(S,S)-eddp)Cl4] on cell growth in HCT-116 cells after 24 and 72 h exposure. The cells were
treated with methanol extract Phellinus linteus in concentration range from 10-250 µg/ml.The
antiproliferative effects was measured by MTT assay. Result were expressed as the means ± SE from
three independent determinations.
Table 1. Growth inhibitory effects - IC50 values (μg/ml) of methanolic extracts on HCT-116 cell line after
24 and 72 h exposure.
Figure 1.
Table 1.
Tested extracts
mushrooms+10 µM
[Pt(dpe-(S,S)-eddp)Cl4]
Phellinus linteus
Dragana Đačić, MSci
IC 50 (µg/ml)
24 h
72 h
111.81±7.19
13.16±2.04
Snežana Marković, Ph.D.