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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
Number
User request
Ch-Pl/07
Milena Ćurčić, Faculty of
Science, University of
Kragujevac
Model system
Material reception /
Start of testing
September, 2011.
Responsible person
Dragana Đačić
Active substance
Analysis
Cisplatin,
Methanolic extract of
leaves from Ligustrum
Immunofluorescence(UM.06)
vulgare
HCT-116
UM.01, UM.02, UM.03, UM.04
Methanolic extract of
fruit from Ligustrum
vulgare
Title
Determination of changes in nuclear morphology and organization of tubulin
in HCT-116 cell line in different treatment
The aim of this study was to determinate the changes in nuclear morphology and
Objective
organization of tubulin in HCT-116 cell line treated with cisplatin and methanolic
extracts from leaves and fruit of Ligustrum vulgare
Material,
Drugs
methods, patients HCT-116 cell line were treated with 2 ml 50 µM of cisplatin, and 50 µg/ml of
methanolic extract of leaves from Ligustrum vulgare, and methanolic extract of fruits
from Ligustrum vulgare for 24h.
Cell preparation and culturing (UM.01, UM.02, UM03)
HCT-116 (human colon cancer) was obtained from American Type Culture
Collection. Cells were maintained in DMEM supplemented by 10% FBS, with 100
units/ml of penicillin and 100 µg/ml of 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
6-well plate (exponentially growing viable cells were used throughout the assay).
Imunofluorescence (UM.06)
HCT-116 cell line were seeded in 6-well plate (50 000 cells per well). After 48 h of
cells incubation, the medium was replaced with 2 ml of medium containing 50 μM
concentrations of cisplatin, 50 µg/ml methanol extract of leaves and 50 µg/ml
methanol extract of fruit from plant Ligustrum vulgare. Untreated cells served as a
control. After 24 h of tretmant we were observated changes by microscpe NIKON
Ti. Imunofluorescence is based on usage of specificity of antibodies to
their antigen to target fluorescent dyes to specific bimolecular targets within a cell,
and therefore allows visualization of the distribution of the target molecule through
the sample. In our experiment, detergent lysis is the most usual way to remove the
cell membrane, thus exposing the cytoskeleton. However, immediately upon
removal of the cell membrane, the potential for extraction and artifact arises. A
relatively high concentration of a non-ionic detergent, e.g. 1% Triton X-100, is
critical for rapid solubilization of the cell membrane and cessation of cell activity.
The detergent is made up in a buffer containing stabilizing supplement. We
Dragana Đačić, MSci
Snežana Marković, PhD
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Results
Discussion
Conclusion
References
Notes
routinely add high molecular weight polyethileneglycol (PEG) for general
preservation on the cytoskeleton. Phallioidin are added for the specific prevent of
actin filaments, respectively. Extraction was made at room temperature. After
extraction, washing and fixation with 0.2% glutaraldehide we added primary
antibody for protein tubulin (mouse anti- tubulin). For visualization cell nuclear
material we used DAPI staining. After incubation and washing we added secondary
antibody anti mouse Cay3. Fixed cover slip with mounting media and viewed under
Nikon inverted fluorescence microscope (Ti-Eclipse) at 600x magnification
Our micrographs clearly showed the separate cells in the control. The analysis of
micrographs showed that cells in control (untreated cells) not had affinity for
associating in clusters. The analysis of images showed that all treatments had highly
affinity for associating of cells compared with control cells as shown in Figures 1-4.
When compared treatments with control (untreated) cells, we can observe that all
treatments showed associating of cells. The analysis of treatments with methanol
extracts of leaves and fruit from Ligustrum vulgare showed reorganization of
cytoskeleton protein tubulin, who as part of cytoskeleton, have role in organization
structure and position of cells organelles. That could be to mean that investigated
active substances induced changes in cells metabolism and cells had highly
communications between them. Both extracts and cisplatin showed highly affinity
for compaction and associating of cells.
We can conclude that cisplatin had effect on reorganization of tubulin in cell
cytoskeleton . All treatments showed significant associating and compaction of cells
compared to untreated cells and could be used in further studies.
Immunocytochemistry Methods and Protocols. Edited by Lorette C. Javois, 2nd
edition, 1999. Human Press.
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
17.10.2011.
Dragana Đačić, MSci
Responsible person for Laboratory
Dr Snežana Marković
Snežana Marković, PhD
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Figure 1. Imunofluorescence images of HCT-116 cell line untreated cells (control) a) protein tubulin Cy3staining, b) nuclear-DAPI staining
Figure 2. Imunofluorescence image of HCT-116 cell line treated with 50 μM concentrations of cisplatin
for 24 h a) protein tubulin -Cy3staining, b) nuclear-DAPI staining
Figure 3. Imunofluorescence images of HCT-116 cell line treated with 50 μg/ml concentrations of
methanolic extract of leaves from Ligustrum vulgare for 24 h a) protein tubulin -Cy3staining, b) nuclearDAPI staining
Figure 4. Imunofluorescence images of HCT-116 cell line treated with 50 μg/ml concentrations of
methanolic extract of fruits from Ligustrum vulgare for 24 h a) protein tubulin -Cy3staining, b) nuclearDAPI staining
Dragana Đačić, MSci
Snežana Marković, PhD
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Figure 1.
Dragana Đačić, MSci
Snežana Marković, PhD
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Figure 2.
Dragana Đačić, MSci
Snežana Marković, PhD
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Figure 3.
Dragana Đačić, MSci
Snežana Marković, PhD
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Figure 4.
Dragana Đačić, MSci
Snežana Marković, PhD