Centre for pre-clinical testing of active substances laboratory for cell and molecular biology




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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

Material reception /

Start of testing

Responsible person


Pl/08

Milan Stanković,

Department for Biology and Ecology, Faculty of Science

University of Kragujevac


May, 2011.


Milena Ćurčić





Active substance

Model system

Analysis

Methanolic extracts of leaves and fruits from Sedum acre



HCT-116 cell line


MTT cell viability assay,

UM.01, UM.02, UM.03, UM.04, UM.05

Title

Antiproliferative activity of methanolic extract from Sedum acre L. on HCT-116 cell line

Objective

The aim of this study was to determinate the antprolifertive effect of methanolic extract from Sedum acre on HCT-116 cell line, human colon cancer.

Material, methods, patients

Drugs

The HCT-116 cells were treated with various concentrations of methanolic extracts ranging from 1 to 500 μg/ml for 24 and 72 h.



Cell preparation and culturing (UM.01, UM.02, UM.03, UM.04)

HCT-116 cell line, human colon cancer was obtained from American Type Culture Collection. Cells were maintained in DMEM supplemented by 10% FBS, with 100 units/ml penicillin and 100 µg/ml 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 96-well plate. All studies were done with cells at 70 to 80% confluence.



Cell viability assay (MTT assay)(UМ.05)

HCT-116 cells were seeded in a 96-well plate (10 000 cells per well). After 24 h of cells incubation, the medium was replaced with 100 μl medium containing various doses of methanolic extracts at different concentrations (1, 10, 50, 100, 250 and 500 μg/ml) for 24 and 72 h. Untreated cells served as the control. After 24 and 72 h of treatment the cell viability was determined by MTT assay (Mosman, 1983). The proliferation test is based on the color reaction of mitochondrial dehydrogenase in living cells by MTT. 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 2-4 h. The colored crystals of produced formazan were dissolved in 150 μl DMSO. The absorbance was measured at 570 nm on Microplate Reader. Cell proliferation was calculated as the ratio of absorbance of treated group divided by the absorbance of control group, multiplied by 100 to give a percentage proliferation.



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. A p value < 0.05 was considered as significant. 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).



Results

Antiproliferative activity

In order to get completly screening of biological activities cell proliferation ability was also examined. To explore the antiproliferative activity of the methanolic extracts from S. acre on HCT-116 cell line, the MTT cell viability assay was used. We treated HCT-116 cells with different concentrations of extract (in concentration range from 1 to 500 μg/ml) and determined cell viability 24 and 72 hours after treatment.

The shape of dose-response curves indicates a significant inhibition of cell growth in dose and time-dependent manner (Figure 1). Cell growth was significantly lower (p<0.05) when extract-treated cells were compared to control cells. The extract exhibited higher cytotoxic effects after longer time of exposure. The effects of extracts were expressed by IC50 values (inhibitory dose inhibited cell growth by 50%). The IC50 value was used as a parameter for cytotoxicity. IC50 values was 281.69±3.87 after 24 hours and 126.57±2.91 after 72 hours of exposure. Methanolic extract from S. acre affects on cell proliferation and inhibits cell growth, but according to IC50 values we can conclude that it has intermediate effect on HCT-116 cells proliferation.

Discussion




Conclusion

According to the results of investigations, our in vitro data indicated the methanolic extract from S. acre inhibited proliferation of HCT-116 cells and has intermediate effect on HCT-116 cells proliferation

References

Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth.1983, 65, 55-63.

Notes

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

Suzana Popović
Milena Ćurčić

Responsible person for Laboratory

Dr Snežana Marković


Date

06.07.2011.





Figure and table legends
Figure 1. The dose-response curves of the effects of S. acre on cell growth in HCT-116 cells. The cells were treated with various concentrations of drugs, after 24 and 72 h of exposure. The antiproliferative effects were measured by MTT assay. Results were expressed as means ± SE for three independent determinations.

Figure 1.





Milena Ćurčić, MSci Snežana Marković, Ph.D.


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