1
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: [email protected]
User request
Material reception / Responsible person
Start of testing
Marko Živanović,
Prof. Zorica Bugarčić
MSci
Faculty of Science, University
October, 2011.
Danijela Cvetković,
of Kragujevac
Msci
Number
Ch/08
Jelena Košarić,
Msci
Active substance
Phenylselenoether of
pent-4-en-1-ol,
and hex-5-en-1-ol
Title
Objective
Material,
methods,
patients
Model system
Analysis
MDA-MB-231
HCT-116
UM.01, UM.02, UM.03, UM.04,
UM.16 NBT Assay
EFFECTS OF PHENYLSELENOETHER OF PENT-4-EN-1-OL AND
HEX-5-EN-1-OL ON SUPEROXIDE ANION PRODUCTION IN
MDA-MB-231 AND HCT-116 CELL LINE
The aim of this study was to evaluate the effects of phenylselenoether of pent-4en-1-ol, and hex-5-en-1-ol on superoxide anion production in human cancer
MDA-MB-231 and HCT-116 cell line.
Drugs
The MDA-MB-231 and HCT-116 cells were treated with various concentrations
of phenylselenoether of pent-4-en-1-ol, and hex-5-en-1-ol (ranging from 0.1 to
500 μM) for 24 h.
Cell preparation and culturing (UM.01, UM.02, UM.03, UM.04)
The cancer cell line MDA-MB-231 and HCT-116 were 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. All studies were done with cells at 70 to 80% confluence.
Determination of superoxide anion radical (NBT assay) UM.16
The concentration of superoxide anion radical (O2.-) in the sample was
determined by spectrophotometric method (Esfandiari, N et al, 2003), and is
based on the reduction of nitroblue tetrazolium (NBT) to nitroblue-formazan in
the presence of O2.-. MDA-MB-231 and HCT-116 cells were seeded in triplicates
in a 96-well plate (10 000 cells per well). After 24 h of cells incubation, the
medium was replaced with 100 μl of medium containing various doses of
phenylselenoether of pent-4-en-1-ol, and hex-5-en-1-ol at different
concentrations (0.1, 1, 10, 50, 100 and 500 μM) for 24 h. Assay was performed
by adding 100 μl of 5 mg/ml NBT to each well and then cells were incubated for
3 h at 37 oC in 5% CO2. To quantify the formazan product, formazan was
solubilized in 60 μl of 2M KOH and DMSO and the resulting colour reaction
was measured spectrophotometrically on microplate reader at 570 nm (ELISA
Marko Živanović, Msci; Danijela Cvetković, Msci
Jelena Košarić, Msci
Snežana Marković, PhD
2
Results
Discussion
Conclusion
References
Notes
Sign
Date
2100C). The amount of reduced NBT was determined by the change in
absorbance at 560 nm, based on molar extinction coefficient for monoformazan
which is 15,000.M-1 cm-1 and the results were expressed as nmol/ml of cells.
Data presented in pictures and tables show releasing of O2.- as nmol/ml after 24 h
of incubation with phenylselenoether of pent-4-en-1-ol, and hex-5-en-1-ol.
MDA-MB-231 cells exhibit increasing of O2.- concentration after treatement with
both selenium complexes. Statisticaly significant difference is observed with
phenylselenoether of pent-4-en-1-ol at concentrations of 1 and 50 μM, while
phenylselenoether of hex-5-en-1-ol exhibits statisticaly significant difference at
all concenrations except the concentration of 50 μM.
HCT-116 cells in comparison to MDA-MB-231 cells show different behaviour,
namely phenylselenoether of pent-4-en-1-ol induce decreasing in O2.concentration at all concentrations except the concentrations of 10 and 500 μM
with no statistically significant difference. Phenylselenoether of hex-5-en-1-ol
express increasing of O2.- in all applied concentrations with statistically
significant difference at concentration of 100 μM.
From the data shown below one could assume that investigated selenium
complexes at given conditions produce more O2.- radical in MDA-MB-231 cells
than in HCT-116 cells. It is clearly that in both cell lines phenylselenoether of
hex-5-en-1-ol induced greater production of O2.- radical in comparison with
phenylselenoether of pent-4-en-1-ol which actually induce decreasing the
production of O2.- radical in HCT-116 cells.
From data shown in pictures 1a and 2a we observed the greatest changes in O2.radical concentrations with selenium complexes concentration alterations in
concentration range from 1 to 100 μM. Due to the greatest changes in above
mentioned concentration range we will put more efforts in some future
experiments to elucidate this phenomenon.
As in the MDA-MB-231 cell line also in the HCT-116 cell line it is reported that
phenylselenoether of hex-5-en-1-ol shown much greater release of O2.- than
phenylselenoether of pent-4-en-1-ol.
Esfandiari, N.; Sharma, R.K.; Saleh, R.A.; Thomas, A.J.Jr.; Agarwal. A. Utility
of nitroblue tetrazolium reduction test for assessment of reactive oxigen species
production by seminal leukocytes and spermatozoa. J. Androl. 2003, 24, 862870.
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.
Responsible person for testing
Marko Živanović, MSci
Danijela Cvetković, Msci
Jelena Košarić, Msci
07.10.2011.
Marko Živanović, Msci; Danijela Cvetković, Msci
Jelena Košarić, Msci
Responsible person for Laboratory
Snežana Marković, Ph. D.
Snežana Marković, PhD
3
Table 1. Effect of phenylselenoether of pent-4-en-1-ol, and hex-5-en-1-ol, on MDA-MB-231 cell line
after 24 h of exposure, on superoxide anion radical (O2.-) production expressed as nmol/ml. All values are
mean ± SEM, n=3, *p < 0.05 as compared with control.
Table 2. Effect of phenylselenoether of pent-4-en-1-ol, and hex-5-en-1-ol, on HCT-116 cell line after 24
h of exposure, on superoxide anion radical (O2.-) production expressed as nmol/ml. All values are mean ±
SEM, n=3, *p < 0.05 as compared with control.
Figure 1. Effect of phenylselenoether of pent-4-en-1-ol, and hex-5-en-1-ol, on MDA-MB-231 cell line
after 24 h of exposure, on superoxide anion radical (O2.-) production expressed as nmol/ml.
Figure 1a. Extended X-axes
Figure 2. Effect of phenylselenoether of pent-4-en-1-ol, and hex-5-en-1-ol, on HCT-116 cell line after
24 h of exposure, on superoxide anion radical (O2.-) production expressed as nmol/ml.
Figure 2a. Extended X-axes
Marko Živanović, Msci; Danijela Cvetković, Msci
Jelena Košarić, Msci
Snežana Marković, PhD
4
Table 1
MDA –MB- 231
after 24 h
chemical
0 μM
0.1 μM
1 μM
10 μM
50 μM
100 μM
500 μM
Phenylselenoether
of pent-4-en-1-ol
(Selenium 1)
299.7 ± 0.3
310.6 ±
6.4
322.1 ±
5.5*
305.55 ±
3.25
319.75 ±
4.75*
295.7 ±
2.7
306.7 ±
4.2
Phenylselenoether
of hex-5-en-1-ol
(Selenium 2)
299.7 ± 0.3
320.65 ±
1.65*
319.35 ±
8.31*
322.2 ±
1.9*
303.1 ±
0.4
341.0 ±
5.5*
326.65 ±
0.35*
Figure 1.
Selen1
Selen2
kontrola
MDA-MB-231
350
330
-
nmol O 2 * /ml
340
320
310
300
290
0
07.10.2011.
0.1
1
10
50
100
concentration (M)
500
Figure 1a.
MDA-MB-231
350
Selen1
Selen2
kontrola
-
nmol O 2 * /ml
340
330
320
310
300
290
1 10 50 100
07.10.2011.
500
concentration (M)
Marko Živanović, Msci; Danijela Cvetković, Msci
Jelena Košarić, Msci
Snežana Marković, PhD
5
Table 2
HCT
after 24 h
0 μM
0.1 μM
1 μM
10 μM
50 μM
100 μM
500 μM
299.7 ±
8.1
282.05 ±
1.85*
284.4 ±
2.0
301.7
± 7.7
294.15
± 3.45
295.5 ±
0.1
310.95 ±
6.75
299.7 ±
8.1
296.7 ±
1.1
307.1 ±
1.1
315.3
± 0.1
308.8
± 0.6
323.9 ±
7.3*
304.3 ±
9.7
chemical
Phenylselenoether
of pent-4-en-1-ol
(Selenium 1)
Phenylselenoether
of hex-5-en-1-ol
(Selenium 2)
Figure 2.
Selen1
Selen2
kontrola
HCT-116
330
-
nmol O 2 * /ml
320
310
300
290
280
0
1
0.1
10
50
100
500
concentration (M)
Figure 2a.
HCT-116
Selen1
Selen2
kontrola
330
-
nmol O 2 * /ml
320
310
300
290
280
1 10 50 100
07.10.2011.
500
concentration (M)
Marko Živanović, Msci; Danijela Cvetković, Msci
Jelena Košarić, Msci
Snežana Marković, PhD