CYTO2018 PRAGUE APRIL 28 â MAY 2. LOVO cells control. Celastrol. (CEL). Resveratrol. (RSV). SIRT 1. 1,0. (0,37). 0,29. (0,05). 13,3. (2,13). SIRT 2. 1,0. (0,30).
Effect of celastrol and resveratrol on spontaneous DNA damages in LOVO cell cultures. Cytophotometric approach and SIRT genes’ expression study. Helena Moreira1, Anna Szyjka1, Justyna Gołuchowska1, Justyna Mierziak-Derecka2, Anna Kulma2, Kazimierz Gąsiorowski1 1Department of Basic Medical Sciences, Wroclaw Medical University, Wroclaw, Poland 2 Laboratory of Genetic Biochemistry, University of Wroclaw, Wroclaw, Poland
METHODS
INTRODUCTION Sirtuins (SIRT) are class III deacetylases involved in DNA repair, energy metabolism, apoptosis, cell survival and their stimulating as well as inhibiting effects on tumors' growth have been described. Celastrol (CEL) and resveratrol (RSV) are plant polyphenols that modulate SIRT genes’ expression. Learning the relationship between SIRT expression and cancer cell response to DNA damage in human colon cancer can help to develop new potential therapeutic strategies for this cancer.
AIM Estimation of the level of spontaneous DNA damage in Lovo cells and the effect of celastrol and resveratrol on the repair of DNA damage and on the expression of sirtuin genes.
RESULTS Expression of SIRT genes
16
Human colon cancer cells (Lovo cell line) were incubated for 18hrs with celastrol and resveratrol [2,5 μM]. The rate of apoptotic cells was estimated with Dead Cell/Apoptosis Kit (Thermo Fisher) and cell cycle analysis was done by the IP staining method. The percentage of H2A.X activated cells was evaluated using anti-phospho-ATM (Ser1981)-PE and anti-phospho-Histone H2A.X (Ser139)-PECy5 (Muse Multi-Color DNA Damage kit, Millipore). Flow cytometric measurements were done with CyFlow®Space cytometer (Sysmex-Partec) equipped with FlowMax software (Sysmex-Partec). The cell cycle profile was analysed using MultiCycle™ DNA analysis model (FCS 4 Express, DeNovo Software). Expression of sirtuin genes was measured by real-time PCR (StepOnePlus™ Real-Time PCR Systems, Applied Biosystems).
H2A.X phosphorylation and ATM activation
C o n tro l
Expression of sirtuin genes (SIRT1, SIRT2, SIRT3) was significantly decreased in cell cultures incubated with celastrol (up to 10-fold decrease for SIRT1), while it was considerably increased in cultures incubated with resveratrol (even 14-folds for SIRT1 and SIRT2). SIRT6 gene was overexpressed both in the presence of celastrol (3-fold increase) and resveratrol (9,5fold increase). Control: LOVO cells culturesd without polyphenols.
CEL 12
RSV
10
CEL 2.5µM 50
6
C o n tro l c e lls
4
2 0 S IR T 1
S IR T 2
S IR T 3
S IR T 6
control
Celastrol (CEL)
Resveratrol (RSV)
1,0 (0,37)
0,29 (0,05)
13,3 (2,13)
SIRT 2
1,0 (0,30)
0,54 (0,14)
13,9 (1,71)
SIRT 3
1,0 (0,23)
0,73 (0,09)
2,55 (0,13)
SIRT 6
1,0 (0,01)
3,0 (0,29)
9,5 (0,71)
LOVO cells SIRT 1
Significant decrease in expression
C e la s tro l 2 .5 M
40
R e s v e ra tro l 2 .5 M 30
20
10
0 H 2 A.X
+
AT M
+
Celastrol caused a 3.7-fold increase of γH2A.X+ frequency in Lovo cell cultures, together with a marked (12-fold) decrease of cell frequency containing activated ATM. Resveratrol did not influence on γH2A.X+ cell frequency, whereas it caused 1,62-fold decrease in the percentage of ATM+ cells.
Significant increase in expression
Cell apoptosis estimation
Cell cycle analysis with PI
Control 80
S G 2 /M
40
20
2431
C o n tro l
C E L 2 .5 M
Control 1823
1216
RSV 2.5µM
CEL 2.5µM 1602
%G1=51,858 %S=24,405 %G2/M=23,737
1268
%G1=36,874 %S=48,388 %G2/M=14,738
1068
0
423
1024
2048
DNA content (PI)
3072
4096
0
1024
2048
DNA content (PI)
3072
4096
E A R L Y A P O P T O S IS L A T E A P O P T O S IS
N E C R O S IS
40
20
0
0
0 0
%G1=32,976 %S=56,860 %G2/M=10,164
846
534
608
Cells incubation of with celastrol and resveratrol led to an significant increase (up to about 50%) in the percentage of cells in apoptosis, mainly in late apoptosis.
1691
2136
R S V 2 .5 M
RSV 2.5µM
60
0
% O F TO TAL CELLS
% O F TO TAL CELLS
G1
CEL 2.5µM
Both, celastrol and resveratrol increased the number of cells occupying the S phase of the cell cycle (by 55% and 31% respectively), while the number of cells in the G2 phase was reduced.
60
RSV 2.5µM
8
% O F TO TAL CELLS
g e n e e x p r e s s io n
14
0
1024
2048
3072
4096
C o n tro l
DNA content (PI)
C E L 2 .5 M
R S V 2 .5 M
Conclusions [1.] Lovo cells cultured in the presence of celastrol showed reduced expression of SIRT1-3 genes which coexisted with increased H2AX phosphorylation, a marker of double-stranded DNA strand breaks. [2.] Expression of cancer-suppressing sirtuin the SIRT6 was elevated in the presence of each of the polyphenols tested. [3.] Despite the different effects of tested polyphenols on sirtuin gene expression and on the cellular content of phosphorylated H2AX histone, the level of final cellular response (cell cycle delay) and cell fate (apoptosis frequency) were similar. This indicates that the cells also have other mechanisms to repair DNA damage, independent of H2AX phosphorylation. This work was supported by Wroclaw Medical University Grant Nr. ST.D130.16.009
CYTO2018 PRAGUE APRIL 28 – MAY 2
