1. Material
In this study we used ovarian cancer tissue samples of 123 patients who underwent surgery for ovarian cancer from 1990 to 2002 at the Department of Gynecology and Obstetrics, Ludwig-Maximilians-University of Munich, Germany. Patients who underwent surgery due to serous or mucinous ovarian cancer were included while other histological subtypes were excluded due to low number. The median age was 59 years (range 20–88 years) and median overall survival was 2.67 years. The distribution of clinic-pathological variables can be seen in table 1. As positive controls for immunohistochemical staining, we utilized palatine tonsil for Sirt1 staining and first trimester placenta for RXR staining, both received from the Department of Obstetrics and Gynecology of the Ludwig-Maximilians-University of Munich. Clinical and follow-up data for statistical analyses were provided by the Munich cancer registry and retrieved from medical records.
Table 1. Patients’ characteristics.
|
N
|
%
|
Subtype
|
|
|
serous
|
110
|
89.4
|
mucinous
|
13
|
10.6
|
Age
|
|
|
≥60
|
61
|
49.6
|
<60
|
62
|
50.4
|
FIGO
|
|
|
I/II
|
29
|
23.6
|
III/IV
|
92
|
74.8
|
NA’s
|
2
|
01.6
|
Grading
|
|
|
1/2
|
76
|
61.8
|
3
|
40
|
32.5
|
NA’s
|
7
|
05.7
|
Progression (18 years)
No progression
Progression
NA’s
|
101
21
1
|
82.1%
17.1%
0.1%
|
Overall-Survival (18 years)
Right censured
Died
NA’s
|
38
84
1
|
30.1%
68.3%
0.1%
|
2. Ethics Approval
All ovarian cancer specimens had been collected for histopathological diagnostics during surgery. They were no longer used for clinical tests. Patients’ data were anonymized and authors were blinded for clinical information during experimental analyses. The study was conducted in consent to the Declaration of Helsinki and was approved by the local ethics committee of the Ludwig-Maximilians University of Munich (reference number 227-09 and 18-392).
3. Immunohistochemistry
Paraffin-embedded slides of 3µm were dewaxed in xylol and washed in 100% ethanol. For inhibition of the endogen peroxidases, tissue samples were incubated in 3% methanol/H2O2 and rehydrated in a descending alcohol series. Slides were afterwards heated in a pressure cooker using sodium citrate buffer (pH = 6.0; containing 0.1 M citric acid and 0.1 M sodium citrate in distilled water). After cooling and washing in PBS (phosphate-buffered saline), all slides were incubated with blocking solution to avoid non-specific binding of the primary antibodies. Subsequently, the slides were stained with the primary antibodies anti-Sirt1 and anti-RXRα (table 2) and incubated. After washing, the secondary complexes of the ABC detection kits were applied following the manufacturer’s protocols to detect reactivity. Immunostaining was visualized with the substrate and the chromogen-3, 3′-diaminobenzidine (DAB) for 1 min. For exact staining protocol see table 2.
For the light microscopy analysis, the semi-quantitative immune-reactive score (IRS) is calculated via the multiplication of optical staining intensity (grades: 0 = no, 1 = weak, 2 = moderate and 3 = strong staining) and the percentage range of positive stained cells (0 = no staining, 1 = ≤10% of the cells; 2 = 11–50% of the cells; 3 = 51–80% of the cells and 4 = ≥81% of the cells were stained for the antibody, respectively). Palatine tonsil was used as control for Sirt1 and first trimester placenta was used as control for RXR staining.
Table 2. Antibodies and chemicals used for the immunohistochemistry
anti-Sirt11
|
anti-RXRα2
|
PBS3
|
PBS3
|
Blocking solution4: 20 min
|
Blocking solution4: 20 min
|
primary antibody1: 1:1000
incubation: 16 h, 4◦C min
|
primary antibody2: 1:200
incubation: 16 h, 4◦C
|
ABC detection kid 5
|
ABC detection kid 5
|
Chromogen: DAB 6 (1 min)
|
Chromogen: DAB 5 (1 min)
|
1 anti-Sirt1 rabbit IgG, polyclonal antibody, concentration: 1:1000; Atlas Antibody, Sweden; order number: SHPA006295.
2 anti-RXRα rabbit IgG, polyclonal antibody, concentration: 1:200; PPMX, Japan; order number: pp-k8508-10.
3 HRP-Polymer-Kit (mouse/rabbit); Zytomed Systems, Germany; order number: POLHRP-100.
4 ABC detection kid; Vectastain, USA; order number: AK-6401.
5 Dulbecco’s Phosphate Buffered Saline; Gibco, USA; order number: 14190-094.
4. Cell culture
Human ovarian cancer cell lines with different characteristics (A2780, UWB1.289 and cisA2780, see table 3) were used in the study. The cell lines were ordered from Gibco (see table 3).
Table 3. Cell lines
|
A2780 1
|
UWB1.289 2
|
cisA2780 3
|
Cell type
|
Epithelial ovarian cancer cell
|
Epithelial ovarian cancer cell
|
Epithelial ovarian cancer cell
|
Characteristics
|
Mucinous
|
Serous brca1-null
|
Mucinous, carboplatin-resistant
|
Culture medium
|
RPMI 16404 + 10% FBS 5
|
RPMI 16404 + 10% FBS 5
|
RPMI 16404 + 10% FBS 5
|
1/2/3 Gibco. 4 Gibco, USA; Order number: 21875-034. 5 Foetal Bovine Serum, biochrom, Germany; order number S0615
Cell lines were cultured (see table 3) and seeded into 96-well plates for MTT and BrdU (see below) and 6-well plates for Western blotting. After 20 h, cell culture medium was replaced with fresh culture medium with resveratrol (50 and 100µM RSV; Sigma, America; order number: R5010-100MG) for the remaining 24 h, which included dimethyl sulfoxide (DMSO; concentration: 0.5%; SERVA, Germany; order number: 20385, 0.5%) as vehicle control.
ELISAs
Cell viability assay
A2780, UWB1.289 and cisA2780 ovarian cancer cells were seeded at the density of 1.5 × 104 cells/well in 96-well plates with 200μl medium. After 20 h cells were incubated with 50µM and 100µM of resveratrol for 24 h. Untreated control cells were plated in medium only. To each well, 20μg MTT (Sigma, USA; order number: M-5655) were added for 1.5 h at 37°C in order to show viability. After removing MTT from the plates, 200μL DMSO were added and mixed thoroughly on the shaker for 5 min at room temperature. The optical density was examined at 595 nm using Elx800 universal Microplate Reader. Each experiment was carried out in triplicate.
Marker of proliferation: BrdU
A2780, UWB1.289 and cisA2780 ovarian cancer cells were cultured at the density of 1.0 × 104 cells/well together with various dilutions (50/100µM) of resveratrol in 96-well plates. For the labelling of DNA replication BrdU (Bromodeoxyuridine; Roche, Switzerland; order number: 11647229001) was added to the culture medium for 2h. The final concentration of BrdU was 10μM. After removal of BrdU by pipette, 200µl/well FixDenat (Bromodeoxyuridine; Roche, Switzerland; order number: 11647229001) were added and cells were incubated for 30 min at room temperature. Afterwards, FixDenat solution had to be removed thoroughly and 100µl/well anti-BrdU-POD (Bromodeoxyuridine; Roche, Switzerland; order number: 11647229001) working solution were added. Cells were then incubated for approximately 90 min at room temperature and washed 3 times with PBS. 100µ/well substrate solution BrdU was added and incubation for 20 minutes was performed. To each well 25µl 1M H2SO4 were added and the absorbance of the samples was measured by an ELISA reader at 450nm.
Apoptosis assay
Apoptosis was evaluated by measuring the level of caspase-cleaved cytokeratin 18 (M30, Roche, Switzerland; order number: 121140322001). The ovarian cell lines A2780, cisA2780 and UWB1.289 were seeded at a density of 1.0 × 104 cells/well on 96-well plates in 200μl medium. After 20 h, 50 or 100µM RSV were added and cells were incubated for 24 h. M30 CytoDeath (Roche, Switzerland; order number: 121140322001, dilution 1:1000) was used to detect the apoptotic cells.
TUNEL
Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis. TUNEL staining was performed to assess in situ DNA fragmentation using a commercial kit (FragELTM DNA Fragmentation Detection Kit, Colorimeric- TdT Enzyme , USA; order number: Qia33-1EA) following the manufacturer’s protocol.
Western blotting
Cell lysates were extracted from A2780, cisA2780 and UWB1.289 cells with radio-immuno-precipitation assay buffer (RIPA, Sigma-Aldrich, St. Luis, USA; order-number: R0278-50ML). For Western blotting, 20µg of cell lysates were first separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked in 10% casein and then incubated with the primary antibodies for 16 h at room temperature. We used the same antibodies as for immunohistochemistry (see table 2).
GAPDH was used as a housekeeping gene and mouse monoclonal anti-GAPDH antibody (GeneTex, America; order number: GTX277408) was diluted 1:1000 in 10% CASEIN (Vector, Germany; order number: ZE0925). Afterwards, the membrane was incubated with the goat-anti-rabbit secondary antibody (Vector; bioZol; Germany; order number: VEC-BA-1000, dilution 1:1000) conjugated with alkaline phosphatase, and detected with 5-bromo-4-chloro-3′-indolylphosphate/nitro-blue tetrazolium (BCIP/NBT) -chromogen substrate solution (Vector; bioZol; Germany; order number: Vec-SP-5020). Western blots were scanned and quantified using the GelScan V6.0 1D Analysis Software (SERVA, Electrophoresis GmbH, Heidelberg, Germany). Band intensities of Sirt1 and RXRα were normalized with band intensities of GAPDH. The blots were repeated three times.
5. Statistics
SPSS Statistics 25 was used for data collection, processing and analysis. The Wilcoxon test was used for the evaluation of Sirt1, RXR, and GAPDH values between related groups. Spearman’s test was applied to compare the IRS of Sirt1 and RXR staining in the ovarian cancer patients. Survival rates were shown by Kaplan-Meier curves. P-value <0.05 was considered as statistically significant.