A total of 27 breast cancer patients were included in this study between 2007 and 2008. Tumor tissues and the paired normal breast tissues were immediately frozen after collection. The cases were diagnosed as infiltrating ductal carcinoma (IDC; n=27) according to the World Health Organization classification system (26). Clinical data were collected retrospectively. All patients provided informed consent and the study was approved by the Institutional Review Board of Changhua Christian Hospital.

The antibodies used for western blotting were: anti-SIRT1 (ab53517; Abcam, Cambridge, UK), anti-β-actin (A-5441) and anti-Bcl-2 (B-3170) (both from Sigma-Aldrich Co., MI, USA). The primary antibodies used for immunohistochemical analysis were: anti-SIRT1 (ab7343; Abcam) and anti-Ki67 (RM-9106-S1; Lab Vision, Fremont, CA, USA). Chemicals and reagents were purchased from Sigma-Aldrich Co., unless otherwise indicated.

The 5-μm sections of formalin-fixed, paraffin-embedded tissues were deparaffinized, rehydrated and the antigen retrieved. The tissue sections were incubated with hydrogen peroxide for 5 min at room temperature to quench the endogenous peroxidase. After blocking in normal goat serum, the tissue sections were incubated for 1 h at room temperature with the primary antibodies anti-SIRT1 (1:100 dilution) and anti-Ki67 (1:50 dilution), respectively. The slides were then washed with 0.5% Triton X-100 in PBS and incubated with HRP-conjugated secondary antibody for 1 h (Vector Laboratories, Burlingame CA, USA). Nuclei were counterstained with hematoxylin. As a negative control, normal goat serum was substituted for the primary antibody. All slides were examined by two independent investigators who were blind to the clinical data. To evaluate the expression of SIRT1 and Ki67 in tumor breast tissue and matched normal breast tissue, the staining intensity was scored from 0 to 3. The ratio of the positively stained area to the total section area was assessed.

Total RNA was isolated from breast tissue using TRIzol Reagent and cDNA was synthesized by MMLV Reverse transcriptase (both from Invitrogen, Carlsbad, CA, USA). The primer sequences used for PCR are shown in Table I. Synthesized cDNA was amplified and the PCR product was then visualized on 1% agarose gel. The intensity of the PCR product was determined by densitometry using the Gel Image Analysis System (Alpha Innotech Co., San Leandro, CA, USA).

The human breast cancer cell lines and MCF-7 and MDA-MB-231 cell lines were grown in Dulbecco's modified Eagle's medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Invitrogen Ltd., Carlsbad, CA, USA) in a humidified atmosphere containing 5% CO₂ at 37°C.

The cytotoxicity effect of sirtinol on MCF-7 and MDA-MB-231 cells was determined by trypan blue dye exclusion and Alamar-blue assays (AbD Serotec, Kidlington, UK). Briefly, the cells were plated at a density of 2×10⁵ cells in 6-well plates with an increasing dose of sirtinol (10–50 μM in DMSO) or vehicle alone. Following incubation for 24 or 48 h, cells were collected and an aliquot of cell suspension was mixed with an equal volume of trypan blue, and cells were then counted under the microscope. The cell viability assay was performed following the instructions of the Alamar blue assay kit. In brief, cells were plated at a density of 2×10³ cells in 200 μl of complete medium in a 96-well plate and treated with increasing doses of sirtinol or vehicle alone. Each treatment was performed in triplicate. The cells were incubated for 24 or 48 h at 37°C in 5% CO₂. Five hours before the end of the culture, the Alamar blue reagent (20 μl in phenol red free RPMI-1640 medium) was added into each well and incubated up to 24 or 48 h. Absorbance of 590 nm wavelength emissions was recorded.

Cell cycle analysis was performed with flow cytometry. Cells were grown at a density of 5×10⁵ cells in 6-well culture plates, treated with different concentrations of sirtinol for 24 and 48 h, and maintained at 37°C in a 5% CO₂ incubator. Following treatment, the cells were gently trypsinized, added to the culture medium, and pelleted by centrifugation. The cell pellet was resuspended in 1 ml PBS, washed at 1,500 × g for 30 min at 4°C, and fixed in ethanol (75% v/v). The cells were then washed with PBS and labeled with propidium iodide/RNase A solution. Labeled cells were analyzed by flow cytometry (Epics XL-MCL Flow Cytometry; Beckman Coulter, Inc., Fullerton, CA, USA) and the analyses were performed using WinMDI 2.9 software.

The cells were harvested at 24 and 48 h following sirtinol treatment, as described above, and washed with PBS, respectively. The cells were lysed with ice-cold RIPA lysis buffer (1X TBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide with protease inhibitor) and harvested. The lysate was centrifuged at 14,000 × g for 30 min at 4°C, and the supernatant was collected and stored at −20°C until analysis. The protein concentration was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Fifty micrograms of protein were separated by SDS-PAGE and then transferred onto a PVDF membrane (0.4 μm; Millipore, Billerica, MA, USA). Non-specific interactions were blocked by incubating the membrane with 5% non-fat dry milk in 0.5% PBST for 1 h at room temperature. The membrane was then blotted with anti-SIRT1, Bcl-2, or β-actin antibodies at 4°C overnight. HRP-conjugated secondary antibodies were added for 1 h, and the immunoreactive signals were then detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce). The level of protein expression was analyzed using the Gel Image Analysis System (Alpha Innotech Co.).

Statistical analyses were performed using the Student's t-test. A P-value of <0.05 was considered to indicate a statistically significant difference. All data are shown as the means ± SEM. Statistical calculations were performed using SPSS software (SPSS, Chicago, IL, USA).

The results of immunohistochemical analysis revealed that SIRT1, Ki67 and p53 were localized in tumor and in normal cells with varying intensities (Fig. 1). The expression of SIRT1 and Ki67 was significantly higher in tumor tissue than in normal tissue (P<0.001 and P<0.005 for SIRT1 and Ki67, respectively). Twenty-three (82.1%) tumor cases had a higher level of SIRT1 and Ki67 proteins. Furthermore, the RT-PCR results showed that the mRNA level of SIRT1 was overexpressed in breast tumor tissues (29%), compared to the paired normal tissues (Fig. 2).

When cells were treated with sirtinol at higher concentrations (30–40 μM), cell shrinkage was observed (Fig. 3A). The cell viability was reduced by sirtinol in a time- and dose-dependent manner in both MCF-7 and MDA-MB-23 cells (Fig. 3B). Flow cytometry analysis was used to examine the effects of sirtinol on the cell cycle progression of breast cancer cells. As shown in Fig. 4, sirtinol induced a cell cycle arrest in the G0/G1 phase in both cell lines. The proportion of the sub-G1 population also increased slightly in MDA-MB-231 cells, but not in MCF-7 cells.

To characterize the molecular mechanisms underlying these different behaviors, we analyzed the protein expression of Bcl-2 by western blotting. The Bcl-2 protein level in MCF-7 and MDA-MB-231 cells was significantly decreased with sirtinol treatment, particularly with 48-h treatment (Fig. 5).