Materials
EGCG and thiazolyl blue were purchased from Sigma (St. Louis, MO, USA). RPMI-1640 basal medium and fetal bovine serum (FBS) was produced by HyClone (Logan, UT, USA). RPMI-1640 basal medium and EGCG were used to prepare a 10 mmol/l storage solution. It was sterilized by filtration (0.22 µm) and stored at −80°C in the dark. MTT was dissolved in phosphate-buffered saline (PBS) solution at 5 mg/ml, and was stored at −20°C in the dark after sterilization. Mouse anti-human P53, Bcl-2 and GAPDH were all purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and the RT-PCR kit was purchased from Takara (Otsu, Shiga, Japan). P53, Bcl-2 and GAPDH primers were produced by Invitrogen (Carlsbad, CA, USA) (Table I) and si-P53 was supplied by Gemma (Shanghai, China).
Methods
MTT detection of the sensitivity of MCF-7 cells to EGCG
The EGCG concentration gradient was 0, 2, 4, 8, 16, 32, 64 and 128 µmol/l. MCF-7 cells (2×103) were seeded in a 96-well plate with blank control wells. After cells adhered to the walls, EGCG of the concentration gradient of 0, 2, 4, 8, 16, 32, 64 and 128 µmol/l was added. After incubation for 72 h, the supernatant was discarded and 20 µl of MTT was added to each well. After incubation for 4 h, the liquid was discarded and 150 µl of dimethyl sulfoxide (DMSO) was added to each well of the EGCG. The optical density (OD) of each well at 490 nm was measured by a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The inhibition rate was calculated as: (control group OD - experimental group OD/control group OD) × 100%.
Effect of EGCG on MCF-7 cell apoptosis measured by flow cytometry (FCM)
MCF-7 cells were seeded in a 6-well cell culture plate. After adherence, 30 µmol/l EGCG was added and the cells were stained after 48 h with Annexin V-FITC kit. The change in apoptotic rate was measured, and the experiment was repeated three times.
RT-qPCR and western blot analysis determine how EGCG treatment on MCF-7 affects the mRNA and protein levels of P53 and Bcl-2
The experiment was conducted on the control and EGCG groups. EGCG was added to fresh MCF-7 cells in the experimental group after cell adhesion (EGCG at a final concentration of 30 µmol/l), but the control group was not treated. After 72 h of incubation, total RNA was extracted using TRIzol reagent. The RT-qPCR assay was performed using the Takara kit, and the GAPDH gene was used as an internal control. The conditions for RT-qPCR were: Preheating at 95°C for 2 min, denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 40 sec. The experiment was repeated three times. The results were expressed as 2−ΔΔCq. MCF-7 cells were treated with 30 µmol/l EGCG for 48 h. The cells were then rinsed three times with PBS and 100 µl of RIPA lysate was added. The cells were placed on ice for 30 min and centrifuged at 12,000 × g for 20 min at 4°C. The supernatant was collected and stored at −80°C. Protein concentration was determined using the BCA protein method. The protein extract was subjected to SDS-PAGE (10%) and transferred to a PVDF membrane. The membrane was blocked with 3% BSA solution for 2 h. Then, primary rabbit monoclonal P53 antibody (dilution, 1:500; cat. no. ab32049), rabbit polyclonal Bcl-2 antibody (dilution, 1:500; cat. no. ab59348) and rabbit polyclonal GAPDH antiboody (dilution, 1:500; cat. no. ab37168), purchased from Abcam (Cambridge, MA, USA), were added at 1:1,000 and incubated at 4°C overnight. The membrane was washed with PBS three times (5 min each time). Then secondary goat anti-rabbit (HRP) IgG antibody (dilution, 1:2,000; cat. no. ab6721) was added followed by 2-h incubation and washing with PBS three times (5 min each time). Finally, it was placed on the ECL for exposure and development.
RT-qPCR and western blot analysis of si-P53 interference effect
The cells were divided into the si-P53, si-NC and NC groups (si-P53 was added with small fragments of interference P53, with si-NC as a positive control and NC as a negative control). Cells in the logarithmic growth phase were inoculated into the culture flask and transiently transfected according to the protocol of the Lipofectamine™ 2000 kit. After 48 h, the RNA was extracted and purified according to the kit protocol, and RNA purity and concentration was determined by a spectrophotometer (Hitachi, Tokyo, Japan). The P53 mRNA expression was evaluated using RT-qPCR, and the P53 protein expression level was measured using western blot analysis. The three groups were compared and the transfection efficiency was calculated.
Alterations in the expression of P53 and Bcl-2 proteins in MCF-7 cells transfected with si-P53 detected by western blot analysis
The cells were divided into the si-P53, EGCG and EGCG + si-P53, (si-P53 plus interference P53 small fragments, si-P53 for the positive control, NC for the negative control) groups. The P53 and Bcl-2 protein expression levels in the three groups were evaluated using western blot analysis using the same method explained before.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) and SPSS 20.0 (Chicago, IL, USA) statistical software. The measurement data were presented as mean ± standard deviation. Comparison between two groups was conducted using a t-test. The one-way analysis of variance (ANOVA) test was used for comparison among three groups. P<0.05 was considered to indicate a statistically significant difference.