Cell culture and reagents
Human breast cancer cell lines MCF-7, MDAMB231, Hs578t, and human embryonic kidney (HEK) 293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human breast cancer multidrug resistance cell MCF-7/MDR was purchased from the Cell Bank of Xiangya Medical College, Central South University. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, California, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Beijing, China), 10 mmol/l HEPES, at 37 °C and 5% CO2. Adriamycin (ADM), 5-fluorouracil (5-FU), and cisplatin (DDP) were purchased from sigma (Sigma, MO, USA).
Plasmid
CLDN6 shRNA plasmid, i.e. pGCsilencer-U6/Neo/GFP was constructed which also expresses Green Fluorescent Protein (GFP) (KeyGEN Biotech, Nanjing, China). Target sequence is GGCAAGGTGTACGACTCA. A functional non-targeting siRNA was used as a control.
A CLDN6 overexpression plasmid was generated by sub-cloning human CLDN6 cDNA (NCBI RefSeq record: NM_021195) into the mammalian expression plasmid EX-LV201 (GeneCopoeia, Guangzhou, China) with FLAGs SV40-eGFP-IRES-puromycin.
Lentivirus production and stable knockdown of the GSTP1 genes shRNA plasmid (sh-GSTP1–21:5′-GCCCTACACCGTGGTCTATTT-3′,sh-GSTP1–22:5′-AGGACCTCCGCTGCAAATACA-3′ and sh-GSTP1–23:5′-GGCAAGGATGACTATGTGAAG-3′) and negative-shRNA as shRNA-control (sh-GSTP1-CT) were purchased from GeneCopoeia (GeneCopoeia, Guangzhou, China). Silencer negative control with no significant homology to any known human genes was used as a negative control shRNA with FLAGs U6-mCherry-Hygromycin.
Lentivirus production and stable knockdown of the TP53 gene shRNA interference (shRNAi) plasmids were inserted into the psi-LVRU6MH vector downstream of the U6 promoter with FLAGs U6-mCherry-Hygromycin (GeneCopoeia, Guangzhou, China). Plasmids sh-TP53–32 (5′-GGAAATTTGCGTGTGGAGTAT-3′, sh-TP53–33 (5′-CCACTACAACTACATGTGTAA-3′) and sh-TP53–34 (5′-GGACTTCCATTTGCTTTGTCC-3′) were used to knockdown the TP53 gene and a functional non-targeting shRNA (sh-TP53-CT) was served as a negative control clone. For stable RNAi, lentiviral particles were produced.
A GSTP1 overexpression plasmid was generated by subcloning human GSTP1 cDNA (NCBI RefSeq record: NM_000852) into the mammalian expression plasmid EX-LV206 with FLAGs SV40-mCherry-IRES-puromycin (GeneCopoeia, Guangzhou, China).
Lentiviral packaging plasmids expressing gag-pol, rev, and pVSVG genes were obtained from Institute of Biochemistry and Cell Biology of Shanghai Life Science Research Institute, Chinese Academy of Sciences. These vectors were transfected into 293T cells by FuGene HD (Roche Applied Science, Basel, Switzerland). Viral supernatants were harvested at 48 h and 72 h after transfection and concentrated by ultracentrifugation. Viruses were transduced in the presence of 5 μg/mL polybrene.
Reverse-transcription PCR (RT-PCR) and quantitative RT-PCR (qRT-PCR) analysis
Total RNA was isolated from 5 × 106 cells using TRIzol reagent (Invitrogen, California, USA), according to the manufacturer’s instructions. Total RNA concentration and purity were analyzed in duplicate samples using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). cDNA was synthesized from the qualified RNA using an RT-PCR reverse transcription kit (TransGen Biotech, Beijing, China). 1000 ng of total RNA was reverse transcribed into cDNA under the condition: 25 °C 10 min, 42 °C 30 min, and 85 °C 5 s, as manufacturer’s recommendations. Then the cDNA was stored at −20 °C until use. The PCR was performed using a PCR kit (TransGen Biotech, Beijing, China). The PCR product was electrophoresed on 1.5% agarose gels. Primers were synthesized by Sangon (Sangon, Shanghai, China). Quantitative PCR was carried out with either Taq-Man or SYBR Green PCR reagents on an ABI Prism 7300 detection system (all from Applied Biosystems, Foster City, CA). The reaction program was consisted of 95 °C for 3 min followed by 40 cycles of 95 °C 30 s, 55 °C 20 s, and 72 °C 15 s. GAPDH gene was served as internal control and the relative mRNA levels were calculated by 2−ΔΔCt. Primers designed were synthesized by Sangon (Shanghai, China).
Western blot analysis
When cells reach 80% confluence, cells were harvested and washed with PBS. Total protein was extracted with 200 μL RIPA Lysis Buffer (Beyotime, Shanghai, China) with 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma, MO, USA) and 1 mM Phosphatase inhibitor (Solarbio, Beijing, China), followed with centrifugation 12,000 g for 20 min. The protein concentration was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). 50 μg of denatured protein was applied to 12% SDS-PAGE gels. After electrophoresis the proteins on the gel were then transferred onto a nitrocellulose membrane (Millipore, California, USA). The membrane was blocked with 5% defatted milk at 37 °C for 1 h and incubated overnight at 4 °C with the primary antibodies. The membrane was incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Finally the immunoreactive bands were visualized using an ECL western blotting system (Beyotime, Shanghai, China). The following antibodies were used: a monoclonal mouse anti-β-actin antibody (Santa Cruz Biotechnology, California, USA), a polyclonal rabbit anti-CLDN6 antibody (Santa Cruz Biotechnology, California, USA), a polyclonal rabbit anti-cleaved-caspase-9 antibody (Cell Signaling Technology, MA, USA), a polyclonal rabbit anti-cleaved-PARP antibody (Cell Signaling Technology, MA, USA), a polyclonal rabbit anti-γH2AX antibody (Cell Signaling Technology, MA, USA), a polyclonal rabbit anti-p-Ser15-p53 antibody (Cell Signaling Technology, MA, USA), and a polyclonal mouse anti-GSTP1 antibody (Cell Signaling Technology, MA, USA).
In vitro drug sensitivity assay
In vitro drug cytotoxicity was measured by Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan). The cells were seeded into 96-well plates (3 × 103 cells/well) and then treated for 48 h in 100 μL of medium with anticancer drugs. The cells incubated without drugs (i.e. control wells) were set at 100% survival and were utilized to calculate the concentration of each cytostatic drug lethal to 50% of the cells (IC50). CCK-8 reagent was then added and then incubated at 37 °C for 2 h. The optical density (OD) of each well at 450 nm was recorded on a Microplate Reader (Thermo, Schwerte, Germany). The cell viability (% of control) is expressed as the percentage of (ODtest − ODblank)/(ODcontrol − ODblank). The assay was conducted in three replicate wells for each sample and three parallel experiments were performed.
Apoptosis assay
4′,6-diamidino-2-phenylindole (DAPI) staining was used to detect apoptosis in vitro. Cells were harvested when grown to 60-80% confluency, and treated with ADM for 48 h, then fixed with 4% paraformaldehyde, stained with the 1 mg/mL DAPI (Sigma, MO, USA) for 15 min and examined by fluorescence microscopy to determine the fraction of apoptotic cells. Apoptotic cells were recognized as chromatin condensed, punctate nuclear ghosts with stained, degraded nuclei when examined by fluorescence microscopy. The incidence of apoptosis was analyzed by counting nuclear deep dyeing cells with condensed chromatin, and determining the percentage of apoptotic cells.
GST activity assay
GST activity was measured using a GST activity kit (Solarbio, Beijing, China) according to the manufacturer’s protocol. It was defined as the amount of enzyme that was required to reflect the ability to reduce GSH and 1-chloro-2, 4-dinitrobenzene (CDNB). The changes in absorbance of the GSH and CDNB were recorded at 340 nm for 10 s and 310 s respectively. GST activity was expressed as nmol per min per mg of total protein concentration.
Nuclear/cytosol fractionation
To monitor the nuclear and cytosol p53 protein level after CLDN6 overexpression, nuclear/cytosol fractionation along with immunoblotting analysis were performed. 1 × 106 cells were needed. Nuclear/Cytosol Fractionation Kit (TransGen Biotech, Beijing, China) was applied to isolate nucleus and cytosol protein according to the manufacturer’s instructions.
Immunoprecipitation–western blots
The cells were lysed in IP lysis buffer (Beyotime, Shanghai, China) for 30 min on ice, vortex for 10 s interval of 5 min, then transferred to a 1.5 mL microcentrifuge tube and centrifuged for 20 min at 14,000 g to remove cellular debris. The supernatants were analyzed for total protein content, and 300 μg of total protein was incubated with 25 μL of agarose-immobilized goat polyclonal anti-rabbit antibody in a final volume of 500 μL, adjusted with lysis buffer. Immunoprecipitation was carried out with gentle rocking, overnight at 4 °C. The agarose beads were pelleted by centrifugation at 3000 rpm for 5 min, and then washed 3 times with 1 mL lysis buffer, with each wash followed by a 3 min centrifugation at 3000 rpm. After the final wash, 24 μL lysis buffer and 6 μL of 5× SDS sample buffer was added to the beads, the samples were boiled and then loaded onto 12% SDS-PAGE gels. Following protein transfer to PVDF membrane (Millipore, California, USA), p53 and CLDN6 expression were detected by western blotting as described earlier.
Immunohistochemistry
Immunohistochemistry of tumor tissues collected from human patients breast cancer samples were performed as we described elsewhere [2]. 40 patients with breast cancer at the department of pathology of the second hospital of Jilin university who had not been treated with any chemotherapy and those received neoadjuvant chemotherapy for relapsed disease after initial biopsy either for organ preservation or for unresectable disease. Formalin-fixed, paraffin-embedded biopsy tissues were available. Immunohistochemistry was performed as described elsewhere. Tissue sections were immunostained with CLDN6 antibody (Abcam, MA, USA) and GSTP1 antibody (Cell Signaling Technology, MA, USA). Diaminobenzidine (DAB) was used for color development. CLDN6 expression is indicated in brown and is expressed in the membrane of breast cancer cells and GSTP1 is indicated in brown and expressed in the nuclear of breast cancer cells.
Immunoreactivity was evaluated independently by two observers without specific knowledge of the patients. The staining was graded for intensity (0-negative, 1-weak, 2-moderate, and 3-strong) and percentage of positive cells as follows: 0, <5% positive tumor cells; 1, 5%–25% positive tumor cells; 2, 26%–50% positive tumor cells; 3, 51%–75% positive tumor cells; 4, >75% positive tumor cells. The grades were multiplied to determine an H-score. The H-scores for tumors with multiple scores were averaged. Protein expression was then defined as negative (H-score = 0), weakly positive (H-score = 1–4), moderately positive (H-score = 5–8), and strong (H-score = 9–12). Positive or negative reactions were determined in five random fields of each sample with image processing software Image-Pro Plus 6.0.
Statistical analysis
All experiments were performed in triplicate and data was reported as means ± SD. Statistical analysis was performed with SPSS 19.0 software. Statistical significance was determined by Student’s t-test or one-way analysis. Correlation between CLDN6 and GSTP1 was evaluated using Spearman’s test. P < 0.05 was considered statistically significant.