Human ovarian cancer cell lines, SKOV3 and A2780, as well as their cisplatin resistant cohorts, SKOV3/DDP and A2780/DDP, were maintained in RPMI1640 (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Sigma), 100 IU/ml of penicillin, and 100 µg/ml of streptomycin (Sigma). The cells were maintained in a humidified atmosphere at 37°C with 5% CO2.
24 patients with 43 to 69 years old (58 on the 9 average) from June 2014 to July 2017 who underwent surgical resection at Shengjing Hospital of China Medical University were recruited in this study. The collected tissues were snap frozen in liquid nitrogen and stored in −80 °C freezer for further analysis. The patients were divided into two groups, platinum-sensitive (sixteen patients) and platinum resistant (eight patients) groups according whether or not recurrence 6 months after first cisplatin-based chemotherapy. None of the patients had received chemotherapy or radiotherapy prior to the operation. The project was approved by Institutional Review Board of China Medical University that informed consent was not needed to obtain from the patients or their family.
Tissue microarray and immunohistochemical staining
Tissue microarray sections (Shanghai Outdo Biotech Co., LTD) were used for immunohistochemical staining using an antibody against BAG5, Rictor or Bcl6. A semi-quantitative H-score was used for each specimen as previously reported. Briefly, immunohistochemical intensity was assessed by multiplying the distribution areas (0-100%) at each staining intensity level by the intensities (0: negative; 1: weak staining; 2: moderate staining; 3: strong staining).
Colony formation assay
For the plate colony formation assay, 200 cells/well were seeded into six-well plates and cultured for 2 weeks. The cells were fixed with 4% paraformaldehyde for 15 min, then stained with 0.1% crystal violet. The colony number was determined under an optical microscopy.
Cells were suspended in serum-free medium, seeded at 1 × 104 cells per well in the upper chamber with 100 μL. 600 μL of RPMI1640 containing 10% FBS was added into the bottom chamber. After incubation for 24 hours, the cells remaining in the upper chamber were removed. The cells invaded through the Matrigel matrix membrane were fixed with 4% paraformaldehyde for 15 min, then stained with 0.1% crystal violet.
Cell viability assay
Cells were seeded at 1.0 × 104 per well into 96-well plates. After adhesion, cells were treated with the indicated concentrations of cisplatin. The CCK-8 cell proliferation kit was used to measure cell viability after 2 days. For each test, 10 μL of the CCK-8 reagent was added to the cells, which were incubated at 37°C for 3 h. Absorbance at 450 nm was then measured using a microplate reader.
Measurement of glucose consumption
Cells were plated in 24-well plates at the density of 3 x 104 cells per well in 0.5 ml RPMI1640. A blank well without cells was included as the control. After 24 h of incubation, glucose in the culture medium was analyzed using a Glucose Assay Kit (Biovison, Milpitas, CA). The levels of glucose consumption were determined by subtracting the concentration of glucose. The numbers of cells in each well were counted and used to normalize the data.
Determination of the extracellular lactate
Cells were seeded into 24-well plates. Cells were washed with PBS, and then incubated with a KRPH buffer supplemented with 0.2% BSA and 10 mM glucose for 2 h. Next, supernatants were analyzed for their lactate content via an enzyme-coupled fluorescent assay using a L-Lactate Assay Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. In the assay, lactate dehydrogenase catalyzes the oxidation of lactate to pyruvate, along with the concomitant reduction of NAD+ to NADH. NADH then reacts with the fluorescent substrate to generate a highly fluorescent product, which is analyzed with an excitation wavelength of 530 nm and an emission wavelength of 585 nm. Cell numbers were accounted to normalize extracellular lactate production.
Oxygen consumption rate (OCR) and Extracellular acidification rate (ECAR)
15,000 cells were seeded into each well of a 24-well assay plate (Seahorse Bioscience) and grown overnight. OCR and ECAR was performed using the Extracellular Flux Analyzer XF24 from Seahorse Bioscience according to the manufacturer’s protocol.
Label and capture nascent RNA
Newly synthesized RNA was labeled and isolated using Click-iT Nascent RNA Capture kit (Invitrogen). In brief, cells were incubated with 0.2 mM of 5-ethymyl uridine for 4 h to label nascent RNA. Total RNA was isolated using TRIzol reagent, and the 5-ethymyluridine–labeled nascent RNA was biotinylated in Click-iT reaction buffer with 0.5 mM of biotin azide. The biotinylated nascent RNA was subsequently captured on streptavidin magnetic beads.
Western blot and immunoprecipitation
Total cellular proteins were extracted using a lysis buffer containing 20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100 and a protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO). Extracted proteins were quantified using the BCA protein assay kit. Next, 20 μg total proteins were separated using 10% SDS-PAGE, and transferred to a PVDF membrane (Millipore Corporation, Billerica, MA). For immunoprecipitation, cell lysates were pre-cleared with protein A/G magnetic beads, which were treated with various antibodies, then incubated overnight at 4°C. The immunoprecipitants were washed three times with a lysis buffer, and analyzed using Western blot analysis, which was performed with primary antibodies against Flag, and Myc antibodies. The following antibodies were used in this study: BAG5(Sigma); mTOR Pathway Antibody Sampler Kit, Rictor, BcL6, Flag (Cell Signaling Technology), and GAPDH (Millipore).
Nude mouse xenograft experiments
BALB/c-nu/nu mice (4–5 weeks old, female) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. All animal procedures were approved by and compiled with the guidelines of the Institutional Animal Care Committee of China Medical University. Mice were subcutaneously inoculated with the specified number of viable SKOV3/DDP cells. The status of the mice and tumor formation were observed over time, and the mice were sacrificed after 28 days. The subcutaneous tumors were removed and photographed.
The statistical significance of the difference was analyzed by ANOVA and post hoc Dunnett’s test. Statistical significance was defined as P<0.05. All experiments were repeated three times, and data were expressed as the mean ± SD (standard deviation) from a representative experiment.