Cell lines and reagents
Human pancreatic cancer cell line PANC-1, Capan2 cell lines were purchased from the Stem Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). PNAC-1, Capan2 cells were cultured in DMEM or 1640 medium supplemented with 10% FBS in a atmosphere of 5% CO2 at 37℃. Ibrutinib (PCI-32765, 99.2%) was purchased from Selleck, USA. Ibr-7Ibr-7 (Lot: 20161216, > 95%) were provided by Hangzhou Hezheng Pharmaceutical Co., Ltd. Ibrutinib or Ibr-7 were dissolved in DMSO (Sigma-Aldrich, St Louis, Missouri, USA) and stored at -20℃.
Antibodies and reagents
Anti-PARP (46D11, 9532), anti-cleaved caspase3 (CST, D175R, 9661S), anti-caspase3 (CST, 9662S), anti-EGFR (4267s), anti-pEGFR (Tyr1068; 3777S) were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin (J0914) was purchased from Santa Cruz biotechnology (Dallas, TX, USA). PVDF(0.45 µm,Millpore༌CAT. NO: IPVH00010); 5 × Loading༈biosharp, BL502b); Goat anti-mouse IgG, Peroxidase Conjugated, H + L (BL001A) were purchased from biosharpand Goat anti-rabbit IgG, Peroxidase Conjugated, H + L (BL003A). ECL HRP substrates for western blot purchased from CYANAGEN (WESTAR ηC2.0, lot HG13A-CC).
Cell viability assay
Cell proliferation was measured by Cell Counting Kit-8 (CCK-8, Bestbio, Shanghai, China) assay. Cells were cultured in 96-well plates with 3–5 × 103/well. Cells were treated with Ibr-7 (1.56-25 µmol/L) or Ibrutinib (1.56-50 µmol/L) for 24, 48 or 72 h. The cells were measured using CCK-8 solutions at 450 nm by A SpectraMax M2e (Molecular Devices, San Jose, CA, USA). Cell viability was calculated for each well and the 50% growth inhibition (IC50) was determined. Assays were performed on at least three independent experiments.
The pancreatic cancer cells were plated in six-well plates and pretreated with Ibr-7 or DMSO as control for 24 h. Then the cells were exposed to the indicated doses (0, 2, 4, 6 Gy) of radiation respectively. The cells were then incubated with DMEM or RPMI-1640 medium supplementing 10% FBS at 37℃ in a 5% CO2 atmosphere. After 14 days, the colonies were stained with 0.1% crystal violet (Sigma-Aldrich) in absolute methanol for 30 min. Colonies more than 50 cells were counted. The survival fraction (SF) was calculated as described previously. SF = mean number of colonies / (plating efficiency × number of cells inoculated) in treated groups, the plating efficiency= (mean number of colonies / number of cells inoculated) in untreated groups. A multi-target click mathematical model was applied to simulate the cell survival fraction. We then calculated the the sensitization enhancement ratio (SER) as the dose (2 Gy) for the radiation divided by the dose (2 Gy) for radiation plus Ibr-7 (SER = SFIR/SFIR + Ibr-7). The mean lethal dose of cells (D0), extrapolation number (N) values, the quasithreshold dose (Dq) were also calculated according to the curve. Error bars were calculated as SD via the results of three independent experiments.
Cell cycle assay
PANC-1 and Capan2 cells were divided into four groups: The control group (DMSO), the Ibr-7 (2 µM) group, the radiation (2 Gy) group, The combination group. 5 × 105 cells were plated into 6-well plates, exposed to Ibr-7 for 24 h, then the cells exposed to 6-MV X-ray. After another 24 h, the cells were trypsinized and fixed with precooled 70% ethanol at 20℃ overnight. Then the cells were added with PI (supplemented with RNase A) and incubated for 30 min at room temperature. Then the treated cells were detected by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Error bars were calculated as SD via the results of three independent experiments.
Cells were divided into four groups as mentioned above. Cells were plated into 6-well plates and pretreated with Ibr-7 for 24 h, then the cells were exposed at 2 Gy of radiation. After another 24 h, the cells were trypsinized and washed with PBS for twice. Then cell suspension was stained with 5 µL Annexin V-FITC and 5 µL PI solution and analyzed by flowcytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Error bars were calculated as SD via the results of three independent experiments.
3 × 104 cells were plated on coverslips in 24-well plates and pretreated with DMSO or Ibr-7 for 24 h. Then the cells were exposed to radiation (6 Gy). The cells were then incubated for 24 h and collected. The cells were washed in PBS for 3 times and fixed with 4% paraformaldehyde for 30 min. After permeabilizing cells with 0.5% Triton-100 for 15 min, the cells were blocked with 1% BSA, then incubated with anti-γ-H2AX antibody overnight at 4 °C. The cells were incubated with goat anti-rabbit IgG (FITC-labeled) antibody (Beyotime) for 60 min, followed by incubation with DAPI for 5 min. Finally, the cells were photographed with immunofluorescence microscopy
Western blot analysis
Cultured cells were collected and washed with PBS thrice. Total proteins extraction was conducted using RIPA lysing buffer containing phosphatase inhibitors and protease according to manufacturer instructions. A total of 20–40 µg of proteins were submitted to 10% SDS/PAGE and transferred to PVDF membrane. The membranes were then blocked with 5% skim milk for 1 h in room temperature. The membranes were incubated with primary antibodies against EGFR, p-EGFR and β-actin respectively overnight at 4 °C, and then incubated with secondary antibodies for 1 h at room temperature. The membranes were washed with TBST 3 times, the proteins were visualized by adding ECL.
The pcDNA3.1-EGFR plasmid and empty vector were purchased from GenePharma. The plasmid was transfected into PANC-1 and Capan2 cells by jetPRIME according to the manufacturer’s protocol.
The results came from at least three independent experiments. All quantitative values are given as mean ± SD (standard deviation). Student’s t-test was used to determine the significance and statistically significant was defined when p < 0.05. Graphs were performed using GraphPad Software.