Materials
HMGB1 was purchased from Sigma(USA).HMGB1 was dissolved in double distilled water(DDW) to make a stock solution(3.7mg/ml) and stored at -80℃.Fetal bovine serum (FBS), DMEM/F12 were obtained from Gibco (USA). HMGB1 ELISA Kit was purchased from R&D systems (USA). Ethyl pyruvate(EP) which is HMGB1 inhibitor was purchased by Sigma(USA).CFSE was purchased from BD(USA).Tris, glycine, TEMED, SDS, acrylamide were obtained from Amresco (USA).
Cell Culture
Pancreatic cancer cell lines (Panc-1 and SW1990) was purchased from Chinese Academy of Sciences Shanghai Branch Cell Bank (Shanghai, China). Panc-1 and SW1990 cells were cultured in DMEM/F12 medium supplemented with 10% FBS,5% penicillin, and 5% streptomycin at 37℃ in a moist atmosphere with 5% CO2. And cell passage dealt with when confluency of cells reached 70-80% by 0.25% trypsin.
Residual cells isolation
We collected residual Panc-1 cells after 1*4Gy radiation dose 24h later and residual SW1990 cells after 1*10Gy radiation dose 24h later. These still living Panc-1 and SW1990 cells after radiotherapy were defined as residual pancreatic cancer cells. These residual pancreatic cancer cells were cultured in DMEM/F12 medium which is supplemented with 10% FBS and cells grown as monolayers in a humidified atmosphere at 37℃,5% CO2.Then we used these surviving cells to do some experiments at once.
Extraction of supernatant postradiotherapy
Firstly, Panc-1 and SW1990 cells were cultured about 2.5×105 per well. Secondly, Panc-1 and SW1990 cells cultivated 24h after radiotherapy which including 0Gy, 4Gy, 8Gy, 10Gy, and 12Gy. Thirdly, we collected the supernatant of Panc-1 and SW1990 cells using sterile EP tube.
Enzyme linked immunosorbent assay
The content of HMGB1 in the supernatant after radiotherapy was determined by using HMGB1 ELISA kit. All steps are performed in a sterile environment. Prepare all reagent and sample dilution in advance according to the instructions. Blank hole, standard hole and sample hole were set up respectively. The samples were supernatant of Panc-1 and SW1990 cells after radiotherapy which including 0Gy, 4Gy, 8Gy, 10Gy, and 12Gy. Add 100μl standard or sample per hole. Experiments were performed in triplicate.
CFSE staining
After Panc-1 and SW1990 cells stick to the wall, the experiment was divided into three groups (control group, HMGB1 group and HMGB1+EP group). After collect and wash by PBS, cells were incubated for 10min at 37℃ in 5mM CFSE with fetal calf serum(FCS) free RPMI. Labelling was stopped with FBS and cells were washed 3 times prior to use. For the quantification of cell proliferation, cells were analysed by flow cytometry. Experiments were performed in triplicate.
Cell migration assay
We studied the migration of Panc-1 and SW1990 cells by wound scratch assay. Cells seeded in a 6-well plate until the confluence rate up to 60%. Through all night incubation, we made a scratch at the center of each well by a 200μl pipette tip. After the cells were incubated with PBS,100ng/ml HMGB1 and 100ng/ml HMGB1+5μM EP(HMGB1 inhibitor)for 24 hours, we use pipette scratch across the confluent cell layer. Cells were washed twice in order to remove detached cells and debris. Then, the size of wounds were observed and measured after 0h, 12h, and 24h by EMS. Each experiment was manipulated in triplicate.
Western Blot Analysis
Cultured cancer cells were washed three times in phosphate-buffered saline (PBS) and lysed with RIPA buffer. Proteins were isolated by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE). And then transferred to nitrocellulose membranes and incubated with primary antibodies all night at 4℃ followed by secondary antibodies for 1h at room temperature. Blots were visualized using an ECL detection system (Amersham, Piscataway, NJ, USA). LANE-1D Analyzer (Bjsage, BJ, China) was used to quantify the intensity of protein bands. The relative intensity of β-actin, total GSK3β and Histone 4 was calculated by normalization. Histone 4was used as a loading control. Experiments were manipulated in triplicate.
Nude mice
Male nude mice (6 weeks old) were purchased from Yangzhou University in Jiangsu (China). All animal experimental protocols were approved conducted in accordance with the guidelines of the animal care committee of Yangzhou University. All male nude mice were fostered in animal experiment center of Jiangsu University where have the condition of constant temperature 25-27℃,constant humidity(45%-50%),fresh air, and without special pathogenic bacteria. We set up the pancreatic tumor model of nude mice by injecting SW1990 cells (2×106 per mouse) into the flanks of the mice subcutaneously, When the tumor reached about 5 mm in diameter, all subcutaneous tumor of nude mice were treated by radiotherapy (3*10Gy). After radiotherapy for 6 days, nine nude mice were divided into three groups and then received hypodermic injection of physiological saline, 100 ng/ml HMGB1, and 100 ng/ml HMGB1+ 5mmol/L EP around subcutaneous tumor. All tumor were measured with a ruler every 3 days. After 2 weeks treatments, all nude mice were sacrificed and then tumors were collected. Experiments were manipulated in triplicate.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc. San Diego, CA, USA). Data were presented as the mean ± standard deviation (SD). Between two groups, the statistical analysis was performed by using two-tailed unpaired t test. Among three or more group, the statistical analysis was performed by using one-way analysis of variance (ANOVA). And p<0.05 suggested a statistically significant difference.