AZD8055 was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies for mTOR, p-mTOR, Akt, p-Akt (S473), S6 and p-S6 (Ser235/236) were purchased from Cell Signaling Technology (Beverly, MA). Bcl-2, Bcl-XL and Mcl-1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Tumor TACS™ In Situ Apoptosis Detection Kit was purchased from Trevigen, Inc. (Gaithersburg, MD). mTOR shRNA was obtained from Sigma-Aldrich (St. Louis, MO). All other reagents were obtained from stated commercial sources.
Biopsies collection of pancreatic cancer patients
Patients with locally advanced pancreatic cancer were diagnosed by computed tomography (CT) and MRI imaging, and all patients received a comprehensive evaluation and were considered to be unresectable. Eight patients were treated with Intensity-modulated radiation therapy (IMRT) at 50 Gy and responses were evaluated via computed tomography. Five patients who have stable disease (SD) or progressive disease (PD) were resistant to IMRT among total 8 patients. The biopsies were taken by tru-cut needle from these five radiotherapy resistant patients. None of the subjects received other biotherapy or chemotherapy treatments. The study was approved by the ethics committees of the First Hospital of Jilin University and the Fourth Military Medical University. Written informed consents were also obtained from all subjects before study.
Cell culture and sulforhodamine B assay
Human pancreatic cancer cells PANC-1, Capan-2 and BxPC-3 purchased from National Rodent Laboratory Animal Resource (Shanghai, China) were grown as previously described. Briefly, these cell lines were cultured and maintained in exponential growth in Dulbecco’s modified Eagle’s medium (DMEM) containing 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 mM glutamine and 10% heat-inactivated FCS (Atlanta Biologicals, Lawrenceville, GA) in a humidified atmosphere of 5% CO2 at 37°C. For sulforhodamine B (SRB) assay, the exponential growing cells were seeded at 6–8 × 103/well in 96-well plates and cultured overnight. Cells were treated with radiation alone or combined with AZD8055. AZD8055 was added to cultured cells and radiation was applied 4 h later in single doses of 1, 2.5, 5 or 10 Gy. The cells were irradiated using an X-ray machine (X-RAD 320, Precision X-ray) at 320 kV, 10 mA with a 2-mm aluminum filter, and the dose rate was 2 Gy/min. Cells were then cultured at 37°C for 48 h and the surviving fractions were determined using SRB assay as previously described[33, 34]. The absorbance was measured with a spectrophotometer (Bio-Rad Inc) at 510 nm and cell growth inhibition was calculated by using the equation: cell viability (%) = (At/Ac) × 100%, in which At and Ac represent the absorbance in treated and control cultures respectively, as described previously.
Cell lysate and Western blot assay
Cells were lysed in ice-cold EBC buffer (50 mM pH 8.0 Tris, 120 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 μM sodium orthovanadate, 1 × Protease Inhibitors, 1 × Phosphatase Inhibitors) and proteins were quantified and subjected to SDS-PAGE electrophoresis, followed by protein transfer to nitrocellulose membranes. The membranes were incubated with the primary and secondary antibodies, then developed by chemiluminescence.
RNA isolation and quantitative real-time PCR
Total RNA was isolated from cells using Trizol (Invitrogen), 1–10 μg of RNA was used to synthesize cDNA with SuperScript II First-Strand Synthesis System (Invitrogen) or TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). Aliquots of the reaction mixture were used for real-time PCR with Power SYBR Green PCR Master Mix or with the TaqMan® 2 × Universal PCR Master Mix. The reaction conditions: 50°C for 20 s, 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. All real-time PCR experiments were performed in triplicate. A melting curve was obtained to verify the presence of a single amplicon. The primer sequences are as described previously[36–38].
Colony formation assay
PANC-1 cells were seeded in 6-well-plates (1000 /well), and then treated or untreated with radiation and AZD8055, alone or in combination. The medium was replaced with fresh medium containing the reagent and radiation-treatment every three days. After 10 days treatment, the medium was removed and cell colonies were stained with crystal violet (0.1% in 20% methanol). Pictures were taken using a digital camera to record the result as described. To evaluate the colony formation ability of irradiation-resistant cells, PANC-1 irradiation-resistant cell line (PANC-1-RR) was firstly generated by plating PANC-1 cells in 100-mm culture dishes and irradiating with 2 Gy X-ray every three days over a period of 5 months, for a total dose of 100 Gy, and then colony formation assay was used as above mentioned.
PANC-1 cells were suspended in DMEM supplemented with 10% FBS and seed in 6-well plates (1 × 106/well) and transfected with miR-99b precursor or inhibitor (Ambion) with Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instruction. After 48 h of transfection, cells were treated by radiation at 5 Gy, then harvested and lysed for Western blot assay. For mTOR interfering, mTOR shRNA with the sequence of CCGGGCTGTGCTAC ACTACAAACATCTCGAGATGTTTGTAGTGTAGCACAGCTTTTTG was used to transfect PANC-1 cells.
Annexin V/PI Apoptosis Detection kit (Clontech Laboratories) was used for quantification of apoptosis. Cells were seeded in 6-well plates in the absence or presence of AZD8055 (500 nM), then radiation was applied 4 h later. After cultured for 24 h, 0.5-1 × 106 cells were collected into each tube and gently washed with PBS. Cell pellets were suspended in 1 × binding buffer and stained with Annexin V and PI. After incubated for 15 min at RT in the dark, the apoptosis analysis was carried out using a FACScan (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc).
Cell cycle analysis
Cells were synchronized by growing in serum free medium for 48 h and then released into the cell cycle by adding 10% FBS to the medium. The cells were treated with radiation in the absence or presence of AZD8055 (500 nM) for 24 h, harvested, fixed with 70% ethanol, and stained with PI. Data were acquired using flow cytometry and analyzed using FlowJo software.
Pancreatic cancer xenografts and treatments
Animal experiments were careful to follow the protocols approved by Jilin University and the Fourth Military Medical University Institutional Animal Care and Use Committees. PANC-1 cells (7 × 106) were resuspended in HBSS and injected subcutaneously into the flank region of 6-week-old female athymic (nu/nu) mice (Shanghai, China). The tumors were allowed to grow to average volume of 200 mm3 prior to initiation of therapy as described. Then mice were assigned randomly to four groups (n =10) as following: (1) vehicle control (5% DMSO, 100 μl/d p.o.); (2) 8 Gy fractionated radiotherapy (2 Gy for every three days); the radiation was performed using the same X-ray machine with a different filter (1.5 mm aluminum, 0.8 mm tin, and 0.25 mm copper), at a dose rate of 1 Gy/min; (3) AZD8055 (20 mg/kg/d), AZD8055 was dissolved in DMSO and administered by oral gavage (0.1 ml/10 g of body weight); (4) Combination of AZD8055 (20 mg/kg/d) and 8 Gy (2Gy × 4) fractionated radiotherapy. Tumor volumes were measured with a caliper every other day and calculated based on the formula: V = 4/3 × π(length/2 × (width/2)2). After 21 days treatment, mice were sacrificed and the tumors were removed and submerged in 10% neutrally buffered formalin for immunohistochemistry analysis.
Four-μm thick paraffin sections were deparaffinised, rehydrated and stained using the R.T.U.Vectastain kit following the manufacturer’s standard protocol (Vector Laboratories). The sections were incubated with anti-mTOR antibody (1:50) overnight at 4°C, then stained with secondary antibody. Thereafter, the slides were exposed to DAB chromogen for 5 min, then hematoxylin counter stained, dehydrated, and treated with xylene following the approach as earlier reported. Finally all slides were examined and representative pictures were taken using an Olympus BX41 microscope.
TUNEL staining was performed by using Tumor TACS™ In Situ Apoptosis Detection Kit (Trevigen), the specimens were deparaffinised and labeled following the procedure provided by the manufacturer. Finally, DAB staining were visualized under microscopy. For TUNEL assay, ten fields were randomly selected from each slide for measurement, the images were analyzed by MetaMorph software and presented as a percentage of the total number of cells.
Levels of significance were determined by different methods, two-sided unpaired student’s t-test and one-factor ANOVA were used in the comparison between groups, and LSD-t tests was used in multiple comparisons. Results were considered statistically significant at P values < 0.05.