The pancreatic cancer cell lines BxPC-3, HPAF-II, and Panc 10.05 were obtained from the American Type Culture Collection (Manassas, VA, USA). BxPC-3 and Panc 10.05 cells are epithelial cell lines that were derived from pancreatic adenocarcinomas. The HPAF-II cell line consists of epithelial cells derived from ascites that originated from pancreatic adenocarcinomas.
BxPC-3 cells were grown in RPMI-1640 (Life Technologies, Carlsbad, CA, USA) with 10 % (v/v) fetal calf serum. HPAF-II cells were grown in Eagle’s medium (Life Technologies) with 10 % (v/v) fetal calf serum. Panc 10.05 cells were grown in RPMI-1640 (Life Technologies) containing 10 units/ml of human recombinant insulin, and 15 % (v/v) fetal calf serum. All media were supplemented with 50 μg/ml gentamicin. All cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO2.
The oral iron chelator DFX was obtained from Novartis (Basel, Switzerland). For in vitro studies, DFX was dissolved in dimethyl sulfoxide at a stock concentration of 100 mM and was used at the concentrations indicated in the results and figures by dilution in culture media containing 10 % fetal calf serum. For in vivo studies, DFX was dissolved in sodium chloride solution (0.9 % w/v; Chemix Inc., Shinyokohama Kohoku-ku, Yokohama, Japan).
Cellular proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cell suspensions (2,000 cells/100 μl) were added to each well in a 96-multiwell culture plate (BD Bioscience, San Jose, CA, USA) and incubated at 37 °C for 24 h. The indicated concentrations of DFX were then added to each well, and the cells were incubated for a further 72 h. At the end of the culture period, 10 μl of MTS solution (Promega, Madison, WI, USA) was added to each 100 μl of culture media and incubated for 2 h. Absorbance at 490 nm was measured with a multimode reader (Infinite 200 PRO, Tecan Trading, AG, Switzerland), and the results are expressed as the percentage viable with respect to the untreated control.
Cell cycle analysis
Each pancreatic cancer cell line was seeded into 100-mm dishes and cultured with phosphate-buffered saline (PBS) as a vehicle control or DFX at 10, 50, or 100 μM for 72 h. After incubation, the cells were fixed with 70 % ethanol and stored overnight at −20 °C. The cells were washed and then stained with a solution containing 0.1 % Triton® X-100 (Promega), 0.02 mg/ml propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA), and 0.2 mg/ml RNase A (Qiagen, Hilden, Germany) in the dark at 37 °C for 15 min. After staining, the cells were subjected to cellular DNA content examination by a flow cytometer (Gallios, Beckman Coulter, Fullerton, CA, USA). The data were analyzed by Multicycle for Windows software (Beckman Coulter).
Apoptosis analysis by flow cytometry
For the apoptosis analysis, the cells were cultured as described above. After harvesting, apoptosis was evaluated with an apoptosis detection kit (Annexin V Apoptosis Detection Kit APC, eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. After staining, the cells were examined using a flow cytometer (Gallios, Beckman Coulter). The data were analyzed by FlowJo software (Tree star, Ashland, OR, USA).
Apoptosis analysis with the luminescence assay
Cell suspensions (2,000 cells/100 μl) were added to each well of a 96-multiwell culture plate (BD Bioscience) and were incubated at 37 °C for 24 h. PBS as a vehicle control or the indicated concentrations of DFX were then added to each well, and the cells were further incubated for 48 h. Immediately after the incubation, caspase activity was measured using the caspase 3/7 assay kit (Caspase-Glo 3/7 kit, Promega) according to the manufacturer’s instructions.
Tumor xenografts in nude mice and deferasirox administration
Animal care was performed in accordance with the animal ethics requirements at Yamaguchi University School of Medicine, and the experimental protocol was approved (approval ID 21-035). Twenty female BALB/c (nu/nu) mice were purchased from Nippon SLC (Shizuoka, Japan) and were housed in sterile conditions. Experiments commenced when the mice were 8–10 weeks of age. Tumor cells (BxPC-3) in culture were harvested and resuspended in a 1:1 ratio of RPMI-1640 and Matrigel (BD Bioscience). Viable cells (5 × 106 cells) were injected subcutaneously into the backs of the mice. After engraftment, tumor size was measured using Vernier calipers every 2 days, and tumor volume was calculated as follows: tumor volume (mm3) = (the longest diameter) (mm) × (the shortest diameter) (mm)/2. When tumor volumes reached 150 mm3, oral treatment began (day 0). Each group of mice (n = 5) received DFX suspended in saline, which was administered by oral gavage every second day, with three treatments per week, over 21 days at concentrations of 120, 160, or 200 mg/kg. The control mice were treated with the vehicle alone. At the end of the experiment, the mice were sacrificed, and the tumors were excised and processed for immunohistochemistry and genetic analyses. A total of 20 blood samples were collected simultaneously during tumor removal. Serum levels of ferritin were measured using the enzyme-linked immunoassay method (Mouse Ferritin ELISA kit, Kamiya Biochemical Company, Seattle, WA, USA). Serum biochemistry with the exception of ferritin was analyzed by YAMAGUCHI Laboratory Co., Ltd. (Ube, Japan).
The removed tumors were fixed in 4 % paraformaldehyde (Muto-kagaku, Tokyo, Japan), sectioned, and embedded in paraffin. Immunohistochemistry was performed as previously described on the paraffin sections with antibody specific to ferritin-H (Anti-Ferritin Heavy Chain antibody, AbCam, Cambridge, MA, USA) . The slides were scored according to the intensity of the immunoreactivity and the percentage of epithelial cells stained .
The detection of gene expression alternation in resected tumors induced by deferasirox administration
Total RNA isolation
A total of six tumors were genetically analyzed. Of these, three tumors were removed from vehicle-treated mice, and the other three tumors were removed from DFX 200 mg/kg-treated mice. According to the manufacturer’s instructions, total RNA was isolated from the removal tumors using TRIzol Reagent (Invitrogen Corp., CA, USA) and purified using the SV Total RNA Isolation System (Promega). RNA samples were quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA), and RNA quality was checked using an Experion automated electrophoresis station (Bio-Rad Laboratories Inc., Hercules, CA, USA).
Gene expression microarrays
The cRNA was amplified, labeled, and hybridized to a 60K Agilent 60-mer oligomicroarray according to the manufacturer’s instructions. All hybridized microarray slides were scanned by an Agilent scanner. Relative hybridization intensities and background hybridization values were calculated using Agilent Feature Extraction Software (126.96.36.199).
Data analysis and filter criteria
The raw signal intensities of all samples were log2-transformed and normalized with a quantile algorithm from the ‘preprocessCore’ library package  on Bioconductor software . We selected the probes, excluding the control probes, where the detection p-values of all samples were less than 0.05, and used them to identify differentially expressed genes. To determine significant enrichment canonical pathways, we used the tools and data provide by the Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, INC. http://www.ingenuity.com). The results are the comparisons of tumors removed from vehicle-treated mice vs. the tumors removed from DFX 200 mg/kg-treated mice.
All obtained data are calculated and expressed as the mean ± SD. In the in vitro experiments, the differences were analyzed statistically using 1-way ANOVA, followed by Dannett’s test. In the in vivo experiments, the differences were analyzed statistically using the Kruskal-Wallis H test, followed by Steel’s test. JMP 9 statistical software (SAS Institute Inc., Cary, NC, USA) was used in the analysis. Values of p <0.05 were considered significant.