Preparation of AbE
A. blazei powder (A. blazei mycelia-dikaryon, strain my26) was provided by JMCU Center Corporation . The powder (2.5 g) was boiled in 45 mL water for 10 min. The supernatant was recovered by centrifugation at 3300×g for 3 min and sterilized by filtration through a 0.22-μm membrane. The solution obtained (AbE) was used for the following experiments.
Three human pancreatic cancer cell lines, MIA PaCa-2, PCI-35, and PK-8, and the immortalized human pancreatic duct epithelial cell line, HPDE, were used in this study. MIA PaCa-2 was purchased from American Type Culture Collection (Manassas, VA, USA). PCI-35 was obtained from Dr. Hiroshi Ishikura at Department of Pathology, Hokkaido University Graduate School of Medicine, Sapporo, Japan . PK-8 was obtained from Dr. Masao Kobari at the Department of Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan . HPDE was obtained from Dr. Ming-Sound Tsao, Department of Pathology, Princess Margaret Hospital and Toronto University, Toronto, Canada . PCI-35 and PK-8 were cultured in RPMI 1640 (Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France). MIA PaCa-2 was cultured in DMEM (Sigma-Aldrich Corp.) supplemented with 10% fetal bovine serum (Biowest). HPDE was maintained in Medium 154 (Cascade Biologics, Portland, OR, USA) containing 1% human keratinocyte growth supplement (Cascade Biologics). All cells were maintained at 37 °C in a humid atmosphere with 5% CO2.
Cell proliferation assay
For this assay, 5000 cells/well were plated in 100 μL of the appropriate culture medium in 32 wells of a 96-well plate; in total, 5 plates were used. The cells were incubated for 24 h and then the culture medium of 8 wells was replaced with medium supplemented with AbE at final concentrations of 0.005, 0.015%, or 0.045% (w/v). For the control (MOCK), an equal volume of water was added to the medium of the remaining 8 wells. On each assay day, the medium from each of the 32 wells in one plate was replaced with 100 μL of 0.05% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)/PBS(−), and the plate was then incubated for 1 h at 37 °C in an atmosphere containing 5% CO2. After incubation, the MTT solution was removed by aspiration, the cells were resuspended in 100% ethanol, and the absorption of the ethanol solution at 590 nm was measured. The process was repeated for each plate on consecutive days. The 50% growth inhibitory concentration (GI50) values were calculated from the percentage of surviving cells after AbE treatments of 0, 0.00057, 0.0017, 0.005, 0.0085, 0.015, 0.026, and 0.045% by a four-parameter logarithmic method using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA).
The cells were plated in the appropriate culture medium at a density of 2.5 × 105 cells/plate in 10-cm dishes. After incubation for 24 h, the cells were treated with 0.005, 0.015%, or 0.045% (w/v) AbE for 48 h. For the control (MOCK), an equal volume of water as AbE supplement was added to the medium. The cells were washed with PBS(−) and fixed overnight with 70% ethanol at − 20 °C. The fixed cells were resuspended in 100 μL hypotonic citrate buffer (192 mmol/L Na2HPO4, 4 mmol/L citric acid) and incubated for 20 min at room temperature. The cells were then pelleted and resuspended in PI/RNase/PBS (100 μg/mL propidium iodide and 10 μg/mL RNase A in PBS). DNA content was analyzed using the FACSCalibur™ System (BD Biosciences, San Diego, CA, USA).
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay
The cells were plated using the appropriate culture medium at a density of 2.5 × 105 cells/plate in 10 cm dishes. After incubation for 24 h, the cells were treated with 0.045% (w/v) AbE. For the control, an equal volume of water was added to the medium of the MOCK cells. After incubation for 48 h, the floating and adherent cells were collected and fixed with 4% paraformaldehyde/PBS overnight at 4 °C. The cells were washed with PBS and then spread on MAS-coated glass slides (Matsunami Glass Industry Ltd., Tokyo, Japan). The TUNEL assay was performed to identify DNA fragmentation in situ by using a FragEL™ DNA fragmentation Detection Kit, Colorimetric-TdT Enzyme (Calbiochem, San Diego, CA, USA) in accordance with the manufacturer’s protocol. Approximately 500 cells from randomly selected fields were observed at 400× magnification, from which the number of TUNEL-positive cells was counted.
Total RNA was extracted from cells treated with 0.045% (w/v) AbE or water for 48 h by using the RNeasy Midi Kit (QIAGEN, Hilden, Germany) in accordance with the supplier’s instructions. The extraction was repeated independently in triplicate. cRNAs labeled with Cyanine 3 were synthesized via the T7-linear amplification method from 500 ng of total RNA using the Low RNA Input Linear Amplification Kit (Agilent Technologies, Palo Alto, CA, USA), and subsequently purified by using the RNeasy Mini Kit (QIAGEN). For each hybridization, 1.65 μg of cRNAs was fragmented at 60 °C for 30 min using the Gene Expression Hybridization Kit (Agilent Technologies) and hybridized at 65 °C for 17 h to a 4 × 44 K Whole Human Genome Microarray slide (Agilent Technologies) in accordance with the manufacturer’s instructions. The microarrays were scanned and the signals in the scanned image were converted to intensity values that were subsequently normalized by software provided by Hokkaido System Science Co., Ltd. (Sapporo, Japan). The expression levels were normalized to the 75th percentile, and baseline transformations were conducted by using the median of the control samples from each cell line. As a means of quality control, the correlation coefficients were analyzed; one sample of PK-8 cells treated with AbE was excluded because its correlation coefficient with the other replicates was below 0.01. Lists of genes with greater than four-fold change in expression were identified by t-test (P < 0.05) using R (https://www.r-project.org/). Gene Ontological category lists containing genes with statistically significant expression levels were obtained using Fisher’s exact tests, using Benjamini-Yekutieli corrections (P < 0.05). The whole microarray data set was deposited to Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE89396. Over-representation analyses were performed using online the programs of PANTHER (http://www.pantherdb.org)  and REACTOME (https://www.reactome.org) .
Pancreatic cancer cells treated with 0.045% (w/v) AbE or an equal volume of water for 48 h were harvested by mild scraping, centrifuged, and washed with ice-cold PBS(−) containing Complete Mini protease inhibitors (Roche, Basel, Switzerland). The cells were then suspended in an appropriate volume of RIPA buffer (Sigma-Aldrich) plus protease inhibitors (Roche) and lysed by sonication. The lysates (120 mg) were denatured, separated on SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA), and then electroblotted onto Clear Blot membrane-p (ATTO, Tokyo, Japan). An SDS gradient gel of 5–20% polyacrylamide was used for poly (ADP-ribose) polymerase 1 (PARP1) and 10–20% polyacrylamide was used for caspase-3, caspase-9, and β-actin. After non-specific binding to the membrane was blocked by incubation with TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween® 20) containing 5% (w/v) Amersham ECL Blocking Agent (GE Healthcare UK Ltd., Buckinghamshire, UK), the membranes were incubated overnight at 4 °C with Can Get Signal® Solution 1 (TOYOBO, Osaka, Japan) containing the following antibodies, separately: mouse monoclonal anti-PARP1 (clone 42/PARP, dilution 1:125, BD Biosciences, San Jose, CA, USA), mouse monoclonal anti-caspase-3 (clone 3G2, dilution 1:1000, Cell Signaling Technology Inc., Beverly, MA, USA), rabbit polyclonal anti-caspase-9 (dilution 1:1000, Cell Signaling Technology Inc.), or mouse monoclonal anti-β-actin (clone AC-15, dilution 1:1000, Sigma-Aldrich). Then, the membranes were incubated with Can Get Signal® Solution 2 (TOYOBO) containing an appropriate horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit immunoglobulin secondary antibody (dilution 1:10000, GE Healthcare UK Ltd.) at room temperature for 1 h. The bound antibodies were detected by the application of enhanced chemiluminescence (ECL) solution (Amersham ECL Plus™ Western Blotting Detection Reagents, GE Healthcare UK Ltd.) and digitally processed using an LAS-4000 mini image analyzer (Fuji Photo Film Co. Ltd., Minamiashigara, Japan).
Quantitative reverse transcription PCR
Single-stranded cDNA was synthesized from the total RNA used for the microarray analysis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s instructions. Quantitative PCR was performed using the 7500 Real-Time PCR System (Applied Biosystems) with the synthesized cDNAs and Pre-Developed TaqMan® Assay Reagents (Applied Biosystems) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 4326317E) or TaqMan® Gene Expression Assays (Applied Biosystems) for cyclin D1 (CCND1; Hs00277039_m1), cyclin-dependent kinase inhibitor 3 (CDKN3; Hs00193192_m1), cyclin B2 (CCNB2; Hs00270424_m1), cyclin A2 (CCNA2; Hs00996789_g1), cyclin F (CCNF; Hs00171049_m1), CHK2 checkpoint homolog (S. pombe) (CHEK2; Hs00200485_m1), ataxia telangiectasia mutated (ATM; Hs00175892_m1), cell division cycle 25 homolog A (S. pombe) (CDC25A; Hs00153168_m1), breast cancer 1, early onset (BRCA1; Hs00173237_m1), high mobility group (HMG)-box transcription factor 1 (HBP1; Hs00202110_m1), forkhead box O4 (FOXO4; Hs00172973_m1), vasohibin 1 (VASH1; Hs00208609_m1), BRCA1 associated RING domain 1 (BRAD1; Hs00184427_m1), cyclin-dependent kinase 6 (CDK6; Hs00608037_m1), NLR family pyrin domain containing 1 (NLRP1; Hs00248187_m1), death effector domain containing 2 (DEDD2; Hs00370206_m1), or death-associated protein kinase 3 (DAPK3; Hs00154676_m1). The PCR cycling program consisted of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All samples were analyzed in triplicate and gene expression was calculated relative to the reference housekeeping gene, GAPDH.