The BxPC-3 human pancreatic cancer cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Tan-IIA, sodium deoxycholate, leupeptin, Triton X-100, Tris-HCl, sodium pyruvate, HEPES, RPMI-1640, trypsin-EDTA, mouse anti-β-actin antibody (cat. no. A5441; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), penicillin-streptomycin, dimethyl sulfoxide, potassium phosphates and were obtained from Merck KGaA. Fetal bovine serum (FBS) and glutamine were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Tris-glycine-SDS buffer (10X), Tween-20 and glycine were obtained from Ameresco, Inc. (Framingham, MA, USA). BioMax film was obtained from Kodak (Rochester, NY, USA). Anti-protein kinase RNA-like endoplasmic reticulum kinase (PERK) (cat. no. 9956), anti-inositol-requiring enzyme 1α (IRE1α) (cat. no. 9956), anti-phosphorylated (p)-c-Jun N-terminal (JNK) (cat. no. 9910), anti-CCAAT-enhancer-binding protein homologous protein (CHOP) (cat. no 9956) and anti-caspase-3 (cat. no 9661) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-caspase 12 (cat. no. ab62484), anti-activating transcription factor 6 (ATF6) (cat. no. ab37149) and anti-eukaryotic initiation factor 2α (elF2α) (cat. no. ab5369) antibodies were obtained from Abcam (Cambridge, UK). Anti-B-cell lymphoma 2 (Bcl-2) antibody (cat. no. NB100-92142) was obtained from Novus Biologicals, LLC (Littleton, CO, USA).

Human pancreatic adenocarcinoma BxPC-3 cells were cultured as previously described (10,11). Briefly, BxPC-3 cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 10,000 U/ml penicillin and 10 mg/ml streptomycin, at 37°C in a humidified atmosphere containing 5% CO₂.

Cultured BxPC3 cells (2×10⁶/0.2 ml) were implanted into 4-week old, male nude severe combined immunodeficiency (SCID) mice (n=30) via subcutaneous injection over the flank area. Mice were maintained in a pathogen-free environment (Laboratory Animal Center of Tzu Chi University, Hualien, Taiwan). SCID mice implanted with BxPC-3 cells were randomly divided into 3 groups (n=10 per group) to receive 3 different weekly doses of Tan-IIA (0, 30 and 90 mg/kg). Tan-IIA was dissolved in corn oil and administered intraperitoneally on weeks 1, 3 and 5 following xenotransplantation. Volumes of the xenograft tumors were measured every other week. Tumor volume was estimated according to the following formula: Tumor volume (mm³)=LxW²/2, where L refers to tumor length and W refers to tumor width. On day 35 following xenotransplantation SCID mice were sacrificed by CO₂ inhalation, the xenograft tumors were dissected and total protein was extracted from the tumors. Subsequently, protein expression levels of PERK, ATF6, caspase-12/caspase-3, IRE1α, eIF2α, p-JNK, CHOP and Bcl-2 in the xenograft tumors were assessed using western blot analysis.

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Tzu Chi University (approval no. CCH-AE-101-010).

Total protein was extracted from xenograft tumors. Following dissection, tumors were homogenized and lysed in ice-cold whole cell lysis buffer containing protease inhibitors (BioVision, Inc., Milpitas, CA, USA). The lysates were incubated for 30 min at 4°C with agitation and were centrifuged at 12,281 × g for 10 min. Protein concentration was measured using the bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.).

Western blot analysis was conducted as previously described (10,11). Briefly, equal amounts of extracted protein samples (10 µg) were separated by 12% SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred onto polyvinylidene difluoride membranes, which were blocked for 1 h at 4°C with blocking buffer [5% dried skimmed milk in solution containing 50 mM Tris-HCl (pH 8.0), 2 mM CaCl₂, 80 mM sodium chloride, 0.05% Tween-20 and 0.02% sodium azide (Merck KGaA)]. The membranes were incubated for 2 h at room temperature with the following primary antibodies: PERK, ATF6, caspase-3, caspase-12, IRE1α, eIF2α, p-JNK, CHOP, Bcl-2 (all diluted to 1:1,000) and β-actin (diluted to 1:5,000). Subsequently, they were incubated at room temperature for 1 h with anti-rabbit (cat. no. sc-2004) or anti-mouse (cat. no. sc-2005) immunoglobulin G-horseradish peroxidase-conjugated secondary antibodies (1:5,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA). The membranes were washed 3 times for 10 min with 1X PBS with 0.05% Tween-20. The protein bands were visualized on X-ray film using an enhanced chemiluminescence detection system (PerkinElmer, Inc., Waltham, MA, USA) and quantified using ImageJ version 1.44 (National Institute of Health, Bethesda, MD, USA).

The statistical significance of the difference between groups was assessed by one-way analysis of variance followed by Dunnett's test. Data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference. The analysis was performed using IBM SPSS software version 20.0 (IBM SPSS, Armonk, NY, USA).

Mice implanted with BxPC-3-derived tumor xenografts were treated with 3 doses of Tan-IIA (0, 30 and 90 mg/kg) for 4 weeks. Tan-IIA was demonstrated to impair xenograft tumor growth in a dose-dependent manner (Fig. 1). In addition, protein expression of PERK, ATF6, caspase-3, caspase-12, IRE1α, eIF2α, p-JNK, CHOP and Bcl-2 was assessed using western blot analysis, with β-actin as an internal control. The present results revealed that Tan-IIA significantly increased the protein expression levels of PERK (Fig. 2A), ATF6 (Fig. 2B), caspase-12 (Fig. 2C), IRE1α (Fig. 2D), elF2α (Fig. 3A), p-JNK (Fig. 3B), CHOP (Fig. 3C) and caspase-3 (Fig. 4A) in a dose-dependent manner. Conversely, treatment with Tan-IIA resulted in a significant dose-dependent decrease in Bcl-2 protein expression levels (Fig. 4B).