Cell culture reagents used in this investigation were purchased from Invitrogen; Thermo Fisher Scientific, Inc. All other chemicals used were purchased from Sigma-Aldrich; Merck KGaA unless otherwise stated. Primary antibodies [Bax (product no. 2774), Bcl-2 (product no. 2872), cleaved caspase-3 (product no. 9664), cleaved poly(ADP-ribose) polymerase (PARP) (cat. no. 5625S), GAPDH (product no. 5174S), phosphorylated-signal transducer and activator of transcription 3 (p-STAT3) (product no. 9145S), STAT-3 (product no. 9139S), matrix metalloproteinase-2 (MMP2) (product no. 13132S), Snail (product no. 3879S), vimentin (product no. 5741S), p-AMP-activated protein kinase (p-AMPKα) (product no. 2535S), AMPKα (product no. 2532S), Yes-associated protein (YAP) (product no. 4912S), cysteine-rich angiogenic inducer 61 (Cyr61) (product no. 14479S), connective tissue growth factor (CTGF) (product no. 10095S), p-ataxia telangiectasia mutated kinase (p-ATM) (product no. 13050S), and p-checkpoint kinase 2 (p-Chk2) (product no. 2661S) were purchased from Cell Signaling Technology. The primary antibody MMP9 was purchased from EMD Millipore (product no. AB19016). Secondary antibodies [goat anti-rabbit (cat. no. PI-1000-1) and horse anti-mouse immunoglobin (Ig)G (cat. no. PI-2000-1)] were purchased from Vector Laboratories, Inc. The BS ECL Plus kit (cat. no. W6002) was purchased from Biosesang, Inc.

Four human pancreatic cancer cell lines, Aspc-1, MiaPaCa2, Panc-1, and SNU-213, were obtained from the laboratory of Professor Jae-Hoon Kim at the Faculty of Biotechnology, Jeju National University, Republic of Korea. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and an antibiotic cocktail (100 µg/ml streptomycin and 100 U/ml penicillin) and incubated at 37°C under 5% CO₂. Dermal fibroblasts (13) were cultured in DMEM under similar culture conditions. Cells were sub-cultured upon 80% confluency.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay was used as the cell viability assay in the present investigation. Prior to the MTT assay, four types of pancreatic cancer cells (Aspc-1, MiaPaCa2, Panc-1 and SNU-213) were cultured in 96-well culture plates (5×10³ cells/well) and incubated for 48 h at 37°C. Following incubation, the cells were exposed to different doses (12.5–200 µM) of catechol and incubated again for 48 h at 37°C. In chemosensitivity evaluation experiments of catechol, Panc-1 cells were co-treated with catechol (50 or 100 µM) and gemcitabine (0.25–1 µM) for 48 h. The MTT assay was then conducted to assess cell viability. Dimethyl sulfoxide (DMSO) was used to dissolve purple formazan and the absorbance was recorded at 540 nm using a micro-plate reader (Sunrise; Tecan Group, Ltd.). The percentage of cell viability was determined using the formula: [(A_T-A_B)/(A_C-A_B)] ×100, where A_T denotes the absorbance value of the treated group, A_B denotes the absorbance value of the blank, and A_C denotes the absorbance value of the untreated control. GraphPad Prism 7.00 (GraphPad Software, Inc.) was used to determine the half maximum inhibitory concentration (IC₅₀) of catechol in each pancreatic cancer cell line.

Prior to the colony formation assay, Panc-1 cells (200 cells/dish) were seeded in cell culture dishes and exposed to catechol alone, or to a combination of catechol (50 µM) and ionizing radiation (2 or 4 Gy). After 10 days, the cells were fixed with 4% formaldehyde at room temperature for 30 min, stained with crystal violet (0.5% crystal violet), incubated at room temperature for 30 min and washed. NIST's Integrated Colony Enumerator software (https://www.nist.gov/services-resources/software/nists-integrated-colony-enumerator-nice) was used to count the number of colonies in the culture dishes. Irradiation was carried out at the Applied Radiological Science Institute at Jeju National University using a ⁶⁰CO Theratron-780 teletherapy unit.

Panc-1 cells (1×10⁵/well) were cultured in 6-well plates and incubated until the cells reached confluence. A uniform scratch was then made through the cell monolayer using a sterilized 200-µl pipette tip. After washing with phosphate-buffered saline (PBS), different doses (25 or 50 µM) of catechol were added to the wells with DMEM containing 5% FBS (14), and the cells were incubated for 24 h. Following incubation, the width of the wound was measured under a phase-contrast microscope (×100).

Prior to the invasion assay, cells were exposed to different concentrations (12.5, 25, or 50 µM) of catechol and incubated for 24 h. For this assay, a Transwell system (24-well plate; 0.2 µm pore; Corning, Inc.) was used. The upper chambers were coated with 1% Matrigel by incubating at 37°C for 1 h. The upper chambers received 200 µl of DMEM and 1×10⁵ cells (without FBS) supplemented with or without catechol (12.5, 25 and 50 µM), while the lower chambers received DMEM supplemented with 10% FBS. Following 24 h of incubation at 37°C, invading Panc-1 cells were first fixed with methanol and then 4% paraformaldehyde at room temperature for 30 min, stained with 2% crystal violet for 30 min at room temperature, washed with distilled water and observed under a phase contrast microscope (×100).

Panc-1 cells (5×10⁴/well) were cultured on coverslips and incubated for 24 h. Following incubation cells were exposed to catechol for 48 h. Cells were then fixed with 4% formaldehyde for 30 min at room temperature and stained with Hoechst 33342 solution (0.01 mg/ml) for 30 min at room temperature. Stained Panc-1 cells were observed and images were captured under a fluorescence microscope (×100) (IX73; Olympus Corporation).

The effects of catechol on the cell cycle and apoptosis were investigated using flow cytometry at the Bio-Health Materials Core-Facility at Jeju National University. Flow cytometric analysis was performed with BD FACSDiva™ Software (BD Biosciences). Prior to the analysis, the cells (3×10⁴ cells) were washed twice with PBS and fixed in 70% ethanol for 30 min at 37°C. Following fixation, the cells were treated with RNase A (25 ng/ml) and staining with propidium iodide (40 µg/ml) and subjected to cell cycle analysis. The Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) was used according to the manufacturer's instructions for apoptosis analysis.

Following treatments, radioimmunoprecipitation assay buffer (RIPA; Thermo Fisher Scientific, Inc.) was used to extract cell lysates. The protein concentrations were determined using a BCA assay. Upon quantification of proteins, 40 µg of proteins were electrophoresed (10-12.5% gels) and transferred to PVDF membranes. After blocking with 5% dried non-fat milk overnight at 4°C, membranes were exposed to different primary antibodies overnight at 4°C. Most of the primary antibodies and the horseradish peroxidase-conjugated (HRP) goat anti-rabbit IgG secondary antibodies were diluted prior to the experiments (primary antibodies to 1:1,000 and secondary antibodies to 1:5,000). GAPDH or β-actin (reference protein) was diluted to 1:10,000 prior to the experiments. Protein bands were detected using the BS ECL-Plus kit (Biosesang, Inc.), and the ImageJ software (version 1.48; National Institutes of Health) was used to quantify bands.

Statistical analysis. Group comparisons were performed using the paired Student's t-test and one-way analysis of variance (with Duncan's post hoc test) with SPSS software (ver. 18.0; IBM Corp.). P-values <0.05 were considered to indicate a statistically significant difference.

The MTT assay was used to evaluate the effects of catechol on pancreatic cancer cell proliferation. As revealed in Fig. 1A, catechol inhibited the proliferation of pancreatic cancer cell lines (Aspc-1, MiaPaCa-2, Panc-1 and SNU-213) in a dose-dependent manner at 48 h post-incubation. Catechol had the greatest anti-proliferative effect (IC₅₀: 91.71±5.14 µM) on Panc-1 cells among the four pancreatic cancer cell lines tested. As catechol exerted the strongest anti-proliferative effect on Panc-1 cells, this cell line was selected for other experiments in the present study. Moreover, it has been demonstrated that Panc-1 cells are resistant to gemcitabine, a commonly used chemotherapeutic drug in pancreatic cancer treatments, compared to Aspc-1 cells (15). Catechol displayed an IC₅₀ value of 162.6±15.6 µM in dermal fibroblasts following 48 h of exposure (Fig. S1). It was then assessed whether the anti-proliferative effects of catechol were associated with Panc-1 cell cycle arrest. As revealed in Fig. 1B, catechol treatment caused the sub-G1 population to increase, from 4.27±0.31% (0 µM) to 16.33±1.13% (200 µM). Moreover, as revealed in Fig. 1C, morphological changes associated with apoptosis such as condensed and fragmented chromatin and apoptotic bodies (arrows) were evident in catechol-treated (50, 100 and 200 µM) Panc-1 cells. Next, we assessed the effects of catechol on the expression of apoptosis-associated proteins (cleaved PARP, caspase-3, and cleaved caspase-3) and STAT3 via western blot experiments. The data revealed that catechol increased cleaved PARP and cleaved caspase-3 protein levels, confirming the apoptosis-inducing effects of catechol on Panc-1 cells (Fig. 1D). Catechol also decreased the phosphorylation of STAT3 in a dose-dependent manner (Fig. 1D). The transcription factor STAT3 has been reported to play a key role in tumorigenesis (11). This observation is also consistent with findings from a previous study, which reported that catechol inhibited STAT3 activation in breast cancer stem cells (11). Collectively, the results demonstrated that catechol inhibited the proliferation of Panc-1 cells and promoted apoptosis through the inactivation of STAT3, one of the known targets of apoptosis (16).

Next, two marginally cytotoxic doses (12.5 and 50 µM) of catechol were selected for migration and invasion assays to examine whether catechol could suppress EMT in Panc-1 cells. The results of the migration and invasion assays revealed that catechol reduced cell migration (Fig. 2A) and invasion (Fig. 2B) in Panc-1 cells at 24 h post-incubation. Furthermore, catechol treatment caused an increase in the expression levels of E-cadherin protein along with a gradual decrease in the expression of two EMT markers, Snail and vimentin, in Panc-1 cells (Fig. 2C). MMP2 expression was also decreased following catechol exposure in Panc-1 cells (Fig. 2C). The expression of MMP9 was slightly affected by catechol exposure (Fig. 2C). These data indicated that catechol suppressed Panc-1 cell migration and invasion by reducing the expression of EMT-related proteins.

The Hippo signaling pathway has been reported to drive tumorigenesis and play a role in developing chemo- and radio-resistance in a range of human cancers (17). YAP is a transcription co-activator in the Hippo signaling pathway (17), and both CYR61 and CTGF are major downstream targets of YAP (18). In addition, activated AMPK has been reported to be associated with YAP inhibition (19). Therefore, the regulatory effects of catechol on the AMPK/Hippo signaling pathway in Panc-1 cells were investigated. It was observed that catechol induced the phosphorylation of AMPK in a dose-dependent manner with a concomitant decrease in YAP, CYR61, and CTGF protein levels (Fig. 3), indicating that catechol could effectively target oncogenic Hippo signaling in Panc-1 cells.

To assess whether catechol enhances the in vitro cytotoxic efficacy of gemcitabine. Cell viability was then analyzed using an MTT assay. Combining catechol and gemcitabine resulted in the reduction of Panc-1 cell viability compared to the gemcitabine treatment alone (Fig. 4A). To investigate whether catechol and gemcitabine had synergistic cytotoxic effects in Panc-1 cells, combination index (CI) values were calculated. The CI values for drug combinations in Panc-1 cells ranged from 0.43 to 0.68. Combined treatment comprising 50 µM of catechol and 0.25 µM of gemcitabine exerted the highest synergistic inhibitory effects with a CI value of 0.43 (Fig. 4B). In addition, as revealed in Fig. 4C, condensed and fragmented chromatin and apoptotic bodies (arrows) were prominent in the combined-treatment groups. As the combined treatment (50 µM of catechol and 0.25 µM of gemcitabine) demonstrated the highest synergistic inhibitory effects, this combination was used in the following experiments to explore the mechanisms by which catechol synergizes with gemcitabine in Panc-1 cells. Cell cycle analysis revealed that sub-G1 populations of Panc-1 cells exposed to catechol and gemcitabine increased in a dose-dependent manner (Fig. 4D). The largest sub-G1 population (10.05±4.40%) of Panc-1 cells was observed in the treatment combining 50 µM of catechol and 0.25 µM of gemcitabine (Fig. 4D). Furthermore, the combined treatment increased p-AMPK expression, cleavage of PARP, and the Bax/Bcl-2 ratio, as well as decreased YAP, CYR61, and p-STAT3 levels (Fig. 4E). Based on these observations, it was concluded that 50 µM of catechol can enhance the in vitro cytotoxic efficacy of gemcitabine and promote gemcitabine-induced apoptosis in Panc-1 cells.

The response of Panc-1 cells to radiation treatment in the presence or absence of catechol was examined by performing a colony formation assay. Results of the colony formation assay revealed that Panc-1 cell survival rates markedly decreased after exposure to catechol and radiation treatments (Fig. 5A). The linear-quadratic model was applied to generate radiosensitivity curves for each experimental group to represent the results of the colony formation assay (Fig. 5B). Catechol-treated cells were revealed to be more sensitive to radiation compared to control cells (Fig. 5B). Cell cycle analysis (Fig. 5C) revealed that combined catechol-exposure (50 µM) and radiation treatment (4 Gy) increased the sub-G1 population of Panc-1 cells compared to the control (radiation only). Furthermore, western blotting revealed that the co-treatment group (50 µM catechol and 4 Gy radiation dose) had higher expression levels of p-ATM, p-Chk2, and cleaved PARP, known markers for radiation-induced DNA damage (20), compared to the untreated control (Fig. 5D). Increased AMPK levels have been reported to be associated with enhanced radiosensitivity (21). In the present results, increased p-AMPK levels in the combined treatment group were observed, indicating that catechol could promote radiosensitivity by activating p-AMPK. Moreover, the expression of YAP and CYR61 was reduced in the combined treatment group (Fig. 5D). These results indicated that catechol enhanced the effects of radiation treatments in Panc-1 cells.