The human pancreatic carcinoma cell line, PANC-1, was obtained from the Korean Cell Line Bank; Korean Cell Line Research Foundation (cat. no. 21469). PANC-1 cells were cultured in DMEM (Welgene, Inc.) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% heat-inactivated FBS (Welgene, Inc.). The culture was maintained at 37°С in a humidified atmosphere of 5% CO₂.

C5E consisted of ten traditional Chinese medicinal herbs. The proportions, manufacturing process and analyses of the major components were previously reported (8). C5E was prepared using 16.6% Panax ginseng (w/w; supplied from Kyung-dong market, Seoul, Republic of Korea), 16.6% Inonotus obliquus, 11.1% Pinellia ternata, 5.6% rhizome of Sparganium stoloniferum Buchanan-Hamilton, 5.6% Alpinia galanga, 5.6% Cinnamomum cassia, 5.6% Astragalus membranaceus, 11.1% Psoralea corylifolia L., 11.1% Tetradium ruticarpum and 11.1% Melia azedarach L. The medicinal herbs were obtained from the Oriental Medical Hospital, Dongguk University (Ilsan, Republic of Korea). C5E was prepared as follows: The dried and pulverized herbs were mixed and a 1 kg batch was soaked and slowly stirred overnight at 25°C with 3 l ethanol (40%). The ethanol extract was concentrated using a rotary evaporator to remove solvent, lyophilized at −80°C using a freeze-dryer (FDU-2110; Eyela) and reconstituted in Milli-Q water (18.2 MΩ; EMD Millipore) for in vitro studies.

PANC-1 cells were seeded at a density of 5×10⁵ cells/ml and subjected to three different treatments and control: i) 10 nM gemcitabine (Sigma-Aldrich; Merck KGaA); ii) 30 µg/ml C5E; and iii) pretreatment with 15 µg/ml C5E for 2 h before treatment with 5 nM gemcitabine; iv) untreated control. After 72 h of treatment at 37°C, the cells were harvested at the logarithmic growth stage by treatment with 0.05% trypsin-EDTA and centrifugation (220 × g; 3 min; 25°C). Cells were resuspended in an equal volume of DMEM (Welgene, Inc.), then a 10 µl sample was stained for 10 sec with 10 µl 0.4% trypan blue dye at 25°C. Viability was determined by cell counting using hemocytometer and the half maximal inhibitory concentration (IC₅₀) values were calculated as previously described (24).

Cell cycle progression was analyzed by flow cytometry. Following 72 h incubation at 37°C, PANC-1 cells were harvested using trypsin-EDTA treatment as described above and washed twice with PBS. When cells were harvested for SP analysis, the population was divided in half: One half was used for SP analysis, and the other half was used to examine cell cycle arrest. The cells were fixed in ice-cold 70% ethanol at −20°C for 24 h. Prior to flow cytometric analysis, the ethanol was removed by centrifugation (250 × g, 5 min, 25°C) and the cells were washed and resuspended in 5 ml PBS. 100 µl propidium iodide (PI) staining solution containing 50 µg/ml PI (Sigma-Aldrich; Merck KGaA) diluted in 1X PBS, 1 mg/ml RNase (Sigma-Aldrich; Merck KGaA) diluted in 1X PBS and 0.1% Triton X-100 was added to a FACS tube and incubated in the dark at room temperature for 30 min. Cell cycle analysis was performed using a FACSCalibur flow cytometer (BD Biosciences) at an excitation wavelength of 488 nm and an emission wavelength of 610 nm by measuring the amount of PI-labeled DNA in the cells. Data were analyzed using ModFit LT™ version 3.0 software (Verity Software House, Inc.).

Flow cytometric analysis was performed using an FITC Annexin V Apoptosis Detection kit І (BD Biosciences). PANC-1 cells were harvested via trypsin-EDTA treatment as described above, washed twice with PBS and centrifuged (250 × g, 5 min, 25°C). The pellets were resuspended in 100 µl Annexin V binding buffer, and then incubated with 5 µl Annexin V-FITC and 5 µl PI for 15 min at room temperature in the dark. For the analysis, 300 µl binding buffer was added to each mixture and analyzed using a FACSCalibur flow cytometer. The fluorescence intensity of Annexin V-FITC and PI was analyzed at an excitation/emission wavelength of 488/530 nm and 488/617 nm, respectively. The data were analyzed using BD CellQuest Pro software version 5.2.1 (Becton Dickinson, Inc.) and apoptotic rate was calculated by the percentage of each of early and late apoptotic cells.

PANC-1 cells were harvested via treatment with trypsin-EDTA as described above and labeled with Hoechst 33342 dye (Sigma-Aldrich; Merck KGaA), using the methods described by Goodell et al (11). Briefly, the cells were resuspended at a density of 1×10⁶ cells/ml in pre-warmed DMEM (phenol red-free) containing 2% FBS and 10 mM HEPES buffer. Hoechst 33342 was added at a final concentration of 5 µg/ml in the presence or absence of 50 µM verapamil (Sigma-Aldrich; Merck KGaA) which inhibits ABC transporter to prevent generation of SP cells, and the cells were incubated at 37°С for 90 min with intermittent mixing. Following the incubation, the cells were centrifuged (250 × g; 5 min; 25°C) and resuspended in ice-cold HBSS (Welgene, Inc.) containing 2% FBS and 10 mM HEPES buffer. The cells were then analyzed using a FACSAria™ flow cytometer (BD Biosciences). Hoechst 33342 was excited at 357 nm, and its fluorescence was analyzed using a dual-wavelength (blue, 402–446 nm; red, 650–670 nm). The data were analyzed using BD CellQuest Pro software (version 5.2.1; Becton Dickinson, Inc.).

Total RNA was extracted from cells using the RNeasy Mini kit (Qiagen, Inc.), according to the manufacturer's instructions, followed by processing with the QIAshredder homogenizer (Qiagen, Inc.). The RNase-free DNase set (Qiagen, Inc.) was used for further DNA removal. Oligo (dT)₁₄ single-stranded cDNA was synthesized from the RNA using AMV Reverse Transcriptase (Promega Corporation). The following conditions were used for cDNA synthesis: Initial incubation at 65°C for 5 min; followed by 42°C incubation for 60 min and termination at 70°C for 5 min. qPCR was subsequently performed on a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories, Inc.) using a TaqMan PreAmp Master Mix kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Commercially available primers were obtained for Sonic hedgehog (SHH) (cat. no. Hs00179843_m1; Applied Biosystems; Thermo Fisher Scientific, Inc.) and GAPDH (cat. no. Hs02786624_g1; Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation for 10 min at 95°C; followed by 45 cycles of denaturation for 15 sec at 95°C and annealing/elongation at 60°C for 60 sec. The housekeeping gene, GAPDH, was used as an internal control. The expression levels were measured using the 2^−ΔΔCq method (25). Data are expressed as the fold change relative to the expression levels in the control group.

Statistical analyses were performed using SPSS version 25.0 software (IBM Corp.). Data are expressed as the mean ± SD of ≥3 independent experiments. Significant differences were analyzed using a one-way ANOVA followed by a Tukey's test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The viability of PANC-1 cells was evaluated using the trypan blue exclusion assay 72 h after C5E treatment. The results revealed that C5E treatment inhibited cell viability in a dose-dependent manner (Fig. S1A). The IC₅₀ of C5E was calculated to be 30 µg/ml for PANC-1 cells. Gemcitabine also inhibited the cell viability of PANC-1 cells in a dose-dependent manner; the IC₅₀ value of gemcitabine was calculated to be 10 nM (Fig. S1B). Additionally, following the co-treatment with C5E and gemcitabine, the cell viability was also inhibited in a dose-dependent manner. The IC₅₀ value of co-treatment with C5E and gemcitabine decreased to 15 µg/ml for C5E and 5 nM for gemcitabine (Fig. S1C).

To determine the distribution of the cell cycle in cells following C5E and gemcitabine treatment, cells were treated with C5E and/or gemcitabine at their IC₅₀ values. The treatment of PANC-1 cells with 30 µg/ml C5E for 72 h significantly increased the percentage of cells in the S phase (65.8±0.8%) compared with the control group (40.2±0.8%), while concomitantly significantly reducing the percentage of cells in the G₀/G₁ and G₂/M phases (Fig. 1A and B). The relative percentage increase in S indicated that the cell cycle progress was blocked by treatment (26,27). In addition, the increase in the percentage of S phase cells following the treatment with gemcitabine (49.2±2.5%) was significantly lower compared with the C5E treatment. Notably, the percentage of S phase cells following the co-treatment (67.8±1.2%) was significantly increased compared with the treatment with gemcitabine-alone (49.2±2.5%). These findings suggested that the co-treatment may have a synergistic effect on cell cycle arrest compared with the treatment with C5E or gemcitabine alone. In addition, the inhibition of cell viability following the treatment with C5E and/or gemcitabine may be related to S phase cell cycle arrest.

To analyze the effect of C5E on apoptosis, PANC-1 cells were treated with C5E and/or gemcitabine for 72 h, and the number of apoptotic cells was analyzed using Annexin V/PI double staining. The levels of early apoptosis in PANC-1 cells following C5E treatment were significantly increased (7.3±0.7%) compared with the control group (1.9±0.5%) (Fig. 2A and B). In addition, the levels of early apoptosis following C5E treatment (7.3±0.7%) were more than double that of the gemcitabine-induced levels of early apoptosis (2.5±0.3%). Moreover, the levels of early apoptosis following the co-treatment were increased (12.0±0.9%) by 10.1% compared with the control and by 4.7% compared with the C5E treatment alone (Fig. 2A and B). Late apoptosis was also assessed following the treatment with C5E and/or gemcitabine. The levels of late apoptosis following the co-treatment were 17.1±1.6%, which were increased compared with C5E treatment alone (14.2±3.5%) and the control treatment (3.2±0.6%). The overall trend was very similar to that observed for early apoptosis. Thus, these results suggested that the co-treatment may have a synergistic effect on apoptosis, as well as cell cycle progression.

Subsequently, the proportion of SP cells following the treatment with C5E, gemcitabine or C5E plus gemcitabine was analyzed by Hoechst 33342 staining. As expected, verapamil, which is an inhibitor of ABC transporter, inhibited generation of SP cells. Flow cytometry analysis showed 8.2±0.2% SP cells in the control group without verapamil treatment (Fig. 3A and B). A decrease in the percentage of SP cells in the PANC-1 cell culture was induced by C5E and/or gemcitabine in the absence of verapamil treatment. The percentage of SP cells significantly decreased to 3.9±0.3% following treatment with C5E-alone and 7.2±0.3% following treatment with gemcitabine-alone compared with the control group. Notably, the percentage of SP cells was also significantly decreased to 5.1±0.7% following the co-treatment with C5E and gemcitabine compared with the control group (Fig. 3). The percentage of SP cells with verapamil treatment were <0.2% (Fig. 3B). Thus, the percentage of SP cells following the co-treatment decreased by less than C5E treatment alone. These results suggested that C5E may have a higher influence than gemcitabine on the reduction of the SP cell population. Subsequently, the changes in the percentage of SP cells at varying concentrations of C5E were investigated. The percentage of SP cells was 7.9±0.1, 4.3±1.2 and 4.2±1.5%, respectively, at the IC₂₅ (10 µg/ml), IC₅₀ (30 µg/ml) and the IC₇₅ (50 µg/ml) doses, indicating that the proportion of SP cells decreased with increasing concentration of C5E (Fig. 4A and B). Thus, the percentage of SP cells was significantly decreased at IC₅₀ and IC₇₅ compared with the control. However, the difference in the percentages was much smaller between the IC₅₀ and IC₇₅ doses compared with between the IC₅₀ and IC₂₅ doses.

SHH is a stem cell-associated gene, which has been implicated in the development of pancreatic cancer cells (28). The expression levels of SHH were analyzed following the treatment with C5E and/or gemcitabine using RT-qPCR. The results revealed that SHH expression levels were downregulated by ~20% following the treatment with C5E or gemcitabine alone compared with the control group (Fig. 5). However, the expression levels of SHH were significantly downregulated by ~60% following the co-treatment with C5E and gemcitabine compared with the control group (Fig. 5). Thus, the downregulation in SHH mRNA expression levels was more significant upon the co-treatment with C5E and gemcitabine compared with the treatment with each individual compound. These data indicated that C5E and gemcitabine may exert a synergistic effect with respect to the inhibition of SHH mRNA expression levels.