FYGL was extracted from Ganderma lucidum in our laboratory (5). The human pancreatic cancer cell line, PANC-1, and the human liver cancer cell line, HepG2, were obtained from Procell Life Science & Technology Co., Ltd., while the human pancreatic cancer cell line, Mia PaCa-2, was donated by Professor Wuli Yang (Fudan University, Shanghai, China). The human pancreatic cancer cell line, BxPC-3, was donated by Doctor Yiqun Ma (Zhongshan Hospital, Shanghai, China). The PANC-1, Mia PaCa-2 and HepG2 cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin/streptomycin and 1% L-glutamine (Thermo Fisher Scientific, Inc.). The BxPC-3 cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine. Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂. The following antibodies: Caspase-3 (dilution 1:1,000; cat. no. 9662S), cleaved-caspase-3 (dilution 1:1,000; cat. no. 9661S), Bcl-2 (dilution 1:1,000; cat. no. 4223S), P62 (dilution 1:1,000; cat. no. 39749S), LC3A/B (dilution 1:1,000; cat. no. 12741S), β-actin (dilution 1:1,000; cat. no. 4970S), GAPDH (dilution 1:1,000; cat. no. 5174S), and HRP-conjugated secondary anti-rabbit (dilution 1:2,000; cat. no. 7074S) were all purchased from Cell Signaling Inc.

The cells were seeded in 96-well plates at 37°C in a humidified atmosphere with 5% CO₂, at a density of 5×10⁴ cells/well. After 24 h, the cells in each well were treated with FYGL at a concentration range of 0–1,000 µg/ml, and gemcitabine (Eli Lilly and Company) as a positive control at a concentration range of 5–20 µM for 24 and 48 h, then, 10 µl Cell Counting Kit-8 (CCK-8) reagent (Shanghai Yeasen Biotechnology Co., Ltd.) was added into each well. The absorbance was measured at 450 nm, using a microplate reader (Cytation 3; BioTek Instruments, Inc.), 1–4 h later.

The cells were seeded in cell culture dishes (Wuxi NEST Biotechnology Co., Ltd.) at a density of 1×10⁵ cells/well at 37°C in a humidified atmosphere with 5% CO₂. After treatment with 0 (control) and 150 µg/ml FYGL, the cells were fixed with 4% paraformaldehyde (PFA) for 10 min at 37°C, then permeabilized with 0.3% Triton X-100 (Sinopharm Chemical Reagent Co., Ltd.). FYGL was stained with a green fluorescent agent, fluorescein isothiocyanate (FITC), and the cell nucleus and cytoskeleton were stained with a blue fluorescent agent, 4′,6-diamidino-2-phenylindole (DAPI), and a red fluorescent agent, rhodamine-labeled phalloidin (all from Shanghai Yeasen Biotech Co., Ltd.), respectively for 10 min at 37°C. Images were obtained using a laser scanning confocal microscope (magnification, ×100; LSCM; Nikon C2+; Nikon Corporation).

The cells were seeded (5×10⁵ cells/well) in 6-well plates at 37°C in a humidified atmosphere with 5% CO₂, and the cell monolayer was scratched with a pipette tip when the cells grew to a single layer. Following which, the cells were treated with FYGL at a concentration range of 0–1,000 µg/ml in DMEM medium with 3% FBS for 48 h. Cell images were obtained using an inverted optical microscope (Nikon ECLIPSE Ts2; Nikon Corporation) to observe the migration of the cells across the wound (magnification, ×4). ImageJ software (v1.51j8; National Institutes of Health) was used for digital analysis of the wound healing area.

The cells were seeded in 6-well culture plates at 37°C in a humidified atmosphere with 5% CO₂, at a density of 200 cells/well and incubated for 24 h. Then, the cells in each well were treated with FYGL at a concentration range of 0–1,000 µg/ml for 12 days. All colonies were fixed with 4% PFA for 10 min at 37°C, and washed with PBS (Sangon Biotech Co., Ltd.) subsequently, the cells in each well were stained with a purple dye, giemsa (Shanghai Yeasen Biotechnology Co., Ltd.) for 10 min at 37°C. Cell images were obtained using an optical camera (magnification, ×1; Sony α6400; Sony Corporation).

The percentage of apoptotic cells was determined using an Annexin V-FITC/PI kit (Shanghai Yeasen Biotechnology, Co., Ltd.). Cells were seeded in 6-well plates, at a density of 5×10⁵ cells/well. After incubation for 24 h, the cells were treated with FYGL at a concentration range of 0–500 µg/ml for 24 h and harvested using 0.25% trypsin (Sangon Biotech Co., Ltd.), then the cells were incubated with 5 µl Annexin V-FITC and 10 µl PI working solution for 30 min at 37°C. Finally, the stained cells were analyzed using flow cytometry (Beckman Coulter, Inc.), and the data were analyzed using FlowJo software (version 10.4; BD Biosciences).

The fluorescent agent, 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA; Shanghai Yeasen Biotech Co., Ltd.) was used to investigate ROS production. The cells were seeded in 6-well plates at 37°C in a humidified atmosphere with 5% CO₂, at a density of 5×10⁵ cells/well. After incubation for 24 h, the cells were treated with FYGL at a concentration range of 0–500 µg/ml for 24 h. Following which, the cells were washed with PBS and added to 1 ml serum-free medium with 10 µl DCFH-DA per well. After incubation for 30 min at 37°C, the level of ROS in the cells were identified using DCFH, and analyzed using flow cytometry (Beckman Coulter, Inc.), and the data were analyzed using FlowJo software (version 10.4; BD Biosciences).

The cells were seeded in cell culture dishes at 37°C in a humidified atmosphere with 5% CO₂, at a density of 1×10⁵ cells/well and incubated for 24 h, then, the cells were treated with FYGL at a concentration range of 0–1,000 µg/ml for 24 h. The medium was removed and the cells were washed with PBS, then stained with 10 µl rhodamine 123 (Rh-123; a green fluorescent agent; 5 mg/ml; Shanghai Yeasen Biotech Co. Ltd.) for 30 min in the dark at 37°C, to detect the mitochondrial membrane potential. Following which, the cells were fixed with 4% PFA for 10 min at 37°C, and permeabilized with 0.3% Triton X-100. Subsequently, the cell nucleus and cytoskeleton were stained blue with DAPI and red with rhodamine-labeled phalloidin for 10 min at 37°C, respectively. Images were taken by LSCM (magnification, ×60; Nikon C2+, Nikon Corporation).

The cells were seeded in cell culture dishes at 37°C in a humidified atmosphere with 5% CO₂, at a density of 1×10⁵ cells/well then, treated with FYGL at a concentration range of 0–1,000 µg/ml for 24 h. After washing with PBS three times, the cells were incubated with a Cyto-ID autophagy detection kit (Enzo Life Sciences, Inc.) for 30 min at 37°C, which including a mitochondrial staining reagent Cyto-ID and nuclear staining reagent Hoechst 33258, subsequently were washed with PBS and fixed with 4% PFA for 15 min at 37°C. Images were obtained using LSCM (magnification, ×60; Nikon C2+; Nikon Corporation).

Total protein was lysed from the cells using a RIPA containing phenylmethylsulfonyl fluoride, then the concentration was measured using a BCA protein assay kit (all from Shanghai Yeasen Biotech Co., Ltd.). Following which, the lysed proteins were separated using a 10–15% SDS-PAGE and transferred to PVDF membranes (Beyotime Institute of Biotechnology). The membranes were blocked with a buffer containing 10 mM Tris-HCl, (pH 7.4), 150 mM NaCl, 0.1% Tween-20, and 5% skimmed milk (Beyotime Institute of Biotechnology), then incubated with the primary antibodies for 12 h at 4°C, followed by incubation with the HRP-conjugated secondary antibody for 1 h at 25°C. The antibodies were detected using a super enhanced chemiluminescence detection reagent (Shanghai Yeasen Biotech Co., Ltd.) and a chemiluminescent imaging system (Bio-Rad ChemiDoc MP; Bio-Rad Laboratories, Inc.). GAPDH and β-actin were used as the loading controls.

The adenovirus expressing (Ad)-mCherry-GFP-LC3 fusion protein (Beyotime Institute of Biotechnology) was used to investigate the binding of autophagosomes and lysosomes. The PANC-1 cell line was transfected with ad-mCherry-GFP-LC3 adenovirus, at 20 multiplicity of infection for 24 h at 37°C. Then, the culture medium was removed and fresh DMEM with FYGL at a concentration range of 0–500 µg/ml was added. After 24 h, the cells were detected with LSCM (magnification, ×60; Nikon C2+; Nikon Corporation).

The results are presented as the mean ± standard deviation (n=3). SPSS software (v20.0; IBM Corp.) was used for statistical analyses. ImageJ software (v1.51j8; National Institutes of Health) was used for digital analysis of the western blots. One-way ANOVA was performed to compare the mean values among multiple groups followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of FYGL on the viability of 2 different cancer cell lines, three pancreatic cancer cell lines, PANC-1, BxPC-3, and Mia PaCa-2, and one hepatic cell line, HepG2, were treated with FYGL, at a concentration range of 50–1,000 µg/ml, with gemcitabine, as a positive control, at a concentration range of 5–20 µM. All the cell lines were treated for 24 (Fig. 1A) and 48 h (Fig. 1B), then the viability was investigated using a CCK-8 assay. The results indicated that FYGL had different effects on cell viability in the 2 different cancer cell lines. The viability of PANC-1 and BxPC-3 cells was markedly decreased by the treatment of FYGL in a dose dependent manner at both 24 and 48 h, comparable with the cells treated with the positive agent, gemcitabine. The viability of the Mia PaCa-2 cells was not affected by FYGL treatment in a dose dependent manner, both at 24 and 48 h. As well as in HepG2 cells, however, gemcitabine was markedly toxic for all the cell lines following treatment for 48 h. The results, therefore, indicated that FYGL was moderate, safe, and selective for the given cell lines.

To understand the functional mechanism of FYGL in the cells, the PANC-1 and Mia PaCa-2 cell lines were selected for further research. First, the absorption of FYGL into the cells was investigated. The FYGL, cell nucleus, and cytoskeleton were all labeled with fluorescent agents namely: FITC (green), DAPI (blue), and rhodamine-labeled phalloidin (red). As shown in Fig. 2, the green fluorescence from FYGL overlapped with the red fluorescence from the cytoskeleton. Furthermore, some green fluorescent areas were found between the red and the blue fluorescent areas (from the nucleus labelled with DAPI), indicating that FYGL was encapsulated by vesicles and dispersed into the cytoplasm. Some green fluorescent areas were found to be outside of the cells, indicating not all of the FYGL was absorbed into the cells; however, most of the FYGL was absorbed in the cells.

Cell migration in cell treated with FYGL was determined using a wound healing assay. Both PANC-1 and Mia PaCa-2 cells were treated with FYGL for 48 h. The cell migration of PANC-1 cells into the wound area was halted by the increased concentrations of FYGL (Fig. 3A). The wound healing percentage of PANC-1 cells significantly decreased at 250, 500 and 1,000 µg/ml concentrations of FYGL compared with that in the control group (Fig. 3B). However, the wound healing percentage of Mia PaCa-2 cells increased at 250 µg/ml concentrations of FYGL and decreased at 1,000 µg/ml concentrations of FYGL, but no significant differences were observed compared with that in the control group, which indicated that cell migration was not affected by FYGL in the Mia PaCa-2 cells (Fig. 3C).

Fig. 3D and E showed the colony formation of the PANC-1 and Mia PaCa-2 cells treated with different concentrations of FYGL. A total of 200 cells, in each well, were incubated with FYGL for 12 days. The colony formation of the PANC-1 cells was low at higher concentrations, indicating sensitivity to FYGL treatment, while there was a high number of colonies with the Mia PaCa-2 cell line indicating insensitivity to FYGL treatment.

Apoptosis of cancer cells is a critical index for an anticancer drug (23), and is typically investigated using an Annexin V-FITC/PI kit and measured using flow cytometry (24). The apoptotic rate of the PANC-1 cells increased to 23%, as the concentration of FYGL increased to 500 µg/ml (Fig. 4A), however, the apoptotic rate of Mia PaCa-2 cells was unaffected (Fig. 4B).

Furthermore, western blot analysis was used to analyze the expression levels of key proteins for example, caspase-3, Bcl-2 and cleaved-caspase-3, which are involved in apoptosis. The production of cleaved caspase-3 from caspase-3 promotes cell apoptosis, while the expression level of Bcl-2 prevents the cells from undergoing apoptosis (25,26). Fig. 5A shows that the protein expression levels of caspase-3 and Bcl-2 in the PANC-1 cells significantly decreased at 250, 500 and 1,000 µg/ml concentrations of FYGL compared with that in the control group, while Fig. 5B shows that the protein expression level of cleaved-caspase-3 in the PANC-1 cells was significantly increased, at 500 and 1,000 µg/ml concentrations of FYGL, compared with that in the control group. The results indicated that cell apoptosis in the PANC-1 cells was induced by FYGL.

However, Fig. 5C and D demonstrated that the protein expression levels of caspase-3 in the Mia PaCa-2 cells decreased at 50 and 100 µg/ml concentrations of FYGL and increased at 500 and 1,000 µg/ml concentrations of FYGL, but no significant differences were observed compared with that in the control group. The protein expression levels of Bcl-2 in the Mia PaCa-2 cells increased at 50, 100 and 500 µg/ml concentrations of FYGL, but no significant differences were observed compared with that in the control group. The protein expression levels of cleaved-caspase-3 in the Mia PaCa-2 cells increased at 50 µg/ml concentrations of FYGL and decreased at 250, 500 and 1,000 µg/ml concentrations of FYGL, but no significant differences were observed compared with that in the control group. These results indicated that FYGL does not induce Mia PaCa-2 cell apoptosis.

The fluorescent probe, DCFH-DA was used to detect ROS in the cells using flow cytometry. Fig. 6A showed that the ROS level in the PANC-1 cells significantly increased as the concentration of FYGL increased from 0 to 500 µg/ml; however, there was no increase in the Mia PaCa-2 cells (Fig. 6B). The decrease of MMP has been associated with the increase of ROS, and with cell apoptosis (11,27).

Therefore, in the subsequent experiments, the green fluorescent agent, Rh-123 was used to detect the MMP in the PANC-1 and Mia PaCa-2 cell lines, treated with FYGL for 24 h and analyzed using confocal microscopy. Fig. 7A and B showed the fluorescent images of the PANC-1 and Mia PaCa-2 cell lines, respectively; wherein blue, green, and red represent the cell nucleus (DAPI), MMP (Rh-23), and cytoskeleton (rhodamine-labeled phalloidin), respectively. The MMP was found to be deceased in the PANC-1 cells, as the concentrations of FYGL increased; however, there was no marked change in the Mia PaCa-2 cells, which were consistent with the results from the ROS experiments. The results indicated that FYGL could selectively induce the production of ROS, leading to the reduction of the MMP and apoptosis in the PANC-1 cells line.

The cell autophagy process occurs from autophagosome formation, along with organelle damage, to the movement of autophagosomes into lysosomes for the recycling of damaged organelles (18). The polyubiquitinated protein, LC3-I/II, and the polyubiquitin-binding protein, P62, are autophagic effector proteins. During the cell autophagy process, LC3-I is covalently bound to phosphatidylethanolamine (PE) to form LC3-II, which then localizes on the autophagosome membrane (28). Once the autophagy process has been completed, the P62 protein is degraded, and the autophagosomes enter the lysosomes (29). Fig. 8A showed that the autophagosomes, labeled with the green fluorescent agent, cyto-ID, increased, as the concentration of FYGL increased, in the PANC-1 cells treated with FYGL for 24 h, suggesting that FYGL promoted autophagosome formation in the PANC-1 cell line. FYGL had almost no effect on the fluorescence intensity in the Mia PaCa-2 cell line, indicating that FYGL did not induce autophagosome formation in the Mia PaCa-2 cell line (Fig. 8B).

Fig. 9A and B showed the protein expression level of LC3-I/II and P62 in the PANC-1 and Mia PaCa-2 cells, respectively, treated with different concentrations of FYGL for 24 h. In the PANC-1 cells, the cascade protein ratio of LC3-I/II significantly decreased at 50, 100, 250, 500 and 1,000 µg/ml concentrations of FYGL compared with that in the control group further indicating that there was an increase in the number of autophagosomes in the PANC-1 cells. However, it was found that in the PANC-1 cells treated with different concentrations of FYGL, the relative protein expression level of P62 was significantly increased, at 500 and 1,000 µg/ml compared with that in the control group, indicating that the cell autophagy process was halted, and not completed in the signaling pathway. In the Mia PaCa-2 cell line, the ratio of LC3-I/II decreased at 50, 250, 500 and 1,000 µg/ml concentrations of FYGL and increased at 100 µg/ml concentrations of FYGL, but no significant differences were observed compared with that in the control group. The protein expression levels of P62 in the Mia PaCa-2 cells increased at 500 and 1,000 µg/ml concentrations of FYGL, but no significant differences were observed compared with that in the control group. These results indicated that FYGL had almost no effect on the autophagy of Mia PaCa-2 cells.

In addition, the fluorescent adenovirus, ad-mCherry-GFP-LC3 was used to confirm that the autophagosomes enter the lysosomes. When the ad-mCherry-GFP-LC3 adenovirus, bound to the autophagosomes, are present in the lysosomes of cells, the GFP in the fusion protein is quenched. Fig. 9C shows the fluorescence images of the PANC-1 cells infected with Ad-mCherry-GFP-LC3 and treated with (500 µg/ml) or without (control) FYGL for 24 h. It was found that the fluorescent intensity of GFP in FYGL-treated cells was markedly higher compared with that in the control cells, suggesting that FYGL prevented autophagosomes from entering the lysosomes.