KFX (Good Doctor Pharmaceutical Group, Sichuan, China) was dissolved in cell culture medium to the required concentration before use. 4-phenylbutyric acid (4-PBA; Aladdin, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) to make a 2 mmol/l stock solution and was diluted in cell culture medium to the required concentration before use. A FITC-Annexin V Apoptosis detection kit (BD Biosciences, San Jose, CA, USA) were prepared for the apoptosis assay. All chemicals in the present study were used for experimental research only.

A human gastric cancer cell line, SGC-7901, was purchased from the Shanghai Institute of Cell Biology (Shanghai, China). Cells were routinely maintained in RPMI-1640 (31800–022; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, SV30087.02; HyClone; GE Healthcare Life Sciences, Logan, UT, USA), penicillin (100 U/ml, P3032) and streptomycin (100 U/ml, S9137) (both from Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) at 37°C in a humidified 5% CO₂ incubator.

To detect the anticancer activity of different concentrations of KFX, the SGC-7901 cell viability in different treatment groups was assessed by using a CCK-8 kit. CCK-8 was used to measure cell viability and evaluate the survival rate of SGC-7901 cells by KFX treatment. Cells were seeded in a 96-well plate at a density of 2×10³ cells/well and incubated for 12 h in 0.1 ml RPMI-1640 medium supplemented with 10% FBS at 37°C. Then, the medium was replaced with fresh medium containing KFX at various concentrations ranging from 0.1 to 5 µg/ml. The untreated cells were used as controls. After 24, 48 and 72 h incubation, 10 µl of CCK-8 was added into each well. After 4 h, absorption in each well was observed by a micro plate reader at 450 nm (Multiskan MK3; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

SGC-7901 cells were seeded into 6-well plates at 3×10⁵ cells/well and were incubated for 12 h. A scratch was created with a sterile clear tip (200 µl). Then, the cells were washed twice with PBS to remove the debris and detached cells. Scratch pictures were recorded immediately using an inverted fluorescence microscope (Nikon, Tokyo, Japan). Afterwards, the cells in the wells were cultured with various concentrations of KFX in medium, separately. The only cells treated with medium were the control group. After 24 or 48 h of treatment, cell migration was observed by using an inverted fluorescence microscope.

SGC-7901 cells were seeded in 6-well plates at a density of 3×10⁵ cells/well and incubated overnight at 37°C. Cells were treated with a different concentration of KFX with the untreated cells as the controls. After 48 h treatment, the cells were collected and analysed. For quantitative assays, the cells were harvested by trypsinisation, then centrifuged at 1,000 rpm for 3 min and suspended in binding buffer. After adding 5 µl of Annexin V and 5 µl propidium iodide (50 µg/ml), the cells were incubated at room temperature for 5 min in the dark using the FITC Annexin V Apoptosis detection kit (BD Biosciences). Cell apoptosis was analysed by flow cytometry (FACSCalibur FCM; Becton-Dickinson, San Jose, CA, USA).

Cells were washed with PBS and lysed in Lysis-M reagent supplemented with complete mini-protease inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN, USA). The equivalent of 60 µg of protein was separated on a 12% gel and then transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% (w/v) non-fat milk for 2 h, the membranes were incubated with the following antibodies: Bax, Bcl2, GRP78, Chop, LC3, Beclin1 (1:000; Abcam, Cambridge, UK) and GAPDH (1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Next, the membranes were incubated with a goat-anti-rabbit secondary antibody for 2 h at room temperature. Signals were visualised using the ChemiDoc™ XRS+ Imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

SGC-7901 cells were seeded in 6-well plates at 3×10⁵ cells/well and incubated for 12 h. The cells were treated with various concentrations KFX or without KFX for 48 h. After treatment, the cells were washed with PBS three times, fixed with 4% paraformaldehyde in PBS for 20 min, permeabilised with 0.5% Triton X-100 in PBS for 15 min and incubated with 5% bovine serum albumin (BSA) in PBS for 1 h to block nonspecific antibody binding at 37°C. After blocking with 1% BSA for 1 h, the cells were incubated with anti-CHOP (1:100; Santa Cruz Biotechnology, Inc.) antibody. Then, the cells were incubated with goat-anti-rabbit secondary fluorescence antibody for 2 h at room temperature. The nuclei were stained with DAPI. Fluorescence images were obtained using a positive position fluorescence microscope or confocal laser microscope (both from Nikon).

After fixation in 2.5% (w/v) glutaraldehyde overnight, cells were post-fixed in 2% (v/v) osmium tetroxide and blocked with 2% (v/v) uranyl acetate. Following dehydration in a series of acetone washes, the cells were embedded in Araldite. Semi-thin sectioning and toluidine blue staining were performed to observe the location. Finally, ultra-thin sections of at least three blocks per sample were cut and observed using a Hitachi TEM.

Statistical analyses were carried out using SPSS 20.0 statistical software (SPSS, Inc., Chicago, IL, USA). All values are presented as the means ± standard error of the mean (SEM). Statistical evaluation of the data was performed using one-way analysis of variance (ANOVA) and Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

In the CCK-8 assay, the SGC-7901 cells were treated with KFX for 24, 48 and 72 h at different concentrations (0.1, 0.25, 0.5, 1, 2, 5 µg/ml) to evaluate the optimised KFX treatment on the inhibition of cell growth. In each group, 1 µg/ml of KFX showed substantial inhibition in SGC-7901 cells, whereas there was no concentration dependence over 1 µg/ml (Fig. 1). From the results of the cell viability test, KFX showed the strongest inhibition on SGC-7901 cell survival at 48 h, while the cancer cell inhibition of KFX treatment at 24 h was much lower than that at 48 h. However, there was no difference at 48 and 72 h. Thus, in the CCK-8 analysis, 48 h of KFX treatment was the optimal administration time. Consistent with the CCK-8 results, the scratch assay revealed that KFX could achieve the strongly inhibition on cell migration at 24 h. However, the inhibition on cell migration at 48 h was similar or even stronger than that at 24 h. Considering the optimized drug bioavailability, the treatment time at 48 h was better than 24 h (Fig. 2). The semi-quantification bar chart was show in Fig. 2. According to those two assays, the optimum treatment concentration (1 µg/ml) and time (48 h) for the KFX anticancer effect on SGC-7901 cells were used in the following studies.

SGC-7901 cells were treated with various concentrations of KFX and apoptosis was investigated using a FITC Annexin V apoptosis detection kit using flow cytometry. Compared with the control, treatment of SGC-7901 cells with different doses of KFX could induce different stages of apoptosis. Among all the experimental groups, the cells showed a progressive increase in apoptosis with increasing amounts of KFX treatment. In detail, the cells treated with 1 µg/ml of KFX showed nearly 2.53- and 2.75-fold higher apoptosis than those treated with 0.5 µg/ml KFX and 0.1 µg/ml KFX, respectively, suggesting the strongest and most effective tumour inhibition at 1 µg/ml of KFX (Fig. 3A). Furthermore, the apoptosis effect of KFX was validated by western blot analysis. Compared with the control, both 0.1 and 1 µg/ml of KFX upregulated the expression of Bax and downregulated the expression of Bcl-2, and the 1 µg/ml KFX treatment group had the strongest apoptosis effect compared to the 0.1 µg/ml KFX treatment group (Fig. 3B). The quantitative analysis of the western blot results are shown in Fig. 3C and D.

It is known that the ER can induce ER stress marker proteins (such as GRP78 and phospho-eIF2a), which upregulates the expression of CHOP (16–18). CHOP can influence the expression of Bcl −2 family proteins and inhibit the anti-apoptotic protein expression of Bcl-2 (19). Regarding this study, many studies have proven that many TCMs inhibit cancers via activating ER stress (20–23). Therefore, we hypothesized that ER stress was involved in the anti-cancer effect of KFX and whether ER stress was associated with the mechanism of KFX-induced apoptosis in SGC-7901 cells. We examined the expression of ER stress proteins in the cells after treatment with KFX (Fig. 4). The Western blot results showed that the expressions of CHOP, GRP78 and caspase-12 were increased with KFX treatment group, and the 1 µg/ml KFX group was stronger than 0.1 µg/ml KFX group (Fig. 4A). To further confirm that KFX promoted apoptosis through ER stress, the ER stress inhibitor 4-PBA (2 mM) was used (24). 4-PBA is a chemical chaperone that contributes to protein folding and trafficking within the ER, alleviating ER stress and acting as an ER stress inhibitor (24). After exposure to 4-PBA for 6 h, the expressions of CHOP, GRP78 and caspase-12 were significantly suppressed by 4-PBA, whereas these protein expressions were significantly restored after KFX treatment (Fig. 4C). Quantitative analysis of the western blot analysis is shown in Fig. 4B and D. In the immunofluorescence results, the CHOP expression in the cells in the 4-PBA+KFX group was stronger than the 4-PBA group (Fig. 4E). All these results demonstrated that KFX could reverse the expressions of the 4-PBA-inhibited ER-related proteins. ER-stress played a critical role in the KFX anticancer effect.

In addition to ER stress, autophagy may also be involved in SGC-7901 cell apoptosis after KFX treatment. TEM images verified that KFX activated autophagy. As shown in Fig. 5A, the KFX-treated group had many autophagosomes compared with the control group. These data revealed that KFX could activate autophagy in SGC-7901 cells, resulting in an increase in autophagosomes in cells. In addition, we further examined the expression of the protein Beclin-1, which is a key protein in autophagy. SGC-7901 cells were treated with 0.1 and 1 µg/ml KFX for 48 h. As shown in Fig. 5B, the expression of Beclin-1 was significantly upregulated in the KFX groups compared with the control group. Moreover, autophagosome formation is mediated by the conjugation systems composed of ATG proteins, which culminate in the conjugation of ATG12 to ATG5 and conversion of a soluble form of microtubule-associated protein 1 light chain 3 (LC3)-I to another bound form, LC3-II (25). Thus, the ratio of LC3-II/LC3-I is a marker of autophagy in some studies (26,27). The ratio of LC3-II/LC3-I in the KFX-treated groups was significantly higher than the control group, and the 1 µg/ml KFX group showed the greatest effect (Fig. 5B and C). Furthermore, the autophagy activation in SGC-7901 cells was suppressed when treated with the ER stress inhibitor 4-PBA. Interestingly, the expressions of Beclin-1 and LC3-II/LC3-I were slightly increased in 4-PBA+KFX group (Fig. 5D). These results illustrated that KFX induces apoptosis through activation of ER stress and autophagy. However, the KFX autophagy effect may be controlled by ER stress activation. Quantitative analysis of the Western blot is shown in Fig. 5C and E.