br Results br Bufothionine reduces cell viability in
3.1. Bufothionine reduces cell viability in MKN28 and AGS cells
The chemical structure of bufothionine is displayed in Fig. 1A. Based on the preliminary experimental results, doses of 20, 50 and 100 μg/ml were chosen to test the eﬀect of bufothionine on the cell viability of MKN28 and AGS cells. The results showed that MKN28 3X FLAG were more resistant to bufothionine-induced cell death. As shown in Fig. 1B, MKN28 cells displayed a marked loss in cell viability following bufothionine treatment at 50 and 100 μg/ml for 24 h. As for the case of 48 h treatment, bufothionine treatment at 20, 50 and 100 μg/ml caused significant reduction in cell viability. With regard to AGS cells, bu-fothionine treatment at 20, 50 and 100 μg/ml produced a dose-depen-dent cell death at both 24 and 48 h (Fig. 1B). The eﬀect of bufothionine on the viability of normal gastric epithelial cells GES-1 was then de-termined. As shown in Fig. 1C, bufothionine treatment at 100 μg/ml significantly decreased the cell viability at 48 h. Similarly, bufothionine also eﬀectively suppressed the clongenic potential of MKN28 and AGS cells in a dose-dependent manner (S2).Based on these findings, bu-fothionine treatment at 20 and 50 μg/ml for 48 h was chosen for the subsequent experiment. Compromised cell membrane integrity is a
feature of cell death. Hence, LDH leakage assay was performed to ex-amine whether bufothionine could destroy the cell membrane. As shown in Fig. 1D, bufothionine promoted LDH leakage in both MKN28 and AGS cells in a concentration-dependent manner. These findings supported the hypothesis that bufothionine promoted GC cell death.
3.2. Bufothionine facilitates caspase-dependent apoptosis in MKN28 and AGS cells
The role of apoptosis in bufothionine-induced cell death was first examined by Hoechst/PI staining. As shown in Fig. 2A, fragmented nuclei and condensed chromatins were observed in MKN28 and AGS cells following bufothionine treatment for 48 h. The percentage of apoptotic cell was then determined by flow cytometry. As shown in Fig. 2B, bufothionine treatment at 20 and 50 μg/ml significantly in-creased the apoptotic cell population in MKN28 cells to almost 10% and 20%, respectively. The results also showed that the level of cell apop-tosis in AGS cells induced by bufothionine treatment was higher com-pared to that ofMKN28 cells (Fig. 2B). The pro-apoptotic activities of bufothionine on MKN28 and AGS cells were therefore further validated using apoptosis-related proteins. Elevated cleavage of caspase-3, cas-pase-8 and caspase-9 was observed in GC cells following bufothionine treatment (S3). In addition, bufothionine treatment led to a con-centration-dependent upregulationofBcl-2 and downregulation of Bax, which further revealed the pro-apoptotic activities of bufothionine (Fig. 2C). Furthermore, bufothionine did cause obvious change on the
Fig. 5. Eﬀect of bufothionine on MKN28 and AGS cells after transfection with PIM3 overexpressing vector. GC cells were transfected with PIM3 overexpressing vector and bufothionine treatment was started 48 h after transfection. A. 48 h post transfection, the protein level of PIM3 was assessed by western blotting. B. Eﬀect of the combination of bufothionine and ectopic PIM3 expression on cell viability was assessed by CCK-8 assay. C. Cytotoxicity of the combination of bufothionine and ectopic PIM3 expression was assessed by LDH assay of cell viability. D. Pro-apoptotic activity of the combination of bufothionine and ectopic PIM3 expression was examined using Annexin V-FITC/PI apoptosis kit. E. Eﬀect of the combination of bufothionine and ectopic PIM3 expression on the protein levels of cleaved caspase-3, Bax and Bcl-2 was examined by western blotting. **P < 0.01.
expression levels of apoptosis inducing factor (AIF) and endonuclease G (Endo G) (S3), which are two key genes that involved in caspase-in-dependent manner. MKN28 and AGS cells were pretreated with specific caspase inhibitor z-VAD-FMK at 10 μM for 2 h before incubation with bufothionine at 50 μg/ml for 48 h. As shown in Fig. 2D, z-VAD-FMK did not cause any significant change on the number of apoptotic cells. However, pretreatment with z-VAD-FMK significantly blocked the pro-apoptotic activities of bufothionine in both MKN28 and AGS cells, which indicated that bufothionine promoted apoptotic cell death in a caspase-dependent fashion.
3.3. Bufothionine downregulates PIM3 in MKN28 and AGS cells
ITraq proteomic analysis was conducted to identify the potential target of bufothionine. After incubation with 50 μg/ml bufothionine for 48 h, the diﬀerentially expressed proteins in AGS cells are listed in Fig. 4A. The results from iTraq proteomic analysis showed that PIM3 expression was remarkably suppressed by bufothionine. Hence, PIM3 mRNA level was determined by qRT-PCR in both MKN28 and AGS cells. After bufothionine treatment, the mRNA level of PIM3 in GC cells was downregulated in a concentration dependent manner (Fig. 3B). Corre-spondingly, the PIM3 protein level was also repressed by bufothionine treatment (Fig. 3C).