Figure 1.
β,β-Dimethylacrylshikonin inhibits SGC-7901 cells viability.
(A) The chemical structure of β,β-dimethylacrylshikonin (MW. 370.4). (B) Effects of β,β-dimethylacrylshikonin on cell viability inhibition of SGC-7901 cells. Cells were treated with different concentrations of β,β-dimethylacrylshikonin for 24 h and 48 h. The related cell viability was determined by MTT assay. The viability of the control group (0.1% DMSO) was set to 100%. Data represent the mean±SD obtained from three independent experiments. * indicated p<0.05 compared to control group respectively.
Figure 2.
β,β-Dimethylacrylshikonin induced apoptosis in SGC-7901 cells.
(A) Cell morphology was detected by using an invert microscope with 200×magnification. (B) DNA condensation (white arrow) was measured by DAPI stain and analyzed under a fluorescent microscope with 200×magnification. (C) The apoptotic status was evaluated by Annexin V-FITC binding assay. The lower right part (Annexin V-FITC+/PI−) was considered as early stage of apoptotic cells and top right part (Annexin V-FITC+/PI+) was considered as late stage of apoptotic cells. The lower left part (Annexin V-FITC−/PI−) was considered as viable cells and the upper left part (Annexin V-FITC−/PI+) was considered as necrotic cells.
Figure 3.
β,β-Dimethylacrylshikonin induced mitochondrial events associated with apoptosis in SGC-7901 cells.
Western blotting analysis for anti-apoptotic proteins: Bcl-2, Bcl-xL, cIAP-2, XIAP and survivin(A), and pro-apoptotic proteins: Bax, Bak (B), and cleaved PARP, cleaved caspase-9, cleaved caspase-8, cleaved caspase-3(C) in whole cell extracts after the SGC-7901 cells were treated with β,β-dimethylacrylshikonin for 24 h. Protein levels of actin were also measured as controls. (D) Detection of mitochondrial membrane potential by flow cytometry. SGC-7901 cells were treated with or without β,β-dimethylacrylshikonin (10 µmol/L) for 24 h and were stained with JC-1 for 15 min at 37°C and subjected to flow cytometry. (E) SGC-7901 cells were fixed and labeled for cytochrome c (green) and DNA (blue). (F) SGC-7901 cells were treated with β,β-dimethylacrylshikonin in the presence or absence of Z-VAD-FMK (10 µmol/L) for 24 h. Protein extracts were prepared and subjected to Western blot assay using antibody against cleaved caspase-3, cleaved PARP. Protein levels of actin were also measured as controls. (G) SGC-7901 cells were treated with β,β-dimethylacrylshikonin in the presence or absence of Z-VAD-FMK (10 µmol/L) for 24 h. The apoptotic status was determined by Annexin V-FITC binding assay. The lower right part (Annexin V-FITC+/PI−) was considered as early stage of apoptotic cells and top right part (Annexin V-FITC+/PI+) was considered as late stage of apoptotic cells. The lower left part (Annexin V-FITC−/PI−) was considered as viable cells and the upper left part (Annexin V-FITC−/PI+) was considered as necrotic cells.
Figure 4.
Effects of β,β-Dimethylacrylshikonin on MAPK pathways.
SGC-7901 cells were treated with the indicated β,β-dimethylacrylshikonin concentration or the indicated interval, total cellular extracts were prepared and subjected to Western blot assay to measure levels of phosphorylated forms of ERK(A), JNK(B) and p38(C). Membranes were reprobed with antibodies against total ERK, JNK and p38 for normalization.
Figure 5.
β,β-Dimethylacrylshikonin induced SGC-7901 cells apoptosis was mediated through ERK activation.
(A) SGC-7901 cells were pretreated or not pretreated with U0126 (10 µmol/L) then added with DMSO (vehicle) or β,β-dimethylacrylshikonin (10 µmol/L) for 2 h, 24 h respectively. Protein extracts were prepared and subjected to Western blot assay to measure levels of phosphorylated ERK. Protein levels of total ERK were also measure as controls. (B) SGC-7901 cells were pretreated or not pretreated with U0126 (10 µmol/L) then added with DMSO (vehicle) or β,β-dimethylacrylshikonin (10 µmol/L) for 24 h. Protein extracts were prepared and subjected to Western blot assay using antibody against cleaved caspase-3, cleaved PARP. Protein levels of actin were also measured as controls. (C) SGC-7901 cells were treated with β,β-dimethylacrylshikonin in the presence or absence of U0126 (10 µmol/L) for 24 h. The apoptotic status was determined by Annexin V-FITC binding assay. The lower right part (Annexin V-FITC+/PI−) was considered as early stage of apoptotic cells and top right part (Annexin V-FITC+/PI+) was considered as late stage of apoptotic cells. The lower left part (Annexin V-FITC−/PI−) was considered as viable cells and the upper left part (Annexin V-FITC−/PI+) was considered as necrotic cells. (D) SGC-7901 cells were pretreated with or without Z-VAD-FMK (10 µmol/L) were further incubated with β,β-dimethylacrylshikonin (10 µmol/L) for 24 h. Protein extracts were prepared and subjected to Western blot assay to measure levels of phosphorylated ERK. Protein levels of ERK were also measured as controls.