RESUMO
OBJECTIVE:To study the effect and mechanism of physcion 8-O-β-glucopyranoside(PG)on the apoptosis of skin melanoma A375 cells. METHODS:After A375 cells were treated by PG with 0,10,20,50 μg/mL for 24,48,72 h,CCK-8 method was adopted to determine the survival rate of cells. After A375 cells were treated by PG with 0(control),20,50 μg/mL for 48 h,flow cytometry was used to detect the apoptosis rate of cells with membrane protein Ⅴ/propidium iodide (PI) double staining. Immunoblotting was used to detect the protein expressions of Caspase-3 and polyadenyl adenine diphosphate ribose poly-merase (PARP) and protein expressions of cytochrome C inside and outside mitochondria. After A375 cells were treated by PG with 0 (control),5,10 μmol/L for 48 h,enzyme substrate method was used to determine the activities of Caspase-8 and Cas-pase-9. RESULTS:PG can effectively decrease the survival rate of A375 cells. Compared with control,apoptosis rate of cells was obviously increased after treated by PG with 20,50 μg/mL(P<0.01);protein expressions of Caspase-3,PARP in cells and cyto-chrome C in cell matrix were obviously enhanced(P<0.05 or P<0.01);and protein expression of cytochrome C in mitochondria was obviously weakened(P<0.05 or P<0.01). Caspase-9 activity in cells was obviously enhanced after treated by PG with 5,10μmol/L(P<0.05 or P<0.01);and Caspase-8 activity had no obvious changes. CONCLUSIONS:PG can inhibit activity of A375 cells and promote its apoptosis,and its pro-apoptotic effects is achieved by destructing mitochondrial membrane potential and pro-moting cytochrome C outflow.
RESUMO
OBJECTIVE:To study the effect and mechanism of physcion 8-O-β-glucopyranoside(PG)on the apoptosis of skin melanoma A375 cells. METHODS:After A375 cells were treated by PG with 0,10,20,50 μg/mL for 24,48,72 h,CCK-8 method was adopted to determine the survival rate of cells. After A375 cells were treated by PG with 0(control),20,50 μg/mL for 48 h,flow cytometry was used to detect the apoptosis rate of cells with membrane protein Ⅴ/propidium iodide (PI) double staining. Immunoblotting was used to detect the protein expressions of Caspase-3 and polyadenyl adenine diphosphate ribose poly-merase (PARP) and protein expressions of cytochrome C inside and outside mitochondria. After A375 cells were treated by PG with 0 (control),5,10 μmol/L for 48 h,enzyme substrate method was used to determine the activities of Caspase-8 and Cas-pase-9. RESULTS:PG can effectively decrease the survival rate of A375 cells. Compared with control,apoptosis rate of cells was obviously increased after treated by PG with 20,50 μg/mL(P<0.01);protein expressions of Caspase-3,PARP in cells and cyto-chrome C in cell matrix were obviously enhanced(P<0.05 or P<0.01);and protein expression of cytochrome C in mitochondria was obviously weakened(P<0.05 or P<0.01). Caspase-9 activity in cells was obviously enhanced after treated by PG with 5,10μmol/L(P<0.05 or P<0.01);and Caspase-8 activity had no obvious changes. CONCLUSIONS:PG can inhibit activity of A375 cells and promote its apoptosis,and its pro-apoptotic effects is achieved by destructing mitochondrial membrane potential and pro-moting cytochrome C outflow.