| Objective:To study the effects and mechanisms of Ginkgo biloba exocarp extracts(GBEE)inducing apoptosis and autophagy in Lewis lung cancer(LLC)cells by establishing LLC solid tumor model in C57BL/6J mice and culturing LLC cells in vitro.Methods:The LLC solid tumor model was established in C57BL/6J mice.The tumor-bearing mice were randomly divided into 5 groups.A normal control group without tumor cells was established additionally.There were 10 mice in each group,and they were dosed 24 h after inoculation.The GBEE(50,100,200 mg/kg b.w.)groups were dosed by intragastric gavage(i.g.).The mice in positive control group were intraperitoneal(i.p.)injected with 20 mg/kg(b.w.)cyclophosphamide(CPA).The model control group and the normal control group were both given normal saline(NS)by i.g..All the groups were dosed at a volume of 0.1mL/10g(b.w.),once a day for 18d.The day after the last administration,the transplanted tumors were stripped and the inhibition rate was detected.In vitro experiments,MTT method was applied to detect the effects of GBEE on LLC cells and primary cultured mouse lung cells.Annexin V-FITC/PI method was used to detect the apoptosis rate of LLC cells.Rhodamine 123 method was used to detect the Mitochondrial transmembrane potential(MTP).Monodansylcadaverine(MDC)staining method was used to detect the acidic vacuoles in cells.The transmission electron microscope(TEM)to confirm the formation of autophagosomes.Quantify reverse transcription-polymerase chain reaction(qRT-PCR)was applied to detect the mRNA levels of Beclinl and Atg5.Western Blot was used to detect the expression of Bax,Bcl-2,Cyt C,Caspase-3,Fas,FasL,Beclinl,Atg5,LC3 Ⅰ/Ⅱ,p-AMPK,AMPK,p-mTOR,mTOR,p-p70S6k,p70S6K and MAPK proteins in the corresponding parts of LLC cells.Results:GBEE(50-200mg/kg)inhibited the growth of LLC transplanted tumors with a dose-effect relationship.GBEE(5-160 μg/mL)inhibited the proliferation of LLC cells in vitro with the half maximal inhibitory concentration(IC50)value of 162.43 μg/mL,while it had no significant inhibitory effects on the primary cultured mouse lung cells.After GBEE(10,20 and 40 μg/mL)acted on the LLC cells,the apoptosis rate was increased and the MTP was decreased.The ratio of Bax/Bcl-2 was increased in the cells.Meanwhile,it also promoted the translocation of Bax/Bcl-2 in mitochondrial membrane and the release of Cyt C from mitochondria to cytosol.In addition,it up-regulated the cleaved-Caspase-3 protein expression.The protein levels of Fas and FasL were both increased.The formation and activation of acidic vacuoles were increased by the action of GBEE(10,20 and 40 μg/mL)on LLC cells.The autophagosomes were also increased.Meanwhile,it up-regulated both the mRNA and protein levels of Beclinl and Atg5.The ratio of LC3-I/LC3-Ⅱ protein was down-regulated.In addition,the protein levels of p-p38 and p-AMPK was increased,and the p-ERK1/2,p-JNK1/2.p-mTOR and p-p70S6K was decreased.But the p38.ERK1/2,JNK1/2,AMPK,mTOR and p70S6K proteins were not significantly changed.Conclusions:’I he inhibitory effect of GBEE on LLC is associate with inducing apoptosis and autophagy in LLC cells.Those effects may involve the MAPK signaling pathways and AMPK/mTOR/p70S6k signaling pathway. |
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