| Objective: To study the effects of Ar-Turmerone on proliferation,migration,invasion and apoptosis of human squamous cell carcinoma A431 cells,and to clarify the activity of Ar-Turmerone against squamous cell carcinoma of the skin.Whether its pro-apoptotic mechanism is achieved by the Notch1/Hes1/PTEN pathway.Methods:(1)Cell Counting Kit-8(CCK-8)method was used to detect the inhibition rate of proliferation: the concentration of 0,2.5,5,10,20,40,80,160 mg/L of Ar-Turmerone was applied to A431 cells.At 24,48,and 72 h,CCK-8 method was used to detect the proliferation inhibition rate of cells,and the half-inhibitory concentration(IC 50)of Ar-Turmerone on proliferation of A431 cells was calculated.(2)Giemsa staining observed apoptosis morphology: according to proliferation A431 cells were treated with 20,40,80 mg/L Ar-Turmerone for 24 h,and morphological changes were observed by Giemsa staining.(3)Scratch test and Transwell invasion assay were used to detect cell migration and invasion in vitro: According to the results of the CCK-8 experiment,the concentration of the drug which has less inhibition on cell proliferation is selected.5,10 mg / L of Ar-Turmerone was applied to A431 cells for 24 and 48 h.The in vitro migration ability of the cells was observed by scratch test.The in vitro invasion ability of cells was evaluated by Transwell chamber test.(4)Flow cytometry was used to detect cell withering death rate: 20,40,80 mg / L of Ar-Turmerone applied to cells for 48 h,flow cytometry to detect apoptosis rate;(5)real-time fluorescence quantitative polymerase chain reaction(real-time PCR,rt-PCR)and Western blotting(WB)were used to detect mRNA and protein expression: 20,40,80 mg/L of Ar-Turmerone were applied to cells for 48 h,and rt-PCR was used to detect the expression of Notch1,Hes1 and PTEN mRNA,WB detects the protein expression;(6)Ar-Turmerone regulate apoptosis through Notch1/Hes1/PTEN pathway:(1)Grouping and treatment: divided into blank group(A431 cells without any treatment),negative control group(negative siRNA Transfected A431 cells),Ar-Turmerone group(20 mg/L Ar-Turmerone applied to A431 cells for 48 h),siRNA group(siRNA transfected cells),siRNA + Ar-Turmerone group(with 20 mg/L Ar-Turmerone acting on transfected A431 cells for 48 h);(2)Transfecting A431 cells with Notch1 siRNA,The expression of Hes1 mRNA and protein was detected by grouping and processing.(3)A431 cells were also transfected with Hes1 siRNA,and the mRNA and protein expression of PTEN were detected by grouping and processing.(The above mRNA expression was detected by rt-PCR method.Protein expression was detected by WB method);(4)A431 cells were transfected with PTEN siRNA,grouped and processed according to 1 time,and apoptosis rate was detected by flow cytometry again.Results:(1)The IC 50 of Ar-Turmerone at 24,48 and 72 h were 124.67,98.82 and 72.16 mg/L,respectively.With the increase of the concentration of Ar-Turmerone and the prolongation of action time,A431 The proliferation inhibition rate of cells also increased gradually(P<0.05 and P<0.01),and it was time-dose-dependent.(2)After treatment of A431 cells with Ar-Turmerone,Giemsa staining showed the disappearance of normal cell morphology and cell gap.With the increase of Ar-Turmerone concentration,cell adherence decreased,nuclear fragmentation and cell morphology changed more obviously.(3)Ar-Turmerone 5,10 mg / L acted on A431 cells for 24 and 48 h,and inhibited in vitro.The ability of A431 cells to invade and migrate(P<0.05).(4)The apoptotic rate of A431 cells treated with 20,40,80 mg/L Ar-Turmerone was(8.30±0.36)%,(16.37±0.46)%,(30.48±1.08)%,compared with the control group(0.02±0.01)%,the apoptotic rate increased(P<0.01);(5)120,40,80 mg/L Ar-Turmerone acted on A431 cells for 48 h,Notch1,The mRNA and protein expressions of Hes1 and PTEN were higher than the control group in a dose-dependent manner(P<0.01).(6)(1)After silencing Notch1,the mRNA and protein expression of Hes1 in siRNA+ Ar-Turmerone group was lower than that of simple administration.(2)The expression of PTEN mRNA and protein in the siRNA+ Ar-Turmerone group was lower than that in the simple administration group(P<0.01).(3)After silencing PTEN,siRNA+ Ar-Turmerone group and simple administration group In comparison,the apoptotic rate was reduced(P < 0.01).Conclusion: Ar-Turmerone can inhibit the proliferation of human squamous cell carcinoma A431 cells and promote cell apoptosis;inhibit the migration and invasion in vitro;the mechanism of Ar-Turmerone promoting apoptosis of human squamous cell carcinoma A431 cells One is achieved through the path of Notch1/Hes1/PTEN. |
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