Honokiol Inhibits The Growth Of SK-BR-3 Cells And Its Mechanism

Objective:To study the effects of honokiol on the proliferation,apoptosis,invasion and migration of human SK-BR-3 cells and the potential molecular mechanism.Methods:1.Treat human breast cancer SK-BR-3 cells with different concentrations of honokiol for 48 h and 72 h,and detect the total number of living cells and changes in proliferation by crystal violet staining and MTT methods,and select the best concentration range and drug effect time,while using MCF-10 A and HK-2 cells to detect drug toxicity.Changes in cell apoptosis rate are detected by flow cytometry and Hoechst assay.Wound healing analysis was performed to detect the migration ability of SK-BR-3cells.The invasion ability of cells was studied by Transwell assay.2.Western blot was used to detect the expression levels of proliferationassociated protein(PCNA),migration and invasion-related protein matrix metalloproteinase-2(MMP-2),Vimentin,E-cadherin.Apoptosis-related proteins Bcl-xl,Bax,caspase 3,and cleaved caspase 3(CC3).At the same time,the luciferase reporter assay(Luciferase assay)was used to analyze the activation degree of the relevant signal pathways and screen out the signal pathways that may be involved in this inhibition process.Western blot was used to detect the expression levels of β-catenin,c-myc,Gsk-3,p-Gsk3β,ERK1/2,p-ERK1/2.Results:1.Crystal violet staining,MTT assay revealed that honokiol could significantly inhibit the proliferation of breast cancer cells SK-BR-3,but had less toxicity to normal cells(MCF-10 A,HK-2).Flow cytometry and Hoechst staining experiments confirmed that honokiol can promote the apoptosis of SK-BR-3 cells.The cell cycle was significantly blocked in G0/G1 phase.Scratch healing experiment and Transwell chamber method show that honokiol can inhibit the migration and invasion of SK-BR-3 cells.The above inhibitory effects were concentration-dependent.2.The expressions of Proliferation-related protein(PCNA)and cyclerelated protein Cyclin D1 were significantly decreased.At the same time,the expression of anti-apoptotic protein Bcl-xl reduced,while the expressions of Bax and cleaved-caspase3 increased.We also found that the expression levels of invasion related protein matrix metalloproteinase-2(matrix metalloproteinase-2,MMP-2)and Vimentin were decreased,while the expression of E-cadherin was increased.Luciferase reporter gene assay indicated that the activity of transcription factors of Wnt/β-catenin and ERK1/2/MAPK signaling pathways decreased after honokiol treatment.We also found a decrease in the expression levels of the Wnt/β-catenin signaling pathway-related proteins β-catenin and c-Myc.The expression level of upstream Gsk-3β remained unchanged while the expression level of p-Gsk-3β decreased.The protein level of total ERK1/2 was not significantly changed,but the levels of p-ERK1/2(T202/Y204)were strikingly decreased.Conclusion:Honokiol can promote the apoptosis of SK-BR-3 cells and inhibit the proliferation,migration and invasion of human breast cancer SK-BR-3 cells.The underlying mechanism may be through inhibiting the activation of the Wnt and MAPK/ERK signaling pathway.