Effects Of Sulforaphane On Proliferation And Apoptosis Of Acute Myeloid Leukemia Cells Through Notch Signaling Pathway And Its Mechanism

BackgroundAcute myeloid leukemia（AML）is a malignant proliferative disease of the hematologic system.It is expensive to treat and has a high mortality rate,which brings a huge economic and social burden to human beings.With the increase in the global incidence of tumors year by year,whether it is the treatment of tumor drugs,surgical options and multi-mode treatment methods have shown a higher and more urgent demand,researchers and clinical staff have been actively exploring the treatment methods and means of acute myeloid leukemia.In recent years,the induced therapeutic effect of phytochemical drugs on cancer has attracted more and more attention.Sulforaphane（SFN）is a natural active substance isothiocyanate,which exists in a variety of forms and is mainly rich in cruciferous plants.Years of research have found that SFN shows great potential in the prevention and treatment of cancer,but there is less research in the treatment of leukemia.The Notch signaling pathway is a conserved receptor-ligand signaling pathway that plays an important regulatory role in cell proliferation and apoptosis,affecting the formation and function of tissues and organs.Studies have demonstrated that abnormal activation of the Notch pathway is closely related to the onset and development of tumors,and blocking or inhibiting the abnormally activated Notch pathway is expected to become a method of treating malignant tumors.（3,5-Difluorophenylacetyl）-L-alanine-L-2-phenylglycine tert-butyl ester（DAPT）is an effective inhibitor of the Notch pathway,a potent inhibitor of γ secretory enzymes in the Notch pathway,which can block the shearing of γ secretion enzymes on Notch1,thereby blocking its activation of downstream genes HES1 and c-MYC.In this study,in vitro and in vivo experiments were used to systematically explore the regulatory role of SFN on proliferation and apoptosis of acute myeloid leukemia cells and the role of the Notch pathway in it.The effects of SFN on cell proliferation and apoptosis were further investigated through the application of Notch pathway blockers.It is initially revealed that SFN provides new drugs,new targets and new theoretical support for the treatment of acute myeloid leukemia through the Notch pathway.ObjectiveBy studying the effect of SFN on the proliferation and apoptosis of acute myeloid leukemia cells KG1 a and KG1 in vitro and in vivo,this experiment clarifies its potential mechanism of action,and provides new adjuvant therapeutic drugs,new targets and new theoretical support for the treatment of leukemia.Method1.Cell Counting Kit-8（CCK8）experiments detected the effect of SFN on the proliferation viability of KG1 and KG1 a cells.2.Colony formation experiments detect the effect of SFN on the colony formation of leukemia cells.3.Flow cytometry was used to detect the effect of SFN on the apoptosis of acute myeloid leukemia KG1 and KG1 a cells.4.Transcriptome sequencing to detect difference genes before and after SFN action KG1 a cells.5.Real-time PCR（qRT-PCR）experiments detected the m RNA levels of Notch pathway correlated factors Notch1,RBPJ,HES1,and c-MYC after SFN action.6.Western Blot（WB）experiments detected the expression of Notch pathway-related proteins Notch1,RBPJ,HES1,c-MYC,and apoptotic-related proteins Bax,Cleaved Caspase-3,and Bcl-2 after SFN acted on KG1 and KG1 a.7.The Notch signaling pathway inhibitor DAPT was used to block the Notch pathway,and the effects of SFN on the proliferation and apoptosis of KG1 and KG1 a cells were detected again by CCK8 experiment,colony formation experiment and flow cytometry.8.Mouse subcutaneous tumor model was constructed to detect the effect of SFN on tumor proliferation,and immunohistochemistry（IHC）staining detected the effect of SFN on the expression of Notch pathway-related proteins Notch1,RBPJ,HES1,and apoptoticrelated proteins Bax and Bcl-2.Result1.SFN inhibited cell viability（P < 0.05）and cell colony formation（P < 0.01）of KG1 and KG1a cells,and there were statistical differences between 4,8 and 12 μM compared with the control group.2.The results of flow cytometry and WB experiments showed that SFN could induce apoptosis of KG1 a and KG1 cells in a dose-dependent manner,and SFN could upregulate the expression of the pro-apoptotic proteins Bax and Cleaved Caspase-3 in the intrinsic apoptosis pathway of KG1 a and KG1（P < 0.05）,and down-regulate the expression of the anti-apoptotic protein Bcl-2（P < 0.05）。3.The transcriptome sequencing results showed that 207 genes were significantly upregulated and 237 genes were significantly down-regulated after SFN treatment in KG1 a cells.Notch pathway-related factors Notch1,HES1,c-MYC and RBPJ were significantly different.4.Real-time PCR and Western Blot experimental results showed that SFN can inhibited the expression of Notch pathway related factors Notch1,HES1 and c-MYC at m RNA and protein levels in a dose-dependent manner（P < 0.05）.5.After the Notch pathway was blocked by an inhibitor（DAPT）,the regulatory ability of SFN to KG1 and KG1 a cell proliferation（P < 0.05）,colony formation（P < 0.01）and apoptosis was weakened（P < 0.05）.6.In vivo experiments showed that SFN could inhibit tumor growth（P < 0.05）;IHC staining showed that the expression of Notch1,HES1,Bcl-2 decreased after SFN action,and the expression of Bax increased after SFN action（P < 0.01）.ConclusionSFN induces apoptosis of acute myeloid leukemia KG1 and KG1 a cells by regulating Notch pathway and inhibits proliferation of acute myeloid leukemia cells.