Mechanism Of Annonaceous Acetogenins(ACGs) Inhibiting The Proliferation Of Oral Squamous Cell Carcinoma SCC15 Cells

BackgroudOral Squamous Cell Carcinoma(OSCC),accounting for over 90%of Head and Neck Squamous Cell Carcinoma(HNSCC).is one of the most common human malignancies,and its pathogenesis is still unclear.TCGA database showed that epigenetic modification proteins NSD1,EZH2 and cell fate determinant Notchl were found to have high frequency mutations in HNSCC,indicating that they may play a crucial role in the formation and development of OSCC.OSCC has the characteristics of strong infiltration,early cervical lymph node metastasis and high recurrence rate.Surgical resection is the main clinical treatment,supplemented by radiotherapy and chemotherapy.Patients with OSCC have a poor quality of postoperative life and poor outcome.Therefore,it is important to explore new drugs with clinical value.Our previous studies have shown that Annonaceous acetogenins(ACGs)inhibited OSCC cells'proliferation,but the mechanism is unclear.PurposeTo elucidate the role and mechanism of NSD1,EZH2 and Notch1 in ACGs-mediated OSCC cell proliferation and provide new strategies and targets for the diagnosis and treatment of OSCC.Methods1.Analysis the effects of ACGs on the proliferation,apoptosis,cycle,cloning and migration of SCC15 cells in vitro.2.qRT-PCR and Western Blot were used to verify the effect of ACGs on the expression of NSD1,EZH2 and Notchl in SCC15 cells.3.The stable OSCC cell lines with knockdown or overexpressed of NSD1,EZH2 and Notchl were constructed,and MTT and flow cytometry were used to analysis the effects of ACGs on stable cells' proliferation,apoptosis and cell cycle.The differential expression of NSD1,EZH2 and Notchl in the above stable strains was detected by Western Blot4.MTT and Western Blot were used to analysis the proliferation and expression of NSD1,EZH2 and Notchl in SCC15 cells treated with EZH2 inhibitor EPZ6438.5,Whole transcriptome sequencing was used to analyze the effect of ACGs on transcriptome level of SCC15 cells.Results1.ACGs significantly inhibited the proliferation,cloning and migration of SCC15 cells.2.qRT-PCR and Western Blot results showed that in SCC15 cells,ACGs up-regulated the expression of NSD1,inhibited the expression of EZH2 and its downstream target gene c-Myc,and inhibited the activation of Notchl signaling pathway.3.Down-regulation of NSD1 antagonized proliferation inhibition and apoptosis mediated by ACGs on SCC 15 cells.4.Overexpression of EZH2 antagonized ACGs mediated proliferation inhibition and cell cycle arrest on SCC15 cells and promoted apoptosis.Overexpression of NlICD can antagonize ACGs mediated proliferation inhibition,and it inhibited both apoptosis and cell cycle arrest.5.After NSD1 was knockdown in SCC15 cells,H3K36me2 level decreased,while EZH2 and H3K27me3 level increased,and Notchl signaling pathway was activated.Overexpression of EZH2 in SCC 15 cells increased H3K27me3 level and reduced the expression of NSD1 and H3K36me2 level,inhibited Notchl signaling pathway.Overexpression of N1ICD in SCC15 cells activated Notchl signaling pathway,reduced the expression of NSD1 and H3K36me2 level,and although the expression of EZH2 did not increase and H3K27me3 level was increased.6.EPZ6438,an inhibitor of EZH2,inhibited the proliferation of SCC15 cells,and significantly inhibited the expression of EZH2 and H3K27me3 level,increased the expression of NSD1 and H3K36me2 level,and inhibited the activation of Notch1 signaling pathway.7.The whole transcriptome sequencing showed that ACGs significantly up-regulated 5,315 genes and significantly down-regulated 5,193 genes,which mainly involved in Hippo,Hedgehog,TNF,apoptosis and cell cycle signaling pathways.Conclusions1.In vitro experiments showed that ACGs inhibited the proliferation of SCC15 cells by up-regulating NSD1 and down-regulating EZH2 and Notchl,and that NSD1,EZH2 and Notchl regulated each other and mediated the inhibition of ACGs on the proliferation of SCC15 cells.2.ACGs up-regulate genes related to the biological process such as ribosome,oxidative phosphorylation andendoplasmic reticulum protein synthesis and down-regulate DNA repair,ribosome biosynthesis,RNA methylation and other process-related genes.The differential gene mainly involved in TNF,cell apoptosis and cell cycle signal pathways.