Evaluation The Activity And Molecular Mechanism Of Sulforaphene Blocking Esophageal Squamous Cell Carcinoma

Esophageal cancer is the sixth leading cause of cancer-related deaths worldwide,with a high incidence in many Asian regions,including China.Esophageal cancer is principally comprised of two pathologically distinct diseases,esophageal adenocarcinoma(EAC)and esophageal squamous cell cancer(ESCC),which accounts for 70%of esophageal cancer cases.The most typical symptoms of ESCC are pain behind the breastbone or between the shoulder blades,dysphagia,and the resulting weight loss and malnutrition.As symptoms may be noticeable only when the tumor has reached a relatively advanced stage,the overall prognosis is disappointing,and the 5-year survival probability is barely 10%-15%.Therefore,comprehension of the epidemiology,pathology,and molecular mechanisms of ESCC,and developing new strategies are earnestly required to raise patient prognosis.A number of studies have confirmed that isothiocyanates from cruciferous vegetables have anti-cancer effects,which can suppress the growth of various tumor cells and induce cell apoptosis.Sulforahene(SFE),as an isothiocyanate,has received more and more attention in recent years.It can block multiple cancer-promoting signaling pathways,and upregulate key antitumor factors that are inactivated in cancer cells to promote cell cycle arrest and apoptosis,achieving the effects of inhibiting tumor proliferation and metastasis.This research mainly focuses on the activity and mechanism of SFE against the occurrence and development of ESCC.We first performed the xenograft tumor assay,and noticed that there was no obvious difference between the body weight of mice,while the volume and weight of tumor lumps in SFE-treated mice were clearly lighter than that of mice treated with saline,which proved SFE could successfully stop the growth of ESCC in vivo without harming the health of nude mice.We then examined the role of SFE in two ESCC cell lines with cell viability and colony formation assays,finding a time-dependent and dose-dependent inhibitory effect of SFE on cell proliferation.FACS analyses and western blotting results indicated that SFE inhibition of ESCC cells proliferation was achieved by inducing G2/M cell cycle arrest and cell apoptosis.Subsequently,we proved SFE could also significantly inhibit cell invasion and migration through scratch healing and transwell assays.In view of the strong anticancer activity of SFE,we then tried to identify genes and signaling pathways that connected SFE with the progression of ESCC cells through microarrays to detect changes in gene expression in ESCC cells with or without SFE treatment.The microarray analyses and subsequent verification experiments showed that SCD,CDH3 and p53 pathways were the genes and pathway with the most significant changes in expression after SFE treatment,which were most likely to be involved in SFE inhibiting ESCC progression.We confirmed through rescue experiments that SFE actually suppressed ESCC metastasis by regulating the expression of SCD and CDH3.Databases predicted and subsequent western blotting proved that the activity of the Wnt/?-catenin pathway is regulated by the two genes,and once SFE inhibits gene expression,the Wnt/?-catenin pathway is inactivated.We found that SCD and CDH3 had no obvious effects on cell proliferation,which meant that there were other targets of SFE involved in this process.The post-transcriptional regulation of p53 by SFE reminded us of a common p53 regulator,the p38 pathway,and MAP2K3 and GADD45B which are the activators of this pathway were significantly increased by SFE treatment.Decreasing the expression of MAP2K3 and GADD45B reversed the inhibitory effect of SFE on ESCC cell proliferation,and co-IP results indicated that GADD45B,a known target of p53,could promote the phosphorylation level of MAP2K3.Thus,we could drew a conclusion that SFE activated the positive feedback loop GADD45B-MAP2K3-p38-p53 to suppress ESCC proliferation.Overexpression of SCD and CDH3 or downregulation of GADD45B and MAP2K3 could affect the inhibition of SFE on ESCC progression,yet not fully offset.Considering there must be other vital effectors,we reprocessed the microarray data by GSEA,finding that the NF?B pathway was associated with SFE inhibition of ESCC proliferation and metastasis.We downloaded and processed two datasets from the Gene Expression Omnibus(GEO)database,and identified CXCL10,INHBA,TNFAIP3 and PLAU as the target genes of SFE in ESCC.The Cistrome database predicts that p65,a key regulatory protein in the NF?B pathway,plays a transcriptional activation effect by binding to the promoters or enhancers of these four genes.We selected TNFAIP3 and PLAU with the most obvious changes in expression after SFE treatment for verification,confirming that SFE regulated gene expression by blocking the transcription-promoting activity of the NF?B pathway and eventually inhibited ESCC progression.All the results suggest that SFE has a strong inhibitory effect on ESCC development.In this study,we shed light on the mechanism involved in the regulation of SFE on ESCC cell progression,proving that SFE is a promising chemotherapeutic agent that may be used to treat ESCC in the future.