title: Luteolin inhibits colorectal cancer cell epithelial-to-mesenchymal transition by suppressing CREB1 expression revealed by comparative proteomics study.
abstract: The cyclic AMP (cAMP) response element binding protein 1 (CREB1) is a promising target for cancer therapy. Here, we report that luteolin, a natural product, inhibits the expression of CREB1 at the transcriptional level and blocks epithelial-to-mesenchymal transition (EMT) of colorectal cancer cells. Treatment of colorectal cancer cells with luteolin induced mesenchymal-to-epithelial transition, reduced the expressions of mesenchymal markers and inhibited cell mobility in vitro. Through comparison of the proteomic profile of HCT-116 cells with and without luteolin treatment, we identified 366 differentially expressed proteins. Bioinformatics analysis revealed that downregulation of CREB1 plays a central role in this process. Immunoblot analysis verified that the protein levels of CREB1 and its downstream target genes were decreased in luteolin-treated cells. Moreover, forced expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells, suggesting the important role of CREB1 in this process. Furthermore, luciferase reporter assay and examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX) revealed that luteolin inhibits the expression of CREB1 at the transcriptional level. In summary, our results demonstrated that suppressing the expression of CREB1 is crucial in the mechanism-of-action of luteolin inhibiting EMT of colorectal cancer cells.
  
  
  
  It is no doubt that understanding the mechanism-of-action of natural products at the molecular level is important for their translational application. Proteomics is a powerful platform to explore the effects of natural products on the cells. In this study, we compared the proteomic profile of HCT-116 colorectal cancer cells with and without luteolin treatment to investigate the mechanism-of-action of luteolin against colorectal cancer cells. Subsequent bioinformatics analysis revealed that CREB1 could be one of the main targets of luteolin against colorectal cancer cells. Downregulation of CREB1 by luteolin affects glucagon signaling pathway and cAMP signaling pathway. The proteomics findings were verified with mechanistic analyses. We first identified that luteolin decreased the mRNA and protein levels of CREB1 and its downstream target genes. We then found that luteolin inhibits CREB1 expression at the transcriptional level by real-time PCR and luciferase reporter assay which confirmed by examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX). Finally, we generated CREB1-overexpressing stable cell line and showed that ectopic expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells and restored the expression levels of CREB1 target genes in colorectal cancer cells, and thereby demonstrated the critical role of CREB1 in the mechanism-of-action of luteolin against colorectal cancer. In summary, we revealed a novel mechanism-of-action of luteolin against colorectal cancer cell by the combination of proteomics discovery and mechanistic analyses.
authors.affiliation: Shanghai University of Medicine & Health Sciences Shanghai Sixth People's Hospital East Campus, Shanghai 201306, PR China; Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics (Ministry of Education of China), Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Tingyuan
authors.initials: T
authors.lastname: Lang
authors.affiliation: Shanghai University of Medicine & Health Sciences Shanghai Sixth People's Hospital East Campus, Shanghai 201306, PR China.
authors.forename: Bingwei
authors.initials: B
authors.lastname: Jin
authors.affiliation: Shanghai University of Medicine & Health Sciences Shanghai Sixth People's Hospital East Campus, Shanghai 201306, PR China.
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics (Ministry of Education of China), Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Singapore Eye Research Institute, Singapore 169856, Singapore; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore; Ophthalmology and Visual Sciences Academic Clinical Research Program, Duke-NUS Graduate Medical School, Singapore 169857, Singapore.
authors.forename: Roger W
authors.initials: RW
authors.lastname: Beuerman
authors.affiliation: Singapore Eye Research Institute, Singapore 169856, Singapore; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore; Ophthalmology and Visual Sciences Academic Clinical Research Program, Duke-NUS Graduate Medical School, Singapore 169857, Singapore.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Shanghai University of Medicine & Health Sciences Shanghai Sixth People's Hospital East Campus, Shanghai 201306, PR China; Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China. Electronic address: zzq72@163.com.
authors.forename: Zhiqi
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D002465:Cell Movement / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000473:pathology*; D019295:Computational Biology / Q000379:methods; D017362:Cyclic AMP Response Element-Binding Protein / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D015536:Down-Regulation / Q000187:drug effects; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D045325:HCT116 Cells; D006801:Humans; D047311:Luteolin / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000187:drug effects
chemical_list: C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; D047311:Luteolin
keywords: CREB1; Colorectal cancer; Epithelial-to-mesenchymal transition; Luteolin; iTRAQ quantitative proteomics