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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Luteolin inhibits colorectal cancer cell epithelial-to-mesenchymal transition by suppressing CREB1 expression
Yuan Liu1, Tingyuan Lang2, Bingwei Jin3
1Shanghai 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.
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.
Significance:
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.
Insights
Luteolin, a natural compound, inhibits colorectal cancer progression by suppressing cyclic AMP (cAMP) response element binding protein 1 (CREB1) expression at the transcriptional level. This action blocks epithelial-to-mesenchymal transition (EMT), offering a potential therapeutic strategy.
Area of Science:
- Molecular Oncology
- Natural Product Chemistry
- Cancer Therapeutics
Background:
- The cyclic AMP (cAMP) response element binding protein 1 (CREB1) is a key regulator implicated in cancer development and progression.
- Natural products offer a rich source of potential therapeutic agents for cancer treatment.
- Understanding the molecular mechanisms of natural products is crucial for their clinical application.
Purpose of the Study:
- To investigate the mechanism of action of luteolin, a natural product, against colorectal cancer cells.
- To determine the role of CREB1 in luteolin's anti-cancer effects.
- To elucidate how luteolin affects epithelial-to-mesenchymal transition (EMT) in colorectal cancer.
Main Methods:
- Proteomic profiling of HCT-116 cells treated with luteolin.
- Bioinformatics analysis to identify key molecular targets.
- Validation using immunoblot analysis, real-time PCR, luciferase reporter assays, and cell-based assays with CREB1 overexpression.
Main Results:
- Luteolin treatment led to mesenchymal-to-epithelial transition (MET) in colorectal cancer cells, reducing mesenchymal markers and inhibiting cell migration.
- Proteomic and mechanistic analyses identified CREB1 as a primary target, with luteolin downregulating its expression transcriptionally.
- Forced expression of CREB1 counteracted luteolin's inhibitory effects, confirming CREB1's critical role in luteolin's anti-cancer activity.
Conclusions:
- Luteolin effectively inhibits colorectal cancer cell progression by suppressing CREB1 expression at the transcriptional level.
- The downregulation of CREB1 by luteolin is a key mechanism underlying its ability to block EMT and induce MET.
- These findings highlight luteolin as a potential therapeutic agent for colorectal cancer, targeting the CREB1 pathway.
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