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Published on: January 11, 2019
Transcriptome analysis upon potassium usnate exposure reveals ATF3-induced apoptosis in human gastric and colon
Kyung Hyun Yoo1, Dae-Hwan Kim2, Sumin Oh1
1Department of Biological Science, Sookmyung Women's University, Seoul, Republic of Korea.
Background:
Potassium usnate (KU), a water-soluble form of usnic acid, shows anticancer activity. However, the underlying mechanisms have not been fully elucidated.
Purpose:
We aimed to identify the pathways involved in anticancer effects of KU in human gastric cancer (GC) and colorectal cancer (CRC) cells using RNA-sequencing (RNA-seq) based transcriptome analysis.
Study Design:
We analyzed the cytotoxic effects of KU to identify the common molecular events in GC and CRC cells upon KU exposure using unbiased approaches.
Methods:
Cell viability assays and western blot experiments were used to examine apoptotic changes, cell cycle arrest, and endoplasmic reticulum (ER) stress-induced cellular responses in KU-treated cells. Total RNA from KU-treated human GC and CRC cells was prepared for RNA-seq analysis. Gene ontology term and gene set enrichment analyses were used to identify the key mediators of the cytotoxic effects of KU. The expression of ER stress-induced apoptotic markers was evaluated using quantitative reverse-transcription PCR and western blot analysis. Chromatin immunoprecipitation assays for ATF3 and H3K27ac, and ATF3 knockdown were employed to verify the underlying molecular mechanisms. The inhibitory effect of KU on tumor growth in vivo was validated with metastatic tumor nodule formations in a mouse liver model.
Results:
KU exerted cytotoxicity in human GC and CRC cells through the activation of the ER stress-induced apoptotic pathway. KU stimulated ATF3 expression, an important mediator of molecular events of apoptosis. ATF3 binds to the promoter region of ATF3, CHOP, GADD34, GADD45A, DR5, and PUMA genes and subsequently promoted apoptotic events. Knockdown of ATF3 significantly reduced the expression of ATF3 target genes and the cytotoxic effects of KU. The intraperitoneal injection of KU induced ATF3 and the apoptosis of implanted colon cancer cells, resulting in reduced metastatic tumor growth in the mouse livers.
Conclusion:
KU exerts cytotoxic effects in human GC and CRC cells by triggering ER stress-induced apoptosis via an ATF3 dependent pathway.
Insights
Potassium usnate (KU) triggers cancer cell death by activating endoplasmic reticulum (ER) stress-induced apoptosis. This process is mediated by the ATF3 pathway, which is crucial for KU's anticancer effects in gastric and colorectal cancer cells.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- Potassium usnate (KU), a derivative of usnic acid, exhibits anticancer properties.
- The precise molecular mechanisms underlying KU's anticancer activity remain largely uncharacterized.
Purpose of the Study:
- To elucidate the molecular pathways responsible for the anticancer effects of KU in human gastric cancer (GC) and colorectal cancer (CRC) cells.
- To utilize RNA-sequencing (RNA-seq) based transcriptome analysis for pathway identification.
Main Methods:
- RNA-sequencing (RNA-seq) was employed to analyze transcriptomic changes in KU-treated GC and CRC cells.
- Cell viability assays, western blotting, and quantitative reverse-transcription PCR were used to assess apoptosis, cell cycle arrest, and ER stress.
- Chromatin immunoprecipitation assays and ATF3 knockdown were performed to validate molecular mechanisms.
- In vivo studies evaluated KU's inhibitory effect on tumor growth in a mouse liver model.
Main Results:
- KU induced cytotoxicity in GC and CRC cells by activating the endoplasmic reticulum (ER) stress-induced apoptotic pathway.
- KU significantly increased the expression of ATF3, a key mediator of apoptosis, which binds to and promotes the expression of downstream apoptotic genes.
- ATF3 knockdown abrogated KU's cytotoxic effects and the expression of its target genes.
- In vivo, KU administration reduced metastatic tumor growth in mouse livers by inducing ATF3 and apoptosis.
Conclusions:
- Potassium usnate (KU) exerts cytotoxic effects on human gastric and colorectal cancer cells.
- The mechanism involves the induction of endoplasmic reticulum (ER) stress-mediated apoptosis through an ATF3-dependent pathway.
