Transcriptome analysis upon potassium usnate exposure reveals ATF3-induced apoptosis in human gastric and colon cancer cells

Abstract

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.

Keywords: Activating Transcription Factor 3; Apoptosis; Endoplasmic Reticulum Stress; Potassium usnate; RNA-Seq; Usnic acid.