Phenylacetaldehyde induces apoptosis through endoplasmic reticulum stress and potentiates the effect of 5-FU treatment in colorectal cancer

Introduction: Phenylacetaldehyde (PAA), a product of phenylalanine metabolism, is generated by microbiota and has demonstrated anticancer properties in breast cancer. As intestinal homeostasis is influenced by the gut microbiome, we investigated the effect of PAA on colorectal cancer (CRC). Methods: Serum PAA levels and feces microbiota from a test cohort of 30 CRC patients and 30 healthy control patients were analyzed by targeted GC-MS/MS and metagenomic sequencing. A validation cohort of 75 CRC patients and 53 healthy control patients was analyzed for PAA levels in serum. CRC cell lines and CRC-patient-derived organoids were cultured and treated with PAA alone or in combination with 5-fluorouracil (5-FU), then evaluated for viability, proliferation, , and cell death in vitro . The effect of PAA alone or in combination with 5-FU on tumor growth was also tested in vivo . We identified the mechanisms induced by PAA treatment using RNA sequencing, western blot, immunostaining, ROS quantification assay, cysteine and glutathione quantification. Results: PAA levels were significantly decreased in the circulation of CRC patients compared to healthy controls in our study population and the validation cohort. Metagenomic sequencing indicated the bacteria that positively correlated with PAA levels were significantly more abundant in healthy controls; while bacteria that negatively correlated with PAA were more abundant in CRC. In vitro , PAA inhibited the viability and colony formation of CRC cell lines as well as human-derived organoids in a concentration-dependent manner. Mechanistically, PAA treatment induced generation of ROS leading to cysteine and reduced glutathione depletion. An investigation by RNA sequencing and immunoblotting showed that PAA inhibits CRC by inducing endoplasmic reticulum stress-induced autophagy accompanied by downregulation in PI3K/AKT/mTOR and ERK pathways. When combined with 5-FU, PAA enhanced the potency of 5-FU on viability, cell death, DNA damage and tumor growth. Conclusions: The bacteria metabolite PAA increased CRC cell death by endoplasmic reticulum stress-induced autophagy and downregulation of the PI3K/AKT/mTOR and ERK pathways. PAA also potentiated the chemotherapeutic efficacy of 5-FU through induction of DNA damages. These findings warrant further exploration of PAA as a potential CRC biomarker and novel therapeutic agent.