Effects of Everolimus on Circadian Gene Expression and Cell Fate in Synchronized Caco-2 Cells

Abstract

Circadian rhythms regulate key biological processes, including cell proliferation and metabolism, and their disruption is implicated in colorectal cancer (CRC). mTOR signaling interacts bidirectionally with the circadian clock, yet how mTOR inhibition modulates clock gene dynamics and cellular behavior in intestinal models remains unclear. This study aimed to investigate the effects of everolimus, an mTOR inhibitor, on circadian gene expression, cell viability, apoptosis, and cell cycle progression in synchronized Caco-2 cells, with consideration of cell confluency and Circadian Time (CT). Methods: Caco-2 cells were synchronized using serum shock at 20 % and 70 % confluency. Time-series samples were collected across multiple CTs (CT6–CT60). Gene expression (BMAL1, PER2, mTOR) was assessed by qRTPCR using ACTB and RPLP0 as reference genes. Rhythmicity was evaluated via Cosinor analysis. Cell viability, apoptosis, and cell cycle dynamics were analyzed using the Muse™ Cell Analyzer following everolimus treatment (1–50 μM). Results: RPLP0 proved to be a more stable reference gene than ACTB. BMAL1 exhibited stronger rhythmic expression than PER2, particularly at 20 % confluency. Everolimus (50 μM) significantly reduced cell viability in a time-dependent manner, with the greatest effect at CT6 and CT18 (p < 0.0001). Apoptosis was markedly increased at CT6 (+38.5 %) and moderate at CT18, indicating circadian modulation of drug sensitivity. Serum shock alone shifted cell cycle distribution, decreasing G0/G1 and increasing G2/M phase populations (p < 0.01). Everolimus altered BMAL1 and PER2 expression rhythms and significantly reduced mTOR expression at CT30, where baseline mTOR levels were highest. Cosinor analysis confirmed rhythmicity in BMAL1/RPLP0 and mTOR/ RPLP0 profiles under low confluency. Conclusion: Our findings demonstrate that everolimus influences circadian gene expression and exerts timedependent antiproliferative and pro-apoptotic effects in Caco-2 cells. These results support the potential of circadian timing as a strategy to enhance mTOR-targeted therapies in CRC.