Related Experiment Video
Updated: Jan 11, 2026
Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures
Published on: November 11, 2016
Effects of everolimus on circadian gene expression and cell fate in synchronized Caco-2 cells
Dilek Ozturk Civelek1, Beyza Goncu2, Sadullah Goncu3
1Istanbul University-Cerrahpasa, Faculty of Pharmacy, Department of Pharmacology, Istanbul, Turkiye; Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Istanbul, Turkiye.
Objectives:
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 qRT-PCR 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 time-dependent 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.
Insights
Everolimus, an mTOR inhibitor, impacts circadian gene expression and shows time-dependent effects on cell viability and apoptosis in colorectal cancer cells. This suggests circadian timing may enhance mTOR-targeted therapies.
Area of Science:
- Chronobiology
- Cancer Biology
- Pharmacology
Background:
- Circadian rhythms are crucial for cell proliferation and metabolism, and their disruption is linked to colorectal cancer (CRC).
- The mechanistic target of rapamycin (mTOR) signaling pathway interacts with the circadian clock, but its inhibition's effects on clock gene dynamics in intestinal models are not fully understood.
Purpose of the Study:
- To investigate the impact of everolimus, an mTOR inhibitor, on circadian gene expression, cell viability, apoptosis, and cell cycle progression.
- To analyze these effects in synchronized Caco-2 cells, considering cell confluency and circadian time (CT).
Main Methods:
- Caco-2 cells were synchronized and sampled across multiple circadian times (CT6-CT60).
- Gene expression of BMAL1, PER2, and mTOR was measured using qRT-PCR.
- Cell viability, apoptosis, and cell cycle were assessed using the Muse™ Cell Analyzer after everolimus treatment.
Main Results:
- Everolimus (50 μM) significantly reduced cell viability and increased apoptosis in a time-dependent manner, with greatest effects at CT6 and CT18.
- Drug sensitivity was modulated by circadian timing, with increased apoptosis observed at CT6.
- Everolimus altered BMAL1 and PER2 expression rhythms and reduced mTOR expression, particularly when baseline mTOR levels were highest.
Conclusions:
- Everolimus influences circadian gene expression and exhibits time-dependent antiproliferative and pro-apoptotic effects in Caco-2 cells.
- These findings highlight the potential of leveraging circadian timing to optimize mTOR-targeted therapies for colorectal cancer.
Related Concept Videos
02:19Circadian Rhythms and Gene Regulation
01:15Chronopharmacokinetics: Circadian Rhythms and Influence on Drug Response
The time of drug administration is an important factor to consider, as it can influence the toxic dose of a drug. For example, a study conducted by Prins et al. in 1997 examined the effects of the timing of...
01:58Cell Specific Gene Expression
01:58Cell Specific Gene Expression
02:34Inhibition of Cdk Activity
01:22PI3K/mTOR/AKT Signaling Pathway