Oncogenic and Circadian Effects of Small Molecules Directly and Indirectly Targeting the Core Circadian Clock

Abstract

Circadian rhythms are essential for controlling the cell cycle, cellular proliferation, and apoptosis, and hence, are tightly linked to cell fate. Disruption of circadian rhythms has been shown to trigger various pathological developments, including cancer. Several recent studies have used a variety of small molecules to affect circadian oscillations, however, their concomitant cellular effects were not assessed. Here, we use five molecules, grouped into direct versus indirect effectors of the circadian clock, to modulate periods in a human osteosarcoma cell line (U2OS), and determined their influences on cellular behaviors, including motility and colony formation. Luciferase reporters, whose expression were driven via Bmal1and Per2-promoters (positive and negative protein components of the core clock), were used to facilitate the visualization and quantitative analysis of circadian oscillations. We show that all molecules significantly increase or decrease the circadian periods of Bmal1 and Per2 in a dosedependent manner, but period length does not correlate with the extent of cell migration or proliferation. We observed that only molecules that affected circadian oscillations to a greater extent showed significant influence on cell functions (e.g. motility and colony formation).Because it is important to consider the likelihood of biological effects resulting from noncircadian targets, we also provide a thorough discussion of potential modes of action. Future studies should employ additional compounds that directly target circadian proteins and/or have different circadian effects, and evaluation in other cancer models to determine whether results obtained here remain consistent. proteins aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL/BMAL1) and circadian locomotor output cycles kaput (CLOCK) heterodimerize in the morning and bind to the E-box promoter, which initiates the expression of Period (Per) and Cryptochrome (Cry). PER and CRY accumulate in the evening and form a heterodimer, which translocates back into the nucleus and inhibits Bmal1 and Clock activity. 18 As a result, the core circadian machinery oscillates with a period of approximately 24 h. In cell culture, intrinsic and self-sustained circadian clocks are persistent even in the absence of external time cues. 19 To synchronize clocks in cultured cells, treatments of high concentration serum 20 or chemical reagents (e.g. dexamethasone 21 or forskolin 22 ) are frequently used. Circadian rhythmicity is tightly associated with post-translational modifications of clock proteins. 23 Phosphorylation of most clock proteins occurs in a rhythmic manner; thus, alteration of clock protein phosphorylation can result in changes to circadian periods. 24 Genetic manipulations of the post-translational regulators of clock proteins have been shown to affect circadian functions and periodicity. 24-25 However, conventional genetic approaches also result in fatality, pleiotropy, and functional redundancy. Alternatively, chemical modulation of these...

Nobiletin affects circadian rhythms and oncogenic characteristics in a cell-dependent manner

“…Both cell lines were tested for mycoplasma via PCR detection. U2OS cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco), with 10% Fetal Bovine Serum (FBS; Corning), 1% L-Glutamine (L-glut; Gibco), 1% penicillin-streptomycin (Gibco), 1% Non-Essential Amino Acids (HyClone), and 1% Sodium Pyruvate (Gibco) [36]. MCF7 and MDA-MB-231 cells were maintained in DMEM (Gibco), supplemented with 10% FBS (Corning), 1% penicillin-streptomycin (Gibco), and 1% L-glut (Gibco) [32].…”

“…The generation of Bmal1:luc and Per2:luc plasmids [37] and their subsequent stable transfection into U2OS [36], MCF7, and MDA-MB-231 [32] cells have been described previously.…”

“…To assess the circadian effects of nobiletin in a detailed approach, we used luciferase-reporter cells for Bmal1 and Per2 promoter activity previously generated in our lab [37]. Here, we employed U2OS, MCF7, and MDA-MB-231 cell lines separately transfected with Bmal1:luc and Per2:luc [32,36]. Prior to initiating luminometry experiments, we evaluated potential viability effects of treatments to be used.…”

“…In both cases, we also found increased colony numbers (Fig 5F). Decreased colony sizes may be accompanied by increased numbers because it is possible that nobiletin prevents the aggregation of smaller colonies into larger ones, which can result in higher numbers of smaller colonies but fewer larger ones [36].…”

Nobiletin Affects Circadian Rhythms and Oncogenic Characteristics in a Cell-Dependent Manner

“…33 Here, we employed U2OS, MCF7 and MDA-MB-231 cell lines separately transfected with Bmal1:luc and Per2:luc. 32,34 Prior to initiating luminometry experiments, we evaluated potential viability effects of treatments to be used. The highest and lowest concentrations used in this study were determined based on the concentration required for the 50% induction of activity in MCF7 cells (44 µM).…”