Modulatory effects of 5-fluorouracil on the rhythmic expression of circadian clock genes: A possible mechanism of chemotherapy-induced circadian rhythm disturbances

doi:10.1016/j.bcp.2008.01.011

Biochemical Pharmacology

Volume 75, Issue 8, 15 April 2008, Pages 1616-1622

https://doi.org/10.1016/j.bcp.2008.01.011 Get rights and content

Abstract

The circadian clock system is necessary to adapt endogenous physiological functions to daily variations in environmental conditions. Abnormality in circadian rhythms, such as the sleep–wake cycle and the timing of hormonal secretions, is implicated in various physiological and psychiatrical disorders. Recent molecular studies have revealed that oscillation in the transcription of specific clock genes plays a central role in the generation of 24 h cycles of physiology and behavior. It has been noticed that patients receiving chemotherapeutic agents experience disturbances in their behavioral and physical performances, including circadian rhythms. To explore the underlying mechanism of chemotherapeutic agent-induced disturbance of these rhythms, we investigated the influence of 5-fluorouracil (5-FU), one of the most widely used chemotherapeutic agents for the treatment of cancers, on the expression of clock genes. Treatment of cultured NIH3T3 cells with 5-FU for 48 h resulted in a significant reduction of mRNA levels of Period1 (Per1) and Period2 (Per2) without affecting cell viability; however, treatment with the same amount of uracil, a structural analog of 5-FU, had little effect on the expression of clock genes. Consistent with its inhibitory actions, continuous administration of 5-FU (2 mg/kg/h) to mice attenuated the oscillation in the expressions of Per1 and Per2 in the liver and suprachiasmatic nuclei, the center of the mammalian circadian clock. These results reveal a possible pharmacological action by the chemotherapeutic agent 5-FU on the circadian clock mechanism, which is the underlying cause of its adverse effects on 24-h rhythms of physiology and behavior.

Introduction

Anticancer chemotherapeutic medication in either curative or palliative settings is associated with various undesirable side effects, and the patient's quality of life is remarkably decreased [1]. These side effects may include acute and delayed nausea, vomiting, and anorexia [2], [3], [4]. Decreased whole body weight, disrupted gastrointestinal tract function, including dyspepsia and diarrhea, and also fatigue are accompanied in many cases [5], [6].

Cancer chemotherapeutic drug-associated fatigue has subjective (self-reported) and objective (reduced physical activity or capacity to undertake physical and mental tasks) dimensions. It differs from body tiredness and the feeling of fatigue by sleep deprivation or excessive physical exercise in that this fatigue is not relieved by rest or sleep [5]. Measuring the amount of physical activity using “Actigraphy” in patients receiving chemotherapeutic drugs and comparing with the results of normal people showed that physical activity is not only decreased but also that there is a disturbance of the circadian rhythm [7]. However, the detailed mechanism of this disturbance of the circadian rhythm has not been clarified.

The mammalian circadian system is hierarchically organized by central and peripheral oscillators. An ensemble of coupled oscillators in the suprachiasmatic nucleus (SCN) of the hypothalamus is entrained to a 24-h period by daily light input from the visual system. Neural and humoral output signals from the SCN coordinate the phase of independent circadian oscillators in peripheral tissues throughout the organism [8], [9]. Self-sustaining circadian oscillators in the SCN use a molecular mechanism similar to that used in subsidiary oscillators present in all cell types in the organism [10]. Recent molecular dissection of the circadian biological clock system has revealed that oscillation in the transcription of specific clock genes plays a central role in the generation of circadian rhythms. Gene products of Clock and Bmal1 form a heterodimer that activates the transcription of Period (Per) and Cryptochrome (Cry) genes. Once PER and CRY proteins have reached a critical concentration, they attenuate CLOCK/BMAL1 transactivation, thereby generating circadian oscillation in their own transcription [11], [12]. The clock genes, consisting of the core oscillation loop, control downstream events by regulating the rhythmic expression of clock-controlled genes [13], [14], [15].

To address the mechanism underlying chemotherapeutic agent-induced circadian rhythm disruption, we investigated whether oscillation in the expression of clock genes was influenced by chronic treatment with 5-fluorouracil (5-FU), one of the most frequently used chemotherapeutic agents for the treatment of cancers. Furthermore, we also explored how treatment with 5-FU affected the SCN oscillatory function and locomotor activity rhythm in mice.

Section snippets

Materials

The following materials were commercially obtained: 5-fluorouracil, uracil and dexamethasone (Dex) from Wako Pure Chemical Industries Ltd. (Tokyo, Japan); 2-deoxy-[³H]-glucose ([³H]-2-DG) and [¹⁴C]-Leucine ([¹⁴C]-Leu) from GE Healthcare (Chalfont St. Giles, UK); Dulbecco's modified Eagle's medium (DMEM; product #D6046) and fetal bovine serum (FBS), sometimes called fetal calf serum (FCS) from Sigma–Aldrich (St. Louis, MO).

Cells and treatment

Mouse NIH3T3 fibroblasts were obtained from the Cell Resource Center for

Influence of 5-FU on mRNA levels of clock gene in NIH3T3 cells

To test the possibility that chemotherapeutic agents affect the expression of clock genes, we investigated the influence of 5-FU on mRNA levels of Per1 and Per2 in NIH3T3 cells. Treatment of confluent cultured cells with 5-FU for 48 h resulted in a reduction of mRNA levels of Per1 and Per2 in a dose-dependent manner (Fig. 1A). The most significant reduction was seen at a concentration of 10 μM (P < 0.05); however, the same concentration of uracil had little effect on mRNA levels of these clock

Discussion

In this study, we showed that chronic treatment with a chemotherapeutic agent, 5-FU, modulated circadian clock function at the molecular level. Treatment of cultured mouse NIH3T3 cells with 5-FU resulted in a significant reduction of mRNA levels of Per1 and Per2 expression, and also attenuated the oscillation of reporter luciferase bioluminescence driven by the mouse Per2 promoter in rat C6 glioma cells. 5-FU is widely used for several types of cancer. The principal mechanism of 5-FU

Acknowledgments

This study was partially supported by a Grant-in-Aid for Scientific Research on Priority Areas ‘Cancer’ (S.O., 18014020) from the Ministry of Education, Culture, Sports, Science and Technology, a Grant-in-Aid for Scientific Research (B) (S.O., 18390050) and a Grant-in-Aid for the Encouragement of Young Scientists (S.K., 18790692) from the Japan Society for the Promotion of Science.

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Mammalians' circadian pacemaker resides in the paired suprachiasmatic nuclei (SCN). SCN control biological rhythms such as the sleep-wake rhythm and homeostatic functions of steroid hormones and their receptors. Alterations in these biological rhythms are implicated in the outcomes of pathogenic conditions such as depression, diabetes, and cancer. Chronotherapy is about optimizing treatment to combat risks and intensity of the disease symptoms that vary depending on the time of day. Thus, conditions/diseases such as allergic rhinitis, arthritis, asthma, myocardial infarction, congestive heart failure, stroke, and peptic ulcer disease, prone to manifest severe symptoms depending on the time of day, would be benefited from chronotherapy. Monitoring rhythm, overcoming rhythm disruption, and manipulating the rhythms from the viewpoints of underlying molecular clocks are essential to enhanced chronopharmacotherapy. New drugs focused on molecular clocks are being developed to improve therapeutics. In this review, we provide a critical summary of literature reports concerning (a) the rationale/mechanisms for time-dependent dosing differences in therapeutic outcomes and safety of antitumor drugs, (b) the molecular pathways underlying biological rhythms, and (c) the possibility of pharmacotherapy based on the intra- and inter-individual variabilities from the viewpoints of the clock genes.
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Thus, alteration of the clock function, a new concept of adverse effects, can be overcome by devising a dosing regimen that minimizes adverse drug effects on clock function. Also, the influence of 5-fluorouracil (5-FU) on the expression of clock genes is studied to explore the mechanism underlying chemotherapeutic agent-induced disturbance of circadian rhythms.68 Continuous administration of 5-FU to mice suppresses the oscillation in the expressions of Per1 and Per2 mRNA in the liver and SCN.
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¹: Present address: Institute of Biomaterials and Bioengineering, Department of Biomedical Information, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.

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Modulatory effects of 5-fluorouracil on the rhythmic expression of circadian clock genes: A possible mechanism of chemotherapy-induced circadian rhythm disturbances

Abstract

Introduction

Section snippets

Materials

Cells and treatment

Influence of 5-FU on mRNA levels of clock gene in NIH3T3 cells

Discussion

Acknowledgments

Surg Clin North Am

Eur J Cancer

Eur J Cancer

Lancet

Cell

Cell

Biochem Biophys Res Commun

Brain Res

Neurosci Lett

Brain Res

Pharmacol Ther