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Transferrin receptor 1(TfR1) is a key cell surface molecule that regulates the uptake of iron-bound transferrin. TfR1 expression is higher in tumor cells than in normal cells. Thus, intracellular targeting using iron-saturated Tf as a ligand for TfR-mediated endocytosis has attracted attention. TfR1 in colon cancer-bearing mice exhibits a 24-hour rhythm in mRNA and protein levels. The clock-controlled gene c-MYC rhythmically activate the transcription of the TfR1 gene. In addition, the cyclical accumulation of TfR1 causes dosing time-dependent changes in the intratumoral delivery of drug by receptor-mediated endocytosis. Identification of the circadian properties of molecules that are targeted by ligand-directed DDS may aid the choice of the most appropriate time of day for their administration.

The expression of genes involved in xenobiotic detoxification is under the control of the circadian clock. The aryl hydrocarbon receptor (AhR) is one of the transcription factors responsible for the induction of detoxification enzymes in response to xenobiotic toxins, and the expression of AhR has been suggested to be regulated by a circadian oscillator. In this study, we investigated whether toxin-mediated activation of the AhR signaling pathway was modulated by CLOCK protein, a key component of the mammalian circadian clock. The expression of AhR and its DNA binding ability in the lungs of wild-type mice showed significant 24-h oscillation. Clock mutant (Clk/Clk) mice, producing CLOCK protein deficient in transcriptional activity, failed to show significant oscillation in the expression of AhR. The mRNA levels of AhR in the lungs of Clk/Clk mice were significantly lower than in wild-type mice. A single intraperitoneal injection of benzo[α]pyrene, a ligand of AhR, induced the expression of Cyp1a1 in the lungs of wild-type mice, but the induction varied depending on the benzo[α]pyrene injection time. The dosing time-dependency of benzo[α]pyrene-induced Cyp1a1 expression was also modulated by Clock gene mutation. These findings suggest that CLOCK protein affects the toxin-induced expression of detoxification enzymes through modulating the activity of AhR. Our present findings provide a molecular link between the circadian clock and xenobiotic detoxification., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Many pathophysiological circumstances vary during 24 h periods. Many physiologic processes undergo biological rhythms, including the sleep-wake rhythm and metabolism. Disruptive effect in the 24 h variations can manifest as the emergence or exacerbation of pathological conditions. So, chronotherapeutics is gaining increasing interest in experimental biology, medicine, pharmacy, and drug delivery. This science and the plethora of information should be used intelligently for optimizing the effectiveness and safety of the drug, relying on the timing of drug intake. These chronopharmacological findings are affected by not only the pharmacodynamics but also pharmacokinetics of drugs. The mammalian circadian pacemaker is located in the suprachiasmatic nucleus. The molecular mechanisms are associated with Clock genes that control the circadian rhythms in physiology, pathology, and behavior. Clock controls several diseases such as metabolic syndrome, cancer, and so on. CLOCK mutation influences the expression of both rhythmic and nonrhythmic genes in wild-type tissues. These genotypic changes lead to phenotypic changes, affecting the drug pharmacokinetic and pharmacodynamic parameters. This review is intended to elaborate system regulating biological rhythms and the applicability in pharmaceutics from viewpoints of the intraindividual and interindividual variabilities of Clock genes., (Copyright © 2011 Wiley-Liss, Inc.)

The Clock gene is a core clock factor that plays an essential role in generating circadian rhythms. In the present study, it was investigated whether the Clock gene affects the response to diethylnitrosamine (DEN)-induced cytotoxicity using mouse primary hepatocytes. DEN-induced cytotoxicity, after 24h exposure, was caused by apoptosis in hepatocytes isolated from wild-type mouse. On the other hand, Clock mutant mouse (Clk/Clk) hepatocytes showed resistance to apoptosis. Because apoptosis is an important pathway for suppressing carcinogenesis after genomic DNA damage, the mechanisms that underlie resistance to DEN-induced apoptosis were examined in Clk/Clk mouse hepatocytes. The mRNA levels of metabolic enzymes bioactivating DEN and apoptosis-inducing factors before DEN exposure were lower in Clk/Clk cells than in wild-type cells. The accumulation of p53 and Ser15 phosphorylated p53 after 8h DEN exposure was seen in wild-type cells but not in Clk/Clk cells. Caspase-3/7 activity was elevated during 24h DEN exposure in wild-type cells but not in Clk/Clk cells. In addition, resistance to DEN-induced apoptosis in Clk/Clk cells affected the cell viability. These studies suggested that the lower expression levels of metabolic enzymes bioactivating DEN and apoptosis inducing factors affected the resistance to DEN-induced apoptosis in Clk/Clk cells, and the Clock gene plays an important role in cytotoxicity induced by DEN., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Hypoxia-induced gene expression frequently occurs in malignant solid tumors because they often have hypoxic areas in which circulation is compromised due to structurally disorganized blood vessels. Hypoxia-response elements (HREs) are responsible for activating gene transcription in response to hypoxia. In this study, we constructed a hypoxia-response plasmid vector producing short hairpin RNA (shRNA) against B-cell leukemia/lymphoma-2 (bcl-2), an anti-apoptotic factor. The hypoxia-response promoter was made by inserting tandem repeats of HREs upstream of cytomegalovirus (CMV) promoter (HRE-CMV). HRE-CMV shbcl-2 vector consisted of bcl-2 shRNA under the control of HRE-CMV promoter. In hypoxic mouse rectum carcinoma cells (colon-26), the production of bcl-2 shRNA driven by HRE-CMV promoter was approximately 2-fold greater than that driven by CMV promoter. A single intratumoral (i.t.) injection of 40 microg HRE-CMV shbcl-2 to colon-26 tumor-bearing mice caused apoptotic cell death, and repetitive treatment with HRE-CMV shbcl-2 (40 microg/mouse, i.t.) also significantly suppressed the growth of colon-26 tumor cells implanted in mice. Apoptotic and anti-tumor effects were not observed in tumor-bearing mice treated with CMV shbcl-2. These results reveal the ability of HRE-CMV shbcl-2 vector to suppress the expression of bcl-2 in hypoxic tumor cells and suggest the usefulness of our constructed hypoxia-response plasmid vector to treat malignant tumors. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10054FP].