Iron is an important biological catalyst and is critical for DNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding to RNA stem-loop structures known as iron-response elements. We also found that Irp2 mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(⌬19) tumors expressing the clock-mutant protein (CLOCK ⌬19 ) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCK ⌬19 expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.Circadian rhythms affect blood pressure, locomotor activity, core body temperature, and the sleep-wake cycle. These circadian controls of physiology and behavior are driven by a master pacemaker located in the suprachiasmatic nucleus of the hypothalamus, which relies on the interplay of interconnected transcriptional and translational feedback loops (1). The brain and muscle Arnt-like 1 (BMAL1) 3 and circadian locomotor output cycles kaput (CLOCK) heterodimer drive the transcription of cryptochrome (CRY) and period (PER) genes by binding to the E-box in their promoter regions. CRY and PER homodimers and heterodimers inhibit the function of BMAL1/CLOCK, thus decreasing their own expression (2-5). This core loop creates a 24-h rhythmic oscillation of clock-controlled gene expression in normal and tumor cells.Iron is an important metal for cell proliferation, metabolism, respiration, and DNA synthesis (6 -8). Iron homeostasis is dependent on the expression of transferrin receptor 1 (TfR1) (9, 10). TfR1 is a membrane receptor responsible for iron uptake and ferritin is an intracellular protein that stores iron. When present in excess, cellular iron is toxic (11-13). Therefore, iron concentration must be tightly regulated. In general, the expression of TfR1 is regulated by iron-regulatory proteins (IRPs), which are sensors of intracellular iron levels. IRPs control TfR1 and divalent metal transporter 1 (Dmt1) at the post-transcriptional level by binding to RNA stem-loop structures known as iron-responsive elements (IREs) in the 3Ј-untranslated region (UTR) of mRNA. IRP binding stabilizes transcripts in these genes (14 -16).Iron metabolism is critical for the rapid growth of tumors and iron is also required for DNA synthesis in tumor cells. Therefor...

Circadian clock systems regulate many biologic functions, including cell division and hormone secretion in mammals. In this study, we explored the effects of circadian control on the pivot cell growth regulatory mTOR, the activity of which is deregulated in tumor cells compared with normal cells. Specifically, we investigated whether the antitumor effect of an mTOR inhibitor could be improved by changing its dosing schedule in RenCa tumorbearing mice. Active, phosphorylated mTOR displayed a 24-hour rhythm, and levels of total mTOR protein (but not mRNA) also showed a circadian rhythm in RenCa tumor masses. Through investigations of the oscillation mechanism for mTOR expression, we identified the ubiquitination factor Fbxw7 as an mTOR regulator that oscillated in its expression in a manner opposite from mTOR. Fbxw7 transcription was regulated by the circadian regulator D-site-binding protein. Notably, administration of the mTOR inhibitor everolimus during periods of elevated mTOR improved survival in tumor-bearing mice. Our findings demonstrate that the circadian oscillation of mTOR activity is regulated by circadian clock systems, which influence the antitumor effect of mTOR inhibitors. Cancer Res; 74(2); 543-51. Ó2013 AACR.

Cisplatin, cis-diamminedichloro-platinum (CDDP), is a widely used anticancer agent, the clinical applications of which have been limited by severe nephrotoxicity. Although dosing timedependent differences in CDDP-induced nephrotoxicity have been reported in both humans and laboratory animals, the underlying mechanism remains unknown. In the present study, we investigated the molecular mechanism for the dosing-time dependency of the nephrotoxic effect of CDDP in mice. CDDPinduced nephrotoxicity was significantly attenuated by injecting CDDP at times of the day when its renal clearance was enhanced. The dosing-time dependency of the nephrotoxic effect was parallel to that of CDDP incorporation into renal DNA. Two types of transporters, organic cation transporter 2 (OCT2, encoded by Slc22a2) and multidrug and toxin extrusion 1 (MATE1, encoded by Slc47a1), are responsible for the renal excretion of CDDP. The expression of OCT2, but not MATE1, exhibited a significant time-dependent oscillation in the kidneys of mice. The circadian expression of OCT2 was closely related to the dosing-time dependency of CDDP incorporation into renal DNA. Molecular components of the circadian clock regulated the renal expression of Slc22a2 mRNA by mediating peroxisome proliferator-activated receptor-a, which resulted in rhythmic oscillations in OCT2 protein levels. These findings indicate a clock-regulated mechanism of dosing time-dependent changes in CDDP-induced nephrotoxicity and also suggest a molecular link between the circadian clock and renal xenobiotic excretion.

Bisphosphonates and statins are known to have antitumor activities against different types of cancer cell lines. In the present study, we investigated the antiproliferative effects of the combination of zoledronic acid (ZOL), a bisphophosphonate, and fluvastatin (FLU), a statin, in vitro on two types of human pancreatic cancer cell lines, Mia PaCa-2 and Suit-2. The pancreatic cancer cell lines were treated with ZOL and FLU both individually and in combination to evaluate their antiproliferative effects using WST-8 cell proliferation assay. In this study, we demonstrated a potent synergistic antiproliferative effect of both drugs when used in combination in both cell lines. Moreover, we studied the molecular mechanism behind this synergistic effect, which was inhibited by the addition of the mevalonate pathway products, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Furthermore, we aimed to determine the effect of ZOL and FLU combination on RhoA and Ras guanosine 5′-triphosphate (GTP)-proteins. The combination induced a marked accumulation in RhoA and unprenylated Ras. GGPP and FPP reversed the increase in the amount of both proteins. These results indicated that the combination treatment impaired RhoA and Ras signaling pathway by the inhibition of geranylgeranylation and/or farnesylation. This study provides a potentially effective approach for the treatment of pancreatic cancer using a combination treatment of ZOL and FLU.Key words pancreatic cancer; zoledronic acid; fluvastatin; combination treatment; synergistic Pancreatic cancer is known to be the fifth leading cause of cancer-related mortality in Japan and considered to be one of the most aggressive malignancies. 1) Moreover, patients treated with surgical interventions usually develop tumor relapse and/or liver metastasis.2) Gene mutations, including K-ras, CDKN2A (p16), and TRP53, are often associated with pancreatic cancer.3) Previous studies have shown that blocking and/or depriving cells of K-ras may be a promising way to treat pancreatic cancer. 4,5) Although gemcitabine has been considered the standard care treatment for pancreatic cancer, 6) its clinical use remains limited. Therefore, the development of new therapeutic strategies is needed.The third-generation nitrogen bisphosphonates (NBPs) are currently considered a key therapy for the treatment of osteoclast-mediated bone resorption, bone metastasis, and malignant skeletal-related diseases. 7) NBPs have also shown direct antiproliferative and apoptotic effects on solid tumors [8][9][10][11][12] by inhibiting the farnesyl pyrophosphate (FPP) synthase, a key regulatory enzyme in the mevalonate pathways.13) Impairing the prenylation of small guanosine 5′-triphosphate (GTP) proteins such as Ras, Rab, Rho, and Rac by FPP synthase inhibitors may lead to the loss of many cellular processes. 14)Zoledronic acid (ZOL) is one of the most potent drugs owing to its direct and indirect antitumor effects, 15) and it has been effectively used for the treatment of several cancer cell types...

Xanthine oxidase (XOD), also known as xanthine dehydrogenase, is a rate-limiting enzyme in purine nucleotide degradation, which produces uric acid. Uric acid concentrations in the blood and liver exhibit circadian oscillations in both humans and rodents; however, the underlying mechanisms remain unclear. Here, we demonstrate that XOD expression and enzymatic activity exhibit circadian oscillations in the mouse liver. We found that the orphan nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) transcriptionally activated the mouse gene and that bile acids suppressed transactivation. The synthesis of bile acids is known to be under the control of the circadian clock, and we observed that the time-dependent accumulation of bile acids in hepatic cells interfered with the recruitment of the co-transcriptional activator p300 to PPARα, thereby repressing expression. This time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the hepatic expression of XOD, which, in turn, led to circadian alterations in uric acid production. Finally, we also demonstrated that the anti-hyperuricemic effect of the XOD inhibitor febuxostat was enhanced by administering it at the time of day before hepatic XOD activity increased. These results suggest an underlying mechanism for the circadian alterations in uric acid production and also underscore the importance of selecting an appropriate time of day for administering XOD inhibitors.