Time dependent cisplatin dosing differences on hypoalgesia focusing on oxidative stress.

Although cisplatin is a key drug in cancer chemotherapy, it often causes sensory peripheral neuropathy, presenting as allodynia in the early stage and hypoalgesia in the serious stage. Chronotherapy has previously been shown to ameliorate cisplatin-induced peripheral neuropathy that was severe enough to cause hypoalgesia in rats. It also has adverse effects such as renal dysfunction and ototoxicity, which are induced by oxidative stress. Here, we show that oxidative stress causes severe cisplatin-induced peripheral neuropathy, and that differences in oxidative stress occur depending on the dosing time of cisplatin. Cisplatin was administered to rats at 5:00 or 17:00 every seven days for four weeks. The antioxidant agent, 1,3-Dimethylthiourea (DMTU), was administered before and after the administration of cisplatin. The hot plate test was used to assess hypoalgesia. Oxidative stress in the sciatic nerve was assessed from thiobarbituric acid reactive substances (TBARs) and superoxide dismutase (SOD) activity. Nerve apoptosis was analysed with qRT-PCR. We observed an increase in TBARs and a decrease in SOD activity with the development of cisplatin-induced hypoalgesia, which was ameliorated by DMTU treatment. Furthermore, differences in the dosing time of cisplatin caused differences in oxidative stress which were correlated with cisplatin-induced hypoalgesia. Severe oxidative stress caused cisplatin-induced hypoalgesia, and chronotherapy with cisplatin ameliorated hypoalgesia by reducing oxidative stress. In the future, chronotherapy with cisplatin may contribute to the treatment of cancer in humans.

Effects of a rifampicin pre-treatment on linezolid pharmacokinetics.

Linezolid is an oxazolidinone antibiotic that effectively treats methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Since rifampicin induces other antibiotic effects, it is combined with linezolid in therapeutic regimes. However, linezolid blood concentrations are reduced by this combination, which increases the risk of the emergence of antibiotic-resistant bacteria. We herein demonstrated that the combination of linezolid with rifampicin inhibited its absorption and promoted its elimination, but not through microsomal enzymes. Our results indicate that the combination of linezolid with rifampicin reduces linezolid blood concentrations via metabolic enzymes.

Lamellarin-inspired potent topoisomerase I inhibitors with the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one scaffold.

A new class of topoisomerase I inhibitors containing the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (abbreviated as BBPI) ring system have been developed based on structure-activity relationship studies of the cytotoxic marine alkaloid lamellarin D. The pentacyclic BBPI scaffold was constructed from N-tert-butoxycarbonylpyrrole by sequential and regioselective functionalization of the pyrrole core using directed lithiation, conventional electrophilic substitution, and palladium-catalyzed cross-coupling reactions. Further N-alkylation of the scaffold followed by selective deprotection of the O-isopropyl group produced a range of N-substituted BBPI derivatives. The BBPIs thus prepared exhibited potent topoisomerase I inhibitory activity in DNA relaxation assays. The activities of BBPIs were higher than those of lamellarin D and camptothecin; they showed potent and selective antiproliferative activity in the panel of 39 human cancer cell lines established by Japanese Foundation for Cancer Research. COMPARE analyses indicated that the inhibition patterns of the BBPIs correlated well with those of the known topoisomerase I inhibitors such as SN-38 and TAS-103. The water-soluble valine ester derivative exhibited antitumor activity in vivo against murine colon carcinoma colon 26. The activity was comparable to that of the approved anticancer agent irinotecan.

Administering xCT Inhibitors Based on Circadian Clock Improves Antitumor Effects.

Clock genes encoding transcription factors that regulate circadian rhythms may inform chronomodulated chemotherapy, where time-dependent dose alterations might affect drug efficacy and reduce side effects. For example, inhibiting the essential cystine transporter xCT with sulfasalazine induces growth arrest in cancer cells. Although the anticancer effects of sulfasalazine have been studied extensively, its effects on transcriptional control of xCT expression have not been studied. Here, we show that sulfasalazine administration during the period of increased xCT expression improves its anticancer effects and that the Clock gene itself induces xCT expression and regulates its circadian rhythm. Our findings highlight the clinical potential of chronomodulated chemotherapy and the importance of xCT-mediated transcriptional regulation in the utility of such strategies. Cancer Res; 77(23); 6603-13. ©2017 AACR.

Mechanism Underlying Linezolid-induced Thrombocytopenia in a Chronic Kidney Failure Mouse Model.

OBJECTIVE: To investigate the relationship between renal function and linezolid (LZD)-induced thrombocytopenia and elucidate the underlying mechanism using a chronic renal disease (CRD) mouse model. MATERIALS AND METHODS: CRD was induced in 5-week-old male Institute of Cancer Research (ICR) mice by 5/6 nephrectomy. After this procedure, LZD (25 and 100 mg/kg) was administered intraperitoneally once every day for 28 days. Platelet counts, white blood cell (WBC) counts, and hematocrit (HCT) levels were measured every 7 days. 2-14C-thymidine (0.185 MBq) was administrated intravenously to LZD-administered mice to evaluate the thymidine uptake ability of bone marrow. RESULTS: Platelet counts were significantly lower in the LZD-administered CRD group than in the LZD-nonadministered groups at 14, 21, and 28 days (P < 0.05); however, these changes were not observed in LZD-administered mice with normal renal function, regardless of the duration of LZD administration. No significant changes were observed in WBC counts or HCT levels in any LZD-administered CRD mouse. Moreover, radioactive levels in bone marrow were not significantly different in each group. CONCLUSIONS: These results indicate that LZD-induced decreases in platelet counts were enhanced by renal impairment in vivo, suggesting that LZD-induced thrombocytopenia is not caused by nonimmune-mediated bone marrow suppression.

Influence of dosing times on cisplatin-induced peripheral neuropathy in rats.

BACKGROUND: Although cis-diamminedichloro-platinum (CDDP) exhibits strong therapeutic effects in cancer chemotherapy, its adverse effects such as peripheral neuropathy, nephropathy, and vomiting are dose-limiting factors. Previous studies reported that chronotherapy decreased CDDP-induced nephropathy and vomiting. In the present study, we investigated the influence of dosing times on CDDP-induced peripheral neuropathy in rats. METHODS: CDDP (4 mg/kg) was administered intravenously at 5:00 or 17:00 every 7 days for 4 weeks to male Sprague-Dawley rats, and saline was given to the control group. To assess the dosing time dependency of peripheral neuropathy, von-Frey test and hot-plate test were performed. RESULTS: In order to estimate hypoalgesia, the hot-plate test was performed in rats administered CDDP weekly for 4 weeks. On day 28, the withdrawal latency to thermal stimulation was significantly prolonged in the 17:00-treated group than in the control and 5:00-treated groups. When the von-Frey test was performed to assess mechanical allodynia, the withdrawal threshold was significantly lower in the 5:00 and 17:00-treated groups than in the control group on day 6 after the first CDDP dose. The 5:00-treated group maintained allodynia throughout the experiment with the repeated administration of CDDP, whereas the 17:00-treated group deteriorated from allodynia to hypoalgesia. CONCLUSIONS: It was revealed that the severe of CDDP-induced peripheral neuropathy was inhibited in the 5:00-treated group, whereas CDDP-treated groups exhibited mechanical allodynia. These results suggested that the selection of an optimal dosing time ameliorated CDDP-induced peripheral neuropathy.

Circadian Clock in a Mouse Colon Tumor Regulates Intracellular Iron Levels to Promote Tumor Progression.

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 thatIrp2mRNA 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(Δ19) tumors expressing the clock-mutant protein (CLOCK(Δ19)) 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(Δ19)expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.

Mechanism of the 24-hour rhythm of tumor necrosis factor-alpha formed by onset of rheumatoid arthritis.

OBJECTIVE: Morning stiffness and plasma cytokine levels in rheumatoid arthritis (RA) patients exhibit 24-hour variations. Tumor necrosis factor-α (TNF-α) plays a central role in RA clinical conditions, including the invasion of inflammatory cells, destruction of cartilage, systemic inflammatory response and its levels show a 24-hour rhythm after the onset of RA. In this study, we investigated what cytokines and/or transcriptional factors are involved in the formation of 24-hour variations in TNF-α levels after the onset of RA using MRL/Mpj-Tnfrsf6(lpr) (MRL/lpr) mice. METHOD: Blood was drawn at six different times from MRL/lpr mice to measure cytokines, serum amyloid A (SAA), IgG rheumatoid factor (IgG-RF) and corticosterone levels. Cytokine and transcriptional factor levels at the different times were measured in 10- and/or 15-week-old MRL/lpr mice. The promoter activity of TNF-α by lymphotoxins (LTs) was investigated using a dual-luciferase assay. RESULTS: SAA and TNF-α concentrations clearly exhibited 24-hour rhythms with higher levels at the light phase and lower levels at the dark phase after RA crisis. The expression of LT-α and LT-ß showed significant 24-hour rhythms in 15-week-old MRL/lpr mice and the phases of LT-α and LT-ß levels were antiphase compared with that of TNF-α. AP-1 binding sites were found in LT-α and LT-ß promoter regions, and jun mRNA expression corresponded to LT-α and LT-ß levels. TNF-α promoter activity was decreased due to the co-transfection of LT-α and LT-ß. CONCLUSION: LT-α and LT-ß controls the 24-hour rhythm in TNF-α levels after the onset of RA in order to suppress TNF-α promoter activity.

System A amino acid transporter SNAT2 shows subtype-specific affinity for betaine and hyperosmotic inducibility in placental trophoblasts.

Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [(14)C]betaine uptake by HEK293 cells transiently transfected with human or rat sodium-coupled neutral amino acid transporters (SNATs), SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (Km) of betaine uptake by human and rat SNAT2 were estimated to be 5.3 mM and 4.6 mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts.

Circadian regulation of mTOR by the ubiquitin pathway in renal cell carcinoma.

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 tumor-bearing 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.

[Chrono-drug delivery system based on the circadian rhythm of transferrin receptor].

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.

Circadian rhythm of transferrin receptor 1 gene expression controlled by c-Myc in colon cancer-bearing mice.

The abundance of cell surface levels of transferrin receptor 1 (TfR1), which regulates the uptake of iron-bound transferring, correlates with the rate of cell proliferation. Because TfR1 expression is higher in cancer cells than in normal cells, it offers a target for cancer therapy. In this study, we found that the expression of TfR1 in mouse colon cancer cells was affected by the circadian organization of the molecular clock. The core circadian oscillator is composed of an autoregulatory transcription-translation feedback loop, in which CLOCK and BMAL1 are positive regulators and the Period (Per), Cryptochrome (Cry), and Dec genes act as negative regulators. TfR1 in colon cancer-bearing mice exhibited a 24-hour rhythm in mRNA and protein levels. Luciferase reporter analysis and chromatin immunoprecipitation experiments suggested that the clock-controlled gene c-MYC rhythmically activated the transcription of the TfR1 gene. Platinum incorporation into tumor DNA and the antitumor efficacy of transferrin-conjugated liposome-delivered oxaliplatin could be enhanced by drug administration at times when TfR1 expression increased. Our findings suggest that the 24-hour rhythm of TfR1 expression may form an important aspect of strategies for TfR1-targeted cancer therapy.

Influence of intermittent hypoxia on the signal transduction pathways to inflammatory response and circadian clock regulation.

AIMS: Obstructive sleep apnea syndrome (OSAS), characterized by intermittent hypoxia/reoxygenation (IHR), is often associated with changing levels of circulating inflammatory cytokines and causes excessive daytime sleepiness, mood disturbances, and cardiovascular disease. An abnormal rhythm in the expression of circadian clock genes is observed in OSAS patients, and is also implicated in OSAS-related clinical symptoms. IHR-induced signal transduction is thought to underlie OSAS-associated complications. The aim of this study is to elucidate the influence of IHR on signal transduction pathways to inflammatory response and circadian clock regulation. MAIN METHODS: To evaluate the direct action of IHR on intracellular signaling, we used a cell culture model to explore the underlying transcriptional events initiated by IHR. KEY FINDINGS: Treatment of cultured human lung adenocarcinoma epithelial cells (A549) with IHR resulted in the elevation of mRNA levels of an inflammation cytokine interleukin-6 (IL-6), due to activation of the signaling pathway of nuclear factor-kappaB, a potent transcriptional activator of IL-6. On the other hand, the treatment of cells with IHR had little effect on clock gene response element-driven transcription. As a consequence, there was no significant change in mRNA levels of clock genes in IHR-treated cells. SIGNIFICANCE: These results suggest that IHR can activate signal transduction to an inflammatory response, but not to circadian clock regulation. The abnormal rhythm in the expression of clock genes in OSAS patients is attributable to the changed levels of circulating factors that have the ability to modulate clock gene expression.