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Cytotoxic metabolites originating from the peripheral circulation can induce central nervous system complications associated with diabetes. Since a large proportion of these metabolites bind to plasma albumin, mechanisms for transporting albumin-metabolite complexes into the brain exist under diabetic conditions. Secreted protein acidic and rich in cysteine (SPARC) is one of the vesicular transport receptors responsible for albumin transport. This study aimed to investigate the changes in SPARC expression and cellular albumin transfer under high-glucose conditions and evaluate the permeability of molecules with high protein-bound properties to the brain tissue. Glucose (30 mM) increased SPARC expression, and intracellular albumin accumulation in NIH3T3 cells. In addition, these changes were observed in the brain of ob/ob mice. Brain microvessels function as a physiological barrier to limit the penetration of molecules from the peripheral blood circulation into the brain by forming tight junctions. Although protein expression of molecules involved in tight junction formation and cell adhesion was increased in the brain microvessels of ob/ob mice, molecular transfer into the brain through cellular junctions was not enhanced. However, Evans blue dye injected into the peripheral vein and endogenous advanced glycation end-products, exerted a high protein-binding property and accumulated in their brains. These observations indicate that peripheral molecules with high protein-binding properties invade the brain tissue and bind to albumin through transcytosis mediated by SPARC.
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The retina is the only organ projecting external light to the suprachiasmatic nucleus. Cholecystokinin receptor type A (Cckar/Cckar) is one of the essential factors for light reception in retinal cells. As there was a lack of literature on the matter, we aimed to elucidate the cause of the time-dependent phase change in clock gene expression. We found that Cckar mRNA expression in retinal cells exhibited diurnal variations. The rhythm of expression of the clock gene Per1/Per2 in retinal cells was altered in Cckar -/- mice. The light sensitivity of retinal cells was evaluated in wild-type mice, which showed c-Fos was activated in the ganglion cell layer more than in the inner granular layer. This increase in the number of c-Fos-positive cells was suppressed by lorglumide, a Cckar antagonist. Treatment of rat retina primary cells with lorglumide suppressed Per2 transcription, which was altered in a time-dependent manner relative to the Per2 expression. Light irradiation studies in Cckar -/- mice did not exhibit an increase in Period expression in the suprachiasmatic nucleus. These results indicate that Cckar is among the factors that regulate the cycle of clock genes on the retina. Cckar knockout attenuates the light responsiveness of suprachiasmatic nucleus and reduces the expression amplitude of Period genes in the retina. Thus, Cckar may contribute to entrainment of the light environment and maintenance of the expression cycle of Period gene, which is one of the core clock genes.
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BACKGROUND: The metabolic reprogramming of amino acids is critical for cancer cell growth and survival. Notably, intracellular accumulation of cysteine is often observed in various cancers, suggesting its potential role in alleviating the oxidative stress associated with rapid proliferation. The liver is the primary organ for cysteine biosynthesis, but much remains unknown about the metabolic alterations of cysteine and their mechanisms in hepatocellular carcinoma cells. METHODS: RNA-seq data from patients with hepatocarcinoma were analyzed using the TNMplot database. The underlying mechanism of the oncogenic alteration of cysteine metabolism was studied in mice implanted with BNL 1ME A.7 R.1 hepatocarcinoma. RESULTS: Database analysis of patients with hepatocellular carcinoma revealed that the expression of enzymes involved in de novo cysteine synthesis was down-regulated accompanying with increased expression of the cystine uptake transporter xCT. Similar alterations in gene expression have also been observed in a syngeneic mouse model of hepatocarcinoma. The enhanced expression of DNA methyltransferase in murine hepatocarcinoma cells caused methylation of the upstream regions of cysteine synthesis genes, thereby repressing their expression. Conversely, suppression of de novo cysteine synthesis in healthy liver cells induced xCT expression by up-regulating the oxidative-stress response factor NRF2, indicating that reduced de novo cysteine synthesis repulsively increases cystine uptake via enhanced xCT expression, leading to intracellular cysteine accumulation. Furthermore, the pharmacological inhibition of xCT activity decreased intracellular cysteine levels and suppressed hepatocarcinoma tumor growth in mice. CONCLUSIONS: Our findings indicate an underlying mechanism of the oncogenic alteration of cysteine metabolism in hepatocarcinoma and highlight the efficacy of alteration of cysteine metabolism as a viable therapeutic target in cancer.
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Prostaglandins are bioactive compounds, and the activation of their receptors affects the expression of clock genes. However, the prostaglandin F receptor (Ptgfr) has no known relationship with biological rhythms. Here, we first measured the locomotor period lengths of Ptgfr-KO (B6.129-Ptgfrtm1Sna) mice and found that they were longer under constant dark conditions (DD) than those of wild-type (C57BL/6J) mice. We then investigated the clock gene patterns within the suprachiasmatic nucleus in Ptgfr-KO mice under DD and observed a decrease in the expression of the clock gene cryptochrome 1 (Cry1), which is related to the circadian cycle. Moreover, the expression of Cry1, Cry2, and Period2 (Per2) mRNA were significantly altered in the mouse liver in Ptgfr-KO mice under DD. In the wild-type mouse, the plasma prostaglandin F2α (PGF2α) levels showed a circadian rhythm under a 12 h cycle of light-dark conditions. In addition, in vitro experiments showed that the addition of PTGFR agonists altered the amplitude of Per2::luc activity, and this alteration differed with the timing of the agonist addition. These results lead us to hypothesize that the plasma rhythm of PGF2α is important for driving clock genes, thus suggesting the involvement of PGF2α- and Ptgfr-targeting drugs in the biological clock cycle.
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Ritmo Circadiano , Dinoprost , Ratones , Animales , Dinoprost/metabolismo , Ratones Endogámicos C57BL , Ritmo Circadiano/genética , Relojes Biológicos , Núcleo Supraquiasmático/metabolismo , Expresión Génica , Criptocromos/genética , Criptocromos/metabolismoRESUMEN
Neuropathic pain often results from injuries and diseases that affect the somatosensory system. Disruption of the circadian clock has been implicated in the exacerbation of the neuropathic pain state. However, in this study, we report that mice deficient in a core clock component Period2 (Per2m/m mice) fail to develop tactile pain hypersensitivity even following peripheral nerve injury. Similar to male wild-type mice, partial sciatic nerve ligation (PSL)-Per2m/m male mice showed activation of glial cells in the dorsal horn of the spinal cord and increased expression of pain-related genes. Interestingly, α1D-adrenergic receptor (α1D-AR) expression was up-regulated in the spinal cord of Per2m/m mice, leading to increased production of 2-arachidonoylglycerol (2-AG), an endocannabinoid receptor ligand. This increase in 2-AG suppressed the PSL-induced tactile pain hypersensitivity. Furthermore, intraspinal dorsal horn injection of adeno-associated viral vectors expressing α1D-AR also attenuated pain hypersensitivity in PSL-wild-type male mice by increasing 2-AG production. Our findings reveal an uncovered role of the circadian clock in neuropathic pain disorders and suggest a link between α1D-AR signaling and the endocannabinoid system.
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Neuropathic pain associated with cancers is caused by tumor growth compressing and damaging nerves, which would also be enhanced by inflammatory factors through sensitizing nociceptor neurons. A troublesome hallmark symptom of neuropathic pain is hypersensitivity to innocuous stimuli, a condition known as "tactile allodynia", which is often refractory to NSAIDs and opioids. The involvement of chemokine CCL2 (monocyte chemoattractant protein-1) in cancer-evoked neuropathic pain is well established, but opinions remain divided as to whether CCL2 is involved in the production of tactile allodynia with tumor growth. In this study, we constructed Ccl2 knockout NCTC 2472 (Ccl2-KO NCTC) fibrosarcoma cells and conducted pain behavioral test using Ccl2-KO NCTC-implanted mice. Implantation of naïve NCTC cells around the sciatic nerves of mice produced tactile allodynia in the inoculated paw. Although the growth of Ccl2 KO NCTC-formed tumors was comparable to that of naïve NCTC-formed tumors, Ccl2-KO NCTC-bearing mice failed to show tactile pain hypersensitivity, suggesting the involvement of CCL2 in cancer-induced allodynia. Subcutaneous administration of controlled-release nanoparticles containing the CCL2 expression inhibitor NS-3-008 (1-benzyl-3-hexylguanidine) significantly attenuated tactile allodynia in naïve NCTC-bearing mice accompanied by a reduction of CCL2 content in tumor masses. Our present findings suggest that inhibition of CCL2 expression in cancer cells is a useful strategy to attenuate tactile allodynia induced by tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for the treatment of cancer-evoked neuropathic pain. SIGNIFICANCE STATEMENT: The blockade of chemokine/receptor signaling, particularly for C-C motif chemokine ligand 2 (CCL2) and its high-affinity receptor C-C chemokine receptor type 2 (CCR2), has been implicated to attenuate cancer-induced inflammatory and nociceptive pain. This study demonstrated that continuous inhibition of CCL2 production from cancer cells also prevents the development of tactile allodynia associated with tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for management of cancer-evoked tactile allodynia.
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Fibrosarcoma , Neuralgia , Animales , Ratones , Quimiocina CCL2/metabolismo , Quimiocina CCL2/uso terapéutico , Preparaciones de Acción Retardada , Fibrosarcoma/complicaciones , Fibrosarcoma/tratamiento farmacológico , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Ligandos , Neuralgia/tratamiento farmacológicoRESUMEN
Diurnal oscillations in the expression of several types of cell surface transporters have been demonstrated in the intestinal epithelial cells, which are mainly generated at transcriptional or degradation processes. Concentrative nucleoside transporter-2 (CNT2) is expressed at the apical site of intestinal epithelial cells and contributes to the uptake of nucleosides and their analogs from the intestinal lumen into the epithelial cells. In this study, we demonstrated that the localization of CNT2 protein in the plasma membrane of mouse intestinal epithelial cells exhibited a diurnal oscillation without changing its protein level in the whole cell. The scaffold protein PDZK1 interacted with CNT2 and stabilized its plasmalemmal localization. The expression of PDZK1 was under the control of molecular components of the circadian clock. Temporal accumulation of PDZK1 protein in intestinal epithelial cells enhanced the plasmalemmal localization of CNT2 at certain times of the day. The temporal increase in CNT2 protein levels at the plasma membrane also facilitated the uptake of adenosine into the intestinal epithelial cells. These results suggest a novel molecular mechanism for the diurnal localization of cell surface transporters and extend our understanding of the biological clock system that generates apparent physiological rhythms.
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Proteínas Portadoras , Nucleósidos , Animales , Ratones , Transporte Biológico , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Células Epiteliales/metabolismoRESUMEN
Type 1 diabetes mellitus (T1DM) is characterized by pancreatic beta cell destruction by autoantibodies and other factors, resulting in insulin secretion deficiency. Therefore, beta cell regeneration would be necessary to cure the disease. Nevertheless, the impact of type 1 diabetes on the stemness and transplantation efficiency of stem cells has not been previously described. In this study, we used next-generation sequencing to identify genes differentially expressed in T1DM adipose-derived stem cells (T1DM ADSCs) that originate from patients with type 1 diabetes. Furthermore, we evaluated their effects on transplantation efficiency following xenotransplantation into immunodeficient mice. In the T1DM ADSCs transplant group, the volume and weight of the graft were significantly reduced and the transplant efficiency was reduced. Next-generation sequencing and quantitative PCR results showed that T1DM ADSCs had significantly increased expression of AMFR and DCTN2. AMFR and DCTN2 gene knockdown in T1DM ADSC significantly restored cell proliferation and stem cell marker expression. Therefore, transplantation of T1DM ADSCs, in which AMFR and DCTN2 were knocked down, into immunodeficient mice improved transplant efficiency. This study revealed that AMFR and DCTN2 can reduce transplantation efficiency of T1DM ADSCs. Focusing on AMFR and DCTN2 is expected to increase the efficiency of stem cell transplantation therapy for diabetic patients.
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Diabetes patients are at a high risk of developing complications related to angiopathy and disruption of the signal transduction system. The liver is one of the multiple organs damaged during diabetes. Few studies have evaluated the morphological effects of adhesion factors in diabetic liver. The influence of diurnal variation has been observed in the expression and functioning of adhesion molecules to maintain tissue homeostasis associated with nutrient uptake. The present study demonstrated that the rhythm-influenced functioning of tight junction was impaired in the liver of ob/ob mice. The tight junctions of hepatocytes were loosened during the dark period in control mice compared to those in ob/ob mice, where the hepatocyte gaps remained open throughout the day. The time-dependent expression of zonula occludens 1 (ZO1, encoded by Tjp1 gene) in the liver plays a vital role in the functioning of the tight junction. The time-dependent expression of ZO1 was nullified and its expression was attenuated in the liver of ob/ob mice. ZO1 expression was inhibited at the mRNA and protein levels. The expression rhythm of ZO1 was found to be regulated by heat shock factor (HSF)1/2, the expression of which was reduced in the liver of ob/ob mice. The DNA-binding ability of HSF1/2 was decreased in the liver of ob/ob mice compared to that in control mice. These findings suggest the involvement of impaired expression and functioning of adhesion factors in diabetic liver complications.
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Moléculas de Adhesión Celular , Uniones Estrechas , Proteína de la Zonula Occludens-1 , Animales , Ratones , Moléculas de Adhesión Celular/análisis , Hepatocitos/metabolismo , Hígado , Uniones Estrechas/química , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/análisis , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Eating during a rest phase disrupts the biological clock system and leads to obesity and metabolic diseases. Although a rest phase restricted feeding (RF) is reported to enhance hepatic lipid accumulation, the mechanism(s) of the phenomenon is still unknown. This study evaluated the potential involvement of the CD36-related transport of lipids into the liver in mice with the RF procedure. This study showed that hepatic lipid accumulation was more significant in the RF group compared with mice under an active phase restricted feeding (AF). The RF procedure also elevated the expression of CD36 mRNA and its protein on the cellular membrane throughout the day. The transcription factor profiling array revealed that the RF activated the proliferator-activated receptor-γ (PPARγ), one of the CD36 transcript enhancers. In the liver of RF mice, the expression of miR-27b-3p, which is known to interfere with PPARγ gene expression, significantly decreased. These results suggest that the RF procedure inhibits the expression of miR-27b-3p in the liver and subsequently elevates PPARγ activity. Activated PPARγ might lead to CD36 upregulation, which, in turn, stimulates the transport of lipids into the liver.
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MicroARNs , PPAR gamma , Animales , Ritmo Circadiano , Lípidos , Hígado/metabolismo , Ratones , MicroARNs/genética , PPAR gamma/metabolismoRESUMEN
Regenerative therapeutic approaches involving the transplantation of stem cells differentiated into insulin-producing cells are being studied in patients with rapidly progressing severe diabetes. Adipose-derived mesenchymal stem cells have been reported to have varied cellular characteristics depending on the biological environment of the location from which they were harvested. However, the characteristics of mesenchymal stem cells in type II diabetes have not been clarified. In this study, we observed the organelles of mesenchymal stem cells from patients with type II diabetes under a transmission electron microscope to determine the structure of stem cells in type II diabetes. Transmission electron microscopic observation of mesenchymal stem cells from healthy volunteers (N-ADSC) and those from patients with type II diabetes (T2DM-ADSC) revealed enlarged nuclei and degenerated mitochondrial cristae in T2DM-ADSCs. Moreover, T2DM-ADSCs were shown to exhibit a lower expression of Emerin, a constituent protein of the nuclear membrane, and a decreased level of mitochondrial enzyme activity. In this study, we successfully demonstrated the altered structure of nuclear membrane and the decreased mitochondrial enzyme activity in adipose-derived mesenchymal cells from patients with type II diabetes. These findings have contributed to the understanding of type II diabetes-associated changes in mesenchymal stem cells used for regenerative therapy.
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Diferenciación Celular , Diabetes Mellitus Tipo 2/fisiopatología , Células Madre Mesenquimatosas/patología , Mitocondrias/patología , Membrana Nuclear/patología , Adulto , Células Cultivadas , Femenino , Humanos , Células Madre Mesenquimatosas/metabolismo , Mitocondrias/metabolismo , Membrana Nuclear/metabolismo , Adulto JovenRESUMEN
The expression and function of some xenobiotic transporters varies according to the time of day, causing the dosing time-dependent changes in drug disposition and toxicity. Multidrug resistance-associated protein-4 (MRP4), an ATP-binding cassette (ABC) efflux transporter encoded by the Abcc4 gene, is highly expressed in bone marrow cells (BMCs) and protects them against xenobiotics, including chemotherapeutic drugs. In this study, we demonstrated that MRP4 was responsible for the extrusion of oxaliplatin (L-OHP), a platinum (Pt)-based chemotherapeutic drug, from BMCs of mice, and that the efflux transporter expression exhibited significant diurnal variation. Therefore, we investigated the relevance of the diurnal expression of MRP4 in BMCs for L-OHP-induced myelotoxicity in mice maintained under standardized light/dark cycle conditions. After intravenous injection of L-OHP, the Pt content in BMCs varied according to the injection time. Lower Pt accumulation in BMCs was detected in mice after injection of L-OHP at the mid-dark phase, during which the expression levels of MRP4 increased. Consistent with these observations, the myelotoxic effects of L-OHP were attenuated when mice were injected with L-OHP during the dark phase. This dosing schedule also alleviated the L-OHP-induced reduction of the peripheral white blood cell count. The present results suggest that the myelotoxicity of L-OHP is attenuated by optimizing the dosing schedule. Diurnal expression of MRP4 in BMCs is associated with the dosing time-dependent changes in L-OHP-induced myelotoxicity.
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Ritmo Circadiano/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antineoplásicos/farmacología , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos ICR , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Compuestos Organoplatinos/farmacología , Oxaliplatino/farmacologíaRESUMEN
The expression levels of many cell-surface proteins vary with the time of day. Glycoprotein 2 (Gp2), specifically expressed on the apical surface of M cells in Peyer's patches, functions as a transcytotic receptor for mucosal antigens. We report that cAMP response element-binding protein (CREB) regulates the transcription of the Gp2 gene, thereby generating the circadian change in its expression in mouse Peyer's patches. The transcytotic receptor activity of Gp2 was increased during the dark phase when the Gp2 protein abundance increased. Rhythmic expression of clock gene mRNA was observed in mouse Peyer's patches, and expression levels of Gp2 mRNA also exhibited circadian oscillation, with peak levels during the early dark phase. The promoter region of the mouse Gp2 gene contains several cAMP response elements (CREs). Chromatin immunoprecipitation assays revealed that CREB bound to the CREs in the Gp2 gene in Peyer's patches. Forskolin, which promotes CREB phosphorylation, increased the transcription of the Gp2 gene in Peyer's patches. As phosphorylation of CREB protein was increased when Gp2 gene transcription was activated, CREB may regulate the rhythmic expression of Gp2 mRNA in Peyer's patches. These findings suggest that intestinal immunity is controlled by the circadian clock system.
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Relojes Biológicos , Ritmo Circadiano , Proteínas Ligadas a GPI/metabolismo , Ganglios Linfáticos Agregados/metabolismo , Animales , Proteínas Ligadas a GPI/genética , Masculino , Ratones , Ratones Endogámicos ICR , Ratones MutantesRESUMEN
P-glycoprotein (P-gp/ABCB1) is an ATP-binding cassette drug efflux transporter expressed in a variety of tissues that affects the pharmacokinetic disposition of many drugs. Although several studies have reported gender-dependent differences in the expression of P-gp, the role of sex hormones in regulating the expression of P-gp and its transport activity has not been well understood. In this study, we demonstrated that 17ß-estradiol has the ability to induce the expression of P-pg in mouse kidneys and cultured human renal proximal tubular epithelial cells. After intravenous injection of a typical P-gp substrate, digoxin, renal clearance in female mice was approximately 2-fold higher than that in male mice. The expression of murine P-gp and its mRNA (Abcb1a and Abcb1b) were also higher in female mice than in male mice. The expression of P-gp in cultured renal tissues prepared from female and male mice was significantly increased by 17ß-estradiol, but not testosterone. Similar 17ß-estradiol-induced expression of P-gp was also detected in cultured human tubular epithelial cells, accompanied by the enhancement of its transport activity of digoxin. The present findings suggest the contribution of estradiol to female-predominant expression of P-gp in renal cells, which is associated with sex-related disparities in the renal elimination of digoxin.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Digoxina/farmacocinética , Células Epiteliales/efectos de los fármacos , Estradiol/farmacología , Estrógenos/farmacología , Túbulos Renales/efectos de los fármacos , Riñón/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Células Cultivadas , Digoxina/administración & dosificación , Digoxina/análisis , Células Epiteliales/metabolismo , Femenino , Humanos , Inyecciones Intravenosas , Riñón/metabolismo , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Distribución TisularRESUMEN
A number of diverse cell-surface proteins are anchored to the cytoskeleton via scaffold proteins. Na+/H+ exchanger regulatory factor-1 (NHERF1), encoded by the Slc9a3r1 gene, functions as a scaffold protein, which is implicated in the regulation of membrane expression of various cell-surface proteins. Here, we demonstrate that the circadian clock component PERIOD2 (PER2) modulates transcription of the mouse Slc9a3r1 gene, generating diurnal accumulation of NHERF1 in the mouse liver. Basal expression of Slc9a3r1 was dependent on transcriptional activation by p65/p50. PER2 bound to p65 protein and prevented p65/p50-mediated transactivation of Slc9a3r1. The time-dependent interaction between PER2 and p65 underlay diurnal oscillation in the hepatic expression of Slc9a3r1/NHERF1. The results of immunoprecipitation experiments and liquid chromatography-mass spectrometry analysis of mouse liver revealed that NHERF1 time-dependently interacted with fatty acid transport protein-5 (FATP5). Temporary accumulation of NHERF1 protein stabilized plasmalemmal localization of FATP5, thereby enhancing hepatic uptake of fatty acids at certain times of the day. Our results suggest an unacknowledged role for PER2 in regulating the diurnal expression of NHERF1 in mouse liver. This machinery also contributed to diurnal changes in the ability of hepatic cells to uptake fatty acids.
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Relojes Circadianos/genética , Ritmo Circadiano/genética , Proteínas Circadianas Period/genética , Fosfoproteínas/genética , Intercambiadores de Sodio-Hidrógeno/genética , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Transporte de Ácidos Grasos/genética , Proteínas de Transporte de Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Noqueados , Células 3T3 NIH , Proteínas Circadianas Period/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
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 XOD gene and that bile acids suppressed XOD 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 XOD 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.
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Ácidos y Sales Biliares/metabolismo , PPAR alfa/metabolismo , Xantina Oxidasa/metabolismo , Animales , Ritmo Circadiano/fisiología , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Receptores Nucleares Huérfanos/metabolismo , Purinas/metabolismo , Ácido Úrico/metabolismo , Xantina Deshidrogenasa/metabolismo , Xantina Oxidasa/genéticaRESUMEN
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.
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Antineoplásicos/farmacología , Difosfonatos/farmacología , Ácidos Grasos Monoinsaturados/farmacología , Imidazoles/farmacología , Indoles/farmacología , Anticolesterolemiantes/farmacología , Antioxidantes/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fluvastatina , Humanos , Ácido Mevalónico/metabolismo , Neoplasias Pancreáticas/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Ácido Zoledrónico , Proteínas ras/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
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.