Insulin-like growth factor-1 directly mediates expression of mitochondrial uncoupling protein 3 via forkhead box O4.

OBJECTIVE: The objective of our study was to examine the direct action of insulin-like growth factor-1(IGF-1) signaling on energy homeostasis in myocytes. DESIGN: We studied the IGF-1 stimulation of mitochondrial uncoupling protein 3 (UCP3) expression in the HEK 293 derived cell line TSA201, murine C2C12 skeletal muscle myoblasts, and rat L6 skeletal myoblasts. We also investigated the direct effect of IGF-1 on the Insulin/IGF-1 receptor (IGF-1R)/phosphatidylinositol 3 (PI3)-Akt/forkhead box O4 (FOXO4) pathway using a combination of a reporter assay, semi-quantitative polymerase chain reaction, western blotting, and animal experiments. RESULTS: We demonstrated that IGF-1 regulates UCP3 expression via phosphorylation of FOXO4, which is a downstream signal transducer of IGF-1. UCP3 expression increased with activated FOXO4 in a dose-dependent manner. We also examined the functional FOXO4 binding site consensus sequences and identified it as the -1922 bp site in the UCP3 promoter region. UCP3 was also found to be concomitantly expressed with IGF-1 during differentiation of C2C12 myoblasts. Our animal experiments showed that high fat diet induced IGF-1 levels which likely influenced UCP3 expression in the skeletal muscle. CONCLUSION: Our findings demonstrate that that IGF-1 directly stimulates UCP3 expression via the IGF-1/IGF-1R/PI3-Akt/FOXO4 pathway.

IGF-1 regulate the expression of uncoupling protein 2 via FOXO1.

Mitochondria uncoupling protein2 (UCP2) expressed ubiquitously is a key molecule of energy metabolism. Insulin-like growth factor-1 (IGF-1) is a hormone, a target molecule of growth hormone (GH) signal pathway, which is also known as the drug "mecasermin" for clinical usages. IGF-1 is seemed to be closely related to metabolic diseases, such as adult GH deficiency. However, there has not been reports depicted possible relationship with each other. So, we sought to elucidate the mechanisms by which expression of UCP2 is regulated by IGF-1 via FOXO1. The findings suggested that three sequences in the consensus UCP2 promoter play complementary functional roles in the functional expression of FOXO1. So, we found that FOXO1 is involved in IGF-1-mediated energy metabolism greater than that of direct action of GH via STAT5. Our findings suggested that IGF-1 was involved in energy metabolism by regulating the expression of UCP2 via the PI3K/Akt/FOXO1 pathway.

Verification of surface contamination of Japanese cyclophosphamide vials and an example of exposure by handling.

PURPOSE: Cyclophosphamide (CP) contamination has been detected in Japanese hospitals. In other countries, the surface contamination of CP vials has been reported; however, the manufacturing process of Japanese CP vials is unknown, so the conditions are not necessarily the same as in other countries. This study aimed to establish whether vial surface contamination also occurs in Japan. METHOD: Contamination of vial surfaces was examined with a wipe test. Urine samples were taken from a pharmacist, engaged solely in dispensing work, for 29 h. It was also investigated whether CP vials were dispensed during the urine sampling period. In addition, vial surfaces, purposely coated with CP and then washed, were examined using wipe tests. RESULT: CP was detected at 30-60% in vials, which was 11-62 ng (0.10-0.54 ng/cm(2)). One of the urine samples was contaminated (CP 13.5 ng); this was taken on Day 2 (11:35 AM). CP was not detected among the washed vials. DISCUSSION: This study shows that the surface of Japanese CP vials was contaminated and that it was probable that healthcare workers were exposed to CP. CP absorption by the pharmacist was probably due to dermal uptake while dispensing. Washing the vial is considered effective to avoid CP exposure. Manufacturers should be more proactive to prevent contamination and healthcare workers should comply with exposure prevention rules. Cytotoxic drugs should be included in institution monitoring lists.

Investigation on residual-related error and the effect of solution properties using protective devices for the reconstitution of cytotoxic agents in actual situations.

PURPOSE: Three products can be used in Japan for the reconstitution of cytotoxic agents: PhaSeal, Chemo CLAVE and Chemo Mini Spike (CMS). The low preparation volume may be affected by residual-related volume in their devices. In this study, the residual-related error in their devices was examined and compared. METHOD: The blank of each component of these devices was weighed using a precision electric balance. After ejecting distilled water (DW) for injection, each was weighed again with the balance. In addition, for etoposide in the cases of PhaSeal and Chemo CLAVE, the components of the devices were similarly weighed. RESULT: The weight gains of each device after ejecting DW were as follows: CMS-V (440 mg) greater than the combined components of Chemo CLAVE (128-171 mg)/CMS-MT (123 mg) greater than the combined components of PhaSeal (13-56 mg). For etoposide, the weight gains of PhaSeal (208 mg) and Chemo CLAVE (223 mg) showed no significant difference. The priming volume of each device was calculated from the specific gravity of water. The residual-related volume was 'CMS-V > Chemo CLAVE, CMS-MT > PhaSeal', although this was very slight in actual situations. CONCLUSION: The residual-related volume was marked in its low preparation volume. In water-soluble drugs, the residual volume of PhaSeal was lowest of the devices in this study, but in viscous drugs, such as etoposide, the residual volume of PhaSeal was almost identical to Chemo CLAVE; that is, the residual volume of these devices was affected by the solution property. The residual-related volume in the devices will lead to errors; therefore, residual-related errors need to be considered in the use of these devices.

Effect of dose-escalation of 5-fluorouracil on circadian variability of its pharmacokinetics in Japanese patients with Stage III/IVa esophageal squamous cell carcinoma.

OBJECTIVE: The effects of dose-escalation of 5-fluorouracil (5-FU) on the clinical outcome and pharmacokinetics of 5-FU were investigated in Japanese patients with Stage III/IVa esophageal squamous cell carcinoma. METHODS: Thirty-five patients with Stage III/IVa were enrolled, who were treated with a definitive 5-FU/cisplatin-based chemoradiotherapy. A course consisted of continuous infusion of 5-FU at 400 mg/m(2)/day (the standard dose group, N=27) or 500-550 mg/m(2)/day (the high dose group, N=8) for days 1-5 and 8-12, infusion of cisplatin at 40 mg/m(2)/day on days 1 and 8, and radiation at 2 Gy/day on days 1 to 5, 8 to 12, and 15 to 19, with a second course repeated after a 2-week interval. Plasma concentrations of 5-FU were determined by high performance liquid chromatography at 5:00 PM on days 3, 10, 38 and 45, and at 5:00 AM on days 4, 11, 39 and 46. RESULTS AND CONCLUSIONS: No patient with Stage IVa achieved a complete response in the standard dose group, whereas a complete response was observed at a rate of 50% in the high dose group, and this can be explained by a higher plasma concentration of 5-FU. The circadian rhythm in the concentrations found at the standard dose was not observed for a higher dose.

Replacement of cisplatin with nedaplatin in a definitive 5-fluorouracil/cisplatin-based chemoradiotherapy in Japanese patients with esophageal squamous cell carcinoma.

OBJECTIVE: The effects of replacing cisplatin (CDDP) with cis-diammineglycolatoplatinum (nedaplatin, NDP), a second-generation platinum complex, on the pharmacokinetics of 5-fluorouracil (5-FU) were investigated in Japanese patients with esophageal squamous cell carcinoma, who were treated with a definitive 5-FU/CDDP-based chemoradiotherapy. METHODS: Fifty-six patients were enrolled, 49 treated with CDDP and 7 treated with NDP. A course consisted of continuous infusion of 5-FU at 400 mg/m(2)/day for days 1-5 and 8-12, infusion of CDDP or NDP at 40 mg/m(2)/day on days 1 and 8, and radiation at 2 Gy/day on days 1 to 5, 8 to 12, and 15 to 19, with a second course repeated after a 2-week interval. Plasma concentrations of 5-FU were determined by high performance liquid chromatography at 5 PM on days 3, 10, 38 and 45, and at 5 AM on days 4, 11, 39 and 46. RESULTS AND CONCLUSIONS: The circadian rhythm in plasma concentrations of 5-FU observed in the case of CDDP was altered when NDP was used instead. The clinical response can be predicted by monitoring plasma concentrations of 5-FU in the CDDP group, but not in the NDP group.

Regression of abdominal aortic aneurysms by simultaneous inhibition of nuclear factor kappaB and ets in a rabbit model.

Because current therapy to treat abdominal aortic aneurysm (AAA), and particularly to manage small AAA, is limited to elective surgical repair, we explored less invasive molecular therapy by simultaneous inhibition of the transcription factors nuclear factor (NF)kappaB and ets using a decoy strategy. Both NFkappaB and ets were shown to be markedly activated in human AAA. In addition, NFkappaB- and ets-positive cells were increased in the aneurysm wall, and a part of the expression of NFkappaB and ets was detected in migrating macrophages. Thus, we used chimeric decoy oligodeoxynucleotides (ODNs) containing consensus sequences of both NFkappaB and ets binding sites to treat AAA. Inhibitory effects of chimeric decoy ODNs on matrix metalloproteinase-1 and -9 expression were confirmed by ex vivo experiments using a human aorta organ culture. To examine the regressive effect in a rabbit already-formed AAA model, transfection by wrapping a delivery sheet containing chimeric decoy ODNs around the aneurysm was performed 1 week after incubation with elastase. Importantly, treatment with chimeric decoy ODNs significantly decreased the size of AAA. Interestingly, significant preservation of elastic fibers was observed with chimeric decoy ODN treatment, accompanied by a reduction of matrix metalloproteinase-2 and -9 and induction of macrophage apoptosis. Regression of AAA was also associated with an increase in elastin and collagen type I and III synthesis in the aneurysm wall. Minimally invasive molecular therapy targeted to the inhibition of NFkappaB and ets is expected to be useful for AAA through the rebalance of matrix synthesis and degradation.

Hypertension accelerated experimental abdominal aortic aneurysm through upregulation of nuclear factor kappaB and Ets.

In this study, we focused on the effect of hypertension on the transcription factors nuclear factor kappaB (NFkappaB) and ets in the mechanisms of abdominal aortic aneurysm (AAA), and we investigated how hypertension affects the progression of AAA. AAA was produced by elastase perfusion in hypertensive rats and normotensive rats. The size of AAA rapidly increased in hypertensive rats as compared with normotensive rats. Western blot analysis demonstrated that the expression of matrix metalloproteinase (MMP)-2, -3 , -9, and -12, as well as intercellular adhesion molecule, was increased in hypertensive AAA rats, accompanied by upregulation of NFkappaB and ets. Moreover, in situ zymography showed that the activity of MMPs was increased in the aorta of a hypertensive AAA model as compared with that in a normotensive AAA model. Interestingly, transfection of chimeric decoy oligodeoxynucleotide (ODN) resulted in significant inhibition of aortic dilatation both in normotensive and hypertensive rats at 4 weeks after transfection. Destruction of elastic fibers was also significantly inhibited by transfection of chimeric decoy ODN in both hypertensive rats and normotensive rats. The expression of MMP-2, -3, -9, and -12, as well as intercellular adhesion molecule, was significantly attenuated by the chimeric decoy ODN, accompanied by inhibition of the migration of macrophages. Also, the effect of chimeric decoy ODN was confirmed in an organ culture. The present study demonstrated that hypertension accelerated the progression of experimental AAA through upregulation of NFkappaB and ets. Inhibition of NFkappaB and ets could be a novel therapeutic strategy to treat AAA in hypertensive patients.

Inhibition of experimental abdominal aortic aneurysm in the rat by use of decoy oligodeoxynucleotides suppressing activity of nuclear factor kappaB and ets transcription factors.

BACKGROUND: Two phenomena, inflammation and matrix degradation, contribute to the progression of abdominal aortic aneurysm (AAA). Importantly, the inflammation is regulated by the transcription factor nuclear factor (NF)-kappaB, whereas the destruction and degradation of elastin fibers by matrix metalloproteinases (MMP) are regulated by ets. Thus, we developed a novel strategy to treat AAA by simultaneous inhibition of both NF-kappaB and ets by using chimeric decoy oligodeoxynucleotides (ODN). METHODS AND RESULTS: AAA was induced in rats by transient aortic perfusion with elastase, whereas transfection of decoy ODN was performed by wrapping a delivery sheet containing decoy ODN around the aorta. Gel-mobility shift assay at 7 days after treatment demonstrated that both NF-kappaB and ets binding activity were simultaneously inhibited by chimeric decoy ODN. Transfection of chimeric decoy ODN resulted in significant inhibition of the progression of AAA such as aneurysmal dilation at 4 weeks after treatment as compared with control, accompanied by a reduction of MMP expression. Moreover, the destruction of elastin fibers was inhibited in the aorta transfected with chimeric decoy ODN. Importantly, transfection of chimeric decoy ODN demonstrated potent inhibition of aneurysmal dilatation compared with NF-kappaB decoy ODN alone, whereas scrambled decoy ODN had no effects. Interestingly, the migration of macrophages was significantly inhibited by chimeric decoy ODN. CONCLUSIONS: We demonstrated that inhibition of the progression of AAA was achieved by a novel strategy with chimeric decoy ODN used against NF-kappaB and ets in rat model. NF-kappaB and ets are considered to play an important role in the pathogenesis of AAA.