In mammalian cultured cells, the cystine/glutamate exchange transport mediated by system x c ؊ is important to maintain intracellular GSH levels. System x c ؊ consists of two protein components, xCT and the heavy chain of 4F2 antigen. The activity of system x c ؊ is induced by various stimuli, including electrophilic agents like diethyl maleate. In the present study, we have investigated the mechanism of the transcriptional regulation of xCT mRNA by diethyl maleate. The xCT gene consisted of twelve exons and sequence analysis identified four electrophile response element (EpRE)-like sequences between ؊230 and ؊1 in the 5-flanking region, designated EpRE-1 to EpRE-4. To identify sequences mediating the constitutive and induced expression of xCT, a series of 5-deletion mutants created from the 5-flanking region were cloned into a luciferase reproter vector and transfected into BHK21 cells. The 5-deletion analysis revealed that the sequence between ؊116 and ؊82 is essential for the basal expression and the sequence between ؊226 and ؊116 containing EpRE-1 is essential in response to diethyl maleate. Mutational analysis demonstrated that EpRE-1 is critically involved in the response to diethyl maleate. Other stress agents like arsenite, cadmium, and hydroquinone seemed to induce system x c ؊ activity via the same sequence. Furthermore, the experiments using the mouse embryonic fibroblasts derived from the Nrf2-deficient mice revealed that the induction of xCT gene by electrophilic agents is mediated by Nrf2. EpRE occurs in a broad spectrum of genes for the proteins that are involved in the defense against xenobiotics and regulates their expression. The present results have demonstrated that xCT is a novel member of this protein family.
Twenty-one patients with pancreatic cancer pain were studied to evaluate the effectiveness of celiac plexus block (CPB) on pain relief and quality of life (QOL), compared to the traditional NSAID-morphine treatment. The criteria were morphine consumption, visual analogue pain scale (VAS), performance status (PS) determined by medical and nursing staffs, and answers to QOL questionnaires. Morphine consumption, VAS, PS, and self-assessed QOL scores were taken when the administration of morphine was necessary for pain relief and those scores were used as control. Morphine consumption and the VAS score were recorded at regular weekly intervals and the PS and QOL scores were measured every 2 weeks thereafter. CPB was performed within 2-3 days after the control measurement. The VAS scores of the patients receiving CPB (n = 10) were statistically lower for the first 4 weeks after the procedure than those of the patients receiving the standard NSAID-morphine treatment (n = 11) during the same time period after the control measurement. Morphine consumption was significantly lower in weeks 4-7 (inclusive) following the procedure in the CPB group and continued to be lower thereafter, though not significantly so. Although the PS score slightly improved at the 2nd week after CPB, it was not improved by the start of the NSAID-morphine treatment. Self-assessed QOL scores did not ameliorate statistically after CPB; however, they did deteriorate remarkably in the patients treated only with morphine-NSAID during their survival periods, while they deteriorated only slightly in the CPB group. There were fewer side effects after CPB. These results indicate CPB does not directly improve QOL in patients with pancreatic cancer pain, but it may prevent deterioration in QOL by the long-lasting analgesic effect, limitation of side effects and the reduction of morphine consumption, compared to treatment only with NSAID-morphine.
The study shows constitutive activation of the Notch pathway in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of osteosarcoma. We investigated the expression of the Notch pathway molecules in osteosarcoma biopsy specimens and examined the effect of Notch pathway inhibition. Real-time PCR revealed overexpression of Notch2, Jagged1, HEY1, and HEY2. On the other hand, Notch1 and DLL1 were downregulated in biopsy specimens. Notch pathway inhibition using g-secretase inhibitor and CBF1 siRNA slowed the growth of osteosarcomas in vitro. In addition, g-secretase inhibitortreated xenograft models exhibited significantly slower osteosarcoma growth. Cell cycle analysis revealed that g-secretase inhibitor promoted G1 arrest. Real-time PCR and western blot revealed that g-secretase inhibitor reduced the expression of accelerators of the cell cycle, including cyclin D1, cyclin E1, E2, and SKP2. On the other hand, p21cip1 protein, a cell cycle suppressor, was upregulated by g-secretase inhibitor treatment. These findings suggest that inhibition of Notch pathway suppresses osteosarcoma growth by regulation of cell cycle regulator expression and that the inactivation of the Notch pathway may be a useful approach to the treatment of patients with osteosarcoma.
BackgroundThe Hedgehog signaling pathway functions as an organizer in embryonic development. Recent studies have demonstrated constitutive activation of Hedgehog pathway in various types of malignancies. However, it remains unclear how Hedgehog pathway is involved in the pathogenesis of osteosarcoma. To explore the involvement of aberrant Hedgehog pathway in the pathogenesis of osteosarcoma, we investigated the expression and activation of Hedgehog pathway in osteosarcoma and examined the effect of SMOOTHENED (SMO) inhibition.ResultsTo evaluate the expression of genes of Hedgehog pathway, we performed real-time PCR and immunohistochemistry using osteosarcoma cell lines and osteosarcoma biopsy specimens. To evaluate the effect of SMO inhibition, we did cell viability, colony formation, cell cycle in vitro and xenograft model in vivo. Real-time PCR revealed that osteosarcoma cell lines over-expressed Sonic hedgehog, Indian hedgehog, PTCH1, SMO, and GLI. Real-time PCR revealed over-expression of SMO, PTCH1, and GLI2 in osteosarcoma biopsy specimens. These findings showed that Hedgehog pathway is activated in osteosarcomas. Inhibition of SMO by cyclopamine, a specific inhibitor of SMO, slowed the growth of osteosarcoma in vitro. Cell cycle analysis revealed that cyclopamine promoted G1 arrest. Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb. On the other hand, p21cip1 wprotein was up-regulated by cyclopamine treatment. In addition, knockdown of SMO by SMO shRNA prevents osteosarcoma growth in vitro and in vivo.ConclusionsThese findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma.
The absorption and excretion of paeoniflorin after intravenous and oral administration was studied in rats to evaluate the significance of paeoniflorin in the pharmacological action of Paeony root. The plasma concentration of paeoniflorin after intravenous administration at the doses of 0.5, 2.0 and 5.0 mg kg-1 rapidly decreased, simulated by a biexponential curve, with mean terminal half-lives of 11.0, 9.9 and 12.6 min, respectively. The Vdss values were 0.332, 0. 384 and 0.423 L kg-1 and the CLtot values were 26.1, 31.2 and 30.3 mL min-1 kg-1 at each dose. When given orally at the same doses, the absolute bioavailability values (F) determined by the AUC were 0.032, 0.033 and 0.038, respectively. The cumulative urinary and faecal excretions of paeoniflorin at the dose of 5 mg kg-1 after intravenous administration were 50.5 and 0.22% of the dose within 72 h, and 1.0 and 0.08% of the dose after oral administration within 48 h, respectively. Cumulative biliary excretion after intravenous or oral administration at a dose of 0.5 mg kg-1 was 6.9 and 1.3% of the dose within 24 h, respectively. The total CLR and CLB value after intravenous dosing was less than the CLtot value. These findings suggest that paeoniflorin is metabolized in other organs as well as in the liver. We conclude that paeoniflorin absorbed is excreted mainly in urine, it has a low bioavailability and the metabolites may be involved in the pharmacological action of Paeony root.
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