METHODS. Antiproliferation effects of PROG on 3AO and AO ovarian carcinomaShanghai Institute of Cell Biology, Chinese cells were determined by 3 H-thymidine incorporation. Apoptosis of the PROGAcademy of Sciences, Shanghai, China. treated cells was determined by DNA laddering analysis and was quantitated by both nuclear condensation and flow cytometry after cells were stained with propidium iodide. Cell cycle analysis was also performed by flow cytometry. The expression of p53, bcl-2, and c-myc after 72 hours of PROG treatment was detected by Northern blot analysis. RESULTS. In both 3AO and AO cell lines, cells proliferation was maximally inhibitedby PROG after 72 hours of treatment at 10 mM concentration. Under the same conditions, more than 50% of PROG-treated cells had undergone apoptosis, whereas less than 3% of the cells were apoptotic in untreated cell cultures. After exposure to PROG for 72 hours, cells were arrested in the G 1 phase of the cell cycle, and the levels of p53 mRNA were remarkably increased in both cell lines.No changes in expression of bcl-2 or c-myc were detected.CONCLUSIONS. PROG significantly inhibited cell proliferation and induced apoptosis in both of the ovarian carcinoma cell lines tested in this study. PROG treatment markedly up-regulated p53 expression in these cells, indicating involvement of p53 in PROG-induced apoptosis.

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Intracellular glucocorticoid (GC) receptor (GR) function determines tissue sensitivity to GCs and strongly affects the development of type 2 diabetes and obesity. 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1) mediates intracellular steroid exposure to mouse liver GR by prereceptor reactivation of GCs and is crucially dependent on hexose-6-phosphate dehydrogenase (H6PDH)-generating NADPH system. Pharmacological inhibition of 11b-HSD1 improves insulin intolerance and obesity. Here, we evaluated the potential beneficial effects of 11b-HSD1 inhibitor carbenoxolone (CBX) in diet-induced obese (DIO) and insulin-resistant mice by examining the possible influence of CBX on the expression of GR, 11b-HSD1, and H6PDH in vivo and in vitro in hepatocytes. Treatment of DIO mice with CBX markedly reduced hepatic GR mRNA levels and reduced weight gain, hyperglycemia, and insulin resistance. The reduction of hepatic GR gene expression was accompanied by CBX-induced inhibition of both 11b-HSD1 and H6PDH activity and mRNA in the liver. Moreover, CBX treatment also suppressed the expression of both phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase enzyme (G6Pase) mRNA and improved hepatic [1,[2][3] H] deoxy-D-glucose uptake in DIO mice. In addition, the treatment of primary cultures of hepatocytes with increasing concentrations of CBX led to a dose-dependent downregulation of GR mRNA levels, which correlated with the suppression of both 11b-HSD1 and H6PDH activity and their gene expression. Addition of CBX to primary hepatocytes also resulted in suppression of both PEPCK and G6Pase mRNA levels. These findings suggest that CBX exerts some of its beneficial effects, at least in part, by inhibiting hepatic GR and H6PDH expression.

Inhibition of integrins IntroductionAngiogenesis and endothelial cell responses are essential processes in diseases, such as cancer, and ischemic conditions. Integrins, heterodimeric cell-surface receptors composed of ␣ and ␤ subunits, are central regulators of angiogenesis and endothelial cell functions. 1 Integrins enable cells to adhere to extracellular matrix (ECM), migrate over ECM substrates, and respond to ECM contact by proliferation, differentiation, and protection from apoptosis mediated by regulation of a number of intracellular signaling pathways. 1-3 Inhibition of integrins ␣v␤3 and ␣v␤5, which are preferentially expressed and activated on angiogenic endothelial cells, induces endothelial apoptosis and impairs tumor angiogenesis. 4,5 ␣v␤3/␣v␤5-integrin signaling is mediated through interactions with an arginine-glycine-aspartic acid (RGD) peptide sequence in matrix proteins, such as vitronectin (VN), and can be abrogated by soluble function-blocking RGD peptides, such as cyclic RGDfV. 4 Indeed, inhibitors of integrin ␣v␤3 are undergoing clinical trials in cancer patients, and cilengitide (EMD 121974; Merck KGaA), an integrin ␣v␤3/␣v␤5 function-blocking RGDfV peptide, has encouraging activity in phase 1 and 2 trials against brain tumors in children and adult cancer patients. 6,7 Integrin ␣v␤3 mechanism of action is complex. ␣v␤3 participates in pathologic angiogenesis, 4,8 supporting its development as a target for therapy. To mediate its function, integrin ␣v␤3 requires activation and phosphorylation of the ␤3 integrin tail on Tyr747 and Tyr459, which signal downstream to pathways involving, among others, Src, FAK, Shc, p53, and p21 WAF . 8,9 Complicating matters, integrin ␣v␤3 cosignals with growth factor receptors, such as vascular endothelial growth factor receptor-2 and others. 8 The intracellular signaling events mediating outside-in and inside-out signaling are complex and depend on the context of activation of the integrin and the cell type studied. Therefore, it is not surprising that the precise molecular mechanisms induced by engagement, crosstalk, or inhibition of ␣v␤3 integrin remain only partially understood.Engagement of integrins with the ECM allows cells to adhere and spread, inducing changes in the actin cytoskeleton. Actin is an abundant cytoskeletal protein important in cell spreading and motility. 10,11 Engagement of integrins with the ECM generates complex bidirectional signaling cascades between integrins and the actin cytoskeleton, which serve to transmit both force and biochemical signals. The interaction of integrins with actin is mostly through a number of intermediary proteins that can be cell-specific and/or stimulus-specific. 11 A critical molecule that interacts with F-actin in fibroblasts is c-Abl. c-Abl integrates multiple signals to coordinate F-actin dynamics, whereas F-actin itself has an inhibitory effect on c-Abl kinase activity. 12-14 Therefore, it is anticipated that actindependent signaling, including that by c-Abl, may mediate at least some of the phenotypes ...

Purpose We present here the first report of PID1 (Phosphotyrosine Interaction Domain containing 1; NYGGF4) in cancer. PID1 was identified in 2006 as a gene that modulates insulin signaling and mitochondrial function in adipocytes and muscle cells. Experimental Design and Results Using four independent medulloblastoma datasets, we show that mean PID1 mRNA levels were lower in unfavorable medulloblastomas (Groups 3 and 4, and anaplastic histology) compared with favorable medulloblastomas (SHH and WNT groups, and desmoplastic/nodular histology) and with fetal cerebellum. In two large independent glioma datasets PID1 mRNA was lower in glioblastomas (GBMs), the most malignant gliomas, compared to other astrocytomas, oligodendrogliomas and non-tumor brains. Neural and proneural GBM subtypes had higher PID1 mRNA compared to classical and mesenchymal GBM. Importantly, overall survival and radiation-free progression-free survival were longer in medulloblastoma patients with higher PID1 mRNA (univariate and multivariate analyses). Higher PID1 mRNA also correlated with longer overall survival in glioma and GBM patients. In cell culture, overexpression of PID1 inhibited colony formation in medulloblastoma, atypical teratoid rhabdoid tumor (ATRT) and GBM cell lines. Increasing PID1 also increased cell death and apoptosis, inhibited proliferation, induced mitochondrial depolarization, and decreased serum-mediated phosphorylation of AKT and ERK in medulloblastoma, ATRT and/or GBM cell lines, whereas siRNA to PID1 diminished mitochondrial depolarization. Conclusions These data are the first to link PID1 to cancer and suggest that PID1 may have a tumor inhibitory function in these pediatric and adult brain tumors.

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Aims/hypothesis Tissue-specific amplification of glucocorticoid action through 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) affects the development of the metabolic syndrome. Hexose-6-phosphate dehydrogenase (H6PDH) mediates intracellular NADPH availability for 11β-HSD1 and depends on the glucose-6-phosphate transporter (G6PT). Little is known about the tissue-specific alterations of H6PDH and G6PT and their contributions to local glucocorticoid action in db/db mice. Methods We characterised the role of H6PDH and G6PT in pre-receptor metabolism of glucocorticoids by examining the production of the hepatic 11β-HSD1-H6PDH–G6PT system in db/db mice. Results We observed that increased production of hepatic H6PDH in db/db mice was paralleled by upregulation of hepatic G6PT production and responded to elevated circulating levels of corticosterone. Treatment of db/db mice with the glucocorticoid antagonist RU486 markedly reduced production of both H6PDH and 11β-HSD1 and improved hyperglycaemia and insulin resistance. The reduction of H6PDH and 11β-HSD1 production by RU486 was accompanied by RU486-induced suppression of hepatic G6pt (also known as Slc37a4) mRNA. Incubation of mouse primary hepatocytes with corticosterone enhanced G6PT and H6PDH production with corresponding activation of 11β-HSD1 and PEPCK: effects that were blocked by RU486. Knockdown of H6pd by small interfering RNA showed effects comparable with those of RU486 for attenuating the corticosterone-induced H6PDH production and 11β-HSD1 reductase activity in these intact cells. Addition of the G6PT inhibitor chlorogenic acid to primary hepatocytes suppressed H6PDH production. Conclusions/interpretation These findings suggest that increased hepatic H6PDH and G6PT production contribute to 11β-HSD1 upregulation of local glucocorticoid action that may be related to the development of type 2 diabetes.

Here we report the involvement of nociceptin receptor in tolerance to morphine-induced antinociception and in morphine dependence. There was no different nociceptive perception and antinociceptive effects of morphine between wild-type and the nociceptin receptor knockout mice. Tolerance to morphine (10 mg/kg)-induced antinociception was developed in both wild-type and the nociceptin receptor knockout mice after administration of morphine (10 mg/kg) twice a day for 5 days. When naloxone (5 mg/kg) was administered to mice treated with morphine repeatedly on the 6th day, morphine withdrawal syndrome was observed in both wild-type and the nociceptin receptor knockout mice, which were accompanied by the elevation of cyclic AMP levels. While naloxone benzoylhydrazone (1 mg/kg), a putative antagonist for nociceptin receptor/naloxone benzoylhydrazone-sensitive sites, also induced the morphine withdrawal signs in both wild-type and the nociceptin receptor knockout mice, the jumping signs in the nociceptin receptor knockout mice were less severe than those in wild-type mice. Treatment with naloxone benzoylhydrazone in morphine-dependent wild-type mice caused a significant increase in cyclic AMP levels in the thalamus while it had no effect in the nociceptin receptor knockout mice. The analysis of opioid mu-receptor binding showed no difference between wild-type and the nociceptin receptor knockout mice. These results suggest that the nociceptin receptor/naloxone benzoylhydrazone-sensitive sites contribute to the induction of morphine withdrawal syndrome in part. Furthermore, it is demonstrated that morphine withdrawal syndrome excepting jumping can be induced by naloxone benzoylhydrazone without any changes in the cyclic AMP levels in the thalamus.