Increasing the intracellular availability of all-trans retinoic acid in neuroblastoma cells

Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 μM) or R116010 (1 or 10 μM) in combination with either 10 μM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma

Molecular targeting of retinoic acid metabolism in neuroblastoma: the role of the CYP26 inhibitor R116010 in vitro and in vivo

Isomerisation to all-trans-retinoic acid (ATRA) is widely accepted as the key mechanism underlying the favourable clinical properties of 13-cis-retinoic acid (13cisRA). As intracellular metabolism of ATRA by CYP26 may result in clinical resistance to 13cisRA, an increase in efficacy may be achieved through modulation of this metabolic pathway. We have evaluated the effect of the CYP26 inhibitor R116010 on retinoid metabolism in neuroblastoma cell lines and a xenograft model. In neuroblastoma cells, which showed a high level of CYP26 induction in response to ATRA, R116010 selectively inhibited ATRA metabolism. In addition, siRNA-mediated knockdown of CYP26 selectively increased ATRA levels and the expression of retinoid-responsive marker genes was potentiated by R116010. Treatment of mice bearing SH-SY5Y xenografts with 13cisRA (100 mg kg−1) revealed substantial levels (16%) of intratumoral ATRA after 6 h, despite plasma ATRA levels representing only 1% total retinoids under these conditions. Co-administration of R116010 with 13cisRA in this mouse model resulted in significant increases in plasma ATRA and 13cisRA concentrations. Furthermore, R116010 induced significant decreases in levels of 4-oxo metabolites in hepatic tissue after co-administration with either ATRA or 13cisRA. These data suggest considerable potential for CYP26 inhibitors in the future treatment of neuroblastoma with 13cisRA

A novel method to compare protein structures using local descriptors

<p>Abstract</p> <p>Background</p> <p>Protein structure comparison is one of the most widely performed tasks in bioinformatics. However, currently used methods have problems with the so-called "difficult similarities", including considerable shifts and distortions of structure, sequential swaps and circular permutations. There is a demand for efficient and automated systems capable of overcoming these difficulties, which may lead to the discovery of previously unknown structural relationships.</p> <p>Results</p> <p>We present a novel method for protein structure comparison based on the formalism of local descriptors of protein structure - DEscriptor Defined Alignment (DEDAL). Local similarities identified by pairs of similar descriptors are extended into global structural alignments. We demonstrate the method's capability by aligning structures in difficult benchmark sets: curated alignments in the SISYPHUS database, as well as SISY and RIPC sets, including non-sequential and non-rigid-body alignments. On the most difficult RIPC set of sequence alignment pairs the method achieves an accuracy of 77% (the second best method tested achieves 60% accuracy).</p> <p>Conclusions</p> <p>DEDAL is fast enough to be used in whole proteome applications, and by lowering the threshold of detectable structure similarity it may shed additional light on molecular evolution processes. It is well suited to improving automatic classification of structure domains, helping analyze protein fold space, or to improving protein classification schemes. DEDAL is available online at <url>http://bioexploratorium.pl/EP/DEDAL</url>.</p

Mineral Composition is Altered by Osteoblast Expression of an Engineered Gs-Coupled Receptor

Activation of the Gs G protein–coupled receptor Rs1 in osteoblasts increases bone mineral density by 5- to 15-fold in mice and recapitulates histologic aspects of fibrous dysplasia of the bone. However, the effects of constitutive Gs signaling on bone tissue quality are not known. The goal of this study was to determine bone tissue quality in mice resulting from osteoblast-specific constitutive Gs activation, by the complementary techniques of FTIR spectroscopy and synchrotron radiation micro-computed tomography (SRμCT). Col1(2.3)-tTA/TetO-Rs1 double transgenic (DT) mice, which showed osteoblast-specific constitutive Gs signaling activity by the Rs1 receptor, were created. Femora and calvariae of DT and wild-type (WT) mice (6 and 15 weeks old) were analyzed by FTIR spectroscopy. WT and DT femora (3 and 9 weeks old) were imaged by SRμCT. Mineral-to-matrix ratio was 25% lower (P = 0.010), carbonate-to-phosphate ratio was 20% higher (P = 0.025), crystallinity was 4% lower (P = 0.004), and cross-link ratio was 11% lower (P = 0.025) in 6-week DT bone. Differences persisted in 15-week animals. Quantitative SRμCT analysis revealed substantial differences in mean values and heterogeneity of tissue mineral density (TMD). TMD values were 1,156 ± 100 and 711 ± 251 mg/cm3 (mean ± SD) in WT and DT femoral diaphyses, respectively, at 3 weeks. Similar differences were found in 9-week animals. These results demonstrate that continuous Gs activation in murine osteoblasts leads to deposition of immature bone tissue with reduced mineralization. Our findings suggest that bone tissue quality may be an important contributor to increased fracture risk in fibrous dysplasia patients

Growth and DNA damage-inducible transcription factor 153 mediates apoptosis in response to fenretinide but not synergy between fenretinide and chemotherapeutic drugs in neuroblastoma.

Fenretinide induces apoptosis of neuroblastoma cells in vitro and interacts synergistically with the chemotherapeutic drugs cisplatin and etoposide. The stress-inducible transcription factor known as growth and DNA damage (GADD)-inducible transcription factor 153 is induced in response to fenretinide and in other cell types modulates apoptosis via pro- and antiapoptotic members of the BCL2 family. Because BCL2-family proteins are important in apoptosis induced by chemotherapeutic drugs, GADD153 may be a key mediator of synergy between fenretinide and chemotherapeutic drugs. To investigate this, GADD153 cDNA in sense and antisense orientations was stably transfected into SH-SY5Y neuroblastoma cells using a tetracycline-inducible vector. Increased expression of GADD153 raised the background level of apoptosis and increased apoptosis induced by fenretinide or the chemotherapeutic drugs cisplatin and etoposide. However, there was no increase in synergy between fenretinide and chemotherapeutic drugs. Conversely, expression of antisense-GADD153 virtually abolished the induction of apoptosis in response to fenretinide but overall had no significant effect on apoptosis induced by chemotherapeutic drugs. The effect of antisense-GADD153 on synergy between chemotherapeutic drugs and fenretinide varied with the drug used: there was no effect on synergy between fenretinide and cisplatin, but the combination of fenretinide with etoposide became antagonistic. These results suggest that mechanisms mediating synergy between fenretinide and chemotherapeutic drugs lie upstream of GADD153