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

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

Cloning and Functional Studies of a Splice Variant of CYP26B1 Expressed in Vascular Cells

Background: All-trans retinoic acid (atRA) plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development but may in turn be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis. The cellular atRA levels are under strict control involving several cytochromes P450 isoforms (CYPs). CYP26 may be the most important regulator of atRA catabolism in vascular cells. The present study describes the molecular cloning, characterization and function of atRA-induced expression of a spliced variant of the CYP26B1 gene. Methodology/Principal Findings: The coding region of the spliced CYP26B1 lacking exon 2 was amplified from cDNA synthesized from atRA-treated human aortic smooth muscle cells and sequenced. Both the spliced variant and full length CYP26B1 was found to be expressed in cultured human endothelial and smooth muscle cells, and in normal and atherosclerotic vessel. atRA induced both variants of CYP26B1 in cultured vascular cells. Furthermore, the levels of spliced mRNA transcript were 4.5 times higher in the atherosclerotic lesion compared to normal arteries and the expression in the lesions was increased 20-fold upon atRA treatment. The spliced CYP26B1 still has the capability to degrade atRA, but at an initial rate one-third that of the corresponding full length enzyme. Transfection of COS-1 and THP-1 cells with the CYP26B1 spliced variant indicated either an increase or a decrease in the catabolism of atRA, probably depending on the expression of other atRA catabolizing enzymes in the cells. Conclusions/Significance: Vascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions. The spliced variant displays a slower and reduced degradation of atRA as compared to the full-length enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1 splice variant in health and disease

Feature Selection via Chaotic Antlion Optimization

Selecting a subset of relevant properties from a large set of features that describe a dataset is a challenging machine learning task. In biology, for instance, the advances in the available technologies enable the generation of a very large number of biomarkers that describe the data. Choosing the more informative markers along with performing a high-accuracy classification over the data can be a daunting task, particularly if the data are high dimensional. An often adopted approach is to formulate the feature selection problem as a biobjective optimization problem, with the aim of maximizing the performance of the data analysis model (the quality of the data training fitting) while minimizing the number of features used.This work was partially supported by the IPROCOM Marie Curie initial training network, funded through the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grants agreement No. 316555, and by the Romanian National Authority for Scientific Research, CNDIUEFISCDI, project number PN-II-PT-PCCA-2011-3.2- 0917. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Cyp26b1 Regulates Retinoic Acid-Dependent Signals in T Cells and Its Expression Is Inhibited by Transforming Growth Factor-β

The vitamin A metabolite, retinoic acid (RA), plays important roles in the regulation of lymphocyte properties. Dendritic cells in gut-related lymphoid organs can produce RA, thereby imprinting gut-homing specificity on T cells and enhancing transforming growth factor (TGF)-β-dependent induction of Foxp3+ regulatory T cells upon antigen presentation. In general, RA concentrations in cells and tissues are regulated by its degradation as well. However, it remained unclear if T cells could actively catabolize RA.We assessed the expression of known RA-catabolizing enzymes in T cells from mouse lymphoid tissues. Antigen-experienced CD44+ T cells in gut-related lymphoid organs selectively expressed Cyp26b1, a member of the cytochrome P450 family 26. However, T cells in the spleen or skin-draining lymph nodes did not significantly express Cyp26b1. Accordingly, physiological levels of RA (1-10 nM) could induce Cyp26b1 expression in naïve T cells upon activation in vitro, but could not do so in the presence of TGF-β. Overexpression of Cyp26b1 significantly suppressed the RA effect to induce expression of the gut-homing receptor CCR9 on T cells. On the other hand, knocking down Cyp26b1 gene expression with small interfering RNA or inhibiting CYP26 enzymatic activity led to enhancement of the RA-induced CCR9 expression.Our data demonstrate a role for CYP26B1 in regulating RA-dependent signals in activated T cells but not during TGF-β-dependent differentiation to Foxp3+ regulatory T cells. Aberrant expression of CYP26B1 may disturb T cell trafficking and differentiation in the gut and its related lymphoid organs

A comparison of the radiosensitisation ability of 22 different element metal oxide nanoparticles using clinical megavoltage X-rays

Background: A wide range of nanoparticles (NPs), composed of different elements and their compounds, are being developed by several groups as possible radiosensitisers, with some already in clinical trials. However, no systematic experimental survey of the clinical X-ray radiosensitising potential of different element nanoparticles has been made. Here, we directly compare the irradiation-induced (10 Gy of 6-MV X-ray photon) production of hydroxyl radicals, superoxide anion radicals and singlet oxygen in aqueous solutions of the following metal oxide nanoparticles: Al2O3, SiO2, Sc2O3, TiO2, V2O5, Cr2O3, MnO2, Fe3O4, CoO, NiO, CuO, ZnO, ZrO2, MoO3, Nd2O3, Sm2O3, Eu2O3, Gd2O3, Tb4O7, Dy2O3, Er2O3 and HfO2. We also examine DNA damage due to these NPs in unirradiated and irradiated conditions. Results: Without any X-rays, several NPs produced more radicals than water alone. Thus, V2O5 NPs produced around 5-times more hydroxyl radicals and superoxide radicals. MnO2 NPs produced around 10-times more superoxide anions and Tb4O7 produced around 3-times more singlet oxygen. Lanthanides produce fewer hydroxyl radicals than water. Following irradiation, V2O5 NPs produced nearly 10-times more hydroxyl radicals than water. Changes in radical concentrations were determined by subtracting unirradiated values from irradiated values. These were then compared with irradiation-induced changes in water only. Irradiation-specific increases in hydroxyl radical were seen with most NPs, but these were only significantly above the values of water for V2O5, while the Lanthanides showed irradiation-specific decreases in hydroxyl radical, compared to water. Only TiO2 showed a trend of irradiation-specific increase in superoxides, while V2O5, MnO2, CoO, CuO, MoO3 and Tb4O7 all demonstrated significant irradiation-specific decreases in superoxide, compared to water. No irradiation-specific increases in singlet oxygen were seen, but V2O5, NiO, CuO, MoO3 and the lanthanides demonstrated irradiation-specific decreases in singlet oxygen, compared to water. MoO3 and CuO produced DNA damage in the absence of radiation, while the highest irradiation-specific DNA damage was observed with CuO. In contrast, MnO2, Fe3O4 and CoO were slightly protective against irradiation-induced DNA damage. Conclusions: Beyond identifying promising metal oxide NP radiosensitisers and radioprotectors, our broad comparisons reveal unexpected differences that suggest the surface chemistry of NP radiosensitisers is an important criterion for their success

Prediction of Glioblastoma Multiform Response to Bevacizumab Treatment Using Multi-Parametric MRI

Glioblastoma multiform (GBM) is a highly malignant brain tumor. Bevacizumab is a recent therapy for stopping tumor growth and even shrinking tumor through inhibition of vascular development (angiogenesis). This paper presents a non-invasive approach based on image analysis of multi-parametric magnetic resonance images (MRI) to predict response of GBM to this treatment. The resulting prediction system has potential to be used by physicians to optimize treatment plans of the GBM patients. The proposed method applies signal decomposition and histogram analysis methods to extract statistical features from Gd-enhanced regions of tumor that quantify its microstructural characteristics. MRI studies of 12 patients at multiple time points before and up to four months after treatment are used in this work. Changes in the Gd-enhancement as well as necrosis and edema after treatment are used to evaluate the response. Leave-one-out cross validation method is applied to evaluate prediction quality of the models. Predictive models developed in this work have large regression coefficients (maximum R2 = 0.95) indicating their capability to predict response to therapy

The varied roles of nuclear receptors during vertebrate embryonic development

Nuclear receptors comprise a superfamily of sequence-specific transcription factors whose members have diverse roles during development. This review will summarize the developmental roles of selected members of the nuclear receptor superfamily

Inhibitory effects of retinoic acid metabolism blocking agents (RAMBAs) on the growth of human prostate cancer cells and LNCaP prostate tumour xenografts in SCID mice

In recent studies, we have identified several highly potent all-trans-retinoic acid (ATRA) metabolism blocking agents (RAMBAs). On the basis of previous effects of liarozole (a first-generation RAMBA) on the catabolism of ATRA and on growth of rat Dunning R3227G prostate tumours, we assessed the effects of our novel RAMBAs on human prostate tumour (PCA) cell lines. We examined three different PCA cell lines to determine their capacity to induce P450-mediated oxidation of ATRA. Among the three different cell lines, enhanced catabolism was detected in LNCaP, whereas it was not found in PC-3 and DU-145. This catabolism was strongly inhibited by our RAMBAs, the most potent being VN/14-1, VN/50-1, VN/66-1, and VN/69-1 with IC50 values of 6.5, 90.0, 62.5, and 90.0 nM, respectively. The RAMBAs inhibited the growth of LNCaP cells with IC50 values in the μM-range. In LNCaP cell proliferation assays, VN/14-1, VN/50-1, VN/66-1, and VN/69-1 also enhanced by 47-, 60-, 70-, and 65-fold, respectively, the ATRA-mediated antiproliferative activity. We then examined the molecular mechanism underlying the growth inhibitory properties of ATRA alone and in combination with RAMBAs. The mechanism appeared to involve the induction of differentiation, cell-cycle arrest, and induction of apoptosis (TUNEL), involving increase in Bad expression and decrease in Bcl-2 expression. Treatment of LNCaP tumours growing in SCID mice with VN/66-1 and VN/69-1 resulted in modest but statistically significant tumour growth inhibition of 44 and 47%, respectively, while treatment with VN/14-1 was unexpectedly ineffective. These results suggest that some of our novel RAMBAs may be useful agents for the treatment of prostate cancer