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Lawrence I. Gilbert - One of the best experts on this subject based on the ideXlab platform.

  • identification characterization and developmental expression of halloween genes encoding p450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm manduca sexta
    Insect Biochemistry and Molecular Biology, 2006
    Co-Authors: Kim F Rewitz, James T Warren, Lawrence I. Gilbert
    Abstract:

    The Insect Molting Hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-Hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this work to the tobacco hornworm Manduca sexta, an established model for endocrinological and developmental studies. cDNA clones were obtained for three Manduca orthologs of CYP306A1 (phantom; phm, the 25-hydroxylase), CYP302A1 (disembodied; dib, the 22-hydroxylase) and CYP315A1 (shadow; sad, the 2-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the developmentally varying steroidogenic capacities of the prothoracic glands during the fifth instar. The consistent expression of the Halloween genes confirms the importance of the prothoracic glands in pupal-adult development. These studies establish Manduca as an excellent model for examining the regulation of the Halloween genes.

  • Developmental expression of Manduca shade, the P450 mediating the final step in Molting Hormone synthesis.
    Molecular and cellular endocrinology, 2006
    Co-Authors: Kim F Rewitz, James T Warren, Lawrence I. Gilbert
    Abstract:

    The ecdysone 20-monooxygenase (E20MO; 20-hydroxylase) is the enzyme that mediates the conversion of ecdysone (E) to the active Insect Molting Hormone, 20-hydroxyecdysone (20E), which coordinates developmental progression. We report the identification and developmental expression of the Halloween gene shade (shd; CYP314A1) that encodes the E20MO in the tobacco hornworm, Manduca sexta. Manduca Shd (MsShd) mediates the conversion of E to 20E when expressed in Drosophila S2 cells. In accord with the central dogma, the data show that Msshd is expressed mainly in the midgut, Malpighian tubules, fat body and epidermis with very low expression in the prothoracic gland and nervous system. Developmental variations in E20MO enzymatic activity are almost perfectly correlated with comparable changes in the gene expression of Msshd in the fat body and midgut during the fifth instar and the beginning of pupal-adult development. The results indicate three successive and overlapping peaks of expression in the fat body, midgut and Malpighian tubules, respectively, during the fifth larval instar. The data suggest that precise tissue-specific transcriptional regulation controls the levels, and thereby the activity, of the Manduca E20MO.

  • shade is the drosophila p450 enzyme that mediates the hydroxylation of ecdysone to the steroid Insect Molting Hormone 20 hydroxyecdysone
    Proceedings of the National Academy of Sciences of the United States of America, 2003
    Co-Authors: Anna Petryk, Lawrence I. Gilbert, James T Warren, Guillermo Marques, Michael Jarcho, Jonathan Kahler, Jeanphilippe Parvy, Chantal Dauphinvillemant
    Abstract:

    The steroid 20-hydroxyecdysone (20E) is the primary regulatory Hormone that mediates developmental transitions in Insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any Insect. We show here that the Drosophila E-20-monooxygenase (E20MO) is the product of the shade (shd) locus (cytochrome p450, CYP314a1). When shd is transfected into Drosophila S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous shd embryos, no E20MO activity is apparent either in vivo or in vitro. Mutations in shd lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in disembodied (dib) and shadow (sad) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike dib and sad, shd is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of Insects. However, the tissue in which shd is expressed does not appear to be important for developmental function because misexpression of shd in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which shd is expressed, rescues embryonic lethality.

James T Warren - One of the best experts on this subject based on the ideXlab platform.

  • identification characterization and developmental expression of halloween genes encoding p450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm manduca sexta
    Insect Biochemistry and Molecular Biology, 2006
    Co-Authors: Kim F Rewitz, James T Warren, Lawrence I. Gilbert
    Abstract:

    The Insect Molting Hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-Hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this work to the tobacco hornworm Manduca sexta, an established model for endocrinological and developmental studies. cDNA clones were obtained for three Manduca orthologs of CYP306A1 (phantom; phm, the 25-hydroxylase), CYP302A1 (disembodied; dib, the 22-hydroxylase) and CYP315A1 (shadow; sad, the 2-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the developmentally varying steroidogenic capacities of the prothoracic glands during the fifth instar. The consistent expression of the Halloween genes confirms the importance of the prothoracic glands in pupal-adult development. These studies establish Manduca as an excellent model for examining the regulation of the Halloween genes.

  • Developmental expression of Manduca shade, the P450 mediating the final step in Molting Hormone synthesis.
    Molecular and cellular endocrinology, 2006
    Co-Authors: Kim F Rewitz, James T Warren, Lawrence I. Gilbert
    Abstract:

    The ecdysone 20-monooxygenase (E20MO; 20-hydroxylase) is the enzyme that mediates the conversion of ecdysone (E) to the active Insect Molting Hormone, 20-hydroxyecdysone (20E), which coordinates developmental progression. We report the identification and developmental expression of the Halloween gene shade (shd; CYP314A1) that encodes the E20MO in the tobacco hornworm, Manduca sexta. Manduca Shd (MsShd) mediates the conversion of E to 20E when expressed in Drosophila S2 cells. In accord with the central dogma, the data show that Msshd is expressed mainly in the midgut, Malpighian tubules, fat body and epidermis with very low expression in the prothoracic gland and nervous system. Developmental variations in E20MO enzymatic activity are almost perfectly correlated with comparable changes in the gene expression of Msshd in the fat body and midgut during the fifth instar and the beginning of pupal-adult development. The results indicate three successive and overlapping peaks of expression in the fat body, midgut and Malpighian tubules, respectively, during the fifth larval instar. The data suggest that precise tissue-specific transcriptional regulation controls the levels, and thereby the activity, of the Manduca E20MO.

  • shade is the drosophila p450 enzyme that mediates the hydroxylation of ecdysone to the steroid Insect Molting Hormone 20 hydroxyecdysone
    Proceedings of the National Academy of Sciences of the United States of America, 2003
    Co-Authors: Anna Petryk, Lawrence I. Gilbert, James T Warren, Guillermo Marques, Michael Jarcho, Jonathan Kahler, Jeanphilippe Parvy, Chantal Dauphinvillemant
    Abstract:

    The steroid 20-hydroxyecdysone (20E) is the primary regulatory Hormone that mediates developmental transitions in Insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any Insect. We show here that the Drosophila E-20-monooxygenase (E20MO) is the product of the shade (shd) locus (cytochrome p450, CYP314a1). When shd is transfected into Drosophila S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous shd embryos, no E20MO activity is apparent either in vivo or in vitro. Mutations in shd lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in disembodied (dib) and shadow (sad) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike dib and sad, shd is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of Insects. However, the tissue in which shd is expressed does not appear to be important for developmental function because misexpression of shd in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which shd is expressed, rescues embryonic lethality.

Attila Hunyadi - One of the best experts on this subject based on the ideXlab platform.

  • significant activity of ecdysteroids on the resistance to doxorubicin in mammalian cancer cells expressing the human abcb1 transporter
    Journal of Medicinal Chemistry, 2012
    Co-Authors: Ana Martins, Mária Báthori, Noémi Tóth, Attila Vanyolos, Zoltan Beni, Istvan Zupko, Jozsef Molnar, Attila Hunyadi
    Abstract:

    Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy. Fifty-eight ecdysteroids, herbal analogues of the Insect Molting Hormone and their semisynthetic derivatives, were tested for their activity against L5178 mouse T-cell lymphoma cells (non-MDR) and their subcell line transfected with pHa MDR1/A retrovirus overexpressing the human ABCB1 efflux pump (MDR cell line). The compounds showed very low antiproliferative activities but modulated the efflux of rhodamine 123 mediated by the ABCB1 transporter. Roughly depending on the polarity, mild to strong synergism or antagonism was observed by combining ecdysteroids with doxorubicin, and specific structure-activity relationships were also found. Our results show the effect of ecdysteroids on MDR cancer cells for the first time. Less polar derivatives may serve as valuable leads toward a potent and safe resistance modulator. Biological significance of the resistance-increasing activity of the most abundant phytoecdysteroids including 20-hydroxyecdysone is yet to be clarified.

  • Significant Activity of Ecdysteroids on the Resistance to Doxorubicin in Mammalian Cancer Cells Expressing the Human ABCB1 Transporter
    2012
    Co-Authors: Ana Martins, Noémi Tóth, Attila Ványolós, Zoltán Béni, István Zupkó, József Molnár, Mária Báthori, Attila Hunyadi
    Abstract:

    Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy. Fifty-eight ecdysteroids, herbal analogues of the Insect Molting Hormone and their semisynthetic derivatives, were tested for their activity against L5178 mouse T-cell lymphoma cells (non-MDR) and their subcell line transfected with pHa MDR1/A retrovirus overexpressing the human ABCB1 efflux pump (MDR cell line). The compounds showed very low antiproliferative activities but modulated the efflux of rhodamine 123 mediated by the ABCB1 transporter. Roughly depending on the polarity, mild to strong synergism or antagonism was observed by combining ecdysteroids with doxorubicin, and specific structure–activity relationships were also found. Our results show the effect of ecdysteroids on MDR cancer cells for the first time. Less polar derivatives may serve as valuable leads toward a potent and safe resistance modulator. Biological significance of the resistance-increasing activity of the most abundant phytoecdysteroids including 20-hydroxyecdysone is yet to be clarified

  • Phytoecdysteroids and Anabolic-Androgenic Steroids - Structure and Effects on Humans
    Current medicinal chemistry, 2008
    Co-Authors: Mária Báthori, Noémi Tóth, Attila Hunyadi, Árpád Márki, Ernö Zádor
    Abstract:

    Phytoecdysteroids are structural analogs of the Insect Molting Hormone ecdysone. Plants comprise rich sources of ecdysteroids in high concentration and with broad structural diversity. Ecdysteroids have a number of proven beneficial effects on mammals but the hormonal effects of ecdysteroids have been proven only in arthropods. Their structures are somewhat similar to those of the vertebrate steroid Hormones but there are several structural differences between the two steroid groups. Despite of these essential structural differences, ecdysteroids exert numerous effects in vertebrates that are similar to those of vertebrate hormonal steroids, and they may serve as effective anabolic, hepatoprotective, immunoprotective, antioxidant and hypoglycemic agents. Ecdysteroids do not bind to the cytosolic steroid receptors, instead, they are likely to influence signal transduction pathways, like the anabolic steroids, possibly via membrane bound receptors. The application of phytoecdysteroids is a promising alternative to the use of anabolic-androgenic steroids because of the apparent lack of adverse effects. The prospective use of phytoecdysteroids may extend to treatments of pathological conditions where anabolic steroids are routinely applied. One of the most cited aspects of phytoecdysteroid application (on the Internet) is the increase of muscle size. However in this field too stringent research is needed as an adequate cytological explanation is not yet available for the anabolic. This paper reports on the most important structural differences between androgenic Hormones, their synthetic analogs and ecdysteroids. The anabolic/hormonal effects and the possible mechanisms of action of these compounds are also discussed as concerns the skeletal muscle.

E. E. Ball - One of the best experts on this subject based on the ideXlab platform.

  • the structure of the usp rxr of xenos pecki indicates that strepsiptera are not closely related to diptera
    Development Genes and Evolution, 2005
    Co-Authors: D. C. Hayward, Michael J Bastiani, John W. H. Trueman, E. E. Ball
    Abstract:

    The receptor for the Insect Molting Hormone, ecdysone, is a heterodimer consisting of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The ligand binding domain sequences of arthropod USPs divide into two distinct groups. One group consists of sequences from members of the holometabolous Lepidoptera and Diptera, while the other arthropod sequences group with vertebrate retinoid-X-receptors (RXRs). We therefore wondered whether USP/RXR structure could be used to clarify the contentious phylogenetic position of the order Strepsiptera, which has proposed affinities with either Diptera or Coleoptera. We have cloned and sequenced the USP/RXR from the strepsipteran Xenos pecki. Phylogenetic analyses are not consistent with a close affinity between Strepsiptera and Diptera.

  • the sequence of locusta rxr homologous to drosophila ultraspiracle and its evolutionary implications
    Development Genes and Evolution, 1999
    Co-Authors: D. C. Hayward, Michael J Bastiani, John W. H. Trueman, Lynn M Riddiford, James W Truman, E. E. Ball
    Abstract:

    The cellular response to steroid Hormones is mediated by nuclear receptors which act by regulating transcription. In Drosophila melanogaster, the receptor for the Insect Molting Hormone, 20-hydroxyecdysone, is a heterodimer composed of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The DNA binding domains of arthropod USPs and their vertebrate homologs, the retinoid X receptor (RXR) family, are highly conserved. The ligand binding domain sequences, however, divide into two distinct groups. One group consists of sequences from members of the holometabolous higher Insect orders Diptera and Lepidoptera, the other of sequences from vertebrates, a crab and a tick. We here report the sequence of an RXR/USP from the hemimetabolous orthopteran, Locusta migratoria. The locust RXR/USP ligand binding domain clearly falls in the vertebrate-crab-tick rather than the dipteran-lepidopteran group. The reason for the evolutionarily abrupt divergence of the dipteran and lepidopteran sequences is unknown, but it could be a change in the type of ligand bound or the loss of ligand altogether.

Ana Martins - One of the best experts on this subject based on the ideXlab platform.

  • ecdysteroids sensitize mdr and non mdr cancer cell lines to doxorubicin paclitaxel and vincristine but tend to protect them from cisplatin
    BioMed Research International, 2015
    Co-Authors: Ana Martins, Peter Sipos, Katalin Der, Jozsef Csabi, Walter Miklos, Walter Berger, Attila Zalatnai, Leonard Amaral, Joseph Molnar, Piroska Szaborevesz
    Abstract:

    Ecdysteroids, analogs of the Insect Molting Hormone, are known for their various mild, nonhormonal bioactivities in mammals. Previously, we reported that less-polar ecdysteroids can modulate the doxorubicin resistance of a multidrug resistant (MDR) mouse lymphoma cell line expressing the human ABCB1 transporter. Here, we describe the ability of 20-hydroxyecdysone (1) and its mono- (2) and diacetonide (3) derivatives to sensitize various MDR and non-MDR cancer cell lines towards doxorubicin, paclitaxel, vincristine, or cisplatin. Drug IC50 values with or without ecdysteroid were determined by MTT assay. Compound 3 significantly sensitized all cell lines to each chemotherapeutic except for cisplatin, whose activity was decreased. In order to overcome solubility and stability issues for the future in vivo administration of compound 3, liposomal formulations were developed. By means of their combination index values obtained via checkerboard microplate method, a formulation showed superior activity to that of compound 3 alone. Because ecdysteroids act also on non-ABCB1 expressing (sensitive) cell lines, our results demonstrate that they do not or not exclusively exert their adjuvant anticancer activity as ABCB1 inhibitors, but other mechanisms must be involved, and they opened the way towards their in vivo bioactivity testing against various cancer xenografts.

  • Ecdysteroids Sensitize MDR and Non-MDR Cancer Cell Lines to Doxorubicin, Paclitaxel, and Vincristine but Tend to Protect Them from Cisplatin
    Hindawi Limited, 2015
    Co-Authors: Ana Martins, Peter Sipos, Katalin Der, Jozsef Csabi, Walter Miklos, Walter Berger, Attila Zalatnai, Leonard Amaral, Joseph Molnar, Piroska Szabó-révész
    Abstract:

    Ecdysteroids, analogs of the Insect Molting Hormone, are known for their various mild, nonhormonal bioactivities in mammals. Previously, we reported that less-polar ecdysteroids can modulate the doxorubicin resistance of a multidrug resistant (MDR) mouse lymphoma cell line expressing the human ABCB1 transporter. Here, we describe the ability of 20-hydroxyecdysone (1) and its mono- (2) and diacetonide (3) derivatives to sensitize various MDR and non-MDR cancer cell lines towards doxorubicin, paclitaxel, vincristine, or cisplatin. Drug IC50 values with or without ecdysteroid were determined by MTT assay. Compound 3 significantly sensitized all cell lines to each chemotherapeutic except for cisplatin, whose activity was decreased. In order to overcome solubility and stability issues for the future in vivo administration of compound 3, liposomal formulations were developed. By means of their combination index values obtained via checkerboard microplate method, a formulation showed superior activity to that of compound 3 alone. Because ecdysteroids act also on non-ABCB1 expressing (sensitive) cell lines, our results demonstrate that they do not or not exclusively exert their adjuvant anticancer activity as ABCB1 inhibitors, but other mechanisms must be involved, and they opened the way towards their in vivo bioactivity testing against various cancer xenografts

  • significant activity of ecdysteroids on the resistance to doxorubicin in mammalian cancer cells expressing the human abcb1 transporter
    Journal of Medicinal Chemistry, 2012
    Co-Authors: Ana Martins, Mária Báthori, Noémi Tóth, Attila Vanyolos, Zoltan Beni, Istvan Zupko, Jozsef Molnar, Attila Hunyadi
    Abstract:

    Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy. Fifty-eight ecdysteroids, herbal analogues of the Insect Molting Hormone and their semisynthetic derivatives, were tested for their activity against L5178 mouse T-cell lymphoma cells (non-MDR) and their subcell line transfected with pHa MDR1/A retrovirus overexpressing the human ABCB1 efflux pump (MDR cell line). The compounds showed very low antiproliferative activities but modulated the efflux of rhodamine 123 mediated by the ABCB1 transporter. Roughly depending on the polarity, mild to strong synergism or antagonism was observed by combining ecdysteroids with doxorubicin, and specific structure-activity relationships were also found. Our results show the effect of ecdysteroids on MDR cancer cells for the first time. Less polar derivatives may serve as valuable leads toward a potent and safe resistance modulator. Biological significance of the resistance-increasing activity of the most abundant phytoecdysteroids including 20-hydroxyecdysone is yet to be clarified.

  • Significant Activity of Ecdysteroids on the Resistance to Doxorubicin in Mammalian Cancer Cells Expressing the Human ABCB1 Transporter
    2012
    Co-Authors: Ana Martins, Noémi Tóth, Attila Ványolós, Zoltán Béni, István Zupkó, József Molnár, Mária Báthori, Attila Hunyadi
    Abstract:

    Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy. Fifty-eight ecdysteroids, herbal analogues of the Insect Molting Hormone and their semisynthetic derivatives, were tested for their activity against L5178 mouse T-cell lymphoma cells (non-MDR) and their subcell line transfected with pHa MDR1/A retrovirus overexpressing the human ABCB1 efflux pump (MDR cell line). The compounds showed very low antiproliferative activities but modulated the efflux of rhodamine 123 mediated by the ABCB1 transporter. Roughly depending on the polarity, mild to strong synergism or antagonism was observed by combining ecdysteroids with doxorubicin, and specific structure–activity relationships were also found. Our results show the effect of ecdysteroids on MDR cancer cells for the first time. Less polar derivatives may serve as valuable leads toward a potent and safe resistance modulator. Biological significance of the resistance-increasing activity of the most abundant phytoecdysteroids including 20-hydroxyecdysone is yet to be clarified