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Otmar Spring - One of the best experts on this subject based on the ideXlab platform.
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Costunolide Influences Germ Tube Orientation in Sunflower Broomrape – A First Step Toward Understanding Chemotropism
Frontiers in Plant Science, 2021Co-Authors: Anna Krupp, Barbara Bertsch, Otmar SpringAbstract:Orobanche cumana WALLR. is a host-specific root parasite of cultivated sunflowers with increasing economic importance in Europe, North Africa, and parts of Asia. While sesquiterpene lactones (STLs) released from sunflower roots were identified as natural germination stimulants of O. cumana seeds in the soil, the chemical nature of the signals guiding the emerging germ tube toward the host root has remained unknown hitherto. Thus, we designed a bioassay that allowed the observation of broomrape germination and subsequent germ tube development in the presence of substances with putative chemotropic activity. Root exudates and sunflower oil extracts, both containing STLs in micromolar concentrations, caused the positive chemotropic orientation of germ tubes. A similar positive chemotropic effect was achieved with Costunolide, one of the four STLs of sunflower present in the exudate and oil extracts. In contrast, GR24, a synthetic strigolactone (SL) with germination-inducing activity on O. cumana seeds, showed no effect on the germ tube orientation. The effect of Costunolide was concentration-dependent and within the range of its natural micromolar occurrence in roots. We assume that an STL gradient is responsible for the stronger inhibition of elongation growth on the host-facing flank of the germ tube compared with the far side flank. This would confer a double role of STLs from sunflower root exudates in the sunflower–broomrape interaction, namely, as germination stimulants and as chemotropic signals.
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Costunolide influences germ tube orientation in sunflower broomrape a first step toward understanding chemotropism
Frontiers in Plant Science, 2021Co-Authors: Anna Krupp, Barbara Bertsch, Otmar SpringAbstract:Orobanche cumana WALLR. is a host specific root parasite of cultivated sunflower with increasing economic importance in Europe, North Africa and parts of Asia. While sesquiterpene lactones (STLs) released from sunflower roots were identified as the natural germination stimulants of O. cumana seeds in soil, the chemical nature of signals guiding the emerging germ-tube towards the host root remained unknown hitherto. We designed a bioassay which allowed observation of broomrape germination and subsequent germ-tube development in the presence of substances with putative chemotropic activity. Root exudates and sunflower oil extracts, both containing STLs in micromolar concentrations, caused positive chemotropic orientation of germ-tubes. A similar positive chemotropic effect was achieved with Costunolide, one of the four STLs of sunflower present in the exudate and oil extract. In contrast, GR24, a synthetic strigolactone (SL) with germination inducing activity on O. cumana seeds, showed no effect on the germ-tube orientation. The effect of Costunolide was concentration dependent and in the range of its natural micromolar occurrence in roots. We assume that a STL gradient is responsible for a stronger inhibition of elongation growth on the host-facing flank of the germ-tube compared to the far side flank. This would confer a double role of STLs from sunflower root exudates in the sunflower-broomrape-interaction, namely as germination stimulants and as chemotropic signals.
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Table_1_Costunolide Influences Germ Tube Orientation in Sunflower Broomrape – A First Step Toward Understanding Chemotropism.XLSX
'Frontiers Media SA', 2021Co-Authors: Anna Krupp, Barbara Bertsch, Otmar SpringAbstract:Orobanche cumana WALLR. is a host-specific root parasite of cultivated sunflowers with increasing economic importance in Europe, North Africa, and parts of Asia. While sesquiterpene lactones (STLs) released from sunflower roots were identified as natural germination stimulants of O. cumana seeds in the soil, the chemical nature of the signals guiding the emerging germ tube toward the host root has remained unknown hitherto. Thus, we designed a bioassay that allowed the observation of broomrape germination and subsequent germ tube development in the presence of substances with putative chemotropic activity. Root exudates and sunflower oil extracts, both containing STLs in micromolar concentrations, caused the positive chemotropic orientation of germ tubes. A similar positive chemotropic effect was achieved with Costunolide, one of the four STLs of sunflower present in the exudate and oil extracts. In contrast, GR24, a synthetic strigolactone (SL) with germination-inducing activity on O. cumana seeds, showed no effect on the germ tube orientation. The effect of Costunolide was concentration-dependent and within the range of its natural micromolar occurrence in roots. We assume that an STL gradient is responsible for the stronger inhibition of elongation growth on the host-facing flank of the germ tube compared with the far side flank. This would confer a double role of STLs from sunflower root exudates in the sunflower–broomrape interaction, namely, as germination stimulants and as chemotropic signals.
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Image_1_Costunolide Influences Germ Tube Orientation in Sunflower Broomrape – A First Step Toward Understanding Chemotropism.TIF
'Frontiers Media SA', 2021Co-Authors: Anna Krupp, Barbara Bertsch, Otmar SpringAbstract:Orobanche cumana WALLR. is a host-specific root parasite of cultivated sunflowers with increasing economic importance in Europe, North Africa, and parts of Asia. While sesquiterpene lactones (STLs) released from sunflower roots were identified as natural germination stimulants of O. cumana seeds in the soil, the chemical nature of the signals guiding the emerging germ tube toward the host root has remained unknown hitherto. Thus, we designed a bioassay that allowed the observation of broomrape germination and subsequent germ tube development in the presence of substances with putative chemotropic activity. Root exudates and sunflower oil extracts, both containing STLs in micromolar concentrations, caused the positive chemotropic orientation of germ tubes. A similar positive chemotropic effect was achieved with Costunolide, one of the four STLs of sunflower present in the exudate and oil extracts. In contrast, GR24, a synthetic strigolactone (SL) with germination-inducing activity on O. cumana seeds, showed no effect on the germ tube orientation. The effect of Costunolide was concentration-dependent and within the range of its natural micromolar occurrence in roots. We assume that an STL gradient is responsible for the stronger inhibition of elongation growth on the host-facing flank of the germ tube compared with the far side flank. This would confer a double role of STLs from sunflower root exudates in the sunflower–broomrape interaction, namely, as germination stimulants and as chemotropic signals.
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biosynthesis of eupatolide a metabolic route for sesquiterpene lactone formation involving the p450 enzyme cyp71dd6
ACS Chemical Biology, 2018Co-Authors: Maximilian Frey, Katharina Schmauder, Irini Pateraki, Otmar SpringAbstract:Sesquiterpene lactones are a class of natural compounds well-known for their bioactivity and are characteristic for the Asteraceae family. Most sesquiterpene lactones are considered derivatives of germacrene A acid (GAA). GAA can be stereospecifically hydroxylated by the cytochrome P450 enzymes (CYP) Lactuca sativa Costunolide synthase CYP71BL2 (LsCOS) and Helianthus annuus GAA 8β-hydroxylase CYP71BL1 (HaG8H) at C6 (in α-orientation) or C8 (in β-orientation), respectively. Spontaneous subsequent lactonization of the resulting 6α-hydroxy-GAA leads to Costunolide, whereas 8β-hydroxy-GAA has not yet been reported to cyclize to a sesquiterpene lactone. Sunflower and related species of the Heliantheae tribe contain sesquiterpene lactones mainly derived from inunolide (7,8-cis lactone) and eupatolide (8β-hydroxy-Costunolide) precursors. However, the mechanism of 7,8-cis lactonization in general, and the 6,7-trans lactone formation in the sunflower tribe, remain elusive. Here, we show that, in plant cells, heterologous expression of CYP71BL1 leads to the formation of inunolide. Using a phylogenetic analysis of enzymes from the CYP71 family involved in sesquiterpenoid metabolism, we identified the CYP71DD6 gene, which was able to catalyze the 6,7-trans lactonization in sunflowers, using as a substrate 8β-hydroxy-GAA. Consequently, CYP71DD6 resulted in the synthesis of eupatolide, thus called HaES ( Helianthus annuus eupatolide synthase). Thus, our study shows the entry point for the biosynthesis of two distinct types of sesquiterpene lactones in sunflowers: the 6,7-trans lactones derived from eupatolide and the 7,8-cis lactones derived from inunolide. The implications for tissue-specific localization, based on expression studies, are discussed.
Rudolf Bauer - One of the best experts on this subject based on the ideXlab platform.
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sesquiterpene lactones downregulate g2 m cell cycle regulator proteins and affect the invasive potential of human soft tissue sarcoma cells
PLOS ONE, 2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Eva Bernhart, Ehsan Bonyadi Rad, Andreas Leithner, Bibiane Steineckerfrohnwieser, Annelie Weinberg, Rudolf BauerAbstract:Soft tissue sarcomas (STS) represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. Many studies have demonstrated the great potential of plant-derived agents in the treatment of various malignant entities. The present study investigates the effects of the sesquiterpene lactones Costunolide and dehydrocostus lactone on cell cycle, MMP expression, and invasive potential of three human STS cell lines of various origins. Both compounds reduced cell proliferation in a time- and dose-dependent manner. Dehydrocostus lactone significantly inhibited cell proliferation, arrested the cells at the G2/M interface and caused a decrease in the expression of the cyclin-dependent kinase CDK2 and the cyclin-dependent kinase inhibitor p27 Kip1 . In addition, accumulation of cells at the G2/M phase transition interface resulted in a significant decrease in cdc2 (CDK1) together with cyclin B1. Costunolide had no effect on the cell cycle. Based on the fact that STS tend to form daughter cell nests and metastasize, the expression levels of matrix metalloproteinases (MMPs), which play a crucial role in extracellular matrix degradation and metastasis, were investigated by LuminexH technology and real-time RT-PCR. In the presence of Costunolide, MMP-2 and -9 levels were significantly increased in SW-982 and TE-671 cells. Dehydrocostus lactone treatment significantly reduced MMP-2 and -9 expression in TE-671 cells, but increased MMP-9 level in SW-982 cells. In addition, the invasion potential was significantly reduced after treatment with both sesquiterpene lactones as investigated by the HTS FluoroBlock TM insert system.
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MMP-1, -2, and -9 expression measured with the Luminex® system.
2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Bibiane Steinecker-frohnwieser, Eva Bernhart, Ehsan Bonyadi Rad, Annelie Martina Weinberg, Andreas Leithner, Rudolf BauerAbstract:STS cells were treated with the IC50 concentrations of Costunolide or dehydrocostus lactone for 24 and 48 h. Results are represented by mean fluorescence intensity (MFI) and p-values. Bold numbers represent significant differences between control and treated cells (p>0.05).
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Sesquiterpene lactones downregulate G2/M cell cycle regulator proteins and affect the invasive potential of human soft tissue sarcoma cells. PLoS One 8: e66300
2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Eva Bernhart, Ehsan Bonyadi Rad, Annelie Martina Weinberg, Andreas Leithner, Bibiane Steinecker, Rudolf BauerAbstract:Soft tissue sarcomas (STS) represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. Many studies have demonstrated the great potential of plant-derived agents in the treatment of various malignant entities. The present study investigates the effects of the sesquiterpene lactones Costunolide and dehydrocostus lactone on cell cycle, MMP expression, and invasive potential of three human STS cell lines of various origins. Both compounds reduced cell proliferation in a time- and dose-dependent manner. Dehydrocostus lactone significantly inhibited cell proliferation, arrested the cells at the G2/M interface and caused a decrease in the expression of the cyclin-dependent kinase CDK2 and the cyclin-dependent kinase inhibitor p27Kip1. In addition, accumulation of cells at the G2/M phase transition interface resulted in a significant decrease in cdc2 (CDK1) together with cyclin B1. Costunolide had no effect on the cell cycle. Based on the fact that STS tend to form daughter cell nests and metastasize, the expressio
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Western blot analysis of G2/M arrest regulator proteins.
2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Bibiane Steinecker-frohnwieser, Eva Bernhart, Ehsan Bonyadi Rad, Annelie Martina Weinberg, Andreas Leithner, Rudolf BauerAbstract:Total protein analysis revealed a significant downregulation of cdc2 and cyclin B1 in dehydrocostus lactone treated STS cells, whereas, Costunolide treatment did not affect the protein levels of these G2/M checkpoint regulators. β-actin was used as loading control. Data shown are representative from at least three independent experiments.
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Western blot analysis of p53 and the CDK inhibitor p27Kip1 proteins.
2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Bibiane Steinecker-frohnwieser, Eva Bernhart, Ehsan Bonyadi Rad, Annelie Martina Weinberg, Andreas Leithner, Rudolf BauerAbstract:Total protein analysis confirmed no significant changes in the p53 expression after Costunolide or dehydrocostus lactone treatment. In dehydrocostus lactone treated TE-671 and SW-872 cells, the expression of the CDK inhibitor p27Kip1 was reduced. β-actin was used as loading control. Data shown are representative from at least three independent experiments.
Harro J Bouwmeester - One of the best experts on this subject based on the ideXlab platform.
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Costunolide-glutathione and Costunolide-cysteine conjugate identification by GST enzyme assay and LC-MS analysis.
2013Co-Authors: Qing Liu, Katarina Cankar, Jules Beekwilder, Mohammad Majdi, Tatsiana Charnikhova, Miriam Goedbloed, Francel W. A. Verstappen, Ric C. H. De Vos, Sander Van Der Krol, Harro J BouwmeesterAbstract:A. LC-MS chromatograms of [m/z]− = 538 of extracts of N. benthamiana leaves agro-infiltrated with TpGAS+CiGAO+CiCOS, Costunolide-GSH conjugate formed in an enzyme assay of Costunolide and GSH with GST, Costunolide-GSH conjugate formed by non-enzymatic conjugation of Costunolide and GSH. B. LC-MS chromatograms of [m/z]− = 352 of extracts of N. benthamiana leaves agro-infiltrated with TpGAS+CiGAO+CiCOS, Costunolide-cysteine (Cys) conjugate formed in an enzyme assay of Costunolide and Cys with GST, Costunolide-Cys conjugate formed non enzymatically from Costunolide and Cys. C. [m/z]− spectrum of peak 22.48 and Costunolide-GSH conjugate (RT = 22.52). Arrows indicate parent ions of GSH-glutathione. D. [m/z]− spectrum of peak 22.30 and Costunolide-Cys conjugate (RT = 22.30). Arrows indicate parent ions of GSH-cysteine. E. Presumed molecular structure of Costunolide-GSH (a) and Costunolide-Cys (b) conjugates. GSH, glutathione; Cys, cysteine; GST, glutathione S-transferase. RT, retention time. Y-axis scale is identical in all chromatograms.
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Phylogenetic analysis of Asteraceae GAO genes and five chicory CYP71 P450 ESTs.
2013Co-Authors: Qing Liu, Katarina Cankar, Jules Beekwilder, Mohammad Majdi, Tatsiana Charnikhova, Miriam Goedbloed, Francel W. A. Verstappen, Ric C. H. De Vos, Sander Van Der Krol, Harro J BouwmeesterAbstract:Chicory candidate 3368 was later identified as Cichorium intybus Costunolide synthase (CiCOS). Amino acid seuqences of GAOs were obtained from cDNAs deposited at the NCBI. LsGAO germacrene A oxidase from Lactuca sativa (GU198171) or from Cichorium intybus (Ci; GU256644), Helianthus annuus (Ha; GU256646), Saussurea costus (Sc; GU256645) and Barnadesia. spinosa (Bs; GU256647). Bootstrap values are shown in frequency values from 1000 replicates.
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Phylogenetic analyses of the germacrene A synthases and oxidases.
2013Co-Authors: Aldana M. Ramirez, Maurice C R Franssen, Harro J Bouwmeester, Nils Saillard, Ting Yang, Maarten A. JongsmaAbstract:A, tree based on the deduced amino acid sequences of the putative pyrethrum germacrene A synthase (TcGAS) and other characterized plant GAS genes. B, tree based on the putative pyrethrum germacrene A oxidase (TcGAO) and Costunolide synthase (TcCOS) and other characterized plant GAO and COS genes. The tree was constructed by Neighbour-Joining method using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2). The species abbreviations are Ci, Cichorium intybus; Ha, Helianthus annuus; Ls, Lactuca sativa; Tp, Tanacetum parthenium; Tc, Tanacetum cinerariifolium.
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reconstitution of the Costunolide biosynthetic pathway in yeast and nicotiana benthamiana
PLOS ONE, 2011Co-Authors: Qing Liu, Katarina Cankar, Jules Beekwilder, Mohammad Majdi, Tatsiana Charnikhova, Miriam Goedbloed, Francel W. A. Verstappen, Ric C. H. De Vos, Sander Van Der Krol, Harro J BouwmeesterAbstract:The sesquiterpene Costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to Costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward Costunolide has not been characterized until now. Here we show that chicory Costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield Costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of Costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in Costunolide production of up to 60 ng.g−1 FW. In addition, two new compounds were formed that were identified as Costunolide-glutathione and Costunolide-cysteine conjugates.
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biosynthesis of Costunolide dihydroCostunolide and leucodin demonstration of cytochrome p450 catalyzed formation of the lactone ring present in sesquiterpene lactones of chicory
Plant Physiology, 2002Co-Authors: De J W Kraker, Maurice C R Franssen, M Joerink, De Ae Groot, Harro J BouwmeesterAbstract:Chicory ( Cichorium intybus ) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-Costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-Costunolide. This (+)-Costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18 O 2 resulted in the incorporation of one atom of 18 O into (+)-Costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C 6 -hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C 12 -atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-Costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-Costunolide into 11( S ),13-dihydroCostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-Costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme.
Birgit Lohberger - One of the best experts on this subject based on the ideXlab platform.
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sesquiterpene lactones downregulate g2 m cell cycle regulator proteins and affect the invasive potential of human soft tissue sarcoma cells
PLOS ONE, 2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Eva Bernhart, Ehsan Bonyadi Rad, Andreas Leithner, Bibiane Steineckerfrohnwieser, Annelie Weinberg, Rudolf BauerAbstract:Soft tissue sarcomas (STS) represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. Many studies have demonstrated the great potential of plant-derived agents in the treatment of various malignant entities. The present study investigates the effects of the sesquiterpene lactones Costunolide and dehydrocostus lactone on cell cycle, MMP expression, and invasive potential of three human STS cell lines of various origins. Both compounds reduced cell proliferation in a time- and dose-dependent manner. Dehydrocostus lactone significantly inhibited cell proliferation, arrested the cells at the G2/M interface and caused a decrease in the expression of the cyclin-dependent kinase CDK2 and the cyclin-dependent kinase inhibitor p27 Kip1 . In addition, accumulation of cells at the G2/M phase transition interface resulted in a significant decrease in cdc2 (CDK1) together with cyclin B1. Costunolide had no effect on the cell cycle. Based on the fact that STS tend to form daughter cell nests and metastasize, the expression levels of matrix metalloproteinases (MMPs), which play a crucial role in extracellular matrix degradation and metastasis, were investigated by LuminexH technology and real-time RT-PCR. In the presence of Costunolide, MMP-2 and -9 levels were significantly increased in SW-982 and TE-671 cells. Dehydrocostus lactone treatment significantly reduced MMP-2 and -9 expression in TE-671 cells, but increased MMP-9 level in SW-982 cells. In addition, the invasion potential was significantly reduced after treatment with both sesquiterpene lactones as investigated by the HTS FluoroBlock TM insert system.
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MMP-1, -2, and -9 expression measured with the Luminex® system.
2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Bibiane Steinecker-frohnwieser, Eva Bernhart, Ehsan Bonyadi Rad, Annelie Martina Weinberg, Andreas Leithner, Rudolf BauerAbstract:STS cells were treated with the IC50 concentrations of Costunolide or dehydrocostus lactone for 24 and 48 h. Results are represented by mean fluorescence intensity (MFI) and p-values. Bold numbers represent significant differences between control and treated cells (p>0.05).
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Sesquiterpene lactones downregulate G2/M cell cycle regulator proteins and affect the invasive potential of human soft tissue sarcoma cells. PLoS One 8: e66300
2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Eva Bernhart, Ehsan Bonyadi Rad, Annelie Martina Weinberg, Andreas Leithner, Bibiane Steinecker, Rudolf BauerAbstract:Soft tissue sarcomas (STS) represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. Many studies have demonstrated the great potential of plant-derived agents in the treatment of various malignant entities. The present study investigates the effects of the sesquiterpene lactones Costunolide and dehydrocostus lactone on cell cycle, MMP expression, and invasive potential of three human STS cell lines of various origins. Both compounds reduced cell proliferation in a time- and dose-dependent manner. Dehydrocostus lactone significantly inhibited cell proliferation, arrested the cells at the G2/M interface and caused a decrease in the expression of the cyclin-dependent kinase CDK2 and the cyclin-dependent kinase inhibitor p27Kip1. In addition, accumulation of cells at the G2/M phase transition interface resulted in a significant decrease in cdc2 (CDK1) together with cyclin B1. Costunolide had no effect on the cell cycle. Based on the fact that STS tend to form daughter cell nests and metastasize, the expressio
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Western blot analysis of G2/M arrest regulator proteins.
2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Bibiane Steinecker-frohnwieser, Eva Bernhart, Ehsan Bonyadi Rad, Annelie Martina Weinberg, Andreas Leithner, Rudolf BauerAbstract:Total protein analysis revealed a significant downregulation of cdc2 and cyclin B1 in dehydrocostus lactone treated STS cells, whereas, Costunolide treatment did not affect the protein levels of these G2/M checkpoint regulators. β-actin was used as loading control. Data shown are representative from at least three independent experiments.
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Western blot analysis of p53 and the CDK inhibitor p27Kip1 proteins.
2013Co-Authors: Birgit Lohberger, Beate Rinner, Nicole Stuendl, Heike Kaltenegger, Bibiane Steinecker-frohnwieser, Eva Bernhart, Ehsan Bonyadi Rad, Annelie Martina Weinberg, Andreas Leithner, Rudolf BauerAbstract:Total protein analysis confirmed no significant changes in the p53 expression after Costunolide or dehydrocostus lactone treatment. In dehydrocostus lactone treated TE-671 and SW-872 cells, the expression of the CDK inhibitor p27Kip1 was reduced. β-actin was used as loading control. Data shown are representative from at least three independent experiments.
Jules Beekwilder - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome P450s from Cynara cardunculus L. CYP71AV9 and CYP71BL5, catalyze distinct hydroxylations in the sesquiterpene lactone biosynthetic pathway
Plant Science, 2014Co-Authors: Kaouthar Eljounaidi, Barbara Menin, Andrea Moglia, Cinzia Comino, Katarina Cankar, Sergio Lanteri, Alain Hehn, Frédéric Bourgaud, Harro Bouwmeester, Jules BeekwilderAbstract:Cynara cardunculus (Asteraceae) is a cross pollinated perennial crop which includes the two cultivated taxa globe artichoke and cultivated cardoon. The leaves of these plants contain high concentrations of sesquiterpene lactones (STLs) among which cynaropicrin is the most represented, and has recently attracted attention because of its therapeutic potential as anti-tumor and anti-photoaging agent. Costunolide is considered the common precursor of the STLs and three enzymes are involved in its biosynthetic pathway: i.e. the germacrene A synthase (GAS), the germacrene A oxidase (GAO) and the Costunolide synthase (COS). Here we report on the isolation of two P450 genes, (i.e. CYP71AV9 and CYP71BL5), in a set of 19,000 C cardunculus unigenes, and their functional characterization in yeast and in planta. The metabolite analyses revealed that the co-expression of CYP71AV9 together with GAS resulted in the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid in yeast. The co-expression of CYP71BL5 and CYP71AV9 with GAS led to biosynthesis of the free Costunolide in yeast and Costunolide conjugates in Nicotiana benthamiana, demonstrating their involvement in STL biosynthesis as GAO and COS enzymes. The substrate specificity of CYP71AV9 was investigated by testing its ability to convert amorpha-4,11-diene, (+)-germacrene D and cascarilladiene to their oxidized products when co-expressed in yeast with the corresponding terpene synthases.
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Reconstitution of the Costunolide Biosynthetic Pathway in Yeast and Nicotiana benthamiana
2013Co-Authors: Qing Liu, Katarina Cankar, Jules Beekwilder, Mohammad Majdi, Tatsiana Charnikhova, Miriam Goedbloed, Ric C. H. De Vos, Harro JAbstract:The sesquiterpene Costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to Costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward Costunolide has not been characterized until now. Here we show that chicory Costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield Costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of Costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in Costunolide production of up to 60 ng.g 21 FW. In addition, two new compounds wer
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Phylogenetic analysis of Asteraceae GAO genes and five chicory CYP71 P450 ESTs.
2013Co-Authors: Qing Liu, Katarina Cankar, Jules Beekwilder, Mohammad Majdi, Tatsiana Charnikhova, Miriam Goedbloed, Francel W. A. Verstappen, Ric C. H. De Vos, Sander Van Der Krol, Harro J BouwmeesterAbstract:Chicory candidate 3368 was later identified as Cichorium intybus Costunolide synthase (CiCOS). Amino acid seuqences of GAOs were obtained from cDNAs deposited at the NCBI. LsGAO germacrene A oxidase from Lactuca sativa (GU198171) or from Cichorium intybus (Ci; GU256644), Helianthus annuus (Ha; GU256646), Saussurea costus (Sc; GU256645) and Barnadesia. spinosa (Bs; GU256647). Bootstrap values are shown in frequency values from 1000 replicates.
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Costunolide-glutathione and Costunolide-cysteine conjugate identification by GST enzyme assay and LC-MS analysis.
2013Co-Authors: Qing Liu, Katarina Cankar, Jules Beekwilder, Mohammad Majdi, Tatsiana Charnikhova, Miriam Goedbloed, Francel W. A. Verstappen, Ric C. H. De Vos, Sander Van Der Krol, Harro J BouwmeesterAbstract:A. LC-MS chromatograms of [m/z]− = 538 of extracts of N. benthamiana leaves agro-infiltrated with TpGAS+CiGAO+CiCOS, Costunolide-GSH conjugate formed in an enzyme assay of Costunolide and GSH with GST, Costunolide-GSH conjugate formed by non-enzymatic conjugation of Costunolide and GSH. B. LC-MS chromatograms of [m/z]− = 352 of extracts of N. benthamiana leaves agro-infiltrated with TpGAS+CiGAO+CiCOS, Costunolide-cysteine (Cys) conjugate formed in an enzyme assay of Costunolide and Cys with GST, Costunolide-Cys conjugate formed non enzymatically from Costunolide and Cys. C. [m/z]− spectrum of peak 22.48 and Costunolide-GSH conjugate (RT = 22.52). Arrows indicate parent ions of GSH-glutathione. D. [m/z]− spectrum of peak 22.30 and Costunolide-Cys conjugate (RT = 22.30). Arrows indicate parent ions of GSH-cysteine. E. Presumed molecular structure of Costunolide-GSH (a) and Costunolide-Cys (b) conjugates. GSH, glutathione; Cys, cysteine; GST, glutathione S-transferase. RT, retention time. Y-axis scale is identical in all chromatograms.
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reconstitution of the Costunolide biosynthetic pathway in yeast and nicotiana benthamiana
PLOS ONE, 2011Co-Authors: Qing Liu, Katarina Cankar, Jules Beekwilder, Mohammad Majdi, Tatsiana Charnikhova, Miriam Goedbloed, Francel W. A. Verstappen, Ric C. H. De Vos, Sander Van Der Krol, Harro J BouwmeesterAbstract:The sesquiterpene Costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to Costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward Costunolide has not been characterized until now. Here we show that chicory Costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield Costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of Costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in Costunolide production of up to 60 ng.g−1 FW. In addition, two new compounds were formed that were identified as Costunolide-glutathione and Costunolide-cysteine conjugates.
