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Catherine H. Kaschula - One of the best experts on this subject based on the ideXlab platform.
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The Garlic Compound Z-Ajoene, S-Thiolates COX2 and STAT3 and Dampens the Inflammatory Response in RAW264.7 Macrophages.
Molecular nutrition & food research, 2020Co-Authors: Jessica K. Hitchcock, Lisa M. Graham, Arieh A. Katz, Christopher B. Barnett, Roger Hunter, Georgia Schäfer, Nonkululeko Mkwanazi, Catherine H. KaschulaAbstract:Scope Garlic (Allium sativum) has been used for centuries as both a prophylactic and therapeutic medicinal agent to control inflammation-associated pathologies. To investigate the underlying mechanisms, we established an in vitro inflammatory model using RAW264.7 murine macrophages exposed to low-doses of lipopolysaccharide (LPS) in the presence of the garlic compounds allicin and Z-Ajoene, mimicking regular garlic consumption. Methods and results Both allicin and Z-Ajoene dampened both transcript and protein expression of the pro-inflammatory cytokines IL1β, IL6 and IL12β, and upregulated the expression of the anti-inflammatory cytokine IL10. Protein arrays of selected secreted inflammatory mediators confirmed that Z-Ajoene has a pronounced down-regulatory effect on LPS-induced inflammatory cytokines and chemokines. Many of these proteins are known targets of the transcription factor STAT3; and indeed, Z-Ajoene or its analogue dansyl-Ajoene (DP) was found to decrease the phosphorylation and nuclear translocation of STAT3, and to covalently modify the protein by S-thiolation at Cys108, Cys367 and Cys687. It was further found that Z-Ajoene dose-dependently and non-competitively inhibited the activity of COX2, possibly attributed to S-thiolation at Cys9 and Cys299. Conclusion The characterisation of Z-Ajoene's activity of targeting and covalently modifying STAT3 and COX2, both important regulators of inflammation, may contribute to the health benefits of regular dietary garlic consumption. This article is protected by copyright. All rights reserved.
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The garlic compound Ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells
BMC Cancer, 2019Co-Authors: Catherine H. Kaschula, Rosanna Tuveri, Ellen Ngarande, Daniel A. Kusza, Lisa M. Graham, Arieh A. Katz, Mohamed Suhail Rafudeen, Kevin Dzobo, Christopher Barnett, M. Iqbal ParkerAbstract:Background Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide Ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S -thiolating reactive cysteines in target proteins. Methods Using our fluorescently labelled Ajoene analogue called dansyl-Ajoene, Ajoene’s protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to Ajoene was performed using Schrödinger and p K _a calculations by Epik software. Cytotoxicity of Ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in Ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. Results The dominant protein tagged by dansyl-Ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that Ajoene and dansyl-Ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of Ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by Ajoene. Conclusions The garlic-derived phytochemical Ajoene targets and covalently modifies vimentin in cancer cells by S -thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of Ajoene in cancer cells.
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The garlic compound Ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells
BMC cancer, 2019Co-Authors: Catherine H. Kaschula, Rosanna Tuveri, Ellen Ngarande, Daniel A. Kusza, Lisa M. Graham, Arieh A. Katz, Mohamed Suhail Rafudeen, Kevin Dzobo, Christopher B. Barnett, M. Iqbal ParkerAbstract:Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide Ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S-thiolating reactive cysteines in target proteins. Using our fluorescently labelled Ajoene analogue called dansyl-Ajoene, Ajoene’s protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to Ajoene was performed using Schrodinger and pKa calculations by Epik software. Cytotoxicity of Ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in Ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. The dominant protein tagged by dansyl-Ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that Ajoene and dansyl-Ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of Ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by Ajoene. The garlic-derived phytochemical Ajoene targets and covalently modifies vimentin in cancer cells by S-thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of Ajoene in cancer cells.
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the cytotoxicity of the Ajoene analogue bispmb in whco1 oesophageal cancer cells is mediated by chop gadd153
Molecules, 2017Co-Authors: Vuyolwethu Siyo, Arieh A. Katz, Roger Hunter, Georgia Schäfer, Iqbal M Parker, Andriy Grafov, Iryna Grafova, Martin Nieger, Catherine H. KaschulaAbstract:Garlic is a food and medicinal plant that has been used in folk medicine since ancient times for its beneficial health effects, which include protection against cancer. Crushed garlic cloves contain an array of small sulfur-rich compounds such as Ajoene. Ajoene is able to interfere with biological processes and is cytotoxic to cancer cells in the low micromolar range. BisPMB is a synthetic Ajoene analogue that has been shown in our laboratory to have superior cytotoxicity to Ajoene. In the current study we have performed a DNA microarray analysis of bisPMB-treated WHCO1 oesophageal cancer cells to identify pathways and processes that are affected by bisPMB. The most significantly enriched biological pathways as assessed by gene ontology, KEGG and ingenuity pathway analysis were those involving protein processing in the endoplasmic reticulum (ER) and the unfolded protein response. In support of these pathways, bisPMB was found to inhibit global protein synthesis and lead to increased levels of ubiquitinated proteins. BisPMB also induced alternate splicing of the transcription factor XBP-1; increased the expression of the ER stress sensor GRP78 and induced expression of the ER stress marker CHOP/GADD153. CHOP expression was found to be central to the cytotoxicity of bisPMB as its silencing with siRNA rendered the cells resistant to bisPMB. The MAPK proteins, JNK and ERK1/2 were activated following bisPMB treatment. However JNK activation was not critical in the cytotoxicity of bisPMB, and ERK1/2 activation was found to play a pro-survival role. Overall the Ajoene analogue bisPMB appears to induce cytotoxicity in WHCO1 cells by activating the unfolded protein response through CHOP/GADD153.
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The Cytotoxicity of the Ajoene Analogue BisPMB in WHCO1 Oesophageal Cancer Cells Is Mediated by CHOP/GADD153
Molecules (Basel Switzerland), 2017Co-Authors: Vuyolwethu Siyo, Arieh A. Katz, M. Iqbal Parker, Roger Hunter, Georgia Schäfer, Andriy Grafov, Iryna Grafova, Martin Nieger, Catherine H. KaschulaAbstract:Garlic is a food and medicinal plant that has been used in folk medicine since ancient times for its beneficial health effects, which include protection against cancer. Crushed garlic cloves contain an array of small sulfur-rich compounds such as Ajoene. Ajoene is able to interfere with biological processes and is cytotoxic to cancer cells in the low micromolar range. BisPMB is a synthetic Ajoene analogue that has been shown in our laboratory to have superior cytotoxicity to Ajoene. In the current study we have performed a DNA microarray analysis of bisPMB-treated WHCO1 oesophageal cancer cells to identify pathways and processes that are affected by bisPMB. The most significantly enriched biological pathways as assessed by gene ontology, KEGG and ingenuity pathway analysis were those involving protein processing in the endoplasmic reticulum (ER) and the unfolded protein response. In support of these pathways, bisPMB was found to inhibit global protein synthesis and lead to increased levels of ubiquitinated proteins. BisPMB also induced alternate splicing of the transcription factor XBP-1; increased the expression of the ER stress sensor GRP78 and induced expression of the ER stress marker CHOP/GADD153. CHOP expression was found to be central to the cytotoxicity of bisPMB as its silencing with siRNA rendered the cells resistant to bisPMB. The MAPK proteins, JNK and ERK1/2 were activated following bisPMB treatment. However JNK activation was not critical in the cytotoxicity of bisPMB, and ERK1/2 activation was found to play a pro-survival role. Overall the Ajoene analogue bisPMB appears to induce cytotoxicity in WHCO1 cells by activating the unfolded protein response through CHOP/GADD153.
R L Krauthsiegel - One of the best experts on this subject based on the ideXlab platform.
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Ajoene is an inhibitor and subversive substrate of human glutathione reductase and trypanosoma cruzi trypanothione reductase crystallographic kinetic and spectroscopic studies
Journal of Medicinal Chemistry, 1999Co-Authors: H Gallwitz, S Bonse, A Martinezcruz, Ilme Schlichting, K Schumacher, R L KrauthsiegelAbstract:Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-Ajoene revealed a mixed disulfide between the active site Cys58 and the CH2=CH-CH2-SO-CH2-CH=CH-S moiety of Ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-Ajoene with a kcat/Km of 6.8 x 10(3) M-1 s-1 yielding 4,5,9-trithiadodeca-1, 6,11-triene (deoxyAjoene) and 4,8,9,13-tetrathiahexadeca-1,6,10, 15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and Ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of Ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism.
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Ajoene is an inhibitor and subversive substrate of human glutathione reductase and trypanosoma cruzi trypanothione reductase crystallographic kinetic and spectroscopic studies
Journal of Medicinal Chemistry, 1999Co-Authors: H Gallwitz, S Bonse, A Martinezcruz, Ilme Schlichting, K Schumacher, R L KrauthsiegelAbstract:Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-Ajoene revealed a mixed disulfide between the active site Cys58 and the CH2CH−CH2−SO−CH2−CHCH−S moiety of Ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-Ajoene with a kcat/Km of 6.8 × 103 M-1 s-1 yielding 4,5,9-trithiadodeca-1,6,11-triene (deoxyAjoene) and 4,8,9,13-tetrathiahexadeca-1,6,10,15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and Ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and ...
Angelika M. Vollmar - One of the best experts on this subject based on the ideXlab platform.
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Ajoene-induced cell death in human promyeloleukemic cells does not require JNK but is amplified by the inhibition of ERK
Oncogene, 2003Co-Authors: Dorothee Antlsperger, Verena M. Dirsch, Dulce Ferreira, Ming Liang Kuo, Angelika M. VollmarAbstract:Treatment of human promyeloleukemic HL-60 cells with the experimental antileukemic drug Ajoene induces the activation of the mitogen-activated protein kinases (MAPKs) c-Jun NH2-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK) 1/2 as well as the survival kinase Akt. JNK activation occurred in HL-60/neo, HL-60/bcl-xL, and in HL-60 cells pretreated with the pan-caspase inhibitor zVAD-fmk, indicating that JNK activation is not dependent on Ajoene-induced mitochondria perturbation and subsequent caspase activation. Cells overexpressing a dominant-negative JNK showed no altered sensitivity towards Ajoene suggesting that the activation of JNK is not necessary for Ajoene-induced cell death. Inhibition of p38 MAPK by SB 203580 had no influence on Ajoene-mediated apoptosis. In contrast, inhibition of ERK1/2 vastly enhanced Ajoene-induced cell death. The survival kinase Akt, in contrast, did not participate in Ajoene-induced death signaling as shown by the use of the phosphatidylinositol-3-kinase inhibitor wortmannin. Thus in contrast to the previous findings regarding stress-induced cell death, Ajoene-mediated activation of JNK and p38 has no impact on Ajoene-induced apoptosis in HL-60 cells. Blockade of ERK1/2 but not Akt pathways leads to sensitization of cells against Ajoene-mediated apoptosis supporting the view that inhibition of ERK1/2 is a valuable strategy to increase the sensitivity of promyeloleukemic cells towards Ajoene.
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Ajoene, an experimental anti-leukemic drug: mechanism of cell death.
Leukemia, 2002Co-Authors: Verena M. Dirsch, Dorothee Antlsperger, Hannes Hentze, Angelika M. VollmarAbstract:The organosulfur compound Ajoene, a constitutent of garlic, has been shown to induce apoptosis in a leukemic cell line as well as in blood cells of a leukemic patient. The mechanisms of action of Ajoene, however, are unknown. The present study aims to characterize the molecular events leading to Ajoene-triggered apoptosis. We show here that Ajoene (20 μM) leads to a time-dependent activation of caspase-3-like activity as well as to the proteolytic processing of procaspase-3 and -8. Activation of caspases was necessary for Ajoene-induced apoptosis since the broad-range caspase inhibitor zVAD-fmk completely abrogated Ajoene-mediated DNA fragmentation. Although the initiator caspase-8 was activated, the CD95 death receptor was not involved in death signaling since the HL-60 clone used was shown to express a functionally inactive CD95 receptor. Furthermore, Ajoene induced the release of cytochrome c, which was not inhibited by zVAD-fmk indicating that cytochrome c release precedes caspase activation. Ajoene also led to a dissipation of the mitochondrial transmembrane potential. Overexpression of Bcl-xL clearly diminished Ajoene-induced caspase activation as well as apoptosis. These results indicate that apoptosis in leukemia cells triggered by Ajoene is based on the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.
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Ajoene, a natural product with non-steroidal anti-inflammatory drug (NSAID)-like properties?
Biochemical pharmacology, 2001Co-Authors: Verena M. Dirsch, Angelika M. VollmarAbstract:Abstract The inducible isoform of cyclooxygenase (COX-2) is implicated in the pathogenesis of various inflammatory diseases as well as in carcinogenesis, especially of gastrointestinal tumors. Epidemiological as well as experimental data support a role for constituents of allium vegetables, such as garlic and onions, in the prevention of gastrointestinal cancer. Therefore, the aim of the present study was to examine whether the garlic-derived natural product Ajoene interferes with the COX-2 pathway by using lipopolysaccharide (LPS)-activated RAW 264.7 cells as in vitro model. Ajoene was shown to dose-dependently inhibit the release of LPS (1 μg/mL)-induced prostaglandin E2 in RAW 264.7 macrophages ( ic 50 value: 2.4 μM). This effect was found to be due to an inhibition of COX-2 enzyme activity by Ajoene ( ic 50 value: 3.4 μM). Ajoene did not reduce COX-2 expression, but rather increased LPS-induced COX-2 protein and mRNA expression compared to LPS-stimulated cells only. In the absence of LPS, however, Ajoene was unable to induce COX-2. The non-steroidal anti-inflammatory drug indomethacin was shown to act similarly in LPS-activated RAW 264.7 cells. These data suggest that Ajoene works by a mechanism of action similar to that attributed to non-steroidal anti-inflammatory drugs. This finding may add a novel aspect to the biological profile of the garlic-derived natural product Ajoene which might be important for understanding the usefulness of garlic for chemoprevention of gastrointestinal carcinomas.
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Ajoene, a Compound of Garlic, Induces Apoptosis in Human Promyeloleukemic Cells, Accompanied by Generation of Reactive Oxygen Species and Activation of Nuclear Factor κB
Molecular pharmacology, 1998Co-Authors: Verena M. Dirsch, Alexander L. Gerbes, Angelika M. VollmarAbstract:The pharmacological role of garlic in prevention and treatment of cancer has received increasing attention, but thorough investigations into the molecular mechanisms of action of garlic compounds are rare. The present study demonstrates that Ajoene, a major compound of garlic induces apoptosis in human leukemic cells, but not in peripheral mononuclear blood cells of healthy donors. The effect was dose and time dependent. Apoptosis was judged by three criteria, morphology of cells, quantification of subdiploid DNA content by flow cytometry, and detection of DNA fragmentation by gel electrophoresis. Ajoene increased the production of intracellular peroxide in a dose- and time-dependent fashion, which could be partially blocked by preincubation of the human leukemic cells with the antioxidant N -acetylcysteine. Interestingly, N -acetylcysteine-treated cells showed a 50% loss of Ajoene-induced apoptosis. Moreover, Ajoene was demonstrated to activate nuclear translocation of the transcription factor nuclear factor κB, an effect that was abrogated in N -acetylcysteine-loaded cells. These results suggested that Ajoene might induce apoptosis in human leukemic cells via stimulation of peroxide production and activation of nuclear factor κB. This is a novel aspect in the biological profile of this garlic compound and an important step in elucidating the underlying molecular mechanisms of its antitumor action.
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Effect of allicin and Ajoene, two compounds of garlic, on inducible nitric oxide synthase.
Atherosclerosis, 1998Co-Authors: Verena M. Dirsch, Hildebert Wagner, Alexandra K. Kiemer, Angelika M. VollmarAbstract:Inducible nitric oxide synthase (iNOS) has recently been shown to be present in human atherosclerotic lesions and to promote the formation of deleterious peroxynitrite. Allicin and Ajoene are discussed as active compounds with regard to the beneficial effects of garlic in atherosclerosis. The aim of this study was to investigate the effect of allicin and Ajoene on the iNOS system in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ajoene (IC50 2.5-5 microM) and allicin (IC50 15-20 microM) dose dependently reduced nitrite accumulation, a parameter for NO synthesis, in supernatants of LPS-stimulated (1 microg/ml, 20 h) macrophages. Accordingly, reduced iNOS enzyme activities were measured by conversion of L-[3H]arginine to L-[3H]citrulline in homogenates of LPS-activated cells treated with Ajoene or allicin. None of these compounds, however, showed a direct effect on the catalytic-activity of iNOS. Consequently, iNOS protein and mRNA expression in Ajoene (10 microM) or allicin (50 microM) treated cells were evaluated by Western blot and Northern blot analysis, respectively. Markedly reduced iNOS protein as well as mRNA levels were demonstrated. These observations indicate that allicin and Ajoene inhibit the expression of iNOS in activated macrophages. The possible link of this effect to the beneficial features attributed to garlic is discussed.
M. Iqbal Parker - One of the best experts on this subject based on the ideXlab platform.
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The garlic compound Ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells
BMC Cancer, 2019Co-Authors: Catherine H. Kaschula, Rosanna Tuveri, Ellen Ngarande, Daniel A. Kusza, Lisa M. Graham, Arieh A. Katz, Mohamed Suhail Rafudeen, Kevin Dzobo, Christopher Barnett, M. Iqbal ParkerAbstract:Background Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide Ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S -thiolating reactive cysteines in target proteins. Methods Using our fluorescently labelled Ajoene analogue called dansyl-Ajoene, Ajoene’s protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to Ajoene was performed using Schrödinger and p K _a calculations by Epik software. Cytotoxicity of Ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in Ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. Results The dominant protein tagged by dansyl-Ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that Ajoene and dansyl-Ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of Ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by Ajoene. Conclusions The garlic-derived phytochemical Ajoene targets and covalently modifies vimentin in cancer cells by S -thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of Ajoene in cancer cells.
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The garlic compound Ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells
BMC cancer, 2019Co-Authors: Catherine H. Kaschula, Rosanna Tuveri, Ellen Ngarande, Daniel A. Kusza, Lisa M. Graham, Arieh A. Katz, Mohamed Suhail Rafudeen, Kevin Dzobo, Christopher B. Barnett, M. Iqbal ParkerAbstract:Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide Ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S-thiolating reactive cysteines in target proteins. Using our fluorescently labelled Ajoene analogue called dansyl-Ajoene, Ajoene’s protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to Ajoene was performed using Schrodinger and pKa calculations by Epik software. Cytotoxicity of Ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in Ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. The dominant protein tagged by dansyl-Ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that Ajoene and dansyl-Ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of Ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by Ajoene. The garlic-derived phytochemical Ajoene targets and covalently modifies vimentin in cancer cells by S-thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of Ajoene in cancer cells.
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The Cytotoxicity of the Ajoene Analogue BisPMB in WHCO1 Oesophageal Cancer Cells Is Mediated by CHOP/GADD153
Molecules (Basel Switzerland), 2017Co-Authors: Vuyolwethu Siyo, Arieh A. Katz, M. Iqbal Parker, Roger Hunter, Georgia Schäfer, Andriy Grafov, Iryna Grafova, Martin Nieger, Catherine H. KaschulaAbstract:Garlic is a food and medicinal plant that has been used in folk medicine since ancient times for its beneficial health effects, which include protection against cancer. Crushed garlic cloves contain an array of small sulfur-rich compounds such as Ajoene. Ajoene is able to interfere with biological processes and is cytotoxic to cancer cells in the low micromolar range. BisPMB is a synthetic Ajoene analogue that has been shown in our laboratory to have superior cytotoxicity to Ajoene. In the current study we have performed a DNA microarray analysis of bisPMB-treated WHCO1 oesophageal cancer cells to identify pathways and processes that are affected by bisPMB. The most significantly enriched biological pathways as assessed by gene ontology, KEGG and ingenuity pathway analysis were those involving protein processing in the endoplasmic reticulum (ER) and the unfolded protein response. In support of these pathways, bisPMB was found to inhibit global protein synthesis and lead to increased levels of ubiquitinated proteins. BisPMB also induced alternate splicing of the transcription factor XBP-1; increased the expression of the ER stress sensor GRP78 and induced expression of the ER stress marker CHOP/GADD153. CHOP expression was found to be central to the cytotoxicity of bisPMB as its silencing with siRNA rendered the cells resistant to bisPMB. The MAPK proteins, JNK and ERK1/2 were activated following bisPMB treatment. However JNK activation was not critical in the cytotoxicity of bisPMB, and ERK1/2 activation was found to play a pro-survival role. Overall the Ajoene analogue bisPMB appears to induce cytotoxicity in WHCO1 cells by activating the unfolded protein response through CHOP/GADD153.
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Anti-Proliferative Activity of Synthetic Ajoene Analogues on Cancer Cell-Lines
Anti-cancer agents in medicinal chemistry, 2011Co-Authors: Catherine H. Kaschula, Roger Hunter, Jonathan Cotton, Hassan T. Hassan, Nashia Stellenboom, Xiao Q. Zhai, M. Iqbal ParkerAbstract:The ability of garlic preparations to inhibit cancer cell-growth has been attributed to a group of structurally-related organosulfur compounds found in the crushed clove. Historically, interest has centred on three such compounds as allicin, diallyl disulfide and diallyl trisulfide, with less interest on E- and Z-Ajoene. A recently developed synthetic route from our laboratory for preparing Ajoene analogues allows access to derivatives containing the sulfoxide / vinyl disulfide core whilst varying the terminal end-group functionality. A small library has been synthesized and an advanced lead with p-methoxybenzyl end groups (8) identified. Data on the in vitro anti-proliferation activity of compound (8) is presented here on six cancer cell-lines in comparison with that of Z- and E-Ajoene to reveal an enhancement in activity of up to twelvefold. In addition, a modest selectivity is observed for tumour over normal cell-lines of up to threefold. Data on Ajoene and its derivatives is presented in the context of chemosensitization in drug-resistance, and ideas on Ajoenes mode of action at the molecular level are presented and discussed.
H Gallwitz - One of the best experts on this subject based on the ideXlab platform.
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Ajoene is an inhibitor and subversive substrate of human glutathione reductase and trypanosoma cruzi trypanothione reductase crystallographic kinetic and spectroscopic studies
Journal of Medicinal Chemistry, 1999Co-Authors: H Gallwitz, S Bonse, A Martinezcruz, Ilme Schlichting, K Schumacher, R L KrauthsiegelAbstract:Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-Ajoene revealed a mixed disulfide between the active site Cys58 and the CH2=CH-CH2-SO-CH2-CH=CH-S moiety of Ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-Ajoene with a kcat/Km of 6.8 x 10(3) M-1 s-1 yielding 4,5,9-trithiadodeca-1, 6,11-triene (deoxyAjoene) and 4,8,9,13-tetrathiahexadeca-1,6,10, 15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and Ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of Ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism.
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Ajoene is an inhibitor and subversive substrate of human glutathione reductase and trypanosoma cruzi trypanothione reductase crystallographic kinetic and spectroscopic studies
Journal of Medicinal Chemistry, 1999Co-Authors: H Gallwitz, S Bonse, A Martinezcruz, Ilme Schlichting, K Schumacher, R L KrauthsiegelAbstract:Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-Ajoene revealed a mixed disulfide between the active site Cys58 and the CH2CH−CH2−SO−CH2−CHCH−S moiety of Ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-Ajoene with a kcat/Km of 6.8 × 103 M-1 s-1 yielding 4,5,9-trithiadodeca-1,6,11-triene (deoxyAjoene) and 4,8,9,13-tetrathiahexadeca-1,6,10,15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and Ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and ...