RESUMEN
Garlic is a medicinal plant and spice that has been used for millennia for its health-promoting effects. These medicinal properties are associated with low molecular weight organosulfur compounds, produced following the crushing of garlic cloves. One of these compounds, ajoene, is proposed to act by S-thioallylating cysteine residues on target proteins whose identification in cancer cells holds great promise for understanding mechanistic aspects of ajoene's cancer cell cytotoxicity. To this end, an ajoene analogue (called biotin-ajoene, BA), containing a biotin affinity tag, was designed as an activity-based probe specific for the protein targets of ajoene in MDA-MB-231 breast cancer cells. BA was synthesized via a convergent "click" strategy and found to retain its cytotoxicity against MDA-MB-231 cells compared to ajoene. Widespread biotinylation of proteins was found to occur via disulfide bond formation in a dose-dependent manner, and the biotin-ajoene probe was found to share the same protein targets as its parent compound, ajoene. The biotinylated proteins were affinity-purified from the treated MDA-MB-231 cell lysate using streptavidin-coated magnetic beads followed by an on-bead reduction, alkylation, and digestion to liberate the peptide fragments, which were analyzed by liquid chromatography tandem mass chromatography. A total of 600 protein targets were identified, among which 91% overlapped with proteins with known protein cysteine modification (PCM) sites. The specific sites were enriched for those susceptible to S-glutathionylation (-SSG) (16%), S-sulfhydration (-SSH) (20%), S-sulfenylation (-SOH) (22%), and S-nitrosylation (-SNO) (31%). As target validation, both ajoene and a dansylated ajoene (DP) were found to S-thiolate the pure recombinant forms of glutathione S-transferase pi 1 (GSTP1) and protein disulfide isomerase (PDI), and the ajoene analogue DP was found to be a more potent inhibitor than 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Pathway analysis elucidated that ajoene targets functional and signaling pathways that are implicated in cancer cell survival, specifically cellular processes, metabolism, and genetic information processing pathways. The results of this study provide mechanistic insights into the character of the anti-cancer activity of the natural dietary compound ajoene.
Asunto(s)
Neoplasias de la Mama , Ajo , Humanos , Femenino , Proteómica , Cisteína/metabolismo , Biotina , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Disulfuros/farmacología , Disulfuros/química , Sulfóxidos , Ajo/química , AntioxidantesRESUMEN
SCOPE: Garlic (Allium sativum) has been used for centuries as a prophylactic and therapeutic medicinal agent to control inflammation-associated pathologies. To investigate the underlying mechanisms, an in vitro inflammatory model is established using RAW264.7 murine macrophages exposed to low-doses of lipopolysaccharide (LPS) in the presence of garlic compounds allicin and Z-ajoene (ZA), mimicking regular garlic consumption. METHODS AND RESULTS: Both allicin and Z-ajoene dampen both transcript and protein expression of the pro-inflammatory cytokines IL1ß, IL6, and IL12ß, and upregulate the expression of the anti-inflammatory cytokine IL10. Protein arrays of selected secreted inflammatory mediators confirm 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 signal transducer and activator of transcription 3 (STAT3); and indeed, Z-ajoene or its analogue dansyl-ajoene is found to decrease phosphorylation and nuclear translocation of STAT3, and to covalently modify the protein by S-thiolation at Cys108, Cys367, and Cys687. Z-Ajoene dose-dependently and non-competitively inhibit the activity of cyclooxygenase 2 (COX2), possibly attributed to S-thiolation at Cys9 and Cys299. CONCLUSION: The characterization 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.
Asunto(s)
Disulfuros/farmacología , Ajo/química , Inflamación/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Sulfóxidos/farmacología , Animales , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Citocinas/genética , Citocinas/metabolismo , Inflamación/metabolismo , Inflamación/patología , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Células RAW 264.7 , Factor de Transcripción STAT3/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Ácidos Sulfínicos/farmacologíaRESUMEN
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 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. 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.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Disulfuros/farmacología , Ajo/química , Neoplasias/tratamiento farmacológico , Vimentina/metabolismo , Línea Celular Tumoral , Simulación por Computador , Disulfuros/metabolismo , Disulfuros/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Invasividad Neoplásica/prevención & control , Neoplasias/patología , Unión Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sulfóxidos , Vimentina/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Disulfuros/farmacología , Neoplasias Esofágicas/metabolismo , Factor de Transcripción CHOP/metabolismo , Línea Celular Tumoral , Disulfuros/química , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Sulfóxidos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Ajoene is a natural allylsulfur compound found in crushed garlic that arrests growth and induces apoptosis in cancer cells. To gain mechanistic insights into the cytotoxicity of ajoene in cancer cells, two fluorescently labelled ajoene analogs with dansyl- (DP) and fluorescein- (FOX) tags were synthesized. The tagged ajoenes were found to retain their activity at inhibiting proliferation and inducing apoptosis in MDA-MB-231 human breast-cancer and WHCO1 human esophageal-cancer cells. Both tagged ajoenes localized to the endoplasmic reticulum (ER) in MDA-MB-231 cells as observed by live cell confocal laser scanning microscopy (CLSM) and confirmed by generating an MDA-MB-231 cell line expressing yellow fluorescent protein (YFP) in the ER. DP appears to S-thiolate multiple protein targets in MDA-MB-231 cells as observed by immunoblotting under non-reducing conditions only; and a competition assay demonstrated that DP and Z-ajoene in fact share the same target. Ajoene S-thiolation interfered with protein folding and led to an accumulation of misfolded protein aggregates and activated the unfolded protein response (UPR). Consistent with this mechanism, increased levels of GRP78 and total ubiquitinated proteins were observed; and an ER-folded protein, type-1 collagen, was tracked to the proteasome following ajoene treatment. The intracellular protein aggregates were observed by CLSM and transmission electron microscopy (TEM). This is the first time that ajoene has been shown to target protein folding in the ER of cancer cells. © 2015 Wiley Periodicals, Inc.
Asunto(s)
Disulfuros/farmacología , Proteínas de Choque Térmico/metabolismo , Neoplasias/metabolismo , Extractos Vegetales/farmacología , Pliegue de Proteína/efectos de los fármacos , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Disulfuros/química , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Fluoresceína/química , Humanos , Microscopía Electrónica de Transmisión , Neoplasias/tratamiento farmacológico , Fosfatidilcolinas/química , Sulfóxidos , Ubiquitinación , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Garlic (Allium sativum) has been used for centuries as a prophylactic and therapeutic medicinal agent. Importantly, garlic has been suggested to have both cancer-preventive potential as well as significant enhancing effects on the immune system. While these observations are supported experimentally both in vitro and in vivo, the impact of garlic in assisting the immune system in the prevention of cancer still lacks experimental confirmation. Studies addressing the immunomodulatory effects of garlic reveal conflicting data as to pro- or anti-inflammatory responses depending on the particular experimental set-ups and the garlic preparation used (i.e. garlic extract versus chemically pure garlic compounds). Here we provide an overview of the chemistry of the major garlic organosulfur compounds, summarize the current understanding and propose a link between the immunomodulating activity of garlic and the prevention of cancer. We hypothesize that garlic rather elicits anti-inflammatory and anti-oxidative responses that aid in priming the organism towards eradication of an emerging tumor.