Abstract
BackgroundGarlic 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.MethodsUsing 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.ResultsThe 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.ConclusionsThe 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.
Highlights
Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer
We show that the covalent modification of vimentin by ajoene disrupts the vimentin filamentous network that in turn counters the metastatic phenotype of MDA-MB-231 and HeLa cancer cells
Ajoene targets vimentin in MDA-MB-231 cells Previous structure-activity studies in our laboratory have identified the vinyl disulfide functional group to be the ajoene pharmacophore that is responsible for cancer cell cytotoxicity [21]
Summary
Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. Ajoene and its related polysulfane family members have been shown to counter the different stages of cancer. In this regard, they inhibit tumour initiation by various chemical carcinogens (reviewed in [4]), and counter tumour growth by inhibiting proliferation and inducing apoptosis in growing cancer cells (reviewed in [5, 6]). Some of the garlic polysulfanes have been shown to inhibit the more advanced stages of cancer by countering the metastatic process [7,8,9,10]. Ajoene displays attractive therapeutic properties, being cytotoxic to cultured cancer cells in the low micromolar range [9, 11,12,13], and showing a level of selectivity for cancer over normal cells [11,12,13] while being relatively non-toxic in vivo [14]
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