The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells
- Resource Type
- Authors
- Mohamed Suhail Rafudeen; Christopher B. Barnett; Ellen Ngarande; Kevin Dzobo; Lisa M. Graham; Georgia Schäfer; Catherine H. Kaschula; Rosanna Tuveri; M. Iqbal Parker; Arieh A. Katz; Daniel A. Kusza; Roger Hunter
- Source
- BMC Cancer, Vol 19, Iss 1, Pp 1-16 (2019)
BMC Cancer
- Subject
- Models, Molecular
0301 basic medicine
Cancer Research
Vimentin
macromolecular substances
Immunofluorescence
lcsh:RC254-282
Natural product
HeLa
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Ajoene
Cell Movement
Cell Line, Tumor
Neoplasms
Genetics
medicine
Humans
Computer Simulation
Neoplasm Invasiveness
MTT assay
Disulfides
Cytotoxicity
Garlic
Cancer
biology
medicine.diagnostic_test
EMT
biology.organism_classification
Antimetastatic
lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens
Molecular biology
Recombinant Proteins
030104 developmental biology
Oncology
chemistry
Cell culture
Sulfoxides
030220 oncology & carcinogenesis
Cancer cell
biology.protein
Drug Screening Assays, Antitumor
Research Article
Protein Binding
- Language
- English
- ISSN
- 1471-2407
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.