The cytotoxicity of garlic-related disulphides and thiosulfonates in WHCO1 oesophageal cancer cells is dependent on S-thiolation and not production of ROS

• Published in: Biochimica et biophysica acta Vol. 1860; no. 7; pp. 1439 - 1449
• Main Authors: Smith, Muneerah, Hunter, Roger, Stellenboom, Nashia, Kusza, Daniel A., Parker, M. Iqbal, Hammouda, Ahmed N.H., Jackson, Graham, Kaschula, Catherine H.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2016
• Subjects: Antineoplastic Agents, Phytogenic - chemical synthesis; Antineoplastic Agents, Phytogenic - pharmacology; Antioxidants - pharmacology; apoptosis; Apoptosis - drug effects; ascorbic acid; Carcinoma, Squamous Cell - drug therapy; Carcinoma, Squamous Cell - metabolism; Carcinoma, Squamous Cell - pathology; cell cycle checkpoints; Cell Line, Tumor; cell proliferation; Cell Proliferation - drug effects; Cell Survival - drug effects; cysteine; cytotoxicity; Disulfides - chemical synthesis; Disulfides - pharmacology; Dose-Response Relationship, Drug; Esophageal Neoplasms - drug therapy; Esophageal Neoplasms - metabolism; Esophageal Neoplasms - pathology; Esophageal Squamous Cell Carcinoma; G2 Phase Cell Cycle Checkpoints - drug effects; Garlic; health promotion; Humans; inhibitory concentration 50; Molecular Structure; neoplasm cells; neoplasms; Reactive Oxygen Species - metabolism; Signal Transduction - drug effects; Structure-Activity Relationship; Sulfhydryl Compounds - metabolism; sulfides; sulfur; thermodynamics; thiolysis; Thiosulfonic Acids - chemical synthesis; Thiosulfonic Acids - pharmacology; Time Factors; traditional medicine
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2016.03.032

Garlic has been used for centuries in folk medicine for its health promoting and cancer preventative properties. The bioactive principles in crushed garlic are allyl sulphur compounds which are proposed to chemically react through (i) protein S-thiolation and (ii) production of ROS. A collection of R-propyl disulphide and R-thiosulfonate compounds were synthesised to probe the importance of thiolysis and ROS generation in the cytotoxicity of garlic-related compounds in WHCO1 oesophageal cancer cells. A significant correlation (R2=0.78, Fcrit (7,1) α=0.005) was found between the cytotoxicity IC50 and the leaving group pKa of the R-propyl disulphides and thiosulfonates, supporting a mechanism that relies on the thermodynamics of a mixed disulphide exchange reaction. Disulphide (1) and thiosulfonate (11) were further evaluated mechanistically and found to induce G2/M cell-cycle arrest and apoptosis, inhibit cell proliferation, and generate ROS. When the ROS produced by 1 and 11 were quenched with Trolox, ascorbic acid or N-acetyl cysteine (NAC), only NAC was found to counter the cytotoxicity of both compounds. However, NAC was found to chemically react with 11 through mixed disulphide formation, providing an explanation for this apparent inhibitory result. Cellular S-thiolation by garlic related disulphides appears to be the cause of cytotoxicity in WHCO1 cells. Generation of ROS appears to only play a secondary role. Our findings do not support ROS production causing the cytotoxicity of garlic-related disulphides in WHCO1 cells. Importantly, it was found that the popular ROS inhibitor NAC interferes with the assay. •Disulphide induced cytotoxicity in WHCO1 cells correlates to leaving group pKa.•Findings support thiolysis exchange with glutathione or a protein thiol.•ROS production does not lead to cytotoxicity in WHCO1 cells.•ROS inhibitor N-acetyl cysteine reacts directly with the disulphide test compound.