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

Abstract

Background: 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.

Methods: 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.

Results: A significant correlation (R(2)=0.78, Fcrit (7,1) α=0.005) was found between the cytotoxicity IC(50) and the leaving group pK(a) 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 G(2)/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.

Conclusion: 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.

General significance: 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.