Browsing by Author "Klinck, Johan"

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    Investigations into the cytotoxic mechanism of action for the garlic compound ajoene and its derivatives in breast cancer cells
    (Stellenbosch : Stellenbosch University, 2022-04) Klinck, Johan; Kaschula, Catherine H.; Engelbrecht, Anna-Mart; Loos, Ben; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Breast cancer continues to place a severe burden on women globally. There has been a growing interest in natural compounds for cancer prevention, with several phytochemicals possessing anti-cancer properties. These compounds typically have little toxicity and are available readily. Garlic has been considered to have health benefits, throughout history attributed in part to several organosulfur compounds (OSC’s). Ajoenes are a subset of OSC’s that occur naturally in crushed and cooked cloves. We investigated the mechanism of action of ajoene and its analogues Z-ajoene, Z- and E- propyl ajoene and E/Z-dansyl ajoene on the breast cancer cell lines MDA-MB-231 and MCF7. We found that ajoene and its analogues are cytotoxic to both cell lines with IC50 values between 18 and 78 μM, which is comparable to chemotherapeutic drugs. MDA-MB-231 cells were found to be 2-times more sensitive to ajoene than MCF7 cells. Ajoene is proposed to act by S-thiolating cysteine residues of target proteins. Z-ajoene stereoisomers are approximately 1.5-fold more active than E-stereoisomers. Using computational modelling, we were able to rule out potential energy, nucleophile approach, and allyl-sulfur electrophilicity as explaining the differences in this cytotoxicity. We observed for the first time the “U- shape” of propyl ajoene in their most stable conformations in which the hydrophobic tails stabilise both the Z- and E-conformations such that the potential energy is similar, being -89.175 kJ/mol and -91.676 kJ/mol respectively. We also found vinyl-sulfur to be more electrophilic than allyl-sulfur. This suggests the thiolysis reaction is driven by the stability of the vinyl-sulfur-leaving group and thus driven by thermodynamics and not kinetics. In support of our proposed mechanism of action for ajoene, we found that many proteins were dansylated when treated with E/Z-dansyl ajoene, which were found in the membrane, structural and nuclear fractions, but not in the cytosolic fraction. This supports our proposal that dansyl is linked to proteins via disulfide bonds, as the cytosolic environment is hostile towards disulfide bonds. In support of ajoene inducing ER stress we found an increases in cytosolic Ca2+ in MDA-MB-231 cells treated with ajoene. Furthermore, increased expression was found for chaperones (BiP 1.6-fold, PDI 2-fold and Calnexin 2-fold) and UPR transducers (PERK 2.6-fold, IRE1α 2.7-fold) as well as an increase in autophagic activity (LC3-II 2.9-fold and p62 0.2-fold). In MCF7 cells chaperone expression increased (BiP 1.8-fold, PDI 2-fold, Calnexin 2-fold) indicating ER stress was induced, however no increase was observed for UPR transducers and protein levels for autophagy markers fluctuated. Ajoene and its analogues appear to exert their cytotoxicity in breast cancer cells by interfering with protein folding via protein S-thiolation. This induces misfolded proteins and triggers ER stress which activates the UPR and mobilises autophagy machinery to remove misfolded protein aggregates. Chronic or intense ER stress activates the pro-apoptotic branch of the UPR leading to apoptosis. Garlic intake supports breast cancer prevention where cancer cells have basally high levels of ER stress which can be exploited to activate the pro-apoptotic component of the UPR and autophagy.