Investigations into the cytotoxic mechanism of action for the garlic compound ajoene and its derivatives in breast cancer cells

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dc.contributor.advisorKaschula, Catherine H.en_ZA
dc.contributor.advisorEngelbrecht, Anna-Marten_ZA
dc.contributor.advisorLoos, Benen_ZA
dc.contributor.authorKlinck, Johanen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Physiological Sciences.en_ZA
dc.date.accessioned2022-03-11T16:50:27Z
dc.date.accessioned2022-04-29T09:31:18Z
dc.date.available2022-03-11T16:50:27Z
dc.date.available2022-04-29T09:31:18Z
dc.date.issued2022-04
dc.descriptionThesis (MSc)--Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH 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.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Borskanker veroorsaak ‘n voortdurende swaar las op vrouens en huishoudings wêreldwyd. Voorkoming, asook vroeë diagnose is noodsaaklik, siende dat beperkte behandelingsopsies beskikbaar is en langtermyn oorlewingskoerse laag is indien die siekte eers op ‘n laat stadium gediagnoseer word. Daar is ‘n groeiende belangstelling in die gebruik van natuurlike middels ten einde kanker te voorkom. Daar is verskeie fitochemikalieë met natuurlike anti-kanker eienskappe. Hierdie verbindings het normaalweg geen toksisiteit of ongewenste newe-effekte nie en is maklik verkrygbaar in jou dieet. Knoffel (Allium sativum) word as medisinale kruie geag vir die behandeling van verskeie mediese kwale. Hierdie medisinale eienskappe kan toegeskryf word aan verskeie organoswael verbindings. Ajoene-tipe verbindings vorm deel van die verbindings wat natuurlik voorkom in fyngedrukte en gekookte knoffelhuisies. In hierdie studie het ons die sitotoksiese meganisme van aksie van ajoene en sy analoë Z- ajoene, Z- en E-propiel ajoene en E/Z-dansiel ajoene op die borskanker sellyne MDA-MB-231 en MCF7 ondersoek. Deur rekenaarmodellering kon ons potensiële energie, nukleofiele benadering en alliel-swael elektrofilisiteit as moontlikhede om die verskil in aktiwiteit van Z- teenoor E-ajoene isomere te verklaar uit skakel. Ons het vir die eerste keer die U-vorm van propiel ajoene in hul mees stabiele bouvorm gesien. In kontras met vorige bevindinge het ons ook viniel-swael meer elektrofilies as alliel-swael gevind. Dit dui daarop dat die tiolise-reaksie deur die stabiliteit van die viniel wat die groep verlaat en nie reaksiekinetika gedryf word. Ons het gevind dat ajoene en sy analoë sitotoksies is vir beide MDA-MB-231 en MCF7 selle met IC50 waardes wat wissel tussen 18 en 78 μM, wat vergelykbaar is met chemoterapeutiese middels. MDA-MB- 231 selle was 2 keer meer sensitief vir ajoene as MCF7-selle. Ons het bevind dat sitotoksiese konsentrasies van Z-propiel ajoene Ca2+ uitvloei in MDA-MB-231 selle teweegbring. Ons kon die raporterende dansielgroepe aan proteïene in elke selfraksie koppel, uitgesonder die sitosoliese fraksie. Dit is aanduidend dat ajoene reageer met proteïene deur die proteïene via alliel-swael te S-tioleer. Verhoogde uitdrukking of aktiwiteit van die begeleiers (BiP 1.6-voudig, PDI 2-voudig and Calnexin 2- voudig), UPR omskakelaars (PERK, IRE1α) en proteïene verbonde aan autofagie (LC3-II en p62), het aangedui dat Z-propiel ajoene ER stres, UPR aktivering en outofagie in MDA-MB-231 selle teweegbring. In MCF7 selle was slegs die begeleiers (BiP 1.8-voudig, PDI 2-voudig, Calnexin 2-voudig) se uitdrukking opgereguleer terwyl geen verandering vir UPR omskakelaars waargeneem is nie en vlakke van outofagie merkers fluktueer. Ajoene en sy analoë blyk dus in borskankerselle op te tree deur hul betrokenheid met proteïenvouing via S- tiolering, deur dat proteïene wat verkeerd vou ER stres veroorsaak wat daarna die UPR aktiveer en outofagie mobiliseer om proteïene wat verkeerd gevou het, te verwyder. Chroniese of intense ER stres kan die pro- apoptotiese vertakking van die UPR mobiliseer wat tot die dood van die sel lei. Dit ondersteun die voorkoming van borskanker, omdat kanker hoë basale vlakke van ER stres het, wat uitgebuit kan word deur die pro-apoptotiese vertakking van die UPR te aktiveer, wat op sy beurt outofagie aktiveer wat bekend is om anti-kanker werking in die vroeë stadiums van die vorming van kankergewasse te hê.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxvii, 135 pages : illustrations (some color)en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/124763
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectGarlic -- Therapeutic useen_ZA
dc.subjectBreast -- Cancer -- Chemotherapyen_ZA
dc.subjectChemopreventionen_ZA
dc.subjectCancer cells -- Effect of drugs onen_ZA
dc.subjectUCTDen_ZA
dc.titleInvestigations into the cytotoxic mechanism of action for the garlic compound ajoene and its derivatives in breast cancer cellsen_ZA
dc.typeThesisen_ZA
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