Browsing by Author "Kaschula, Catherine"

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    An Activity-Based Proteomics Approach for Identifying Ajoene's S-thiolation Protein Targets in Blood and Cancer Cells
    (2021) Kusza, Daniel Andreas; Hunter, Roger; Kaschula, Catherine
    Garlic has been used as a medicinal plant since ancient times and is well-documented to produce several organosulfur compounds (OSCs) that show promising chemopreventative and therapeutic properties. The vinyl disulfide sulfoxide garlic rearrangement product, ajoene, is one of the phytochemicals in garlic that possesses a broad spectrum of biological activities against a variety of cancers both in vitro and in vivo. Our group's previous investigations into ajoene's cytotoxicity have shown that it modifies proteins by S-thioallylating redox-sensitive cysteine residues through a thioldisulfide exchange reaction. Further investigations into this exchange have been the primary aim of this PhD thesis using a biotinylation protocol for trapping out the said protein targets of ajoene to link them to anticancer signalling pathways. The thesis begins in Chapter One with an overview of the chemopreventative and antitumour activity of OSCs from garlic in which a particular emphasis is placed on OSC structure and mechanistic aspects of their cancer biology. Chapter Two discusses the various aspects of ajoene as the target natural product of the thesis. These include its biosynthesis and synthesis, in both native form and as derivatives, for SAR studies, for which a UCT synthesis is described. Mechanistic aspects of ajoene's cytotoxicity towards cancer cells in terms of S-thioallylation via disulfide exchange are then discussed at length. Of crucial importance for the chemical biology studies to follow was the conclusion that S-thioallylation by ajoene is regioselective at the vinyl sulfur, as well as effectively irreversible with suitably reactive cysteine thiol groups. Chapter Three presents an in-depth Chapter on the metabolism and pharmacokinetic properties of ajoene and selected derivatives in a murine xenograft model for human oesophageal cancer (WHCO1). This concluded, disappointingly, that no significant differences in terms of tumour volume, mass and growth rate were observed compared to an untreated control. A follow-up study using a small library of eight ajoene derivatives varying ajoene's different functional groups in a blood stability study, revealed a proportional relationship between the in vitro half-life in blood and the IC50 value for WHCO1 cancer cells. This led to mass spectrometry studies showing that ajoene Sthioallylates the βCys-93 residues in haemoglobin, a chemical modification that most likely explains both ajoene's blood instability and its lack of antitumour activity in vivo. Chapter Four describes the organic synthesis and characterisation of four biotin-ajoene chemical biology probes for chemical biology investigation of S-thiolation in which the background theory of activity-based protein profiling and biotin affinity purification is presented. In the synthesis of these probes, chemical stability emerged as a major stumbling block. Gratifyingly, after several trials, the fourth probe designed and constructed, using a simplified tether involving a convergent “Click”-strategy, turned out to be chemically stable. Subsequent biological validation studies confirmed that the probe retained cytotoxicity against cancer cell lines in vitro (human epithelial mammary gland adenocarcinoma cancer cells (MDA-MB-231) and WHCO1) and shared the same compound–target interaction as its parent ajoene; namely, the regioselective S-thiolation of cellular proteins. Chapter Five constitutes a proteomics study into ajoene's primary protein targets in the MDA-MB-231 cell line using our biotin-ajoene probe. Streptavidin-coated magnetic beads in conjunction with an affinity purification mass spectrometry protocol allowed the isolation and identification of 633 protein targets for ajoene in the MDA-MB-231 proteome. Pathway analyses revealed that ajoene interacts with several targets involved in the control of cell cycles (G2/M cell cycle checkpoint), energy metabolism (glycolysis and pentose phosphate pathway) and the regulation of protein metabolism (translation, folding, quality control and degradation), which supports previously reported cytotoxic modes of action for ajoene against MDA-MB-231 cancer cells. Importantly, we have validated that ajoene S-thioallylates glutathione S-transferase (GSTP1), which is a known cancer-therapy target in breast cancer. Overall, this study complements findings on the cancer-cell protein targets of allicin from crushed garlic, by identifying proteins regulating apoptotic and antiproliferative signalling pathways. These findings support the hypothesis that the anticancer activity of ajoene is due to its S-thioallylation of proteins essential to cellular functions. The thesis concludes with comprehensive experimental and reference sections.
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    The immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammation
    (2015) Hitchcock, Jessica Kaari; Kaschula, Catherine; Schäfer, Georgia; Katz, Arieh A.
    Cancer is a leading cause of death in the modern world. Chronic inflammation facilitates tumourigenesis and cancer progression by providing an environment conducive to cancer. Dysregulation of the immune response, and particularly inflammation, is an important part of this process. Garlic (Allium sativum) has been used for centuries as both a prophylactic and therapeutic medicinal agent, more recent epidemiological and experimental evidence shows that garlic has both cancer-preventative and immune system-enhancing effects. While garlic contains many bioactive compounds, garlic organosulfur compounds (OSCs) have been most widely studied for their anti-cancer properties. In this study, we hypothesize that garlic OSCs modulate the inflammatory immune response by downregulating pro-inflammatory while stimulating anti-inflammatory responses, preventing the formation of a cancer-friendly chronic inflammatory environment. To test this hypothesis we established and optimised an in vitro inflammatory model using lipopolysaccharide-stimulated RAW264.7 murine macrophages. Expression analysis of selected inflammatory genes was performed by qPCR on RNA harvested 4 h and 8 h post treatment, while protein expression was analysed by ELISA using cell culture supernatant samples harvested 8 h and 24 h post treatment. These experiments were complemented by gene and protein arrays. Results showed that allicin had a more pronounced upregulatory effect on LPS-induced gene expression 4 h post-LPS treatment. In contrast, Z-ajoene generally had mild downregulatory effects on the expression of LPS-induced genes. Conversely, Z-ajoene had pronounced downregulatory effects on LPS-induced inflammatory proteins after 24 h, while allicin showed mild up- or downregulatory effects. Overall, we found that allicin induced an initial pro-inflammatory gene response, while Z-ajoene induced a longer-lasting anti-inflammatory response at a protein level. Finally, as many of the inflammatory genes investigated are regulated by the transcription factor STAT3, we investigated the effects of allicin and Z-ajoene on STAT3 phosphorylation and hence activation. Western blot analyses showed that allicin increased LPS-induced STAT3 phosphorylation (2-8 h), while Z-ajoene was found to decrease the phosphorylation of STAT3 after 4 h. These effects on STAT3 phosphorylation are in agreement with the early pro-inflammatory effect of allicin and the later anti-inflammatory effect of Z-ajoene on LPS-induced gene and protein expression. Further, using Western blotting we showed that E/Z-ajoene directly interacts with and reversibly alkylates STAT3 via a thiol-disulfide reaction with a cysteine thiolate on STAT3.
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    Synthesis and mechanistic studies into the cytotoxic activity of garlic-related trisulfides in cancer cells
    (2025) Ali, Doaa; Hunter, Roger; Kaschula, Catherine
    Garlic has long been recognized for its medicinal properties and is well-documented to produce organosulfur compounds (OSCs) with significant chemopreventive and therapeutic potential. Among these, trisulfides are notable phytochemicals that exhibit a broad range of biological activities, including anticancer effects both in vitro and in vivo, as well as antimicrobial, antioxidant, and antithrombotic properties. Recent methodologies have enabled the development of novel synthetic methodologies for synthesizing unsymmetrical organotrisulfides. This thesis explores our contributions to the synthesis of these compounds, their cytotoxicity and their mechanisms of action against cancer cells. Building on our group's previous research on the cytotoxicity of ajoene, which demonstrated its ability to modify proteins by S-thioallylation of cysteine residues through a thiol-disulfide exchange reaction, we extend these findings to trisulfides, providing new insights into their potential as anticancer agents. Chapter One offers an overview of naturally occurring trisulfides, highlighting their significant biological activities. It emphasizes trisulfides from natural sources, particularly diallyl trisulfide (DATS), a garlic-derived compound with notable anticancer potential. This chapter sets the stage for exploring trisulfides therapeutic potential in cancer treatment, a central theme of this dissertation. Chapter Two reviews existing synthetic methods for trisulfide formation, highlighting the need for greener and more sustainable approaches. It discusses current challenges, including the difficulty of synthesizing unsymmetrical trisulfides, and introduces our novel method for achieving these compounds with high yield, purity, and selectivity. This chapter lays the groundwork for the synthetic developments detailed in chapter 4. Chapter Three shifts the focus to the cancer biology of organosulfur compounds (OSCs), with a detailed examination of trisulfides. It provides an in-depth analysis of their extraction, biosynthesis, metabolism, and bioavailability, particularly in relation to cancer chemoprevention. The chapter investigates the role of reactive species, including hydrogen sulfide (H₂S) and perthiols (RSSH), generated through thiolysis exchange of trisulfides with protein thiols. The role of H₂S as a crucial gaseous signaling molecule is examined, highlighting its involvement in various physiological processes. The chapter also explores perthiols, Page | IV characterized by their unique chemical properties, which enable them to participate in redox reactions and radical processes. Chapter Four presents our novel methodology for synthesizing unsymmetrical trisulfides, including details of the optimization conditions and the stability of the products. The methodology yields aliphatic trisulfides in high yield and purity, while aromatic trisulfides are more labile. The trisulfide's distinct chemical shifts in 1H NMR allow for their identification and differentiation from other sulfur species. The applicability of this method extends to a range of functional groups, including those derived from cysteine and sugar moieties. Chapter Five investigates the cytotoxic effects of trisulfides in cancer cells, focusing on a refined library of trisulfides to identify structure-activity relationships, particularly in benzyl derivatives. The chapter examines the impact of various substituents on perthiol stability, demonstrating that electron-donating groups prolong perthiol lifetime and enhance cytotoxicity. This by raising their pKa and reducing their disproportionation to homotrisulfides. The p-OMe derivative, in particular, displayed higher extracellular perthiol concentrations, as measured by Ellman's assay, corroborating initial stability studies. The chapter further extends to monitoring H₂S production in WHCO1 cancer cells using a fluorescent probe, establishing that fluorescence intensity, indicative of H₂S concentration, increases with greater electron release from para-substituents. This relationship between enhanced perthiol stability and increased H₂S production reinforces the thiolysis-based mechanism of trisulfide cytotoxicity and suggests a pathway for designing more effective anticancer agents. Additionally, studies using a dansyl-propyl trisulfide probe indicated localisation in the endoplasmic reticulum (ER), suggesting a role for the unfolded protein response (UPR) in conjunction with H₂S signaling in promoting apoptosis.