Catherine H. Kaschula

The Immunomodulation and Anti-Inflammatory Effects of Garlic Organosulfur Compounds in Cancer Chemoprevention

Garlic (Allium sativum) has been used for centuries as a prophylactic and therapeutic medicinal agent. Importantly, garlic has been suggested to have both cancer-preventive potential as well as significant enhancing effects on the immune system. While these observations are supported experimentally both in vitro and in vivo, the impact of garlic in assisting the immune system in the prevention of cancer still lacks experimental confirmation. Studies addressing the immunomodulatory effects of garlic reveal conflicting data as to pro- or anti-inflammatory responses depending on the particular experimental set-ups and the garlic preparation used (i.e. garlic extract versus chemically pure garlic compounds). Here we provide an overview of the chemistry of the major garlic organosulfur compounds, summarize the current understanding and propose a link between the immunomodulating activity of garlic and the prevention of cancer. We hypothesize that garlic rather elicits anti-inflammatory and anti-oxidative responses that aid in priming the organism towards eradication of an emerging tumor.

Strategies to reverse drug resistance in malaria.

Purpose of review Despite the current success of artemisinin combination therapy, the threat of drug-resistant falciparum malaria remains severe. Reversal of resistance to old drugs remains one strategy to deal with this problem. This review highlights recent significant findings. Recent findings This review provides a brief description of current antimalarials, their known or putative targets and mechanisms of resistance (where applicable). The main focus is recent reports on chloroquine resistance-reversing agents, including primaquine, so-called ‘reversed chloroquines’, novel resistance reversers such as xanthenes and two new mefloquine resistance-reversing compounds. A number of patents also report interesting new chloroquine resistance reversers, most notably HIV protease inhibitors. The review is confined to Plasmodium falciparum. Summary Only chlorpheniramine has so far shown some clinical utility as a chloroquine resistance reverser. Recent observations, however, that both primaquine and HIV protease inhibitors are chloroquine resistance reversers may eventually prove to be of clinical significance. ‘Reversed chloroquines’ are a scientifically innovative new class of antimalarial that both kill malaria parasites and have the potential to reverse resistance to their own antimalarial pharmacophore.

Garlic-derived anticancer agents: Structure and biological activity of ajoene

Garlic has been used throughout the centuries to treat infections, heart disease, and cancer. Ajoene is one of the main compounds formed from heating crushed garlic as a mixture of E‐ and Z‐isomers (E‐ and Z‐4,5,9‐trithiadodeca‐1,6,11‐triene 9‐oxide). Ajoene possesses a broad spectrum of biological activities that include anticancer activity. Its cytotoxicity towards cancer cells is postulated to occur via an apoptotic mechanism involving activation of the mitochondrial‐dependent caspase cascade. Structure‐activity studies on ajoene and ajoene analogues have revealed that the Z‐isomer is moderately more active than the E‐isomer at inhibiting in vitro tumor cell growth, suggesting that specific protein interactions may be important. Substitution of the terminal end allyl groups in ajoene for alkyl, aromatic, or heteroaromatic groups produces some analogs with superior in vitro anticancer activity to ajoene, opening up the way to developing ajoene‐based anticancer agents.

Substituted ajoenes as novel anti-cancer agents

A new synthesis of the ajoene pharmacophore core is presented involving the regioselective radical addition of a thiyl radical to a terminal alkyne as the key step. The synthesis allows structural variation of the two end groups on sulfur, and a range of novel derivatives varying the R(1) group (sulfoxide end) has been prepared and tested against CT-1 transformed fibroblast cells for anti-cancer activity. The results indicate comparable or even improved activity compared to the parent natural product ajoene isomers. This opens up the way to systematically studying the biology of the ajoene core.

Anti-Proliferative Activity of Synthetic Ajoene Analogues on Cancer Cell-Lines

The ability of garlic preparations to inhibit cancer cell-growth has been attributed to a group of structurally-related organosulfur compounds found in the crushed clove. Historically, interest has centred on three such compounds as allicin, diallyl disulfide and diallyl trisulfide, with less interest on E- and Z-ajoene. A recently developed synthetic route from our laboratory for preparing ajoene analogues allows access to derivatives containing the sulfoxide / vinyl disulfide core whilst varying the terminal end-group functionality. A small library has been synthesized and an advanced lead with p-methoxybenzyl end groups (8) identified. Data on the in vitro anti-proliferation activity of compound (8) is presented here on six cancer cell-lines in comparison with that of Z- and E-ajoene to reveal an enhancement in activity of up to twelvefold. In addition, a modest selectivity is observed for tumour over normal cell-lines of up to threefold. Data on ajoene and its derivatives is presented in the context of chemosensitization in drug-resistance, and ideas on ajoenes mode of action at the molecular level are presented and discussed.

Forkhead Box Q1 Is a Novel Target of Breast Cancer Stem Cell Inhibition by Diallyl Trisulfide.

Diallyl trisulfide (DATS), a metabolic byproduct of garlic, is known to inhibit the growth of breast cancer cells in vitro and in vivo. This study demonstrates that DATS targets breast cancer stem cells (bCSC). Exposure of MCF-7 and SUM159 human breast cancer cells to pharmacological concentrations of DATS (2.5 and 5 μm) resulted in dose-dependent inhibition of bCSC, as evidenced by a mammosphere assay and flow cytometric analysis of aldehyde dehydrogenase 1 (ALDH1) activity and the CD44high/CD24low/epithelial specific antigen-positive fraction. DATS-mediated inhibition of bCSC was associated with a decrease in the protein level of FoxQ1. Overexpression of FoxQ1 in MCF-7 and SUM159 cells increased ALDH1 activity and the CD49f+/CD24− fraction. Inhibition of ALDH1 activity and/or mammosphere formation upon DATS treatment was significantly attenuated by overexpression of FoxQ1. In agreement with these results, stable knockdown of FoxQ1 using small hairpin RNA augmented bCSC inhibition by DATS. Expression profiling for cancer stem cell-related genes suggested that FoxQ1 may negatively regulate the expression of Dachshund homolog 1 (DACH1), whose expression is lost in invasive breast cancer. Chromatin immunoprecipitation confirmed recruitment of FoxQ1 at the DACH1 promoter. Moreover, inducible expression of DACH1 augmented DATS-mediated inhibition of bCSC. Expression of FoxQ1 protein was significantly higher in triple-negative breast cancer cases compared with normal mammary tissues. Moreover, an inverse association was observed between FoxQ1 and DACH1 gene expression in breast cancer cell lines and tumors. DATS administration inhibited ALDH1 activity in vivo in SUM159 xenografts. These results indicate that FoxQ1 is a novel target of bCSC inhibition by DATS.

Synthesis and anti-prion activity evaluation of aminoquinoline analogues

Transmissible spongiform encephalopathies form a group of neurodegenerative diseases that affect humans and other mammals. They occur when the native prion protein is converted into an infectious isoform, the scrapie PrP, which aggregates, leading to neurodegeneration. Although several compounds were evaluated for their ability to inhibit this conversion, there is no effective therapy for such diseases. Previous studies have shown that antimalarial compounds, such as quinolines, possess anti-scrapie activity. Here, we report the synthesis and evaluate the effect of aminoquinoline derivatives on the aggregation of a prion peptide. Our results show that 4-amino-7-chloroquinoline and N-(7-chloro-4-quinolinyl)-1,2-ethanediamine inhibit the aggregation significantly. Therefore, such aminoquinolines might be considered as candidates for the further development of therapeutics to prevent the development of prion diseases.

The garlic compound ajoene targets protein folding in the endoplasmic reticulum of cancer cells

Ajoene is a natural allylsulfur compound found in crushed garlic that arrests growth and induces apoptosis in cancer cells. To gain mechanistic insights into the cytotoxicity of ajoene in cancer cells, two fluorescently labelled ajoene analogs with dansyl‐ (DP) and fluorescein‐ (FOX) tags were synthesized. The tagged ajoenes were found to retain their activity at inhibiting proliferation and inducing apoptosis in MDA‐MB‐231 human breast‐cancer and WHCO1 human esophageal‐cancer cells. Both tagged ajoenes localized to the endoplasmic reticulum (ER) in MDA‐MB‐231 cells as observed by live cell confocal laser scanning microscopy (CLSM) and confirmed by generating an MDA‐MB‐231 cell line expressing yellow fluorescent protein (YFP) in the ER. DP appears to S‐thiolate multiple protein targets in MDA‐MB‐231 cells as observed by immunoblotting under non‐reducing conditions only; and a competition assay demonstrated that DP and Z‐ajoene in fact share the same target. Ajoene S‐thiolation interfered with protein folding and led to an accumulation of misfolded protein aggregates and activated the unfolded protein response (UPR). Consistent with this mechanism, increased levels of GRP78 and total ubiquitinated proteins were observed; and an ER‐folded protein, type‐1 collagen, was tracked to the proteasome following ajoene treatment. The intracellular protein aggregates were observed by CLSM and transmission electron microscopy (TEM). This is the first time that ajoene has been shown to target protein folding in the ER of cancer cells.

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

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.

New Excursions Into the Synthesis and Medicinal Chemistry of the Disulfide Bond

Abstract The presentation discusses two themes of current interest in my research group in conjunction with a garlic mimics program. The first involves a new one-pot methodology for preparing unsymmetrical disulfides in high yield using 1-chlorobenzotriazole (BtCl) as oxidant, which can be applied to a range of functional types. The second part of the lecture will discuss the synthesis of derivatives of the garlic-derived natural product ajoene, in conjunction with an anti-cancer screening program. The synthesis has been used to prepare a fluorescently tagged derivative of the natural product that has been used to investigate localization and mode of action within the cancer cell using confocal laser scanning microscopy. GRAPHICAL ABSTRACT