Synthesis and Investigation into the estrogen receptor antagonist activity of isoflavans and their synthetic derivatives

Background: Breast cancer (BC) is the most invasive and prevalent cancer in women in South Africa, with numbers as high as 1 in 8 women in urban areas and with a large percentage being ER-positive (ER+). ER+ BC cells are reliant on the binding of the natural ligand 17β-estradiol (E2) to ERα and ERβ isoforms which drives tumor growth1. ER antagonists and inhibitors of estrogen synthesis are therefore widely used therapeutic agents in the treatment of BC. Isoflavans are natural products found in many dietary plants. They are phytoestrogens, able to act as natural anti-breast cancer agents, acting as ER antagonists 3. Method: The aim of the study is to investigate the ER antagonist activity of natural isoflavans and their synthetic derivatives. We are synthesizing a small library of non-natural isoflavans which have different substituents at the 4’-position of the isoflavan ring. The synthesis makes use of a [4+2] cycloaddition reaction between an o-quinone methide and the aryl-substituted enol ether based on a method by Gharpure et al2. The synthesized compounds will be tested using a luciferase reporter assay to establish if they have antagonist activity in CV1 cells expressing the ER. Results: Results pending. Conclusion: In this project, we aim to develop new chemistry for isoflavans and to establish isoflavan structure-activity relationships. The results of the study may aid in the future design of more potent ER receptor antagonists for breast cancer therapy

The Cytotoxicity of the Ajoene Analogue BisPMB in WHCO1 Oesophageal Cancer Cells Is Mediated by CHOP/GADD153

Garlic is a food and medicinal plant that has been used in folk medicine since ancient times for its beneficial health effects, which include protection against cancer. Crushed garlic cloves contain an array of small sulfur-rich compounds such as ajoene. Ajoene is able to interfere with biological processes and is cytotoxic to cancer cells in the low micromolar range. BisPMB is a synthetic ajoene analogue that has been shown in our laboratory to have superior cytotoxicity to ajoene. In the current study we have performed a DNA microarray analysis of bisPMB-treated WHCO1 oesophageal cancer cells to identify pathways and processes that are affected by bisPMB. The most significantly enriched biological pathways as assessed by gene ontology, KEGG and ingenuity pathway analysis were those involving protein processing in the endoplasmic reticulum (ER) and the unfolded protein response. In support of these pathways, bisPMB was found to inhibit global protein synthesis and lead to increased levels of ubiquitinated proteins. BisPMB also induced alternate splicing of the transcription factor XBP-1; increased the expression of the ER stress sensor GRP78 and induced expression of the ER stress marker CHOP/GADD153. CHOP expression was found to be central to the cytotoxicity of bisPMB as its silencing with siRNA rendered the cells resistant to bisPMB. The MAPK proteins, JNK and ERK1/2 were activated following bisPMB treatment. However JNK activation was not critical in the cytotoxicity of bisPMB, and ERK1/2 activation was found to play a pro-survival role. Overall the ajoene analogue bisPMB appears to induce cytotoxicity in WHCO1 cells by activating the unfolded protein response through CHOP/GADD153.Peer reviewe

The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

Background Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S-thiolating reactive cysteines in target proteins. Methods Using our fluorescently labelled ajoene analogue called dansyl-ajoene, ajoene’s protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to ajoene was performed using Schrödinger and pKa calculations by Epik software. Cytotoxicity of ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. Results The dominant protein tagged by dansyl-ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that ajoene and dansyl-ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by ajoene. Conclusions The garlic-derived phytochemical ajoene targets and covalently modifies vimentin in cancer cells by S-thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of ajoene in cancer cells