RESUMEN
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.
Asunto(s)
Disulfuros/farmacología , Neoplasias Esofágicas/metabolismo , Factor de Transcripción CHOP/metabolismo , Línea Celular Tumoral , Disulfuros/química , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Sulfóxidos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
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. © 2015 Wiley Periodicals, Inc.
Asunto(s)
Disulfuros/farmacología , Proteínas de Choque Térmico/metabolismo , Neoplasias/metabolismo , Extractos Vegetales/farmacología , Pliegue de Proteína/efectos de los fármacos , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Disulfuros/química , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Fluoresceína/química , Humanos , Microscopía Electrónica de Transmisión , Neoplasias/tratamiento farmacológico , Fosfatidilcolinas/química , Sulfóxidos , Ubiquitinación , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
The organosulfur compound ajoene derived from the rearrangement of allicin found in crushed garlic can inhibit the proliferation of tumour cells by inducing G(2)/M cell cycle arrest and apoptosis. We report on the application of a concise four-step synthesis (Hunter et al., 2008 [1]) that allows access to ajoene analogues with the end allyl groups substituted. A library of twelve such derivatives tested for their anti-proliferation activity against WHCO1 oesophageal cancer cells has identified a derivative containing p-methoxybenzyl (PMB)-substituted end groups that is twelve times more active than Z-ajoene, with an IC(50) of 2.1µM (Kaschula et al., 2011 [2]). Structure-activity studies involving modification of the sulfoxide and vinyl disulfide groups of this lead have revealed that the disulfide is the ajoene pharmacophore responsible for inhibiting WHCO1 cell growth, inducing G(2)/M cell cycle arrest and apoptosis by caspase-3 activation, and that the vinyl group serves to enhance the anti-proliferation activity a further eightfold. Reaction of the lead with cysteine in refluxing THF as a model reaction for ajoene's mechanism of action based on a thiol/disulfide exchange reveals that the allylic sulfur of the vinyl disulfide is the site of thiol attack in the exchange.