Benzoquinones from Cyperus spp. trigger IRE1α-independent and PERK-dependent ER stress in human stomach cancer cells and are novel proteasome inhibitors

Background: The roots and tubers of several species of the Cyperus genus are used in several parts of the world as foodstuffs and beverages. The genus is rich in several classes of quinones, however their biological properties have not been studied before. Purpose: We evaluated the anticancer effect of several benzoquinones isolated from the genus and described their mechanism of action towards cancer cells. Methods: The most potent molecules were selected according to their effect upon cell viability. The mechanism of cell death was studied by using pharmacological inhibitors of caspases, caspase-3/4/9 activity assays, annexin-V/7-AAD by flow cytometry and intracellular reactive oxygen species and calcium levels through fluorescence spectroscopy. Elucidation of the involvement of distinct branches of the ER stress pathway was pursued by RT-PCR and WB for mRNA and protein expression levels, respectively, as well as pharmacological inhibitors. Proteasome inhibitory activity was assessed by using purified 20S catalytic subunit with the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC. Results: Cytotoxicity studies against cancer cell lines showed that the human gastric cancer cell line AGS was the most susceptible, the most potent molecule, hydroxycyperaquinone, exhibiting an IC50, close to 1 mu M. Morphological and biochemical traits suggested that a process of regulated cell death was taking place, which was shown to be intrinsic pathway-independent. Results indicated that benzoquinones exert their toxicity by triggering ER stress, as shown by increased expression of CHOP (mRNA and protein levels), intracellular reactive oxygen species, changes in calcium dynamics and caspase-4 activation. Proteasome inhibition by these molecules is described for the first time. Conclusion: Hydroxycyperaquinone is a novel sub-micromolar inhibitor of the 20S catalytic core of the 26S proteasome, causing cell death via IRE1 alpha-independent/PERK-dependent pathways in stomach cancer cells. Its presence in products consumed orally may be of relevance for gastric tumors.