br et al In Spain they are used
et al., 2015). In Spain they are used in the preparation of the horchata drink, owing to their flavour similar to vanilla and almond. The roots are used in the preparation of teas and the burning of the whole plant in the production of salt (Nassar et al., 2002; Peerzada et al., 2015). The family Cyperaceae has also been used for medicinal purposes in stomach and skin diseases, according to ethnopharmacological strate-gies (Morimoto et al., 2001; Nassar et al., 2002).
Abbreviations: ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; CHOP, CCAAT/EBP homologous protein; DNA, deoxyribonucleic acid; eIF2α, eukaryotic initiation factor 2; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum associated proteins degradation; IRE-1α, inositol-requiring enzyme 1 α; PERK, PKR-like ER kinase; PP1, protein phosphatase 1; PKR, protein kinase R; RCD, regulated cell death; ROS, reactive oxygen species; UPR, unfolded protein response; XBP-1, X-box binding protein 1
E-mail address: [email protected] (D.M. Pereira).
Previous studies have elucidated the phytochemical composition of the genus, highlighting the diversity in quinones (Nassar et al., 2002; Seabra et al., 1997), flavonoids and sesquiterpenes. In the specific case of quinones, the genus has been shown to be a prolific source of che-mical diversity, several molecules having been described; however, their biological potential, specifically anticancer, has not been ad-dressed before.
The endoplasmic reticulum (ER) is an organelle pivotal for the synthesis, folding and trafficking of proteins to their target sites, being also a major player in the control of calcium levels within cells. In re-cent years, the discovery of several sensor signalling pathways and even the discovery of ER-resident caspases has shed a light to the contribute of this organelle in several processes vital to 2 , 3Diaminonaphthalene and, also, in cell death (da Silva et al., 2017).
The signalling cascade of the ER begins with the involvement of one or more of the three known sensors: PKR-like ER kinase (PERK), in-ositol-requiring enzyme 1α (IRE1α) and activating transcription factor
6 (ATF6). Two of these sensor proteins also have catalytic activity: PERK has a kinase activity and phosphorylates eukaryotic translation-initiation factor 2α (eIF2α). IRE1α has both kinase and en-doribonuclease (RNase) activity and removes a 26-nucleotide intron from mRNA encoding the X box binding protein (XBP). We report for the first time the anticancer activity of five benzo-quinones isolated from Cyperus species, namely cyperaquinone, hy-droxycyperaquinone, dihydrocyperaquinone, tetrahydrocyperaquinone and scabequinone. Cytotoxicity was evaluated against the human lung cancer cell line A549 and the human gastric adenocarcinoma cell line AGS. Biochemical, pharmacological and morphological assessment of these benzoquinones show that these molecules trigger ER stress with involvement of caspase-4, changes in calcium levels and upregulation of the protein CHOP. Inhibition of the 20S subunit of the 26S proteasome is suggested as an upstream event leading to the onset of ER stress.
Materials and methods
Chemicals and reagents
Benzoquinones (cyperaquinone, dihydrocyperaquinone, tetra-hydrocyperaquinone, hydroxycyperaquinone and scabequinone) were isolated from Cyperus sp. as previously described by Moreira (1990). Briefly, the vegetal material was extracted with petroleum ether, fol-lowed by chloroform extraction. Purification of the obtained extract was performed by preparative thin layer chromatography. For struc-tural elucidation, the spectrum obtained by ultraviolet-visible, infrared, mass spectrometry and proton nuclear magnetic resonance were ana-lysed (Nassar et al., 2002).
Dimethyl sulfoxide (DMSO), trypan blue, sodium pyruvate, 3-(4,5-di-methylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), ethanol, β-nicotinamide adenine dinucleotide (NADH), Triton X-100, thapsigargin, dichlorodihydrofluorescein diacetate (DCDHF-DA), trizma hydrochloride, magnesium chloride (MgCl2), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), sucrose, 1,4-dithiothreitol (DTT), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2,2′, 2″,2′′′-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), 4 μ8C, Trizma® base, palmitic acid, phalloidin–tetramethylrhodamine B isothiocyanate, irinotecan hydrochloride was acquired from Sigma-Aldrich (St. Louis, MO, USA). The Fura-2 AM fluorescent probe was purchased from Abcam Biochemicals. Methanol was purchased from Merck (Darmstadt, Germany). SuperScript™ IV VILO™ Master Mix, Qubit™ RNA HS Assay Kit and Qubit™ RNA IQ Assay Kit, Dulbecco's Modified Eagle Medium (DMEM), Hank's balanced salt solution (HBSS), foetal bovine serum (FBS), penicillin-streptomycin solution (penicillin 5000 units/ml and strepto-mycin 5000 μg/ml) and 0.05% and 0.25% trypsin-EDTA were obtained from GIBCO, Invitrogen™ (Grand Island, NY, USA). Staurosporine and salubrinal were purchased from Santa Cruz Biotechnology, Inc. Caspase-Glo® 3/7 luminescent kit was obtained from Promega Corporation. The