br In light of this information we were
In light of this information, we were interested in clarifying if hy-droxycyperaquinone could be a new proteasome inhibitor. To this end, we have assessed the ability of hydroxycyperaquinone to inhibit the chymotrypsin-like activity of 20S subunit of the 26S proteasome. As shown in Fig. 8, hydroxycyperaquinone is a potent proteasome in-hibitor, causing a decrease in catalytic activity of 50% in concentrations as low as 0.78 μM. The proteasome inhibitor lactacystine was used as positive control, 20 µM being necessary to attain similar inhibition values.
Toxicity caused by benzoquinones involves caspase-4 activity
When ER stress is prolonged, apoptosis is specifically activated by overexpression of CHOP and the activation of the caspase-4. Caspase-4 is an ER-resident and a caspase-1 subfamily member, which is cleaved upon ER stress and is capable of triggering cell death (Jung and Choi, 2016; Pereira et al., 2015a). Bortezomib, a drug well-known as pro-teasome inhibitor, mediated cell death through mitochondrial apoptotic signaling via Bax and Bak (ER-resident). Some results showed that Bax and Bak and ER stress can trigger caspase-4 activation. (Chauhan et al., 2005). Once we had overexpression of CHOP and 20S proteasome in-hibition, to assess if cyperaquinone and hydroxycyperaquinone could cause the activation of caspase-4, the activity of this caspase following incubation with the benzoquinones under study was evaluated. As shown in Fig. 9, both molecules elicited an increase in the activity of caspase-4, palmitic DEA NONOate being used as a positive control.
When integrating all results, we conclude that the benzoquinones presented in this work exert cytotoxicity towards cancer cells by trig-gering ER-stress and subsequent apoptosis mediated by caspase-4 and CHOP upregulation. In the case of hydroxycyperaquinone, the most potent molecule, we have shown that proteasome inhibition may be the initiator factor that triggers ER stress, probably by overload of mis-folded proteins in the ER, as a consequence of deficient protein de-gradation.
This is the first report on the biological properties, specifically an-ticancer, of the benzoquinones cyperaquinone, hydroxycyperaquinone, dihydrocyperaquinone, scabequinone and tetrahydrocyperaquinone.
We have shown that some of these molecules are toxic against cancer cells, the gastric cancer cell line AGS being the most susceptible. Mechanistic studies have shown that a process of regulated cell death was taking place, which was shown to involve ER stress, as re-vealed by increased levels of the CHOP protein, changes in calcium dynamics, caspase-4 activation and upregulation of intracellular re-active oxygen species. Hydroxycyperaquinone is a novel sub-micromolar inhibitor of the 20S catalytic core of the 20S proteasome, causing cell death via IRE1α-independent/PERK-dependent pathways. Despite this benzoquinone Phytomedicine 63 (2019) 153017
displayed toxicity against normal cell lines, sensor is an important source to develop a new drug inspired in their chemical structure in order to be more selective to cancer cells.
Conflict of interest
Nothing to declare.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
da Silva, D.C., Andrade, P.B., Valentão, P., Pereira, D.M., 2017. Neurotoxicity of the steroidal alkaloids tomatine and tomatidine is RIP1 kinase- and caspase-independent and involves the eIF2α branch of the endoplasmic reticulum. J. Steroid Biochem. Mol. Biol. 9. r> Kinupp, V.F., 2008. Plantas alimentícias não-convencionais da região metropolitana de porto alegre, RS. RBA 3.
Moreira, M.M., 1990. Benzoquinonas existentes em espécies de Cyperus da flora
Portuguesa - contribuição para o seu conhecimento. Farmacognosia - Faculdade def
Farmácia. Universidade do Porto, pp. 152.
Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells. BMC Complement. Altern. Med. 13, 1472–6882. Peerzada, A.M., Ali, H.H., Naeem, M., Latif, M., Bukhari, A.H., Tanveer, A., 2015. Cyperus rotundus L.: traditional uses, phytochemistry, and pharmacological activities. J. Ethnopharmacol. 174, 540–560.
Pereira, D., Valentão, P., Andrade, P.B., 2018. Tuning protein folding in lysosomal storage diseases: the chemistry behind pharmacological chaperones. Chem. Sci. 9, 1740–1752. Pereira, D.M., Correia-da-Silva, G., Valentão, P., Teixeira, N., Andrade, P.B., 2014a. Anti-Inflammatory effect of unsaturated fatty acids and ergosta-7,22-dien-3-ol from Marthasterias glacialis: prevention of CHOP-mediated ER-stress and NF-κB activation. PLoS One 9, e88341.