br As shown in Fig all compounds tested significantly increa
As shown in Fig. 4, all compounds tested significantly increased calcium levels.
Taking into account that the ER acts as the major calcium store in EPZ031686 (Pereira et al., 2018, 2015a), we hypothesized that this organelle could be involved in the toxicity elicited by benzoquinones, reason for which we have directed subsequent efforts to elucidate the role of the ER in this process using the two most active molecules, namely cy-peraquinone and hydroxycyperaquinone.
Benzoquinones induce the upregulation of the ER stress-involved gene CHOP
Considering the previous results regarding alteration of calcium dynamics, we evaluated the effect of cyperaquinone and hydro-xycyperaquinone in genes related to ER stress, namely ATF4, EDEM1,
BiP, GRP94 and CHOP. As show in Fig. 5, only CHOP expression was altered by exposing cells to the benzoquinones under study. In light of these results, the levels of the CHOP protein in cyperaquinone- and hydroxycyperaquinone-treated cells were studied. As shown in Fig. 6A, both benzoquinones increased the expression of CHOP.
In light of the previously described link between CHOP upregulation and the onset of oxidative stress (Jung and Choi, 2016) we further evaluated the impact of the molecules upon intracellular ROS levels. Staurosporine was used as positive control for ROS upregulation. As shown in Fig. 6B, increased levels of ROS were found following in-cubation of cells with benzoquinones, although it did not reach statis-tical significance in the case of cyperaquinone. Demir and co-workers attribute the increase in CHOP levels and ER involvement to ROS (Demir et al., 2016). Pharmacologic data from Su and collaborators suggest that there are at least two upstream cascades for CHOP gene
Fig. 8. Effect of hydroxycyperaquinone (HydroxyCyp 3.13μM) on the chemo-trypsin-like activity of the 20S subunit of the 26S proteasome. The data re-present the mean ± standard deviation of the mean of each concentration studied and in triplicate. ****p < 0.0001 compared to the control. Lactacystine (Lact 20μM) was used as a positive control.
induction. One is ROS- and calcium-dependent pathway, while the other is ROS-dependent but calcium-independent pathway (Su et al., 2008).
Benzoquinone toxicity is IRE1α-independent and PERK-dependent
In order to elucidate the involvement of IRE1α in the toxicity dis-played by the benzoquinones, these molecules were incubated in the presence of 4μ8C, a specific IRE1α inhibitor that forms a stable Schiff base with lysine 907 in the IRE1 endonuclease domain, thus inhibiting this activity (Cross et al., 2012). As shown in Fig. 7, inhibition of IRE1α Phytomedicine 63 (2019) 153017
Fig. 9. Effect of benzoquinones for 8 h on the activation of caspases-4 in the AGS cell line. Data represent the mean ± standard error of the mean of five independent experiments, performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, compared to the respective control. Cyp: Cyperaquinone
did not result in any cytoprotective effect, which is in line with the results from gene expression, which revealed no changes in the EDEM1 gene.
During ER stress, PERK phosphorylates eIF2α, which can be later dephosphorylated by GADD34-protein phosphatase 1 (PP1) complex (Hotamisligil, 2010). For this reason, studies aiming to elucidate the contribution of PERK-mediated toxicity frequently use the PP1 inhibitor salubrinal, as it protects from the deleterious effects of ER stress (Komoike et al., 2012; Wang et al., 2018).
The toxicity of cyperaquinone was partly attenuated when co-in-cubated with salubrinal (Fig. 7), which suggests that PERK mediates or
is involved in the toxicity elicited by this benzoquinone, a result that is in line with the known effect that the PERK branch of the UPR has in CHOP levels (Scheuner et al., 2001). Rather unexpectedly, co-incuba-tion of hydroxycyperaquinone with the PP1 inhibitor resulted in en-hanced toxicity of the benzoquinone. This is a relatively rare event that was described for the first time by Drexler, 2009. The author concluded that this process is found in some cell types when proteasome inhibition is simultaneously taking place.
The proteasome is the largest non-lysosomal degradation pathway of proteins in eukaryotic cells (Correia da Silva et al., 2017). This proteolytic machinery is involved in degradation of oxidized proteins, unfolded/misfolded proteins, and in antigen presentation for regulating the levels of proteins and transcription factors (Lu et al., 2011; Noolu et al., 2013). This complex set of subunits regulates cellular processes, such as apoptosis, signal transduction, cell cycle regulation, differ-entiation, cell mitosis and inflammation (Semren et al., 2015).