br Fig Morphological assessment of the effect
Fig. 2. Morphological assessment of the effect of cyperaquinone (Cyp), hydroxycyperaquinone (HydroxyCyp) and dihydrocyperaquinone (DiHCyp) upon AGS EPZ 031686 after 24 h of incubation. Molecules were tested at their IC50 value, as per Table 1. Overall cell morphology was evaluated using phalloidin (cytoplasmic traits) and chromatin status (DAPI). Yellow arrows: karyorrhexis (nuclear fragmentation); white arrows: pyknosis (chromatin condensation). (For interpretation of the refer-ences to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. (A) Flow cytometry of the effect of cyperaquinone (Cyp) and hydroxycyperaquinone (HydroxyCyp) upon AGS cells after 8 h of incubation. Molecules were tested at their IC50 value, as per Table 1. Cell apoptosis was evaluated using Annexin V/7-AAD. (B) Effect of cyperaquinone (Cyp, 6.25 µM), hydroxycyperaquinone (Hydroxy, 6.25 µM), dihydrocyperaquinone (Dihydro, 12.5 µM), tetrahydrocyperaquinone (Tetrahydro, 50μM) and scabequinone (Scab, 25μM) in the activity of caspases-9 and caspase-3 in the AGS cell line following 8 h of incubation. Data represent the mean ± standard error of the mean of five independent experiments, performed in triplicate. ***p < 0.001, compared to the respective control. Staurosporine (Stauro, 500 nM) was used as positive control.
compounds evaluated, it was used in subsequent mechanistic studies with the three most potent molecules. In order to guarantee that ne-crosis was not taking place, the LDH assay was used to assess the impact of the molecules upon membrane integrity, with only the highest con-centration that was LDH-negative being used (Fig. S2).
Benzoquinones trigger morphological changes compatible with regulated cell death
In light of the results from the viability assays, we were interested in evaluating the mechanism of action behind the toxicity observed. For this reason, we have initially assessed the impact of the most potent
molecules upon overall cytoplasmic and chromatin morphology.
As shown in Fig. 2, incubation of cells with cyperaquinone, hydro-xycyperaquinone and dihydrocyperaquinone caused cytoplasmic shrinkage, pyknosis and karyorrhexis which, in tandem with the in-formation that cell retained membrane integrity at these concentrations (Fig. S2, LDH assay), suggests that a process of Regulated Cell Death (RCD) could be taking place, as per the latest guidelines of the No-menclature Committee on Cell Death (Galluzzi et al., 2018). Following these results, we evaluated the proportion of cells in apoptosis with Annexin-V/7-AAD, cyperaquinone and hydroxycyperaquinone causing approximately 80% of cells to be Annexin-V positive, thus indicating that an apoptotic process was taking place (Fig. 3A).
Benzoquinones do not trigger the intrinsic pathway of apoptosis
One of the most widely reported processes of RCD is the intrinsic pathway of apoptosis. In this process, upon an apoptotic stimulus, mi-tochondrial outer membrane permeabilization takes place, resulting in the dissipation of the mitochondrial transmembrane potential and subsequent exit of cytochrome c to the cytosol. In the cytosol, cyto-chrome c will lead to the activation of the apoptosome which, once active induces the activation of activator caspase-9, which subsequently activates executor caspase-3 (Zhivotovsky and Orrenius, 2011). At the highest concentration, none of the molecules studied were able to exert an effect upon caspase-9 activity nor caspase-3 (Fig. 3B). In both cases, staurosporine was used as a positive control for caspase activation (Pereira et al., 2014b).
Benzoquinones interfere with Ca2+ levels
Following the results that showed that caspases-3/9 were not in-volved in the process of RCD elicited by benzoquinones, we shifted our attention to other processes. Disruption of calcium homeostasis is a hallmark of several types of RCD (Galluzzi et al., 2018), reason for which we focused our attention in this parameter in subsequent
Fig. 5. Gene expression analysed by RT-PCR for ATF4, EDEM1, CHOP, BiP and GRP94 in AGS cell line incubated with cyperaquinone (Cyp), hydroxycyperaquinone (Hydroxy) and thapsigargin (Thapsi) was used as a positive control. The data represent the mean ± standard deviation of the mean of three independent ex-periments. mRNA values were normalized to the expression of GAPDH. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to the housekeeping gene.
Fig. 6. Effect of cyperaquinone (Cyp, 6.25 µM), hydroxycyperaquinone (HydroxyCyp, 6.25 µM) and thapsigargin (THG, 2.5 µM) on CHOP ex-pression and intracellular ROS levels. Densitometric analysis of the protein after nor-malization with β-tubulin levels. Data represent the mean ± standard deviation of the mean of each concentration studied and in triplicate. *p < 0.05, ***p < 0.001, ****p < 0.0001. STS: Staurosporine (100 nM).