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  • br Determination of cytosolic Ca levels br

    2020-08-18


    Determination of cytosolic Ca2+ levels
    Cells were plated in black bottom 96-well plates at 1.5 × 104 cells/ well and incubated for 1 h with the fluorescent probe Fura-2/AM (5 Μm), after which the molecules under study were added and further incubated for another hour. The fluorescence was read at 340 nm/ 505 nm and at 380 nm/505 nm in a Cytation™ 3 (BioTek) multi-functional microplate reader. The analysis was performed considering the ratio F340/505/F380/505, as described by da Silva et al. (2017).
    RNA isolation and RT-PCR
    Total RNA was isolated from AGS 2 , 3Diaminonaphthalene using the PureZol™ RNA Isolation Reagent by BioRad. For quantification and evaluation of RNA integrity were used the Qubit® RNA HS and IQ assay kits, according to the manufacturer's instructions. Reverse transcription was obtained from 1 µg of mRNA using the SuperScript™ IV VILO™ Master Mix  Phytomedicine 63 (2019) 153017
    (Invitrogen), as per the manufacturer's guidelines, and cDNA's were amplified in a qPCRsoft 4.0 supplied with the equipment qTOWER3 G (Analytik Jena AG, Germany). At the end of the PCR cycling, melting curves were produced. Results were normalized to GAPDH as en-dogenous control. Relative expression levels were calculated as follows: 2(Ct GAPDH gene−Ct gene of interest) × 1000. The primers used can be consulted in Supplementary material (Table S1).
    Protein extraction and western blot analysis
    Western blot analysis was performed using a previously described method (Pereira et al., 2014a) with some modifications. Cells were plated in 6-well plates at 36 × 104 cells/well. Cells were incubated with hydroxycyperaquinone and cyperaquinone for 18 h at 37 °C at IC50 concentration. After incubation, cells were trypsinized and subjected to centrifugation at 1300 rpm for 3 min. After centrifugation, RIPA lysis buffer containing 1% protease and phosphatase inhibitors cocktail was added for 30 min in ice. After that, cells were centrifuged at 14.500 rpm for 15 min, at 4 °C. Protein content in supernatants was measured by the Bradford assay (Bradford, 1976). Proteins (40 µg) were separated on 12% sodium dodecyl sulphate (SDS) – polyacrylamide gel and trans-ferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% skimmed milk in PBS 0.1% Triton X-100. Subsequently, the membranes were incubated with an antibody against CHOP (1:250) or β-tubulin (1:200), overnight, at 4 °C. Membranes were incubated with secondary antibody (1:1750) after washing at room temperature for 1 h, followed by the addition of ECL reagent on ChemiDoc™ Imaging Systemsc (Bio-Rad, Hercules, CA, USA). The relative optical density of bands was quantified by densitometry and normalized with respect to β-tubulin.
    Assessment of the IRE1α and PERK/eIF2α branches of the UPR
    AGS cells were plated at 1.5 × 104 cells/well and incubated with the benzoquinones under study for 8 h in the presence or absence of 4μ8C (5 µm) or salubrinal (40 µm), IRE1α and PERK reference in-hibitors respectively. The effects produced by these inhibitors were measured by evaluating changes in cell viability by the MTT assay, as previously described (Pereira et al., 2014b).
    Proteasome activity assay
    For the 20S proteasome activity assay, different concentrations of benzoquinones were added to black-bottom 96-well plates with 70 ng of purified human 20S proteasome isolated from erythrocytes (Enzo Life Sciences) and the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC. 20 mM Tris–HCl assay buffer was added to the reaction mixture. Lactacystin was used as positive control for proteasome inhibition. After incubation for 2 h, 37 °C, in the dark, the inhibition of 20S pro-teasome was measured (340 nm/460 nm) in a Cytation™ 3 (BioTek) microplate reader (Pereira et al., 2014b).
    Statistical analysis
    In preliminary assessment of data, outliers were identified by the Grubbs' test. Shapiro–Wilk’s normality test was performed in the data pool to ensure that all data followed a normal distribution. Comparison between the means of controls and each experimental condition was performed using t-test. Data was expressed as the mean ± standard error of the mean of 5 independent experiments, unless otherwise specified, each performed in triplicate. GraphPad Prism software was used and values were considered statistically significant with a p ≤ 0.05.
    Table 1
    Cytotoxicity (IC50, µM) of benzoquinones from Cyperus spp. towards the A549, AGS and MRC-5 cell lines, as evaluated by the MTT assay.
    Benzoquinones A549 AGS MRC-5
    Results and discussion
    Benzoquinones are toxic against cancer cell lines
    No studies are available regarding the toxicity of the benzoquinones studied herein. For this reason, they were initially screened against two
    human cancer cell lines and one normal cell line, namely non-small cell cancer (A549) and gastric adenocarcinoma (AGS) and human fibro-blastic cell line (MRC-5). Cancer cells were chosen as per the latest reports that show that lung and stomach cancer are among the most prevalent cancer worldwide (Global Burden of Disease Cancer, 2018). As shown in Fig. 1, in a general way all molecules displayed toxicity towards the A549 cell line, hydroxycyperaquinone, cyperaquinone and dihydrocyperaquinone being the most potent molecules, with IC50 of 3.0, 11.3 and 45.3 µM, respectively (Table 1). When tested in the AGS cell line the IC50 were considerably lower, 1.7, 3.0 and 16.8 µM (Table 1). For MRC-5 cell line, dihydrocyperaquinone and tetra-hydrocyperaquinone did not display any toxicity, cyperaquinone and scabequinone were toxic at 12.5 µM and hydroxycyperaquinone was the most toxic molecule (Table 1). With the positive control irinotecan hydrochloride an IC50 of 33.6 µM was obtained (Fig. S1). Taking into account that the AGS cell line was shown to be more susceptible to all