Cell culture
In this study, A2780 (chemosensitive) and A2780-AD (chemoresistance) ovarian cancer cell lines were used. These cell lines were generously provided by Dr. Shelly B. Hooks, University of Georgia. These cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum, 5 mM L-glutamine and 5 mM pen/strep, in humidified 5% CO2 incubator at 37ºC. Chemoresistant cell line A2780-AD were continuously grown with 3 µM cisplatin [Ali et al., 2013]. Cisplatin was purchased from Kocak Pharma (Istanbul, Turkey). A2780 cell line has the following characteristics: A2780 (age unspecified, endometrioid histotype, sequence variations: ATM p.Pro604Ser (c.1810C > T), PTEN p.Lys128_Arg130del (c.383_391del9)) [Takenaka et al., 2015.; Beaufort et al., 2014]. A2780 human ovarian cancer cell line was established from an ovarian endometroid adenocarcinoma tumour in an untreated patient. A2780 is the parent line to the doxorubicin (adriablastin; adriamycin)-resistant cell line A2780-AD [Tsuruo et al., 1986; Huxham et al., 1994].
Estrogen, nigericin and disulfiram treatment
Cells were treated with different final concentrations of estrogen (17α-Estradiol, Cayman, Cat # 20776). Estrogen in crystalline solid form was dissolved in DMSO to prepare stock solutions. For RNA isolation, 300.000 cells per well were seeded in 6-well plates. After 24 hours, estrogen was added to the media in wells. For control wells, an equal volume of DMSO was added. Cells were incubated with estrogen / DMSO for 48 hours before RNA isolation.
Nigericin (Cayman, Cat # 11437) was used in the final concentration of 10 µM. Nigericin (sodium salt) in crystalline solid form was dissolved in DMSO to prepare stock solutions. For ELISA and microscopy experiments, cells were incubated with nigericin for 1 or 2 hours. For MTT experiments, cells were incubated with nigericin for 24 hours.
For MTT experiments with disulfiram (N,N,N',N'-tetraethyl-thioperoxydicarbonic diamide; Cayman, Cat # 15303), we first added 50 µM disulfiram (dissolved in DMSO) to the cells seeded 24 h earlier (20.000 cells/well in 96-well plate), and after 1 h incubation, we added 10 µM nigericin. We performed MTT assay 24 hours later. Disulfiram inhibits pyroptosis by blocking gasdermin D pore formation [Hu et al., 2020].
RNA isolation
500.000 cells per well in 1 ml media were seeded in 6-well plates. The following day, certain treatments were performed (for instance, estrogen treatment, GSDMC overexpression, etc.; more details are given elsewhere). 48 hours later, 1 ml ice-cold TRIzol was added to each well, mixed well by pipetting and incubated for 5 minutes at 15-30C (or at room temperature (RT)) to permit complete dissociation of the nucleoproteins complex. Then, it was transferred to a clean (autoclaved and RNase-free) 1.5 ml microcentrifuge tube, and 200 µl ice-cold chloroform was added, shaked for around 15 seconds by turning upside down and incubated at RT for 2–3 minutes. Samples were then centrifuged at 12000 rpm for 10 minutes at 4 C. Following the centrifugation step, upper colorless aqueous phase containing RNA was carefully transferred to a new clean 1.5 ml microcentrifuge tube, and 500 µl isopropanol was added to this tube and mixed by turning the tube a few times. Later, the sample was incubated at 15–30 C (or at RT) for 10 min and then centrifuged at 12000 rpm for 10 min at 4 C. Supernatant was discarded, and 1 ml ice-cold 75% etanol (prepared previously with RNase-free water) was added to RNA pellet to wash. The sample was just slowly mixed by turning the tube a few times. Centrifugation was performed at 12000 rpm at 4 C for 2 mins, and then ethanol was discarded. This washing step was repeated. At the end of the protocol, RNA pellet was air-dried in a cell culture hood and dissolved in ice-cold 50 µl RNase-free water and then kept at -20 C for shorter storage or at -80 C for longer periods. RNA concentration was measured at 260 nm using a spectrophotometer (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer). 1 µl RNA sample was loaded to each aperture on µDrop plate. Obtained absorbance value was multiplied by 40 after substracting the absorbance value of RNase-free water to convert the absorbance value to ng/µl unit. Measurements were performed at least in duplicates. The quality of isolated RNA samples was determined with the ratio of A260/A280, and only high-quality RNA was used in the following experiments.
RT-PCR
For RT-PCR, manufacturers’ protocols were generally followed. For each reaction, 10 ng RNA was used. In each 20 µl PCR reaction; 10 µl master mix, 0.4 µl enzyme mix, 0.6 µl forward and reverse primers were used. Total volume was completed to 20 µl with nuclease-free water. 20 µl reaction mixture was transferred to a LightCycler™ capillary (Roche Diagnostics), and centrifuged at 4 C at 1000 rpm for 10 secs. Then, capilleries were placed in a LightCycler™ 1.5 RT-PCR device (Roche Diagnostics). Reaction conditions were programmed as follows: 50 C for 25 min, 95 C for 15 min, (95 C for 15 sec, 55 C for 30 sec, 72 C for 35 sec) x 40 cycles, and 37 C for 30 sec. RT-PCR experiments were performed in duplicates in 3 independent experiments (i.e. 6 data points for each condition). The relative gene expression was measured and normalized to GAPDH by the 2−ΔΔCt method (2−(Cp – GAPDH_Cp)).
Primers used were:
GSDMC – forward : 5′-CCCATCACCAAACCTGGAAGAC-3′
GSDMC – reverse : 5′-TCAACAGCCTCTGTCACCACGT-3′
GSDMD – forward : 5′-ATGAGGTGCCTCCACAACTTCC-3′
GSDMD – reverse : 5′-CCAGTTCCTTGGAGATGGTCTC-3′
GAPDH – forward : 5′-GTCTCCTCTGACTTCAACAGCG-3′
GAPDH – reverse : 5′- ACCACCCTGTTGCTGTAGCCAA-3′
Overexpression experiments
In overexpression experiments, following non-viral expression vectors from Applied Biological Materials (ABM) Inc. (Richmond, BC, Canada) were used: GSDMC protein vector (Human) (pPM-C-HA) (Cat # 227250210500) (plasmid size: 4765 bp; insert size: 459 bp) and GSDMD protein vector (Human) (pPM-C-HA) (Cat # 227300210500) (plasmid size: 4765 bp; insert size: 1455 bp). For both plasmids, the list of markers are as following: bacterial: kanamycin and mammalian: neomycin. Both have CMV promoter and restriction sites for NheI and XhoI enzymes.
In overexpression experiments, TurboFect transfection reagent (Cat # R0531, Thermo Scientific) was used. For the subsequent RNA/protein isolation experiments, cells were seeded in 24-well plates; for MTT experiments, cells were seeded in 96-well plates.
In 24-well plates, 100.000 cells (A2780 and A2780-AD) were seeded in 1ml serum-free RPMI, 24 hours before transfection. Around 500 ng plasmid DNA was diluted in 100 µl serum-free RPMI. The transfection reagent was briefly vortexed, and 2 µl was added to the diluted DNA and then mixed immediately by vortexing. DNA:transfection reagent mixture was incubated for 20 minutes at RT. Then, 100 µl transfection reagent/DNA mixture was added drop-wise to each well, and plates were gently rocked to achieve even distribution of the complexes immediately after adding the transfection reagent. Afterwards, plates were incubated at 37°C in a humidified CO2 incubator. Transgene expression was analyzed after 48 hours by qRT-PCR.
In overexpression experiments for 96-well format (for MTT Assay), 15.000 per well cells were seeded; and the next day, 100 ng plasmid DNA was diluted in 20 µl serum-free RPMI, and 0.4 µl transfection reagent was added. Other steps were performed as performed for 24-well plates.
Silencing experiments
For silencing experiments, following siRNAs were used: Silencer pre-designed siRNAs (Invitrogen, Life Technologies Corp., CA, USA) (negative control: Cat # AS02K9RH; GSDMC: Cat #AS02KN42; GSDMD: Cat # AS0KN43). As transfection reagent, we used Lipofectamine 3000 (Invitrogen; Cat # L3000001) and followed manufacturer’s protocol. Briefly, we plated cells so they are 70–90% confluent at the time of transfection. Next day, we prepared siRNA-lipid complexes and added siRNA-lipid complexes to the cells. To prepare siRNA-lipid complexes, we first diluted Lipofectamine 3000 reagent in serum-free RPMI-1640 media. Similarly, we diluted siRNAs in serum-free RPMI-1640 media. Then, we added diluted siRNA to diluted transfection reagent at 1:1 ratio and incubated for 10–15 minutes at RT. After adding siRNA-lipid complexes to the cells, we incubated cells for 2 days at 37°C. For 96 well format, we used 3 pmol siRNA and 0.3 µl Lipofectamine 3000 reagent. For 6-well format, we used 75 pmol siRNA and 7.5 µl Lipofectamine 3000 reagent.
Specific caspase inhibition
Following specific caspase inhibitors were used in the experiments: caspase-1 inhibitor (Ac-YVAD-CHO; N-acetyl-L-tyrosyl-L-valyl-N-[(1S)-2-carboxyl-1-formylethyl]-L-alaninamide; Cayman; Cat # 10016), caspase-4 inhibitor (Santa Cruz Biotechnology; Cat # sc-396109), caspase-6 inhibitor (Ac-VEID-FMK; Santa Cruz Biotechnology; Cat # sc-3080) and caspase-8 inhibitor (Ac-IETD-CHO; sc-3082; Santa Cruz Biotechnology).
These specific caspase inhibitors were dissolved in DMSO. Specific caspase inhibitors were used at a final concentration of 50 µM. For ELISA experiments, after 2 hours of incubation with caspase inhibitors, nigericin was applied and cells were further incubated for 1 hour. For MTT Assay, cells were incubated with specific caspase inhibitors and nigericin for 24 hours.
Enzyme-Linked ImmunoSorbent Assay (ELISA) for IL-18 and HMGB1
For ELISA experiments, 20.000 cells per well were seeded in 96-well plates. Next day, specific caspase inhibitors were added to wells at a final concentration of 50 µM. After 2 hours of incubation with caspase inhibitors, nigericin was applied and cells were further incubated for 1 hour. Then, cell supernatants were collected to be used in ELISA experiments to measure the levels of IL-18 and HMGB1 released from cells. As control for caspase inhibitors and nigericin, an equal volume of DMSO was added to the wells.
In ELISA experiments, cell culture supernatants were centrifuged at 3570 rpm (1000 g, with 7 cm radius of the rotor of the centrifuge) at 4°C to remove insoluble impurity and cell debris. The clear supernatants collected were then diluted with dilution buffer at 1:2 to be used in the ELISA experiments.
In these experiments, following ELISA kits from FineTest (Wuhan, China) were used: Human IL-18(Interleukin 18) ELISA Kit (Cat # EH0011) and Human HMGB1(High mobility group protein B1) ELISA Kit (Cat # EH0884). Manufacturer’s protocol was followed in both experiments. Briefly, plates were washed 2 times with wash buffer before the addition of samples. Then, 100 µl of properly diluted samples (supernatants from 96-well plates; diluted previously at 1:2 with sample dilution buffer provided with the kit) was added into wells of pre-coated plates, and plates were sealed with the cover and incubated at 37°C for 90 min. Later, the cover was removed and plate content was discarded. Plates were washed with wash buffer 2 times, and 100 µl biotin-labeled antibody working solution was added at the bottom of each well. After covering the plate with the seal, plates were incubated for 60 min at 37°C. At the end of incubation period, cover was removed and plates were washed with wash buffer 3 times, by letting the wash buffer stay in the wells for 2 min each time. Then, 100 µl SABC (HRP-Streptavidin conjugate) working solution was added into each well, plates were covered and incubated for 30 min at 37°C. Afterwards, the cover was removed and plates were washed 5 times with wash buffer, again letting the wash buffer stay in the wells for 2 min each time. Next, 90 µl TMB subtrate was added into each well, plates were covered and incubated at dark at 37°C for 20 min. Finally, 50 µl stop solution was added into each well and the absorbance was read at 450 nm in a microplate reader.
Cell Viability Analysis
For cell viability analysis, MTT Assay was performed. We first seeded cells at a density of 15.000–20.000 cells/well in 96-well plates. 24 hours later, we discarded the growth media from wells, and added 50 µl of serum-free RPMI-1640 (without phenol red) and 50 µl of MTT solution (Thiazolyl blue tetrazolium bromide (Bio Basic, Cat # 298-93-1), prepared as 5 mg/mL solution in PBS) into each well. Then, we incubated the plate at 37°C for 3 hours. After incubation, we added 150 µl DMSO as MTT solvent into each well and wrapped the plate in foil and shaked on an orbital shaker for around 15 minutes. Afterwards, we did pipetting of the liquid to fully dissolve the MTT formazan formed and finally read the absorbance at OD = 590 nm in a microplate reader (Multiskan GO Microplate Spectrophotometer, Thermo Scientific).