Paclitaxel induces pyroptosis by inhibiting the volume‑sensitive chloride channel leucine‑rich repeat‑containing 8a in ovarian cancer cells
- Authors:
- Published online on: April 20, 2023 https://doi.org/10.3892/or.2023.8552
- Article Number: 115
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Copyright: © Yang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Pyroptosis, as a lytic programmed cell death, is mediated by the gasdermin family of proteins and is common in inflammatory cells (1). Cell pyroptosis is mainly formed by caspase-1/3/4/5/11 cutting the gasdermin family of proteins to form an N-terminal structure, and is then transferred to the cell membrane for cutting and perforation, in order that cell content can be released (2–4). It is characterized by nuclear aggregation, formation of a large number of 1–2-nm holes on the cell membrane, cell swelling, dissolution and rupture of the plasma membrane, as well as release of inflammatory factors (such as IL-1β and IL-18) to further amplify the inflammatory response (5,6).
As aforementioned, one of the significant morphological features of pyroptosis is excessive cell swelling, which signifies that the capacity of cell volume regulation, particularly regulatory volume decrease (RVD), is impaired. Several studies, including a previous study by the authors, verified that cells could regulate their volume to counteract osmotic swelling, as well as during cell division, growth, migration and cell death by RVD (7–11). Volume-regulated anion channels (VRACs) were activated by hypotonic-stimulus and played pivotal roles in RVD (12). VRAC inhibition could decrease the capacity of RVD, ultimately resulting in cell swelling (8,9,13–15). At present, the involvement of chloride channels in cell pyroptosis is poorly understood. Recently, Ye et al (16) proposed that lipopolysaccharides (LPS) and nigericin, the classic pyroptosis inducers, can decrease the hypotonicity-activated chloride currents and the capability of RVD in bone marrow-derived macrophages (BMDMs). In addition, leucine-rich repeat-containing 8a (LRRC8A), the constituent protein of VRACs, was significantly downregulated in pyroptotic BMDMs treated with LPS and nigericin. These results indicated that VRAC/LRRC8A was involved in the pyroptosis of BMDM cells induced by LPS and nigericin.
The mechanisms of pyroptosis in cancer and immune cells appear to be different. Recently, the roles of pyroptosis in cancer and the developments in inducing pyroptosis for cancer therapy have attracted the attention of researchers (17). Paclitaxel (PTX) plus platinum were used as first-line chemotherapy against ovarian cancer (18). To date, PTX has been proven to be closely associated with cell pyroptosis in a number of studies. On the one hand, PTX can induce pyroptosis in nasopharyngeal carcinoma cells through the caspase-1/gasdermin D (GSDMD) pathway (19), and in lung cancer through caspase-3/gasdermin E (GSDME) activation (20). On the other hand, PTX can enhance innate immunity by promoting PYD domains-containing protein 3 (NLRP3) inflammasome activation in macrophages and inducing cell pyroptosis by enhancing the cleavage of GSDMD (21). A previous study by the authors reported that PTX inhibited the background chloride currents, hypotonicity- and cisplatin-activated chloride currents in A2780 ovarian cancer cells (22). It is well known that blocking chloride channels inhibited the RVD, ultimately causing cell swelling (23,24). Therefore, in the present study, whether PTX could induce cell swelling and finally lead to ovarian cancer cell pyroptosis by inhibiting chloride channels was investigated.
Materials and methods
Cell culture
A2780 (cat. no. CC0803; http://www.culturecollections.org.uk/collections/ecacc.aspx) and SKOV3 (cat. no. CC0801; http://www.ATCC.org/) human ovarian cancer cell lines were purchased from Guangzhou Cellcook Biotech Co., Ltd. A2780 and SKOV3 cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Biological Industries) and 100 µg/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. A2780 was subcultured every 2 days, and SKOV3 was subcultured every 3 days. In subsequent cell experiments, DMSO was used as the solvent control by default for PTX.
Cell viability assay and half maximal inhibitory concentration (IC50) measurement
The effect of PTX on A2780 and SKOV3 cell viability was evaluated by MTT assay. Cells were seeded in 96-well plates with a density of 7,000-8,500 cells/well and cultured overnight at 37°C with 5% CO2. The cells were exposed to PTX (0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 and 256 nM) in culture medium for 24 and 48 h respectively. Next, 20 µl of 5 mg/µl MTT was added to each well and incubated for 4 h. After the supernatant was removed, 150 µl dimethyl sulfoxide was added to each well. Finally, the plates were shaken for 10 min to fully dissolve the formation crystals. All experiments were carried out in the dark. Subsequently, an infinite M200PRO microplate reader (Bio-Rad Laboratories, Inc.) was used to measure the optical density (OD) value at an absorbance of 490 nm. The IC50 values for each group of cells were determined using the following formula: Inhibition (%)=(ODcontrol-ODtest)/ODcontrol ×100%.
Cell apoptosis assay by flow cytometry and Annexin V-FITC-A double staining
For cell apoptosis analysis, A2780 and SKOV3 cells were seeded in 6-well plates at a density of 15,000 cells/well overnight. A2780 was treated with 10 nM PTX for 24 h at 37°C and SKOV3 was treated with 10 and 250 nM PTX for 48 h at 37°C. Following collection, the cells were washed with ice-cold PBS twice, and fixed with 70% ethanol at −20°C for >4 h. Next, the collected cells were stained with 400 µl propidium iodide (PI) at room temperature for 30 min. Finally, cell apoptosis distribution was examined by flow cytometry (BD FACSCanto 8.0; Beckman Coulter, Inc.). Data were analyzed using the Modfit program (version 8.0; BD Biosciences).
Cell volume assay by flow cytometry
As for volume analysis, cells were seeded in six-well plates with a density of 15,000 cells/well overnight. A2780 was treated with 10 nM PTX for 0–24 h at 37°C and SKOV3 was treated with 250 nM PTX for 0–48 h at 37°C. Following collection, the cells were washed with ice-cold PBS twice, and fixed with 70% ethanol at −20°C for >4 h. Following fixation, the fixation solution was removed and the cells were collected again and resuspended in PBS. The forward scattered light (FSC) was proportional to the cell volume. Finally, FSC was examined by flow cytometry (BD FACSCanto 8.0; Beckman Coulter, Inc.). All data were analyzed using the Modfit program (version 8.0; BD Biosciences).
Lactate dehydrogenase (LDH) release assay and PI staining
LDH release in cells was assessed using an LDH Cytotoxicity Assay Kit (cat. no. C0017; Beyotime Institute of Biotechnology) with a full-wavelength enzyme reader (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. In brief, 120 µl of the supernatant culture medium of A2780 and SKOV3 cells treated with PTX were collected in a 96-well plate, and the response mixture was added and incubated in the dark for 30 min at room temperature. The absorbance value at 490 nm was then measured, referenced by 600 nm.
The A2780 and SKOV3 cells were cultured for 15 min with 2.5 µg/ml PI at 20°C (cat. no. 556463; BD Biosciences). At the end of the incubation, cells were slowly washed with PBS twice. The cells were then exposed to 1 µg/ml Hoechst 33342 (cat. no. C1022; Beyotime Institute of Biotechnology) for 5 min at 20°C, and observed under a fluorescence microscope.
Scanning electron microscopy (SEM)
Cell morphology was monitored by SEM: Following fixing in 2.5% glutaraldehyde overnight at 20°C, cells were dehydrated through a graded series of ethanol (30, 50, 70, 95 and 100%) and treated for 5 min at each concentration. The alcohol in the sample was gradually replaced with isoamyl acetate, then were critical-point dried. Dried specimens were chilled at −80°C and transferred to a freezing dryer overnight. Each sample was coated with gold-palladium and imaged by SEM (S-3400 N; Hitachi Science Systems Ltd.).
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA from A2780 cells was obtained using Total RNA Extraction Kit (cat. no. RIJ26-01; Magen Biotechnology Co., Ltd.) and reversed-transcribed to cDNA with a Prime ScriptRT reagent (cat. no. RR037A; Takara Bio, Inc.), according to the manufacturer's instructions. The amplification of cDNA was performed using TB Green Premix Ex Taq (cat. no. RR820A; Takara Bio, Inc). The parameters of a real-time system (CFX Connect; Bio-Rad Laboratories, Inc.) were set at 95°C for 30 sec, followed by 40 cycles of 5 sec at 95°C and 30 sec at 60°C. The primers used are listed in Table I. Quantification of gene expression was performed by quantification curve. The expression levels of the target genes were quantitated using the 2−ΔΔCq method and GAPDH was used as the internal control to normalize the qPCR data (25).
Western blotting for protein expression
Cells were washed with ice-cold PBS, and lysed with cell lysis buffer for western blotting and IP (cat. no. P0013; Beyotime Institute of Biotechnology) containing 1% PMSF. Protein lysates were quantified using the BCA Protein Assay Kit (cat. no. KGP903; Nanjing KeyGEN Biotech Co., Ltd.). Equal amounts of protein (20 µg) were separated using 10% SDS-PAGE and transferred onto PVDF membranes (cat. no. PR 05505; MilliporeSigma). Following blocking with 5% non-fat milk in Tris-buffered saline with Tween-20 (0.05%) at room temperature for 2 h, the membranes were incubated with rabbit anti-LRRC8A primary antibody (cat. no. 24979S; dilution, 1:800; Cell Signaling Technology, Inc.), rabbit anti-ClC-2 primary antibody (cat. no. ab192526; dilution, 1:800; Abcam), rabbit anti-ClC-3 primary antibody (cat. no. ab28736; dilution, 1:1,000; Abcam), rabbit anti-anoctamin-1 (ANO1) primary antibody (cat. no. ab64085; dilution, 1:800; Abcam), rabbit anti-caspase-3 primary antibody (cat. no. ab49822; dilution, 1:600; Abcam), rabbit anti-GSDME primary antibody (cat. no. ab215191; dilution, 1:800; Abcam) and rabbit anti-GAPDH antibody (cat. no. RIP008; dilution, 1:1,000; Beijing Dingguo Changsheng Biotechnology Co. Ltd.) overnight at 4°C. Membranes were then incubated for 2 h at room temperature with HRP-linked goat anti-rabbit secondary antibody (dilution, 1:10,000; cat. no. IH-0011; Beijing Dingguo Changsheng Biotechnology Co. Ltd.). Proteins were visualized using Ultra High Sensitivity ECL Kit (GlpBio Technology, Inc.). Images were acquired and quantified using the gel imaging system (Alliance 4.7; Bio-Rad Laboratories, Inc.).
Whole-cell current recording
Whole-cell chloride currents were measured using a patch clamp amplifier system (EPC-10; HEKA GmbH Technology & Trade). The pipette solution contained 70 mM N-methyl-d-glucamine, 1.2 mM MgCl2, 10 mM N-2-hydroxyethylpiperazine-N‧-2-ethanesulfonic acid (HEPES), 1 mM ethylene glycol tetraacetic acid, 140 mM D-mannitol and 2 mM adenosine triphosphate, with a pH of 7.25 and osmolality of 300 mOsm/kg·H2O. The isotonic bath solution contained 70 mM NaCl, 0.5 mM MgCl2, 2 mM CaCl2, 10 mM HEPES and 140 mM D-mannitol, pH 7.4, osmolality 300 mOsm/kg·H2O. The 47% hypotonic bath solution was similar to the isotonic bath solution, except for D-mannitol, and its osmolality was 160 mOsm/kg·H2O. All chemicals were the highest grade available and purchased from Sigma-Aldrich (Merck KGaA).
A2780 cells (20,000 cells/ml) plated on coverslips were washed with the isotonic bath solution. The whole-cell current was recorded with a resistance of 4–5 MΩ under the aforementioned patch-clamp amplifier (EPC-10) using patch-clamp pipettes, which were made from glass microtubules on a two-stage vertical pipette puller (PC-10; Sutter Instrument Company). Cell membrane potential was held at the Cl− equilibrium potential (0 mV) and repeatedly stepped to 0, ±40 and ±80 mV for 200 msec pulses in 4 sec throughout the experiments. A computer recorded the whole-cell currents using a laboratory interface (MICRO 1401-4; Cambridge Electronic Design, Ltd.) at a sampling rate of 3 kHz. All experiments were processed at room temperature (20–24°C). The inhibition rate of PTX of the background Cl− current was calculated using the following equation: Inhibition (%)=(CIso-CIso + PTX)/CIso ×100%. The inhibition rate of PTX of the hypotonicity-activated Cl− current was calculated using the following equation: Inhibition (%)=(Chypo-Chypo + PTX)/Chypo ×100%, where CIso is the background current under isotonic condition, Chypo is the peak value of hypo-activated currents, and CIso + PTX and Chypo + PTX are the currents recorded after adding PTX in isotonic or hypotonic solution.
Measurement of intracellular chloride ion concentration ([Cl−]i)
Ovarian cancer cells A2780 were washed with Krebs-HEPES buffer (20 mM HEPES, 128 mM NaCl, 2.5 mM KCl, 2.7 mM CaCl2, 1 mM MgCl2, 16 mM glucose, pH 7.4) and exposed to 10 mM N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE; Beyotime Institute of Biotechnology), a novel fluorescent probe for Cl− whose fluorescence intensity quenches when it binds to Cl−, for 60 min at 37°C and washed again with Krebs-HEPES buffer. 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on5-yl) oxobutyric acid (DCPIB; MedChemExpress) was configured as a 5 mM high-concentration storage solution with DMSO. This was then further diluted with PBS to a concentration of 1 µM. Fluorescence emission was used to detect the fluorescence intensity of MQAE and quantify the concentration of Cl−, including inverted fluorescence photography and real-time fluorescence detection. Real-time fluorescence detection used fast laser scanning confocal microscopy (cat. no. SP8; Leica Microsystems, Inc.) to detect cellular fluorescence. The instrument first recorded the fluorescence intensity of the cells without drug stimulation for ~5 min, added the Krebs-Hepes buffer containing purple stripes according to the experimental needs and recorded the changes of MQAE fluorescence intensity following drug stimulation.
Small interfering RNA (siRNA) transfection
A2780 ovarian cancer cells were transfected (37°C for 8 h) using GP-transfect-mate (4 µl in 500 µl serum-free medium; Shanghai GenePharma Co., Ltd.) transfection reagents with siRNA (0.16 µM) targeting human LRRC8A (Shanghai GenePharma Co., Ltd.). The time interval between transfection and subsequent experiments was 48 h. The siRNA target sequences used are presented in Table II.
Statistical analysis
Data are presented as the mean ± SEM. Comparative studies of means were performed using one-way analysis of variance (ANOVA) followed by a post hoc test (projected least significant difference Fisher). Differences between two groups were assessed using an unpaired Student's t-test, and between paired variables were assessed using a paired Student's t-test. Dunnett's t-test was used to compare the differences between the control group and multiple experimental groups. P<0.05 was considered to indicate a statistically significant difference.
Results
PTX induces pyroptosis-like cell death in A2780 ovarian cancer cells
The cell viability of A2780 and SKOV3 ovarian cancer cell lines was inhibited to various degrees following treatment with PTX at concentrations of 0.5–256 nM for 24–48 h. As shown in Fig. 1A and B, the IC50 in A2780 cells following treatment with PTX for 24 h was 9.67±1.46 nM, while little inhibition can be observed in SKOV3 cells. Following treatment with PTX for 48 h, the IC50 in A2780 cells and SKOV3 cells were 3.23±0.33 and 221.9±31.72 nM (P<0.001 vs. A2780, n=4), respectively. Flow cytometry revealed a higher level of necrosis (Annexin V+/PI+ cells) as compared with that of apoptosis (Annexin V+/PI− cells) in A2780 cells (Fig. 1C and D) following 10 nM PTX treatment for 24 h. However, apoptosis was dominantly exhibited in SKOV3 cells treated with 250 nM PTX for 48 h (Fig. 1C and E).
Similarly, Hoechst 33342/PI staining also demonstrated that the ratio of necrosis (PI+ cells) was markedly elevated in A2780 cells following treatment with PTX (Fig. 1F-G; P<0.001; n=3). The effect of PTX on plasma membrane damage was evaluated by measuring LDH release in the culture supernatant. LDH release was significantly increased in A2780 cells following treatment with 10 nM PTX compared with the control group (Fig. 1H; P<0.001; n=3), while it was increased in SKOV3 cells at a PTX concentration of 250 nM. These results indicated that PTX induced cell death by causing plasma membrane damage in ovarian cancer cells.
PTX-induced cell swelling and cleavage of caspase-3/GSDME in A2780 cells
Membrane rupture and cell swelling induced by cleaved gasdermin family members are the main morphological characteristics of pyroptosis (26). In the present study, the volume of A2780 cells was gradually increased following treatment with PTX 10 nM for 1.5–24 h (Fig. 2A), suggesting cell swelling, while no obvious changes were observed in SKOV3 cells (Fig. 2B). The volume only increased after 12 h of 250 nM PTX treatment in SKOV3 cells (Fig. 2C). Therefore, the next experiments focused on A2780 cells.
The morphological changes of A2780 cells were then examined by SEM. As revealed in Fig. 2D, the membrane surface of normal cells was intact and relatively smooth. Following treatment with PTX, the cells were swollen with pores and evident large bubbles extending from the plasma membrane, suggesting the occurrence of a distinct morphological change characteristic of pyroptosis (5).
GSDMD and GSDME are common executive proteins in pyroptosis, and the GSDME protein is highly expressed in ovarian tissue (27,28). However, it is unclear which member of the gasdermin family is involved in the induction of pyroptosis by PTX. In the present study, the genes associated with pyroptosis were screened using RT-qPCR. The results showed that GSDME, caspase-3 and caspase-4 genes were mainly expressed in A2780 cells, and the expression of both caspase-3 and GSDME were increased while that of caspase-4 was significantly decreased following treatment with PTX (Fig. 2E and F). At the protein level, PTX also induced the cleavage of caspase-3 and GSDME, creating cleaved caspase-3 (cl-caspase-3) and GSDME N-terminal fragment (GSDME-N), respectively, in A2780 cells (Fig. 2G and H). These results indicated that PTX induced pyroptosis through the caspase-3/GSDME pathway in A2780 cells.
PTX inhibits chloride currents and increases [Cl−]i in A2780 cells
The above data showed that PTX can cause cell swelling. A previous study by the authors revealed that PTX inhibited background- and hypotonicity-activated chloride currents in A2780 cells (22). In addition, it has previously been revealed that the inhibition of the chloride channel caused an increase in the cell volume (23,29). Herein, it was also confirmed that the inhibition of background- and hypotonicity-activated chloride currents by PTX was similar to the effect of classic VRAC inhibitor, DCPIB (Fig. 3A and B). It is known that the inhibition of chloride currents leads to a decrease in Cl− efflux. In the present study, the MQAE fluorescence intensity was significantly decreased following treatment with DCPIB and PTX, which indicated that the [Cl−]i increased (Fig. 3C and D). Real-time fluorescence assay also showed that the [Cl−]i increased following treatment with PTX and DCPIB (Fig. 3E and F). The statistical analysis results are shown in Fig. 3G. These results suggested that PTX can inhibit the volume-sensitive chloride channels in the same way as DCPIB, and cause an increase in the [Cl−]i.
The component of volume-activated chloride channels, LRRC8A, is influenced by PTX in A2780 cells
There are several molecular categories of volume-activated chloride channel proteins (7). In the present study, RT-qPCR was used to detect the mRNA expression of the chloride channels of ClC-2/ClC-3/ANO1/LRRC8A in A2780 cells. As revealed in Fig. 4A and B, volume-sensitive or volume-related chloride channel genes expressed in A2780 cells mainly included ClC-2, ClC-3 and LRRC8A. Only the LRRC8A mRNA expression was increased, while that of the other genes exhibited no significant changes following treatment with PTX. Similar to the mRNA expression results, the protein expression of LRRC8A was significantly increased following PTX treatment, while the expression of the other proteins did not exhibit any significant changes (Fig. 4C-G). It was therefore inferred that LRRC8A may be involved in PTX-induced pyroptosis in A2780 cells.
The VRAC inhibitor, DCPIB, or the downregulation of LRRC8A protein enhance the efficacy of PTX-induced pyroptosis in A2780 cells
VRAC plays a major role in cell volume regulation, and its main component is LRRC8A (30). To further investigate the role of VRAC in cell pyroptosis, PTX combined with VRAC inhibitor, DCPIB, or LRRC8A siRNA (Fig. S1; the si-L-3 sequence was selected by default for the subsequent downregulation of LRRC8A) was used in the experiment. In the experimental group, the concentration of PTX-treated cells was 10 nM and the treatment time was 24 h. As shown in Fig. 5A and B, DCPIB or si-LRRC8A alone had no effect on LDH release. However, both markedly enhanced the PTX-induced LDH release. The effect of DCPIB and si-LRRC8A on cell volume was investigated, and the cells in the combination group were found to be larger, while DCPIB or siRNA alone had little effect on cell volume (Fig. 5C-F). Next, the morphological changes of the cells were examined using SEM. Consistently, it was found that A2780 cells treated with DCPIB or si-LRRC8A combined with PTX for 24 h exhibited more severe cell membrane damage and could not maintain a normal morphology compared with cells treated with PTX alone (Fig. 5G). However, the treatment of cells with DCPIB or si-LRRC8A alone had little effect on cell morphology. According to these results, the inhibition of VRAC function or the decrease in LRRC8A protein expression could enhance the degree of PTX-induced pyroptosis.
DCPIB or si-LRRC8A increases the expression of pyroptosis-related proteins in A2780 cells
Given that the inhibition of VRAC combined with PTX can increase pyroptosis in A2780 cells, the changes in pyroptosis-related proteins, including caspase-3, cl-caspase-3, GSDME and GSDME-N were further investigated. The levels of pyroptosis proteins, such as cl-caspase-3 and GSDME-N, were significantly increased by DCPIB/si-LRRC8A and PTX combination therapy, as compared with those following treatment with PTX alone (Fig. 6A-D and F and G). However, there was no significant change in pyroptosis-related protein following treatment with DCPIB or si-LRRC8A alone. In addition, cells treated with si-LRRC8 combined with PTX showed an increase in LRRC8 protein compared with si-LRRC8 alone (Fig. 6E). These results indicated that the inhibition of VRAC or decrease in LRRC8A expression promoted PTX-induced pyroptosis in A2780 cells.
Discussion
PTX was first approved for the treatment of ovarian cancer (31), with its application expanding into various other types of cancer some years later (32–34). To date, it is well known that PTX can induce multiple types of cell death, including mitotic catastrophe, apoptosis, senescence, autophagy and pyroptosis (35). In the present study, it was first observed that A2780 ovarian cancer cells exhibited features of pyroptosis, such as cell swelling, LDH release, deep PI staining and membrane perforation. As shown by SEM, A2780 cells exhibited evident swelling, and characteristic large bubbles appeared from the plasma membrane following PTX treatment. These data demonstrated that pyroptosis may be one of the underlying mechanisms of PTX for the treatment of ovarian cancer.
The gasdermin family of proteins plays a key role in cell pyroptosis. The family comprises six paralogous proteins in humans: GSDMA/B/C/D/E and PJVK (36). GSDMD and GSDME are best known for their functions in pore formation and pyroptosis (17). GSDMD activates cutting via caspases-1/4/5/11 (36), while GSDME activates cutting via caspase-3 (37). PTX has been shown to induce pyroptosis in both caspase-1/GSDMD and caspase-3/GSDME pathways in different types of cancer (19–21). Among ovarian tissues, the gasdermin family proteins mainly express GSDME (27,28). Previous studies have shown that chemotherapy drug-activated caspase-3 can induce secondary necrosis/pyroptosis in both cancer and normal cells that express high levels of GSDME (37–39). In the present study, GSDME was mainly expressed in A2780 cells, while GSDMD expression was very low. The upstream proteins of pyroptosis with the highest expression were caspases-3/4. Caspase-3 and GSDME mRNA expression was increased while caspase-4 mRNA expression was significantly decreased following treatment with PTX. Further western blotting also confirmed that the protein expression of caspase-3 and GSDME-N fragment, which was cleaved by caspase-3, were significantly increased in A2780 cells following treatment with PTX. Therefore, it was inferred that PTX induced pyroptosis mainly by activating caspase-3 to cleave GSDME in A2780 ovarian cancer cells.
Following the activation of caspase proteins, gasdermin family proteins have been revealed to cleave to separate their N-terminal pore-forming domain from the C-terminal repressor domain, and finally the N-terminal is inserted into cell membranes and forms large oligomeric pores, disrupting ion homeostasis and subsequently inducing osmotic swelling (27,36). Several studies, including the present one, have demonstrated that the blockage of chloride channels led to cell swelling in both isotonic and hypotonic conditions (8,23,29,40). It was also demonstrated that PTX could inhibit the background-, hypotonicity- and cisplatin-induced chloride currents in A2780 ovarian cancer cells (22). It was also confirmed herein that PTX inhibited the basic/background- and hypotonic-induced chloride currents of A2780 cells, and increased the [Cl−]i, which may decrease the passive water efflux and cause osmotic swelling. The above phenomena were also induced by DCPIB, a classic blocker of VRACs. It could therefore be deduced that the inhibition of chloride channels by PTX may contribute to the underlying mechanism of cell swelling in the formation of pyroptosis. However, it has also been reported that Cl− outflow can initiate and activate NLRP3 inflammasomes by promoting the polymerization of ASC, and then induce caspase-1/GSDMD pathway-mediated pyroptosis (41,42). Therefore, the flow direction of chloride ions on both sides of the cell membrane is likely to be different during pyroptosis and to depend on different cell types and pyroptosis inducers.
The candidate proteins for VRACs have been debated for years. LRRC8A was identified as an essential component of the VRACs in 2014 (30). In the present study, the expression of LRRC8A, ClC-2, ClC-3 and ANO1, which have been proposed to be associated with VRACs, were measured. Only LRRC8A was overexpressed, which was inconsistent with the findings of a previous study by Ye et al (16), which reported that LRRC8A expression was reduced during pyroptosis in BMDM cells induced by LPS and nigericin. Even in the present study, the increased LRRC8A protein expression was inconsistent with the PTX-induced inhibition of chloride currents. DCPIB is a known specific and potent inhibitor of VRAC (43). Unlike the inhibition by si-RNA, the inhibition by DCPIB is completely reversible (44). In recent years DCPIB has been increasingly used for probing the physiologic and pathologic roles of VRAC. Ye et al (16) reported that VRAC inhibitors, such as DCPIB and tamoxifen, and LRRC8A downregulation by siRNA interference can induce pyroptosis-like phenotypes similar to those by LPS and nigericin in BMDM cells. However, the present findings showed that DCPIB and LRRC8A siRNA alone did not cause pyroptosis in A2780 ovarian cancer cells, but could enhance the activities of PTX in inducing pyroptosis. A reason why VRACs exhibit these differences during the pyroptosis process is that the mechanisms of pyroptosis are different in cancer and immune cells with different stimuli. LPS plus nigericin triggered caspase-1/GSDMD-dependent pyroptosis in BMDM cells (16), while PTX activated caspase-3/GSDME-dependent pyroptosis. It has also been reported that different chemotherapeutic agents may induce different secondary pyroptosis even in the same cell line with GSDME expression (20,45). In addition, the cancer cells exhibited more adaptive changes than normal cells. PTX induced an increase in the expression of LRRC8A which may be associated with its adaption in ovarian cancer cells, since ongoing experiments by the authors found that LRRC8A was overexpressed in PTX-resistant cell lines (data not shown). Therefore, whether individual characteristics of differential chloride channel expression in ovarian cancer are associated with different degrees of PTX-induced pyroptosis will be investigated in the future.
In conclusion, caspase-3 was activated and specifically cleaved GSDME following PTX treatment to form a large number of membrane pores, and water molecules entered the cell causing cell swelling and inducing pyroptosis-like phenotypes. In addition, PTX and DCPIB increase the intracellular [Cl-]i by blocking the VRAC channel (LRRC8), which further promotes water entry and reduces water outflow, disrupting the osmotic balance and ultimately leading to persistent cell swelling (Fig. S2). PTX-induced pyroptosis was more effective when combined with the VRAC blocker or LRRC8A knockdown, the essential component of VRACs in A2780 ovarian cancer cells. These findings revealed a potential mechanism of PTX, and identified a combination of PTX and VRAC blockers that can synergistically treat ovarian cancer. However, in order to optimize their use in cancer treatment, whether the combination of PTX and VRAC blockers can harm non-tumorous cells needs to be determined.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was supported by the National Natural Science Foundation of China (grant nos. 81872133 and 81372382), the Basic Research Funds for Central Universities of Jinan University (grant no. 21620408).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
XY and CL contributed equally to this work. XY and CL designed the study, performed the experiments and interpreted the data. XY wrote the manuscript. SL and XLiao performed statistical analyses. KW and XLi revised the manuscript. LZ, GL and HY contributed to the conception of the work, supervised the study design, and corrected the manuscript. XY and CL confirm the authenticity of all the raw data. All authors have read and agreed to the published version of the manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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