ASX (cat. no. SML0982), necrotatin-1 (Nec-1; the stable variant; cat. no. 5042970001), z-VAD-fmk (cat. no. 627610), ML171 (cat. no. 492002) and N-acetylcysteine (NAC; cat. no. A7250) were purchased from Sigma-Aldrich (Merck KGaA). Dichlorofluorescein diacetate (DCF-DA; cat. no. D399) was purchased from Molecular Probes (Thermo Fisher Scientific, Inc.). RPMI-1640 medium (cat. no. 31800022) was purchased from Gibco (Thermo Fisher Scientific, Inc.). MuLV reverse transcriptase (cat. no. M1705) was obtained from Promega Corporation. The protease inhibitor complex (cOmplete™; cat. no. 11697498001) was obtained from Roche Diagnostics GmbH. Antibodies against caspase-9 (cat. no. sc-81663), Bcl-2 (cat. no. sc-492), Bax (cat. no. sc-526) and actin (cat. no. sc-47778) were obtained from Santa Cruz Biotechnology, Inc. Antibodies against phosphorylated (p)-RIP1 (cat. no. 65746) and p-MLKL (cat. no. 91689) were obtained from Cell Signaling Technology, Inc. The antibody against RIP1 (cat. no. 610458) was obtained from BD Pharmingen (BD Biosciences). Antibodies against RIP3 (cat. no. ab56164), p-RIP3 (cat. no. ab209384) and MLKL (cat. no. ab183770) were obtained from Abcam. All other reagents were obtained from Sigma-Aldrich (Merck KGaA). ASX, NEC-1, z-VAD-fmk and ML171 were dissolved in dimethyl sulfoxide (DMSO). Cells incubated with DMSO alone (<0.3%) served as controls (vehicle alone).

The human gastric cancer cell line AGS (ATCC CRL-1739; gastric adenocarcinoma) was purchased from the American Type Culture Collection and cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 2 mM glutamine and antibiotics (100 U•ml⁻¹ penicillin and 100 µg•ml⁻¹ streptomycin). The cells were cultured at 37°C in a humidified atmosphere with 95% air and 5% CO2.

The normal rat gastric epithelial cell line, RGM-1, was obtained from the RIKEN BioResource Center. RGM-1 cells were grown in a 1:1 mixture of Dulbecco's modified Eagles medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) and Ham's F-12 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% newborn calf serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U•ml⁻¹ penicillin and 100 µg•ml⁻¹ streptomycin. The cells were cultured at 37°C in a humidified atmosphere with 95% air and 5% CO2.

For all treatments, the cells were cultured at 37°C. For the time-course experiment to determine ROS levels and necroptotic markers, the cells were treated with 20 µM ASX and cultured for 0.5, 1 and 2 h (for ROS levels) or 2, 4 and 6 h (for the mRNA and protein expression levels of RIP1, RIP3, MLKL, and p-RIP1, p-RIP3 and p-MLKL levels).

For the concentration-course experiment to assess ROS levels and cell viability, the cells were treated with ASX (5, 10 or 20 µM) for 2 h (for ROS determination) and 24 h (for analysis of cell viability).

For the assessment of ROS levels, NADPH oxidase activity, cell viability, LDH release and Hoechst 33342/propidium oxide (PI) double staining, the cells were pretreated with ML171 (2 µM), NAC (1 mM), Nec-1 (25 µM) or z-VAD-fmk (10 µM) for 1 h and treated with 20 µM ASX for 2 h (to measure levels of ROS and NADPH oxidase activity) or 24 h (for cell viability, LDH release and Hoechst 33342/PI double staining).

To assess the role of RIP1 in ASX-induced cell death, cells were transfected with RIP1 small interfering (si)RNA or negative control (NC) siRNA and treated with ASX (20 µM) for 24 h, and the number of viable cells was counted.

To determine the effect of ASX on caspase-9 activation, the levels of Bax and Bcl-2, and Annexin V-FITC/PI double staining, the cells were treated with 20 µM ASX for 24 h. Western blot analysis was used to determine the levels of procaspase-9 and cleaved caspase-9. Fluorescence images of AGS cells were obtained by Annexin V-FITC/PI double staining.

To determine whether ASX induced cell death and activated NADPH oxidase in normal cells, normal rat gastric epithelial cells (RGM-1) were treated with 20 µM ASX for 2 h (to measure NADPH oxidase activity) or different concentrations (5, 10 or 20 µM) of ASX for 24 h (for cell viability).

2′-7′-Dichlorofluorescin diacetate (DCFH-DA) was used to measure intracellular levels of ROS. This method is used to detect hydrogen peroxide and hydroxyl radicals (25). The cells were seeded (6×10⁴ cells per well) in 6-well plates and incubated with 10 µM DCF-DA for 30 min. The cells were then washed and scraped into 1 ml PBS. DCF was measured (excitation/emission at 495/535 nm) using a VICTOR5 multilabel counter (PerkinElmer, Inc.). Intracellular ROS levels were expressed in terms of relative increases.

The cells were plated in a 24-well plate (1×10⁴ cells per well) and cultured for 24 h with or without treatment. Viable cell numbers were determined by direct counting using a hemocytometer based on a trypan blue exclusion test (0.2% trypan blue; Sigma-Aldrich; Merck KGaA). The mean number of viable cells that were not treated with ASX (untreated) was set to 100%.

Total RNA was isolated using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was converted to cDNA by reverse transcription using MuLV reverse transcriptase. The reaction conditions were as follows: 23°C for 10 min, 37°C for 60 min and 95°C for 5 min. cDNA was used for qPCR. qPCR was conducted in triplicate using SYBR-Green Real-time PCR Master Mix (Toyobo Life Science). RIP1 primers (forward, 5′-GGCATTGAAGAAAAATTTAGGC-3′ and reverse, 5′-TCACAACTGCATTTTCGTTTG-3′) were used to generate a 109-bp PCR product, RIP3 primers (forward, 5′-GACTCCCGGCTTAGAAGGACT-3′ and reverse, 5′-CTGCTCTTGAGCTGAGACAGG-3′) were used to generate a 180-bp PCR product and MLKL primers (forward, 5′-AGAGCTCCAGTGGCCATAAA-3′ and reverse, 5′-TACGCAGGATGTTGGGAGAT-3′) were used to generate a 124-bp PCR product. For β-actin, the forward primer was 5′-ACCAACTGGGACGACATGGAG-3′ and the reverse primer was 5′-GTGAGGATCTTCATGAGGTAGTC-3′, yielding a 349-bp PCR product. cDNA was amplified by 45 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec. During the first cycle, the 95°C step was extended to 3 min. Amplification specificity was validated by melting curve analysis generated at the end of each PCR reaction. All genes presented a single peak in the melting curve, indicating the absence of primer-dimer formation during the reaction and specificity of the amplification. Relative changes in gene expression between untreated cells and cells treated with ASX were determined using the 2^−ΔΔCq method (26). Levels of the target transcript were normalized to β-actin endogenous control and were constantly expressed in the group.

Preparation of whole-cell extracts, membrane fractions and cytosolic fractions was performed as previously described (27). Briefly, the cells were harvested by treatment with trypsin/EDTA, washed and centrifuged at 1,000 × g at 21–23°C for 5 min. The cell pellets were resuspended in lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 25 mM Tris (pH 7.4) and protease inhibitor complex. The resulting mixture was centrifuged at 10,000 × g at 21–23°C for 15 min. The supernatants were collected and used as whole-cell extracts. To prepare the cytosolic and membrane fractions, the supernatant was separated by centrifugation at 100,000 × g at 21–23°C for 1 h. The membrane fraction was obtained by resuspending the pellet in lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA and 10% glycerol. The supernatant was used as the cytosolic fraction. The protein concentration was determined using the Bradford assay.

Whole cell extracts were isolated from cells by using lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 25 mM Tris (pH 7.4) and protease inhibitor complex (Complete; Roche Diagnostics GmbH). The protein concentration was determined using the Bradford assay. Whole cell extracts were loaded onto 8–10% SDS-PAGE gels (40–60 µg protein per lane) and separated by electrophoresis under reducing conditions. Proteins were transferred onto nitrocellulose membranes via electroblotting. The membranes were blocked using 3% non-fat dry milk in Tris-buffered saline and 0.2% Tween-20 (TBST) for 1 h at room temperature. The membrane was incubated with antibodies against caspase-9 (1:1,000), actin (1:4,000), RIP1 (1:1,000), p-RIP1 (1:1,000), RIP3 (1:1,500), p-RIP3 (1:1,000), MLKL (1:1,500) and p-MLKL (1:1,000) in TBST solution containing 3% dry milk overnight at 4°C. After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse (1:3,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) or anti-rabbit (1:4,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) secondary antibodies in TBST solution containing 3% dry milk and anti-rabbit) for 2 h at room temperature. The proteins were visualized by exposure to X-ray film by using an enhanced chemiluminescence (ECL) detection system (cat. no. sc-2048; Santa Cruz Biotechnology, Inc.). Actin was used as the loading control. ImageJ software version 1.52a (National Institutes of Health) was used for densitometry analysis of western blots.

A luciferase assay was used to measure NADPH oxidase activity in the membrane and cytosolic fractions (28). The assay was performed in 50 mM Tris-MES buffer (pH 7.0) containing 2 mM KCN, 10 µM lucigenin and 100 µM NADPH. The reaction was initiated by the addition of 10 µg membrane-extract protein. Photon emission was measured using a microplate reader (Molecular Devices, LLC). For negative control experiments, cytosolic fraction protein was used instead of the membrane-fraction protein.

PI is permeable to necrotic cells and is visible as red fluorescence in nuclear DNA (29). Hoechst staining is a cell-permeable nuclear counterstain that emits blue fluorescence when combined with double-stranded DNA (29). Hoechst dyes are very sensitive to DNA conformation and chromatin status in cells and are, therefore, used to detect nuclear damage, such as distinguishing condensed pycnotic nuclei in apoptotic cells. PI and Hoechst 33342 double staining was used to determine necrosis or apoptosis.

The cells were seeded (6.0×10⁴ cells per well in a 6-well plate) and cultured overnight. PI and Hoechst 33342 were prepared in PBS to serve as the staining solutions (0.5 mg•ml⁻¹). The cells were treated with 20 µl staining solution in the dark and then incubated for 10 min at 37°C. The cells were removed from the medium and fixed for 10 min with 2 ml cold methanol at 20–22°C. After the removal of methanol, the cells were washed with PBS (1 ml) for 5 min, examined under a laser scanning confocal microscope (Zeiss LSM 880; Carl Zeiss AG), and photographed. The optical filters for excitation were 490–630 nm for PI and 350–460 nm for Hoechst. The ratio of red and blue fluorescence density was quantified by ZEN 2.3 (blue edition) software (Carl Zeiss Microscopy GmbH) and expressed as the percentage of PI-positive cells.

Annexin V and PI double staining is used to discriminate between apoptosis and necrosis; PI-positive staining reflects necrosis and Annexin V-positive/PI-negative staining reflects apoptosis (30). The cells were plated (6.0×104 cells per well) in a 6-well culture plate and then cultured overnight. The cells were then treated with 20 µM ASX for 24 h. The treated cells were then washed with ice-cold PBS and incubated with Annexin V-FITC and PI (FITC Annexin V Apoptosis Detection Kit; BD Pharmingen; BD Biosciences) in the dark according to the manufacturer's recommendations. The cells were fixed in 2% paraformaldehyde for 10 min at 20–22°C, washed three times with 1 ml PBS for 5 min, and then examined under a laser scanning confocal microscope (Zeiss LSM 880; Carl Zeiss AG) and photographed.

siGenome SMART pool siRNA for human RIP1 (5′-CCACUAGUCUGACGGAUAA-3, 5-UGAAUGACGUCAACGCAAA-3′, 5′-GCACAAAUACGAACUUCAA-3′ and 5′-GAUGAAAUCCAGUGACUUC-3′) and the NC siRNA (siGenome None-Targeting siRNA Pool #1; 5′-UAGCGACUAAACACAUCAA, 5′-UAAGGCUAUGAAGAGAUAC-3′, 5′-AUGUAUUGGCCUGUAUUAG-3′ and 5′-AUGAACGUGAAUUGCUCAA-3′) were obtained from GE Healthcare Dharmacon, Inc. Cells were transfected with 50 nmol RIP1 or NC siRNAs by using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), following the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.), and then cultured for 24 h. Then, 24 h after transfection of RIP1 or NC siRNAs, the cells were treated with 20 µM ASX.

LDH release represents the permeabilization of the plasma membrane, a necrosis marker (31). LDH release was measured using an LDH assay kit (cat. no. ab102526; Abcam). Cell lysates were prepared using lysis buffer containing 0.1 M Tris (pH 7.4) and 10% Triton X-100. Cell lysates were centrifuged at 10,000 × g for 15 min at 4°C. LDH activity in the culture medium and the cells was measured, as previously described (32). LDH release was quantified as a percentage of the total LDH content (LDH in the supernatant + LDH inside the cells).

All data are expressed as the mean ± standard error of the mean. All experiments were repeated three times. Number of each group was ten (n=10). The statistical differences among groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's test. Statistical analysis was performed using SPSS 22.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

To assess the appropriate culture time and concentration of ASX for ROS production in AGS cells, ROS levels were determined using different culture times and various concentrations of ASX. The cells were treated with ASX (20 µM) and cultured for 2 h. As shown in Fig. 1A, 20 µM ASX increased ROS levels in a time-dependent manner. Furthermore, ROS levels were highest at 2 h of culture. When the cells were treated with various concentrations (5, 10 and 20 µM) of ASX and cultured for 2 h, the highest level of ROS in AGS cells was obtained with 20 µM ASX (Fig. 1B). Thus, for further studies on ROS levels, cells were treated with 20 µM ASX for 2 h.

To determine whether ASX induces cell death and activates NADPH oxidase in normal cells, normal rat gastric epithelial RGM-1 cells were treated with 20 µM ASX for 2 h (for determination of NADPH oxidase activity) or different concentrations (5, 10 or 20 µM) of ASX for 24 h (for cell viability). ASX had no effect on cell viability or NADPH oxidase in RGM-1 cells (Fig. 1C and D). The results showed that ASX did not induce death of normal gastric epithelial cells.

To determine whether ASX-induced ROS production was linked to NADPH oxidase activation in AGS cells, the cells were pretreated with ML171, a specific inhibitor of NADPH oxidase 1, or an antioxidant, NAC, for 1 h, and then treated with ASX (20 µM) for 2 h. The ASX-induced increase in ROS levels was decreased by treatment with ML171 and NAC (Fig. 1E). As shown in Fig. 1F, ASX increased NADPH oxidase activity, which was reduced by ML171 in AGS cells. These results indicated that ASX induced NADPH oxidase activation, leading to increased ROS levels in AGS cells.

To examine whether ASX induces cell death, cell viability was assessed after 24 h of culture with various concentrations of ASX. ASX reduced cell viability in a dose-dependent manner (Fig. 2A).

Further, to determine whether NADPH oxidase and ROS induced ASX-associated cell death, the cells were pretreated with ML171 or NAC for 1 h followed by treatment with ASX for 24 h. As presented in Fig. 2B, both ML171 and NAC inhibited the ASX-induced decrease in cell viability.

To determine whether ML171 or NAC inhibited ASX-induced necrosis in AGS cells, PI and Hoechst 33342 double staining was performed (Fig. 2C). The density ratio of the red and blue fluorescence was quantified and expressed as PI-positive cells (%) (Fig. 2D). The number of necrotic AGS cells increased after ASX treatment, as indicated by the increase in the ratio of PI-positive cells. Both ML171 and NAC reduced the number of PI-positive cells, indicating that NAC and ML171 blocked ASX-induced necrotic cell death. These results suggested that ASX induced necrosis, which may be mediated by NADPH oxidase and increased ROS levels.

During necroptosis, the mRNA and protein levels of necroptotic proteins, including RIP1, RIP3 and MLKL, are upregulated (33,34). In the present study, ASX significantly increased the mRNA expression of RIP1 and RIP3 at 4 h compared with the untreated group. However, there were no significant changes in mRNA expression of MLKL by ASX treatment (Fig. 3A).

Total and phospho-specific forms of RIP1, RIP3 and MLKL were confirmed via western blotting to determine whether the effect of ASX on cell death involves necroptosis (Fig. 3B). It was found that ASX increased the protein expression of RIP1, RIP3 and MLKL. Moreover, ASX augmented the activation of RIP1, RIP3 and MLKL, as indicated by the increased levels of the phosphorylated forms of RIP1 and RIP3. These results suggested the involvement of RIP1/RIP3/MLKL in ASX-induced necroptosis in AGS cells.

To further investigate the role of RIP1 in ASX-induced cell death, a gene silencing approach was used to specifically knock down RIP1. To confirm the inhibitory effect of RIP1 siRNA, the protein expression of RIP1 in AGS cells was measured after transfection with RIP1 or NC siRNA. The protein levels of RIP1 were notably suppressed in cells transfected with RIP1 siRNA compared with those transfected with the NC siRNA (Fig. 3C). ASX did not induce cell death in cells transfected with RIP1 siRNA, whereas ASX reduced the viability of cells transfected with the NC siRNA compared with the untreated group (Fig. 3D). These results indicated that ASX-induced cell death may occur through RIP1-mediated necroptosis.

To determine whether ASX-induced cell death was necroptotic, the cells were treated with Nec-1 or z-VAD for 1 h prior to treatment with ASX for 24 h. Nec-1 is known to be a selective inhibitor of RIP1 (35). As shown in Fig. 4A and B, ASX-induced cell death and LDH release were inhibited by the necroptosis inhibitor Nec-1. A pan-caspase inhibitor, z-VAD, as an inhibitor of apoptosis, did not affect the ASX-induced increase in LDH release and cell death. These results suggested that ASX may induce necroptosis, but not apoptosis.

To determine whether ASX triggered necrotic cell death in AGS cells, PI and Hoechst 33342 double staining was performed (Fig. 4C). Hoechst 33342 is a bis-benzamide derivative that binds to AT-rich sequences in the minor groove of double-stranded DNA. Thus, this fluorescent stain is commonly used to visualize nuclei in microscopic studies (27). The plasma membrane-permeable dye PI can be detected in the necrotic cells. In the present study, Hoechst 33342 stained the nuclei of all cells, including healthy cells, producing blue fluorescence. However, live or apoptotic cells are impermeable to PI, which intensely stains necrotic cells red. As presented in Fig. 4C, AGS cells that underwent ASX treatment showed more necrotic cells, as determined by the increased ratio of PI-positive cells (Fig. 4D). Nec-1, a necroptosis inhibitor, decreased the number of PI-positive cells. However, z-VAD did not affect the ASX-induced increase in PI-positive cells, as determined by the ratio of red to blue fluorescence. These results showed that Nec-1 blocked ASX-induced necrotic cell death in AGS cells, suggesting that ASX may induce necroptosis in AGS cells.

Caspase-9 activation amplifies the apoptosis cascade by activation of caspase-3 and other apoptosis mediators (36). Thus, the effect of ASX on caspase-9 activation in cells was determined. ASX did not increase the levels of active caspase-9 (Fig. 4E). As an index of apoptosis, the ratio of Bax/Bcl-2 was determined in cells treated with or without ASX (Fig. 4F). The Bax/Bcl-2 ratio was not affected by ASX. To determine whether ASX triggered apoptotic or necrotic cell death, double staining with Annexin V-FITC and PI was performed (Fig. 4G). Annexin V is a sensitive marker for detecting early apoptosis(30). Apoptotic cells can be distinguished from necrotic cells by double staining with PI. Annexin V-FITC and PI double staining easily distinguished apoptotic cells (shown as green fluorescence) from necrotic cells (shown as red fluorescence). ASX did not cause increased green fluorescence, but resulted in increased red fluorescence, indicating that ASX induced necrosis, but not apoptosis. Taken together, these results indicated that ASX-mediated cell death was related to necrosis/necroptosis.