HNK powder was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China; >99% purity) and was dissolved as 20 mg/ml in dimethyl sulfoxide (75 mM). RIP1 inhibitor, Nec-1, and pan-caspase inhibitor, z-VAD-fmk, were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). CsA, Hoechst 33342, dimethyl sulfoxide and VP-16 were purchased from Sigma-Aldrich (St. Louis, MO, USA). the CCK-8 Cell Counting kit, DNA Ladder Detection kit and MitoTracker probes (MitoRed) were purchased from Nanjing KeyGEN Biotech Co., Ltd. (Nanjing, China). M-PER Mammalian Protein Extraction Reagent, Halt Protease and Phosphatase Inhibitor Cocktails were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit II was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Cleaved caspase-3 and -8, B-cell lymphoma 2 (Bcl-2), RIP1, phosphorylated (p)-Akt (T308), p-Akt (S473), Akt, p-eIF4E-binding protein 1 (4E-BP1), 4E-BP1, p-S6 kinase (S6K), S6K and GAPDH antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Millicell EZ Slides were purchased from Merck Millipore (Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc.). Enhanced BCA Protein Assay Kit (P0009) was purchased from Beyotime Institute of Biotechnology (Haimen, China). Polyvinylidene difluoride (PVDF) membranes were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The HEK-293 human embryonic kidney cell line was obtained from the Cancer Institution of Zhejiang University (Hangzhou, China). The HEK-293 cells were cultured in DMEM supplemented with 10% FBS. HEK-293 cella were cultured at 7°C and passaged every 3–4 days.

HEK-293 cells were seeded in 96-well culture plates at density of 6,000 cells/per well. Following culture for 24 h, cells were pretreated with DMEM/FBS, 25 µM CsA (2 h) or 60 µM Nec-1 (1 h). Then cells were incubated with the drug-free medium, HNK of different concentrations (30 or 40 µg/ml) for different durations (1, 2 and 4 h). Subsequently, the CCK-8 kit was added to the culture medium at 10 µl per 100 µl medium. Following culture with CCK-8 for 1 h, the optical density at a wavelength of 450 nm (OD₄₅₀₎ was tested using a spectrophotometer (Model 680; Bio-Rad Laboratoeis, Inc.). The relative cell viability was calculated using the OD₄₅₀ values.

HEK-293 cells were cultured in 6-cm culture dishes at density of 1.2×10⁶ in 3 ml medium for 24 h. Cells were pretreated with drug-free medium, 25 µM CsA (2 h), 60 µMNec-1 (1 h) or 50 µM z-VAD-fmk (0.5 h). The cells were treated with drug-free medium or 30 µg/ml HNK (liquid volume, 3 ml) for varying durations. Following treatment, the cells were harvested and stained with Annexin V-FITC/propidium iodide (PI) and assayed by flow cytometry, as previously described (7,8).

Cells were treated as described above for flow cytometric analysis. DNA extractions from HEK-293 cells from each treatment group were performed according to the manufacturer's instructions of the DNA Ladder Detection Kit. HL-60 cells that had been treated with 20 µg/ml VP-16 for 6 h served as a positive control.

Cells were cultured on Millicell EZ Slides at a density of 4×10⁴ cells/slide and treated as described above for flow cytometric analysis. Following treatment, cells were stained with Hoechst 33342 and MitoRed according to the manufacturer's instructions. The cells were examined under a confocal microscope (TCS SP5; Leica Camera, Wetzlar, Germany), as previously described (8).

Cells were treated and harvested as described above for flow cytometric analysis. Proteins were extracted using M-PER^® Mammalian Protein Extraction Reagent supplemented with Halt Protease and Phosphatase Inhibitor Cocktail. Protein concentrations were determined using the BCA method. Samples (20 µg of each) were loaded in 10 or 8% sodium dodecyl sulfate polyacrylamide gel and electrophoresis was performed using PVDF membranes (100–150 V; 0.5–2 h), followed by western blotting. Images were obtained by chemiluminesence using ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Inc.). The procedures were performed as previously described (8).

SPSS version 20.0 (IBM SPSS, Amronk, NY, USA) was used to perform all statistical analyses. All the experiments were performed in triplicate, and comparable results were obtained from the three different experimental setups. The data are presented as means ± standard deviation. Paired student's t-test was used in single group comparisons and P<0.01 was considered to indicate a statistically significant difference.

In the current study, 30 µg/ml HNK, a lower concentration than previously reported (40 µg/ml) (6), was used to demonstrate its effect on HEK-293 cells. HEK-293 cells were incubated with 30 µg/ml HNK for 4 h, and labeled with Annexin V-FITC and PI (Fig. 1). Cell viability was significantly reduced following treatment with 30 µg/ml HNK. And the majority of HNK-triggered cell deaths were represented by positive staining for PI, which indicated necrotic cell death (Fig. 1Ac and B). Subsequently, whether the PI-stained cells had undergone necrosis or were composed of necrotic cells and undergoing advance-stage apoptosis was investigated. Internucleosomal DNA fragmentation is a morphological characteristic of apoptosis at the advanced stage (or degradation phase), which can be detected as a ladder pattern by electrophoresis of isolated DNA. Apoptotic bodies are another morphological characteristic of apoptosis and are detectable using nuclei staining with Hoechst 33342. DNA laddering and apoptotic bodies are frequently used to determine the existence of apoptosis. DNA laddering was performed on DNA extracted from HEK-293 cells after exposure to 30 µg/ml HNK. No obvious ladder pattern was observed, however a diffuse fractured pattern was detected following HNK exposure (Fig. 2). Confocal imaging of Hoechst 33342-stained nuclei was used to determine apoptotic bodies during the process of HNK-triggered necrotic cell death. Confocal imaging did not detect any nuclear chromatin condensation or apoptotic bodies in the HEK-293 cells after exposure to 30 µg/ml HNK for 0.5, 1 or 2 h (Fig. 3). In addition, DNA laddering and Hoechst 33342 staining revealed no morphological manifestation of apoptosis in HNK-triggered cell death of HEK-293 cells. Caspase-8 is an important initiator caspase, and it is activated during the initiation phase of extrinsic apoptosis. Activated caspase-8 leads to the release of its active fragments, p18 and p10. It cleaves and activates downstream effector caspases (including caspase-3, -6, and -7), which in turn execute apoptosis. The present study demonstrated that in the ultra early stage of HNK-induced necrosis (0.25 h), the cleaved caspase-8 level was increased. Subsequently, its expression level decreased in parallel with the increase in necrosis. Caspase-3 is a critical activator of intrinsic and extrinsic apoptosis. Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. And active level of cleaved caspase-3 is frequently used as a chemical manifestation to determine apoptosis. Western blot analysis revealed that the protein levels of cleaved caspase-3 had not elevated though the process of HNK-triggered cell death (0.25, 0.5, 1 and 2 h; Fig. 4). Bcl-2 is a pro-survival protein of the Bcl-2 family. It exerts a survival function in response to a wide range of apoptotic stimuli via inhibition of mitochondrial cytochrome C release. In the process of HNK-triggered cell death, no obvious changes in expression levels of Bcl-2 were detected with increasing treatment durations using western blotting (Fig. 4). Conversely, pan-caspase inhibitor, z-VAD-fmk did not reverse HNK-induced cell death (Fig. 1), which also demonstrated that HNK-induced cell death was caspase-independent and did not result from apoptosis. Therefore, the morphological and chemical manifestations reveal that apoptosis was not induced in HEK-293 cells by HNK at a dose of 30 µg/ml. However, 30 µg/ml HNK did trigger simple necrotic cell death in HEK-293 cells.

Regulated necrosis is defined as a genetically controlled cell death process, which is morphologically characterized by cytoplasmic granulation, and organelle and/or cellular swelling (2). Previous reports demonstrated that morphological manifestations of HNK-induced regulated necrotic cell death included increased cell volume (due to swelling of cytoplasmic organelles), dilatation of mitochondria and the endoplasmic reticulum, vacuoles and nuclear modifications (6,7). Among the morphological manifestations (vacuoles presenting as autophagic vacuoles containing a ruptured plasma membrane, manifesting as membranous whorls), mitochondrial dilatation was the most characteristic. MitoRed was used to stain the mitochondria of HEK-293 cells, and confocal imaging was performed to detect morphological changes of the mitochondria. Intense staining of the mitochondria was observed in the early stage of HNK-induced necrosis, and gradually weakened during the necrotic process (Fig. 3). This indicated that the mitochondria underwent swelling and subsequently disintegration during HNK-induced necrosis from the early to late stages. MPT is an initiator of the schematic steps of regulated necrosis (2). It was previously reported that CypD, a vital protein involved in MPT, was a critical regulator in HNK-induced regulated necrotic cell death (6–8). CypD levels gradually increased in parallel with HNK-triggered regulated necrosis. Furthermore, CypD inhibitors (CsA) or CypD siRNA depletion completely blocked CypD, which significantly attenuated HNK-induced necrotic cell death (6,8). In the current study, 25 µM CsA pretreatment prior to HNK treatment was performed to determine whether CsA pretreatment inhibits HNK-induced necrosis. The results indicated that 25 µM CsA pretreatment for 2 h significantly increased cell viabilities following HNK treatment for 1, 2 and 4 h (Fig. 5). In addition, it completely blocked HNK-induced necrotic cell death, which returned the levels of PI-stained cell fractions to the control level. The PI-stained cell fractions in the control, CsA, HNK and CsA + HNK groups were 10.79±0.19, 4.16±0.07, 61.33±1.42 and 3.33±0.09%, respectively (P<0.001; Fig. 6). Conversely, 25 µM CsA pretreatment blocked DNA diffuse fracture (Fig. 2) and mitochondrial dilatation (Fig. 3). The results indicated that HNK-triggered regulated necrotic cell death could be completely blocked by CsA, which indirectly indicates that CypD was a central initiator during the process of HNK-induced regulated necrosis in HEK-293 cells.

The most characteristic form of regulated necrosis is termed necroptosis. In human cells, the RIP1-RIP3-mixed lineage kinase domain-like (MLKL)-PGAM family member 5, Ser/Thr protein phosphatase, mitochondrial (PGAM5)-dynamin-related protein 1 (Drp1) axis drives tumor necrosis factor (TNF)-induced necroptosis via mitochondrial fission (9). Necroptosis is characterized as RIP1-dependent and can be specifically inhibited by RIP1 inhibitor, Nec-1. In previous studies, RIP1 expression levels were demonstrated to be downregulated in HNK-induced regulated necrosis in the MCF-7 breast cancer cell line throughout treatment durations of 1–4 h (8). In the current study, RIP1 expression levels and functions were detected at the ultra early stage in HNK-induced regulated necrosis in HEK-293 cells. Contrary to our previous report, RIP1 expression levels were observed to be upregulated in the ultra early stage (0.25–1 h) of regulated necrosis (Fig. 4). Subsequent results revealed that HNK-induced cell death was partly reversed by pretreatment with 60 µM Nec-1 for 1 h [PI-stained cell fractions in the control, Nec-1, HNK and Nec-1 + HNK groups were 10.79±0.19, 4.36±0.12, 61.33±1.42 and 46.73±1.15%, respectively (P<0.001; Fig. 7)]. In addition, the inhibition effects of Nec-1 on HNK-induced regulated necrosis gradually reduced with the elongation of HNK incubating time (1–4 h; Fig. 5). The results indicated that RIP1 probably participated in the initiation stage of HNK-induced regulated necrosis. However, it was not a pivotal initiator and its effects in the process of HNK-induced regulated necrosis were limited.

mTOR is a highly conserved Ser/Thr kinase, which integrates diverse signals to control cell growth, proliferation, survival and metabolism. mTOR assembles into two functionally different complexes, mTORC1 and mTORC2, with distinct inputs and downstream effects. mTORC1 regulates cell growth by promoting translation, ribosome biogenesis and autophagy via phosphorylation of its substrates, including 4E-BP1 and ribosomal S6K. mTORC2 promotes cell cycle entry, cell survival, actin cytoskeleton polarization and anabolic output though phosphorylation of its substrates, the Ser/Thr protein kinases, Akt, protein kinase A (PKA) and PKC (10,11). Previous findings demonstrate that aberrant activation of the mTOR signaling pathway is vital in tumorigenesis, and promotes growth and survival of various types of cancer cells (12). Due to its high biological relevance to cancer, different therapeutic strategies have been developed to target this signaling cascade, such as mTOR inhibitors Everolimus and Temsirolimus (13). In the current study, the activation levels of the mTOR signaling pathway during HNK-triggered regulated necrosis in HEK-293 cells was determined. Western blot analysis was performed to determine the phosphorylation and expression levels of core mTOR substrates, Akt, 4E-BP1 and S6K. The phosphorylation levels of 4E-BP1 and S6K were observed to gradually decline with the elongation of incubating duration (0.25, 0.5, 1 and 2 h) during the process of HNK-triggered regulated necrosis. Phosphorylation of Akt on Thr308 and Ser473 were also gradually downregulated. Conversely, the protein expression levels of Akt, 4E-BP1 and S6K were stable (Fig. 8A). Figs. 5 and 6 demonstrate that CsA markedly inhibited HNK-induced regulated necrosis. Therefore whether pretreatment with CsA could reverse dephosphorylation of mTOR substrates was investigated. Pretreatment with CsA prominently reversed dephosphorylation of Akt on Ser473, however, only partially reversed dephosphorylation of 4E-BP1, S6K and Akt on Thr308 (Fig. 8B). Collectively, the above results reveal that the mTOR signaling pathways were inhibited during the process of HNK-triggered regulated necrosis in HEK-293 cells. Furthermore, CypD may act downstream of mTOR signaling pathways, as CsA blocked HNK-triggered regulated necrosis, although it only partially reversed the dephosphorylation of mTOR substrates.