This article is mentioned in:

Introduction

In recent decades, apoptosis has been targeted as a therapeutic strategy to induce cancer cell death; therefore, several currently available anticancer drugs are designed to activate the apoptotic pathway directly or indirectly. However, numerous cancer cell types have acquired resistance to apoptosis by altering apoptosis-associated signaling pathways (e.g., loss of caspase 3), which has been a major obstacle to traditional chemotherapy. Therefore, other types of programmed cell death (PCD) have been explored. Dysregulation of PCD may induce various diseases, including numerous types of cancer. Initially, cell death has traditionally been divided into two types: Regulated cell death, i.e., apoptosis, and unregulated and accidental cell death, i.e., necrosis. However, more recent work has led to further information regarding the concept of cell death. One conceptual alteration is that another type of necrosis has been identified, which is considered to be a type of PCD; this type of necrosis has been named necroptosis (1-4). Therefore, it has been accepted that two types of necrosis exist, unregulated necrosis and regulated necroptosis. The present study focused on targeting necroptosis as an alternative strategy for treating cancer.

Necroptosis is a caspase-independent form of cell death, which is morphologically characterized by a loss of physical integrity, including swelling of organelles, increase in cellular volume, disruption of plasma and mitochondrial membranes, and release of intracellular contents. The most well studied signaling pathway for the induction of necroptosis is tumor necrosis factor-mediated necroptosis, which is mainly mediated by activation of three core regulators: Receptor-interacting serine/threonine protein kinase (RIP)1, RIP3 and mixed lineage kinase domain-like protein (MLKL) (1,5-9). These regulators form three complexes (membrane-bound complex I, complex II and necrosome) sequentially, by changing binding partners (1,5-7,10). Necroptosis can be induced by various types of stress, including extreme nutrient deficiency. Unlike normal cells, cancer cells require unusually large amounts of nutrients to support their uncontrolled growth and proliferation; therefore, they are more easily exposed to severe nutrient deficiencies compared with normal cells. However, many cancer cells develop tolerance and overcome nutrient limitations by increasing nutrient uptake through alterations in metabolic signaling pathways or by activating angiogenesis (11-16). Therefore, it is important to elucidate these mechanisms, in order to develop novel therapeutic approaches. Therefore, this study initially focused on p53 and AMP-activated protein kinase (AMPK), as they are well known to serve various important roles under nutrient-deprived conditions and are considered promising therapeutic targets for cancer treatment.

One of the most powerful tumor suppressors, p53, is considered an appealing target for effective cancer therapy. Notably, approximately half of human cancers are known to have defective p53 expression, and p53 has been reported to be involved in several types of cell death [e.g., apoptosis, caspase-independent apoptosis (CIA), autophagy-mediated cell death, necroptosis, entosis, anoikis, paraptosis, pyroptosis, ferroptosis, mitotic catastrophe and efferocytosis] (17). It is widely accepted that p53 sensitizes normal cells to stressful conditions, including nutrient starvation. Mutations or deletions of p53 in cancer cells may overcome cell death pathways; therefore, these cells may exhibit resistance to chemotherapeutic drugs. Considering its vital roles in the regulation of cell death in response to various stressors, determining the role of p53 in the regulation of necroptosis may present a novel therapeutic strategy for cancer treatment.

AMPK also serves several vital roles in the regulation of metabolism and energy homeostasis. Cellular stress, such as nutrient starvation, induces the activation of AMPK, which is important for restoring intracellular energy balance. Activated AMPK inhibits energy-consuming biosynthetic processes and activates ATP biosynthesis (18). The most well known function of AMPK is its involvement in autophagy, in which it directly or indirectly promotes autophagy. In normal cells, AMPK acts as a metabolic tumor suppressor protein by activating autophagy to remove damaged or unnecessary proteins and organelles (19-25). However, it also functions as an oncogenic protein by protecting cancer cells from cell death by supporting their uncontrolled growth and proliferation (22,26,27). In contrast to its well-known function in autophagy, few studies have reported on its role in necroptosis.

The present study aimed to determine how cancer cells evade necroptosis under nutrient starvation, and revealed that enhanced AMPK activation may suppress necroptosis in p53-null cells (p53−/−) but not in wild type cells (p53+/+) under the same conditions.

Materials and methods

Cell lines, cell culture and cell treatment

The colorectal carcinoma cell lines, HCT116 p53+/+ and HCT116 p53−/−, were a gift from Dr B. Vogelstein (Johns Hopkins University, Baltimore, MD, USA) (28). The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (WelGENE, Inc., Gyeongsan, South Korea) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% Antibiotic-Antimycotic solution (cat. no. 15240-06; Gibco; Thermo Fisher Scientific, Inc.). Both cell lines were grown at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Dorsomorphin dihydrochloride (DM; cat. no. sc-361173; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; cat. no. BML-EI330; Enzo Life Sciences, Inc., Farmingdale, NY, USA) were used to inhibit and activate AMPK, respectively. Cells were treated with 0, 5 and 10 µM DM for 48 h, or with 0, 75, 150 and 300 µM AICAR for 48 h at 37°C.

Cell viability and proliferation analysis

Cell viability was analyzed using the Cell Viability, Proliferation & Cytotoxicity Assay kit (EZ-CYTOX, cat. no. EZ-3000; DoGenBio, Seoul, South Korea), according to the manufacturer's protocol, in three replicates. Briefly, HCT116 p53+/+ and HCT116 p53−/− cells (~2×104 cells/well) were seeded in 96-well plates for 24 h, and the media were replaced with RPMI 1640 media with or without 10% FBS for 24, 48, or 72 h. Cell viability was evaluated by measuring absorbance at 450 nm using a Gemini XPA Microplate Reader. Cell proliferation was analyzed by Trypan blue (EBT-001; NanoEnTek, Inc., Seoul, South Korea) staining under the same conditions; 10 µl cells (~105 cells) and 10 µl Trypan blue were mixed in 1.5 ml microcentrifuge tubes at room temperature and were then placed into an EVE™ cell counting slide (NanoEnTek, Inc.), in order to count the number of live or dead cells. Cell proliferation was determined by counting the number of live cells at various time points (0, 24, 48 and 72 h) after culturing in complete media (CM) or serum-starved media (SS). Morphological images of cells were captured using an optical microscope with IS capture software (KI-400F; Korea Lab Tech, Seongnam, South Korea) at ×100 magnification.

Analysis of necroptosis, apoptosis and autophagy

Necroptosis, apoptosis and autophagy were detected using western blotting for the detection of the following corresponding biomarkers: Cyclophilin (Cyp)A and high mobility group box 1 (HMGB1) for necroptosis (29); cleaved poly (ADP-ribose) polymerase (PARP) for apoptosis; and p62/sequestome 1 (SQSTM1) and microtubule-associated protein 1A/1B-light chain 3 (LC3) for autophagy. Necroptosis was also analyzed using the Green Fluorescent Protein-Certified Apoptosis/Necrosis Detection kit (cat. no. ENZ-51002; Enzo Life Sciences, Inc.), according to the manufacturer's protocol. This kit contained 7-amino-actinomycin D (7-AAD) and Annexin V, which were used to detect necroptosis and apoptosis, respectively.

Western blotting

HCT116 p53+/+ and HCT116 p53−/− cells (~5x106/ml) were centrifuged, (Smart R17; Hanil Scientific, Inc., Gimpo, South Korea) at 3,000 × g for 3 min at 4°C, washed in ice-cold PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris, pH 7.5; 48 mM NaCl; 1% Triton X-100 and 1 mM EGTA) with 0.5 mM Na3VO4, 1 mM DTT and 1 µl premade protease inhibitor cocktail III (cat. no. P-1512; AG Scientific, Inc., San Diego, CA, USA)/ml RIPA solution. Protein concentration was quantified using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and 10 or 30 µg protein was loaded into each lane. Subsequently, proteins were separated by 10 or 12% SDS-PAGE and were then transferred onto an Immobilon-P® polyvinylidene fluoride transfer membrane (cat. no. IPVH00010; EMD Millipore, Billerica, MA, USA). Membranes were then blocked using 5% skimmed milk in PBS for 30-60 min at 20-25°C, and incubated overnight at 4°C with the primary antibodies. After washing with Tris-buffered saline containing 1% Tween-20 (cat. no. T1027; Biosesang, Seongnam, Korea), membranes were incubated with the corresponding secondary antibodies: Anti-rabbit (cat. no. 7074S, 1:5,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-mouse (cat. no. sc-516102, 1:5,000; Santa Cruz Biotechnology, Inc.) and anti-goat (cat. no. sc-2020, 1:5,000; Santa Cruz Biotechnology, Inc.). Immunodetection was performed using the PowerOpti-ECL western blotting detection reagent (cat. no. LR01-02; BioNote, Inc., Hwaseong, South Korea). The antibodies used in this study were as follows: Mouse monoclonal anti-p53 (cat. no. sc-126, 1:10,000; Santa Cruz Biotechnology, Inc.), rabbit monoclonal anti-AMPKα (cat. no. 2603P, 1:3,000) and anti-phosphorylated (p)-AMPKα (Thr172) (cat. no. 2535S, 1:3,000) (both Cell Signaling Technology, Inc.), rabbit polyclonal anti-CypA (cat. no. BML-SA296, 1:3,000), rabbit anti-HMGB1 (1:3,000, cat. no. ALX-210-964) (both Enzo Life Sciences, Inc.), rabbit polyclonal anti-PARP (cat. no. 9542S, 1:3,000), rabbit poly-clonal anti-p62/SQSTM1 (cat. no. 5114, 1:3,000) (both Cell Signaling Technology, Inc.), mouse monoclonal anti-LC3 (cat. no. ALX-803-082, 1:2,500; Enzo Life Sciences, Inc.) and mouse monoclonal anti-β-actin (cat. no. sc-47778, 1:5,000; Santa Cruz Biotechnology, Inc.). β-actin was used as a loading control. To measure the extracellular levels of necroptosis biomarkers (CypA and HMGB1), 80 µl media were collected from 10 ml cell culture media and mixed with 20 µl 5X SDS-PAGE loading buffer (cat. no. S2002; Biosesang), of which 20 µl was separated by 12% SDS-PAGE and underwent western blotting. The results of western blot analysis were semi-quantified using ImageJ software (version 1.51u; https://imagej.nih.gov/ij/) supplied by the National Institutes of Health (Bethesda, MD, USA) and relative intensity compared with β-actin is presented in the bar graphs.

Measurement of reactive oxygen species (ROS)

HCT116 p53+/+ and HCT116 p53−/− cells were cultured in RPMI media with or without 10% FBS for 24, 48 or 72 h. The cells were then collected in 15 ml conical tubes by centrifugation at ~200 × g for 2 min. Subsequently, ~5x104 cells/50 µl PBS were added to each well of a 96-well plate and 50 µl 200 µM 2',7'-dichlo-rodihydrofluorescein diacetate (DCFH-DA; cat. no. D399; Invitrogen; Thermo Fisher Scientific, Inc.) was added into each well using a multichannel-pipette (PIPETMAN; Gilson Incorporated, Middleton, WI, USA), in order to achieve a final concentration of 100 µM DCFH-DA ROS levels were detected by measuring fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 535 nm every 5 min for 30 min with a Gemini XPA microplate reader.

Measurement of mitochondrial membrane potential (MMP)

MMP was assessed using a Mito-ID Membrane Potential Cytotoxicity kit (cat. no. ENZ-51019; Enzo Life Sciences, Inc.), according to the manufacturer's protocol. Briefly, HCT116 p53+/+ and HCT116 p53−/− cells (~2×105 cells/well) were cultured in 96-well plates (BD Falcon; BD Biosciences, Franklin Lakes, NJ, USA) in RPMI media with or without 10% FBS for 48 or 72 h. Subsequently, Mito-ID membrane potential dye loading solution was added to each well and was incubated for 3 h at room temperature. MMP was assessed by measuring the resulting fluorescence with a Gemini XPA microplate reader, at an excitation wavelength of 480 nm and an emission wavelength of 590 nm.

Measurement of cellular ATP levels

Cellular ATP levels were measured using the Luminescent ATP Detection Assay kit (cat. no. ab113849; Abcam, Cambridge, MA, USA), according to the manufacturer's protocol. Briefly, HCT116 p53+/+ and HCT116 p53−/− cells were seeded at ~2×104/well in 96-well plates for 24 h. Subsequently, cells were incubated in RPMI media with or without 10% FBS and were cultured for 24 h. Detergent solution (50 µl) was then added to each well and the plate was agitated for 5 min at 600-700 rpm in an orbital shaker, in order to lyse the cells. Subsequently, 50 µl reconstituted substrate solution was added and the plate was agitated for a further 5 min. After 10 min in the dark, cellular ATP levels were measured by detecting luminescence using the GloMax® Discover multimode microplate reader (Promega Corporation, Madison, WI, USA).

Statistical analysis

Data are presented as the means ± standard deviation from three independent experiments. S tatistical analysis was performed using Student's t-test to compare two different groups or one-way analysis of variance, followed by Bonferroni's multiple comparisons test, to compare multiple groups. SPSS statistics version 23 was used to analyze data (IBM Corporation, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Cell proliferation is increased and cell death is reduced in HCT116 p53−/− human colon cancer cells under nutrient/serum starvation

To explore the effects of p53 on necroptosis under nutrient/serum starvation, the present study compared cell proliferation and cell death between HCT116 p53+/+ and HCT116 p53−/− cells after culturing them in CM or SS for 24, 48 or 72 h. HCT116 p53−/− cells exhibited a higher growth rate compared with HCT116 p53+/+ cells under both conditions (Fig. 1A). Cell images were also obtained under an optical microscope. Microscope images revealed that, in response to serum starvation, HCT116 p53+/+ cells stopped proliferating and exhibited the typical morphological alterations of cell death, including detachment from the bottom of the plate and generation of round-shaped, shrunk or swollen cells, whereas HCT116 p53−/− cells exhibited far fewer morphological alterations, although proliferation was greatly decreased (Fig. 1B). The cell viability assay also revealed a lower level of cell growth and viability in HCT116 p53+/+ cells compared with HCT116 p53−/− cells following serum deprivation for 24, 48 and 72 h (Fig. 1C). Taken together, these results suggested that p53 may suppress cell proliferation and enhance cell death under the stress of nutrient/serum starvation.

Figure 1 Effects of p53 on the proliferation and viability of HCT116 cells under conditions of serum starvation. (A) HCT116 p53+/+ and HCT116 p53−/− cells were cultured in RPMI-1640 with or without serum for the indicated durations (0, 24, 48 and 72 h). Growth rates were compared by Trypan blue staining following an initial plating density of ~106 cells per 100-mm plate; cells were counted for 4 days. Data are presented as the means ± standard deviation from three independent experiments. (B) HCT116 p53+/+ and HCT116 p53−/− cells were seeded at a density of ~106 cells/100-mm plate and were grown in RPMI-1640 media with or without serum. Morphological images were captured under an optical microscope at ×100 magnification. (C) Viable cells were evaluated by measuring the absorbance at 450 nm. Data are presented as the means ± standard deviation from three independent experiments. *P<0.05. CM, complete media; SS, serum-starved media.

Necroptosis is reduced in HCT116 p53−/− cells in response to nutrient/serum starvation

HCT116 p53−/− cells exhibited reduced cell death under serum deprivation. To investigate whether loss of p53 exerts inhibitory effects on necroptosis, the extracellular levels of necroptosis biomarkers, CypA and HMGB1, were detected using western blotting after HCT116 p53+/+ and HCT116 p53−/− cells were grown in RPMI media with or without serum for 24, 48 and 72 h (Fig. 2). The export of both proteins into the extracellular media was significantly increased after 48 h serum starvation in both cell lines, indicating the induction of necroptosis (Fig. 2A and B). However, the extracellular levels were much higher for HCT116 p53+/+ cells than for HCT116 p53−/− cells after 72 h, thus indicating that necroptosis was suppressed in HCT116 p53−/− cells (Fig. 2A and B). In addition, PARP cleavage was increased in HCT116 p53+/+ cells compared with in HCT116 p53−/− cells, thus suggesting that the loss of p53 suppressed apoptotic cell death, as well as necroptosis (Fig. 2A and B). The expression levels of p53 were decreased in a time-dependent manner in HCT116 p53+/+ cells under the conditions of serum starvation (Fig. 2A and B).

Figure 2 Necroptosis is reduced in HCT116 p53−/− cells under serum starvation. (A) HCT116 p53+/+ and HCT116 p53−/− cells were grown in CM or SS for 24, 48 and 72 h. Western blotting was performed to detect representative biomarkers: CypA and HMGB1 for necroptosis, and PARP for apoptosis. β-actin was used as a loading control. All experiments were performed independently at least three times and representative data are shown. (B) Western blotting was semi-quantified using ImageJ. *P<0.05. (C) Cells (2x105) were cultured in CM or SS for 72 h after seeding in a 35-mm confocal dish. Cells were observed under a confocal microscope (magnification, ×200) after staining with Annexin V-EnzoGold (enhanced Cyanine-3) to detect apoptosis (yellow staining) and (7-AAD) to detect necroptosis (red staining). (D) Results were semi-quantified using ImageJ. *P<0.05. 7-AAD, 7-aminoactinomycin D; CM, complete media; CypA, cyclophilin A; HMGB1, high mobility group box 1; PARP, poly (ADP-ribose) polymerase; SS, serum-starved media.

The induction of necroptosis and apoptosis in HCT116 p53+/+ and HCT116 p53−/− cells was examined by confocal microscopy; 7-AAD and Annexin V staining were used to detect necroptosis and apoptosis, respectively (Fig. 2C and D). In response to serum starvation for 72 h, marked 7-AAD and Annexin V fluorescence was detected in both cell lines, thus indicating that necroptosis and apoptotic cell death pathways were induced; much higher levels of necroptosis and apoptosis were detected in HCT116 p53+/+ cells compared with HCT116 p53−/− cells (Fig. 2C and 2D).

Taken together, these results suggested that the absence of p53 in HCT116 cells may suppress necroptosis to a higher degree than in p53+/+ cells under serum starvation.

Effects of p53 on mitochondria-mediated necroptosis under serum starvation

To determine the role of p53 in necroptosis, it was hypothesized that p53 may induce it by regulating mitochondrial functions, including the production of ROS, maintenance of the MMP, or generation of ATP. These functions are understood to be strongly associated with several types of cell death; very high levels of ROS, and low levels of MMP and ATP, are known to accelerate cell death pathways (30-39). Therefore, the present study detected the cellular levels of ROS and ATP, as well as the MMP, in HCT116 p53+/+ and HCT116 p53−/− cells under serum starvation.

Cellular ROS levels were detected in HCT116 p53+/+ and HCT116 p53−/− cells cultured in media with or without serum for various durations. The results revealed that there was no significant difference between the cell lines until 48 h (data not shown). However, ROS levels were significantly increased after 72 h in HCT116 p53+/+ cells, whereas they were slightly decreased in HCT116 p53−/− cells (Fig. 3A). These data suggested that p53-mediated necroptosis, at least in part, may occur by increasing cellular ROS levels under serum starvation.

Figure 3 Effects of p53 on cellular levels of ROS and ATP, as well as the MMP, in response to serum starvation. HCT116 p53+/+ and HCT116 p53−/− cells were incubated in media with or without serum for the indicated durations, and (A) cellular levels of ROS, (B) MMP and (C) cellular levels of ATP were measured. Data are presented as the means ± standard deviation from three independent experiments. *P<0.05. CM, complete media; DCF, dichlorofluorescein; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; SS, serum-starved media.

Another mitochondrial mediator of cell death, MMP, was also compared between HCT116 p53+/+ and HCT116 p53−/− cells. Cells were grown in SS for 24, 48 and 72 h. MMP was markedly decreased after 72 h in HCT116 p53+/+ cells, whereas no significant difference was observed in HCT116 p53−/− cells (Fig. 3B). These data indicated that p53 may induce necroptosis by decreasing MMP in response to serum starvation.

Notably, several types of cell death are accompanied by ATP depletion (40-43). Therefore, the effects of p53 on cellular ATP levels were assessed in response to serum starvation. The results indicated that ATP levels were decreased in both cell lines. However, it was unexpectedly observed that ATP levels were more markedly decreased in HCT116 p53−/− cells, whereas they were only slightly reduced in HCT116 p53+/+ cells after 24 h (Fig. 3C).

Taken together, these findings supported the hypothesis that p53 may accelerate necroptosis, at least partially, by increasing ROS levels and decreasing MMP in HCT116 cells under serum starvation.

AMPK activation is increased in p53−/− cells under serum starvation

It is well known that nutrient starvation (e.g., depletion of glucose, amino acids or serum) leads to decreased intracellular ATP levels and that further extreme depletion may eventually induce cell death. Based on the importance of cellular ATP levels with regards to cell survival, it was expected that ATP levels would be lower in HCT116 p53+/+ cells, due to the higher levels of necroptosis. However, the present data revealed that ATP levels were lower in HCT116 p53−/− cells in both CM and SS (Fig. 3C). In addition, much higher levels of proliferation were detected in HCT116 p53−/− cells compared with in HCT116 p53+/+ cells under serum starvation (Fig. 1). Therefore, it was hypothesized that ATP levels may be lower in p53−/− cells due to uncontrolled rapid cell proliferation and growth, which may cause much higher consumption of ATP. ATP depletion is the main signal that induces AMPK activation by phosphorylation of Thr172 (44-46). The present study demonstrated that serum starvation markedly increased phosphorylation of AMPK at Thr172 in HCT116 p53−/− cells compared with in HCT116 p53+/+ cells. Even though there was a slight variation in the expression levels of total AMPK (Fig. 4A and B), the ratio of p-AMPK (Thr172) to total AMPK (p-AMPK/AMPK) indicated the significant increase in levels of AMPK phosphorylation (Fig. 4B). Therefore, it was hypothesized that AMPK activation may be involved in necroptotic cell death in a p53-dependent manner in response to serum depletion.

Figure 4 Negative effects of p53 on AMPK activation in response to serum starvation. (A) HCT116 p53+/+ and HCT116 p53−/− cells were grown in CM or SS for 24, 48 and 72 h. Western blotting was used to determine the expression levels of AMPK, p-AMPK (Thr172), CypA, p62/SQSTM1 and LC3. β-actin was used as a loading control. All experiments were performed independently at least three times and representative data are shown. (B) Western blots were semi-quantified using ImageJ. *P<0.05. The ratio of p-AMPK (Thr172) to total AMPK is shown as p-AMPK/AMPK. AMPK, AMP-activated protein kinase; CM, complete media; CypA, cyclophilin A; LC3, microtubule-associated protein 1A/1B-light chain 3; p, phosphorylated; SQSTM1, sequestome 1; SS, serum-starved media.

A well-known function of activated AMPK is the induction of autophagy, as one of the master regulators. Accordingly, autophagy was analyzed by detecting two well-known biomarkers, p62/SQSTM1 and LC3. The results revealed that serum deficiency decreased the expression levels of p62/SQSTM1 and increased the conversion ratio of LC3-I to LC3-II in HCT116 p53−/− and HCT116 p53+/+ cells, thus indicating autophagic induction (Fig. 4A and B). However, p62/SQSTM1 protein levels were decreased to a slightly higher level in HCT116 p53−/− cells after 24 h (Fig. 4A and B). In addition, the conversion ratio from LC3-I to LC3-II was also greater in HCT116 p53−/− cells after 48 h (Fig. 4A and B). These data suggested that AMPK may be activated in HCT116 p53−/− cells at a higher level compared with in HCT116 p53+/+ cells.

Taken together, these results indicated that AMPK activation was highly induced in p53−/− compared with in p53+/+ cells, probably due to reduced ATP levels.

AMPK activation alleviates necroptosis in HCT116 p53−/− cells but not in HCT116 p53+/+ cells under serum starvation

To investigate whether activated AMPK is responsible for the inhibition of necroptotic cell death in p53−/− cells, the effects of AMPK activation on necroptosis were examined using an AMPK inhibitor and AMPK activator.

AMPK activity was suppressed using the potent and selective AMPK inhibitor, DM. HCT116 p53+/+ and HCT116 p53−/− cells were exposed to CM or SS accompanied by DM at various doses (0, 5 and 10 µM) for 48 h (Fig. 5A and B). Phosphorylation of AMPK at residue Thr172 was significantly decreased following treatment with 5 and 10 μM DM, thus indicating that AMPK activation was successfully inhibited (Fig. 5A and B). Notably, the export of CypA was markedly increased in HCT116 p53−/− cells in response to DM, even in CM (Fig. 5A and B); in addition, no significant differences were detected in CypA export between HCT116 p53+/+ and HCT116 p53−/− cells following DM treatment (Fig. 5A and B). These findings strongly suggested that serum starvation-induced AMPK activation effectively suppressed necroptosis in the p53−/− cells.

Figure 5 AMPK activation alleviates necroptosis in HCT116 p53−/− cells but not in HCT116 p53+/+ cells under serum starvation. HCT116 p53+/+ and HCT116 p53−/− cells were cultured in CM or SS for the indicated durations. Cells were treated with (A and B) 0, 5 and 10 µM DM for 48 h or (C and D) 0, 75, 150 and 300 µM AICAR for 48 h. (A and C) Lysates were obtained and analyzed by western blotting, in order to detect phosphorylation of AMPK on the Thr172 residue, and the expression levels of CypA and p53. β-actin was used as a loading control. (B and D) Western blotting results were semi-quantified using ImageJ. The experiment was performed independently at least three times and representative data are shown. *P<0.05. Numbers represent relative intensity versus β-actin. The ratio of p-AMPK (Thr172) to total AMPK is shown as p-AMPK/AMPK. AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK, AMP-activated protein kinase; CM, complete media; CypA, cyclophilin A; DM, dorsomorphin dihydrochloride; p, phosphorylated; SS, serum-starved media.

Secondly, AMPK activation was enhanced by AICAR, which mimics the effects of AMP (21,47-49). HCT116 p53+/+ and HCT116 p53−/− cells were cultured in CM and SS with various concentrations of AICAR (75, 150 and 300 µM) for 48 h (Fig. 5C and D). The results revealed that phosphorylation of AMPK at residue Thr172 was increased in HCT116 p53+/+ cells following AICAR treatment, thus indicating that AMPK activity was successfully enhanced (Fig. 5C and D). AMPK activation was consistently high in HCT116 p53−/− cells even prior to AICAR treatment. AICAR treatment slightly increased CypA export into the extracellular media in HCT116 p53+/+ and HCT116 p53−/− cells maintained in CM (Fig. 5C and D). Notably, extracellular CypA levels were not decreased but were markedly increased in HCT116 p53+/+ cells, whereas no alteration was detected in HCT116 p53−/− cells under SS, indicating that enhanced AMPK activation by AICAR can inhibit necroptosis only in HCT116 p53−/− cells (Fig. 5C and D). These findings strongly suggested that AMPK activation may suppress necroptosis only in p53−/− cells in response to serum starvation.

The present study revealed that inhibition and activation of AMPK promoted necroptosis in HCT116 p53+/+ cells under serum starvation, in which p53 protein expression levels were increased (Fig. 5). This may be because abnormal AMPK activity triggered upregulation of p53 and induced necroptosis.

Taken together, these observations suggested that AMPK activation through phosphorylation of Thr172 enhanced inhibition of necroptosis in p53−/− cells, but not in p53+/+ cells, under nutrient/serum starvation.

AMPK inhibits necroptosis through the suppression of ROS generation in p53−/− cells

To address how AMPK activation suppresses necroptosis in HCT116 p53−/− cells, the effects of AMPK on cellular levels of ROS and ATP, and on the MMP, were assessed in HCT116 p53+/+ and HCT116 p53−/− cells. The results indicated that AMPK inhibition by DM significantly increased ROS generation in HCT116 p53−/− cells (Fig. 6A). However, no significant difference was detected in MMP between the HCT116 p53+/+ and HCT116 p53−/− cells, thus indicating that reductions in MMP by AMPK inhibition are independent of p53 (Fig. 6B). Furthermore, DM reduced ATP levels more than SS alone in both cell lines; however, ATP levels were slightly higher in HCT116 p53−/− cells compared with in HCT116 p53+/+ cells following DM treatment (Fig. 6C).

Figure 6 Inhibitory role of AMP-activated protein kinase on ROS generation in HCT116 p53−/− cells in response to serum starvation. HCT116 p53+/+ and HCT116 p53−/− cells were cultured in CM or SS for 24 or 48 h. Cells were treated with 10 µM DM for the indicated durations, and (A) cellular ROS, (B) MMP and (C) ATP levels were measured. The experiment was performed independently at least three times and representative data are shown. *P<0.05. CM, complete media; DCF, dichlorofluorescein; DM, dorsomorphin dihydrochloride; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; SS, serum-starved media.

Taken together, these data indicated that highly activated AMPK may be at least partially responsible for the inhibition of necroptosis via suppression of ROS generation in p53−/− cells.

Discussion

Escape from cell death, particularly apoptosis, is a serious problem in current cancer research. Therefore, it might be considered an effective therapeutic strategy to trigger several types of cell death at the same time. The present study focused on necroptosis, in addition to apoptosis, with the eventual aim of identifying a novel class of combined targeted therapeutics. The present study revealed that AMPK serves a different role in the regulation of necroptosis in the presence and absence of p53. It was demonstrated that a much lower level of necroptosis was detected in p53−/− cells, and serum starvation-induced necroptosis was suppressed by enhancing AMPK activation.

p53 is involved in the regulation of several types of cell death, including apoptosis, CIA, autophagy-mediated cell death, necroptosis, entosis, anoikis, paraptosis, pyroptosis, ferroptosis, mitotic catastrophe and efferocytosis (17). Among these types of cell death, several studies have reported vital roles for p53 in necroptosis. Tu et al demonstrated that p53 can activate necroptosis in response to ROS-induced DNA damage through the activation of the lysosomal cysteine protease, cathepsin Q (50). Furthermore, it has been suggested that p53 accumulates in the mitochondrial matrix, complexes with CypD, and induces necroptosis in response to oxidative stress (31,32). Vaseva et al suggested that p53 causes necroptosis by controlling the opening of the mitochondrial permeability transition pore (32). Montero et al reported that p53 triggers necroptosis by increasing PARP-1 activity, and inducing depletion of NAD and ATP (33). In spite of these studies, the role of p53 in necroptosis remains unclear and more information is required.

There are discordant results regarding the functions of p53 in cell death regulation. Different combinations of master regulators may explain it, each of which has various functions, such as AMPK. The present results revealed that serum starvation-induced AMPK activation suppressed necroptosis in p53−/− cells but not in p53+/+ cells. In contrast to these data, two research groups proposed that AMPK induced necroptosis and apoptosis (51-53). For example, Koo et al suggested that AMPK activation, resulting from impaired mitochondrial oxidative phosphorylation, induces necroptosis in human lung epithelial cells (51). Other research groups revealed that AMPK activation induces apoptosis in liver cells by activating signaling pathways, including c-Jun N-terminal kinase and caspase-3 (52,53). However, they did not consider the effects of p53 in their research. It has been reported that the metabolic environmental conditions and pathways that activate AMPK differentially regulate cell proliferation or viability in cancer and normal cells (19,54). Only a few studies have reported on the highly complex interrelationship between p53 and AMPK in cell death. For example, Okoshi et al proposed that AMPK activation induces p53-dependent apoptosis in response to energetic stress (53). Huang et al revealed that AMPK inhibition by DM induces apoptosis in skin cancer cells via a p53-dependent pathway (55). The same effect of DM was reported in human colorectal cancer cells, with regards to the induction of apoptosis and necroptosis (18). Lee et al also demonstrated that AMPK induces p53 acetylation via phosphorylation and inactivation of sirtuin 1 in liver cancer cells (56). In addition, p53 can regulate autophagy, even though its regulation is controversial, indicating an effect of p53 on AMPK (57-59). However, the exact mechanism underlying the interaction between p53 and AMPK remains elusive.

The present study demonstrated that autophagy was induced at a higher level in HCT116 p53−/− cells, probably due to enhanced AMPK activation. Numerous studies have reported that autophagy protects cells from cell death under various types of cell death-inducing stress, such as anticancer treatment and severe nutrient starvation (60-62). These data suggested that increased activation of AMPK in HCT116 p53−/− cells may induce higher levels of autophagy and eventually suppress other types of cell death in response to serum starvation; however, the association between autophagy and necroptosis requires further study.

RIP1-RIP3-MLKL is known to be a core signaling pathway in necroptosis. The RIP1-RIP3-MLKL-dependent necroptosis machinery may be considered a primary mechanism for suppressing tumorigenesis and cancer progression. Cancer cells may suppress necroptosis by downregulating or inducing functional mutations in RIP1, RIP3 and MLKL. Previous studies indicated that RIP1 expression levels are decreased in only a few cancer cells, whereas RIP3 expression is downregulated in numerous cancer cells (5,63-65). For example, HeLa cervical cancer cells are well known for their resistance to necroptosis, and they express normal levels of RIP1 but no RIP3 expression (66,67). Notably, HCT116 cells have also been reported to lack RIP3 protein (5,68). However, in the present study, HCT116 p53+/+ cells underwent necroptosis under the stress of serum starvation; therefore, it remains to be determined as to how HCT116 cells undergo necroptosis. Elucidating the mechanism underlying necroptosis will be of great interest.

In conclusion, the present study proposed a plausible model, in which activation of AMPK through phosphorylation of Thr172 may inhibit necroptosis in the absence of p53 in response to serum starvation (Fig. 7). This study proposed that AMPK may be a combined target to efficiently kill cancer cells with defective p53 under low-nutrient conditions. These data provided information regarding a signaling network between p53 and AMPK in necroptosis, which may be provoked in response to serum starvation.

Figure 7 Summary of the inhibitory role of AMPK in necroptosis in p53−/− cells under the stressful condition of nutrient/serum starvation. Nutrient/serum depletion increased ROS generation, and decreased MMP and ATP levels in HCT116 p53+/+ cells, resulting in necroptosis. Notably, cellular ATP levels were markedly decreased in HCT116 p53−/− cells at a much higher level, compared with in HCT116 p53+/+ cells. This may lead to increased phosphorylation of AMPK on residue Thr172; ultimately, activation of AMPK inhibited necroptosis by negatively regulating cellular ROS production. AMPK, AMP-activated protein kinase; MMP, mitochondrial membrane potential; ROS, reactive oxygen species.

Funding

The present study was supported by Sookmyung Women's University (2015).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

DDTL conducted the majority of the experiments, analyzed data and drafted the manuscript. SJ conceptualized the study, developed the general experimental design, analyzed data and revised the manuscript. NTNQ was involved in the use of ImageJ, statistical analysis and manuscript preparation. ZS assisted in developing the study design and preparing the manuscript. BSL contributed to confocal analysis and preparation of the manuscript. SK, HGL and HJL assisted in experimental preparation, cell culturing and maintenance, and preparation of manuscript. MSL made substantial contributions to conception and experimental design, revised the manuscript and gave final approval of the version to be published. All authors read and approved the manuscript and agree to be accountable for all aspects of this research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated.

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.

Abbreviations:

Acknowledgments

The authors of this study are grateful to Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD, USA) for generously donating the colorectal carcinoma cell lines, HCT116 p53+/+ and HCT116 p53−/−, for use in the experiments.

References