A high dependence on aerobic glycolysis, known as the Warburg effect, is one of the metabolic features exhibited by tumor cells. Therefore, targeting glycolysis is becoming a very promising strategy for the development of anticancer drugs. In the present study, it was investigated whether pre-adaptation of malignant mesothelioma (MM) cells to an acidic environment was associated with a metabolic shift to the Warburg phenotype in energy production, and whether apigenin targets acidosis-driven metabolic reprogramming. Cell viability, glycolytic activity, Annexin V-PE binding activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential, ATP content, western blot analysis and spheroid viability were assessed in the present study. MM cells pre-adapted to lactic acid were resistant to the anticancer drug gemcitabine, increased Akt activation, downregulated p53 expression, and upregulated rate-limiting enzymes in glucose metabolism compared with their parental cells. Apigenin treatment increased cytotoxicity, Akt inactivation and p53 upregulation. Apigenin also reduced glucose uptake along with downregulation of key regulatory enzymes in glycolysis, increased ROS levels with loss of mitochondrial membrane potential, and downregulated the levels of complexes I, III and IV in the mitochondrial electron transport chain with intracellular ATP depletion, resulting in upregulation of molecules mediating apoptosis and necroptosis. Apigenin-induced alterations of cellular responses were similar to those of Akt inactivation by Ly294002. Overall, the present results provide mechanistic evidence supporting the anti-glycolytic and cytotoxic role of apigenin via inhibition of the PI3K/Akt signaling pathway and p53 upregulation.
Living organisms require a continuous influx of energy to perform the various physiological activities necessary for cell growth and survival. Adenosine triphosphate (ATP), the energy currency of cells, is produced by glycolysis in the cytoplasm and oxidative phosphorylation in the mitochondria. Both processes occur essentially in most cells. However, oxidative phosphorylation predominates, and glycolysis is increased when there is insufficient oxygen supply, such as in muscle cells during exercise. However, tumor cells rely more on glycolysis than oxidative phosphorylation for ATP production even under adequate oxygenation, which is called the Warburg effect (
The phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway regulates various cellular processes including cell survival, proliferation, metabolism, motility and apoptosis (
Apigenin (4′,5,7-trihydroxyflavone) not only induces apoptosis, autophagy, cell cycle arrest, but also inhibits angiogenesis and metastasis in different types of cancer cells (
A variety of evidence indicates that inhibition of glycolysis in cancer cells results in ATP depletion and cell death, which is particularly effective in cells with high glycolytic activity and resistance to anticancer drugs (
In the present study, it was aimed to extend the previous investigation into the anticancer role of apigenin and to evaluate its effects on energy metabolism and cell death using two human MM cell lines pre-adapted to lactate. The cell lines were established by continuously exposing MSTO-211H and H2452 cells to 3.8 µM lactic acid through 4 serial passages for 15 days. The current data provide mechanistic evidence suggesting a close link between the PI3K/Akt pathway, p53 and glycolysis in the induction of apoptosis and necroptosis. The current study deepens our understanding of the novel role of apigenin in MM cells.
Human MM cell lines MSTO-211H and H2452 were purchased from the American Type Culture Collection. Acidic pre-adapted cells designated as MSTO-211HAcT (cat. no. CRL-2081) and H2452AcT (cat. no. CRL-5946) were established by continuously exposing MSTO-211H and H2452 cells, respectively, to 3.8 µM lactic acid through 4 passages for 15 days. Cells were cultured in in RPMI-1640 medium (Welgene, Inc.) containing 3.8 µM lactic acid and 5% fetal bovine serum (Welgene, Inc.), and then treated with increasing concentrations (10, 20, 40, 80 and 160 µM) of gemcitabine for cell viability assay. Cells were treated with 20 µM Ly294002 (Merck KGaA) and 30 µM apigenin (MilliporeSigma) alone or in combination for 48 h. For combination treatment, pretreatment with Ly294002 for 2 h followed by treatment with apigenin without removing Ly294002. As a negative control, cells were treated with 0.1% dimethyl sulfoxide. Cell viability was measured by MTT assay as previously described (
Total cell lysates were extracted with 1X RIPA buffer and protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, Inc.). The extracted proteins (40 µg/well) were separated on 4–12% NuPAGE gels (Thermo Fisher Scientific, Inc.) and then transferred to polyvinylidene fluoride membrane (GE Healthcare Life Sciences). The membranes were blocked with 1X casein solution (cat. no. 37528; Thermo Fisher Scientific, Inc.) for 2 h at room temperature, incubated overnight at 4°C with primary antibodies, and then with horseradish-peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Reactive proteins were visualized with an enhanced chemiluminescence detection kit (Cyanagen Srl) using X-ray film. TINA 2.09 software (Raytest Isotopenmessgeraete GmbH) was used for densitometric analysis of protein bands in western blots. The following antibodies were used to detect each protein. Oxphos human WB antibody cocktail (cat. no. 45-8199) and antibodies to HK-I (1:500; cat. no. 2024), HK-II (1:500; cat. no. 2867), PFKP (1:500; cat. no. 8164), pyruvate dehydrogenase (1:500; cat. no. 3205), phosphorylated (p)-MLKL (1:500; cat. no. 91689), p-RIP3 (1:500; cat. no. 93654; 1:500), p-Akt (1:500; cat. no. 9271), Akt (1:500; cat. no. 9272), PARP (1:500; cat. no. 9542), cleaved PARP (1:500; cat. no. 9541), caspase-3 (1:500; cat. no. 14220) and cleaved caspase-3 (1:500; cat. no. 9664) were all purchased from Cell Signaling Technology, Inc. and used for antigen detection. Goat anti-rabbit IgG-HRP (1:5,000; cat. no. sc-2004), goat anti-mouse IgG-HRP (1:5,000; cat. no. sc-2005) and anti-p53 antibody (1:500 cat. no. sc-126) were all purchased from Santa-Cruz Biotechnology, Inc. The membranes were re-probed using anti-β-actin (1:10,000; cat. no. A2228; Sigma-Aldrich; Merck KGaA), anti-RIP3 (1:1,000; cat. no. 13526; Cell Signaling Technology, Inc.), and anti-MLKL (1:1,000; cat. no. 14993; Cell Signaling Technology, Inc.) as the loading controls.
Cells (105 cells/well) were seeded on six-well culture plates and incubated overnight in lactic acid-containing RPMI-1640 medium. Cells were treated with or without 20 µM Ly294002 for 2 h, then 30 µM apigenin was added to each well without removing Ly294002 and incubated for 48 h. After trypsinization, cells were harvested by centrifugation at 500 × g for 7 min, and then resuspended in serum-free RPMI-1640 medium containing 2′,7′-dichlorodihydrofluorescein diacetate (10 µM) and rhodamine 123 (30 nM; both from Sigma-Aldrich; Merck KGaA) in the dark at 37°C for 30 min to measure the levels of ROS and mitochondrial membrane potential, respectively. The fluorescence intensity of the cells was measured with a MACSQuant analyzer and MACSQuantify software version 2.5 (Miltenyi Biotec GmbH).
Analysis of apoptotic and necrotic cell distribution was performed according to the instructions provided with the Muse Annexin V & Dead Cell Assay Kit (cat. no. MCH100105; Merck KGaA). Briefly, cells were treated with or without 20 µM Ly294002 for 2 h, then 30 µM apigenin was added to each well without removing Ly294002 and incubated for 48 h. Cells were trypsinized, and collected in a culture medium supplemented with Muse Annexin V & Dead Cell reagent. The cells were then analyzed with Muse cell analyzer (Merck KGaA). Annexin V-phycoerythrin (PE)-positive apoptotic and 7-AAD-positive necrotic cells were detected using Annexin V-PE and 7-amino-actinomycin D (7-AAD) double staining.
Cell cycle distribution at each phase was determined via propidium iodide (PI) staining as previously described (
Spheroid culture was performed in an ultra-low attachment 96-well plates as previously described (
SPSS version 17.0 software (SPSS, Inc.) was used for statistical analysis of experimental data. Statistical analysis was performed by one-way ANOVA and Tukey's post hoc correction. Data are presented as the mean ± standard deviation (S.D.) for three independent experiments. P<0.05 was considered to indicate a statistically significant difference.
To evaluate the effect of pre-adaptation to an acidic environment, MSTO-211HAcT and H2452AcT cells were cultured for 24, 48 and 72 h in media containing 3.8 µM lactic acid. During this period, the proliferative status was measured and compared with their parental MSTO-211H and H2452 cells. As demonstrated in
To determine the role of Akt as an upstream signaling molecule regulated by apigenin in MSTO-211HAcT and H2452AcT cells, the additive effect of PI3-kinase/Akt inhibition on apigenin-induced cell death was investigated after pre-inhibition of the PI3-kinase using Ly294002 in cultures containing 3.8 µM lactic acid. An MTT assay was performed to determine the viability of these cells upon treatment with increasing concentrations of apigenin for 48 h. As revealed in
The majority of control cells adhered to cell culture plates, but when cells were exposed to Ly294002 and/or apigenin for 48 h, the cells detached from the surface of the culture plate, increasing the number of cells floating in the culture medium (
Next, the effect of apigenin-induced inhibition of the PI3-kinase/Akt pathway on cellular energy metabolism was investigated. Inhibition of the Akt activity by apigenin and/or Ly294002 downregulated the expression of HK-I, HK-II, and PFKP (
Based on the results of the 2D monolayer cultures, the effect of Akt inhibition on the growth of spheroids and the expression levels of the glycolytic, apoptotic and necroptotic proteins in 3D spheroid cultures were further investigated. A two-color fluorescence assay was used to identify live and dead cells. Cell-permeable FDA is converted into green fluorescent by esterases within living cells, whereas PI enters the nucleus of dead or dying cells and emits red fluorescence upon binding to DNA. As demonstrated in
To further elucidate the cytotoxic mechanism(s) of apigenin, it was investigated whether apigenin targeted enzymes involved in energy metabolism and what the critical upstream signaling pathway is in this process. In the present study, it was found that pre-adaptation of MM cells to an acidic medium containing lactic acid induced a metabolic shift towards enhanced glycolysis, along with Akt activation and p53 downregulation. Apigenin and/or Ly294002 treatment increased both apoptosis and necroptosis of MSTO-211HAcT and H2452AcT cells by downregulating key enzymes of glucose metabolism and inducing mitochondrial dysfunction, at least through Akt inactivation and p53 upregulation. These findings were further validated in 3D spheroid cultures.
Increased cell growth, increased tolerance to the anticancer drug gemcitabine and upregulation of glycolytic enzymes, observed in MM cells pre-adapted to lactic acid-containing medium, suggested the critical role of lactic acid in the metabolic shift to a Warburg phenotype. Recently, it has been demonstrated that pre-culturing of human fibroblasts in medium containing lactic acid promotes the metabolic shift from oxidative phosphorylation to glycolysis by activating transcription of the glycolytic genes through ROS-mediated stabilization of hypoxia inducible factor-1α (HIF-1α) (
Aerobic glycolysis, one of the metabolic features found in tumor cells, generates an excess of lactate and H+ in the cytoplasm, which are released extracellularly, causing local acidification (
Akt inhibition and p53 upregulation in response to apigenin in the current study represent an important mechanism underlying the cytotoxicity of apigenin to MM cells. In line with this finding, blockade of active Akt using Ly294002 demonstrated the importance of Akt-p53 network in understanding the role of apigenin in both targeting glycolysis and inducing apoptosis and necroptosis of MM cells. Notably, exposure to Ly294002 and apigenin, alone or in combination, increased cytotoxicity, as demonstrated by reduced cell viability together with increased floating cells, increased sub-G0/G1 peak with a transient delay in the G2/M phase, increased Annexin V-PE(+) cell fraction, chromatin condensation and nuclear fragmentation on DAPI staining, and upregulation of apoptosis-and necroptosis-inducing molecules. However, the combination of apigenin and Ly294002 further potentiated the apoptotic, necroptotic, and anti-glycolytic effects compared with apigenin or Ly294002 alone, with certain synergistic effects. It suggested that a series of cellular responses to induce apigenin-induced cytotoxicity are mediated primarily through inhibition of the PI3K/Akt pathway, but other factors or potential pathways, including the NF-kB, MAPK/ERK and c-JNK pathways, may also be involved in this process (
Apigenin and/or Ly294002 also downregulated HK-I, HK-II and PFKP, which are rate-limiting enzymes in glycolysis. Increased glucose concentrations in culture medium of apigenin and/or Ly294002-treated group indicates a decrease in glucose utilization, corroborating the results of western blotting showing inhibition of the glycolytic pathway. Downregulation of oxidative phosphorylation enzymes and loss of mitochondrial membrane potential, indicative of mitochondrial dysfunction, limit the ATP production and consequently induce ATP depletion in cells treated with apigenin alone or with Ly294002. Mitochondria play a pivotal role in maintaining cellular redox homeostasis and energy levels and in regulating various types of cell death, including apoptosis and necroptosis (
The results of the present study, for the first time to the best of our knowledge, revealed that apigenin-mediated inhibition of Akt leads to reduced glycolysis, and thereby the concurrent induction of apoptosis and necroptosis (
As aforementioned, cancer cells rely largely on aerobic glycolysis rather than efficient oxidative phosphorylation in glucose metabolism. Given that strategies targeting cancer-specific energy metabolism provide selective growth inhibition in rapidly proliferating cancer cells, as a dual inhibitor targeting both aerobic glycolysis and mitochondrial function, apigenin offers potential clinical advantage as a promising therapeutic candidate for MM. Further studies investigating the metabolic regulation of tumor cells are needed to deepen our understanding of the mechanism(s) involved in apigenin-induced necroptosis.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
SL and SH conceived the present study. YL, MC and SL performed the acquisition, analysis and interpretation of data. YL and KP conducted all the flow cytometric analysis. SL and YL wrote the original draft. YL and SL confirm the authenticity of all the raw data. All authors provided critical feedback, read and approved the final version of the manuscript. SL were in charge of overall direction and planning.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Enhancement of the Warburg-like phenotype in MSTO-211HAcT and H2452AcT cells pre-adapted to lactic acid. Cells were cultured in the complete medium containing 3.8 µM lactic acid for the indicated times, otherwise 48 h. (A) Cell viability was measured by MTT assay during cell culture for 72 h. (B) Cells were treated with increasing concentrations of gemcitabine, followed by MTT assay to measure cell viability. (C) The expression levels of rate-limiting enzymes in glucose metabolism. (D) Activities of hexokinase and pyruvate dehydrogenase. (E) The expression levels of complexes I–V in mitochondrial electron transport chain. (F) The levels of p-Akt, Akt, and p53 proteins. Bar graphs present densitometric analysis of western blot images normalized to β-actin. *P<0.05 vs. respective MSTO-211H or H2452 cells. 1, MSTO-211H; 2, MSTO-211HAcT; 3, H2452; 4, H2452AcT; APG, apigenin; HK, hexokinase; PFKP, phosphofructokinase platelet; PDH, pyruvate dehydrogenase; NDUFB8, NADH-ubiquinone oxidoreductase subunit B8 (complex I); SDHB, succinate dehydrogenase complex iron sulfur subunit B (complex II); UQCRC2, ubiquinone-cytochrome C reductase core protein 2 (complex III); COX II, mitochondrial cytochrome C oxidase subunit II (complex IV); ATP5A, ATP synthase F1 subunit alpha (complex V); p-, phosphorylated.
Cytotoxic effects of apigenin and/or Ly294002 on MSTO-211HAcT and H2452AcT cells. (A) Cells were treated with increasing concentrations of apigenin (0, 10, 20, 30, 40, 50 and 60 µM) for 48 h. The percentage of viable cells was determined by comparison with the results obtained using DMSO-treated control cells (100%). Cells were treated with or without Ly294002 (20 µM, 2 h) prior to apigenin treatment (30 µM, 48 h) in RPMI-1640 medium containing 3.8 µM lactic acid. (B) Cell morphology. (C) Percentage of cell viability. (D) The levels of p-Akt and p53 proteins. (E) Cell cycle distribution at each phase. *P<0.05 vs. respective control cells. Arrows indicate sub-G0/G1 peak. APG, apigenin; LY, Ly294002; p-, phosphorylated.
Concurrent induction of apoptosis and necroptosis by apigenin and/or Ly294002 in MSTO-211HAcT and H2452AcT cells. Cells were treated with or without Ly294002 (20 µM, 2 h) prior to apigenin treatment (30 µM, 48 h) in RPMI-1640 medium containing 3.8 µM lactic acid. (A) Fraction of live and dead cells by Annexin V-PE binding assay. (B) Nuclear morphology by DAPI staining (scale bar, 5 µm). (C) The expression levels of apoptosis- and necroptosis-inducing proteins. *P<0.05 vs. respective control cells. Bar graphs present densitometric analysis of western blot images normalized to β-actin. APG, apigenin; LY, Ly294002; p-, phosphorylated.
Anti-glycolytic effects of apigenin and/or Ly294002 in MSTO-211HAcT and H2452AcT cells. Cells were treated with or without Ly294002 (20 µM, 2 h) prior to apigenin treatment (30 µM, 48 h) in RPMI-1640 medium containing 3.8 µM lactic acid. (A) The levels of rate-limiting enzymes in glucose metabolism. Bar graphs present densitometric analysis of western blot images normalized to β-actin. (B) Activities of HK and PDH. (C) Glucose concentration in the culture medium. *P<0.05 vs. respective control cells. APG, apigenin; LY, Ly294002; PFKP, phosphofructokinase platelet; HK, hexokinase; PDH, pyruvate dehydrogenase.
Effects of apigenin and/or Ly294002 on mitochondrial function in MSTO-211HAcT and H2452AcT cells. Cells were treated with or without Ly294002 (20 µM, 2 h) prior to apigenin treatment (30 µM, 48 h) in RPMI-1640 medium containing 3.8 µM lactic acid. (A) Measurement of mitochondrial membrane potential following staining cells with rhodamine123. (B) Measurement of intracellular ROS levels following staining cells with DCF-DA (10 µM). (C) The expression levels of complexes I–V in mitochondrial electron transport chain. Bar graphs present densitometric analysis of western blot images normalized to β-actin. (D) Intracellular ATP levels. *P<0.05 vs. respective control cells. APG, apigenin; LY, Ly294002; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8 (complex I); SDHB, succinate dehydrogenase complex iron sulfur subunit B (complex II); UQCRC2, ubiquinone-cytochrome C reductase core protein 2 (complex III); COX II, mitochondrial cytochrome C oxidase subunit II (complex IV); ATP5A, ATP synthase F1 subunit alpha (complex V), and DCF-DA, 2′,7′-dichlorodihydrofluorescein diacetate.
Anti-glycolytic and cytotoxic effects of apigenin and Ly294002 in 3D spheroid culture. Spheroids were cultured in ultralow cluster 96-well plates for 5 days and were then treated with or without Ly294002 (20 µM, 2 h) prior to apigenin treatment (30 µM, 48 h) in RPMI-1640 medium containing 3.8 µM lactic acid for 48 h. (A) Vitality staining of spheroids [from left to right: (a) phase-contrast image, (b) fluorescent images of fluorescein diacetate(+) living cells in green, (c) propidium iodide(+) dead cells in red, and (d) merged]. (B) Spheroid viability. (C) The levels of p-Akt and p53 proteins. (D) The levels of marker proteins for apoptosis and necroptosis. (E) The levels of marker proteins for glycolysis. *P<0.05 vs. respective control cells. Bar graphs present densitometric analysis of western blot images normalized to β-actin. APG, apigenin; LY, Ly294002; HK, hexokinase; PFKP, phosphofructokinase platelet; PDH, pyruvate dehydrogenase; p-, phosphorylated.
A scheme for apigenin-induced cytotoxicity in malignant mesothelioma cells pre-adapted to lactic acid. Akt inhibition by apigenin causes p53 upregulation, reduced glycolysis, mitochondrial depolarization and cellular ATP depletion, thereby activating executioners leading to apoptosis and necroptosis.