Korean J Physiol Pharmacol.  2020 Nov;24(6):493-502. 10.4196/kjpp.2020.24.6.493.

Apigenin causes necroptosis by inducing ROS accumulation, mitochondrial dysfunction, and ATP depletion in malignant mesothelioma cells

Affiliations
  • 1Department of Biochemistry, Soonchunhyang University College of Medicine, Cheonan, Korea
  • 2Division of Molecular Cancer Research, Soonchunhyang Medical Research Institute, Soonchunhyang University, Cheonan 31151, Korea

Abstract

Apigenin, a naturally occurring flavonoid, is known to exhibit significant anticancer activity. This study was designed to determine the effects of apigenin on two malignant mesothelioma cell lines, MSTO-211H and H2452, and to explore the underlying mechanism(s). Apigenin significantly inhibited cell viability with a concomitant increase in intracellular reactive oxygen species (ROS) and caused the loss of mitochondrial membrane potential (ΔΨm), and ATP depletion, resulting in apoptosis and necroptosis in monolayer cell culture. Apigenin upregulated DNA damage response proteins, including the DNA double strand break marker phospho (p)-histone H2A.X. and caused a transition delay at the G2 /M phase of cell cycle. Western blot analysis showed that apigenin treatment upregulated protein levels of cleaved caspase-3, cleaved PARP, p-MLKL, and p-RIP3 along with an increased Bax/Bcl-2 ratio. ATP supplementation restored cell viability and levels of DNA damage-, apoptosisand necroptosis-related proteins that apigenin caused. In addition, N-acetylcysteine reduced ROS production and improved ΔΨm loss and cell death that were caused by apigenin. In a 3D spheroid culture model, ROS-dependent necroptosis was found to be a mechanism involved in the anti-cancer activity of apigenin against malignant mesothelioma cells. Taken together, our findings suggest that apigenin can induce ROS-dependent necroptotic cell death due to ATP depletion through mitochondrial dysfunction. This study provides us a possible mechanism underlying why apigenin could be used as a therapeutic candidate for treating malignant mesothelioma.

Keyword

Apigenin; Apoptosis; Mesothelioma; Necroptosis; Reactive oxygen species

Figure

  • Fig. 1 Apigenin-induced cytotoxicity. Two MM cell lines, MSTO-211H and H-2452, and normal mesothelial cell line MeT-5A were treated with increasing concentrations (0, 10, 30, and 50 μM) of apigenin or cisplatin for 48 h and 72 h. (A) Cell viability was measured by MTT assay. (B) Cell cycle distribution was determined by flow cytometry following staining with propidium iodide (20 μg/ml). (C) Apoptotic cell fraction was analyzed using annexin V-PE binding assay. (D) Levels of DNA damage response proteins were assessed by Western blotting. MM, malignant mesothelioma; APG, apigenin; 7-AAD, 7-amino-actinomycin D; Arrows, sub-G0/G1 peak. *p < 0.05 vs. respective control group.

  • Fig. 2 Apigenin-induced apoptosis and necroptosis in MSTO-211H and H-2452 cells. (A) Cells were treated with increasing concentrations (0, 10, 30, and 50 μM) of apigenin for 48 h. Levels of apoptosis- and necroptosis-related proteins were assessed by Western blotting. (B) Cells were pretreated with 25 μM necrostatin-1 and 10 μM Q-VD-Oph-1 2 h prior to treatment with apigenin (30 and 50 μM) for 48 h and 72 h. Cell viability was measured by MTT assay. APG, apigenin. *p < 0.05 vs. respective control group. #p < 0 .05 vs. group treated with APG alone.

  • Fig. 3 Effects of apigenin on mitochondrial function in MSTO-211H and H-2452 cells. (A, B) Cells were treated with indicated concentrations of apigenin for 48 h. Intracellular ROS levels were measured after cells were stained with 10 μM DCF-DA (A). Mitochondrial membrane potential was measured after staining cells with 30 nM rhodamine123 (B). (C–E) Cells were pretreated with 5 mM NAC or 1 mM ATP for 2 h prior to treatment with 30 μM apigenin for 48 h. Cellular ATP levels were measured by CellTiter-Glo luminescent cell viability assay (C). Cell viability was measured by MTT assay (D). Levels of DNA damage response-, apoptosis-, and necroptosis-related proteins were assessed by Western blotting (E). APG, apigenin; NAC, N-acetylcysteine; ROS, reactive oxygen species. *p < 0.05 vs. respective control group. #p < 0.05 vs. group treated with APG alone.

  • Fig. 4 Apigenin-induced oxidative stress in MSTO-211H and H-2452 cells. Cells were pretreated with or without 5 mM NAC for 2 h prior to treatment with 30 μM apigenin for 48 h. (A) Cellular ROS levels were measured by staining cells with 10 μM DCF-DA. (B) Cell cycle distribution was determined by flow cytometry following staining with propidium iodide (20 μg/ml). (C) Apoptotic cell fraction was analyzed using annexin V-PE binding assay. (D) ΔΨm was measured by staining cells with 30 nM rhodamine123. APG, apigenin; NAC, N-acetylcysteine; Arrows, sub-G0/G1 peak. #p < 0.05 vs. group treated with APG alone.

  • Fig. 5 Effects of pretreatment with N-acetylcysteine on apigenin-induced cytotoxicity in 3D cultures of MSTO-211H and H-2452 cells. Spheroids were cultured in ultralow cluster 96-well plate and pretreated with or without 5 mM NAC 2 h prior to treatment with 30 μM apigenin for 48 h. (A) Vitality staining of spheroids (from left to right: phase-contrast image [a], fluorescent images of FDA(+) living cells in green [b], PI(+) dead cells in red [c], and merged [d]) (×100). (B) Cell and spheroid viability were measured by MTT assay and the enhanced cell viability assay kit. (C) The levels of necroptosis- and apoptosis-related proteins were analyzed by Western blotting. APG, apigenin; NAC, N-acetylcysteine. *p < 0.05 vs. respective control group. #p < 0.05 vs. group treated with APG alone.


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