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

Our plain-English summary of Apigenin causes necroptosis by inducing ROS accumulation, mitochondrial dysfunction, and ATP depletion in malignant mesothelioma cells, by Y.J. Lee, K.S. Park, H.S. Nam, et al (The Korean Journal of Physiology & Pharmacology, 2020).

The study

• Authors: Y.J. Lee, K.S. Park, H.S. Nam, M.K. Cho, S.H. Lee
• Journal: The Korean Journal of Physiology & Pharmacology
• Published: 2020;24(6):493-502
• Cited by: 27 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This was a laboratory study using cells grown in dishes. No patients or animals were involved, and no asbestos exposure was studied. The authors treated two human malignant mesothelioma cell lines (MSTO-211H and H-2452) and one normal human mesothelial cell line (MeT-5A) with apigenin, a natural flavonoid found in foods such as parsley and celery. Apigenin reduced the survival of both mesothelioma cell lines, more so at higher doses and longer times. MSTO-211H cells were more sensitive. Normal mesothelial cells kept 90% or more viability at the highest dose. At the same doses, the chemotherapy drug cisplatin showed low toxicity to the mesothelioma cells. Apigenin raised reactive oxygen species, reduced mitochondrial membrane potential and cut cellular ATP. It increased markers of DNA damage and of two kinds of cell death, apoptosis and necroptosis. Pretreatment with N-acetylcysteine or added ATP lessened several of these effects, bringing some measures back to or near normal levels. Similar necroptosis results were seen in three-dimensional cell clusters (spheroids). The authors concluded that apigenin might have potential as a new anti-cancer agent.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell-culture experiment, using flat monolayer cultures and 3D spheroid cultures
• Who was studied: Two human malignant mesothelioma cell lines (MSTO-211H and H-2452) and a normal human mesothelial cell line (MeT-5A), obtained from the American Type Culture Collection and studied at Soonchunhyang University, Korea. The mesothelioma cells were first adapted to an acidic culture medium containing lactic acid. No human subjects or animals were studied.
• Exposure: Apigenin at 0, 10, 30 and 50 micromolar for 48 and 72 hours, compared with cisplatin at the same concentrations. Some experiments added the antioxidant N-acetylcysteine, extra ATP, or cell-death inhibitors (necrostatin-1, Q-VD-Oph-1). No asbestos or fiber exposure was studied.
• Main results:
  • In both mesothelioma cell lines, apigenin reduced cell growth depending on dose and time. MSTO-211H cells were more sensitive than H-2452 cells.
  • In normal mesothelial MeT-5A cells, viability stayed at 90% or more after 48 and 72 hours at 50 micromolar apigenin.
  • Cisplatin at the same concentrations as apigenin showed low toxicity to both mesothelioma cell lines.
  • In MSTO-211H cells treated with 30 micromolar apigenin, the share of cells marked as dying by the annexin V test rose to 21.1%.
  • The corresponding figure for H-2452 cells was 12.04%.
  • Apigenin increased the share of cells delayed in the G2/M phase of the cell cycle, depending on dose. It also increased markers of DNA damage, including phosphorylated histone H2A.X, a double-strand break marker.
  • Apigenin increased proteins linked to apoptosis (cleaved caspase-3, cleaved PARP, a higher Bax/Bcl-2 ratio) and to necroptosis (p-MLKL and p-RIP3). Blocking either type of cell death significantly improved cell survival.
  • Cellular ATP began to fall within 2 hours of apigenin treatment and dropped sharply at 6 hours. Pretreatment with N-acetylcysteine restored ATP levels.
  • Adding ATP improved the viability of apigenin-treated cells. It also brought DNA damage, apoptosis and necroptosis proteins back to near-basal levels.
  • Pretreatment with N-acetylcysteine restored reactive oxygen species levels. It also improved apigenin's effects on the cell cycle, cell death and mitochondrial membrane potential.
  • In 3D spheroids, apigenin lowered viability, and N-acetylcysteine blocked this effect. Viability was slightly higher than in flat cultures. Cleaved caspase-3, an apoptosis marker, was not detected in the 3D cultures.
• The authors' conclusion: The authors concluded that apigenin killed the two mesothelioma cell lines in an acidic environment by inducing necroptosis. They wrote that this effect targets the mitochondria and is driven by excess reactive oxygen species and ATP depletion, and that apigenin might have potential as a new anti-cancer agent.
• Limitations the authors state:
  • The authors stated that the exact mechanism linking the two cell-death modes, apoptosis and necroptosis, remains unclear.
  • The authors stated that specific necroptosis markers and physiologically relevant systems are needed to clearly show the role of necroptosis in anti-cancer therapy.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Reactive Oxygen Species (ROS), Apoptosis.

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