Pifithrin-μ induces necroptosis through oxidative mitochondrial damage but accompanies epithelial-mesenchymal transition-like phenomenon in malignant mesothelioma cells under lactic acidosis

Our plain-English summary of Pifithrin-μ induces necroptosis through oxidative mitochondrial damage but accompanies epithelial-mesenchymal transition-like phenomenon in malignant mesothelioma cells under lactic acidosis, by Y.J. Lee, K.S. Park, S.H. Heo, et al (Archives of Pharmacal Research, 2019).

The study

• Authors: Y.J. Lee, K.S. Park, S.H. Heo, H.S. Nam, M.K. Cho, S.H. Lee
• Journal: Archives of Pharmacal Research
• Published: 2019;42(10):890-901
• Cited by: 9 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Heat shock protein 70 (HSP70), a chaperone protein associated with tumorigenesis and chemoresistance, has attracted significant attention as a potential therapeutic target for the development of anticancer drugs. Here, the effects of pifithrin-μ, an effective dual inhibitor of HSP70 and p53, on anticancer activities and epithelial-mesenchymal transition (EMT) were investigated in malignant mesothelioma (MM) cells. MSTO-211HAcT cells, pre-incubated in a medium containing lactic acid, showed more potent resistance to cisplatin and gemcitabine, compared with their acid-sensitive parental MSTO-211H cells. Pifithrin-μ treatment induced both apoptosis and necroptosis, which were accompanied by an EMT-like phenomenon, as evidenced by an elongated cell morphology, decreased levels of epithelial cell markers including E-cadherin, claudin-1, and β-catenin, increased levels of mesenchymal markers including Snail, Slug, and vimentin, and increased cell migratory property. Moreover, pifithrin-μ increased intracellular ROS levels, which is associated with mitochondrial dysfunction and decreased cellular ATP content. A series of changes caused by pifithrin-μ treatment were effectively restored by lowering the ROS level through pretreatment with N-acetylcysteine. Collectively, our results suggest that pifithrin-μ may promote the metastatic behavior of surviving cells by triggering the EMT, despite its effective cell-killing action against MM cells, possibly linked to oxidative mitochondrial dysfunction and ATP depletion.

In plain English

This laboratory study used the human malignant mesothelioma cell line MSTO-211H and an acid-tolerant version (MSTO-211HAcT) grown repeatedly in lactic acid medium. No patients, animals or asbestos exposures were studied. The acid-tolerant cells resisted the chemotherapy drugs cisplatin and gemcitabine more than parent cells. The authors then tested pifithrin-μ (PES), an experimental HSP70 and p53 inhibitor. PES reduced survival in both lines: the dose needed to halve survival was 39.4 μM in parent cells and 34.6 μM in acid-tolerant cells, and the difference in sensitivity was not statistically significant. Cell death involved apoptosis and necroptosis, two programmed forms of cell death. PES also raised reactive oxygen species, damaged mitochondria and lowered cellular energy (ATP). Surviving cells took on an EMT-like change. Compared with untreated controls, cells migrating through a membrane increased by about 27% in the parent line and 31% in the acid-tolerant line. Pretreatment with the antioxidant N-acetylcysteine reversed many of these changes. The authors concluded that PES killed the cells but may promote metastatic behavior in survivors. They called for studies in more cell lines and in animals.

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
• Who was studied: The human malignant mesothelioma cell line MSTO-211H (from the American Type Culture Collection) and an acid-tolerant subline, MSTO-211HAcT, derived from it by serial passage over four times during a 15-day period in medium containing lactic acid. No people or animals were studied.
• Exposure: Cells were treated with pifithrin-μ (PES), an experimental compound described as an anticancer drug candidate. Most treatments used 20 μM for 48 h, in medium containing lactic acid (printed as 3.8 μM). Cells were also exposed to cisplatin and gemcitabine, with or without pretreatment with N-acetylcysteine (NAC, 5 mM), ATP, or cell-death inhibitors. Asbestos was not studied.
• Main results:
  • Acid-tolerant MSTO-211HAcT cells were more resistant to cisplatin and gemcitabine than parental MSTO-211H cells. In MSTO-211H cells the average IC50 values were 131.7 μM for cisplatin and 125.6 nM for gemcitabine.
  • The IC50 of PES was 39.4 μM for MSTO-211H cells and 34.6 μM for MSTO-211HAcT cells. The difference in sensitivity between the two lines was not statistically significant.
  • PES for 48 h decreased cell viability by 27.2% in MSTO-211H cells and 35.3% in MSTO-211HAcT cells.
  • Pretreatment with a caspase (apoptosis) inhibitor (Q-VD-Oph) or a necroptosis inhibitor (necrostatin-1) partly restored viability: about 8.7% or 15.8% in MSTO-211H cells, and 14.8% or 18.2% in MSTO-211HAcT cells. The autophagy inhibitor spautin-1 gave no significant recovery.
  • PES raised the necroptosis markers p-RIPK3 and p-MLKL in a concentration-dependent manner. Levels were similar in the two cell lines.
  • PES caused an EMT-like change in both cell lines. Cells became elongated and fibroblast-like. Epithelial markers (E-cadherin, claudin-1, β-catenin) fell and mesenchymal markers (Snail, Slug, vimentin) rose.
  • PES increased the number of cells migrating through a polycarbonate membrane by approximately 27% (MSTO-211H) and 31% (MSTO-211HAcT) compared with untreated controls. There was no significant difference between the two cell lines.
  • PES increased ROS production to approximately 25.21% in MSTO-211H cells and 38.20% in MSTO-211HAcT cells. NAC pretreatment lowered this to approximately 12.73% and 16.02%, respectively.
  • PES increased the percentage of cells with loss of mitochondrial membrane potential to 24.52% in MSTO-211H cells and 38.85% in MSTO-211HAcT cells.
  • Compared with PES alone, NAC pretreatment raised ATP levels from 77.2% to 89.5% in MSTO-211H cells and from 58.3% to 80.8% in MSTO-211HAcT cells (percent of control). Adding ATP prevented PES-induced cytotoxicity.
  • NAC pretreatment reversed the PES-induced changes in epithelial and mesenchymal markers and reduced nuclear accumulation of Snail and Slug.
• The authors' conclusion: The authors concluded that PES may promote the metastatic behavior of surviving mesothelioma cells by triggering an EMT-like change, despite killing the cells effectively, possibly linked to oxidative mitochondrial dysfunction and ATP depletion. They added that this may limit the efficacy of PES.
• Limitations the authors state:
  • The authors stated that studies using multiple cell lines and animal models are needed to confirm the connection between PES and EMT.
  • The authors stated that the molecular pathways of PES-induced cell death need further characterization.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Malignant Mesothelioma, Tumorigenesis, Cisplatin, Gemcitabine, Apoptosis.

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