Inhibition of ZL55 cell proliferation by ADP via PKC-dependent signalling pathway

Our plain-English summary of Inhibition of ZL55 cell proliferation by ADP via PKC-dependent signalling pathway, by A. Muscella, L.G. Cossa, C. Vetrugno, et al (Journal of Cellular Physiology, 2018).

The study

• Authors: A. Muscella, L.G. Cossa, C. Vetrugno, G. Antonaci, S. Marsigliante
• Journal: Journal of Cellular Physiology
• Published: 2018;233(3):2526-2536
• Cited by: 10 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Extracellular nucleotides can regulate cell proliferation in both normal and tumorigenic tissues. Here, we studied how extracellular nucleotides regulate the proliferation of ZL55 cells, a mesothelioma-derived cell line obtained from bioptic samples of asbestos-exposed patients. ADP and 2-MeS-ADP inhibited ZL55 cell proliferation, whereas ATP, UTP, and UDP were inactive. The nucleotide potency profile and the blockade of the ADP-mediated inhibitory effect by the phospholipase C inhibitor U-73122 suggest that P2Y1 receptor controls ZL55 cell proliferation. The activation of P2Y1 receptor by ADP leads to activation of intracellular transduction pathways involving [Ca2+ ]i , PKC-δ/PKC-α, and MAPKs, ERK1/2 and JNK1/2. Cell treatment with ADP or 2-MeS-ADP also provokes the activation of p53, causing an accumulation of the G1 cyclin-dependent kinase inhibitors p21WAF1 and p27Kip . Inhibition of ZL55 cell proliferation by ADP was completely reversed by inhibiting MEK1/2, or JNK1/2, or PKC-δ, and PKC-α. Through the inhibition of ADP-activated transductional kinases it was found that PKC-δ was responsible for JNK1/2 activation. JNK1/2 has a role in transcriptional up-regulation of p53, p21WAF1/CIP1 , and p27kip1 . Conversely, the ADP-activated PKC-α provoked ERK1/2 phosphorylation. ERK1/2 increased p53 stabilization, required to G1 arrest of ZL55 cells. Concluding, the importance of the study is twofold: first, results shed light on the mechanism of cell cycle inhibition by ADP; second, results suggest that extracellular ADP may inhibit mesothelioma progression.

© 2017 Wiley Periodicals, Inc.

In plain English

This was a laboratory study on ZL55 cells, a human mesothelioma cell line that the authors describe as obtained from asbestos-exposed patients. The study did not test asbestos. It tested how several nucleotides (small signalling molecules: ADP, 2-MeS-ADP, ATP, UTP and UDP) affected the growth of these cells in culture. ATP had no influence on growth, and UTP and UDP had a modest stimulating effect. ADP and 2-MeS-ADP significantly slowed cell growth. Half-maximal inhibition came at 22.8 µM, and the treated cells built up in an early phase of the cell cycle (G0/G1). ADP also raised calcium levels inside the cells and increased the proteins p53, p21 and p27. Blocking certain enzymes (phospholipase C, PKC, MEK1/2, JNK1/2), or lowering p53, weakened or removed ADP's effect. The authors concluded that their results suggest extracellular ADP may inhibit mesothelioma progression. They stated that studies in living organisms are needed to learn whether ADP could be used as a treatment.

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) study of a single human mesothelioma cell line grown in culture
• Who was studied: ZL55 cells, a human mesothelioma cell line described as obtained from asbestos-exposed patients. The authors present it as a model of epithelial malignant pleural mesothelioma. The work was done at the Università del Salento, Lecce, Italy.
• Exposure: No asbestos exposure was studied. The cells were treated with the nucleotides ADP, 2-MeS-ADP, ATP, UTP and UDP at 1 to 100 µM, and with various signalling-pathway inhibitors.
• Main results:
  • In ZL55 cells, ATP had no influence on proliferation. UTP and UDP, up to 100 µM, had a modest stimulating effect.
  • ADP or 2-MeS-ADP significantly inhibited ZL55 cell proliferation, depending on concentration and time. Half-maximal inhibition was at 22.8 plus or minus 1.9 µM (n=8), and the maximal response came after 48 hours of 50 µM ADP.
  • The phospholipase C inhibitor U-73122 at 10 µM completely reversed ADP's effect on ZL55 cell proliferation. The authors took this to suggest that P2Y receptors, not P2X receptor-channels, are involved.
  • ZL55 cells showed mRNA for the P2Y1, P2Y4, P2Y6 and P2Y11 receptor subtypes. P2Y2, P2Y12, P2Y13 and P2Y14 could not be detected.
  • ADP (1, 10, 50 and 100 µM) for 24 hours caused a concentration-dependent increase in ZL55 cells in the G0/G1 phases of the cell cycle.
  • ADP raised intracellular calcium in ZL55 cells, with a maximum at 100 µM ADP. The level rose from 79.5 plus or minus 5.3 to 598.7 plus or minus 86.3 nM (ANOVA p less than 0.001, n=8).
  • ADP treatment significantly increased p21WAF1 and p27kip1 in ZL55 cells, and p53 levels also increased.
  • Knocking down p53 with siRNA significantly lowered p53 expression and inhibited ADP's anti-proliferative effect.
  • Inhibiting MEK1/2 or JNK1/2 attenuated ADP's inhibition of proliferation, depending on concentration. Inhibiting p38MAPK had no effect: the p38 inhibitor SB203580 at 10 µM reduced ADP's effect by only 1.3 plus or minus 3.8% in Table 1.
  • The MEK inhibitor PD98059 and the JNK inhibitor SP600125 used together completely reversed ADP's effect on ZL55 cell proliferation.
  • Reducing PKC-delta with siRNA blocked ADP-induced JNK1/2 activation and the induction of p53, p21 and p27. Reducing PKC-alpha blocked ERK1/2 activation, p53 stabilization and the inhibition of proliferation.
• The authors' conclusion: The authors concluded that ADP acting through the P2Y1 receptor arrests the ZL55 cell cycle by up-regulating p53, p21 and p27, and that the results suggest extracellular ADP may inhibit mesothelioma progression.
• Limitations the authors state:
  • The authors state that studies in living organisms are required to understand ADP's effects and mechanisms, and to find out whether it can be used as a treatment for malignant pleural mesothelioma.
  • The authors state it remains to be determined whether ADP also affects p21 and p27 at the translational (protein-making) level in ZL55 cells.

Topics

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

Medical terms in this study, explained in our Lexicon: Mesothelioma.

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