Arachidonic acid drives adaptive responses to chemotherapy-induced stress in malignant mesothelioma

Our plain-English summary of Arachidonic acid drives adaptive responses to chemotherapy-induced stress in malignant mesothelioma, by M. Cioce, C. Canino, H. Pass, et al (Journal of Experimental & Clinical Cancer Research, 2021).

The study

• Authors: M. Cioce, C. Canino, H. Pass, G. Blandino, S. Strano, V.M. Fazio
• Journal: Journal of Experimental & Clinical Cancer Research
• Published: 2021;40(1):344
• Cited by: 20 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Unlabelled: Background High resistance to therapy and poor prognosis characterizes malignant pleural mesothelioma (MPM). In fact, the current lines of treatment, based on platinum and pemetrexed, have limited impact on the survival of MPM patients. Adaptive response to therapy-induced stress involves complex rearrangements of the MPM secretome, mediated by the acquisition of a senescence-associated-secretory-phenotype (SASP). This fuels the emergence of chemoresistant cell subpopulations, with specific gene expression traits and protumorigenic features. The SASP-driven rearrangement of MPM secretome takes days to weeks to occur. Thus, we have searched for early mediators of such adaptive process and focused on metabolites differentially released in mesothelioma vs mesothelial cell culture media, after treatment with pemetrexed.

Methods: Mass spectrometry-based (LC/MS and GC/MS) identification of extracellular metabolites and unbiased statistical analysis were performed on the spent media of mesothelial and mesothelioma cell lines, at steady state and after a pulse with pharmacologically relevant doses of the drug. ELISA based evaluation of arachidonic acid (AA) levels and enzyme inhibition assays were used to explore the role of cPLA2 in AA release and that of LOX/COX-mediated processing of AA. QRT-PCR, flow cytometry analysis of ALDH expressing cells and 3D spheroid growth assays were employed to assess the role of AA at mediating chemoresistance features of MPM. ELISA based detection of p65 and IkBalpha were used to interrogate the NFkB pathway activation in AA-treated cells.

Results: We first validated what is known or expected from the mechanism of action of the antifolate. Further, we found increased levels of PUFAs and, more specifically, arachidonic acid (AA), in the transformed cell lines treated with pemetrexed. We showed that pharmacologically relevant doses of AA tightly recapitulated the rearrangement of cell subpopulations and the gene expression changes happening in pemetrexed -treated cultures and related to chemoresistance. Further, we showed that release of AA following pemetrexed treatment was due to cPLA2 and that AA signaling impinged on NFkB activation and largely affected anchorage-independent, 3D growth and the resistance of the MPM 3D cultures to the drug.

Conclusions: AA is an early mediator of the adaptive response to pem in chemoresistant MPM and, possibly, other malignancies.

© 2021. The Author(s).

In plain English

This was a laboratory study of human cells grown in culture. It involved no patients or animals and did not study asbestos exposure. The authors compared untransformed human mesothelial cells with malignant pleural mesothelioma (MPM) cell lines after a short pulse of the chemotherapy drug pemetrexed. They used mass spectrometry to measure substances the cells released into the culture fluid. Pemetrexed raised polyunsaturated fatty acids in the fluid of mesothelioma cells, and arachidonic acid rose in 3 of 3 mesothelioma lines tested this way. A second test found increased arachidonic acid release in ten of eleven cell lines, but no significant increase in untransformed mesothelial or immortalized LP9 cells. Drugs blocking the enzyme cPLA2 strongly reduced this release. Adding arachidonic acid increased a chemoresistant cell group (ALDHbright cells) in 9 of 10 lines and raised expression of genes linked to resistance traits. Blocking cPLA2 reduced the size and number of tumor-cell spheres, mostly at higher pemetrexed doses. The authors concluded that arachidonic acid is an early mediator of the adaptive response to pemetrexed in chemoresistant MPM.

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) experimental study on cultured human cell lines; no human or animal subjects
• Who was studied: Human malignant pleural mesothelioma cell lines (including MSTO-211H, ZL34, SPC-212, MPP-89, HP1, NCI-H2373, NCI-H2052, NCI-H2452, NCI-H28, NCI-H2595, NCI-H2596), compared with untransformed human mesothelial cells (HMC, pooled from four healthy donors) and hTERT-immortalized LP9 mesothelial cells. The mass spectrometry comparison used three mesothelioma cell lines.
• Exposure: No asbestos exposure was studied. Cells were given a short pulse of the chemotherapy drug pemetrexed (42 micrograms/ml for 60 minutes, described as corresponding to a 500 mg/m2 dose). Separately, they were treated with arachidonic acid (3 micromol/L), with or without cPLA2, COX or LOX inhibitors.
• Main results:
  • In the mass spectrometry analysis, pemetrexed increased polyunsaturated fatty acids in mesothelioma cell media; arachidonic acid was increased in 3 of 3 MPM cell lines
  • By ELISA, ten of eleven cell lines increased arachidonic acid release after a pemetrexed pulse
  • Neither untransformed HMC cells nor immortalized LP9 mesothelial cells showed significantly increased arachidonic acid after pemetrexed
  • Pretreatment of four MPM cell lines with two cPLA2 inhibitors (AACOCF3 and AVX-001) strongly reduced pemetrexed-induced arachidonic acid release
  • Across nine MPM cell lines, the amount of arachidonic acid released correlated positively with the cell line's measured CC50 for pemetrexed (r2 = 0.6243; p = 0.015)
  • Arachidonic acid treatment significantly increased the share of ALDHbright (chemoresistant) cells in 9 of 10 MPM cell lines
  • In three MPM cell lines, arachidonic acid raised mRNA levels of cytokines (IL1β, IL-6, IL-8) and of EMT- and cancer-stem-cell-related genes, although the size and timing of the changes varied
  • Both pemetrexed and arachidonic acid lowered levels of IkBα, the inhibitor of NFkB
  • COX and LOX inhibitors did not affect the arachidonic acid-induced rise in ALDHbright cells at 24 hours; COX inhibition had a moderate effect at 72 hours
  • Arachidonic acid significantly increased the number of spheres formed by MSTO-211H and HP1 cells
  • The cPLA2 inhibitor AVX001 reduced the size and number of spheres, mostly at higher pemetrexed doses; adding arachidonic acid back reversed the effect on sphere formation
• The authors' conclusion: The authors concluded that arachidonic acid is an early mediator of the adaptive response to pemetrexed in chemoresistant MPM and possibly other cancers. In their view, its cPLA2-dependent release activates NFkB and triggers changes compatible with a chemoresistant phenotype.
• Limitations the authors state:
  • The authors did not establish what activates cPLA2 after pemetrexed treatment
  • The authors did not test whether arachidonic acid itself stimulates cPLA2 activity in a feed-forward manner

Topics

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

Medical terms in this study, explained in our Lexicon: Chemotherapy, Mesothelioma, Prognosis, Pleural Mesothelioma, Pemetrexed (Alimta), Mesothelial Cells, Antifolate.

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• Production of arachidonic acid metabolites by macrophages exposed in vitro to asbestos, carbonyl iron particles, or calcium ionophore — S. Kouzan, A.R. Brody, et al. (1985)
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• Increased sensitivity to apoptosis upon endoplasmic reticulum stress-induced activation of the unfolded protein response in chemotherapy-resistant malignant pleural mesothelioma — D. Xu, S.Q. Liang, et al. (2018)
• Chemotherapy induced pathologic complete response in malignant pleural mesothelioma: a review and case report — C. Bech, J.B. Sørensen (2010)
• Proteasome stress sensitizes malignant pleural mesothelioma cells to bortezomib-induced apoptosis — F. Cerruti, G. Jocollè, et al. (2017)
• BAMLET kills chemotherapy-resistant mesothelioma cells, holding oleic acid in an activated cytotoxic state — E.M. Rath, Y.Y. Cheng, et al. (2018)

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