Impact of metallothionein-knockdown on cisplatin resistance in malignant pleural mesothelioma

Our plain-English summary of Impact of metallothionein-knockdown on cisplatin resistance in malignant pleural mesothelioma, by S. Borchert, P.M. Suckrau, R.F.H. Walter, et al (Scientific Reports, 2020).

The study

• Authors: S. Borchert, P.M. Suckrau, R.F.H. Walter, M. Wessolly, E. Mairinger, J. Steinborn, B. Hegedus, T. Hager, T. Herold, W.E.E. Eberhardt, J. Wohlschlaeger, C. Aigner, A. Bankfalvi, K.W. Schmid, F.D. Mairinger
• Journal: Scientific Reports
• Published: 2020;10(1):18677
• Cited by: 20 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Malignant pleural mesothelioma (MPM) is a rare, but aggressive tumor with dismal prognosis. Platinum-based chemotherapy is regularly used as part of multimodality therapy. The expression of metallothioneins (MT) has been identified as a reason for cisplatin resistance, which often leads to early therapy failure or relapse. Thus, knockdown of MT expression may improve response to cisplatin treatment. The MT gene- and protein expression of the MPM-cell lines MSTO-211H, NCI-H2052 and NCI-H2452 and the human fibroblast cell line MRC-5, as well as their sensitivity to cisplatin treatment have been evaluated. Knockdown of MT1A, 1B and 2A expression was induced by RNA interference. MT expression was measured using quantitative real-time PCR. An in vitro Assay based on enzyme activity was used to detect cell viability, necrosis and apoptosis before and after incubation with cisplatin. MT2A gene expression could be detected in all MPM cell lines, showing the highest expression in NCI-H2452 and NCI-H2052, whereas gene expression levels of MT1A and MT1B were low or absent. The immunohistochemically protein expression of MT-I/II reflect MT2A gene expression levels. Especially for MSTO-211H cell presenting low initial MT2A levels, a strong induction of MT2A expression could be observed during cisplatin treatment, indicating a cell line-specific and platin-dependent adaption mechanism. Additionally, a MT2A-dependent cellular evasion of apoptosis during cisplatin could be observed, leading to three different MT based phenotypes. MSTO-211H cells showed lower apoptosis rates at an increased expression level of MT2A after cisplatin treatment (from sixfold to fourfold). NCI-H2052 cells showed no changes in MT2A expression, while apoptosis rate is the highest (8-12-fold). NCI-H2452 cells showed neither changes in alteration rate of MT2A expression nor changes in apoptosis rates, indicating an MT2A-independent resistance mechanism. Knockdown of MT2A expression levels resulted in significantly induced apoptotic rates during cisplatin treatment with strongest induction of apoptosis in each of the MPM cell lines, but in different markedness. A therapeutic meaningful effect of MT2A knockdown and subsequent cisplatin treatment could be observed in MSTO-211H cells. The present study showed MT2A to be part of the underlying mechanism of cisplatin resistance in MPM. Especially in MSTO-211H cells we could demonstrate major effects by knockdown of MT2A expression, verifying our hypothesis of an MT driven resistance mechanism. We could prove the inhibition of MT2A as a powerful tool to boost response rates to cisplatin-based therapy in vitro. These data carry the potential to enhance the clinical outcome and management of MPM in the future.

In plain English

This was a laboratory study of cells grown in culture, not of patients, and it did not examine asbestos exposure. The authors studied three malignant pleural mesothelioma cell lines (MSTO-211H, NCI-H2052 and NCI-H2452) and a lung fibroblast cell line, MRC-5, used as a non-tumour control. They measured metallothioneins (metal-binding proteins) and used short interfering RNA to reduce expression of three metallothionein genes (MT1A, MT1B and MT2A) before treating cells with the chemotherapy drug cisplatin. MT2A was found in all three mesothelioma lines. Apoptosis (programmed cell death) was measured relative to untreated cells. After cisplatin, MSTO-211H cells showed more MT2A, and their apoptosis readings were lower at higher MT2A levels, from sixfold to fourfold. NCI-H2052 cells showed no MT2A change and the highest readings (8 to 12-fold). NCI-H2452 cells showed the lowest (1.5-fold). Reducing MT2A gave the highest apoptosis rates after cisplatin. The authors concluded this supported their hypothesis of a metallothionein-driven resistance mechanism, and that inhibiting MT2A may be a promising approach to improve cisplatin response. Some figures in the text and tables disagree.

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: In vitro laboratory experiment on cell lines: gene knockdown with short interfering RNA, followed by cisplatin treatment
• Who was studied: Three commercially available human malignant pleural mesothelioma cell lines and the human lung fibroblast cell line MRC-5 as a control. MSTO-211H is described as biphasic, NCI-H2052 as sarcomatoid, and NCI-H2452 as epithelioid and BAP1-mutant. The work was done at University Hospital Essen, Germany, and no patients were involved.
• Exposure: Laboratory treatment with 10 micromolar cisplatin (48 hours before the cell-death assay), with or without prior short interfering RNA knockdown of the metallothionein genes MT1A, MT1B or MT2A. Asbestos exposure was not studied.
• Main results:
  • Apoptosis, necrosis and viability readings were calculated as a percentage of the signal from positive controls, normalized to untreated cells.
  • On immunohistochemical staining, metallothionein was not detected in the MRC-5 control cells.
  • MT2A was expressed in all three mesothelioma cell lines. MSTO-211H had the lowest baseline level but the highest rate of change after cisplatin.
  • In MSTO-211H cells after cisplatin, apoptosis rates were lower (from sixfold to fourfold) at an increased MT2A expression level.
  • NCI-H2052 cells showed no change in MT2A expression and the highest apoptosis rates (8 to 12-fold) compared with the other cell lines.
  • NCI-H2452 cells showed no change in MT2A expression or apoptosis, and the lowest apoptosis rates after cisplatin (1.5-fold).
  • Knockdown significantly reduced MT2A expression, with a median estimate of 73% (95% confidence interval 64 to 79%). The p-values were adjusted for false discovery rate.
  • One siRNA aimed at MT1A (siRNA2) also reduced MT2A. Most other siRNAs aimed at MT1A or MT1B increased MT2A expression.
  • In the mesothelioma cells, the highest apoptosis rates after cisplatin occurred when MT2A expression was knocked down.
  • According to the authors, metallothionein knockdown alone, without cisplatin, did not change cell state.
• The authors' conclusion: The authors concluded that knockdown of MT2A showed significant effects, especially in MSTO-211H cells, and that this supported their hypothesis of a metallothionein-driven cisplatin resistance mechanism. They also concluded that inhibition of MT2A may be a promising approach to improve response to cisplatin-based therapy.
• Limitations the authors state:
  • The antibody used detected MT1 and MT2 protein together. The authors gave this as a possible reason why the protein scores and the gene expression changes differed.
  • mRNA quantity may not be proportional to protein amounts.
  • One siRNA meant to target MT1A had off-target effects that reduced MT2A, so its effect on apoptosis was attributed to MT2A knockdown.
  • The authors stated that metallothionein expression could not be used as a single biomarker.

Topics

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

Medical terms in this study, explained in our Lexicon: Cisplatin, Pleural Mesothelioma, Prognosis, Chemotherapy, Multimodal Therapy, Fibroblast, Necrosis, Apoptosis.

Related studies

• Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines — M. Kitazono-Saitoh, Y. Takiguchi, et al. (2012)
• Gene expression profiling of homologous recombination repair pathway indicates susceptibility for olaparib treatment in malignant pleural mesothelioma in vitro — S. Borchert, M. Wessolly, et al. (2019)
• Putative cancer stem cells in malignant pleural mesothelioma show resistance to cisplatin and pemetrexed — L. Cortes-Dericks, G.L. Carboni, et al. (2010)
• Resistance to caspase-8 and -9 fragments in a malignant pleural mesothelioma cell line with acquired cisplatin-resistance — V. Janson, A. Johansson, et al. (2010)
• Cisplatin-induced expression of Gb3 enables verotoxin-1 treatment of cisplatin resistance in malignant pleural mesothelioma cells — D. Johansson, C. Andersson, et al. (2010)
• Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin — J. Wörthmüller, V. Salicio, et al. (2019)

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