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Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance

BACKGROUND: Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and...

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Autores principales: Oiwa, Kana, Hosono, Naoko, Nishi, Rie, Scotto, Luigi, O’Connor, Owen A., Yamauchi, Takahiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325835/
https://www.ncbi.nlm.nih.gov/pubmed/34332580
http://dx.doi.org/10.1186/s12885-021-08607-9
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author Oiwa, Kana
Hosono, Naoko
Nishi, Rie
Scotto, Luigi
O’Connor, Owen A.
Yamauchi, Takahiro
author_facet Oiwa, Kana
Hosono, Naoko
Nishi, Rie
Scotto, Luigi
O’Connor, Owen A.
Yamauchi, Takahiro
author_sort Oiwa, Kana
collection PubMed
description BACKGROUND: Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance. METHODS: To investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay. RESULTS: Compared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [(14)C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines. CONCLUSIONS: The resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12885-021-08607-9.
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spelling pubmed-83258352021-08-02 Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance Oiwa, Kana Hosono, Naoko Nishi, Rie Scotto, Luigi O’Connor, Owen A. Yamauchi, Takahiro BMC Cancer Research Article BACKGROUND: Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance. METHODS: To investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay. RESULTS: Compared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [(14)C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines. CONCLUSIONS: The resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12885-021-08607-9. BioMed Central 2021-07-31 /pmc/articles/PMC8325835/ /pubmed/34332580 http://dx.doi.org/10.1186/s12885-021-08607-9 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research Article
Oiwa, Kana
Hosono, Naoko
Nishi, Rie
Scotto, Luigi
O’Connor, Owen A.
Yamauchi, Takahiro
Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance
title Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance
title_full Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance
title_fullStr Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance
title_full_unstemmed Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance
title_short Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance
title_sort characterization of newly established pralatrexate-resistant cell lines and the mechanisms of resistance
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325835/
https://www.ncbi.nlm.nih.gov/pubmed/34332580
http://dx.doi.org/10.1186/s12885-021-08607-9
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