Characterization of carfilzomib-resistant non-small cell lung cancer cell lines

Neale T. Hanke, Elliot Imler, Marilyn T. Marron, Bruce E. Seligmann, Linda L Garland, Amanda F Baker

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Purpose: We previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines. Methods: CFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin. Results: The CFZ IC50 value of the resistant cells increased versus parental lines (2.5-fold for A549, 122-fold for H520). Resistant lines showed reduced expression of apoptosis and autophagy markers and reduced death versus parental lines following CFZ treatment. Both resistant lines exhibited higher P-glycoprotein (Pgp) gene (TempO-Seq® analysis, increased 1.2-fold in A549, > 9000-fold in H520) and protein expression levels versus parental lines. TempO-Seq® analysis indicated other drug resistance pathways were upregulated. The resistant cell lines demonstrated less accumulation of intracellular doxorubicin, and were cross-resistant to other Pgp client drugs: bortezomib, doxorubicin, and paclitaxel, but not cisplatin. Conclusions: Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.

Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalJournal of Cancer Research and Clinical Oncology
DOIs
StateAccepted/In press - May 15 2018

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Non-Small Cell Lung Carcinoma
Cell Line
P-Glycoprotein
Doxorubicin
Autophagy
Lung Neoplasms
Parental Death
Pharmaceutical Preparations
Apoptosis
carfilzomib
Transcriptome
Drug Resistance
Inhibitory Concentration 50
Flow Cytometry
Proteins
Up-Regulation
Western Blotting
Gene Expression
Drug Therapy
Polymerase Chain Reaction

Keywords

  • Carfilzomib
  • Cross-resistance
  • Drug resistance
  • Lung cancer
  • Non-small cell lung cancer
  • Pgp
  • Proteasome inhibitor

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Characterization of carfilzomib-resistant non-small cell lung cancer cell lines. / Hanke, Neale T.; Imler, Elliot; Marron, Marilyn T.; Seligmann, Bruce E.; Garland, Linda L; Baker, Amanda F.

In: Journal of Cancer Research and Clinical Oncology, 15.05.2018, p. 1-11.

Research output: Contribution to journalArticle

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title = "Characterization of carfilzomib-resistant non-small cell lung cancer cell lines",
abstract = "Purpose: We previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines. Methods: CFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin. Results: The CFZ IC50 value of the resistant cells increased versus parental lines (2.5-fold for A549, 122-fold for H520). Resistant lines showed reduced expression of apoptosis and autophagy markers and reduced death versus parental lines following CFZ treatment. Both resistant lines exhibited higher P-glycoprotein (Pgp) gene (TempO-Seq{\circledR} analysis, increased 1.2-fold in A549, > 9000-fold in H520) and protein expression levels versus parental lines. TempO-Seq{\circledR} analysis indicated other drug resistance pathways were upregulated. The resistant cell lines demonstrated less accumulation of intracellular doxorubicin, and were cross-resistant to other Pgp client drugs: bortezomib, doxorubicin, and paclitaxel, but not cisplatin. Conclusions: Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.",
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AU - Hanke, Neale T.

AU - Imler, Elliot

AU - Marron, Marilyn T.

AU - Seligmann, Bruce E.

AU - Garland, Linda L

AU - Baker, Amanda F

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N2 - Purpose: We previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines. Methods: CFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin. Results: The CFZ IC50 value of the resistant cells increased versus parental lines (2.5-fold for A549, 122-fold for H520). Resistant lines showed reduced expression of apoptosis and autophagy markers and reduced death versus parental lines following CFZ treatment. Both resistant lines exhibited higher P-glycoprotein (Pgp) gene (TempO-Seq® analysis, increased 1.2-fold in A549, > 9000-fold in H520) and protein expression levels versus parental lines. TempO-Seq® analysis indicated other drug resistance pathways were upregulated. The resistant cell lines demonstrated less accumulation of intracellular doxorubicin, and were cross-resistant to other Pgp client drugs: bortezomib, doxorubicin, and paclitaxel, but not cisplatin. Conclusions: Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.

AB - Purpose: We previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines. Methods: CFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin. Results: The CFZ IC50 value of the resistant cells increased versus parental lines (2.5-fold for A549, 122-fold for H520). Resistant lines showed reduced expression of apoptosis and autophagy markers and reduced death versus parental lines following CFZ treatment. Both resistant lines exhibited higher P-glycoprotein (Pgp) gene (TempO-Seq® analysis, increased 1.2-fold in A549, > 9000-fold in H520) and protein expression levels versus parental lines. TempO-Seq® analysis indicated other drug resistance pathways were upregulated. The resistant cell lines demonstrated less accumulation of intracellular doxorubicin, and were cross-resistant to other Pgp client drugs: bortezomib, doxorubicin, and paclitaxel, but not cisplatin. Conclusions: Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.

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KW - Non-small cell lung cancer

KW - Pgp

KW - Proteasome inhibitor

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