Journal of Cancer Research and Clinical Oncology

, Volume 144, Issue 7, pp 1317–1327 | Cite as

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. BakerEmail author
Original Article – Cancer Research



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.


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.


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.


Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.


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



The authors also wish to thank Adrianna E. Pulver, BS, for her technical assistance and the University of Arizona Cancer Center/Arizona Research Laboratories Flow Cytometry Core Facility which is partially funded by P30CA023074 from the National Cancer Institute (NCI).


This work was supported by a research collaboration award by Onyx Pharmaceuticals, Inc., an Amgen subsidiary, and a Basic/Clinical Translational Partnership Pilot Grant award from the Arizona Cancer Center Support Grant P30CA023074 from the National Cancer Institute (NCI).

Compliance with ethical standards

Conflict of interest

Authors EI, MTM, and BES are employees of BioSpyder Technologies, Inc., the company that developed and now sells the TempO-Seq kits. The other authors have no conflicts to declare.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

No human participants were used in this study.

Supplementary material

432_2018_2662_MOESM1_ESM.pptx (67 kb)
Supplementary material 1 (PPTX 66 KB)
432_2018_2662_MOESM2_ESM.pptx (939 kb)
Supplemental Figure 1. Upregulation of KDM6A corresponds to SAHA sensitivity in H520 CFZ resistant cells. TempO-Seq gene expression analysis showed upregulation of an HDACi sensitizer gene, KDM6A, in H520 CFZ resistant cells compared to its parent, but not in A549 CFZ resistant cells compared to its parent. To further evaluate, cells were left untreated or treated with CFZ and/or SAHA parent 48 h IC50 doses. Detection of cleaved caspase-3 by immunoblot in total extracts of cells harvested after 24 hours of treatment as indicated. Tubulin was used as a loading control (PPTX 939 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of MedicineUniversity of Arizona Cancer CenterTucsonUSA
  2. 2.BioSpyder Technologies IncCarlsbadUSA
  3. 3.Ventana Medical Systems, Inc., A Member of the Roche GroupTucsonUSA

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