Characterization of carfilzomib-resistant non-small cell lung cancer cell lines
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.
KeywordsCarfilzomib 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.
This article does not contain any studies with human participants or animals performed by any of the authors.
No human participants were used in this study.
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