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Carfilzomib combined with suberanilohydroxamic acid (SAHA) synergistically promotes endoplasmic reticulum stress in non-small cell lung cancer cell lines

  • Original Article – Cancer Research
  • Published:
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Abstract

Purpose

The endoplasmic reticulum (ER) stress response is a therapeutic target for pharmacologic intervention in cancer cells. We hypothesized that combining carfilzomib (CFZ), a proteasome inhibitor, and vorinostat (SAHA), a histone deacetylase (HDAC) inhibitor, would synergistically activate ER stress in non-small cell lung cancer (NSCLC) cell lines, resulting in enhanced anti-tumor activity.

Methods

Five NSCLC cell lines were treated with CFZ, SAHA, or the combination and cell proliferation measured using the MTT assay. Calcusyn software was utilized to determine the combination index as a measure of synergy. Cell viability and cytotoxicity were measured using trypan blue exclusion, CellTiter, and CytoTox assays. Western blot was used to measure markers of apoptosis, ER stress, and oxidative stress-related proteins. Reactive oxygen species (ROS) was measured using the fluorophore CM–H2DCFDA.

Results

Synergistic activity was observed for all cell lines following 48 and 72 h of combined treatment. H520 and A549 cell lines were used to assess viability and apoptosis. In both cell lines, increased death and cleaved caspase-3 were observed following combination treatment as compared with single-agent treatments. Combination therapy was associated with upregulation of ER stress-regulated proteins including activating transcription factor 4, GRP78/BiP, and C/EBP homologous protein. Both cell lines also showed increased ROS and the oxidative stress-related protein, heat shock protein 70.

Conclusion

Combining proteasome inhibition with HDAC inhibition enhances ER stress, which may contribute to the synergistic anticancer activity observed in NSCLC cell lines. Further preclinical and clinical studies of CFZ + SAHA in NSCLC are warranted.

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Abbreviations

ATF:

Activating transcription factor

BiP:

Immunoglobulin-binding protein of B cells

BTZ:

Bortezomib

CFZ:

Carfilzomib

CHOP:

C/EBP homologous protein

CI:

Combination index

DMSO:

Dimethyl sulfoxide

EGFR:

Epidermal growth factor receptor

ER:

Endoplasmic reticulum

HDAC:

Histone deacetylase

HSP70:

Heat shock protein 70

MTT:

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

NAC:

N-acetyl cysteine

NSLC:

Non-small cell lung cancer

PARP:

Poly (ADP-ribose) polymerase

PI:

Proteasome inhibitor

ROS:

Reactive oxygen species

SAHA:

Suberanilohydroxamic acid

SCLC:

Small cell lung cancer

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Acknowledgments

The authors wish to thank the University of Arizona Cancer Center/Arizona Research Laboratories (UACC/ARL) Cytometry Core Facility for their assistance with this study.

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Authors and Affiliations

  1. Section of Hematology/Oncology, College of Medicine, University of Arizona Cancer Center, 1515 N Campbell Ave, Tucson, AZ, USA

    Neale T. Hanke, Linda L. Garland & Amanda F. Baker

Authors
  1. Neale T. Hanke
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  2. Linda L. Garland
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  3. Amanda F. Baker
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Corresponding author

Correspondence to Amanda F. Baker.

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Funding

This work was supported by a generous gift from the late Mrs. Jemmie Helmricks and her husband, 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).

Conflict of interest

All authors declare no conflict of interest.

Ethical approval

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

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No human participants were used in this study.

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Hanke, N.T., Garland, L.L. & Baker, A.F. Carfilzomib combined with suberanilohydroxamic acid (SAHA) synergistically promotes endoplasmic reticulum stress in non-small cell lung cancer cell lines. J Cancer Res Clin Oncol 142, 549–560 (2016). https://doi.org/10.1007/s00432-015-2047-6

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  • DOI: https://doi.org/10.1007/s00432-015-2047-6

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