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Overcoming the acquired resistance to gefitinib in lung cancer brain metastasis in vitro and in vivo

  • Genotoxicity and Carcinogenicity
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Abstract

In our previous work, PC-9-Br, a PC-9 brain seeking line established via a preclinical animal model of lung cancer brain metastasis (LCBM), exhibited not only resistance to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) gefitinib in vitro, but also chemotherapy regimens of cisplatin plus etoposide in vivo. Using this cell line, we investigated novel potential targeted therapeutics for treating LCBM in vitro and in vivo to combat drug resistance. Significant increases in mRNA and protein expression levels of Bcl-2 were found in PC-9-Br compared with parental PC-9 (PC-9-P), but no significant changes of Bcl-XL were observed. A remarkable synergistic effect between EGFR-TKI gefitinib and Bcl-2 inhibitors ABT-263 (0.17 ± 0.010 µM at 48 h and 0.02 ± 0.004 µM at 72 h), or ABT-199 (0.22 ± 0.008 µM at 48 h and 0.02 ± 0.001 µM at 72 h) to overcome acquired resistance to gefitinib (> 0.5 µM at 48 h and 0.10 ± 0.007 µM at 72 h) in PC-9-Br was observed in MTT assays. AZD9291 was also shown to overcome acquired resistance to gefitinib in PC-9-Br in MTT assays (0.23 ± 0.031 µM at 48 h and 0.03 ± 0.008 µM at 72 h). Western blot showed significantly decreased phospho-Erk1/2 and increased cleaved-caspase-3 expressions were potential synergistic mechanisms for gefitinib + ABT263/ABT199 in PC-9-Br. Significantly decreased protein expressions of phospho-EGFR, phospho-Akt, p21, and survivin were specific synergistic mechanism for gefitinib + ABT199 in PC-9-Br. In vivo studies demonstrated afatinib (30 mg/kg) and AZD9291 (25 mg/kg) could significantly reduce the LCBM in vivo and increase survival percentages of treated mice compared with mice treated with vehicle and gefitinib (6.25 mg/kg). In conclusion, our study demonstrated gefitinib + ABT263/ABT199, afatinib, and AZD9291 have clinical potential to treat LCBM.

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Acknowledgements

We would like to thank the WVU HSC Microscope Imaging and the Animal Modeling Imaging Facilities.

Funding

This research was partially supported by the National Institute of Environmental Health Sciences of the National Institutes of Health (R15ES026789), the National Institute of General Medical Sciences (P20GM121322-03), METAvivor (1008553R), and the Mylan Chair Endowment Fund. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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  1. Zhongwei Liu and Neal Shah have contributed equally to the manuscript.

Authors and Affiliations

  1. Department of Occupational and Environmental Health Sciences, School of Public Health, West Virginia University, 64 Medical Center Drive, Morgantown, WV, 26505, USA

    Zhongwei Liu, Kent L. Marshall & Weimin Gao

  2. Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, 108 Biomedical Drive, Morgantown, WV, 26506, USA

    Neal Shah, Samuel A. Sprowls, Pushkar Saralkar, Afroz Mohammad, Kathryn E. Blethen, Tasneem A. Arsiwala, Ross Fladeland & Paul R. Lockman

  3. School of Medicine, West Virginia University, Morgantown, USA

    Neal Shah & Kent L. Marshall

  4. West Virginia Clinical and Translational Science Institute, Morgantown, USA

    Kent L. Marshall

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  1. Zhongwei Liu
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Contributions

Conceptualization PRL, WG, ZL and NS. Methodology ZL, NS, KLM, SAS, PS, AM, KEB, TAA, and RF. Software ZL and NS. Validation ZL and NS. Formal analysis ZL and NS. Investigation ZL, NS, KLM, SAS, PS, AM, KEB, TAA, and RF. Data curation ZL, NS, KLM, SAS, PS, AM, KEB, TAA, and RF. Writing—original draft preparation ZL and NS. Writing—review and editing ZL, NS, KLM, SAS, PS, AM, KEB, TAA, and RF. Supervision PRL and WG. Project administration PRL and WG. Funding acquisition SAS, WG, and PRL.

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Correspondence to Paul R. Lockman or Weimin Gao.

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Institutional review board statement

All animal experiments were approved by the Institutional Animal Care and Use Committee at West Virginia University in Morgantown, West Virginia (Protocol No. 16404001894).

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The interpreted and analyzed data from this study are available from the corresponding author upon reasonable request.

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Liu, Z., Shah, N., Marshall, K.L. et al. Overcoming the acquired resistance to gefitinib in lung cancer brain metastasis in vitro and in vivo. Arch Toxicol 95, 3575–3587 (2021). https://doi.org/10.1007/s00204-021-03147-4

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