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Int J Oncol. 2018 Apr;52(4):1165-1177. doi: 10.3892/ijo.2018.4282. Epub 2018 Feb 23.

Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis.

Author information

1
Department of Otolaryngology (Head and Neck Surgery), Tokyo Medical University, Tokyo 160-8402, Japan.
2
Department of Biochemistry, Tokyo Medical University, Tokyo 160-8402, Japan.
3
Department of Breast Oncology and Surgery, Tokyo Medical University, Tokyo 160-8402, Japan.
4
Department of Joint Research for Basic Medical Science, Institute of Medical Science, Tokyo Medical University, Tokyo 160-8402, Japan.
5
Department of Preventive Medicine and Public Health, Tokyo Medical University, Tokyo 160-8402, Japan.

Abstract

The maintenance of the intracellular level of amino acids is crucial for cellular homeostasis. This is carried out via the regulation of both the influx from the extracellular environment and the recycling of intracellular resources. Since epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors, including gefitinib (GEF) have been reported to induce the apoptosis of several cancer cell lines, in the present study, we examined whether the cytotoxic effects of GEF are further enhanced under amino acid starvation (AAS) culture conditions. Under AAS culture conditions, the cell killing effect of GEF was synergistically pronounced in the EGFR-expressing cell lines, namely, CAL 27, Detroit 562, A549 and PANC-1 cells compared with those treated with either GEF or AAS alone. The addition of essential amino acids, but not non-essential amino acids to the cell culture medium resulted in the cancellation of this pronounced cytotoxicity. The knockdown of L-type amino acid transporter 1 (LAT-1) by siRNA also enhanced GEF-induced cytotoxicity. Therefore, the shortage of the intracellular amino acid pool appears to determine the sensitivity to GEF. Notably, this enhanced cytotoxicity is not mediated by the induction of apoptosis, but is accompanied by the pronounced induction of autophagy. The presence of necrostatin-1, an inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK‑1), but not that of Z-VAD-fmk, attenuated the cytotoxic effects of GEF under AAS culture conditions. Electron microscopy demonstrated that the CAL 27 cells treated with GEF under AAS culture conditions exhibited swelling of the cytosol and organelles with an increased number of autophagosomes and autolysosomes, but without chromatin condensation and nuclear fragmentation. Autophagic cell death was excluded as the inhibition of autophagy did not attenuate the cytotoxicity. These results strongly suggest the induction of necroptosis in response to GEF under AAS culture conditions. However, we could not detect any phosphorylation of RIPK-1 and mixed lineage kinase domain like pseudokinase (MLKL), as well as any necrosome formation. Therefore, the enhanced cytotoxic effect of GEF under AAS culture conditions is thought to be mediated by atypical necroptosis.

PMID:
29484439
PMCID:
PMC5843391
DOI:
10.3892/ijo.2018.4282
[Indexed for MEDLINE]
Free PMC Article

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