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, 52 (4), 1165-1177

Amino Acid Starvation Culture Condition Sensitizes EGFR-expressing Cancer Cell Lines to Gefitinib-Mediated Cytotoxicity by Inducing Atypical Necroptosis

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Amino Acid Starvation Culture Condition Sensitizes EGFR-expressing Cancer Cell Lines to Gefitinib-Mediated Cytotoxicity by Inducing Atypical Necroptosis

Yu Saito et al. Int J Oncol.

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.

Conflict of interest statement

Competing interests

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Cell growth inhibition following treatment with gefitinib (GEF) under amino acid starvation (AAS) culture conditions in EGFR-expressing cancer cell lines. (A) Cellular proteins in the CAL 27, Detroit 562, A549, PC-9, PANC-1 and MEF cells in the exponential growth phase in the complete culture condition containing 10% FBS were lysed as described in Materials and methods. The cellular proteins extracted from the 1×105 cells were loaded on each lane and separated by 11.25% SDS-PAGE and immunoblotted with anti-EGFR antibody (Ab) and anti-phospho-EGFR (Tyr1173) Ab. Immunoblotting with anti-GAPHD mAb was performed as an internal control. The cell lysate of HL-60 leukemia cells was used as a negative control, as previously described (11). Numbers indicate the ratios of EGFR to GAPDH and the ratios of the phospho-EGFR to EGFR in each lane. N.D., not detectable. (B) Cells were cultured under the complete culture condition and AAS culture condition in the presence of GEF (0–25 µM) for 24 and 48 h. Viable cell number was assessed by CellTiter-Blue cell viability assay as described in Materials and methods (*P<0.05 and **P<0.01, complete culture condition vs. AAS by a two-tailed non-paired Student's t-test). By multivariate linear regression analysis using both GEF and AAS as independent variables, the synergistic effect was observed in the 24-h treatment of CAL-27 cells (P<0.001) and PANC-1 cells (P=0.008), as well as in the 48-h treatment of CAL-27 cells (P<0.001), Detroit 562 cells (P=0.023), A549 cells (P<0.001) and PANC-1 cells (P<0.001). In the PC-9 cells, the additive cytotoxic effect was observed in the 48-h exposure (P<0.001), but not in the 24-h exposure (P=0.335).
Figure 1
Figure 1
Cell growth inhibition following treatment with gefitinib (GEF) under amino acid starvation (AAS) culture conditions in EGFR-expressing cancer cell lines. (A) Cellular proteins in the CAL 27, Detroit 562, A549, PC-9, PANC-1 and MEF cells in the exponential growth phase in the complete culture condition containing 10% FBS were lysed as described in Materials and methods. The cellular proteins extracted from the 1×105 cells were loaded on each lane and separated by 11.25% SDS-PAGE and immunoblotted with anti-EGFR antibody (Ab) and anti-phospho-EGFR (Tyr1173) Ab. Immunoblotting with anti-GAPHD mAb was performed as an internal control. The cell lysate of HL-60 leukemia cells was used as a negative control, as previously described (11). Numbers indicate the ratios of EGFR to GAPDH and the ratios of the phospho-EGFR to EGFR in each lane. N.D., not detectable. (B) Cells were cultured under the complete culture condition and AAS culture condition in the presence of GEF (0–25 µM) for 24 and 48 h. Viable cell number was assessed by CellTiter-Blue cell viability assay as described in Materials and methods (*P<0.05 and **P<0.01, complete culture condition vs. AAS by a two-tailed non-paired Student's t-test). By multivariate linear regression analysis using both GEF and AAS as independent variables, the synergistic effect was observed in the 24-h treatment of CAL-27 cells (P<0.001) and PANC-1 cells (P=0.008), as well as in the 48-h treatment of CAL-27 cells (P<0.001), Detroit 562 cells (P=0.023), A549 cells (P<0.001) and PANC-1 cells (P<0.001). In the PC-9 cells, the additive cytotoxic effect was observed in the 48-h exposure (P<0.001), but not in the 24-h exposure (P=0.335).
Figure 2
Figure 2
Shortage of intracellular amino acid pool is involved in the pronounced cytotoxicity of gefitinib (GEF) under amino acid starvation (AAS) culture conditions in CAL 27 cells. (A) CAL 27 cells were cultured under AAS conditions supplemented with/without essential and non-essential amino acids in the presence of GEF (0–20 µM) for 48 h. Viable cell number was assessed by CellTiter-Blue cell viability assay (*P<0.05, vs. AAS). EAA, essential amino acids; NEAA, non-essential amino acids. (B) Following pretreatment with LAT-1 siRNA or control siRNA for 24 h, the CAL 27 cells were further cultured with/without GEF (25 µM) for 48 h in DMEM containing 10% FBS, and viable cell numbers were assessed. Immunoblotting with anti-LAT-1 anibody (Ab) was performed after 24 h of treatment with siRNAs. Immunoblotting with anti-GAPDH monoclonal antibody (mAb) was performed as an internal loading control; *P<0.05, vs. control siRNA.
Figure 2
Figure 2
Shortage of intracellular amino acid pool is involved in the pronounced cytotoxicity of gefitinib (GEF) under amino acid starvation (AAS) culture conditions in CAL 27 cells. (A) CAL 27 cells were cultured under AAS conditions supplemented with/without essential and non-essential amino acids in the presence of GEF (0–20 µM) for 48 h. Viable cell number was assessed by CellTiter-Blue cell viability assay (*P<0.05, vs. AAS). EAA, essential amino acids; NEAA, non-essential amino acids. (B) Following pretreatment with LAT-1 siRNA or control siRNA for 24 h, the CAL 27 cells were further cultured with/without GEF (25 µM) for 48 h in DMEM containing 10% FBS, and viable cell numbers were assessed. Immunoblotting with anti-LAT-1 anibody (Ab) was performed after 24 h of treatment with siRNAs. Immunoblotting with anti-GAPDH monoclonal antibody (mAb) was performed as an internal loading control; *P<0.05, vs. control siRNA.
Figure 3
Figure 3
Non-apoptotic cell death induction following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions in CAL 27 cells. (A) Immunoblotting with caspase-3 antibody (Ab) and PARP Ab from the cell lysates of CAL 27 cells cultured under the complete culture condition or AAS with/without GEF (25 µM) for 24 h. Immunoblotting with anti-β-actin mAb was used as an internal control. Numbers indicate the ratios of the cleaved PARP to β-actin and the cleaved caspase-3 to β-actin in each lane. N.D., not detectable. (B) Flow cytometry with Annexin V/PI double staining after 24 h of treatment of CAL 27 cells with GEF (25 µM) under the complete culture condition or AAS containing 10% FBS. The vertical axis indicates the log fluorescence intensity of propidium iodide (PI) and the horizontal axis indicates the log fluorescence intensity of Annexin V. The numbers indicate the percentage of cells in each area. (C) May-Grünwald-Giemsa staining was performed following treatment with/without GEF (25 µM) under the complete culture condition or AAS for 24 h. As a positive control for apoptosis induction, CAL 27 cells were treated with staurosporine at 1 µM for 4 h. Scale bar, 10 µm.
Figure 3
Figure 3
Non-apoptotic cell death induction following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions in CAL 27 cells. (A) Immunoblotting with caspase-3 antibody (Ab) and PARP Ab from the cell lysates of CAL 27 cells cultured under the complete culture condition or AAS with/without GEF (25 µM) for 24 h. Immunoblotting with anti-β-actin mAb was used as an internal control. Numbers indicate the ratios of the cleaved PARP to β-actin and the cleaved caspase-3 to β-actin in each lane. N.D., not detectable. (B) Flow cytometry with Annexin V/PI double staining after 24 h of treatment of CAL 27 cells with GEF (25 µM) under the complete culture condition or AAS containing 10% FBS. The vertical axis indicates the log fluorescence intensity of propidium iodide (PI) and the horizontal axis indicates the log fluorescence intensity of Annexin V. The numbers indicate the percentage of cells in each area. (C) May-Grünwald-Giemsa staining was performed following treatment with/without GEF (25 µM) under the complete culture condition or AAS for 24 h. As a positive control for apoptosis induction, CAL 27 cells were treated with staurosporine at 1 µM for 4 h. Scale bar, 10 µm.
Figure 3
Figure 3
Non-apoptotic cell death induction following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions in CAL 27 cells. (A) Immunoblotting with caspase-3 antibody (Ab) and PARP Ab from the cell lysates of CAL 27 cells cultured under the complete culture condition or AAS with/without GEF (25 µM) for 24 h. Immunoblotting with anti-β-actin mAb was used as an internal control. Numbers indicate the ratios of the cleaved PARP to β-actin and the cleaved caspase-3 to β-actin in each lane. N.D., not detectable. (B) Flow cytometry with Annexin V/PI double staining after 24 h of treatment of CAL 27 cells with GEF (25 µM) under the complete culture condition or AAS containing 10% FBS. The vertical axis indicates the log fluorescence intensity of propidium iodide (PI) and the horizontal axis indicates the log fluorescence intensity of Annexin V. The numbers indicate the percentage of cells in each area. (C) May-Grünwald-Giemsa staining was performed following treatment with/without GEF (25 µM) under the complete culture condition or AAS for 24 h. As a positive control for apoptosis induction, CAL 27 cells were treated with staurosporine at 1 µM for 4 h. Scale bar, 10 µm.
Figure 4
Figure 4
Induction of autophagy following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions or complete culture condition. (A) CAL 27 cells were treated with GEF at 25 µM for 24 h. Cellular proteins were separated by 15% SDS-PAGE, and immunoblotting was performed using anti-LC3B antibody (Ab) and anti-p62 monoclonal antibody (mAb). Immunoblotting with anti-β-actin mAb was performed as an internal control. As a positive control for p62 and LC3B immunoblotting, the cell lysate derived from MDA-MB-231 cells treated with AZM (50 µM) was usedas described in our literature (29). Numbers indicate the ratios of the p62 to β-actin and the LCB-II to β-actin in each lane. (B) Assessment of the induction of autophagy in A549/pBABE-puro-mCherry-EGFP-LC3B stable transfectants. Following 24 h of culture under the complete culture condition to make the cells adherent, the cells were washed twice with PBS and incubated in either the AAS culture medium containing 10% FBS or control complete DMEM with/without GEF (25 µM) for time-lapse imaging at 5-min interval by confocal laser scanning microscopy. Images were merged with EGFP, mCherry and phase contrast. Scale bar, 10 µm. The image on the left bottom panel is an enlarged image of the cells treated with AAS plus GEF at the 1-h time-point. N, nucleus. The numbers of intracellular autophagosome were assessed by counting the fluorescent puncta (EGFP and/or mCherry alone) in 20 cells in each culture condition, and plotted at the indicated time-point. Data are representative of three independent experiments and values are expressed as the means ± SEM (*P<0.05, AAS plus GEF vs. GEF or AAS alone). CAL 27/pBABE-puro-mCherry-EGFP-LC3B stable transfectants behaved similarly to A549 transfectants in autophagosome formation. However, the precise assessment of autophagosome number was difficult on live imaging as the autophagosomes grow in a lump without forming a fine single cell layer (data not shown).
Figure 4
Figure 4
Induction of autophagy following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions or complete culture condition. (A) CAL 27 cells were treated with GEF at 25 µM for 24 h. Cellular proteins were separated by 15% SDS-PAGE, and immunoblotting was performed using anti-LC3B antibody (Ab) and anti-p62 monoclonal antibody (mAb). Immunoblotting with anti-β-actin mAb was performed as an internal control. As a positive control for p62 and LC3B immunoblotting, the cell lysate derived from MDA-MB-231 cells treated with AZM (50 µM) was usedas described in our literature (29). Numbers indicate the ratios of the p62 to β-actin and the LCB-II to β-actin in each lane. (B) Assessment of the induction of autophagy in A549/pBABE-puro-mCherry-EGFP-LC3B stable transfectants. Following 24 h of culture under the complete culture condition to make the cells adherent, the cells were washed twice with PBS and incubated in either the AAS culture medium containing 10% FBS or control complete DMEM with/without GEF (25 µM) for time-lapse imaging at 5-min interval by confocal laser scanning microscopy. Images were merged with EGFP, mCherry and phase contrast. Scale bar, 10 µm. The image on the left bottom panel is an enlarged image of the cells treated with AAS plus GEF at the 1-h time-point. N, nucleus. The numbers of intracellular autophagosome were assessed by counting the fluorescent puncta (EGFP and/or mCherry alone) in 20 cells in each culture condition, and plotted at the indicated time-point. Data are representative of three independent experiments and values are expressed as the means ± SEM (*P<0.05, AAS plus GEF vs. GEF or AAS alone). CAL 27/pBABE-puro-mCherry-EGFP-LC3B stable transfectants behaved similarly to A549 transfectants in autophagosome formation. However, the precise assessment of autophagosome number was difficult on live imaging as the autophagosomes grow in a lump without forming a fine single cell layer (data not shown).
Figure 5
Figure 5
Effects of autophagy inhibition on gefitinib (GEF)-induced cytotoxicity using the Atg5 tet-off MEF cell line (m5–7). Following pre-treatment with/without doxycycline (Dox, 10 ng/ml) for 4 days, the m5–7 cells were seeded in a 96-well culture plate in pentaplicate for 24 h and washed twice with PBS. The cell culture medium was replaced with complete culture medium or amino acid starvation (AAS) culture medium in the presence or absence of GEF (50 µM, at approximately IC50) for 24 h. Assessment of the viable cell number and immunoblotting with anti-Atg5 antibody were performed as described above. Immunoblotting with GAPDH monoclonal antibody was used as an internal control. Significance is indicated as follows: a vs. a′ shows no statistical significance, and b vs. b′ also shows no statistical significance.
Figure 6
Figure 6
Effects of various inhibitors on the enhanced cytotoxicity by combining gefitinib (GEF) and amino acid starvation (AAS) in CAL 27 and A549 cells. The CAL 27 and A549 cells were treated with 3-MA, SP600125, Z-VAD-fmk and necrostatin-1 (NEC-1) at the indicated concentrations in the presence or absence of GEF (25 µM) under the complete culture condition or AAS for 24 h (*P<0.05 and **P<0.01, vs. AAS without NEC-1).
Figure 7
Figure 7
Electron microscopy of CAL 27 cells following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions. The CAL 27 cells were treated with GEF (25 µM) under AAS culture conditions for 24 h, and electron microscopy was performed in adherent and non-adherent CAL 27 cells. Scale bar represents the length indicated at each magnification. The white square box area in the upper panels was enlarged in the lower panels. N, nucleus; mt, mitochondria; arrowhead, autophagosome/autolysosome; open (white) arrow, swollen mitochondria; closed (black) arrow, membrane rupture.
Figure 8
Figure 8
Assessment of the induction of necroptosis in CAL 27 cells following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions. (A) Following pre-treatment with RIPK1 siRNA or control siRNA for 48 h, the CAL 27 cells were further cultured with/without GEF (25 µM) under either AAS conditions with 10% FBS or DMEM containing 10% FBS, and viable cell number was assessed. Immunoblotting with anti-RIPK1 antibody was performed after 24 h of treatment with siRNAs. Immunoblotting with anti-GAPDH monoclonal antibody was performed as an internal loading control (*P<0.05, control siRNA vs. RIPK1 siRNA). (B) Following treatment of the CAL 27 cells with GEF (25 µM) under AAS culture conditions for the indicated time periods, the cells were lysed and the cellular proteins were separated by SDS-PAGE, and then immunoblotted with anti-RIPK1 antibody (Ab), anti-RIPK3 Ab, anti-MLKL Ab, and phosphor (p)-specific Abs for RIPK1 MLKL Abs. As for the positive control for the typical necroptosis induction, HT-29 cells were pre-treated with Z-VAD-fmk (20 µM) for 30 min followed by an additional treatment with cycloheximide (CHX, 10 µg/ml) and human TNF-α (20 ng/ml) for 8 h as previously reported (28). This treatment is indicated as ZCT. (C) The CAL 27 cells treated with GEF (25 µM) under AAS conditions for 24 h were used for co-immunoprecipitation assay. The CAL 27 cells treated under the complete culture condition without GEF were used as control. Cell lysates were immunoprecipitated with anti-RIPK3 Ab or the isotype matched IgG. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with either anti-p-MLKL, anti-MLKL, anti-p-RIPK1, anti-RIPK1, anti-RIPK3, or anti-p62 Ab. HT-29 cells treated with ZCT as described above were used as positive control for necrosome formation.

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References

    1. Kuma A, Mizushima N. Physiological role of autophagy as an intracellular recycling system: With an emphasis on nutrient metabolism. Semin Cell Dev Biol. 2010;21:683–690. doi: 10.1016/j.semcdb.2010.03.002. - DOI - PubMed
    1. Segawa H, Fukasawa Y, Miyamoto K, Takeda E, Endou H, Kanai Y. Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem. 1999;274:19745–19751. doi: 10.1074/jbc.274.28.19745. - DOI - PubMed
    1. Yanagida O, Kanai Y, Chairoungdua A, Kim DK, Segawa H, Nii T, Cha SH, Matsuo H, Fukushima J, Fukasawa Y, et al. Human L-type amino acid transporter 1 (LAT1): Characterization of function and expression in tumor cell lines. Biochim Biophys Acta. 2001;1514:291–302. doi: 10.1016/S0005-2736(01)00384-4. - DOI - PubMed
    1. Oda K, Hosoda N, Endo H, Saito K, Tsujihara K, Yamamura M, Sakata T, Anzai N, Wempe MF, Kanai Y, et al. L-type amino acid transporter 1 inhibitors inhibit tumor cell growth. Cancer Sci. 2010;101:173–179. doi: 10.1111/j.1349-7006.2009.01386.x. - DOI - PubMed
    1. Sakata T, Ferdous G, Tsuruta T, Satoh T, Baba S, Muto T, Ueno A, Kanai Y, Endou H, Okayasu I. L-type amino-acid transporter 1 as a novel biomarker for high-grade malignancy in prostate cancer. Pathol Int. 2009;59:7–18. doi: 10.1111/j.1440-1827.2008.02319.x. - DOI - PubMed
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