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Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells.

Okita R, Wolf D, Yasuda K, Maeda A, Yukawa T, Saisho S, Shimizu K, Yamaguchi Y, Oka M, Nakayama E, Lundqvist A, Kiessling R, Seliger B, Nakata M - PLoS ONE (2015)

Bottom Line: Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.In contrast, Gefitinib attenuated NKG2D ligand expression.In keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of General Thoracic Surgery, Kawasaki Medical School, Kurashiki, Japan; Department of Oncology and Pathology, Immune and Gene Therapy Laboratory, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Introduction: Several cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.

Methods: This study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.

Results: We demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.

Conclusion: In keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.

No MeSH data available.


Related in: MedlinePlus

Gemcitabine-induced upregulation of the expression of NKG2D ligands is regulated via ATM-ATR pathway in A549 cells.(A): Histograms demonstrating MHC class I molecules and NKG2D ligands in A549 cells treated with 5, 10 mM of Caffeine or 30μM of KU55933 or 5mM of N-acetyl-L-cysteine (NAC) for 24 hours. (B): Histograms demonstrating MHC class I and NKG2D ligands in A549 cells pretreated with Caffeine or KU55933 or NAC for 2 hours followed by Gemcitabine (GEM) at 10 nM for 24 hours. Data are representative of three independent experiments. NT: no treatment control. (Ci) A549 cells were treated with Caffeine or KU55933 or for 24 hours. The phosphorylated-ATM (pATM) was assessed by flow cytometry then the effects on the expressions of pATM treated with Caffeine or KU55933 were shown as the relative MFI (rMFI) mean values of four independent experiments and evaluated with Student t-test. (ii) A549 cells were pretreated with Caffeine or KU55933 for 2 hours followed by Gemcitabine for 24 hours then the pATM was assessed by flow cytometry. The effects on the expressions of pATM were shown as the rMFI mean values of at least three independent experiments and evaluated with Student t-test. (D): MHC class I molecules and NKG2D ligands in A549 cells treated with siRNA of ATM for 24 hours followed by Gemcitabine (GEM) at 10 nM for 24 hours. Representative histograms of three independent experiments are shown. The relative MFI (rMFI) of MHC class I molecules and NKG2D ligands were calculated based on at least three independent experiments and evaluated with a Student t-test. Bars, SEM. * -p<0.05 and ** -p<0.01.
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pone.0139809.g003: Gemcitabine-induced upregulation of the expression of NKG2D ligands is regulated via ATM-ATR pathway in A549 cells.(A): Histograms demonstrating MHC class I molecules and NKG2D ligands in A549 cells treated with 5, 10 mM of Caffeine or 30μM of KU55933 or 5mM of N-acetyl-L-cysteine (NAC) for 24 hours. (B): Histograms demonstrating MHC class I and NKG2D ligands in A549 cells pretreated with Caffeine or KU55933 or NAC for 2 hours followed by Gemcitabine (GEM) at 10 nM for 24 hours. Data are representative of three independent experiments. NT: no treatment control. (Ci) A549 cells were treated with Caffeine or KU55933 or for 24 hours. The phosphorylated-ATM (pATM) was assessed by flow cytometry then the effects on the expressions of pATM treated with Caffeine or KU55933 were shown as the relative MFI (rMFI) mean values of four independent experiments and evaluated with Student t-test. (ii) A549 cells were pretreated with Caffeine or KU55933 for 2 hours followed by Gemcitabine for 24 hours then the pATM was assessed by flow cytometry. The effects on the expressions of pATM were shown as the rMFI mean values of at least three independent experiments and evaluated with Student t-test. (D): MHC class I molecules and NKG2D ligands in A549 cells treated with siRNA of ATM for 24 hours followed by Gemcitabine (GEM) at 10 nM for 24 hours. Representative histograms of three independent experiments are shown. The relative MFI (rMFI) of MHC class I molecules and NKG2D ligands were calculated based on at least three independent experiments and evaluated with a Student t-test. Bars, SEM. * -p<0.05 and ** -p<0.01.

Mentions: It has been demonstrated that NKG2D ligand expression can be induced by activation of the DNA stress sensing ATM-ATR [15] or the NKG2D ligand gene transcription via oxidative stress [30]. To determine whether ATM-ATR pathways regulate the NKG2D ligand expression in NSCLC, A549 cells were treated with the ATM-ATR inhibitors Caffeine [31, 32] or KU55933 [33]. In concordance with recently published reports [15, 17], the baseline expression of NKG2D ligands was downregulated by Caffeine, but was not downregulated by KU55933 or by the anti-oxidative reagent NAC in A549 cells (Fig 3A). The upregulation of NKG2D ligands which was observed in A549 cells following 24 hour exposure to 10 nM of Gemcitabine (Fig 2A and 2B) was blocked both by Caffeine and KU55933 treatment but marginal effect by NAC (Fig 3B), suggested that Gemcitabine-induced NKG2D ligand was mainly regulated by ATM-ATR pathway but not by oxidative stress. In subsequent experiments, we explored whether the ATM-ATR signalling would affect NKG2D ligand expression to delineate why Caffeine blocked both basal and Gemcitabine-induced NKG2D ligands while KU55933 blocked only Gemcitabine-induced NKG2D ligands. Flow cytometric analysis of phosphorylated ATM showed that the ATM-ATR inhibitors KU55933 clearly blocked phosphorylation of ATM, while Caffeine had a smaller effect on basal (Fig 3Ci) and Gemcitabine-induced (Fig 3Cii) phosphorylation of ATM than KU55933. To investigate this further, the expression of NKG2D ligands were analyzed in A549 cells treated with ATM-specific siRNA. As shown by Western blot analysis, the ATM expression in A549 cells was diminished by ATM siRNA (S3 Fig). In line with the experiments using the ATM inhibitor KU55933, ATM-specific siRNA did not decrease basal expression of NKG2D ligands, but clearly blocked Gemcitabine-induced NKG2D ligands in A549 cells (Fig 3D).


Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells.

Okita R, Wolf D, Yasuda K, Maeda A, Yukawa T, Saisho S, Shimizu K, Yamaguchi Y, Oka M, Nakayama E, Lundqvist A, Kiessling R, Seliger B, Nakata M - PLoS ONE (2015)

Gemcitabine-induced upregulation of the expression of NKG2D ligands is regulated via ATM-ATR pathway in A549 cells.(A): Histograms demonstrating MHC class I molecules and NKG2D ligands in A549 cells treated with 5, 10 mM of Caffeine or 30μM of KU55933 or 5mM of N-acetyl-L-cysteine (NAC) for 24 hours. (B): Histograms demonstrating MHC class I and NKG2D ligands in A549 cells pretreated with Caffeine or KU55933 or NAC for 2 hours followed by Gemcitabine (GEM) at 10 nM for 24 hours. Data are representative of three independent experiments. NT: no treatment control. (Ci) A549 cells were treated with Caffeine or KU55933 or for 24 hours. The phosphorylated-ATM (pATM) was assessed by flow cytometry then the effects on the expressions of pATM treated with Caffeine or KU55933 were shown as the relative MFI (rMFI) mean values of four independent experiments and evaluated with Student t-test. (ii) A549 cells were pretreated with Caffeine or KU55933 for 2 hours followed by Gemcitabine for 24 hours then the pATM was assessed by flow cytometry. The effects on the expressions of pATM were shown as the rMFI mean values of at least three independent experiments and evaluated with Student t-test. (D): MHC class I molecules and NKG2D ligands in A549 cells treated with siRNA of ATM for 24 hours followed by Gemcitabine (GEM) at 10 nM for 24 hours. Representative histograms of three independent experiments are shown. The relative MFI (rMFI) of MHC class I molecules and NKG2D ligands were calculated based on at least three independent experiments and evaluated with a Student t-test. Bars, SEM. * -p<0.05 and ** -p<0.01.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4595469&req=5

pone.0139809.g003: Gemcitabine-induced upregulation of the expression of NKG2D ligands is regulated via ATM-ATR pathway in A549 cells.(A): Histograms demonstrating MHC class I molecules and NKG2D ligands in A549 cells treated with 5, 10 mM of Caffeine or 30μM of KU55933 or 5mM of N-acetyl-L-cysteine (NAC) for 24 hours. (B): Histograms demonstrating MHC class I and NKG2D ligands in A549 cells pretreated with Caffeine or KU55933 or NAC for 2 hours followed by Gemcitabine (GEM) at 10 nM for 24 hours. Data are representative of three independent experiments. NT: no treatment control. (Ci) A549 cells were treated with Caffeine or KU55933 or for 24 hours. The phosphorylated-ATM (pATM) was assessed by flow cytometry then the effects on the expressions of pATM treated with Caffeine or KU55933 were shown as the relative MFI (rMFI) mean values of four independent experiments and evaluated with Student t-test. (ii) A549 cells were pretreated with Caffeine or KU55933 for 2 hours followed by Gemcitabine for 24 hours then the pATM was assessed by flow cytometry. The effects on the expressions of pATM were shown as the rMFI mean values of at least three independent experiments and evaluated with Student t-test. (D): MHC class I molecules and NKG2D ligands in A549 cells treated with siRNA of ATM for 24 hours followed by Gemcitabine (GEM) at 10 nM for 24 hours. Representative histograms of three independent experiments are shown. The relative MFI (rMFI) of MHC class I molecules and NKG2D ligands were calculated based on at least three independent experiments and evaluated with a Student t-test. Bars, SEM. * -p<0.05 and ** -p<0.01.
Mentions: It has been demonstrated that NKG2D ligand expression can be induced by activation of the DNA stress sensing ATM-ATR [15] or the NKG2D ligand gene transcription via oxidative stress [30]. To determine whether ATM-ATR pathways regulate the NKG2D ligand expression in NSCLC, A549 cells were treated with the ATM-ATR inhibitors Caffeine [31, 32] or KU55933 [33]. In concordance with recently published reports [15, 17], the baseline expression of NKG2D ligands was downregulated by Caffeine, but was not downregulated by KU55933 or by the anti-oxidative reagent NAC in A549 cells (Fig 3A). The upregulation of NKG2D ligands which was observed in A549 cells following 24 hour exposure to 10 nM of Gemcitabine (Fig 2A and 2B) was blocked both by Caffeine and KU55933 treatment but marginal effect by NAC (Fig 3B), suggested that Gemcitabine-induced NKG2D ligand was mainly regulated by ATM-ATR pathway but not by oxidative stress. In subsequent experiments, we explored whether the ATM-ATR signalling would affect NKG2D ligand expression to delineate why Caffeine blocked both basal and Gemcitabine-induced NKG2D ligands while KU55933 blocked only Gemcitabine-induced NKG2D ligands. Flow cytometric analysis of phosphorylated ATM showed that the ATM-ATR inhibitors KU55933 clearly blocked phosphorylation of ATM, while Caffeine had a smaller effect on basal (Fig 3Ci) and Gemcitabine-induced (Fig 3Cii) phosphorylation of ATM than KU55933. To investigate this further, the expression of NKG2D ligands were analyzed in A549 cells treated with ATM-specific siRNA. As shown by Western blot analysis, the ATM expression in A549 cells was diminished by ATM siRNA (S3 Fig). In line with the experiments using the ATM inhibitor KU55933, ATM-specific siRNA did not decrease basal expression of NKG2D ligands, but clearly blocked Gemcitabine-induced NKG2D ligands in A549 cells (Fig 3D).

Bottom Line: Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.In contrast, Gefitinib attenuated NKG2D ligand expression.In keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of General Thoracic Surgery, Kawasaki Medical School, Kurashiki, Japan; Department of Oncology and Pathology, Immune and Gene Therapy Laboratory, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Introduction: Several cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.

Methods: This study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.

Results: We demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.

Conclusion: In keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.

No MeSH data available.


Related in: MedlinePlus