Fig 1.
The expressions of MHC class I, MICA, MICB, ULBP1, ULBP2/5/6, and ULBP3 in non-small-cell lung cancer cell lines.
The basal expression of each cell surface molecules was assessed by flow cytometric analysis. The cells were stained with isotype control (white histograms) or specific antibody for the indicated molecule (gray histograms).
Fig 2.
The expression of NKG2D ligands are upregulated by Gemcitabine in A549 cells.
(A): A549 cells were treated with or without 1 to 10nM of Gemcitabine (GEM), Pemetrexed (PEM), Docetaxel (DTX) or Vinorelbine (VNR) for 24 hours, then the expression of MHC class I molecules and NKG2D ligands were assessed by flow cytometry. (B): 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.
Fig 3.
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.
Fig 4.
The expression of NKG2D ligands are regulated by EGFR/PI3K/AKT pathway in PC–9 cells.
(Ai): The basal expression of EGFR was assessed by flow cytometry in PC–9 cells. (ii): WST cell proliferation assay showed PC–9 cells were sensitive to Gefitinib (Gef). (B): PC–9 cells were treated with or without 1 μM of Gef for 24 hours. MHC class I molecules and NKG2D ligands were assessed by flow cytometry. The representative histograms from three independent experiments were shown. The relative MFI (rMFI) of MHC class I molecules, MICB, and ULBP–2/5/6 were calculated based on at least three independent experiments and evaluated with a Student t-test. (C): PC–9 cells were transfected with siRNA targeting EGFR (siEGFR) or control siRNA (siCtr) as control for 48 hours. The expressions of EGFR, phosphorylated EGFR (pEGFR) and β-actin were assessed by Western blot analyses. Data are presented as representatives of three independent experiments. (D): The expressions of MHC class I, MICB and ULBP2/5/6 were assessed by flow cytometry, then the effects on the expressions of these molecules treated with siRNA of EGFR were shown as the rMFI mean values of three independent experiments and evaluated with Student t-test. (E): PC–9 cells were cultured with various concentration of the PI3K inhibitor LY294002, MEK1 inhibitor PD98059 or DMSO (0.01%) as control. The effects on the expressions of MICB and ULBP2/5/6 treated with each inhibitor are shown as the rMFI mean values of three independent experiments and evaluated with Student t-test. Bars, SEM. * -p<0.05
Fig 5.
The expressions of miRs in NSCLC cells treated with Gemcitabine or Gefitinib.
(A): A549 cells were treated with Gemcitabine for 24h then miR10b, miR20a, miR34c and miR106 expression was evaluated using quantitative real-time PCR as described in Materials and Methods. The results represent the mean of duplicates of 3 independent experiments. (B): PC–9 cells were treated with Gefitinib for 24h then miR10b, miR20a, miR34c and miR106 were evaluated using quantitative real-time PCR for 3 independent experiments. Differences in means were evaluated with Student t-test. (C): NKG2D ligands MICA and ULBP–2/5/6 were assessed by flow cytometry in A549 cells treated with miR20a mimic for 48 hours followed by Gemcitabine (GEM) at 10 nM for 24 hours. Representative histograms of three independent experiments and the relative MFI (rMFI) of NKG2D ligands were calculated based on three independent experiments and evaluated with a Student t-test. (D): NKG2D ligands MICB and ULBP–2/5/6 were assessed by flow cytometry in PC–9 cells treated with miR20a inhibitor for 24 hours followed by Gefitinib (Gef) at 1 μM for 24 hours. Representative histograms of three independent experiments and the relative MFI (rMFI) of NKG2D ligands were calculated based on three independent experiments and evaluated with a Student t-test. Bars, SEM. * -p<0.05 and ** -p<0.01.
Fig 6.
Effects of Gemcitabine or Gefitinib on NK cell-mediated cytotoxicity and NK cell degranulation.
(Ai): A549 cells with or without Gemcitabine (GEM) (10 nM) for 24 hours were subjected to LDH release assay for 4 hours using IL–2 activated NK cells as effector cells. The IL–2 activated NK cells were pretreated with blocking antibody of NKG2D or isotype control for 30 min before the cytotoxicity assay. Data are presented as mean of triplicate samples and are representative of three independent experiments. (ii): IL–2 activated NK cells were pretreated with blocking antibody of NKG2D or isotype control before NK cell degranulation assay, then coincubated with A549 cells treated with or without GEM for 4 hours, and NK cell degranulation was evaluated. (Bi): PC–9 cells with or without Gefitinib (Gef) (1 μM) were subjected to LDH release assay for 4 hours using IL–2 activated NK cells as effector cells. Data are presented as mean of triplicate samples and are representative of three independent experiments. (ii): IL–2 activated NK cells were coincubated for 4 hours with PC–9 cells pretreated with or without Gefitinib, and NK cell-degranulation was evaluated. Data are representative of three independent experiments. E:T ratio; effector/target ratio.
Fig 7.
Schematic diagram summarized how ATM-ATR and EGFR pathway regulate MICA/B expression in NSCLC cells.