Figure 1.
The peripheral mu opioid receptor antagonist, methylnaltrexone (MNTX), inhibits epidermal growth factor (EGF)-induced proliferation and migration of human lung cancer cells in a dose-dependent manner.
Panel A: Human H358 non-small cell lung cancer (NSCLC) cells were analyzed for methylnaltrexone (MNTX) inhibition of EGF-mediated proliferation using a MTS proliferation assay. Cells were growth in the presence of 100 ng/ml EGF and/or 0–250 nM MNTX for 72 hours. MNTX There is a statistically significant difference (p<0.05, indicated by an asterisks) between control and MNTX (10, 50, 100, 250 nM) treatment with n = 3 per condition and error bars = standard deviation. See the Methods section for experimental details. Panel B: Human H358 non-small cell lung cancer (NSCLC) cells were analyzed for methylnaltrexone (MNTX) inhibition of EGF-mediated migration using a transwell assay (8 uM pore size). Cells were allowed to migrate in the presence of 100 ng/ml EGF and/or 0–250 nM MNTX for 18 hours. There is a statistically significant difference (p<0.05, indicated by an asterisks) between control and MNTX (50, 100, 250 nM) treatment with n = 3 per condition and error bars = standard deviation. See the Methods section for experimental details.
Figure 2.
The mu opioid receptor (MOR) is recruited to the EGF receptor with EGF stimulation but does not regulate EGF receptor phosphorylation.
Panel A: Human H358 non-small cell lung cancer (NSCLC) cells were treated with no (control) or 100 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoprecipitated with anti-EGFR antibody. Immunoblots were performed on total cell lysates (left) and immunoprecipitated material (right) using anti-MOR (a) and anti-EGFR (b) antibodies. The mu opioid receptor is recruited to the EGFR with EGF stimulation. Panel B: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone, 10 ng/ml EGF for 5, 15 or 30 minutes, or 100 nM MNTX and 10 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoblotted using anti-pY845 EGFR (a), anti-pY992 EGFR (b), anti-pY1045 EGFR (c), anti-pY1068 EGFR (d), anti-EGFR (e) and anti-actin (e) antibodies. MNTX does not inhibit EGF-induced EGFR tyrosine phosphorylation.
Figure 3.
The peripheral mu opioid receptor antagonist, methylnaltrexone (MNTX), inhibits EGF-induced recruitment/activation of the linker protein, Grb-2, and the scaffolding protein, Gab-1, in human lung cancer cells.
Panel A: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX (1 hour pre-incubation), 10 ng/ml EGF for 15 minutes, or 100 nM MNTX and 10 ng/ml EGF. Cell lysates were obtained and immunoprecipitated with anti-EGFR antibody. Immunoblots were performed on total cell lysates and immunoprecipitated material using anti-Grb-2 (a,c) and anti-EGFR (b,d) antibody. MNTX inhibits EGF-induced recruitment of Grb-2 to the EGFR. Panel B: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone, 10 ng/ml EGF for 5, 15 or 30 minutes, or 100 nM MNTX and 10 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoblotted using anti-pY627 Gab-1 (a), anti-pY307 Gab-1 (b) and anti-actin (c) antibodies. MNTX attenuates EGF-induced Gab-1 tyrosine phosphorylation.
Figure 4.
The peripheral mu opioid receptor antagonist, methylnaltrexone (MNTX), inhibits EGF-induced phosphorylation of Src, PI3 kinase and STAT3 in human lung cancer cells.
Panel A: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone, 10 ng/ml EGF for 5, 15 or 30 minutes, or 100 nM MNTX and 10 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoblotted using anti-phospho-Src (pY416) (a), anti-phospho-p85/p55 PI3 kinase (pY458/pY199) (b,c), anti-phospho-STAT3 (pY705) (d) and anti-actin (e) antibodies. Panel B: Graphical quantitation of immunoreactivity of experiments performed described in Figure 3-B and Panel A with normalization to total specific protein and n = 3 independent experiments per condition. An asterisk (*) indicates a statistically significant difference (p<0.05) from control. The error bars = standard deviation.
Figure 5.
Inhibiting the mu opioid receptor (MOR), Gab-1, Src, PI3 kinase, Akt or STAT3 attenuates EGF and DAMGO-induced proliferation and migration in human lung cancer cells.
Panel A: Human H358 non-small cell lung cancer (NSCLC) cells were analyzed for EGF and DAMGO-mediated proliferation using a MTS proliferation assay. H358 human NSCLC cells were either untreated (control) or treated with 100 ng/ml epidermal growth factor (EGF) or 100 nM DAMGO for 72 hours with or without pretreatment of cells with the peripheral MOR antagonist, methylnaltrexone (MNTX, 100 nM), MOR siRNA, Gab-1 siRNA, Src siRNA, the PI3 kinase inhibitor LY294002 (10 uM), Akt Inhibitor X (5 uM), the Src family kinase inhibitor PP2 (100 nM) or the STAT3 inhibitor Stattic (10 uM). There is a statistically significant difference (p<0.05, indicated by an asterisks) between control and treatment groups with n = 3 independent experiments per condition and error bars = standard deviation. See the Methods section for experimental details. Panel B: Human H358 non-small cell lung cancer (NSCLC) cells were analyzed for EGF and DAMGO-mediated migration using a transwell assay (8 uM pore size). H358 human NSCLC cells were either untreated (control) or treated with 100 ng/ml epidermal growth factor (EGF) or 100 nM DAMGO for 18 hours with or without pretreatment of cells with the peripheral MOR antagonist, methylnaltrexone (MNTX, 100 nM), MOR siRNA, Gab-1 siRNA, Src siRNA, the PI3 kinase inhibitor LY294002 (10 uM), Akt Inhibitor X (5 uM), the Src family kinase inhibitor PP2 (100 nM) or the STAT3 inhibitor Stattic (10 uM). There is a statistically significant difference (p<0.05, indicated by an asterisks) between control and treatment groups with n = 3 independent experiments per condition and error bars = standard deviation. See the Methods section for experimental details.
Figure 6.
Overexpression or silencing of the mu opioid receptor (MOR) or addition of opioids in human lung cancer cells regulates epithelial mesenchymal transition (EMT).
Panel A: Control (non-transfected)(C), stable vector control (VC) and MOR1 overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with EMT markers anti-vimentin (a), anti-Snail (b), anti-Slug (c), anti-claudin-1 (d), anti-ZO-1 (e), anti-MOR (f) and anti-actin (g) antibodies. An increase in vimentin, Snail and Slug expression and a decrease in claudin-1 and ZO-1 expression suggest an epithelial mesenchymal transition. Panel B: Graphical quantitation of immunoreactivity of experiments performed described in Panel A with n = 3 independent experiments per condition. An asterisk (*) indicates a statistically significant difference (p<0.05) from control with error bars = standard deviation. Panel C: H358 human NSCLC cells were either untreated, treated with 100 ng/ml epidermal growth factor (EGF), 100 nM DAMGO, morphine or fentanyl or 100 ng/ml insulin growth factor (IGF) for 96 hours, cell lysates obtained and immunoblotted with EMT markers anti-vimentin (a), anti-Snail (b), anti-Slug (c), anti-claudin-1 (d), anti-ZO-1 (e), anti-MOR (f) and anti-actin (g) antibodies. A decrease in vimentin, Snail and Slug expression and an increase in claudin-1 and ZO-1 expression suggest inhibition of epithelial mesenchymal transition. Panel D: Control shRNA or MOR shRNA H358 cell lines were generated, cell lysates obtained and immunoblotted with EMT markers anti-vimentin (a), anti-Snail (b), anti-Slug (c), anti-claudin-1 (d), anti-ZO-1 (e), anti-MOR (f) and anti-actin (g) antibodies. An increase in vimentin, Snail and Slug expression and a decrease in claudin-1 and ZO-1 expression suggest an epithelial mesenchymal transition.
Figure 7.
Morphologic and epithelial mesenchymal transition (EMT) marker changes in human lung cancer cells with opioids, growth factors and signal transduction pathway inhibitors.
Panel A: H358 human NSCLC cells were either untreated (control), treated with 100 nm morphine or 100 ng/ml insulin growth factor (IGF) for 96 hours and brightfield images were obtained (20×). H358 cells grow in colonies with well demarcated borders and strong cell-cell adhesions. In contrast, morphine or IGF-treated H358 cells show a loss of cell-cell adhesions and a change from cuboidal to an elongated phenotype with several cellular projections visible. These changes are consistent with an epithelial mesenchymal transition (EMT). Panel B: H358 human NSCLC cells were either untreated, treated with 100 ng/ml epidermal growth factor (EGF), 100 nM DAMGO, morphine or fentanyl or 100 ng/ml insulin growth factor (IGF) for 96 hours with or without pretreatment with the peripheral MOR antagonist, methylnaltrexone (MNTX, 100 nM), the Src family kinase inhibitor PP2 (100 nM) or the STAT3 inhibitor Stattic (10 uM). Cell lysates were then obtained and immunoblotted with EMT markers anti-vimentin (a,c,e) or anti-claudin-1 (b,d,f) antibodies. An increase in vimentin expression and a decrease in claudin-1 expression suggest an epithelial mesenchymal transition.
Figure 8.
Inhibiting the mu opioid receptor (MOR), Src, Gab-1, PI3 kinase or STAT3 attenuates opioid and growth factor-induced epithelial mesenchymal transition (EMT) in human lung cancer cells.
Panel A: Graphical representation of the % control vimentin expression. H358 human NSCLC cells were either untreated, treated with 100 ng/ml epidermal growth factor (EGF), 100 nM DAMGO, morphine or fentanyl or 100 ng/ml insulin growth factor (IGF) for 96 hours with or without pretreatment of cells with the peripheral MOR antagonist, methylnaltrexone (MNTX, 100 nM), MOR siRNA, Gab-1 siRNA, Src siRNA, the PI3 kinase inhibitor LY294002 (10 uM), Akt Inhibitor X (5 uM), the Src family kinase inhibitor PP2 (100 nM) or the STAT3 inhibitor Stattic (10 uM). Cell lysates were then obtained and immunoblotted with the EMT marker anti-vimentin antibody. Experiments were repeated in triplicate and immunoreactive bands were analyzed using computer-assisted densitometry. There is a statistically significant difference (p<0.05 indicated by asterisks (*)) between control and treatment groups with error bars = standard deviation. An increase in vimentin expression is suggestive of an epithelial mesenchymal transition. Panel B: Graphical representation of the % control claudin-1 expression. H358 human NSCLC cells were either untreated, treated with 100 ng/ml epidermal growth factor (EGF), 100 nM DAMGO, morphine or fentanyl or 100 ng/ml insulin growth factor (IGF) for 96 hours with or without pretreatment of cells with the peripheral MOR antagonist, methylnaltrexone (MNTX, 100 nM), MOR siRNA, Gab-1 siRNA, Src siRNA, the PI3 kinase inhibitor LY294002 (10 uM), Akt Inhibitor X (5 uM), the Src family kinase inhibitor PP2 (100 nM) or the STAT3 inhibitor Stattic (10 uM). Cell lysates were then obtained and immunoblotted with the EMT marker anti-claudin-1 antibody. Three independent experiments per condition were performed and immunoreactive bands were analyzed using computer-assisted densitometry. There is a statistically significant difference (p<0.05 indicated by asterisks (*)) between control and treatment groups with error bars = standard deviation. A decrease in claudin-1 expression is suggestive of an epithelial mesenchymal transition.
Figure 9.
Schematic diagram illustrating mu opioid receptor (MOR) regulation of growth factor receptor signaling and human lung cancer proliferation, migration and epithelial mesenchymal transition (EMT).
Activation of growth factor receptors promotes complex formation with the mu opioid receptor (MOR) and Grb-2 as well as Src activation (1). These events induce recruitment of the scaffolding protein, Gab1, to the plasma membrane (2) and consequent recruitment/activation of PI3 kinase, Akt and STAT3 signaling (3) which are required for human lung cancer proliferation, migration and epithelial mesenchymal transition (EMT) (4). Inhibition of MOR (siRNA, shRNA and/or the peripheral MOR antagonist, methylnaltrexone (MNTX)) blocks morphine and DAMGO binding to the mu opioid receptor (5). In addition, inhibiting MOR attenuates growth factor-induced phosphorylation/activation of Src, Gab-1, PI3 kinase, Akt and STAT3 with consequent inhibition of human lung cancer proliferation, migration and EMT (6).