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Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation.

Hattar K, Savai R, Subtil FS, Wilhelm J, Schmall A, Lang DS, Goldmann T, Eul B, Dahlem G, Fink L, Schermuly RT, Banat GA, Sibelius U, Grimminger F, Vollmer E, Seeger W, Grandel U - Cancer Immunol. Immunother. (2012)

Bottom Line: Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4.Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells.Synthesis of PGE(2) was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine IV/V, University of Giessen and Marburg Lung Center (UGMLC), Klinikstrasse 33, Giessen, Germany.

ABSTRACT
Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E(2) were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE(2) release and proliferation in response to LPS. Synthesis of PGE(2) was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.

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Mechanisms of LPS-induced A549 proliferation. A549 cells were either sham-incubated (control) or exposed to 10 μg/ml of LPS (n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 or EGFR. After 24 h of incubation proliferation was quantified by determining MTS activity. All data are expressed as percentage of unstimulated cells (control). Mean ± SEM of six independent experiments are given
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Fig2: Mechanisms of LPS-induced A549 proliferation. A549 cells were either sham-incubated (control) or exposed to 10 μg/ml of LPS (n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 or EGFR. After 24 h of incubation proliferation was quantified by determining MTS activity. All data are expressed as percentage of unstimulated cells (control). Mean ± SEM of six independent experiments are given

Mentions: In brief, A549 cells were seeded on 96-well plates (2,500 cells/well) and maintained in culture for 24 h before LPS stimulation. Then, medium was exchanged, and cells were kept in RPMI containing 1 % FCS at a total volume of 200 μl/well. A549 cells were stimulated with different concentrations of LPS (E. coli LPS 0111:B4, Sigma, Deisenhofen, Germany or highly purified E.coli LPS F515) for various time periods or sham incubation (control) was performed. For Fig. 1a with LPS from E. coli 0111:B4, at least five independent experiments were performed, and for Fig. 1b with LPS from E. coli F515, at least four independent experiments were performed. In an additional series of experiments in Fig. 2, function-blocking antibodies targeting TLR2 (clone TL2.1, e-Bioscience, San Diego, CA, USA), TLR4 (clone HTA 125, e-Bioscience, San Diego, CA, USA), CD14 (MY-4, Coulter Immunotech, Hamburg, Germany), EGFR (Cetuximab, Merck Serono, Germany) or COX inhibitors (indomethacin, Sigma, Deisenhofen, Germany and NS-398, Calbiochem, La Jolla, CA, USA) were applied simultaneously to LPS. For these inhibitor studies, at least six independent experiments were performed (at least 3 both for LPS 0111:B4 and for LPS F515, respectively).Fig. 1


Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation.

Hattar K, Savai R, Subtil FS, Wilhelm J, Schmall A, Lang DS, Goldmann T, Eul B, Dahlem G, Fink L, Schermuly RT, Banat GA, Sibelius U, Grimminger F, Vollmer E, Seeger W, Grandel U - Cancer Immunol. Immunother. (2012)

Mechanisms of LPS-induced A549 proliferation. A549 cells were either sham-incubated (control) or exposed to 10 μg/ml of LPS (n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 or EGFR. After 24 h of incubation proliferation was quantified by determining MTS activity. All data are expressed as percentage of unstimulated cells (control). Mean ± SEM of six independent experiments are given
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569588&req=5

Fig2: Mechanisms of LPS-induced A549 proliferation. A549 cells were either sham-incubated (control) or exposed to 10 μg/ml of LPS (n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 or EGFR. After 24 h of incubation proliferation was quantified by determining MTS activity. All data are expressed as percentage of unstimulated cells (control). Mean ± SEM of six independent experiments are given
Mentions: In brief, A549 cells were seeded on 96-well plates (2,500 cells/well) and maintained in culture for 24 h before LPS stimulation. Then, medium was exchanged, and cells were kept in RPMI containing 1 % FCS at a total volume of 200 μl/well. A549 cells were stimulated with different concentrations of LPS (E. coli LPS 0111:B4, Sigma, Deisenhofen, Germany or highly purified E.coli LPS F515) for various time periods or sham incubation (control) was performed. For Fig. 1a with LPS from E. coli 0111:B4, at least five independent experiments were performed, and for Fig. 1b with LPS from E. coli F515, at least four independent experiments were performed. In an additional series of experiments in Fig. 2, function-blocking antibodies targeting TLR2 (clone TL2.1, e-Bioscience, San Diego, CA, USA), TLR4 (clone HTA 125, e-Bioscience, San Diego, CA, USA), CD14 (MY-4, Coulter Immunotech, Hamburg, Germany), EGFR (Cetuximab, Merck Serono, Germany) or COX inhibitors (indomethacin, Sigma, Deisenhofen, Germany and NS-398, Calbiochem, La Jolla, CA, USA) were applied simultaneously to LPS. For these inhibitor studies, at least six independent experiments were performed (at least 3 both for LPS 0111:B4 and for LPS F515, respectively).Fig. 1

Bottom Line: Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4.Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells.Synthesis of PGE(2) was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine IV/V, University of Giessen and Marburg Lung Center (UGMLC), Klinikstrasse 33, Giessen, Germany.

ABSTRACT
Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E(2) were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE(2) release and proliferation in response to LPS. Synthesis of PGE(2) was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.

Show MeSH
Related in: MedlinePlus