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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 PGE2 synthesis. 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 and EGFR, or the COX inhibitor indomethacin (indo) and the specific COX-2 inhibitor NS-398 for 24 h. 8 h before the end of the incubation period, and AA was added. PGE2 release into the cell supernatant is given in pg/ml. Data are expressed as mean ± SEM of at least six independent experiments
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Fig4: Mechanisms of LPS-induced PGE2 synthesis. 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 and EGFR, or the COX inhibitor indomethacin (indo) and the specific COX-2 inhibitor NS-398 for 24 h. 8 h before the end of the incubation period, and AA was added. PGE2 release into the cell supernatant is given in pg/ml. Data are expressed as mean ± SEM of at least six independent experiments

Mentions: Unlike otherwise indicated, data are given as the relative changes compared to control values and expressed as the mean ± SEM. Raw data were analyzed with R [39]. Linear mixed models were calculated using the package “lme” [40]. Raw data from time series were analyzed using the area-under-the-curve (AUC) approach. AUC values were calculated using the trapezoid rule. Percentages were analyzed using beta regression [41]. Linear mixed models were used for Figs. 1a, b, 2, 3a and 4. Linear models were used for Figs. 1c, d, 3c and 5a. Beta regression was used for Fig. 5b. Residuals of the models were checked for normal distribution, variance homogeneity and influential points. Reported p values are not corrected for multiple testing. Unless otherwise stated, p values below 0.05 keep a family-wise error rate of 5 % (i.e., they would be <0.05 after Bonferroni correction).Fig. 4


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 PGE2 synthesis. 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 and EGFR, or the COX inhibitor indomethacin (indo) and the specific COX-2 inhibitor NS-398 for 24 h. 8 h before the end of the incubation period, and AA was added. PGE2 release into the cell supernatant is given in pg/ml. Data are expressed as mean ± SEM of at least six independent experiments
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Related In: Results  -  Collection

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Fig4: Mechanisms of LPS-induced PGE2 synthesis. 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 and EGFR, or the COX inhibitor indomethacin (indo) and the specific COX-2 inhibitor NS-398 for 24 h. 8 h before the end of the incubation period, and AA was added. PGE2 release into the cell supernatant is given in pg/ml. Data are expressed as mean ± SEM of at least six independent experiments
Mentions: Unlike otherwise indicated, data are given as the relative changes compared to control values and expressed as the mean ± SEM. Raw data were analyzed with R [39]. Linear mixed models were calculated using the package “lme” [40]. Raw data from time series were analyzed using the area-under-the-curve (AUC) approach. AUC values were calculated using the trapezoid rule. Percentages were analyzed using beta regression [41]. Linear mixed models were used for Figs. 1a, b, 2, 3a and 4. Linear models were used for Figs. 1c, d, 3c and 5a. Beta regression was used for Fig. 5b. Residuals of the models were checked for normal distribution, variance homogeneity and influential points. Reported p values are not corrected for multiple testing. Unless otherwise stated, p values below 0.05 keep a family-wise error rate of 5 % (i.e., they would be <0.05 after Bonferroni correction).Fig. 4

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