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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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Activation of COX-2 and release of PGE2 in A549 cells and human lung cancer tissue in response to LPS. a Effect of COX inhibitors on LPS-induced proliferation of A549 cells in vitro. 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 indomethacin (indo) or the COX-2 inhibitor NS-398. 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. b Expression of COX-2 mRNA in response to LPS in A549 cells and human lung cancer tissue. A549 cells and specimen of human adenocarcinoma were either sham-incubated or exposed to 10 μg/ml LPS F515. After 16 h, mRNA was extracted and subjected to quantitative reverse transcriptase polymerase chain reaction. The ΔΔCT values represent relative expression of COX-2 mRNA normalized to the internal reference HPRT mRNA in LPS versus unstimulated A549 cells or lung adenocarcinoma (Adeno) tissue. Mean values ± SEM, originating from four independent experiments, each performed in duplicate are given. c Release of PGE2 in A549 cells in response to LPS. A549 cells were either sham-incubated (control) or exposed to the given concentrations of LPS (at least n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total at least n = 6) for various time periods. 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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Fig3: Activation of COX-2 and release of PGE2 in A549 cells and human lung cancer tissue in response to LPS. a Effect of COX inhibitors on LPS-induced proliferation of A549 cells in vitro. 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 indomethacin (indo) or the COX-2 inhibitor NS-398. 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. b Expression of COX-2 mRNA in response to LPS in A549 cells and human lung cancer tissue. A549 cells and specimen of human adenocarcinoma were either sham-incubated or exposed to 10 μg/ml LPS F515. After 16 h, mRNA was extracted and subjected to quantitative reverse transcriptase polymerase chain reaction. The ΔΔCT values represent relative expression of COX-2 mRNA normalized to the internal reference HPRT mRNA in LPS versus unstimulated A549 cells or lung adenocarcinoma (Adeno) tissue. Mean values ± SEM, originating from four independent experiments, each performed in duplicate are given. c Release of PGE2 in A549 cells in response to LPS. A549 cells were either sham-incubated (control) or exposed to the given concentrations of LPS (at least n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total at least n = 6) for various time periods. 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: PGE2 and thromboxane (Tx)B2, the stable hydrolysis product of TxA2, were quantified in a commercial ELISA system (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. For these experiments, A549 cells (50,000 cells/well) were seeded on 24-well plates and grown to confluence. Confluent monolayers were washed twice and kept in RPMI containing 1 % FCS at a total volume of 500 μl/well. Then, incubation with different concentrations of LPS (from E. coli 0111:B4 or E. coli F515) in the absence or presence of the respective antibodies or COX inhibitors for various time periods, or sham incubation (control) was performed. All samples were performed as duplicate. For the time–response curve in Fig. 3c, as well as for the inhibitor studies in Fig. 3a, at least six independent experiments were performed (at least 3 both for LPS 0111:B4 and for LPS F515, respectively). At the end of the incubation period, medium was exchanged, and cells were washed twice and kept in RPMI containing 1 % FCS and further incubated for 8 h with 5 μM arachidonic acid (AA, Sigma, Deisenhofen, Germany). Then, cell supernatants were harvested, cell debris was removed by centrifugation at 13.000×g, and samples were stored at −20 °C until further processing. The measurement of PGE2 and TxB2 release was taken by ELISA technique, according to the manufacturer’s protocols and is expressed in pg/ml. All samples were performed as duplicate, and each sample was measured twice.Fig. 3


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)

Activation of COX-2 and release of PGE2 in A549 cells and human lung cancer tissue in response to LPS. a Effect of COX inhibitors on LPS-induced proliferation of A549 cells in vitro. 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 indomethacin (indo) or the COX-2 inhibitor NS-398. 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. b Expression of COX-2 mRNA in response to LPS in A549 cells and human lung cancer tissue. A549 cells and specimen of human adenocarcinoma were either sham-incubated or exposed to 10 μg/ml LPS F515. After 16 h, mRNA was extracted and subjected to quantitative reverse transcriptase polymerase chain reaction. The ΔΔCT values represent relative expression of COX-2 mRNA normalized to the internal reference HPRT mRNA in LPS versus unstimulated A549 cells or lung adenocarcinoma (Adeno) tissue. Mean values ± SEM, originating from four independent experiments, each performed in duplicate are given. c Release of PGE2 in A549 cells in response to LPS. A549 cells were either sham-incubated (control) or exposed to the given concentrations of LPS (at least n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total at least n = 6) for various time periods. 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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Fig3: Activation of COX-2 and release of PGE2 in A549 cells and human lung cancer tissue in response to LPS. a Effect of COX inhibitors on LPS-induced proliferation of A549 cells in vitro. 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 indomethacin (indo) or the COX-2 inhibitor NS-398. 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. b Expression of COX-2 mRNA in response to LPS in A549 cells and human lung cancer tissue. A549 cells and specimen of human adenocarcinoma were either sham-incubated or exposed to 10 μg/ml LPS F515. After 16 h, mRNA was extracted and subjected to quantitative reverse transcriptase polymerase chain reaction. The ΔΔCT values represent relative expression of COX-2 mRNA normalized to the internal reference HPRT mRNA in LPS versus unstimulated A549 cells or lung adenocarcinoma (Adeno) tissue. Mean values ± SEM, originating from four independent experiments, each performed in duplicate are given. c Release of PGE2 in A549 cells in response to LPS. A549 cells were either sham-incubated (control) or exposed to the given concentrations of LPS (at least n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total at least n = 6) for various time periods. 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: PGE2 and thromboxane (Tx)B2, the stable hydrolysis product of TxA2, were quantified in a commercial ELISA system (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. For these experiments, A549 cells (50,000 cells/well) were seeded on 24-well plates and grown to confluence. Confluent monolayers were washed twice and kept in RPMI containing 1 % FCS at a total volume of 500 μl/well. Then, incubation with different concentrations of LPS (from E. coli 0111:B4 or E. coli F515) in the absence or presence of the respective antibodies or COX inhibitors for various time periods, or sham incubation (control) was performed. All samples were performed as duplicate. For the time–response curve in Fig. 3c, as well as for the inhibitor studies in Fig. 3a, at least six independent experiments were performed (at least 3 both for LPS 0111:B4 and for LPS F515, respectively). At the end of the incubation period, medium was exchanged, and cells were washed twice and kept in RPMI containing 1 % FCS and further incubated for 8 h with 5 μM arachidonic acid (AA, Sigma, Deisenhofen, Germany). Then, cell supernatants were harvested, cell debris was removed by centrifugation at 13.000×g, and samples were stored at −20 °C until further processing. The measurement of PGE2 and TxB2 release was taken by ELISA technique, according to the manufacturer’s protocols and is expressed in pg/ml. All samples were performed as duplicate, and each sample was measured twice.Fig. 3

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