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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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Proliferative response of A549 cells in vivo. a In vivo tumor growth. A549 cells were exposed to 10 μg/ml of LPS F515, or sham incubation was performed. Immediately after treatment, cells were injected subcutaneously into 8-week-old female BALBc nu/nu mice. At indicated time points, the size of the tumor was measured by Mitutoyo digital calipers and is given in mm3. Data are expressed as mean ± SEM (n = 8 for controls and n = 6 for LPS). b Immunohistofluorescent analysis of Ki-67 in cryosections from A549 tumors. Quantitative analysis of Ki-67 relative to DAPI (%) from the experiments described in (a). Data reflect the mean ± SEM (n = 8 for controls and n = 6 for LPS). c Immunofluorescent and H&E staining. Representative images of untreated (control) versus LPS-stimulated (10 μg/ml) A549 tumors and of the corresponding DAPI, Ki-67 and the H&E staining, respectively. The scale bar corresponds to 500 μm
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Fig5: Proliferative response of A549 cells in vivo. a In vivo tumor growth. A549 cells were exposed to 10 μg/ml of LPS F515, or sham incubation was performed. Immediately after treatment, cells were injected subcutaneously into 8-week-old female BALBc nu/nu mice. At indicated time points, the size of the tumor was measured by Mitutoyo digital calipers and is given in mm3. Data are expressed as mean ± SEM (n = 8 for controls and n = 6 for LPS). b Immunohistofluorescent analysis of Ki-67 in cryosections from A549 tumors. Quantitative analysis of Ki-67 relative to DAPI (%) from the experiments described in (a). Data reflect the mean ± SEM (n = 8 for controls and n = 6 for LPS). c Immunofluorescent and H&E staining. Representative images of untreated (control) versus LPS-stimulated (10 μg/ml) A549 tumors and of the corresponding DAPI, Ki-67 and the H&E staining, respectively. The scale bar corresponds to 500 μm

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)

Proliferative response of A549 cells in vivo. a In vivo tumor growth. A549 cells were exposed to 10 μg/ml of LPS F515, or sham incubation was performed. Immediately after treatment, cells were injected subcutaneously into 8-week-old female BALBc nu/nu mice. At indicated time points, the size of the tumor was measured by Mitutoyo digital calipers and is given in mm3. Data are expressed as mean ± SEM (n = 8 for controls and n = 6 for LPS). b Immunohistofluorescent analysis of Ki-67 in cryosections from A549 tumors. Quantitative analysis of Ki-67 relative to DAPI (%) from the experiments described in (a). Data reflect the mean ± SEM (n = 8 for controls and n = 6 for LPS). c Immunofluorescent and H&E staining. Representative images of untreated (control) versus LPS-stimulated (10 μg/ml) A549 tumors and of the corresponding DAPI, Ki-67 and the H&E staining, respectively. The scale bar corresponds to 500 μm
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Fig5: Proliferative response of A549 cells in vivo. a In vivo tumor growth. A549 cells were exposed to 10 μg/ml of LPS F515, or sham incubation was performed. Immediately after treatment, cells were injected subcutaneously into 8-week-old female BALBc nu/nu mice. At indicated time points, the size of the tumor was measured by Mitutoyo digital calipers and is given in mm3. Data are expressed as mean ± SEM (n = 8 for controls and n = 6 for LPS). b Immunohistofluorescent analysis of Ki-67 in cryosections from A549 tumors. Quantitative analysis of Ki-67 relative to DAPI (%) from the experiments described in (a). Data reflect the mean ± SEM (n = 8 for controls and n = 6 for LPS). c Immunofluorescent and H&E staining. Representative images of untreated (control) versus LPS-stimulated (10 μg/ml) A549 tumors and of the corresponding DAPI, Ki-67 and the H&E staining, respectively. The scale bar corresponds to 500 μm
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.

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Related in: MedlinePlus