PMC

Activation of signaling pathways by nicotine in lung cancer cells. A549 cells were serum starved for 24 h followed by incubation with 5 μmol/L nicotine for different times. A, Western blot analysis of the phosphorylated levels of Akt (p-Akt), ERK1/2 (p-ERK1/2), p70S6K (p-p70S6K), and 4E-BP1 (p-4E-BP1).B, densitometric analysis of results in (A). Results are representative of three independent experiments. Columns, mean; bars, SD.

Induction ofVEGF expression by nicotine is HIF-1αdependent. A549 cells were transfected with siRNA_HIF-1αor nonspecific siRNA and then exposed to 5 μmol/L nicotine for 16 h. A, top,Western blot analysis of HIF-1αprotein levels; bottom, densitometric analysis of the relative density of HIF-1αas in (A), wherein the density of the nontransfection control without nicotine treatment was arbitrarily set as 1.0. B, ELISA assay ofVEGF protein concentration in the conditioned media, which were normalized to cell numbers (1 × 10⁵). Columns, mean; bars, SD. **, P < 0.01, compared with nontransfection control without nicotine treatment; #, P < 0.05; ##, P < 0.01, compared with nontransfected cells exposed to 5 Amol/L nicotine for 16 h. Data represent three independent experiments.

Involvement of α₇-nAChR – mediated activation of signaling cascades in nicotine-induced HIF-1αprotein accumulation andVEGF production. A549 cells were pretreated for 1h with increasing concentrations of various pharmacologic inhibitors, including α-BTX and BAPTA/AM (A),W7 and PP2 (B), U0126 and GF109203 (C), and LY294002 and rapamycin (D). Cells were subsequently incubated in the presence or absence of 5 μmol/L nicotine for 16 h. HIF-1αprotein levels in cell lysates and VEGF production in the conditioned medium were determined by Western blot and ELISA, respectively. *, P < 0.05; **, P < 0.01, compared with cells treated with 5 μmol/L nicotine for 16 h. Data are representative of results from three independent experiments. Columns, mean; bars, SD.

Nicotine induces HIF-1αprotein accumulation andVEGF production in A549 cells. A, top,Western blot analysis of HIF-1αprotein levels in A549 cells treated with increasing concentrations of nicotine for 16 h; bottom, densitometric analysis of the relative density of HIF-1αas in (A), wherein the density of the control was arbitrarily set as 1.0. B, immunofluorescence studies on HIF-1αprotein expression in A549 cells after treatment with 100 μmol/L CoCl₂ and 0.5 and 5 μmol/L of nicotine for 16 h, respectively. Magnification, ×20. C, ELISA assay ofVEGF protein concentration in the conditioned media, which were normalized to cell numbers (1 ×10⁵). Columns, mean; bars, SD. *, P < 0.05; **, P < 0.01, compared with control without nicotine treatment. Data are representative of three independent experiments.

Involvement of HIF-1αin nicotine-stimulated invasion of A549 cells. A, images photographed under a phase-contrast microscope. Magnification, × 10. A549 cells transfected with nonspecific siRNA (b and e) or specific HIF-1αsiRNA (c and f) were seeded onto the QCM Collagen-based Cell Invasion Assay system and cultured the presence or absence of 5 μmol/L nicotine for 48 h, and cell migration from the upper to the lower surface of the membrane was stained and photographed using a computer imaging system, wherein nontransfected cells cultured in the presence or absence of nicotine were used as negative (a) and positive (d) controls. B, quantification of cell invasion by colorimetric measurement at 560 nm. The graph shows the relative A560 values, wherein the A560 value from negative control (a) was arbitrarily set as 1.0. **, P < 0.01 (d compared with a); *, P < 0.05 (f compared with d). Results are representative of three independent experiments. Columns, mean; bars, SD.

In vitro capillary tube formation stimulated by nicotine-treated lung cancer cells was attenuated by transfection with specific HIF-1αsiRNA. HUVECs (5 × 10³ per well) were seeded onto the surface of 96-well cell culture plates precoated with polymerized ECMatrix and then incubated in the conditioned medium for 6 h at 37°C. A, images of capillary tubes photographed under a phase-contrast microscope. Magnification, ×10. a and d, HUVECs were incubated in the conditioned medium derived from nontransfected A549 cells after cultured in the presence (d) or absence (a) of nicotine for 24 h; b and e, HUVECs were incubated in the conditioned medium derived from A549 cells transfected with nonspecific siRNA (NS-siRNA) after cultured in the presence (e) or absence (b) of nicotine for 24 h; c and f, HUVECs were incubated in the conditioned medium derived from A549 cells transfected with specific HIF-1αsiRNA after cultured in the presence (f) or absence (c) of nicotine for 24 h. B, quantification of capillary tube formation. The averaged values of branch points formed were calculated by counting the capillary tube branch points in six random view fields per well. **, P < 0.01 (d compared with a); *, P < 0.05 (f compared with d); #, P < 0.05 (c compared with a). Data are representative of three separate experiments. Columns, mean; bars, SD.