AECS/p-BQ-induced proliferation of human lung epithelial cells (A549) and its prevention by anti-p-BQ antibody. Except in (d), all the treatments were made in 24 hr. Effect of concentration gradient of AECS on proliferation and death of A549 cells in culture, as determined by MTT cytotoxicity assay (a). AECS (2 μL/mL) or p-BQ (200 ng/mL)-induced proliferation in other cell lines, namely. L132, Vero, and HepG2 (b). Effect of concentration gradient of p-BQ on proliferation and death of A549 cells in culture, as determined by MTT cytotoxicity assay (c). Treatment with AECS (2 μL/mL) or p-BQ (200 ng/mL) results in continued proliferation for 24–72 hr (d). AECS/p-BQ-induced proliferation is prevented by anti-p-BQ antibody, as evidenced by MTT assay (e). The inhibitory role of anti-p-BQ antibody on the proliferation of A549 cells as determined by the incorporation of BrdU using flow cytometry assay (f, g). All data are depicted as the mean ± SD for three independent experiments (∗ indicates significant difference, P < .05 in comparison to nontreated control (a, b, c, d); and in comparison to AECS and p-BQ, resp. (e, g)).

Vitamin C prevents AECS/p-BQ-induced proliferation of cells. AECS/p-BQ-induced proliferation is prevented by vitamin C (40 μg/mL), as evidenced by MTT assay (a), as well as BrdU-incorporation assay (b and c). Serum-starved A549 cells (2000 cells/well) grown on 96-well tissue culture plates were either nontreated (NT) or exposed to 2 μL/mL AECS or 200 ng/mL p-BQ for 24 hr with or without vitamin C (40 μg/mL) pretreatment in serum-free medium for 15 min (a, b, and c). Data represent means ± SD for three independent experiments (∗ indicates significant difference, P < .05 in comparison to AECS (2 μL/mL) and p-BQ (200 ng/mL), resp.).

Status of p53, phospho-p53, caspase 3, cleaved-caspase 3 and apoptosis in cultured A549 cells exposed to low or high concentration of AECS/p-BQ. The figure represents immunoblots of phosphorylated p53 and p53 (a) and caspase 3 and cleaved caspase 3 (b). Cell lysate of A549 cells were either nontreated (NT) or exposed to AECS (2 μL or 50 μL/mL) or p-BQ (200 ng or 2.5 μg/mL) for 1 hr followed by incubation in serum containing media for 12 hr. Vitamin C (40 μg/mL) pretreatment of cells in serum-free media for 15 min prevented AECS/p-BQ-induced activation of p53 or cleavage of caspase 3 (a, b). β-actin was used as the loading control. Effect of low or high concentrations of AECS/p-BQ on apoptosis in cultured A549 cells and its prevention by vitamin C as evidenced by flow cytometry (c). A549 cells were grown on 60 mm culture plates and were gradually serum starved for 3 days to synchronize the cells. Then the cells were either nontreated (NT) or exposed to 2 μL/mL or 50 μL/mL AECS for 1 hr, 200 ng/mL or 2.5 μg/mL p-BQ for 1 hr, with or without 40 μg/mL vit C pretreatment in serum-free medium for 15 min. After treatment, cells were incubated in fresh media containing serum for 12 hr, followed by Annexin V-PI assay. Bar graphs show the percentage of normal and apoptotic cells after respective treatments, as evidenced by flow cytometry (d). The numbers within the bars represent percentage of normal and apoptotic cells.

p-BQ mimics AECS in activating EGFR in a time-dependent manner. Serum-starved A549 cells were nontreated (NT) or exposed to 100 ng/mL EGF for 5 min, 2 μL/mL AECS or 200 ng/mL p-BQ for 15 min, 30 min, or 1 hr, respectively (a). Vitamin C (40 μg/mL) pretreatment of A549 cells for 15 min in serum-free media completely prevented AECS/p-BQ-induced EGFR activation (b). 500 nM Tyrphostin AG1478 (EGFR inhibitor) pretreatment of A549 cells for 15 min in serum-free media completely prevented activation of EGFR by 100 ng/mL EGF, 2 μL/mL AECS, or 200 ng/mL p-BQ (c). Anti-p-BQ antibody prevented AECS/p-BQ-induced EGFR activation (d). Cells were lysed, and the EGFR was immunoprecipitated (IP) from the cell lysates using anti-EGFR antibody. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted (IB) with either antiphosphotyrosine (PY20) antibody (a, b, c) or with anti-p-BQ antibody (d).

EGFR exposed to AECS/p-BQ cannot bind c-Cbl and is not ubiquitinated. Serum-starved A549 cells were either nontreated (NT) or exposed to 100 ng/mL EGF for 5 min (used as positive control), 2 μL/mL AECS or 200 ng/mL p-BQ for 1 hr. After treatment, cells were immediately lysed, and the EGFR was immunoprecipitated (IP) from the cell lysates using anti-EGFR antibody. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted (IB) with the indicated antibodies (a). Serum-starved A549 cells were either nontreated (NT) or exposed to 100 ng/mL EGF for 5 min or 2 hr, 2 μL/mL AECS or 200 ng/mL p-BQ for 1 hr. After treatment, cells were washed with PBS and further incubated in fresh serum-free medium for 2 hr at 37°C before lysis. The EGFR was immunoprecipitated (IP) from the cell lysates using anti-EGFR antibody. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted (IB) with antiphosphotyrosine (PY20) antibody (b).

Exposure of A549 cells to AECS/p-BQ results in phosphorylation of c-Myc at Thr 58/Ser 62 and overexpression of c-Fos. Serum-starved A549 cells were either nontreated (NT) or exposed to 2 μL/mL AECS for 1 hr or 200 ng/mL p-BQ for 1 hr followed by incubation in serum containing media for 6 hr. After 6 hr, cells were lysed, and cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with anti-phospho-c-Myc (phospho Thr58 + Ser62), anti-c-Myc, and anti-c-Fos antibodies. β-actin was used as loading control.

Cell cycle analysis of proliferating A549 cells. Histogram plot (Event count versus FL2-H) of AECS/p-BQ-induced proliferation of A549 cells and its prevention by vitamin C as determined by propidium iodide (PI) staining followed by flow cytometry (a). Bar diagram showing mean fluorescence of PI (FL2-H) in cells either nontreated (NT) or exposed to 2 μL/mL AECS or 200 ng/mL p-BQ for 24 hr and its prevention by vitamin C (40 μg/mL) (b). Bar diagram showing the relative cell distribution during various phases of the cell cycle after exposure to 2 μL/mL AECS or 200 ng/mL p-BQ (c). Data are means ± SD for three independent experiments (∗ in (b) and (c) indicates significant difference, P < .05 in comparison to AECS and p-BQ treatment).

Effect of low or high concentration of AECS/p-BQ on reactive oxygen species (ROS) production in cultured A549 cells and its prevention by vit C. Serum-starved A549 cells grown on coverslips were either nontreated (NT) or exposed to 2 μL/mL AECS, 50 μL/mL AECS, 200 ng/mL p-BQ or 2.5 μg/mL p-BQ for 1 hr with or without vitamin C (40 μg/mL) pretreatment in serum-free medium for 15 min. After treatment, cells were incubated in fresh media containing H₂DCFDA for 30 min and PBS washed twice, and fluorescent images were captured.

Effects of concentration gradient of NNN (a), NNK (b), and BP (c) on the growth of cultured A549 cells after 24, 48, and 72 hr as evidenced by MTT assay. Serum-starved A549 cells (2000 cells/well) grown on 96-well tissue culture plates were either nontreated (0) or exposed to 0.025, 0.05, 0.1, 0.5, or 1 μg/mL NNN (a), NNK (b), and BP (c) for 24, 48, and 72 hr. After treatment, MTT cytotoxicity assay was performed. All data are depicted as the means ± SD for four independent experiments.

AECS/p-BQ exposure results in aberrant phosphorylation of the EGFR. Serum-starved A549 cells were either nontreated (NT) or exposed to 100 ng/mL EGF for 5 min, 2 μL/mL AECS or 200 ng/mL p-BQ for 1 hr. Cells were lysed, and the EGFR was immunoprecipitated (IP) from the cell lysates using anti-EGFR antibody. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted (IB) with indicated EGFR phosphotyrosine-specific antibodies or anti-EGFR antibodies.

Exposure of A549 cells to AECS/p-BQ results in overexpression of Hras + Kras and activation of downstream proliferation and survival signaling Akt and ERK 1/2. Serum-starved A549 cells were either nontreated (NT) or exposed to 2 μL/mL AECS or 200 ng/mL p-BQ for 1 hr followed by incubation in serum containing media for 6 hr. After 6 hr, cells were lysed, and cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antiphosphotyrosine (PY20), anti-HRAS + KRAS, anti-phospho-Akt, anti-Akt, anti-phospho-ERK 1/2, and anti-ERK 1/2 antibodies. β-actin was used as the loading control.

Model showing the molecular mechanisms of cigarette smoke-induced proliferation of human lung epithelial cells (A549) and prevention by vitamin C as well as antibody to p-Benzoquinone.