Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells

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  Figure 1. Variation of PON1 protein and gene expression between lung squamous cell carcinoma and lung adenocarcinoma patients (A) Densitometry analysis of lung cancer subtype tissue-based expression of PON1 protein from matched clinical pairs of lung squamous cell carcinoma and lung adenocarcinoma normalized to adjacent normal control. (B, C, D, E, F) Matched tissue samples were either summarized (total) or separated into different groups of: stage, recurrence, age, smoking history, and gender. Presented results were summarized from two biological replicates of the blotting experiment. (G) Representative blots of PON1 protein patient tissue expression selected from 39 matched cases. β-actin variations apparent in some blots were considered in calculating relative densitometry values for each blot using nonparametric Kruskal-Wallis statistical test. (H) Genetic alterations and mRNA expression changes (copy number) of PON1 in the TCGA dataset of lung cancer samples. PON1 genes are represented in rows, and individual cases or patients are represented as columns (upper panel). Higher magnification images are shown in the inset (lower panel). These oncoprints are based on data obtained from the…

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  Figure 2. PON1 is located in the cytosolic fraction and is differentially expressed in various lung normal and tumor cell lines (A) PON1 protein expression in different lung normal (L132) or cancer cell lines (A549, H460, H1299, H358) including another normal cell line (HEK-293). (B) Western blot analysis of nuclear and cytosolic cell extracts (50 μg of each fraction). (C) Live A549 and A549-shPON1 cells stained with reduced FITC (pseudocolored in green) and DAPI. Cells were analyzed by confocal microscopy. Scale bars = 40 μm or 2 μm as indicated. (D, E) Stable overexpression and knockdown of PON1 by lentiviral-induced systems were examined by Western blotting. In PON1 overexpression system (H460- and H1299-PON1), two isoforms of stable PON1 overexpression (#1 and #2, respectively) differ in lentiviral particles used for transfection. (F) Immunoprecipitation of cells with anti-human PON1 antibody. Representative examples of three independent experiments are shown. All experiments were conducted at least in triplicate and biologically repeated at least twice. Statistical analysis was performed using unpaired Student's t test.

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  Figure 3. PON1 regulation impacts lung cancer cell growth and arrest programs (A) Assessment of cell viability. Before day 5, cells were serum starved for 24 h and were subjected to DAPI staining. Subsequent to serum starvation, cells were pre-exposed to 90% O2 level for 12 h and incubated in normoxia for 12 h (displayed as immunofluorescence in right panel of each graph). (B) Colony formation (>50 cells) was assessed by crystal violet staining. (C) Mean G1/S-phase ratio from cell cycle analysis (left panel) and cell cycle progression fractions of >15,000 cells analyzed by FACS (right panel). (D) BrdU-positive spots of cells examined at day 4 at early cell passage in serum-starved medium. (E) Cell passage-dependent viability after pre-culture of cells at low density for 3 days in serum-rich media containing low etoposide concentration and then subsequently starved them for another 6 days. SA-β-gal assay was used to confirm presence of senescent cells after recovery (data not shown). (F) Western blot analysis of whole cell lysates of same samples from E. β-actin served as the loading control. *P<0.05. Error bars are mean ± S.E.M. n=3~5. All experiments were conducted at least in triplicate…

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  Figure 4. PON1 regulation positively impacts lung cancer by antioxidative function-controlled ROS accumulation (A) PON1 immunoblot of A549 (left panel) and L132 (right panel) cells after loading with 100 ng of human rPON1 protein for 24 h. (B) Detection of human rPON1-His-tag protein using His-tag antibody in A549 cells (left panel) and L132 cells (right panel) under oxidative stress induced by H2O2 for 24 h. (C) Assessment of cell viability after treatment with NAC, H2O2, or FeSO4. (D) Representative images showing fluorescence signal after loading with dihydrorhodamine 123. Scale bars = 50 μm. (E, F, G, H, I) Cells were analyzed by FACS for the following: ROS determination (both rhodamine and DCFH fluorescence intensity units), dihydrorhodamine oxidation fluorescence, cyctochrome c release, and mitochondrial membrane. FACS-analyzed cells were 400-1000 in population. In G, representative images showing fluorescence signal after loading with DCFH-DA. Scale bars = 25 μm. (J) FACS analysis of cells loaded with Mito-HE (2 μM), treated with antimycin A (10 and 20 μM). (K) Mitchondrial fragmentation determined by MitoSOX (pseudocolored in orange; 10 μM) after loading cells with either 0.01%…

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  Figure 5. PON1 controls metastatic lung cancer cell behavior by regulating oxidative response (A, B) Invasion and migration counts per field of PON1-overexpressing H460-PON1 and H1299-PON1 cells versus vector control cells for 24 h. (C, D) Invasion and migration counts per field of PON1-knocked down A549-shPON1 cells versus shControl cells for 24 h. (E) Scratch-wound healing ability of cells after pre-exposure to 0.01% DMSO, 10 mM NAC, or 10 mM NAC in combination with 10 μM Akt-inhibitor LY294002. Representative bar graphs show the width in μm of scratch recorded. Representative migration micrographs are shown taken using phase-contrast microscope. (F, G) Western blot analysis of whole cell lysates. Cells were treated with 0.01% DMSO, 10 mM ROS-inhibitor NAC, 1 mM ROS-stimulators H2O2 and 7 mM FeSO4. Cells were treated with or without combination of NAC and Akt-inhibitor LY294002 for G. All experiments were conducted at least in triplicate and biologically repeated at least twice. Statistical analysis was performed using unpaired Student's t test.

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  Figure 6. Glycolytic regulation controls PON1-dependent antioxidative response in lung cancer cells (A) Immunoblot detection of PON1 from whole cell lysates after glucose-enrichment in the media with indicated concentrations for 24 h. (B) Cells were serum-starved, glucose-starved, or glucose-enriched prior to Western blotting with PON1 antibody. (C) Homogenous detection of oxidized and reduced NAD+ and NADPH in H460-PON1, H1299-PON1, A549-shPON1, and their respective controls as indicated after the transfected cells have been exposed to the indicated glucose-deprivation periods. (D) ROS production after cells were either enriched with glucose or glucose-deprived. (E) Immunoblotting and immunoprecipitation of cells after glucose-enrichment or glucose-deprivation. Three independent experiments were summarized. Symbols represent mean ± S.E.M. n = 3~7; n.s., not significant; *P<0.05; **P<0.01.

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  Figure 7. PON1 supports escape from drug- and ligand-induced cell death in lung cancer cells (A) Cell growth inhibition rate after binary dose-dependent treatment with chemotherapeutics. (B, C) FACS-based detection of drug-induced apoptotic fraction (sub-G1 phase) of cells and identification of early and late apoptosis. Symbols represent mean ± S.E.M. (D) Upon induction of apoptosis by various indicated chemotherapeutic drug (0.8 μM STS, 60 μM 5-FU, 60 μM PTX, 60 μM CIS, 10 μM ETOP) for 16 h, cells were assessed for nuclear morphology by DAPI staining shown as representative fluorescent images. (E) The same cells and drug treatment procedures were used in TUNEL assay as in D. Bar graph (right panel) shows representative total count of brightly stained nuclei of apoptotic cells. (F) Western blotting of cells to examine endogenous levels of total and phospho-p53, pro-caspases 3 and 8, p21, p27, total and phospho-ERK, and total and phospho-Akt. β-actin served as the loading control. (G) Cells were either treated with 0.01% DMSO or 1 μM STS and analyzed for Annexin-V-FITC/PI by FACS. (H) Cells were treated with either 0.01% DMSO or 5 μM STS for 48 h and assessed for intracellular ATP levels.…

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  Figure 8. PON1 promotes tumor growth in vivo (A, B), Schematic diagram of tumor inoculation with indicated NSCLC cells subcutaneously injected into nude mice and images taken after removing tumor xenografts grown for 37 days and measured using caliper to quantify tumor size. (C) Representative H&E stained tissues of indicated NSCLC-inoculated tumors. Magnifications, x200. Symbols represent mean ± S.E.M. n=4 mice per group; *P<0.05; **P>0.01. Animal experiments were biologically replicated twice.