Carbon nanotubes induce apoptosis resistance of human lung epithelial cells through FLICE-inhibitory protein

Abstract

Chronic exposure to single-walled carbon nanotubes (SWCNT) has been reported to induce apoptosis resistance of human lung epithelial cells. As resistance to apoptosis is a foundation of neoplastic transformation and cancer development, we evaluated the apoptosis resistance characteristic of the exposed lung cells to understand the pathogenesis mechanism. Passage control and SWCNT-transformed human lung epithelial cells were treated with known inducers of apoptosis via the intrinsic (antimycin A and CDDP) or extrinsic (FasL and TNF-α) pathway and analyzed for apoptosis by DNA fragmentation, annexin-V expression, and caspase activation assays. Whole-genome microarray was performed to aid the analysis of apoptotic gene signaling network. The SWCNT-transformed cells exhibited defective death receptor pathway in association with cellular FLICE-inhibitory protein (c-FLIP) overexpression. Knockdown or chemical inhibition of c-FLIP abrogated the apoptosis resistance of SWCNT-transformed cells. Whole-genome expression signature analysis confirmed these findings. This study is the first to demonstrate carbon nanotube-induced defective death receptor pathway and the role of c-FLIP in the process.

Keywords: apoptosis; c-FLIP; carbon nanotubes; death receptor; lung.

Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.

Figures

FIG. 1.

Acquired resistance to apoptosis of single-walled carbon nanotubes (SWCNT)-transformed cells. a, Subconfluent monolayers of passage-control BEAS-2B and transformed B-SWCNT cells were treated with various surface area doses of SWCNT (0–5 μg/cm2) for 48 h and analyzed for apoptosis by annexin V and propidium iodide (PI) assays. Representative dot plot histograms of annexin V (x-axis) and PI (y-axis) are shown. Early apoptotic cells are in the lower right quadrant with annexin V positive and PI negative. b, Cells were similarly treated with SWCNT (0–8.34 μg/cm2) and analyzed for apoptosis using Hoechst 33342 assay. Apoptotic cells exhibiting condensed or fragmented nuclei with bright nuclear fluorescence were scored under a fluorescence microscope. Data are means ± SD (n = 4). *P ≤ .05 versus passage-matched control.

FIG. 2.

Analysis of mitochondrial pathway of apoptosis in single-walled carbon nanotubes (SWCNT)-transformed cells. a, Subconfluent monolayers of passage-control BEAS-2B and transformed B-SWCNT cells were treated with various concentrations of cisplatin (CDDP, 0–100 μM) or antimycin A (ANA, 0–30 μM), and apoptosis was determined by Hoechst 33342 assay at 12 h post-treatment. Apoptotic cells with condensed or fragmented nuclei were scored under a fluorescence microscope. b, Representative fluorescence micrographs of cells treated with CDDP (100 μM) and ANA (30 μM) stained with Hoechst dye. c, Cells were similarly treated with CDDP (0–100 μM) and ANA (0–30 μM) for 8 h, and cell lysates were prepared and analyzed for caspase-9 and PARP cleavage by Western blotting. Blots were reprobed with β-actin antibody to confirm equal loading of the samples. Data are means ± SD (n = 4). *P ≤ .05 versus passage-matched control.

FIG. 3.

Analysis of death receptor pathway of apoptosis in single-walled carbon nanotubes (SWCNT)-transformed cells. a, Subconfluent monolayers of passage-control BEAS-2B and transformed B-SWCNT cells were treated with various concentrations of TNF-α (0–50 ng/ml) or FasL (0–25 ng/ml), and apoptosis was determined by Hoechst 33342 assay at 12 h post-treatment. Apoptotic cells were scored under a fluorescence microscope. b, Representative fluorescence micrographs of cells treated with TNF-α (100 ng/ml) or FasL (50 ng/ml) stained with Hoechst dye. c, Cells were similarly treated with TNF-α (0–50 ng/ml) or FasL (0–25 ng/ml) for 8 h, and cell lysates were prepared and analyzed for caspase-8 and PARP cleavage by Western blotting. Blots were reprobed with β-actin antibody to confirm equal loading of samples. Data are means ± SD (n = 4). *P ≤ .05 versus passage-matched control.

FIG. 4.

Effects of caspase inhibitors on mitochondrial and death receptor pathways of apoptosis. a, Subconfluent monolayers of passage-control BEAS-2B and transformed B-SWCNT cells were treated with cisplatin (CDDP, 50 μM) or antimycin A (ANA, 30 μM) in the presence or absence of pan-caspase inhibitor zVAD-fmk or caspase-9 inhibitor zLEHD-fmk, or with TNF-α (100 ng/ml) or FasL (50 ng/ml) in the presence or absence of zVAD-fmk or caspase-8 inhibitor zIETD-fmk. Apoptosis was determined by Hoechst 33342 assay at 12 h post-treatment. b, Cells were similarly treated the apoptogens in the presence or absence of specific caspase inhibitors, and caspase-9 and -8 activities were determined at 6 h post-treatment using fluorometric caspase substrates FAM-LEHD-fmk and FAM-LETD-fmk, respectively. Data are means ± SD (n = 4). *P < .05 versus untreated cells. #P ≤ .05 versus treated cells. §P ≤ .05 versus passage-matched control.

FIG. 5.

Analysis of apoptosis-regulatory proteins of the death receptor pathway in single-walled carbon nanotubes (SWCNT)-transformed cells. a, Passage-control BEAS-2B and transformed B-SWCNT cell lysates were prepared and analyzed for c-FLIP, TNFR1, and FasR by Western blotting. b, Subconfluent monolayers of SWCNT-transformed cells were treated with TNF-α (0–100 ng/ml) or FasL (0–50 ng/ml) in the presence or absence of c-FLIP inhibitor 5809354 (5 μM) for 12 h, after which they were analyzed for apoptosis by Hoechst 33342 assay. c, Knockdown of c-FLIP in SWCNT-transformed cells using shRNA lentiviral particles. The expression level of c-FLIP in shRNA control (shCon) and knockdown (shFLIP) B-SWCNT cells was determined by Western blotting using specific antibody against c-FLIP. d, shCon and shFLIP cells were treated with TNF-α (0–100 ng/ml) or FasL (0–50 ng/ml), and analyzed for apoptosis by Hoechst 33342 assay at 12 h post-treatment. Data are means ± SD (n = 4). *P ≤ .05 versus control cells.

FIG. 6.

Apoptosis resistance signaling in single-walled carbon nanotubes (SWCNT)-transformed cells. a, Top-ranked cell death functions in whole genome signature included apoptosis in both epithelial and tumor cell lines. †predicted inhibition (Z = −2.2). Apoptosis in epithelial cell lines possessed a trend for predicted inhibition (Z = −1.96). b, Simulation of c-FLIP (CFLAR)-mediated apoptosis resistance during TNF/FasL exposure in the apoptotic canonical signaling pathway. Both differential gene expression (≥±2-fold and t-test) and prediction signaling (Z ≥ ±2) are shown.