Mechanisms of tumor-promoting activities of nicotine in lung cancer: synergistic effects of cell membrane and mitochondrial nicotinic acetylcholine receptors

Normal human lobar bronchial epithelial cells (BEC) were purchased from Life Technologies (Grand Island, NY) and the established tumorigenic line of grade IV lung squamous cell carcinoma SW900 — from American Type Culture Collection (Catalog # HTB-59; Manassas, VA). BEP2D cells — an established clonal population of HPV-18-immortalized human BEC — was a gift from Dr. Harris (NCI, NIH). For transformation, BEP2D cells were incubated for 48 h with 2 μg/ml of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; Toronto Research Chemicals, North York, ON, Canada) and then grown for 5 passages, which was sufficient to induce malignant transformation evidenced by anchorage-independent growth and tumor formation in Nu/Nu mice [48]. All types of lung cells were grown in the Cambrex bronchial cell medium without retinoic acid and used in experiments at ~80% confluence. The effects of test agents on proliferation were evaluated by directly measuring the number of viable, ie, trypan blue dye (TBD)-negative, cells using a hemocytometer. The nAChR agonist nicotine, as well as α-bungarotoxin (αBtx) — the specific inhibitor of the "central" subtype of the neuronal nAChRs, such as α7 [49], Mecamylamine (Mec) — a preferential blocker of the "ganglionic" nAChR subtypes, such as α3- and α4-made nAChRs [50], the metabolic inhibitor of ACh synthesis hemicholinium-3 (HC-3), which inhibits ACh synthesis by blocking cellular reuptake of its metabolic precursor choline [51], staurosporine, heat-inactivated newborn calf serum and all secondary antibodies were purchased from Sigma-Aldrich Corporation, Inc. (St. Louis, MO). Human recombinant EGF was obtained from R&D Systems, Inc. (Minneapolis, MN), IGF-I — from GenWay Biotech Inc. (San Diego, CA), and VEGF — from Abcam (Cambridge, MA). The human cytochrome c (CytC) immunoassay was purchased from R&D Systems and performed following the protocol provided by the manufacturer. The nicotinic radioligands (—)[N-methyl-³H]nicotine (specific activity 80.4 Ci/mmol), [³H]αBtx (specific activity 73.0 Ci/mmol) and [³H]epibatidine (specific activity 54.0 Ci/mmol) were purchased from GE Healthcare Bio-Sciences (Pittsburgh, PA). The antibodies to human α3, α4, α7, and α9 nAChR subunits were raised and characterized in our previous studies [52-54]. The predesigned and tested small hairpin (sh)RNAs targeting human CHRNA3, CHRNA4, CHRNA7 or CHRNA9, and scrambled shRNA were from OriGene Technologies (Rockville, MD).

For transfection of SW900 cells with the HuSH-29™ predesigned shRNA plasmids specific for human α3, α4, α7 and α9 nAChR subunits, we followed the standard protocol described by us in detail elsewhere [55]. Briefly, SW900 cells were seeded at a density of 1 × 10⁴ cells per well and exposed to experimental, ie, nAChR subunit gene-specific shRNA, or negative control shRNA (shRNA-NC) plasmids in GIBCO™ Opti-MEM I Reduced-Serum Medium (Invitrogen, Carlsbad, CA) with the TransIT®-Keratinocyte Transfection Reagent (Mirus Bio LLC, Madison, WI). The transfection was continued for additional periods of time to determine changes of the relative protein levels of each targeted nAChR subunit by immunoblotting and immunofluorescence. The maximum inhibition was achieved at 72 h after transfection (data not shown), at which point the shRNA-transfected cells were washed and exposed to test nicotine/GF combinations for 24 h. At the end of incubation, alive, ie, TBD-negative, cells were counted with a hemocytometer.

The mitochondrial protein fractions were purified from large quantities of lung cell types used in this study grown in the 225 cm² T-flasks employing the mitochondrial/cytosol fractionation kit from BioVision Research Products (Mountain View, CA), as described by us elsewhere [56]. Briefly, the cells were detached by a brief trypsinization, isolated by centrifugation, washed in PBS, resuspended in the Cytosol Extraction Buffer containing a mix of DTT and protease inhibitors, homogenized in an ice-cold tissue grinderm and centrifuged at 700 × g for 10 min at 4°C. The supernatant was re-centrifuged at 10,000 × g for 30 min at 4°C, and the pelleted mitochondrial fraction was resuspended in 100 μl of the Mitochondrial Extraction Buffer and used in the radioligand-binding assays following the standard protocol detailed by us elsewhere [57]. Depending on the experimental conditions (see Results), the mitochondria were exposed to either increasing concentrations of the pan-nAChR radioligand nicotine or the saturating concentrations of the preferential radioligands of the central and ganglionic nAChR subtypes, αBtx and epibatidine, respectively [58,59]. After incubation, the mitochondria were washed and solubilized with 1% SDS, the protein concentration determined by a Bradford protein assay kit (Bio-Rad Hercules, CA), and the radioactivity counted in a liquid scintillation counter. The specific binding was calculated by subtracting the non-specific binding from total binding.

sELISA was performed as described elsewhere [60]. Briefly, ELISA plates were coated with either α3- or α7-specific rabbit antibody or non-immune rabbit IgG and blocked with 3% BSA. The lysates were applied into the coated wells for 3 h at 37°C, after which the plates were washed and incubated for additional 2 h with biotinylated anti-α3 or anti-α7 antibody (both from Antibodies-online, Inc., Atlanta, GA), followed by ExtrAvidin-Peroxidase conjugate and o- phenylenediamine dihydrochloride. The bound antibody was detected at OD 490 nm using an ELISA plate reader.

All experiments were performed in triplicate or quadruplicate, and results expressed as mean ± SD. Statistical significance was determined using Student's t-test. Differences were deemed significant if the calculated p value was <0.05.