doi: 10.1158/0008-5472.CAN-12-3190. Epub 2012 Dec 7.
Inhibition of cholinergic signaling causes apoptosis in human bronchioalveolar carcinoma
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- PMID:23222296
- PMCID: PMC10461321
- DOI: 10.1158/0008-5472.CAN-12-3190
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Inhibition of cholinergic signaling causes apoptosis in human bronchioalveolar carcinoma
Jamie K Lau et al. Cancer Res..
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Abstract
Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by α7-, α3β2-, and β3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II-induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy.
Conflict of interest statement
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Figures
Human BACs and normal HPAEpiCs express cholinergic proteins. A, ELISA assays show that human BAC cell lines and HPAEpiCs express multiple nAChR subunits. The assay was completed in duplicate, and the whole experiment was carried out 2 independent times. Results indicated by a different letter are significantly different (P < 0.05). B, HPAEpiCs and human BACs express AChE, ChAT, CHT1, and VAChT. H520 human SCC-L cells were used as the positive control for the experiments outlined in A and B. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control, and the results were quantitated by densitometric analysis. The experiment was repeated twice, and the representative data are shown. C and D, immunohistochemistry of the human BAC microarray showed that BAC tumors (isolated from patients) express ChAT and VAChT in the cytoplasm, adjacent to the hematoxylin-counterstained dark nuclei in the tissue samples. A tumor microarray containing 81 cores of human BACs was used for these experiments and 4 panels of representative photos are shown. Scale bar, 20 μm.
Nicotine induces the production of the growth factor ACh in human BACs. A, nicotine caused a concentration-dependent increase in ACh production in A549, H358, and H650 human BAC cell lines. B, time kinetics of nicotine-induced ACh production in A549 human BAC cells. The maximal ACh production was observed at 100 nmol/L nicotine at 36 hours. C, ACh stimulated proliferation of A549 and H358 cells. BrdUrd assays show that 2 μmol/L of ACh (which is approximately the amount of ACh produced in nicotine-treated BAC cells) caused a 4- to 4.5-fold increase in proliferation of A549 and H358 human BACs. Each sample was analyzed in triplicate. Data represent mean ± SEM from 2 independent experiments. Results indicated by a different letter are significantly different (P < 0.05).
Effect of nicotine on cholinergic proteins in human BACs. A, the treatment of A549 and H358 cells with 100 nmol/L nicotine caused 4- to 5-fold increase in VAChT levels. B, nicotine produces a modest increase in ChAT levels. C, nicotine decreases AChE levels in both A549 and H358 cells. The levels of CHT1 are relatively unaffected by nicotine in human BAC cells. Data represent mean ± SEM from 2 independent experiments. Each sample was analyzed in triplicate. Results indicated by a different letter are significantly different (P < 0.05).
Vesamicol caused apoptosis in human BAC cells. A, MTT assays showed that vesamicol decreases the viability of A549 and H358 cells in a concentration-dependent manner. Each sample was analyzed in triplicate. B, the treatment of human BAC cell lines with 50 μmol/L vesamicol caused 2.5- to 3-fold apoptosis in nicotine-treated A549 and H358 cells. C, the apoptotic activity of vesamicol was confirmed with caspase-3 activity assay, and similar results were obtained. D, vesamicol had no effect on EGF-induced proliferation of A549 and H358 cells. BrdUrd assays show that the treatment of A549 and H358 human BAC cells with 10 ng/mL EGF causes robust proliferation. The addition of 50 μmol/L vesamicol along with EGF produced no effect on EGF-induced proliferation of human BACs. E, BrdUrd assays show that vesamicol does not suppress IGF-II–induced proliferation of human BAC cell lines. Each sample was analyzed in duplicate. Data represent mean ± SEM from 2 independent experiments. Results indicated by a different letter are significantly different (P < 0.05).
Vesamicol-induced apoptosis is specifically mediated by VAChT in human BAC cells. A, caspase-3 activity assays showed that the transfection of VAChT-siRNA reversed vesamicol-induced apoptosis in nicotine-treated A549 cells, whereas transfection of sigma-receptor siRNA (SigmaR-siRNA) or control-siRNA has no effect. B, the experiment was repeated in H358 human BAC cells and similar results were obtained. C, the suppression of VAChT, with another independent VAChT-siRNA (VAChT-siRNA-2), abrogated vesamicol-induced apoptosis in A549 human BAC cells. D, the experiment was repeated in H358 cells and similar results were obtained. Each sample was analyzed in duplicate. Data represent mean ± SEM from 2 independent experiments. Results indicated by a different letter are significantly different (P < 0.05).
The apoptotic activity of vesamicol is mediated by the Akt pathway. A, the treatment of A549 and H358 human BAC cells with nicotine and vesamicol inhibited Akt phosphorylation at Thr-308. B, vesamicol decreased the levels of phospho-Akt (Ser-473) in nicotine-treated A549 and H358 cells. C, vesamicol had no effect on the levels of phospho-Akt (Thr-308) in untreated A549 and H358 cells. D, the treatment of quiescent A549 and H358 cells 50 μmol/L vesamicol does not affect phospho-Akt (Ser-473) levels. E, the transfection of pcDNA3-HA-Akt-CA (T308D, S473D) reverses the apoptotic activity of vesamicol, whereas transfection of the empty vector had no effect on vesamicol-induced apoptosis in A549 human BAC cells. F, the transfection experiment was repeated in H358 cells and similar results were obtained. Data represent mean ± SEM from 2 independent experiments. Each sample was analyzed in duplicate. Results indicated by a different letter are significantly different (P < 0.05).
Vesamicol induces apoptosis of human BAC tumorsin vivo. A, the administration of vesamicol (50 mg vesamicol/kg food) decreased the growth rate of A549 human BAC tumors xenografted in nude mice. The nude mice experiments comprised 8 mice per group. B, the tumors from the mice were excised and stained with H&E. The black arrow shows the presence of apoptotic bodies in the tissue sections. The vesamicol-treated tumors (right) showed a greater number of apoptotic bodies relative to the mice administered vehicle (left). Scale bar, 500 μm. C, an aliquot of the tumors from nude mice were frozen and lysates were made. Caspase-3 activity of these lysates was measured. The vesamicol-treated tumors had higher caspase-3 activity (indicating higher apoptosis) than the tumors in the control group. D, Western blotting showed that vesamicol-treated tumors showed lower levels of phospho-Akt (Thr308) and phospho-Akt (Ser473). The Western blotting was quantitated using NIH ImageJ 1.46p. Data represent mean ± SEM. Results indicated by a different letter or an asterisk are significantly different (P < 0.05).
References
- Levy BP, Drilon A, Makarian L, Patel AA, Grossbard ML. Systemic approaches for multifocal bronchioloalveolar carcinoma: is there an appropriate target? Oncology (Williston Park) 2010;24:888–98, 900. - PubMed
- Saintigny P, Wistuba II, Kim ES. Bronchioloalveolar carcinoma: a translational perspective. Oncology (Williston Park) 2010;24:907–8, 14. - PubMed
- Colby TV, Noguchi M, Henschke C, Vazquez MF, Geisinger K, Yokose T, et al. Adenocarcinoma. In:Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC, et al., editors. World health classification of tumours pathology and genetics of tumours of the lung, pleura, thymus and heart. Lyon, France: IARC Press; 2004. p. 35–44.
- The Health Consequences of Smoking. A report of the Surgeon General’s Office on Smoking and Health, DHHS, Washington DC. 2004.
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