Social stress promotes and γ-aminobutyric acid inhibits tumor growth in mouse models of non-small cell lung cancer

Abstract

Psychologic distress is associated with increased lung cancer incidence and mortality. We have shown that non-small cell lung cancer (NSCLC) cells in vitro are stimulated by the cyclic AMP (cAMP)-dependent activation of cAMP-responsive element binding protein (CREB) and extracellular signal-regulated kinase (ERK) downstream of β-adrenergic receptors and that this pathway is inhibited by the neurotransmitter γ-aminobutyric acid (GABA). Because the stress neurotransmitters noradrenalin and adrenalin are β-adrenergic agonists, the current study has tested the hypothesis that social stress stimulates NSCLC growth in vivo and that GABA inhibits this effect. Social stress was induced in mice carrying xenografts from two NSCLC cell lines in the presence and absence of treatment with GABA. Xenograft sizes were measured after 30 days. Noradrenalin, adrenalin, cortisol, GABA, and cAMP were measured in blood and tumor tissues by immunoassays. Expression of nicotinic receptors in the xenografts was assessed by real-time PCR and Western blotting. Protein expression of phospho (p)-CREB, CREB, phospho (p)-ERK, ERK, and glutamate decarboxylase (GAD) 65 and 67 were determined by Western blotting. Xenograft sizes in stress-exposed mice were significantly increased. Nicotinic acetylcholine receptor (nAChR) subunits α3, α4, α5, and α7 in xenograft tissues showed posttranscriptional induction. Noradrenalin, adrenalin, and cortisol were elevated in serum and xenograft tissue whereas GABA was suppressed. Levels of cAMP, p-CREB, and p-ERK were increased whereas GAD65 and GAD67 were suppressed in tumor tissue. Treatment with GABA reversed the effects of stress. Our findings suggest that social stress stimulates NSCLC by increasing nAChR-mediated stress neurotransmitter signaling and that GABA is a promising novel agent for NSCLC intervention.

Figure 1

Effects of social stress on the size of xenografts from human NSCLC cell lines NCI-H322 and NCI-H441 and the effects of GABA treatment on NCI-H322 and NCI-H441 xenografts in unstressed (NS+G) stress exposed (SS+G) mice. The photographs in A exemplify the tumor sizes in the 4 treatment groups. The graph (B) illustrates tumor volumes calculated from two perpendicular diameters of each xenograft. Data in the graph are mean values and standard deviations of xenograft volumes from 20 mice per group expressed as fold change of xenograft volumes from the stress-exposed and GABA treated groups over that from untreated mice not exposed to stress.

Figure 2

Representative examples of Western blots (A) for the assessment of protein expression of the nAChR subunits α3, α4, α5 and α7 and of the two isozymes of the GABA synthesizing enzyme GAD in xenografts from NSCLC cell lines NCI-H322 and NCI-H441 of mice exposed to social stress (SS) and unstressed mice (NS). Columns in the graph (B) represent mean values and standard deviations of four densitometric readings per protein band adjusted for actin from three independent Western blots after background subtraction (n = 12).

Figure 3

Results of immunoassays for the determination of cAMP in the cellular fraction of blood (A) and in xenograft tissues (B) from mice carrying xenografts from NCI-H322 and NCI-H441 cells and exposed to chronic social stress (SS) and from unstressed mice (NS) and GABA treated animals with (SS+G) and without (NS+G) stress. Columns in the graphs represent mean values and standard deviations (n = 5) expressed as fold increase or decrease of values from stress-exposed or GABA treated mice over those from untreated unstressed mice.

Figure 4

Western blots (A) exemplifying the protein expression of p-ERK and p-CREB in xenograft tissues from NCI-H 322 and NCI-H441 cells in untreated mice without stress (NS), social stress exposed mice (SS) and from GABA treated mice without (NS+G) and with (SS+G) stress. Columns in the graph (B) are mean values and standard errors of densitometry ratios of phosphorylated protein/total protein calculated from 4 densitometric readings per band of 3 independent Westerns blots (n = 12) and are expressed as fold increase/decrease of values over untreated animals without stress (NS).