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

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 ß-adrenergic receptors and that this pathway is inhibited by the neurotransmitter γ-aminobutyric acid (GABA). Because the stress neurotransmitters noradrenalin and adrenalin are ß-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.

Regulation of pancreatic cancer by neuropsychological stress responses: a novel target for intervention.

Pancreatic cancer has a poor prognosis and is associated with high levels of psychological stress that may adversely affect clinical outcomes. However, the potential influence of neuropsychological factors on pancreatic cancer has not been investigated to date. Using a mouse model of social stress, we have tested the hypothesis that psychological stress promotes the progression of pancreatic cancer xenografts via neurotransmitter-induced activation of multiple pathways and that the inhibitory neurotransmitter γ-aminobutiric acid (GABA) inhibits these responses. Sytemic and xenograft levels of noradrenalin, adrenalin, GABA, cortisol, vascular endothelial growth factor (VEGF) and cyclic adenosine 3', 5'-monophosphate (cAMP) were measured by immunoassays. Xenograft expression of nicotinic acetylcholine receptors (nAChRs) α3, α4, α5, α6 and α7 and ß-adrenergic receptors 1 and 2 were assessed by real-time PCR and western blots. Expression of glutamate decarboxylases GAD65 and GAD67 and phosphorylated and unphosphorylated signaling proteins of relevance to pancreatic cancer were determined in tumor tissue by western blots. Psychological stress significantly promoted xenograft growth and increased systemic and tumor levels of noradrenalin, adrenalin, cortisol, VEGF and cAMP while GABA and GAD were suppressed. Stress upregulated nAChR proteins but not RNAs and induced phosphorylated ERK, CREB, Src and AKT in xenografts. Reduction of cAMP by treatment with GABA prevented tumor progression and activation of signaling proteins. Our findings suggest that neurotransmitter responses to psychological stress negatively impact clinical outcomes of pancreatic cancer via the activation of multiple pathways and that replacement of the suppressed inhibitory neurotransmitter GABA prevents these effects.

Nicotine stimulates pancreatic cancer xenografts by systemic increase in stress neurotransmitters and suppression of the inhibitory neurotransmitter gamma-aminobutyric acid.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality in Western countries. We have shown previously that four representative human PDAC cell lines were regulated by beta-adrenoreceptors via cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signaling. In the current study, we have tested the hypothesis that nicotine stimulates the growth of PDAC xenografts in nude mice by increasing the systemic levels of the stress neurotransmitters adrenaline and noradrenaline, which are the physiological agonists for beta-adrenoreceptors and that inhibition by gamma-aminobutyric acid (GABA) of the adenylyl cyclase-dependent pathway downstream of adrenoreceptors blocks this effect. The size of xenografts from PDAC cell line Panc-1 was determined 30 days after inoculation of the cancer cells. Stress neurotransmitters in serum as well as cAMP in the cellular fraction of blood and in tumor tissue were assessed by immunoassays. Levels of GABA, its synthesizing enzymes GAD65 and GAD67 and beta-adrenergic signaling proteins in the tumor tissue were determined by western blotting. Nicotine significantly increased the systemic levels of adrenaline, noradrenaline and cAMP while increasing xenograft size and protein levels of cAMP, cyclic AMP response element-binding protein and p-extracellular signal-regulated kinase 1/2 in the tumor tissue. Nicotine additionally reduced the protein levels of both GAD isozymes and GABA in tumor tissue. Treatment with GABA abolished these responses to nicotine and blocked the development of xenografts in mice not exposed to nicotine. These findings suggest that the development and progression of PDAC is subject to significant modulation by stimulatory stress neurotransmitters and inhibitory GABA and that treatment with GABA may be useful for marker-guided cancer intervention of PDAC.

G-Protein Inwardly Rectifying Potassium Channel 1 (GIRK1) Knockdown Decreases Beta-Adrenergic, MAP Kinase and Akt Signaling in the MDA-MB-453 Breast Cancer Cell Line.

Previous data from our laboratory have indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in breast cancer cell lines and that these pathways are involved in growth regulation of these cells. To determine functionality, MDA-MB-453 breast cancer cells were stimulated with ethanol, known to open GIRK channels. Decreased GIRK1 protein levels were seen after treatment with 0.12% ethanol. In addition, serum-free media completely inhibited GIRK1 protein expression. This data indicates that there are functional GIRK channels in breast cancer cells and that these channels are involved in cellular signaling. In the present research, to further define the signaling pathways involved, we performed RNA interference (siRNA) studies. Three stealth siRNA constructs were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence. These constructs were transfected into MDA-MB-453 cells, and both RNA and protein were isolated. GIRK1, ß(2)-adrenergic and 18S control levels were determined using real-time PCR 24 hours after transfection. All three constructs decreased GIRK1 mRNA levels. However, ß(2) mRNA levels were unchanged by the GIRK1 knockdown. GIRK1 protein levels were also reduced by the knockdown, and this knockdown led to decreases in beta-adrenergic, MAP kinase and Akt signaling.

Actin-based features negatively regulated by protein kinase C-epsilon.

Cells exposed to phorbol 12-myristate 13-acetate (PMA) undergo a choreographed sequence of morphological changes. Some of these, including stimulation of membrane ruffles and the later appearance of stress fibers, rely on remodeling of the actin cytoskeleton. Although this process is poorly understood, it is important, because the same features are affected during oncogenic transformation. PMA also activates protein kinase C (PKC). Enzyme activation is followed by degradation. Either process might affect the remodeling of actin. The present studies determined whether any PKC isozymes were subject to degradation in tracheal epithelial cells by quantifying the amount of each isozyme present after PMA exposure. PKC-epsilon was the only isozyme to show declining content correlated with increased stress fiber accumulation. Stress fibers increased between 5 and 10 h, whereas PKC-epsilon declined to 38% of its starting value (95% confidence interval, 10-68%). The relationship could be fit by the function F(x) = 0.683 x exp[-0.841(x - 0.387)], where F is the frequency of fiber-containing cells and x is PKC-epsilon content. Fiber accumulation was further investigated after knockdown of PKC-epsilon with RNA interference and antisense oligodeoxynucleotide. Knockdown enhanced stress fibers in cells not yet exposed to PMA as well as the final frequency of fiber-containing cells after PMA exposure. With knockdown at both transcriptional and protein levels, approximately 15% of the original content was predicted and achieved, as judged from real-time PCR and PKC-epsilon content measurements. The results suggest that PKC-epsilon negatively regulates stress fibers, either by directly turning over one of their components or by regulating an upstream step affecting fiber organization.

Protein expression of G-protein inwardly rectifying potassium channels (GIRK) in breast cancer cells.

BACKGROUND: Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK) channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established risk factor for breast cancer and has been found to open GIRK. In order to further investigate GIRK channels in breast cancer and possible alteration by ethanol, we identified GIRK channel protein expression in breast cancer cells. RESULTS: Cell pellets were collected and membrane protein was isolated to determine GIRK protein expression. GIRK protein was also analyzed by immuno-precipitation. GIRK protein was over-expressed in cells by transfection of GIRK plasmids. Gene expression studies were done by real-time RT-PCR. GIRK protein expression was identified in breast cancer cell lines. Expression of GIRK1 at the indicated molecular weight (MW) (62 kDa) was seen in cell lines MDA-MB-453 and ZR-75-1. In addition, GIRK1 expression was seen at a lower MW (40-42 kDa) in MDA-MB-361, MDA-MB-468, MCF-7, ZR-75-1, and MDA-MB-453 cell lines. To prove the lower MW protein was GIRK1, MDA-MB-453 cells were immuno-precipitated. GIRK2 expression was seen in MDA-MB-468, MCF-7, and ZR-75-1 and was variable in MDA-MB-453, while GIRK4 protein expression was seen in all six cell lines tested. This is the first report indicating GIRK protein expression in breast cancer cells. To determine functionality, MDA-MB-453 cells were stimulated with ethanol. Decreased GIRK1 protein expression levels were seen after treatment with 0.12% ethanol in MDA-MB-453 breast cancer cells. Serum-free media decreased GIRK protein expression, possibly due to lack of estrogen in the media. Transfection of GIRK1 or GIRK4 plasmids increased GIRK1 protein expression and decreased gene expression in MDA-MB-453 breast cancer cells. CONCLUSION: Our data indicates that functional GIRK channels exist in breast cancer cells that are involved in cellular signaling.

Pulmonary fibroblasts stimulate the proliferation of cell lines from human lung adenocarcinomas.

Human lung cancer cell lines are widely used to test anticancer drugs. These in-vitro tests, however, preclude the detection of responses to paracrine factors from surrounding stroma. We have cocultured pulmonary fibroblasts CCD-19Lu, from a healthy donor, or HLF-A, from a patient with epidermoid carcinoma of the lung, with two human pulmonary adenocarcinoma cell lines to test the hypothesis that the fibroblasts stimulate the growth of the tumor cells. Both fibroblast cell lines significantly increased the proliferation of the pulmonary adenocarcinoma cell lines in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assays, with HLF-A fibroblasts yielding the most pronounced responses. The proliferation of the pulmonary adenocarcinoma cell lines in coculture with fibroblasts was blocked by antibodies against the transforming growth factor-alpha and amphiregulin. In addition, reverse transcription-polymerase chain reaction showed expression of mRNA for amphiregulin and transforming growth factor-alpha in all cell lines, whereas mRNA for the epidermal growth factor was detected only in pulmonary adenocarcinoma cell lines. Western blot analysis revealed that medium containing growth factors released by each fibroblast cell line activated extracellular signal-regulated kinase 1/2 in the both tested pulmonary adenocarcinoma cell lines, but activated Akt kinase only in A549 cells. Assessment of protein levels for cyclin D1 and cyclin E by Western blots demonstrated pronounced increases of both proteins in each pulmonary adenocarcinoma cell line, whereas protein levels for cyclin-dependent kinase inhibitor p21 remained unchanged. Immunocytochemical analysis showed positive immunoreactivity for P-extracellular signal-regulated kinase 1/2, cyclin D1 and cyclin E in pulmonary adenocarcinoma cells cocultured with fibroblasts or exposed to fibroblast-conditioned media. Our data suggest that the growth of pulmonary adenocarcinoma is stimulated by amphiregulin and transforming growth factor-alpha released from pulmonary fibroblasts. This may contribute to the disappointing clinical responses to anticancer drugs, which have shown promise in tests with lung cancer cell lines.

Expression of G-protein inwardly rectifying potassium channels (GIRKs) in lung cancer cell lines.

BACKGROUND: Previous data from our laboratory has indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. METHODS: GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay. RESULTS: GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC) cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with beta2-adrenergic antagonist ICI 118,551 (100 microM) daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the beta-adrenergic agonist isoproterenol (10 microM) decreased growth rates in these cells. The GIRK inhibitor U50488H (2 microM) also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2) was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4) mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4) mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein expression was not seen in any non-SCLC cells. CONCLUSION: We feel that this data may indicate that stimulation of GIRK1 or GIRK2 channels may be important in lung cancer. Stimulation of GIRK channels and beta-adrenergic signaling may activate similar signaling pathways in both SCLC and breast cancer, but lead to different results.

Expression of the alpha7 nicotinic acetylcholine receptor in human lung cells.

BACKGROUND: We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the alpha7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a high affinity agonist for the alpha7 nicotinic acetylcholine receptor. In the present study, our goal was to determine the extent of alpha7 mRNA and protein expression in the human lung. METHODS: Experiments were done using reverse transcription polymerase chain reaction (RT-PCR), a nuclease protection assay and western blotting using membrane proteins. RESULTS: We detected mRNA for the neuronal nicotinic acetylcholine receptor alpha7 receptor in seven small cell lung cancer (SCLC) cell lines, in two pulmonary adenocarcinoma cell lines, in cultured normal human small airway epithelial cells (SAEC), one carcinoid cell line, three squamous cell lines and tissue samples from nine patients with various types of lung cancer. A nuclease protection assay showed prominent levels of alpha7 in the NCI-H82 SCLC cell line while alpha7 was not detected in SAEC, suggesting that alpha7 mRNA levels may be higher in SCLC compared to normal cells. Using a specific antibody to the alpha7 nicotinic receptor, protein expression of alpha7 was determined. All SCLC cell lines except NCI-H187 expressed protein for the alpha7 receptor. In the non-SCLC cells and normal cells that express the alpha7 nAChR mRNA, only in SAEC, A549 and NCI-H226 was expression of the alpha7 nicotinic receptor protein shown. When NCI-H69 SCLC cell line was exposed to 100 pm NNK, protein expression of the alpha7 receptor was increased at 60 and 150 min. CONCLUSION: Expression of mRNA for the neuronal nicotinic acetylcholine receptor alpha7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441) as well as normal lung cells. The alpha7 nicotinic receptor protein is expressed in fewer cell lines, and the tobacco carcinogen NNK increases alpha7 nicotinic receptor protein levels.

Expression of inwardly rectifying potassium channels (GIRKs) and beta-adrenergic regulation of breast cancer cell lines.

BACKGROUND: Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling. METHODS: Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting. RESULTS: Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 microM) increased the GIRK1 mRNA levels and decreased beta2-adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K+ flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells. CONCLUSIONS: Our data suggests beta-adrenergic receptors and GIRK channels may play a role in breast cancer.

Receptor-mediated effects of nicotine and its nitrosated derivative NNK on pulmonary neuroendocrine cells.

Pulmonary neuroendocrine cells (PNECs) have been implicated in the development of small cell lung carcinoma (SCLC) and pediatric asthma, and smoking is a risk factor for both diseases. We as well as others have shown that the alpha(7) nicotinic acetylcholine receptor (alpha(7) nAChR) regulates the release of 5-hydroxytryptamine (5-HT, serotonin) in PNECs and SCLC. Serotonin is an autocrine growth factor for PNECs and SCLC and acts as broncho-constrictor. We found that nicotine and its nitrosated carcinogenic derivative 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) bind to the alpha(7) nAChR in SCLC and PNECs, resulting in the influx of Ca(2+), release of 5-HT, and activation of a mitogenic pathway mediated by protein kinase C (PKC), Raf-1, mitogen activated protein kinase (MAPK) and c-myc. Exposure to 10% CO(2) acted synergistically. Unstimulated SCLC cells from smokers demonstrated high base levels of 5-HT release and of individual downstream signaling components in comparison to PNECs. Subchronic exposure of PNECs to NNK up-regulated the alpha(7) nAChR and its associated serotonergic mitogenic pathway in PNECs, an effect that may contribute to the development of SCLC in smokers and pediatric asthma in children of mothers who smoke.