Effect of A549 neuroendocrine differentiation on cytotoxic immune response

in Endocrine Connections

The present study was designed to determine the effects of factors secreted by the lung adenocarcinoma cell line with the neuroendocrine phenotype, A549NED, on cytotoxic T lymphocytes (CTLs) activity in vitro. A perspective that integrates the nervous, endocrine and immune system in cancer research is essential to understand the complexity of dynamic interactions in tumours. Extensive clinical research suggests that neuroendocrine differentiation (NED) is correlated with worse patient outcomes; however, little is known regarding the effects of neuroendocrine factors on the communication between the immune system and neoplastic cells. The human lung cancer cell line A549 was induced to NED (A549NED) using cAMP-elevating agents. The A549NED cells showed changes in cell morphology, an inhibition of proliferation, an overexpression of chromogranin and a differential pattern of biogenic amine production (decreased dopamine and increased serotonin [5-HT] levels). Using co-cultures to determine the cytolytic CTLs activity on target cells, we showed that the acquisition of NED inhibits the decrease in the viability of the target cells and release of fluorescence. Additionally, the conditioned medium of A549NED and 5-HT considerably decreased the viability and proliferation of the Jurkat cells after 24 h. Thus, our study successfully generated a neuroendocrine phenotype from the A549 cell line. In co-cultures with CTLs, the pattern of secretion by A549NED impaired the proliferation and cytotoxic activity of CTLs, which might be partly explained by the increased release of 5-HT.

Abstract

The present study was designed to determine the effects of factors secreted by the lung adenocarcinoma cell line with the neuroendocrine phenotype, A549NED, on cytotoxic T lymphocytes (CTLs) activity in vitro. A perspective that integrates the nervous, endocrine and immune system in cancer research is essential to understand the complexity of dynamic interactions in tumours. Extensive clinical research suggests that neuroendocrine differentiation (NED) is correlated with worse patient outcomes; however, little is known regarding the effects of neuroendocrine factors on the communication between the immune system and neoplastic cells. The human lung cancer cell line A549 was induced to NED (A549NED) using cAMP-elevating agents. The A549NED cells showed changes in cell morphology, an inhibition of proliferation, an overexpression of chromogranin and a differential pattern of biogenic amine production (decreased dopamine and increased serotonin [5-HT] levels). Using co-cultures to determine the cytolytic CTLs activity on target cells, we showed that the acquisition of NED inhibits the decrease in the viability of the target cells and release of fluorescence. Additionally, the conditioned medium of A549NED and 5-HT considerably decreased the viability and proliferation of the Jurkat cells after 24 h. Thus, our study successfully generated a neuroendocrine phenotype from the A549 cell line. In co-cultures with CTLs, the pattern of secretion by A549NED impaired the proliferation and cytotoxic activity of CTLs, which might be partly explained by the increased release of 5-HT.

Introduction

Lung cancer is a leading cause of death due to cancer in both sexes worldwide (1, 2). A primary concern of this disease is the neuroendocrine phenotype linked to decreased survival and increased peripheral tumour cells, drug-resistant tumours and metastasis (3, 4). The main characteristics of tumours with a neuroendocrine phenotype are low proliferation rates, polygonal morphology, dense chromatin and a specific distribution pattern, that is nests, trabecular, acinar and rosette-like structures (5). Such tumours also express neuroendocrine markers such as chromogranin A (CgA) (6), synaptophysin, neuronal enolase and CD56 (7, 8, 9, 10). A key aspect of the neuroendocrine phenotype is the formation of granules that secrete various factors, including hormones and neurotransmitters. In the recent years, there has been an increasing interest in the role of these factors as immunomodulators in bidirectional communication with the immune system (11). In some cancers, it has been proposed that new cells with neuroendocrine-like properties originate from pre-existing epithelial cancer cells via neuroendocrine differentiation (NED) (12). NED can be replicated in vitro by the exposure of cells to agents that elevate or mimic intracellular cAMP (i.e. 8-Br-cAMP, isobutylmethylxanthine and forskolin) (13, 14), androgen depletion (15), interleukin 6 (16), ionising radiation (17) and contact inhibition promoted by a high cellular density (12).

Although several studies have examined NED in lung cancers, the effects of secreted immunomodulators and their associations with antitumour immune responses remain unclear. This study aimed to determine the effects of factors secreted by the lung adenocarcinoma cell line A549 with NED (A549NED) on cytotoxic T lymphocytes (CTLs) activity in vitro.

Materials and methods

Cells and reagents

Jurkat human lymphoblast cells (TIB-152) were purchased from ATCC, and the A549/GFP cell line (AKR-209) from human lung adenocarcinoma was purchased from Cell Biolabs, Inc. (San Diego, CA, USA). DMEM, RPMI-1640, foetal bovine serum (FBS) and phytohaemagglutinin A (PHA) were purchased from Gibco. The monoclonal antibody anti-CgA was purchased from BD Biosciences. 3-isobutyl-1-methylxanthine (I7018), forskolin (F6886) and serotonin (H9523) were purchased from Sigma-Aldrich. All other chemicals were of the highest grade of purity commercially available.

Cell culture

A549/GFP cells and co-cultures were maintained in DMEM, and Jurkat cells were cultured in RPMI-1640. All media were supplemented with 10% FBS, 100 kU/L penicillin and 100 kU/L streptomycin from Gibco at 37°C with a 5% carbon dioxide-enriched air atmosphere.

Neuroendocrine differentiation induced by cAMP-elevating agents

The human lung adenocarcinoma A549 cells were seeded at a density of 2 × 105 cells/mL in 12-well plates in 1000 L triplicate aliquots in DMEM for 24 h. The wells were washed with PBS and then treated for 24, 72, 120 and 144 h with 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM), forskolin (FSK, 0.5 mM) or IBMX + FSK (I + F) in fresh medium. Morphologic changes and proliferation rates were evaluated via light microscopy. The conservation of morphologic changes at 24 or 48 h after treatment withdrawal (WD) was also assessed. Cells were collected to analyse the expression of neuroendocrine markers and evaluate the immunomodulator effects.

Proliferation assay

The proliferation rate of NED was determined by culturing 2 × 105 A549 cells/mL in 12-well plates in 1000 μL triplicate aliquots in DMEM. These cells were stained with 100 μL of 0.04% trypan blue solution to evaluate the cell viability of each treatment at 24, 48, 72, 96 and 120 h.

To evaluate the direct effect of the soluble secretion factors, we used conditioned medium and different concentrations of serotonin (5-HT). The conditioned medium (CM) supplemented with the soluble secretion factors released by A549CTRL and A549NED was collected for 48 h after the treatment for differentiation. CM or 5-HT was added to the Jurkat cells, and proliferation was determined by culturing 2.5 × 105 cells/mL in 12-well plates in 1500 μL triplicate aliquots. Cells were stimulated with 0.02 mL of PHA (Gibco) for 24 h for the activation of CTLs. Naive or activated T-cells were then supplemented with 5-HT at final concentrations ranging from 1 × 10−12 to 1 × 10−3 M for each treatment. Finally, cells were stained with 100 μL of 0.04% trypan blue solution to evaluate the cell viability at 24 and 28 h.

Flow cytometry

Flow cytometry was used to identify the neuroendocrine marker, CgA. Briefly, cells were resuspended in 500 µL PBS, and 20 µL of the anti-CgA-PE monoclonal antibody (clone S21-537, BD) was added. The mixture was incubated for 30 min at room temperature and protected from light. After incubation, the cells were washed twice with PBS and resuspended in 500 µL PBS. Samples were acquired on a FACSVerse flow cytometer (BD Biosciences). Data analysis was performed using FlowJo trial version (18).

Biogenic amine determination

Frozen pellets of cells were used to determine the levels of the neurotransmitters nonadrenaline (NA), adrenaline (AD), dopamine (DA) and 5-HT using reversed-phase chromatographic analysis (RP-HPLC). Briefly, the biogenic amines were extracted using 400 µL of an extraction buffer containing 5% ascorbic acid, 200 mM sodium phosphate, 2.5 mM L-cysteine and 2.5 mM EDTA. The protein was then precipitated by adding 100 μL of 0.4 M perchloric acid and incubated at 20°C for 20 min. Supernatants were collected after centrifugation at 12,000 rpm for 10 min (4°C). The samples were filtered by 0.22 μm before injection. NA, AD, DA and 5-HT concentrations were determined using RP-HPLC in a system integrated with a PU-2089plus pump (Jasco, Inc., Tokyo, Japan), an AS-2057plus autosampler (Jasco) and an X-LC™3120FP fluorescence detector (Jasco). The instruments were controlled using ChromNAV software (Jasco). Chromatographic runs were performed using a Jupiter C18 column (300 A, 5 μ, 4.6 × 250 mm; Phenomenex, Torrance, CA, USA) at 30°C. The column was equilibrated with mobile phase A containing 0.1% trifluoroacetic acid. Mobile phase B (MPB) containing 0.1% trifluoroacetic acid in acetonitrile was used to perform a linear gradient until 20% MPB from min 5 to min 15; 20% MPB was then maintained until min 20. The flow rate was 0.8 mL/min. The fluorescence detector was set at gain 1000, attenuation 32, response 20 s and 280 nm for excitation and 315 nm for emission. Sample injection volume was 50 μL. The order and retention times of the eluted monoamines and their metabolites in the chromatogram were NA (5.5 min), AD (7 min), DA (11.2 min) and 5-HT (16.1 min).

In vitro cytolysis assay

After inducing NED using cAMP-elevating agents, the A549 cells (target cells) with the constitutive expression of GFP were incubated with the Jurkat cells (CTLs) resting or preactivated with 20 μg/mL PHA as effector cells at 37°C for 6 or 24 h in the darkness. Culture medium without phenol red was added to the target cells to determine proliferation, cell viability and the minimum and maximum release of fluorescence. Fluorescence of supernatants was read using the fluorimeter (Varioskan) with excitation at 410 nm and emission at 520 nm.

Receptor detection via western blot

The expression of 5-HT5A and 5-HT7 receptors in the Jurkat cell line was determined via western blot (WB), and the total protein was extracted from the Jurkat cells following the manufacturer’s protocol.

Briefly, the Jurkat cells were solubilised for 30 min at 4°C in a lysis buffer containing 25 mM Tris-HCl, pH 7.1, with a protease inhibitor cocktail (Complete Mini, Roche). After centrifugation, the protein concentration was quantified via the Bradford method. Total proteins were denatured at 85°C for 5 min, subjected to SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline-0.05% Tween 20 PBS containing 5% non-fat dry milk for 1 h at room temperature. The blots were then incubated with polyclonal anti-human receptor antibody (5-HT5A and 5-HT7; Santa Cruz Biotechnology) for 2 h, washed in PBS and incubated overnight with a secondary antibody linked to horseradish peroxidase (Invitrogen). Finally, the bound horseradish peroxidase was visualised using a high-sensitive chemiluminescence system (ECL Kit; GE Healthcare).

Statistical analysis

Data are expressed as mean ± error. Differences between the experimental groups were analysed using one-way ANOVA and Tukey’s significant difference or Dunnett’s test. Differences with P < 0.05 were considered statistically significant.

Results

Consistent with previous studies in which cAMP-elevating agents induced NED in NSCLC cells, we successfully induced NED in A549 (A549NED) via stimulation with IBMX, FSK and I + F (19). Morphologic changes induced by cAMP-elevating agents were observed via light microscopy at 24, 72 and 120 h. Figure 1 shows that treatment with IBMX (0.5 mM) or FSK (0.5 mM) promoted an increase in the cell size as well as the formation of new neurite-like projections after 72 h (20). Treatment with I + F promoted morphology changes after 24 h, including rounding of the cell body and extension of neurite-like cellular processes (21, 22). Lower concentrations (0.25 mM and 0.125 mM) did not promote morphologic changes (data not shown). As shown in Fig. 2, the treatment of cells with FSK or I + F inhibited the proliferation after 48 h, whereas the treatment with IBMX inhibited the proliferation only after 72 h.

Figure 1
Figure 1

Effect of cAMP-elevating agents on A549 cell morphology. A549 cells were treated for 24, 72 or 120 h with IBMX (0.5 mM), FSK (0.5 mM) or I + F. Bars on the photomicrographs correspond to 200 µm. Figures are representative of three independent experiments for triplicate. FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

Figure 2
Figure 2

Effect of cAMP-elevating agents on A549 proliferation rate. The proliferation of cells supplemented with FSK (0.5 mM), IBMX (0.5 mM) or I + F was assessed every 24 h for 144 h. n = 3 for triplicates. One-way ANOVA with Dunnett’s post hoc test (*P < 0.01 compared with A549 control cells). FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

To determine if these changes were due to NED, we quantified the expression of neuroendocrine markers in the cells treated with cAMP-elevating agents. The groups treated with FSK showed a significant overexpression of CgA (Fig. 3B).

Figure 3
Figure 3

Effect of cAMP-elevating agents on neuroendocrine biomarker expression of A549 cell line. A549 cells were exposed to IBMX (0.5 mM), FSK (0.5 mM) or I + F for 120 h. (A) The level of neuroendocrine marker CgA was evaluated using flow cytometry. (B) The results are expressed as the percentage of positive cells to CgA. n = 3 for triplicates. One-way ANOVA with Tukey’s post hoc test. Bars with different letters represent statistical significance (P < 0.01). CgA, chromogranin A; FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

A review by Day and Salzet (23) proposed the main aspects of NED and highlighted the importance of the formation of secretory granules expressing chromogranins. Our results support this proposal and corroborate the acquisition of the neuroendocrine phenotype in A549 cells.

To assess the changes in secretion, A549CTRL and A549NED were compared using HPLC to identify the presence of biogenic amines (Fig. 4). The results revealed that A549CTRL produced EP and DA but not 5-HT or NE. Interestingly, the acquisition of NED in all three groups (i.e. by IBMX, FSK and I + F treatments) changed the pattern of biogenic amine production, showing a reduction of DA (Fig. 4B) and increase of 5-HT (Fig. 4C).

Figure 4
Figure 4

Effect of NED on the pattern of biogenic amine secretion. The A549 cells were exposed to cAMP-elevating agents for 120 h for transdifferentiation. Biogenic amines were then quantified using HPLC in the samples A549CTRL, A549NED (IBMX), A549NED (FSK) and A549 (NED)(i + f) and compared to the standard mixture of biogenic amines: NA, (A) AD, (B) DA and (C) 5-HT. The experiments were repeated thrice with independent samples. ND, not detectable. n = 3 for triplicates. Values are expressed as mean ± error. One-way ANOVA with Tukey’s post hoc test. Bars with different letters represent statistical significance (P < 0.01). DA, dopamine; AD, adrenaline; FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine; NED, neuroendocrine differentiation; NA, nonadrenaline; 5-HT, serotonin.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

Previous studies have reported the ability of some cells to revert to their original phenotype after the acquisition of NED; this occurs in two stages, namely reversible and terminal. To evaluate the cytolytic CTLs activity over the A549 cells after the acquisition of NED, we monitored whether cAMP-elevating agents induced a permanent conversion from epithelial to neuroendocrine morphology. The phenotype obtained after 72 h of treatment was retained for 24 h (not shown). The morphologic changes generated after 120 h of treatment were retained for 48 h, as shown in Fig. 5. These results suggested the generation of a terminal neuroendocrine phenotype (24, 25, 26).

Figure 5
Figure 5

Cell morphology with neuroendocrine differentiation (NED) after withdrawal (WD) of cAMP-elevating agents. The A549 cells were exposed to cAMP-elevating agents for 120 h for transdifferentiation. Cell morphology was assessed 24 and 48 h after WD. Bars on the photomicrographs correspond to 200 µm. Figures are representative of three independent experiments for triplicate.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

To determine the cytolytic activity of the target cells, co-cultures of A549CTRL or A549NED were tested against CTLs (resting and preactivated with PHA Jurkat cells). Figure 6 presents the fluorescence release of the target cells constitutively expressing GFP. The quantification of the viability of the target and cytotoxic cells are separately shown in Fig. 7.

Figure 6
Figure 6

Direct cytolysis in co-cultures of T:C. CTLs (Jurkat) were tested with A549 cells in co-culture. The percentage of direct cytolysis was evaluated as % fluorescence release at (A) different time points and with (B) control and transdifferentiated A549 cell lines. n = 3 for triplicates. One-way ANOVA with Dunnett’s post hoc test (*P < 0.01 compared with A549 control cells; ***P < 0.001 compared with A549 control cells). CTLs, Cytotoxic T lymphocytes; T:C, target and cytotoxic cell lines.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

Figure 7
Figure 7

Effect of NED on the viability of 6 h co-cultures of T:C. Previously, A549 cells were supplemented with cAMP-elevating agents for NED. A549CTRL or A549NED were tested against CTLs (Jurkat) for 6 h in co-culture. The viability of target and cytotoxic cells was separately tested using the MTT assay. n = 2 for quadruplicates. Two-way ANOVA with Dunnett’s post hoc test (***P < 0.01 compared with A549; ****P < 0.001 compared to every group of co-culture (A549:Jurkat). CTLs, Cytotoxic T lymphocytes; NED, neuroendocrine differentiation; T:C, target and cytotoxic cell lines.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

As shown in Fig. 6A, CTLs generated an increased response of fluorescence release in accordance with the effector-to-target ratios and the length of exposure. In addition, there was a significant reduction in the fluorescence release in co-cultures with A549NED than in A549CTRL at 24 h (85.98% ± 6.3% A549NED reversible vs. 83.72% ± 6.3% A549NED terminal vs. 105.7% ± 9.6% A549CTRL). Figure 6B presents an overview of these results.

The viability of the target cells in co-cultures was assessed to corroborate these results. The co-culture of A549CTRL vs the Jurkat cells preactivated with PHA showed a decrease of 54.02 % of viability compared with that of the A549 cells alone (Fig. 7A). In contrast, the previous acquisition of NED in all groups inhibited the decrease of the viability of the target cells in 6 h co-cultures (A549CTRL 54.02% vs. A549NED(i + f) 96.14%), as shown in Fig. 7A.

A further analysis of 6 h co-cultures showed that co-cultures with A549NED significantly decreased the viability of the Jurkat cells preactivated with PHA (Fig. 7B).

In addition, to clearly identify whether this impairment of the viability of the Jurkat cells was due to the secretion of A549NED cells, the 48 h CM from A549CTRL and A549NED was collected to posteriorly assess the influence of secretion on the viability MTT and proliferation (trypan blue) of the Jurkat cells at 24 and 48 h. The results showed that CM decreased the proliferation (Fig. 8A) and viability (Fig. 8B) of the resting Jurkat cells after 24 h (significantly, with forskolin treatments).

Figure 8
Figure 8

Effect of the acquisition of NED on the viability of the cytotoxic cell line (Jurkat). Previously, the medium of A549 cells (CTRL and NED) was conditioned for 48 h and collected. (A) The percentage of proliferation (trypan blue) and (B) percentage of cell viability (MTT assay) of cytotoxic cells were evaluated at 24 and 48 h. n = 3 per triplicates. Two-way ANOVA with Tukey’s post hoc test. Bars with different letters represent statistical significance (P < 0.01 compared with A549 control cells). NED, neuroendocrine differentiation.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

To evaluate the direct effect of 5-HT on the proliferation of the Jurkat cells, we explored the proliferation rate of the resting and preactivated with PHA Jurkat cells at increasing concentrations of 5-HT (from 1 × 10−12 to 1 × 10−3 M) for 24 and 48 h. At 24 h of treatment, there was no significant difference between the control and supplemented groups (Fig. 9A). At 48 h, as shown in Fig. 9B, all 5-HT treatments, except 1 × 10−5 M, significantly inhibited the proliferation rate of the Jurkat cells preactivated with PHA. To confirm the expression of 5-HT receptor, we used WB to examine protein lysates from Jurkat T cells. Figure 9C shows the expression of 5-HT5A and 5-HT7 receptors in the Jurkat cell line.

Figure 9
Figure 9

Antiproliferative effect of 5-HT on the Jurkat cell line. Cells were treated with 5-HT (from 1 × 10−12 to 1 × 10−3 M) alone and with PHA (20 µL/mL) for (A) 24 and (B) 48 h for a proliferation assay with trypan blue. (C) Representative immunoblots showing the relative expression of 5-HT5a and 5-HT7 receptors on Jurkat T cells. Blots were stripped and re-tested for B-actin to ensure that comparable quantities of protein were analysed. n = 3 for triplicates. Values are expressed as mean ± error. Two-way ANOVA with Tukey’s post hoc test. Bars with different letters represent statistical significance (P < 0.01 compared with A549 control cells). PHA, phytohaemagglutinin A; 5-HT, serotonin.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

Discussion

The present study was designed to determine the effects of the factors secreted by the lung adenocarcinoma cell line with the neuroendocrine phenotype A549NED on CTLs (Jurkat) activity in vitro.

The origin of neuroendocrine cells in tumours and the underlying molecular mechanisms remain controversial. Nevertheless, previous studies have reported that the sustained elevation of intracellular cAMP by cAMP analogues, adenylate cyclase agonists (i.e. FSK) or phosphodiesterases (PDEs) inhibitors (i.e. IBMX) may promote the acquisition of neuroendocrine characteristics via two main mechanisms (Fig. 10), namely a classical pathway that involves the release of the catalytic subunits of PKA and an alternative pathway that promotes the activation of the EPAC pathway (13, 14, 26).

Figure 10
Figure 10

Proposed mechanism of neuroendocrine differentiation by cAMP-elevating agents. Solid line indicates the reported pathways in the lungs, and the dashed line indicates the pathways reported in other tissues. ASCL1, achaete-scute homolog like 1; CgA, chromogranin A; Epac1 and Epac2, guanine exchange factor; Egr1, early growth response protein 1; FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine; ND1, NeuroD1; NCAM1, neural cell adhesion molecule 1; PDE, phosphodiesterase; PKA, protein kinase A; REST1, repressor element 1-silencing transcription factor; Rap1, small GTPase Ras-related protein; SYP, synaptophysin; (serine/threonine-protein kinase) B-Raf; MEK, ERK1/2 and p38, mitogen activated protein kinases;Vl2, Vilin2.

Citation: Endocrine Connections 7, 5; 10.1530/EC-18-0145

As shown in Fig. 10, the activation of PKA promotes the phosphorylation of the ser133 in CREB. This phosphorylation stimulates the translocation of bHLH transcription factors such as ND1, ASCL1 and TTF1 that upregulate the expression of functional molecules such as NCAM1 and SYP, respectively. The knockdown of the RE1-silencing transcription factor (REST) also contributes to the upregulation of SYP and expression of neuroendocrine genes with RE1 domains like CgA (13, 20, 21, 26, 27).

The novel mechanism for NED described in PC12 and LNCaP cells states that the elevation of intracellular cAMP simultaneously promotes the binding of cAMP to a Rap1-specific guanine exchange factor, Epac1, which causes a conformational change and leads to an enhanced exchange activity towards Rap1, which is localised on intracellular membranes. The GTP-bound Rap1 then stimulates the effector B-Raf to activate the MAP kinases, MEK and Erk1/2, to upregulate the transcription of two genes, namely Egr1 and Vilin2, which promote neurite growth and increase the cell size, as shown in Fig. 10 (27, 28, 29).

Here, we observed morphological changes in the treated groups after 72 h, in accordance with previous results obtained in NCI-H157 and Lu-CSF1 lung cancer cell lines (13), which might be attributed to the upregulation of Egr1 and Vilin2 (16). Combined treatments produced changes more rapidly than did the individual drugs, suggesting an additive effect due to the mechanism of the elevation of intracellular cAMP. FSK is more specific to activating adenylate cyclase, whereas IBMX may promote a crosstalk via the inhibition of phosphodiesterase. Although cAMP and cGMP are synthesised by different enzymes, they are both degraded by the large superfamily of PDEs, promoting the posterior stimulus of cAMP and cGMP concentration and the posterior activation of PKG (30, 31).

Jones & Palmer have previously demonstrated the ability of FSK to induce early changes in the cell morphology via the phosphorylation of RhoA, stimulating actin polymerisation (21); thus, combined treatments may favour this mechanism via the activation of PKG. Both protein kinases have been reported to phosphorylate active, membrane-bound RhoA and promote its translocation to the cytosol, impairing the activation of its effector Rho kinase, which is well known for its role in the modification of the actin cytoskeleton (32).

The results of this study suggest the generation of a terminal neuroendocrine phenotype after 120 h of treatment. Our finding of a terminal phenotype is consistent with the results of Wang et al. who observed a non-reversible phenotype in the lung cells for 14 days after 120 h of treatment with a mixture of KGF, IBMX, 8-Br-cAMP and dexamethasone (24, 26, 33). Our finding of a decreased proliferation rate corresponds with the findings of Cox et al. (26, 34) who reported a similar inhibition of thymidine incorporation after the exposure of cells to cAMP-elevating agents (dbcAMP, IBMX and FSK) in prostate adenocarcinoma cell lines (LNCaP and C4-2). According to the findings of Pernicová et al. (12) obtained in prostate adenocarcinoma cell lines (LNCaP and LAPC-4), the decreased proliferation rate might be due to more cells in arrest (G0 phase) as a result of NED. The stimulation of Epac2 in bronchoalveolar cells (35) is associated with cell cycle arrest by the phosphorylation of p38 (36) as well as with apoptosis by the activation of Akt (37). Further evidence is necessary to understand the role of these pathways in our results.

The overexpression of the neuroendocrine marker CgA in our study was consistent with the results reported by Chang et al. in the A549 cell line (14) and those reported by Pernicová et al. in LNCaP cells (12). In lung cancers, it has been demonstrated that REST1 is highly expressed in NSCLC cells but transcriptionally repressed in SCLC cells. The inactivation of REST1 via methylation is directly related to the expression of the neuroendocrine biomarkers, synaptophysin and CgA (4). According to Day & Salzet (23), the expression of chromogranin does not imply that the cell has a neuroendocrine origin but that it has acquired a neuroendocrine phenotype. In this sense, our results provide a strong evidence of NED of the A549 cell line. The acquisition of neuroendocrine characteristics could be the result of a genetic switch that induces the expression or inhibits repressors that prevent the inhibition of the neuroendocrine markers (23).

According to Cerasuolo (2015), the ability of neuroendocrine cells to induce an ‘early onset of a hormone-refractory status’ is intriguing and clinically relevant (20). Therefore, the data of the differential pattern of neurotransmitter production support the idea that peptide hormones or biogenic amines can either be released into the bloodstream or can locally act by promoting paracrine interaction with the tumour microenvironment, generating worse prognostic outcomes for patients. Our observations of A549NED showed a different pattern/mixture of secretion compared with that of the control cells, indicating an exacerbated concentration of 5-HT, decreased DA and a different pattern of other components not identified (data not shown). In the future, we aim to identify the composition of this secretion (20, 38).

The decreased DA levels observed in our study was consistent with the data generated in PC12 cells where cAMP induced by forskolin was shown to be associated with neurite growth and decreased intracellular DA levels induced by the reduced phosphorylation of TH (39). The mechanism for the increase in 5-HT levels is unclear, although this phenomenon has been previously observed by Mouillet-Richards in 1C11 cells (40). A possible explanation might be that dopaminergic and serotonergic cells arise from a common progenitor with a dual biogenic amine fate (40), which might explain the clinical reports of neuroendocrine tumours in serotoninergic secretion syndromes (41).

Co-cultures of cytotoxic vs target cells were used to obtain information on the immunomodulatory effects of the soluble factors produced by the A549NED cells (42, 43). The results showed decreased cytolysis in these cells than in the control cells, suggesting the acquisition of NED and that the secreted factors locally act via paracrine and autocrine signalling in the tumour microenvironment, possibly through the adaptation of biogenic amine secretion for the posterior inhibition of the CTLs response. This statement is supported by the decreased viability of the Jurkat cells treated with CM of A549NED compared with those treated with CM of controls, as well as by the 5-HT-mediated inhibition of the proliferation rate in the Jurkat cells preactivated with PHA. As previously reported, the Jurkat cell line expresses 5-HT1A (44), 5-HT1B, 5-HT2B, 5-HT2C, 5-HT2D (45) and 5-HT3 (46) receptors. Our study also provides evidence for the presence of 5-HT5A and 5-HT7 receptors in the Jurkat cell line. Previous research has indicated that the selective activation of 5-HT receptors can enhance or attenuate the activity of lymphocytes and, furthermore, that 5-HT might inhibit the PHA-mediated lymphocyte proliferation, possibly through the reduced expression and distribution of IL-2 receptors (47, 48).

Thus, our results suggest that altered biogenic amine production in A549NED can be used as a defence mechanism against CTLs activity (49).

Conclusion

In conclusion, the present study reported the following findings:

  • The generation of a neuroendocrine phenotype from the A549 cell line (A549NED) that was stable for 48 h after stimuli WD and expressed neuroendocrine protein markers.
  • The transdifferentiated human lung adenocarcinoma cell line (A549NED) acquired a differential pattern of biogenic amine production compared with the control cells, specifically producing less DA and more 5-HT.
  • In co-cultures with the Jurkat cells as CTLs, the acquisition of neuroendocrine characteristics correlated with diminished cytolysis.
  • CM of the transdifferentiated human lung adenocarcinoma cell line (A549NED) independently impaired the viability and proliferation of CTLs.
  • 5-HT alone impaired the proliferation of the PHA-activated CTLs.

Declaration of interest

The authors declare that they have no competing interests. I M performed neuroendocrine transdifferentiation, co-cultures and prepared the manuscript. N A R E participated in the design of the study, analysed the results and performed the statistical analysis. A R C corroborated the effect of 5-HT in the Jurkat cells and determined the presence of receptors. G P S and L P analysed and determined the biogenic amines using HPLC and analysed the results. G G A reviewed the manuscript. L C B provided coordination and helped to design the study. All authors read and approved the final manuscript.

Funding

The authors thank Daniela Eloísa San Juan Mora for technical assistance. This work was financially supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) Mexico and FCQ-2017-01 for Dr Berumen from UAQ. Irasema Mendieta would like to thank CONACYT for the financial support of her doctorate studies with grant number 286837.

References

  • 1

    SongHYaoELinCGacayanRChenMHChuangPT. Functional characterization of pulmonary neuroendocrine cells in lung development, injury, and tumorigenesis. PNAS 2012 109 1753117536. (https://doi.org/10.1073/pnas.1207238109)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2

    RemarkRBeckerCGomezJEDamotteDDieu-NosjeanMCSautes-FridmanCFridmanWHPowellCAAltorkiNKMeradMet al. The non-small cell lung cancer immune contexture. A major determinant of tumor characteristics and patient outcome. American Journal of Respiratory and Critical Care Medicine 2015 191 377390. (https://doi.org/10.1164/rccm.201409-1671PP)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3

    StovoldRMeredithSLBryantJLBaburMWilliamsKJDeanEJDiveCBlackhallFHWhiteA. Neuroendocrine and epithelial phenotypes in small-cell lung cancer: implications for metastasis and survival in patients. British Journal of Cancer 2013 108 17041711. (https://doi.org/10.1038/bjc.2013.112)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4

    YazawaT. Recent advances in histogenesis research of lung neuroendocrine cancers: evidence obtained from functional analyses of primitive neural/neuroendocrine cell-specific transcription factors. Pathology International 2015 65 277285. (https://doi.org/10.1111/pin.12267)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5

    KloppelG. Tumour biology and histopathology of neuroendocrine tumours. Best Practice and Research Clinical Endocrinology and Metabolism 2007 21 1531. (https://doi.org/10.1016/j.beem.2007.01.004)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6

    CampanaDNoriFPiscitelliLMorselli-LabateAMPezzilliRCorinaldesiRTomassettiP. Chromogranin A: is it a useful marker of neuroendocrine tumors? Journal of Clinical Oncology 2007 25 19671973. (https://doi.org/10.1200/JCO.2006.10.1535)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7

    PetrovićMMitrovicSStankovicVJurisicVAtkinsonHD. Neuroendocrine markers–useful predictors of therapeutic responses in non-resectable non-small cell lung cancer. Laboratory Medicine 2012 43 610. (https://doi.org/10.1309/LM0IKAO8Z0MUTVFE)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8

    ModlinIMObergKTaylorADrozdovIBodeiLKiddM. Neuroendocrine tumor biomarkers: current status and perspectives. Neuroendocrinology 2014 100 265277. (https://doi.org/10.1159/000368363)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9

    LindholmDPObergK. Biomarkers and molecular imaging in gastroenteropancreatic neuroendocrine tumors. Hormone and Metabolic Research 2011 43 832837. (https://doi.org/10.1055/s-0031-1287794)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10

    ObergKModlinIMDe HerderWPavelMKlimstraDFrillingAMetzDCHeaneyAKwekkeboomDStrosbergJet al. Consensus on biomarkers for neuroendocrine tumour disease. Lancet Oncology 2015 16 435446. (https://doi.org/10.1016/S1470-2045(15)00186-2)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11

    PavonLBottassoOBesedoskyHLoriaRVelasco-VelazquezMA. Clinical and experimental immunomodulation 2014. Journal of Immunology Research 2015 2015 12. (https://doi.org/10.1155/2015/758328)

    • Search Google Scholar
    • Export Citation
  • 12

    PernicováZSlabákováEFedrRŠimečkováŠJarošJSuchánkováTBouchalJKharaishviliGKrálMKozubíkAet al. The role of high cell density in the promotion of neuroendocrine transdifferentiation of prostate cancer cells. Molecular Cancer 2014 13 113129. (https://doi.org/10.1186/1476-4598-13-113)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    WalkerGEAntonionoRJRossHJPaisleyTEOhY. Neuroendocrine-like differentiation of non-small cell lung carcinoma cells: regulation by cAMP and the interaction of mac25/IGFBP-rP1 and 25.1. Oncogene 2006 25 19431954. (https://doi.org/10.1038/sj.onc.1209213)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    ChangYKimSYChoiYJSoKSRhoJKKimWSLeeJCChungJHChoiCM. Neuroendocrine differentiation in acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor. Tuberculosis and Respiratory Disease 2013 75 95103. (https://doi.org/10.4046/trd.2013.75.3.95)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15

    VanoverbergheKLehen’kyiVThebaultSRaphaelMVanden AbeeleFSlomiannyCMariotPPrevarskayaN. Cytoskeleton reorganization as an alternative mechanism of store-operated calcium entry control in neuroendocrine-differentiated cells. PLoS ONE 2012 7 e45615. (https://doi.org/10.1371/journal.pone.0045615)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    ZhuYLiuCCuiYNadimintyNLouWGaoAC. Interleukin-6 induces neuroendocrine differentiation (NED) through suppression of RE-1 silencing transcription factor (REST). Prostate 2014 74 10861094. (https://doi.org/10.1002/pros.22819)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    DengXElzeyBDPoulsonJMMorrisonWBKoSCHahnNMRatiffTLHuCD. Ionizing radiation induces neuroendocrine differentiation of prostate cancer cells in vitro, in vivo and in prostate cancer patients. American Journal of Cancer Research 2011 1 834844.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18

    GradizRSilvaHCCarvalhoLBotelhoMFMota-PintoA. MIA PaCa-2 and PANC-1 – pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors. Scientific Reports 2016 6 21648. (https://doi.org/10.1038/srep21648)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19

    BurchardtTBurchardtMChenMWCaoYDe la TailleAShabsighAHayekODoraiTButtyanR. Transdifferentiation of prostate cancer cells to a NE cell phenotype, in vitro and in vivo. Journal of Urology 1999 162 18001805. (https://doi.org/10.1016/S0022-5347(05)68241-9)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20

    CerasuoloMParisDIannottiFAMelckDVerdeRMazzarellaEMottaALigrestiA 2015 Neuroendocrine transdifferentiation in human prostate cancer cells: an integrated approach. Cancer Research 1999 75 29752986. (https://doi.org/10.1158/0008-5472.CAN-14-3830)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21

    JonesSEPalmerTM. Protein kinase A-mediated phosphorylation of RhoA on serine 188 triggers the rapid induction of a neuroendocrine-like phenotype in prostate cancer epithelial cells. Cellular Signaling 2012 24 15041514. (https://doi.org/10.1016/j.cellsig.2012.03.018)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22

    DeeblePDMurphyDJParsonsSJCoxME. Interleukin-6- and cyclic AMP-mediated signaling potentiates neuroendocrine differentiation of LNCaP prostate tumor cells. Molecular and Cellular Biology 2001 21 84718482. (https://doi.org/10.1128/MCB.21.24.8471-8482.2001)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23

    DayRSalzetM. The neuroendocrine phenotype, cellular plasticity and the search for genetic switches: redefining the diffuse neuroendocrine system. Neuroendocrinology Letters 2002 23 447451.

    • Search Google Scholar
    • Export Citation
  • 24

    BangYJPirniaFFangWGKangWKSartorOWhitesellLHaMJTsokosMSheahanMDNguyenPet al. 1994 Terminal NE differentiation of human prostate carcinoma cells in response to increased intracellular cAMP. PNAS 91 55305534. (https://doi.org/10.1073/pnas.91.12.5330)

    • Search Google Scholar
    • Export Citation
  • 25

    WangJEdeenKManzerRChangYWangSChenXFunkCJCosgroveGPFangXMasonRJ. Differentiated human alveolar epithelial cells and reversibility of their phenotype in vitro. American Journal of Respiratory: Cell and Molecular Biology 2007 36 661668. (https://doi.org/10.1165/rcmb.2006-0410OC)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26

    CoxMEDeeblePDLakhaniSParsonsSJ. Acquisition of neuroendocrine characteristics by prostate tumor cells is reversible: implications for prostate cancer progression. Cancer Research 1999 59 38213830.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27

    BosJ. EPAC: a new cAMP target and new avenues in cAMP research. Nature Reviews: Molecular Cell Biology 2003 4 733738. (https://doi.org/10.1038/nrm1197)

  • 28

    EmeryACEidenMVEidenLE. Separate cyclic AMP sensors for neuritogenesis, growth arrest, and survival of neuroendocrine cells. Journal of Biological Chemistry 2014 289 1012610139. (https://doi.org/10.1074/jbc.M113.529321)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29

    RavniAVaudryDGerdinMJEidenMVFalluel-MorelAGonzalezBJVaudryHEidenLE. A cAMP-dependent, protein kinase A-independent signaling pathway mediating neuritogenesis through Egr1 in PC12 cells. Molecular Pharmacology 2008 73 16881708. (https://doi.org/10.1124/mol.107.044792)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30

    LugnierC. Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents. Pharmacology and Therapeutics 2006 109 366398. (https://doi.org/10.1016/j.pharmthera.2005.07.003)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31

    CorradiniEMartinNScholtenA. Charting the interactome of PDE3A in human cells using an IBMX based chemical proteomics approach. Molecular Biosystems 2015 11 27862797. (https://doi.org/10.1039/C5MB00142K)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 32

    LuykenaarKDWelshDG. Activators of the PKA and PKG pathways attenuate RhoA-mediated suppression of the KDR current in cerebral arteries. American Journal of Physiology: Heart and Circulatory Physiology 2007 292 H2654H2663. (https://doi.org/10.1152/ajpheart.01255.2006)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33

    WangJEdeenKManzerRChangYWangSChenXFunkCJCosgroveGPFangXMasonRJ. Differentiated human alveolar epithelial cells and reversibility of their phenotype in vitro. American Journal of Respiratory Cell and Molecular Biology 2007 36 661668. (https://doi.org/10.1165/rcmb.2006-0410OC)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 34

    CoxMEDeeblePDBissonetteEAParsonsSJ. Activated 3′,5′-cyclic AMP-dependent protein kinase is sufficient to induce neuroendocrine-like differentiation of the LNCaP prostate tumor cell line. Journal of Biological Chemistry 2000 275 1381213818. (https://doi.org/10.1074/jbc.275.18.13812)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35

    OldenburgerAMaarsinghHSchmidtM. Multiple facets of cAMP signalling and physiological impact: cAMP compartmentalization in the lung. Pharmaceuticals 2012 5 12911331. (https://doi.org/10.3390/ph5121291)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36

    EmeryACXuWEidenMVEidenLE. Guanine nucleotide exchange factor Epac2-dependent activation of the GTP-binding protein Rap2A mediates cAMP-dependent growth arrest in neuroendocrine cells. Journal of Biological Chemistry 2017 292 1222012231. (https://doi.org/10.1074/jbc.M117.790329)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37

    ParkJYJuhnnYS. cAMP signaling increases histone deacetylase 8 expression via the Epac2-Rap1A-Akt pathway in H1299 lung cancer cells. Experimental and Molecular Medicine 2017 49 e297. (https://doi.org/10.1038/emm.2016.152)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38

    YuanTCVeeramaniSLinMF. Neuroendocrine-like prostate cancer cells: neuroendocrine transdifferentiation of prostate adenocarcinoma cells. Endocrine-Related Cancer 2007 14 531547. (https://doi.org/10.1677/ERC-07-0061)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 39

    ParkKHParkHJShinKSChoiHSKaiMLeeMK. Modulation of PC12 cell viability by forskolin-induced cyclic AMP levels through ERK and JNK pathways: an implication for L-DOPA-induced cytotoxicity in nigrostriatal dopamine neurons. Toxicological Sciences 2012 128 247257. (https://doi.org/10.1093/toxsci/kfs139)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40

    Mouillet-RichardsSMutelVLoricSTournoisCLaunayJMKellermannO. Regulation by neurotransmitter receptors of serotonergic or catecholaminergic neuronal cell differentiation. Journal of Biological Chemistry 2000 275 91869192. (https://doi.org/10.1074/jbc.275.13.9186)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41

    ZandeeWTKampKvan AdrichemRCFeeldersRADe HerderWW. Effect of hormone secretory syndromes on neuroendocrine tumor prognosis. Endocrine-Related Cancer 2017 24 R261R274. (https://doi.org/10.1530/ERC-16-0538)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42

    SunSQJiangCGLinYJinYLHuangPL. Enhanced T cell immunity by B7-H downregulation in nonsmall-cell lung cancer cell lines. Journal of International Medical Research 2012 40 497506. (https://doi.org/10.1177/147323001204000211)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43

    WangHZhangGDaiJMinJ. Immune evasion of human lung carcinoma cell A549 suppressed by human lymphoma cell Jurkat via Fas/FasL pathway. Chinese Journal of Lung Cancer 2010 13 681685. (https://doi.org/10.3779/j.issn.1009-3419.2010.07.05)

    • Search Google Scholar
    • Export Citation
  • 44

    AuneTMMcGrathKMSarrTBombaraMPKelleyKA. Expression of 5HT1A receptors on activated Human T cells. Journal of Immunology 1993 151 11751183.

    • Search Google Scholar
    • Export Citation
  • 45

    YinJAlbertRHTretiakovaAPJamesonBA. 5-HT(1B) receptors play a prominent role in the proliferation of T-lymphocytes. Journal of Neuroimmunolology 2006 181 6881. (https://doi.org/10.1016/j.jneuroim.2006.08.004)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46

    VegaLdeLMunozECalzadoMALiebKCandelario-JalilEGschaidmeirHFarberLMuellerWStratzTFiebichBL. The 5-HT3 receptor antagonist tropisetron inhibits T cell activation by targeting the calcineurin pathway. Biochemical Pharmacology 2005 70 369380. (https://doi.org/10.1016/j.bcp.2005.04.031)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 47

    ArreolaRBecerril-VillanuevaECruz-FuentesCVelasco-VelazquezMAGarces-AlvarezMEHurtado-AlvaradoGQuintero-FabianSPavonL. Immunomodulatory effects mediated by serotonin. Journal of Immunology Research 2015 2015 354957. (https://doi.org/10.1155/2015/354957)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 48

    BerumenLCRodriguezAMilediRGarcia-AlcocerG. Serotonin receptors in hippocampus. Scientific World Journal 2012 2012 823493. (http://doi.org/10.1100/2012/823493)

    • Search Google Scholar
    • Export Citation
  • 49

    ZhaoWHuangYLiuZCaoBBPeongYPQiuYH. Dopamine receptors modulate cytotoxicity of NK cells via cAMP PKA CREB signaling pathway. PLoS ONE 2013 8 111. (https://doi.org/10.1371/journal.pone.0065860)

    • Search Google Scholar
    • Export Citation

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    Effect of cAMP-elevating agents on A549 cell morphology. A549 cells were treated for 24, 72 or 120 h with IBMX (0.5 mM), FSK (0.5 mM) or I + F. Bars on the photomicrographs correspond to 200 µm. Figures are representative of three independent experiments for triplicate. FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine.

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    Effect of cAMP-elevating agents on A549 proliferation rate. The proliferation of cells supplemented with FSK (0.5 mM), IBMX (0.5 mM) or I + F was assessed every 24 h for 144 h. n = 3 for triplicates. One-way ANOVA with Dunnett’s post hoc test (*P < 0.01 compared with A549 control cells). FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine.

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    Effect of cAMP-elevating agents on neuroendocrine biomarker expression of A549 cell line. A549 cells were exposed to IBMX (0.5 mM), FSK (0.5 mM) or I + F for 120 h. (A) The level of neuroendocrine marker CgA was evaluated using flow cytometry. (B) The results are expressed as the percentage of positive cells to CgA. n = 3 for triplicates. One-way ANOVA with Tukey’s post hoc test. Bars with different letters represent statistical significance (P < 0.01). CgA, chromogranin A; FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine.

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    Effect of NED on the pattern of biogenic amine secretion. The A549 cells were exposed to cAMP-elevating agents for 120 h for transdifferentiation. Biogenic amines were then quantified using HPLC in the samples A549CTRL, A549NED (IBMX), A549NED (FSK) and A549 (NED)(i + f) and compared to the standard mixture of biogenic amines: NA, (A) AD, (B) DA and (C) 5-HT. The experiments were repeated thrice with independent samples. ND, not detectable. n = 3 for triplicates. Values are expressed as mean ± error. One-way ANOVA with Tukey’s post hoc test. Bars with different letters represent statistical significance (P < 0.01). DA, dopamine; AD, adrenaline; FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine; NED, neuroendocrine differentiation; NA, nonadrenaline; 5-HT, serotonin.

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    Cell morphology with neuroendocrine differentiation (NED) after withdrawal (WD) of cAMP-elevating agents. The A549 cells were exposed to cAMP-elevating agents for 120 h for transdifferentiation. Cell morphology was assessed 24 and 48 h after WD. Bars on the photomicrographs correspond to 200 µm. Figures are representative of three independent experiments for triplicate.

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    Direct cytolysis in co-cultures of T:C. CTLs (Jurkat) were tested with A549 cells in co-culture. The percentage of direct cytolysis was evaluated as % fluorescence release at (A) different time points and with (B) control and transdifferentiated A549 cell lines. n = 3 for triplicates. One-way ANOVA with Dunnett’s post hoc test (*P < 0.01 compared with A549 control cells; ***P < 0.001 compared with A549 control cells). CTLs, Cytotoxic T lymphocytes; T:C, target and cytotoxic cell lines.

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    Effect of NED on the viability of 6 h co-cultures of T:C. Previously, A549 cells were supplemented with cAMP-elevating agents for NED. A549CTRL or A549NED were tested against CTLs (Jurkat) for 6 h in co-culture. The viability of target and cytotoxic cells was separately tested using the MTT assay. n = 2 for quadruplicates. Two-way ANOVA with Dunnett’s post hoc test (***P < 0.01 compared with A549; ****P < 0.001 compared to every group of co-culture (A549:Jurkat). CTLs, Cytotoxic T lymphocytes; NED, neuroendocrine differentiation; T:C, target and cytotoxic cell lines.

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    Effect of the acquisition of NED on the viability of the cytotoxic cell line (Jurkat). Previously, the medium of A549 cells (CTRL and NED) was conditioned for 48 h and collected. (A) The percentage of proliferation (trypan blue) and (B) percentage of cell viability (MTT assay) of cytotoxic cells were evaluated at 24 and 48 h. n = 3 per triplicates. Two-way ANOVA with Tukey’s post hoc test. Bars with different letters represent statistical significance (P < 0.01 compared with A549 control cells). NED, neuroendocrine differentiation.

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    Antiproliferative effect of 5-HT on the Jurkat cell line. Cells were treated with 5-HT (from 1 × 10−12 to 1 × 10−3 M) alone and with PHA (20 µL/mL) for (A) 24 and (B) 48 h for a proliferation assay with trypan blue. (C) Representative immunoblots showing the relative expression of 5-HT5a and 5-HT7 receptors on Jurkat T cells. Blots were stripped and re-tested for B-actin to ensure that comparable quantities of protein were analysed. n = 3 for triplicates. Values are expressed as mean ± error. Two-way ANOVA with Tukey’s post hoc test. Bars with different letters represent statistical significance (P < 0.01 compared with A549 control cells). PHA, phytohaemagglutinin A; 5-HT, serotonin.

  • View in gallery

    Proposed mechanism of neuroendocrine differentiation by cAMP-elevating agents. Solid line indicates the reported pathways in the lungs, and the dashed line indicates the pathways reported in other tissues. ASCL1, achaete-scute homolog like 1; CgA, chromogranin A; Epac1 and Epac2, guanine exchange factor; Egr1, early growth response protein 1; FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine; ND1, NeuroD1; NCAM1, neural cell adhesion molecule 1; PDE, phosphodiesterase; PKA, protein kinase A; REST1, repressor element 1-silencing transcription factor; Rap1, small GTPase Ras-related protein; SYP, synaptophysin; (serine/threonine-protein kinase) B-Raf; MEK, ERK1/2 and p38, mitogen activated protein kinases;Vl2, Vilin2.

  • 1

    SongHYaoELinCGacayanRChenMHChuangPT. Functional characterization of pulmonary neuroendocrine cells in lung development, injury, and tumorigenesis. PNAS 2012 109 1753117536. (https://doi.org/10.1073/pnas.1207238109)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2

    RemarkRBeckerCGomezJEDamotteDDieu-NosjeanMCSautes-FridmanCFridmanWHPowellCAAltorkiNKMeradMet al. The non-small cell lung cancer immune contexture. A major determinant of tumor characteristics and patient outcome. American Journal of Respiratory and Critical Care Medicine 2015 191 377390. (https://doi.org/10.1164/rccm.201409-1671PP)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3

    StovoldRMeredithSLBryantJLBaburMWilliamsKJDeanEJDiveCBlackhallFHWhiteA. Neuroendocrine and epithelial phenotypes in small-cell lung cancer: implications for metastasis and survival in patients. British Journal of Cancer 2013 108 17041711. (https://doi.org/10.1038/bjc.2013.112)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4

    YazawaT. Recent advances in histogenesis research of lung neuroendocrine cancers: evidence obtained from functional analyses of primitive neural/neuroendocrine cell-specific transcription factors. Pathology International 2015 65 277285. (https://doi.org/10.1111/pin.12267)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5

    KloppelG. Tumour biology and histopathology of neuroendocrine tumours. Best Practice and Research Clinical Endocrinology and Metabolism 2007 21 1531. (https://doi.org/10.1016/j.beem.2007.01.004)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6

    CampanaDNoriFPiscitelliLMorselli-LabateAMPezzilliRCorinaldesiRTomassettiP. Chromogranin A: is it a useful marker of neuroendocrine tumors? Journal of Clinical Oncology 2007 25 19671973. (https://doi.org/10.1200/JCO.2006.10.1535)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7

    PetrovićMMitrovicSStankovicVJurisicVAtkinsonHD. Neuroendocrine markers–useful predictors of therapeutic responses in non-resectable non-small cell lung cancer. Laboratory Medicine 2012 43 610. (https://doi.org/10.1309/LM0IKAO8Z0MUTVFE)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8

    ModlinIMObergKTaylorADrozdovIBodeiLKiddM. Neuroendocrine tumor biomarkers: current status and perspectives. Neuroendocrinology 2014 100 265277. (https://doi.org/10.1159/000368363)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9

    LindholmDPObergK. Biomarkers and molecular imaging in gastroenteropancreatic neuroendocrine tumors. Hormone and Metabolic Research 2011 43 832837. (https://doi.org/10.1055/s-0031-1287794)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10

    ObergKModlinIMDe HerderWPavelMKlimstraDFrillingAMetzDCHeaneyAKwekkeboomDStrosbergJet al. Consensus on biomarkers for neuroendocrine tumour disease. Lancet Oncology 2015 16 435446. (https://doi.org/10.1016/S1470-2045(15)00186-2)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11

    PavonLBottassoOBesedoskyHLoriaRVelasco-VelazquezMA. Clinical and experimental immunomodulation 2014. Journal of Immunology Research 2015 2015 12. (https://doi.org/10.1155/2015/758328)

    • Search Google Scholar
    • Export Citation
  • 12

    PernicováZSlabákováEFedrRŠimečkováŠJarošJSuchánkováTBouchalJKharaishviliGKrálMKozubíkAet al. The role of high cell density in the promotion of neuroendocrine transdifferentiation of prostate cancer cells. Molecular Cancer 2014 13 113129. (https://doi.org/10.1186/1476-4598-13-113)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    WalkerGEAntonionoRJRossHJPaisleyTEOhY. Neuroendocrine-like differentiation of non-small cell lung carcinoma cells: regulation by cAMP and the interaction of mac25/IGFBP-rP1 and 25.1. Oncogene 2006 25 19431954. (https://doi.org/10.1038/sj.onc.1209213)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    ChangYKimSYChoiYJSoKSRhoJKKimWSLeeJCChungJHChoiCM. Neuroendocrine differentiation in acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor. Tuberculosis and Respiratory Disease 2013 75 95103. (https://doi.org/10.4046/trd.2013.75.3.95)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15

    VanoverbergheKLehen’kyiVThebaultSRaphaelMVanden AbeeleFSlomiannyCMariotPPrevarskayaN. Cytoskeleton reorganization as an alternative mechanism of store-operated calcium entry control in neuroendocrine-differentiated cells. PLoS ONE 2012 7 e45615. (https://doi.org/10.1371/journal.pone.0045615)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    ZhuYLiuCCuiYNadimintyNLouWGaoAC. Interleukin-6 induces neuroendocrine differentiation (NED) through suppression of RE-1 silencing transcription factor (REST). Prostate 2014 74 10861094. (https://doi.org/10.1002/pros.22819)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    DengXElzeyBDPoulsonJMMorrisonWBKoSCHahnNMRatiffTLHuCD. Ionizing radiation induces neuroendocrine differentiation of prostate cancer cells in vitro, in vivo and in prostate cancer patients. American Journal of Cancer Research 2011 1 834844.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18

    GradizRSilvaHCCarvalhoLBotelhoMFMota-PintoA. MIA PaCa-2 and PANC-1 – pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors. Scientific Reports 2016 6 21648. (https://doi.org/10.1038/srep21648)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19

    BurchardtTBurchardtMChenMWCaoYDe la TailleAShabsighAHayekODoraiTButtyanR. Transdifferentiation of prostate cancer cells to a NE cell phenotype, in vitro and in vivo. Journal of Urology 1999 162 18001805. (https://doi.org/10.1016/S0022-5347(05)68241-9)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20

    CerasuoloMParisDIannottiFAMelckDVerdeRMazzarellaEMottaALigrestiA 2015 Neuroendocrine transdifferentiation in human prostate cancer cells: an integrated approach. Cancer Research 1999 75 29752986. (https://doi.org/10.1158/0008-5472.CAN-14-3830)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21

    JonesSEPalmerTM. Protein kinase A-mediated phosphorylation of RhoA on serine 188 triggers the rapid induction of a neuroendocrine-like phenotype in prostate cancer epithelial cells. Cellular Signaling 2012 24 15041514. (https://doi.org/10.1016/j.cellsig.2012.03.018)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22

    DeeblePDMurphyDJParsonsSJCoxME. Interleukin-6- and cyclic AMP-mediated signaling potentiates neuroendocrine differentiation of LNCaP prostate tumor cells. Molecular and Cellular Biology 2001 21 84718482. (https://doi.org/10.1128/MCB.21.24.8471-8482.2001)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23

    DayRSalzetM. The neuroendocrine phenotype, cellular plasticity and the search for genetic switches: redefining the diffuse neuroendocrine system. Neuroendocrinology Letters 2002 23 447451.

    • Search Google Scholar
    • Export Citation
  • 24

    BangYJPirniaFFangWGKangWKSartorOWhitesellLHaMJTsokosMSheahanMDNguyenPet al. 1994 Terminal NE differentiation of human prostate carcinoma cells in response to increased intracellular cAMP. PNAS 91 55305534. (https://doi.org/10.1073/pnas.91.12.5330)

    • Search Google Scholar
    • Export Citation
  • 25

    WangJEdeenKManzerRChangYWangSChenXFunkCJCosgroveGPFangXMasonRJ. Differentiated human alveolar epithelial cells and reversibility of their phenotype in vitro. American Journal of Respiratory: Cell and Molecular Biology 2007 36 661668. (https://doi.org/10.1165/rcmb.2006-0410OC)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26

    CoxMEDeeblePDLakhaniSParsonsSJ. Acquisition of neuroendocrine characteristics by prostate tumor cells is reversible: implications for prostate cancer progression. Cancer Research 1999 59 38213830.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27

    BosJ. EPAC: a new cAMP target and new avenues in cAMP research. Nature Reviews: Molecular Cell Biology 2003 4 733738. (https://doi.org/10.1038/nrm1197)

  • 28

    EmeryACEidenMVEidenLE. Separate cyclic AMP sensors for neuritogenesis, growth arrest, and survival of neuroendocrine cells. Journal of Biological Chemistry 2014 289 1012610139. (https://doi.org/10.1074/jbc.M113.529321)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29

    RavniAVaudryDGerdinMJEidenMVFalluel-MorelAGonzalezBJVaudryHEidenLE. A cAMP-dependent, protein kinase A-independent signaling pathway mediating neuritogenesis through Egr1 in PC12 cells. Molecular Pharmacology 2008 73 16881708. (https://doi.org/10.1124/mol.107.044792)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30

    LugnierC. Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents. Pharmacology and Therapeutics 2006 109 366398. (https://doi.org/10.1016/j.pharmthera.2005.07.003)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31

    CorradiniEMartinNScholtenA. Charting the interactome of PDE3A in human cells using an IBMX based chemical proteomics approach. Molecular Biosystems 2015 11 27862797. (https://doi.org/10.1039/C5MB00142K)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 32

    LuykenaarKDWelshDG. Activators of the PKA and PKG pathways attenuate RhoA-mediated suppression of the KDR current in cerebral arteries. American Journal of Physiology: Heart and Circulatory Physiology 2007 292 H2654H2663. (https://doi.org/10.1152/ajpheart.01255.2006)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33

    WangJEdeenKManzerRChangYWangSChenXFunkCJCosgroveGPFangXMasonRJ. Differentiated human alveolar epithelial cells and reversibility of their phenotype in vitro. American Journal of Respiratory Cell and Molecular Biology 2007 36 661668. (https://doi.org/10.1165/rcmb.2006-0410OC)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 34

    CoxMEDeeblePDBissonetteEAParsonsSJ. Activated 3′,5′-cyclic AMP-dependent protein kinase is sufficient to induce neuroendocrine-like differentiation of the LNCaP prostate tumor cell line. Journal of Biological Chemistry 2000 275 1381213818. (https://doi.org/10.1074/jbc.275.18.13812)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35

    OldenburgerAMaarsinghHSchmidtM. Multiple facets of cAMP signalling and physiological impact: cAMP compartmentalization in the lung. Pharmaceuticals 2012 5 12911331. (https://doi.org/10.3390/ph5121291)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36

    EmeryACXuWEidenMVEidenLE. Guanine nucleotide exchange factor Epac2-dependent activation of the GTP-binding protein Rap2A mediates cAMP-dependent growth arrest in neuroendocrine cells. Journal of Biological Chemistry 2017 292 1222012231. (https://doi.org/10.1074/jbc.M117.790329)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37

    ParkJYJuhnnYS. cAMP signaling increases histone deacetylase 8 expression via the Epac2-Rap1A-Akt pathway in H1299 lung cancer cells. Experimental and Molecular Medicine 2017 49 e297. (https://doi.org/10.1038/emm.2016.152)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38

    YuanTCVeeramaniSLinMF. Neuroendocrine-like prostate cancer cells: neuroendocrine transdifferentiation of prostate adenocarcinoma cells. Endocrine-Related Cancer 2007 14 531547. (https://doi.org/10.1677/ERC-07-0061)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 39

    ParkKHParkHJShinKSChoiHSKaiMLeeMK. Modulation of PC12 cell viability by forskolin-induced cyclic AMP levels through ERK and JNK pathways: an implication for L-DOPA-induced cytotoxicity in nigrostriatal dopamine neurons. Toxicological Sciences 2012 128 247257. (https://doi.org/10.1093/toxsci/kfs139)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40

    Mouillet-RichardsSMutelVLoricSTournoisCLaunayJMKellermannO. Regulation by neurotransmitter receptors of serotonergic or catecholaminergic neuronal cell differentiation. Journal of Biological Chemistry 2000 275 91869192. (https://doi.org/10.1074/jbc.275.13.9186)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41

    ZandeeWTKampKvan AdrichemRCFeeldersRADe HerderWW. Effect of hormone secretory syndromes on neuroendocrine tumor prognosis. Endocrine-Related Cancer 2017 24 R261R274. (https://doi.org/10.1530/ERC-16-0538)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42

    SunSQJiangCGLinYJinYLHuangPL. Enhanced T cell immunity by B7-H downregulation in nonsmall-cell lung cancer cell lines. Journal of International Medical Research 2012 40 497506. (https://doi.org/10.1177/147323001204000211)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43

    WangHZhangGDaiJMinJ. Immune evasion of human lung carcinoma cell A549 suppressed by human lymphoma cell Jurkat via Fas/FasL pathway. Chinese Journal of Lung Cancer 2010 13 681685. (https://doi.org/10.3779/j.issn.1009-3419.2010.07.05)

    • Search Google Scholar
    • Export Citation
  • 44

    AuneTMMcGrathKMSarrTBombaraMPKelleyKA. Expression of 5HT1A receptors on activated Human T cells. Journal of Immunology 1993 151 11751183.

    • Search Google Scholar
    • Export Citation
  • 45

    YinJAlbertRHTretiakovaAPJamesonBA. 5-HT(1B) receptors play a prominent role in the proliferation of T-lymphocytes. Journal of Neuroimmunolology 2006 181 6881. (https://doi.org/10.1016/j.jneuroim.2006.08.004)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46

    VegaLdeLMunozECalzadoMALiebKCandelario-JalilEGschaidmeirHFarberLMuellerWStratzTFiebichBL. The 5-HT3 receptor antagonist tropisetron inhibits T cell activation by targeting the calcineurin pathway. Biochemical Pharmacology 2005 70 369380. (https://doi.org/10.1016/j.bcp.2005.04.031)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 47

    ArreolaRBecerril-VillanuevaECruz-FuentesCVelasco-VelazquezMAGarces-AlvarezMEHurtado-AlvaradoGQuintero-FabianSPavonL. Immunomodulatory effects mediated by serotonin. Journal of Immunology Research 2015 2015 354957. (https://doi.org/10.1155/2015/354957)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 48

    BerumenLCRodriguezAMilediRGarcia-AlcocerG. Serotonin receptors in hippocampus. Scientific World Journal 2012 2012 823493. (http://doi.org/10.1100/2012/823493)

    • Search Google Scholar
    • Export Citation
  • 49

    ZhaoWHuangYLiuZCaoBBPeongYPQiuYH. Dopamine receptors modulate cytotoxicity of NK cells via cAMP PKA CREB signaling pathway. PLoS ONE 2013 8 111. (https://doi.org/10.1371/journal.pone.0065860)

    • Search Google Scholar
    • Export Citation