PMCID string | Title string | Sentences string |
|---|---|---|
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Another major neuronal cholinergic system is the enteric nervous system (ENS), where cholinergic neurons regulate gastrointestinal motility and secretion. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | It is estimated that over 60% of ENS neurons are cholinergic, and emerging evidence suggests that these circuits are affected in various neurodegenerative diseases . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Non-neuronal cholinergic cells include a diverse array of ChAT-expressing cell types such as epithelial, mesothelial, endothelial, and immune cells . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Notably, ChAT expression increases in lymphocytes and macrophages under inflammatory conditions, forming part of the “cholinergic anti-inflammatory pathway,” in which ACh modulates cytokine release and inflammation . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Impairment of this pathway in aging and disease may contribute to excessive inflammatory responses. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Increasing evidence also points to upregulation of cholinergic machinery in various cancers, particularly small cell lung cancer (SCLC) and colon cancer . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In these contexts, ACh may function as an autocrine growth factor , and nicotinic receptor antagonists have been proposed as potential anticancer agents . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Although ChAT is generally considered a soluble cytosolic enzyme, reports have described intracellular membrane-bound forms of ChAT in synaptosomes and cholinergic nerve terminals across several species . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | As an integral membrane protein, ChAT may exhibit distinct biochemical properties compared to its cytosolic counterpart . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In a recent in vitro study, we demonstrated that human ChAT readily embeds into micellar nanoparticles, resulting in a dramatic 10-fold increase in its catalytic activity . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | ChAT also retains a nuclear localization signal, suggesting potential translocation to the nucleus, although its nuclear function remains unclear . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In this study, we investigated the expression of ChAT and related cholinergic markers in four human tumor cell lines: neuroblastoma (SH-SY5Y), lung adenocarcinoma (A549), and two small cell lung cancer lines (H69 and H82) . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Our aim was to characterize and establish robust cholinergic model systems for future screening and in vitro evaluation of ChAT ligands, including inhibitors and activators previously reported by us , with potential applications as anti-tumor agents. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Additionally, we sought to determine whether ChAT is localized extracellularly as a membrane-anchored enzyme in these tumor cells, as we previously observed in human spermatozoa . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | We further examined the presence and localization—surface versus intracellular—of key components of the cholinergic system across the four cancer cell lines. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Our findings provide novel insights into cholinergic signaling mechanisms that may be relevant to both neurodegeneration and carcinoma pathology. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The small cell lung carcinoma (SCLC) cell lines H69 and H82, the lung adenocarcinoma cell line A549, and the neuroblastoma cell line SH-SY5Y were evaluated for ChAT cell surface expression without fixation or permeabilization (Figure 1). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Cells were simultaneously incubated with both the anti-ChAT antibody and the Live/Dead dye for surface flow cytometry. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This was done to ensure that plasma membrane integrity was not compromised, particularly since two of the cell lines (SH-SY5Y and A549) are adherent and required trypsin treatment to form a suspension. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The Live/Dead dye selectively stains cells with compromised membranes, and the resulting dot plots in Figure 1 show negligible signal in quadrants Q1 and Q2, indicating that SH-SY5Y cells retained intact plasma membranes following trypsin treatment. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The same was observed for the other three cell lines, including A549, which, like SH-SY5Y, grows as an adherent cell line. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Interestingly, under these conditions, surface ChAT localization was exclusively detected in the SH-SY5Y neuroblastoma cell line, but not in the other cell lines. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This surface localization was abolished when the antibody was pre-blocked with recombinant ChAT protein, confirming the specificity of the staining (Figure 1). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Next, whole-cell staining was performed using fixation and permeabilization to assess the total ChAT expression within the cells, encompassing both intracellular and extracellular pools of ChAT. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Positive intracellular ChAT expression and localization were observed across all four tested cell lines (Figure 2), consistent with previous reports. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | ChAT staining using the pre-blocked antibody with recombinant ChAT protein significantly reduced the signal across all cell lines, including H69 and A549 (as shown in Supplementary Figure S1), further confirming the specificity of the antibody for ChAT as the target protein. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Notably, since the intracellular staining approach involves cell permeabilization to allow antibody entry, the resulting signal reflects total ChAT expression, as surface membrane-bound ChAT proteins remain accessible to the antibody. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Therefore, comparing the proportion of cells positive for surface ChAT staining (66 ± 1%) with those positive for total ChAT staining (79 ± 3%) indicates that a major fraction of ChAT protein in SH-SY5Y neuroblastoma cells is localized extracellularly (compare dot plots in Figure 1 vs. Figure 2). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In contrast, in the lung carcinoma cell lines H69, H82, and A549, ChAT staining was only observed following permeabilization, suggesting that ChAT is primarily localized intracellularly in these cells (compare Figure 1 vs. Figure 2). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Notably, the relative fluorescence intensity (RFI) of the anti-ChAT antibody compared to the blocked antibody in H82, H69, and A549 is shown in Figure S1. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The results indicate that antibody blocking was reasonably effective in H69 and A549 cells, although not as efficient as in H82 or SH-SY5Y cells. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Measuring ACh and choline in an experiment is crucial for assessing the functionality of the cellular cholinergic machinery, as these two molecules are intimately linked within a functional signaling circuit. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Their concurrent quantification provides an important measure of the activity and homeostatic state of the cholinergic system, including its potential auto- and paracrine regulatory mechanisms. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In particular, it can reveal whether ChAT protein is functionally intact and whether the cells are capable of synthesizing and releasing ACh. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | We therefore measured the biosynthesis and release levels of ACh in the four cancer cell lines using HPLC-MS/MS analyses. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The analysis was performed on both cell lysates and conditioned media from cultured cells, the latter to assess ACh release. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Cell-free complete medium (RPMI), with or without FBS, served as negative controls. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To preserve biosynthesized ACh during sample preparation, neostigmine—an inhibitor of both AChE and BChE—was used in parallel. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The HPLC-MS/MS results, presented in the upper panel of Figure 3, confirmed that all cell lines were capable of synthesizing ACh, as detected in the cell lysates. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To normalize and compare ACh and choline levels across cell lines, total protein concentrations in the samples were used. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The protein concentration in the cell lysates was 0.58 ± 0.2 mg/mL and did not differ significantly among the cell lines (SH-SY5Y = 0.56 ± 0.10; H82 = 0.52 ± 0.16; H69 = 0.59 ± 0.20; A549 = 0.67 ± 0.28 mg/mL), confirming that the data reflect comparable cell numbers. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Analysis of the conditioned media indicated that SH-SY5Y neuroblastoma cells and the SCLC cell line H82 released substantial amounts of ACh into the medium compared to controls (Figure 3, upper panel, orange bars). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In contrast, only low levels of ACh were detected in the conditioned media from H69 and A549 cells (Figure 3, upper panel, orange bars). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In summary, all tested cancer cell lines were capable of storing and releasing synthesized ACh. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | We further measured choline levels in SH-SY5Y cells and the three lung cancer cell lines, as well as in their conditioned media, using HPLC-MS/MS (Figure 3, lower panel). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The concentration of choline in unused culture medium was determined to be 1.8 ± 0.3 μM. Choline levels were generally much higher in the conditioned media than in the cell lysates, indicating that the culture medium was the primary source of choline and that the cells were capable of choline uptake, likely for ACh biosynthesis (Figure 3, lower panel). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This also suggests that all tested cell lines express either a specific choline transporter or a general organic cation transporter, or both . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The presence or absence of neostigmine had no significant effect on choline levels. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This likely reflects the fact that choline is not metabolized by either AChE or BChE. Notably, the release and hydrolysis of ACh into choline is unlikely to have significantly influenced the observed choline levels, given that the measured concentrations of ACh were in the nanomolar range (Figure 3, upper panel), which is negligible compared to the micromolar levels of choline detected (Figure 3, lower panel). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In addition to ChAT, we performed flow cytometric analyses to assess the expression and localization of selected components of the cholinergic machinery, namely the ACh-hydrolyzing enzymes AChE and BChE, as well as the α7 nicotinic AChR (α7 AChR) and the M1-subtype of muscarinic AChR (M1 AChR) in SH-SY5Y neuroblastoma cells. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The results are shown in Figure 4. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Surface flow cytometric analyses indicated that BChE, α7 AChR, and M1 AChR were present at the cell surface of SH-SY5Y neuroblastoma cells, while only minimal levels of AChE were detected extracellularly or at the cell surface (Figure 4; see also Figure 6). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | These findings partially deviate from prevailing assumptions in the field. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | For instance, the low surface/extracellular staining of AChE, in contrast to the high BChE staining observed in neuroblastoma cells, challenges the canonical view that AChE is the primary membrane-anchored extracellular enzyme responsible for ACh hydrolysis within synapses . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Additionally, BChE is generally regarded as a secreted, soluble enzyme, whereas the current surface flow cytometric data clearly demonstrate that BChE is a major surface protein in all four cell lines (Figure 4). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Furthermore, all cell lines exhibited positive surface staining for α7 and M1 AChRs, supporting the notion that ACh exerts autocrine activity in these cells . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | We next performed flow cytometric analyses following membrane permeabilization of the four cell lines. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | These analyses are expected to detect the total expression of cholinergic markers, encompassing both surface and intracellular protein pools. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The results are presented in Figure 5, which demonstrate that SH-SY5Y neuroblastoma cells were clearly positive for all assessed cholinergic markers (Figure 5A). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Essentially, the same pattern was observed in the lung cancer cell lines H69, H82, and A549 (Figure 5B). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Notably, fluorescence intensities increased significantly in all cell lines following membrane permeabilization compared to surface staining alone. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This is most likely due to the permeabilization step allowing antibodies access to all available binding targets, including those in intracellular compartments. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Furthermore, a comparison of fluorescence intensity profiles for AChE and BChE revealed a markedly higher expression of BChE relative to AChE (see the corresponding fluorescence intensity shifts in the histogram panels in Figure 5), corroborating the observations from the flow cytometric surface expression analyses of these enzymes (Figure 4). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | However, as antibody affinities may differ, this finding should be interpreted with caution. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Similarly, the clear increase in intracellular fluorescence signals for α7- and M1 AChRs compared to non-permeabilized neuroblastoma cells supports the existence of an intracellular pool of these receptors, likely reflecting ongoing synthesis, intracellular trafficking, or receptor internalization processes. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To validate the aforenoted differences in extracellular versus intracellular levels of cholinergic markers in SH-SY5Y neuroblastoma cells, we quantified protein expression using relative fluorescence intensity (RFI), following a previously established protocol , as detailed in Section 4.3 of the Materials and Methods. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The RFI data (Figure 6A,B) confirmed that ChAT is predominantly localized at the cell surface, indicating its extracellular anchoring, while AChE exhibited minimal expression in both extracellular and intracellular compartments. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In contrast, BChE showed notable extracellular presence, although its major fraction was intracellular. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Similarly, both α7-nAChR and M1 mAChR were detected at the cell surface in appreciable amounts, yet their predominant localization was intracellular. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Importantly, with the exception of ChAT, the surface-localized fraction of each cholinergic protein represented only a minor proportion of its total cellular expression, with whole-cell levels exceeding surface expression by factors of 1.12 (ChAT), 2.56 (AChE), 45.85 (BChE), 36.70 (α7-nAChR), and 84.60 (M1 mAChR), respectively (Figure 6B). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The canonical view is that ChAT is a cytosolic enzyme responsible for synthesizing ACh. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | However, we recently reported extracellular localization of ChAT at the surface of human sperm using flow cytometry . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Interestingly, among the four examined cell lines, only the SH-SY5Y neuroblastoma cells exhibited surface ChAT expression (Figure 1). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To further investigate the observed phenomenon, we performed cell surface confocal microscopy targeting ChAT in the same SH-SY5Y neuroblastoma cell line. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Cells were stained under non-permeabilizing conditions to selectively visualize surface-expressed antigens, thereby excluding intracellular signals. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | For this analysis, three distinct anti-ChAT antibodies—different from the one employed in the flow cytometric assay—were independently utilized to validate the presence of surface-localized ChAT. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Confocal imaging of the non-permeabilized cells consistently revealed distinct surface-associated ChAT immunoreactivity with all three antibodies. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | These findings corroborate the flow cytometric data, providing convergent evidence for the extracellular localization of ChAT in SH-SY5Y cells (Figure 7, Figure S2A–C, and Supplementary 3D Videos S1, S3, and S5). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Importantly, the signals observed in these confocal analyses are expected to originate exclusively from surface-localized target proteins, as the cells were not permeabilized during staining. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | As previously noted, Live/Dead staining confirmed that trypsin treatment did not compromise membrane integrity, given that the dye selectively penetrates cells with damaged membranes. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | As shown in Figure 1, negligible fluorescence signals were detected in quadrants Q1 and Q2, indicating that SH-SY5Y cells retained intact plasma membranes following enzymatic treatment. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Nonetheless, to further ensure membrane integrity and eliminate any residual effects of trypsinization, cells were re-seeded directly onto microscopy slides and incubated for 24 h prior to staining, allowing full reattachment and potential membrane repair. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This precautionary step ensured that antibody binding was specific to surface antigens and not confounded by unintended membrane disruption. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Similarly, surface confocal microscopy analysis validated the presence of AChE, BChE, M1-mAChR, and α7-nAChR on the surface of SH-SY5Y neuroblastoma cells (Figure 7). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | These results indicate that SH-SY5Y cells express a functional cholinergic system at the cell surface, suggesting the presence of autocrine ACh activity. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Notably, the data suggest that BChE, rather than AChE, may be the predominant surface-anchored cholinesterase (Figure 7, Figure S2D, Figure S3D and Supplementary 3D-Videos S3 and S4 vs. S5 and S6, respectively). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This observation is in full agreement with the flow cytometry results (Figure 4 and Figure 6). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | We next performed whole-cell confocal analyses on permeabilized cells. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Confocal microscopy images are presented in Figure 8. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | For this analysis, as noted before, three distinct anti-ChAT antibodies—different from the one employed in the flow cytometric assay—were independently utilized to validate the presence ChAT (Figure S3A–C). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | These analyses fully reaffirmed the findings from the whole-cell flow cytometric analysis regarding the expression and cellular distribution of ChAT throughout the cells, including in the cytosol. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To ensure antibody specificity, control experiments were conducted using secondary antibodies alone, which yielded no detectable signal (Figure S4), thereby confirming the specificity of the observed staining. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Similar findings were observed for the M1 and α7 acetylcholine receptors (Figure 8) as well as for the acetylcholine-hydrolyzing enzymes BChE (Figure 8, Figure S3D, and Supplementary 3D-Video S3 vs. S4) and AChE (Figure 8, Supplementary 3D-Video S6 vs. S5). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Notably, signal intensities for all antibodies were markedly enhanced following cell permeabilization compared to non-permeabilized conditions, indicating substantial intracellular localization, in full agreement with the flow cytometric analysis (Figure 6). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This was particularly evident for AChE, which displayed a diffuse intracellular staining pattern post-permeabilization, in contrast to the punctate, dot-like surface expression seen in non-permeabilized cells (compare AChE micrographs in Figure 7 and Figure 8, as well as Supplementary 3D-Video S6 vs. S5). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | These results suggest that AChE expression in these cells is predominantly intracellular, with minimal surface localization. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Importantly, these observations are fully consistent with the flow cytometric data for AChE presented in Figure 4, Figure 5A and Figure 6. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To the best of our knowledge, this study provides the first compelling evidence for the presence of an extracellularly membrane-bound isoform of ChAT in the SH-SY5Y neuroblastoma cell line. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Such an isoform was not observed in the three lung carcinoma cell lines examined. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This was carefully confirmed through confocal microscopy analyses of surface-localized ChAT protein. |
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