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PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
The cells were then detached via trypsinization, pelleted by centrifugation, and washed with PBS.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
After cell counting, the pellets were homogenized using a steel bead homogenizer with a 25 s ON/5 s OFF cycle repeated twice, yielding 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
These lysates were centrifuged at 15,000 rpm for 10 min at 4 °C, and the resulting supernatants were transferred to fresh Eppendorf tubes.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
To precipitate proteins, 100 µL of pre-chilled acetonitrile (stored at −20 °C) was added to both the cell lysates and conditioned media, followed by vortexing until the solutions turned cloudy.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Samples were then centrifuged at 17,000× g for 20 min at 4 °C.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
The clarified supernatants were stored on dry ice until HPLC-MS/MS analysis.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
For quantification, 50 µL of each sample was transferred to injection vials, and 10 µL of a tuning solution containing 1 µg/mL acetylcholine-d9 chloride was added to each vial.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
As controls, samples of complete RPMI with FBS and RPMI without FBS (both without cells) were collected and processed 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
Immunofluorescence staining was performed on SH-SY5Y cells to visualize ChAT and other cholinergic markers.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Once cells reached near confluency, they were trypsinized for five minutes and seeded at a density of 50,000–70,000 cells per 35 mm dish onto 20 mm glass-bottom wells (P35G-1.5-20-C, MatTek Corporation, Ashland, MA, USA).
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 incubated for 24 h to ensure intact plasma membranes prior to surface staining.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Staining was conducted using a two-step optimized protocol described by Vernay and Cosson .
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Initially, cells were fixed in 3.7% paraformaldehyde for 10 min at 37 °C.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Following fixation, cells were placed on ice and incubated with 1% bovine serum albumin (BSA) for 45 min to block nonspecific antibody binding.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
For surface marker analysis, no permeabilization was performed.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
To assess whole-cell protein expression, a second fixation was carried out on ice, followed by permeabilization with 0.05% Triton X-100 (Merck) for 10 min before applying primary and secondary antibodies as described by Vernay and Cosson .
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Three different unconjugated anti-ChAT primary antibodies were used for confocal imaging: a monoclonal mouse anti-ChAT antibody (MAB3447, R&D Systems, Bio-Techne, Stockholm, Sweden), which targets a region between Ala2–Pro630 (Accession # NP_066266); a rabbit polyclonal anti-ChAT antibody (PAB14536, Abnova, Taipei, Taiwan); and another rabbit polyclonal anti-ChAT antibody (AB143, Millipore via Merck).
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Additional primary antibodies included mouse monoclonal anti-AChE (A-11, Santa Cruz), mouse monoclonal anti-BChE (D-5, Santa Cruz Biotechnology (Dallas, TX, USA)), rat monoclonal anti-α7 nicotinic AChR (Santa Cruz Biotechnology), and mouse monoclonal anti-M1 muscarinic AChR (Santa Cruz Biotechnology).
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
All primary antibodies were unconjugated and used at optimized dilutions ranging from 1:50 to 1:300.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Secondary antibodies were fluorophore-conjugated and included goat anti-mouse IgG (FITC, Invitrogen, 1:375), goat anti-rabbit IgG (Alexa Fluor 647, Invitrogen, 1:300), and goat anti-rat IgG (Alexa Fluor 647, Invitrogen, 1:300).
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Nuclear staining was performed using NucBlue DAPI reagent (Invitrogen), and mitochondrial labeling was achieved with Mitotracker Red CMXRos (Invitrogen).
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 was carried out using a Nikon Ti2 inverted microscope equipped with multipoint spinning disk and DeepSIM super-resolution capabilities.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
Z-stack images were captured using 405, 477, 545, and 637 nm lasers.
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 consistency across experiments, microscope settings—including exposure time—were kept constant for all samples, whether stained with primary and secondary antibodies (Figure 7 and Figure 8, Figures S2 and S3, and Supplementary 3D-Video images S1–S6) or with secondary antibodies alone (Figure S4).
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
In conclusion, our findings—consistent with growing evidence—indicate that a functional cholinergic phenotype is a common feature among several carcinoma cell lines, suggesting potential avenues for therapeutic exploitation.
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 identification of an extracellular, membrane-bound form of ChAT uniquely in neuroblastoma SH-SY5Y cells reveals a previously unrecognized mode of in situ ACh signaling.
PMC12608971
Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells
This novel observation warrants further investigation to elucidate its biological significance and potential implications in cancer cell communication and survival.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
1,25-Dihydroxyvitamin D3 is the body's active form of vitamin D and is also known as calcitriol.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
It has an effect on the body's immune system and is involved in the regulation of cell growth, differentiation, and apoptosis.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The effect of 1,25-Dihydroxyvitamin D3 on the viability of human neuroblastoma human neuroblastoma cell line cells was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide assay, the effect on migration was investigated by wound healing assay, and methyltransferase genes (DNA methyltransferase 3 alpha and DNA methyltransferase 3 beta) and phosphatase and tensin homolog gene expression levels were investigated by real time polymerase chain reaction method.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
As a result of the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide assay, 1,25-Dihydroxyvitamin D3 IC50:265.6 nM was found.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
It was observed that 100 and 265.6 nM applied to human neuroblastoma cell line cells had an antiproliferative effect.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In addition, DNA methyltransferase 3 alpha and DNA methyltransferase 3 beta genes, which are effective as methyltransferases, caused a change in expression levels (p<0.05), while phosphatase and tensin homolog expression levels did not change (p>0.05).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
1,25-Dihydroxyvitamin D3 showed antiproliferative, antimigratory activity on neuroblastoma human neuroblastoma cell line cells and decreased the expression levels of genes involved in methylation, which may be evidence of its anticancer effect.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Vitamins are essential for a wide range of biochemical functions and are either insufficiently synthesised or not synthesised at all by the body, so they are obtained from the diet.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Vitamin D is a group of sterols with hormone-like functions.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
It is classified as a fat-soluble vitamin.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The active metabolite of vitamin D is 1,25-dihydroxy vitamin D3 [1,25(OH)2D3] (Calcitriol).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
It binds to cytosolic receptors found in the intestinal, muscle, and haematopoietic cells, as well as in some other tissues such as osteocytes and the brain.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
It is then transported to the cell nucleus, where it interacts with DNA and is involved in the control of more than 900 gene expressions.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Furthermore, vitamin D plays a role in numerous pathological and physiological processes, including calcium metabolism, bone mineralisation, cancer, immune modulation, cardiovascular disease, and metabolic syndrome.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Furthermore, the biological properties of vitamin D include regulation of cell proliferation and increased cell differentiation, effects on apoptosis, and interactions with the epigenome on multiple levels.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Vitamin D is known to alter DNA methylation at the promoter of certain genes.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In mammals, the process of DNA methylation involves the covalent transfer of a methyl group to the C-5 position of the cytosine ring in the CpG dinucleotide region of DNA by DNA methyltransferase.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The genome region with the highest density of CpG dinucleotide repeats is known as a CpG island, which spans 1,000–1,500 base pairs in length.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
There is evidence that 1,25-D3 is capable of inducing DNA demethylation; however, the mechanisms underlying the effect of 1,25-D3 on DNA methylation remain unclear.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Nevertheless, in certain instances, demethylation has been observed to occur within a timeframe of 1–4 h, indicating the potential involvement of an active process.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Notably, only early-stage tumours, but not late-stage tumours, demonstrated a correlation between vitamin D intake and reduced methylation.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In human cancer cells, DNA hypermethylation mainly includes promoter hypermethylation of the tumour suppressor gene (TSG) and promoter hypermethylation of the human DNA mismatch repair system gene.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In conclusion, alterations in DNA methylation may play a significant role in the expression of genes and the disruption of genomic integrity and may be involved in the development and progression of diseases.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The aim of the present study was to investigate the impact of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on methylation mechanisms in the Human neuroblastoma cell line (SH-SY5Y) cell line.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The human neuroblastoma cell line, SH-SY5Y (ATCC, VA, USA), was incubated with Dulbecco's Modified Eagle Medium (Capricorn, Germany) supplemented with 10% foetal bovine serum (Sigma-Aldrich, USA) and 100 IU/mL penicillin, 10 μg/mL streptomycin (Sigma-Aldrich, USA).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
This assay was performed according to Pedron et al. Briefly, a 96-well plate was used for the cytotoxicity assay.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
After cell counting, 10,000 cells/well were seeded.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
After 24 h of incubation, SH-SY5Y cells were treated with 1,25(OH)2D3 (VEM, Turkey) at doses of 0, 5, 10, 25, 50, 50, 100, 250, 500, and 1,000 nM and incubated with these doses for 24 h. The optical densities were recorded at a wavelength of 570 nm using an automated multi-well plate reader (Synergy HTX, BioTek, USA).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In order to evaluate the impact of 1,25(OH)2D3 on cellular migration, a wound healing assay was conducted on SH-SY5Y cells in accordance with the following procedure: 3×10 cells/well were seeded on a 6-well plate.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
To allow the cells to adhere to the surface and form confluent monolayers, they were incubated at 37°C in 5% CO2 for 24 h. The cells were scraped vertically with a 200 μL sterile pipette tip.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Subsequently, the cells were washed with phosphate-buffered saline in order to remove any residual cell debris.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The dose groups were administered doses of 100 and 265.6 nM, while the control group was treated with the culture medium.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The wound size was visualised using an inverted microscope, and the wound closure rate was calculated using the ImageJ software, which is a digital image processing and analysis software.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Effects of 1,25(OH)2D3 on mRNA expression analysis in SH-SY5Y cells were performed by the mRNA expression analysis method described previously.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Briefly, 3×10 cells/well were seeded on a 6-well plate.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
To allow the cells to adhere to the surface and form confluent monolayers, they were incubated at 37°C in 5% CO2 for 24 h. The dose groups were treated with doses of 100 and 265.6 nM for 24 h, while the control group was treated with culture medium.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The cycle threshold values of the target genes were determined, and the relative expression levels were calculated using the Livak method, which employs formula 2ΔΔ.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The examination of each sample was conducted in triplicate.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In Table 1, the primer sequences used for the genes are given.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
F: forward; R: reverse; ACTB: actin beta; DNMT3A: DNA methyltransferase 3 alpha; DNMT3B: DNA methyltransferase 3 beta; PTEN: phosphatase and tensin homolog.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The statistical analysis was conducted using GraphPad Prism v.9 (San Diego, CA, USA).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The differences between the two groups were evaluated using the Student's t-test and one-way analysis of variance (Tukey).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
A p-value of less than 0.05 was considered statistically significant.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
A series of 1,25(OH)2D3 concentrations (0, 5, 10, 25, 50, 100, 250, 500, and 1,000 nM) were subjected to examination.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The viability of SH-SY5Y cells decreased in a concentration-dependent manner and was determined as IC50:265.6 nM (R: 0.695).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The wound-healing assay employed SH-SY5Y cells.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Figure 1 illustrates the relative wound area in distinct groups at 0 and 24 h. The 1,25(OH)2D3 treatment demonstrated a dose-dependent inhibition of the closure of the scratch area.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
These findings indicate that 1,25(OH)2D3 impedes cell migration.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
100 and 265.6 nM doses showed an antiproliferative effect compared to the control group (p<0.05).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The expression levels of genes associated with DNA methylation and TSGs were evaluated and compared between groups to gain insight into the underlying mechanisms of tumour formation.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The decrease in DNA methyltransferase 3 alpha (DNMT3A) expression level was statistically significant at 100 and 256.6 nM concentration levels compared to the control group (p<0.05).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In the DNA methyltransferase 3 beta (DNMT3B) expression level, it was observed that the concentration level of 256.6 nM expressed a statistical significance compared to the control group (p<0.05).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In phosphatase and tensin homolog (PTEN) expression levels, there was no statistical significance between any groups (p>0.05) (Figure 2).
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Neuroblastoma is a common tumour in the paediatric age group.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
It is characterised by a complex management regimen and a higher relapse rate in the post-consolidation phase.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The presence of vitamin D receptor (VDR) in neuroblastoma cell lines, along with its quantities and the impact of the active vitamin D3 metabolite on VDR signalling, have all been linked to the mechanism of action of vitamin D3 in neuroblastoma cell lines.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Vitamin D3 has been demonstrated to exert an antiproliferative effect on a number of different types of cancerous cells, with this effect being achieved through the process of apoptosis, or programmed cell death.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) tetrazolium reduction assay is the inaugural homogeneous cell viability assay that has been developed to be compatible with a 96-well format, thus rendering it suitable for utilisation in high-throughput screening.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The antitumour effects of vitamin D3 have been investigated in a range of tumour types using the MTT assay.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In an MTT assay of a study in ECV-304 (human urinary bladder carcinoma) and T24 (human bladder carcinoma) cells, a combination treatment of 500 nM vitamin D+16.6 μM cisplatin was compared with cisplatin alone.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The results of this experiment showed that only in the T24 cell line did the combination of vitamin D+cisplatin increase antiproliferative activity compared to cisplatin.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Vitamin D3 is believed to exert its protective effects by promoting apoptosis, inhibiting tumour angiogenesis, and regulating antiproliferative, antimetastatic, and prodifferentiation mechanisms in colorectal cancer, breast cancer, prostate cancer, lung cancer, and neuroblastoma cancer cells.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
In this study, it was observed that 1,25(OH)2D3 was effective on SY-SH5Y cell viability and antiproliferative mechanisms with increasing doses, and 100 and 265.6 nM doses were effective on cell migration compared to the control group.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The process of DNA methylation represents an epigenetic mechanism that regulates gene expression.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
There is mounting evidence that vitamin D3 may play an important role in this regulatory process.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Among the epigenetic mechanisms that affect the activity and functionality of specific DNA regions, DNA methylation is provided by DNMTs to direct the transcriptional activity of genes without changing the DNA sequence.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
DNMTs that affect genomic methylation patterns are DNMT1, DNMT3, DNMT3a, and DNMT3b.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The inactivation of TSGs represents a pivotal mechanism underlying the pathogenesis of all common forms of human cancer.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
A study has demonstrated that Schisandrin B, an active substance, has been demonstrated to inhibit Aβ1-42-mediated damage in the SH-SY5Y neuronal cell line.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Furthermore, this inhibition has been found to significantly suppress the induced alterations in mRNA and protein expression of DNMT3A and DNMT3B, with the concentration of the active substance exerting a significant impact on this process.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
The depletion of DNMT3A was observed to result in demethylation of the PTEN promoter in hepatocellular carcinoma (HCC) cells, thereby indicating that DNMT3A silences PTEN through DNA methylation.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
This result provides insight into the mechanisms by which DNMT3A regulates TSGs in HCC through an epigenetic approach.
PMC12172538
Methyltransferase gene-mediated antiproliferative effect of 1,25(OH)2D3 on neuroblastoma cells
Imatinib has been demonstrated to induce epigenetic alterations of the PTEN gene in leukaemia cells, a process that is mediated by the upregulation of DNMT3A.