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PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
MYCN oncogene amplification is the first independent prognostic factor indicating poor clinical outcomes of NB patients.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
However, it occurs in only approximately 18% of neuroblastoma patients, while nearly 80% present with single-copy MYCN—a subgroup that includes low- and intermediate-risk NB and remains comparatively less explored .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Given this clinical distribution, our primary aim was to directly address this research gap and investigate the effects of the JNK inhibitor in the context of the more prevalent MYCN-non-amplified disease, relevant to the broad majority of NB patients .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Treatment of NB remains challenging because of its high genetic, immunological, and clinical heterogeneity, suboptimal response to initial therapy, high risk of relapse, and growing chemoresistance of tumour cells .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
One promising therapeutic strategy against NB is the pharmacological inhibition of c-Jun N-terminal kinases (JNKs), as they converge on many pathways and are considered the main regulators of apoptosis in tumours .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
JNKs constitute a group of Mitogen-Activated Protein Kinases (MAPKs) that regulate cancer cell survival and apoptosis .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
MAPK8, MAPK9, and MAPK10 genes encode the three JNK isoforms, respectively, JNK1, JNK2, and JNK3, which were reported to have multifaceted roles .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The experimental studies on NB indicated that the JNK1 isoform performs a pro-proliferative role, while JNK2 and JNK3 are assumed to play a pro-apoptotic role .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
JNKs control the expression of cell cycle-, apoptosis-, mitochondria-, and ER stress-related genes, playing a crucial role in executing cell death in response to various apoptotic stimuli, both transcriptionally and through transcription-independent mechanisms .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
JNKs undergo mitotic translocation, which results in the discharge of cytochrome c (Cyt c) and the Second Mitochondria-derived Activator of Caspase (SMAC), both of which directly induce apoptosis .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
However, the precise mechanism of the JNK pathway’s role has not been fully elucidated in terms of NB.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Although JNK is known as a well-evidenced apoptosis kinase, under specific stress conditions in the tumours, in which JNK is overexpressed, it may evoke a paradoxical effect and contribute to cancer cell survival and chemotherapy resistance and the inhibition of cancer cell apoptosis .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Emerging evidence indicated that transiently activated JNK promotes cancer cell survival by modulating JNK-Bcl-2/Bcl-xL-Bax/Bak- as well as Cyt-C/caspase-3-dependent pathways .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Studies have demonstrated that both genetic inactivation of JNK signalling and specific pharmacological JNK inhibition can suppress tumorigenesis .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Hence, multiple JNK inhibitors have recently been widely investigated as a potential anticancer treatment, alone and in combination with chemotherapy .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Recent data indicated the enormous potential of JNK inhibitors in NB treatment, as they have been reported to induce NB cell death via direct activation of p53-, JNK-, Bcl-2-, and caspase-dependent pathways .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Based on available evidence, this is the first comprehensive study that investigates the antineoplastic effect of JNK inhibition in the NB in vitro model, a SH-SY5Y human neuroblastoma cell line, using the pharmacological inhibition of JNK signalling by JNK inhibitor AS601245 (JNK inhibitor V, JNK V).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
This selective small-molecule inhibitor exerts the strongest effect on the least-studied isoform, JNK3, which is hypothesised as a target in NB due to its expression in neuronal tissue and potential molecular involvement in apoptosis and oxidative stress response .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The study provides novel preclinical evidence supporting the further development of JNK inhibitors in the treatment of NB.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The cytotoxic effect of JNK V on three cell lines was evaluated using a colorimetric XTT assay (Figure 1).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The cells were then treated and incubated with a particular compound for 24 h to assess the acute short-term effects of the investigated compound.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The cytotoxicity experiment revealed significant inhibition of the proliferation of SH-SY5Y cells following the 24 h incubation with JNK V at concentrations ≥ 0.75 µM compared with the negative control.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The cytotoxic effect was significant, and SH-SY5Y cells’ viability was reduced by almost 60% at 12 µM JNK V. In our experiment on SH-SY5Y cells, the IC50 of JNK V was 9 μM after 24 h of incubation.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Therefore, the particular JNK V concentration ranges for all further experiments were determined based on XTT assay results, using the methods’ dedicated specifications.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Low concentrations of JNK V (0.1–3 μM), reducing cell viability up to 30% after 24 h, were chosen for migration experiments, moderate concentrations for clonogenic assay (0.19–25 μM) and mRNA expression analysis (1–25 μM), and higher concentrations for caspase-3 activity assessment (0.75–100 μM), protein expression, and mitochondrial function analysis (1–100 μM).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
A solvent for JNK V, 0.01% DMSO, did not induce significant toxicity in SH-SY5Y cells after 24 h of incubation compared to the negative control.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Furthermore, on normal cell lines, HSC and BJ, the JNK V did not demonstrate a cytotoxic effect.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Although in HSC, the decrease in cell viability at JNK V ≥ 6 μM was statistically significant compared to the negative control, the viability of HSCs at all tested concentrations was maintained ≥78%, which is above the ISO 10993-5 cytotoxicity threshold .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
HSC is a primary glial cell line characterised by high sensitivity to treatment-induced stress, which may explain the observed but non-cytotoxic reduction in viability.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Moreover, the viability of BJ fibroblasts, which are much more resilient cells, was not significantly affected by JNK V at all tested concentrations, nor at 0.01% DMSO.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Therefore, the XTT assay revealed the selective cytotoxicity of JNK V toward NB cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The morphology of the cells exposed to JNK V, 0.01% DMSO, or 20% DMSO was assessed after 24 h of incubation using an inverted microscope (Nikon, Tokyo, Japan) (Figure 2).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Untreated SH-SY5Y cells (negative control) exhibited a characteristic neuronal-like morphology with elongated neurites, an adherent and polygonal cell shape, and a homogenous cytoplasm.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Cells treated with JNK V demonstrated concentration-dependent morphological alterations, from moderate shrinkage, the loosening of cell–cell contact, and neurite retraction at <3μM JNK V concentrations to increased cell detachment, loss of neurites, and nuclear condensation at 3–12 μM JNK V to loosening of the capacity to attach to the culture vessels and widespread cell lysis at JNK V > 12 μM. The results demonstrated a markedly visible impairment of the morphology of SH-SY5Y cells after treatment with JNK V at a wide concentration range, evidencing the cytotoxic effect of the compound on NB cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
A clonogenic assay was performed in order to assess the proliferation and colony-forming abilities of SH-SY5Y cells, indicating their long-term reproductive capacity and overall viability after 2 weeks of incubation with JNK V at 0.1–25 μM (Figure 3).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Untreated cells maintained undisturbed proliferation, reaching 100% confluence, and properly formed colonies.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The assay results showed that JNK inhibition significantly impaired the proliferation and colony-forming abilities of SH-SY5Y cells in a dose-dependent manner, compared to untreated cells, which reached 47% confluence at the final time point.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Cells exposed to JNK V, even at the very low concentration of 0.1 μM, reached 29% confluence, while the confluence of cells treated with JNK V concentrations ≥1.5 μM was <10% at the final time point.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Treatment with the inhibitor at a high concentration of 25 µM completely suppressed the cells’ proliferation, calculated at 0.3% confluence, and led to no colony formation, indicating the strong cytotoxicity of the compound.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The results showed the impairment of long-term proliferation and survival of SH-SY5Y cells by pharmacological JNK inhibition, showing its cytotoxic and antineoplastic potential against cancerous NB cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The effect of JNK V on the migration ability of SH-SY5Y cells was assessed using a wound healing (scratch) assay (Figure 4).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Cells treated with JNK V at low and moderate concentrations (0.19 µM to 3 µM) closed the mechanically made scratch relatively more slowly compared to untreated cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Phase-contrast microscopy revealed that inhibitor-treated cells retained their morphology but exhibited reduced motility, as indicated by fewer cells at the wound edges and a wider residual gap.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
In the control group (untreated cells), the wound area gradually decreased over time, with 38% wound closure after 24 h and almost complete wound closure (97%) after 48 h, indicating active cell migration.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The treatment with JNK V at all tested concentrations resulted in significantly impaired cell migration across all time points, compared to negative control cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
At all tested JNK V concentrations, cells reached <60% wound closure after 48 h. SH-SY5Y cells reached only 21% of wound closure after 72 h of treatment with JNK V at 3 µM and less than 50% of wound closure after 72 h of incubation with 1.5 µM JNK V, indicating a strong anti-migratory effect of the inhibitor.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
These results suggest that JNK V effectively suppresses SH-SY5Y cells’ invasion abilities in a concentration-dependent manner, indicating the anti-migratory and further anti-metastatic potential of the pharmacological JNK inhibition in NB cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The caspase-3 colorimetric activity assay was conducted to evaluate the activity of the caspase-3 enzyme as an indicator of apoptosis in SH-SY5Y cells treated with JNK V inhibitor at a concentration range of 0.75–100 µM (Figure 5).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Although JNK is typically known to activate caspase-3, its inhibition may paradoxically increase caspase-3 levels in particular cancer and neuronal cells under chronic stress conditions by disinhibiting other pro-apoptotic signals such as p38 or intrinsic mitochondrial pathways, triggering the activation of caspase-9 as well as downstream caspase-3 .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The absorbance of the chromogenic reaction product, p-nitroaniline (pNA), at 405 nm corresponds to the caspase-3 enzymatic activity in cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The obtained results demonstrated a significant elevation in the caspase-3 activity level in cells treated with JNK V inhibitor ≥ 6 µM after 24 h of treatment, compared to the baseline level of caspase-3 in untreated cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The absorbance values in untreated cells showed minimal caspase activity (O.D. = 0.055), whereas treatment with 100 μM of the inhibitor resulted in a 4.3-fold increase in enzyme activity (O.D. = 0.240).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The result evidenced that pharmacological JNK inhibition promotes caspase-dependent apoptosis in NB cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The expression analysis of mRNA of genes associated with apoptosis (Mitogen-activated protein kinase 10/c-Jun N-terminal kinase 3 (MAPK10/JNK3), B-cell lymphoma 2 (BCL2), and BCL2-Associated X, Apoptosis Regulator (BAX)) and anti-oxidative response (nuclear factor erythroid 2-related factor 2 (NRF2)) was performed in SH-SY5Y cells exposed to JNK V (1 μM, 10 μM, 100 μM), 0.01% DMSO (solvent control), and untreated cells (negative control) (Figure 6).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
As NRF2 is a master regulator of antioxidant responses, and JNK can modulate oxidative stress response signalling, the changes in NRF2 mRNA expression could explain whether the observed anti-neoplastic effects could be related to oxidative stress regulation rather than only direct apoptotic signalling .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The obtained results have shown that the expression of MAPK10 mRNA, the less explored JNK isoform with an established neuroprotective effect on normal cells, was significantly reduced by all tested concentrations of JNK V compared to that in the negative control cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
JNK V in all tested concentrations caused a significant decline in the expression of anti-apoptotic BCL2 and a significant increase in the expression of pro-apoptotic BAX mRNA in comparison to that in the negative control.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The expression of the master regulator of anti-oxidative responses, NRF2 mRNA, was significantly reduced by all tested concentrations of JNK V, indicating that JNK inhibition may impair SH-SY5Y resistance to oxidative stress by downregulating Nrf2-dependent protective mechanisms.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
These results suggest that JNK V modulates the expression of the chosen genes associated with apoptosis and oxidative stress response in SH-SY5Y cells, enhancing NB cell apoptosis.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Western blot analysis demonstrated a dose-dependent suppression of JNK pathway activation in SH-SY5Y cells after JNK V treatment (Figure 7).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The investigated compound treatment decreased both JNK isoforms p54 and p46 and modulated p-JNK isoform expression in a dose-dependent manner.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Moreover, the deregulation of chosen key apoptosis- and cell cycle-related protein expression in SH-SY5Y cells by JNK V was demonstrated.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The expression of anti-apoptotic Bcl-2 was significantly downregulated by JNK V at 50 and 100 µM, accompanied by a reduction in p-Bcl2 expression.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The Bim expression levels after JNK V treatment were not changed significantly, but an increasing tendency of protein expression with higher concentrations of JNK V could be observed, which is consistent with a pro-apoptotic shift, since the Bcl-2 level is decreasing.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The expression level of HIF-1α was reduced by all tested concentrations of JNK V, with statistical significance at 50 µM, suggesting a probable effect of JNK V on hypoxia-related signalling or stability.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The p53 protein expression level was significantly increased after JNK V treatment at 50 µM, suggesting that the pro-survival p53 axis is affected by JNK inhibition under these conditions.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The obtained results show that JNK signalling in SH-SY5Y cells can be pharmacologically blocked by JNK V to effectively suppress its downstream phosphorylation events and to promote an apoptotic molecular profile with reduced Bcl-2 signalling and increased p53 expression.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The mitochondrial respiration, including oxidative phosphorylation, defined as oxygen consumption rate (OCR), and glycolysis, defined as extracellular acidification rate (ECAR), was measured using the Seahorse Mitostress Assay (Figure 8).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
SH-SY5Y cells were treated with JNK V inhibitor (1–100 µM) for 24 h. The OCR of untreated cells (control) was the highest, indicating active mitochondrial respiration of the living cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The modulation of OCR in untreated cells is a consequence of injections of the assay reagents.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The ECAR in the control group was relatively high and stable, indicating active glycolysis.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
JNK V treatment showed a significant reduction in OCR in a concentration-dependent manner compared to the control, suggesting that JNK inhibition decreases mitochondrial respiration in a dose-dependent manner.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Similarly to the OCR results, ECAR decreased with increasing JNK inhibitor concentrations.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The drop in both OCR and ECAR suggests that JNK inhibition not only impairs ATP production via mitochondria but also limits the cells’ ability to compensate through glycolysis, ultimately leading to a significant metabolic disruption, preserving only residual OCR while treated with 100 μM JNK V. These findings suggest that JNK activity may be critical for maintaining both oxidative phosphorylation and glycolysis in SH-SY5Y cells.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The treatment of NB remains challenging because of its high genetic, immunological, and clinical heterogeneity; suboptimal response to initial therapy; high risk of relapse; and growing chemoresistance of tumour cells .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Therefore, the development of novel therapies should focus on increasing response rates to first-line therapies (chemo/immunotherapy), developing effective salvage therapies for relapsed and refractory disease, sustaining disease remissions, and enhancing antitumour immune function .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
In infants, NB often regresses spontaneously or matures into ganglioneuroblastoma (GNB) after initial chemotherapy.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
However, the prognosis of NB patients becomes worse as patients age, and the current treatment standards remain ineffective in older patients and those with disseminated or recurrent disease.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Despite advances in therapy, outcomes for high-risk patients remain poor .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
MYCN oncogene amplification is the first independent prognostic factor indicating poor clinical outcomes of NB patients, observed in approximately 18% of cases, accounting for 40% of high-risk neuroblastomas .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The MYCN gene consistently promotes the progression of MYCN-related neuroendocrine tumours by driving dynamic spatial and temporal interactions, including distinct transcriptional programmes, DNA damage repair, resolving torsional stress, regulating R-loops, metabolic networks, stress–response, and apoptosis-related signalling patterns, and contributing to the aggressive phenotype of MYCN-amplified diseases .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
However, despite the extensive study of MYCN-amplified NB, a gap in pre-clinical and clinical research of MYCN non-amplified cases was recently highlighted.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
New large-scale transcriptional subtyping analysis has suggested that MYCN non-amplified NBs are roughly heterogeneous and could be classified into three subgroups according to their transcriptional profiling .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Subgroup 2 had the worst prognosis, presenting an ‘MYCN’ signature that was potentially affected by overexpression of Aurora Kinase A (AURKA), while subgroup 3 showed an ‘inflamed’ immune-related gene signature.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
These findings emphasised the substantial extent of inner heterogeneity within the MYCN non-amplified population and show the vulnerability of stratified subgroups to different therapeutic approaches.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Moreover, this research demonstrated active stress–response pathways and MAPK/JNK signalling within the subtype groups, underscoring that JNK pathway biology is not limited only to MYCN-amplified NB phenotypes.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Consistent with this, JNK activity has been implicated in migration, invasion, and stress-induced apoptosis in both MYCN-amplified and MYCN-non-amplified neuroblastoma models .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Thus, although our study focuses on a non-amplified cell line, it addresses cellular mechanisms relevant to multiple NB subtypes.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Furthermore, NB with MYCN amplification exhibits both transcriptional and functional inhibition of JNK signalling, resulting in low basal JNK activity and a significantly impaired apoptotic response.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Patient-specific modelling consistently places tumours with MYCN amplification in the low JNK activity cluster, and system-level reconstructions show systematic downregulation of upstream JNK circuits in MYCN-induced disease .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Notably, a newly published study has shown that high-risk tumours with MYCN amplification respond better to treatment regimens involving JNK-independent drugs, confirming that the JNK pathways are significantly silenced in this subtype .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Hence, JNK-dependent therapies are much more biologically relevant in MYCN-nonamplified neuroblastoma, where the JNK cascade remains intact.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Elevated JNK activity is an established contributor to malignancy progression and chemo/radiotherapy resistance in many cancers in which JNK is transiently activated, such as B-cell lymphoma, osteosarcoma, breast, lung, skin, and pancreatic cancer .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
JNK exhibits pro-survival function in cancer cells due to its ability to enhance the processes of proliferation, migration, and invasion through a plethora of mechanisms, including synergistic action with p38 MAPKs and nuclear factor kappa B (NF-κB), the upregulation of antiapoptotic gene expression such as BCL2, and the blockade of caspase activation .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Moreover, JNK induces pro-survival autophagy to counteract apoptosis, which, together with immune evasion mechanisms, increases the resistance of cancer cells to chemotherapy .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Therefore, JNK constitutes an interesting molecular anti-neoplastic target for therapeutic intervention with small-molecule kinase inhibitors .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Intriguingly, the JNK inhibitor AS602801, bentamapimod, was evidenced to block gap-junction communication between astrocytes and glioma cells , lung cancer stem cells , and breast cancer cells, and together with modulating Cx43 expression, it inhibited particular cancer metastasis to the brain .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Multiple JNK targeting strategies, including various ATP-competitive, non-kinase, and substrate-competitive inhibitors, have been developed and, due to the anticancer potential demonstrated in preclinical models, several JNK inhibitors have been investigated in the II/III phases of clinical trials .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
However, JNK’s pleiotropic signalling limits the clinical application of direct JNK inhibition, as evidenced by past clinical trials primarily focused on fibrotic and inflammatory diseases .
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The anticancer effects of the inhibition of the JNK signalling pathway have previously been successfully reported in diverse NB in vitro and in vivo models .