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PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The effect of JNK V on the expression of several key apoptosis- and cell cycle-related proteins in SH-SY5Y cells was evaluated by Western blot analysis.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The JNK V inhibitor was applied to the cells for 24 h at doses of 1, 10, 25, 50, and 100 µM. The untreated cells constituted the negative control.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Following treatment, the cells were collected from the wells, and the MinuteTM Total Protein Extraction Kit (Invent Biotechnologies, Plymouth, MN, USA) was used to extract the total protein.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The PierceTM BCA Protein Assay (Thermo Scientific, Waltham, MA, USA) was used to assess and normalise the protein quantities in the samples.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The protein samples were denatured at 70 °C for 10 min, placed into gel wells, and subsequently electrophoresed for 50 min using the NuPageTM/XCell SureLockTM system (Invitrogen, Waltham, MA, USA).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
After that, the proteins were moved to a PVDF membrane (Invitrogen, Waltham, MA, USA) and incubated for one hour.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The membrane was then blocked for one hour in a solution of 5% BSA for phosphoproteins, or skim milk for non-phosphoproteins (BioShop Canada, Burlington, ON, Canada) in 1X TBST (Thermo Scientific Chemicals, Waltham, MA, USA).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Then, the membranes were incubated for 24 h with primary monoclonal antibodies for targeted proteins such as JNK, p-JNK, Bim, Bcl-2, p-Bcl-2, p53, and HIF-1α (dilution 1:1000; Cell Signaling Technology, Danvers, MA, USA).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The antibodies’ ID numbers are listed in Table 1.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The loading control was β-actin.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The next day, secondary HRP-linked antibodies (dilution 1:5000; Cell Signaling Technology, Danvers, MA, USA) were added to the membranes after they had been cleaned three times with 1X TBST.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Following a final wash in TBST, the membrane was exposed to SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) for five minutes in the dark.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA) was used to detect the protein bands using enhanced chemiluminescence.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
NIS-Elements Advanced Research software version 5.42 (Nikon, Tokyo, Japan) was used to conduct the densitometry analysis.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The Agilent Seahorse XFp Real-Time ATP rate assay (Agilent Technologies, Santa Clara, CA, USA) was performed to evaluate the effect of JNK inhibition on SH-SY5Y cells’ cellular respiration.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
After being seeded in Seahorse XF HS Miniplates (Agilent Technologies, Santa Clara, CA, USA), the cells were exposed to 1, 10, 25, 50, and 100 μM of JNK V. Untreated cells constituted the control.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The day before the experiment, the Seahorse XFp Sensor Cartridge was hydrated overnight in a non-CO2 incubator as directed by the manufacturer.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The cartridge was rehydrated using Seahorse XF Calibrant (Agilent Technologies, Santa Clara, CA, USA) and incubated for 45 min on the day of the test.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Following the manufacturer’s instructions, the culture medium was removed from each well, and the cells were washed with preheated assay medium (pH 7.4) that contained Seahorse XF DMEM, 10 mM glucose, 2 mM L-glutamine (Agilent Technologies, Santa Clara, CA, USA), and 1 mM sodium pyruvate.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Cells were cultured at 37 °C in a non-CO2 incubator for 45 min to attain the desired temperature and pH before measurement.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Then, the ATP synthase inhibitor oligomycin (2.5μM) and the mitochondrial complex I/III inhibitors rotenone/antimycin A (0.5 μM) solutions were inserted into suitable cartridge ports, in line with the manufacturer’s instructions.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
After the initial three baseline measurements using a Seahorse XF HS Mini Analyser (Agilent Technologies, Santa Clara, CA, USA), three measurements were taken after each injection of chemical compounds.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The statistical analysis was performed using Statistica (version 13; StatSoft, Kraków, Poland).
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The normality of data distribution was determined by the Shapiro–Wilk test.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
If data were homogeneous and normally distributed, one-way ANOVA with Bonferroni or Tukey’s post hoc testing was performed for comparison between multiple groups.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
Otherwise, the Kruskal–Wallis one-way analysis of variance on ranks with Dunn’s post hoc test with Bonferroni correction was performed.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
All studies were performed in triplicate, and the data are presented as mean ± SD.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
The following symbols indicate statistically significant differences in the graphs: * p < 0.05, ** p < 0.01, *** p < 0.001.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
To our knowledge, this is the first comprehensive study to characterise the acute in vitro effects of JNK inhibitor AS601245 (JNK V) on the human MYCN-non-amplified neuroblastoma cell line SH-SY5Y.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
We demonstrate that JNK V reduces SH-SY5Y cell viability and dysregulates key cellular functions, without exerting cytotoxicity toward normal human Schwann cells and fibroblasts under the conditions tested.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
JNK V induces a dose-dependent inhibition of proliferation, colony formation, and migration, accompanied by increased caspase-3 activity, pro-apoptotic gene and protein expression, and significant disruption of both oxidative phosphorylation and glycolysis.
PMC12732662
Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells
These results provide a detailed description of early cellular responses to pharmacological JNK inhibition in the MYCN-non-amplified neuroblastoma in vitro model.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by memory loss and cognitive decline, primarily due to amyloid β (Aβ) aggregation in the brain.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Astaxanthin (AxN), a xanthophyll carotenoid derived from Haematococcus pluvialis, possesses antioxidant and neuroprotective properties.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
This study investigated the neuroprotective effects of AxN against Aβ aggregation in human neuroblastoma SH-SY5Y cells.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Initially, AxN inhibited Aβ aggregation in DMEM/F12 culture medium but not in PBS, suggesting a medium-dependent effect.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Using quantum dot nanoprobes, Aβ aggregation was visualized in the presence of SH-SY5Y cells.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
AxN treatment (0.032–20 µM) significantly reduced Aβ aggregation and accumulation on SH-SY5Y cells.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
AxN also prevented Aβ-induced early apoptotic cell death but was less effective against late necrosis.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Furthermore, a wound-healing assay showed that AxN restored the impaired cell motility caused by Aβ aggregation.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Thioflavin T staining confirmed the reduction in Aβ fibril formation around the cells following AxN treatment.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
In conclusion, our study suggests that AxN prevents Aβ aggregation and accumulation on the cell surface, thereby restoring cell motility and preventing early apoptosis in neuronal cells.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Alzheimer’s disease (AD) is a chronic neurodegenerative disease caused by—as a complex pathogenesis—the abnormal deposition of amyloid β (Aβ) and hyperphosphorylated tau in senile neurons .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
AD is primarily characterized by memory loss, cognitive decline, and visual–spatial dysfunction .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Misfolded Aβ protein forms amyloid plaques, which cause extracellular deposition in between neurons, and intracellular deposition of neurofibrillary tangles (NFTs) in the neurons are formed by tau proteins, leading to neurodegenerative diseases .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
The progressive decline in cognitive functions leads to acute dementia, while oxidative damage induced by reactive oxygen species disrupts cellular processes, leading to impaired protein function while promoting the aggregation of Aβ plaques, a hallmark of AD .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Aβ is a neurotoxic polypeptide composed of 39–43 amino acid residues that aggregate into soluble oligomers and insoluble amyloid fibrils .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Aβ40 and Aβ42 are the most prevalent species that contribute to Aβ-related neurodegenerative disorders.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Aβ42 accumulates more rapidly than Aβ40, and it is also a primary component of senile plaques, which are characteristic of AD .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Astaxanthin (AxN) is a red-orange pigmented carotenoid that is found in marine organisms and microalgae .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
It is abundantly produced by the microalgal species, Haematococcus pluvialis .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
AxN is also a potent antioxidant that can prevent neuronal cell death and oxidative stress, and improve spatial memory .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
The potent antioxidant effects of AxN are attributed to the stability of its ionone rings and polyene structure, allowing it to effectively counteract damage by free radicals .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
It also enhances mitochondrial functions in neurons, reduces DNA damage, and alleviates cellular stress.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
In recent years, several in vitro and in vivo studies on AxN have demonstrated its neuroprotective effects on brain damage, anti-cancer, anti-inflammatory, anti-diabetic, and immunomodulatory activities .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Although the exact cause of this disease has not yet been identified, the neuronal damage and death caused by Aβ induce the cognitive deficits that characterize AD .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Despite considerable research on AD at present, there is no permanent cure for AD, as the main pathological cause remains unknown.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Therefore, in this study, we investigated the mechanism of neuroprotection of AxN against Aβ-induced cytotoxicity using the SH-SY5Y human neuroblastoma cell line.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Following our previous reports , which demonstrated the inhibitory activity of various natural products on Aβ42 aggregation, this study aimed to evaluate the effect of AxN on 25 µM Aβ42 both in vitro and in a cell line model.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
To achieve this, we assessed Aβ deposition by visualizing and quantifying aggregates using quantum dots (QDs) and demonstrated that AxN inhibited Aβ aggregation in cells and suppressed early apoptosis.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Furthermore, AxN was found to exert a protective effect on Aβ-induced impairments in cell motility and migration dynamics, which are commonly affected in neurodegenerative diseases.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Initially, we investigated the inhibitory effect of 0.32–200 µM AxN on the aggregation of Aβ using the microliter-scale high-throughput screening (MSHTS) system.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
After 24 h of incubation in phosphate-buffered saline (PBS), similar aggregates were observed regardless of the concentration of AxN added, whereas in DMEM/F12, the amount of Aβ aggregates was lower in the presence of 200 µM AxN than in the absence of AxN (Figure 1A).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
We also found that the shape of Aβ aggregates differed between PBS and DMEM/F12, which is presumably due to the various substances found in these media affecting the interaction of Aβ fibrils.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
The amount of Aβ aggregates was estimated from the standard deviation (SD) values of fluorescent intensities of images.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Half-maximal effective concentration (EC50) values clearly determined that Aβ deposition was inhibited by AxN in DMEM/F12 but not in PBS as an MSHTS solvent (Figure 1B,C).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
This result suggests that the inhibitory effect of AxN is enhanced in culture medium but is inefficient in pure buffer conditions.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Similarly, using the QD imaging technique, we investigated the inhibitory effect of Aβ aggregation and deposition on SH-SY5Y neuroblastoma cells in the presence of different concentrations of AxN. Fluorescence images showed the effective reduction in the accumulation of Aβ around the cells in a concentration-dependent manner (Figure 2A).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Quantitative analysis was evaluated using ImageJ 1.53e software and showed a significant decrease in fluorescent intensity, indicated by the mean gray value, in the presence of 0.8 µM or more of AxN (Figure 2B).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
The results show that AxN reduced the deposition of Aβ aggregates around SH-SY5Y cells.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
We performed the Thioflavin T (ThT) assay to study the effect of AxN on the formation and deposition of Aβ fibrils.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
ThT is a fluorescent dye used to detect and quantify the formation of β-sheet-rich Aβ fibrils.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
The decrease in the level of fluorescence intensity of ThT at 24 h demonstrated the inhibitory effect of 20 µM AxN on Aβ deposition (Figure 3A).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Fluorescence images of ThT were also captured using an inverted fluorescence microscope (Figure 3B).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
The images were analyzed using ImageJ software, which demonstrated the inhibition of Aβ deposition by different concentrations of AxN (Figure 3C).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
AxN at 20 µM showed a significant anti-aggregation effect with the greatest reduction in the intensity of ThT fluorescence.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
These results indicate that AxN can reduce the deposition of Aβ fibrils on the cell surface.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
We analyzed in detail the effect of 25 µM Aβ in the presence or absence of AxN (0.032–20 µM) on apoptosis in human neuroblastoma cells.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Cells treated with AxN reduced the number of apoptotic cells in a concentration-dependent manner (Figure 4A, p-SIVA).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Quantification of fluorescence intensity demonstrated that the early apoptotic cell death induced by Aβ was significantly suppressed by the addition of 20 µM AxN (Figure 4B).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
On the other hand, the late necrosis induced by Aβ aggregation was not fully suppressed by AxN (Figure 4C).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
This result suggests that AxN shows a neuroprotective effect in SH-SY5Y cells by inhibiting apoptosis caused by Aβ aggregation and deposition.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
We recently reported that aggregation around cells affected cell motility .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
To elucidate the effect of Aβ on the motility of SH-SY5Y cells, we performed a wound healing assay (WHA).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
The SH-SY5Y cells, which were treated with 0.032–20 µM AxN in the presence of 25 µM Aβ, and were monitored for 24 h, migrated towards the wound area (Figure 5B).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
The difference in area covered by cells over 24 h is shown in Figure 5C.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Although no significant difference was found, this result suggests a tendency for AxN to rescue the defects of cell motility caused by Aβ aggregation and deposition.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Using Hoechst staining, the migration dynamics of individual SH-SY5Y cells were assessed (Figure 6A).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
AxN-treated cells significantly improved cell speed (Figure 6C), and directional persistence was partially restored (Figure 6D).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
This result suggests that 20 µM AxN can attenuate the inhibitory effects of cell migration and its dynamics induced by Aβ aggregation and deposition.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Astaxanthin, a xanthophyll carotenoid primarily found in algae, salmon, shrimps, and other marine organisms, is recognized as a strong and naturally occurring antioxidant.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Its antioxidant activity is superior to that of other carotenoids (fucoxanthin, canthaxanthin, zeaxanthin, β-carotene, and α-tocopherol) .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
In this study, AxN was able to reduce the abnormal deposition of Aβ on human neuroblastoma SH-SY5Y cells.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Although inhibitory effects via cytotoxicity assays were not confirmed, the prevention of early apoptosis and the rescue of cell migration ability were shown.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
First, in this study, we evaluated the Aβ aggregation inhibitory activity of AxN using the MSHTS system (Figure 1), a previously established system to visualize Aβ aggregation and to estimate inhibitory activity using QD nanoprobes .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Interestingly, when PBS and DMEM/F12 were used as solvents for the MSHTS system, AxN was shown to inhibit Aβ aggregation in DMEM/F12 but not in PBS (Figure 1).
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
It has been reported that DMEM/F12 provides a stable environment that enhances the antioxidant property of AxN, whereas PBS does not contain biological components .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
Similarly, DMEM/F12 presumably contains biological components that enhance and stabilize the activity in the inhibition of Aβ aggregation as well.
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
In our previous reports, we elucidated the inhibitory activity of various natural products against Aβ aggregation by estimating EC50 values in PBS .
PMC12608137
Elucidation of the Neuroprotective Effects of Astaxanthin Against Amyloid β Toxicity in the SH-SY5Y Human Neuroblastoma Cell Line
In the future, it will become more important to evaluate the inhibitory activity in a solvent that contains a large number of biological components, such as culture medium.