Langerhans cell histiocytosis (LCH) is rarely encountered in ophthalmology practice. It is a spectrum of disorder characterized by accumulation of histiocytes in various tissues. Diagnosis is challenging as it may simulate periorbital hematoma, rhabdomyosarcoma, and neuroblastoma. We report a case of unifocal LCH with orbital extension. Diagnosis was obtained from incisional biopsy, and histopathological examination showed numerous histiocytes with eosinophilic infiltrations. The presence of Langerhans cells was confirmed by the presence of protein S-100, CD1a, and/or Langerin (CD207). Treatment depends on the degree of organ involvement. She responded well to cytotoxic drugs and steroids. This emphasized that prompt tissue diagnosis is crucial for early management.
The human neuroblastoma cell line, SH-SY5Y cells, derived from the parental SK-N-SH cell line, is commonly used as an in vitro model for neuroscience and neurobiology research. Since SH-SY5Y cells are widely cultured for research, several different culture media have been used to optimize the growth of the cells, including Eagle's Minimum Essential Medium (EMEM), Dulbecco’s modified Eagle’s medium (DMEM) and other recently developed culture media. SH-SY5Y cells has the ability to reach confluency in culture flasks ranges from 5 days to 15 days, depending on the culture media used. Hence, the optimization of the culture media is crucial to achieve the fastest growth rate for the cells. The objective of the study is to evaluate the culture media for the proliferation of SH-SY5Y cells. We compared the growth rate of SH-SY5Y cells cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% heat-inactivated fetal bovine serum (hiFBS), Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (DMEM:F12) + supplemented with 15% hiFBS and DMEM:F12 supplemented with 10% hiFBS. In DMEM:F12 supplemented with 15% hiFBS, cells grew up to 6.67E+05 cells. In DMEM:F12 supplemented with 10% hiFBS, cells grew up to 5.28E+05 cells. In DMEM supplemented with 15% hiFBS, the cells grew up to 4.76E+05 cells. There was a significant difference between culture media DMEM:F12 supplemented with 15% hiFBS as compared to DMEM:F12 supplemented with 10%hiFBS and DMEM supplemented with 15% hiFBS (p0.05). We found that DMEM:F12 supplemented with 15% hiFBS could serve as an optimized culture media for high proliferation rate of SH-SY5Y cells.
The role of the cytoskeleton, actin, and microtubules were examined during the process of Japanese encephalitis (JEV) infection in a human neuroblastoma cell line, IMR32. Cytochalasin D and nocodazole were used to depolymerise the cellular actin and microtubules, respectively, in order to study the effect of JEV infection in the cell. This study shows that depolymerisation of the actin cytoskeleton at early process of infection inhibits JEV infection in the cell; however infection was not inhibited when depolymerisation occurred at the later stage of infection. The microtubules, on the other hand, are required at 2 points in infection. The antigen production in the cells was inhibited when the infected cells were treated at time up to 2 hours after inoculation and there was no significant effect at later times, while the viable virus released continued to be affected until 10 hours after inoculation. In conclusion, infection of JEV in IMR32 cells required actin to facilitate early process in infection and the microtubular network is utilised as the transport system to the virus replication site and the release of mature virus.
Bcl-2 family proteins are crucial regulators of apoptosis. Both pro- and antiapoptotic members exist, and overexpression of the latter facilitates evasion of apoptosis in many cancer types. Bcl-2 homology domain 3 (BH3) mimetics are small molecule inhibitors of antiapoptotic Bcl-2 family members, and these inhibitors are promising anticancer agents. In this study, we report that gamma-tocotrienol (γT3), an isomer of vitamin E, can inhibit Bcl-2 to induce apoptosis. We demonstrate that γT3 induces cell death in human neuroblastoma SH-SY5Y cells by depolarising the mitochondrial membrane potential, enabling release of cytochrome c to the cytosol and increasing the activities of caspases-9 and -3. Treatment of cells with inhibitors of Bax or caspase-9 attenuated the cell death induced by γT3. Simulated docking analysis suggested that γT3 binds at the hydrophobic groove of Bcl-2, while a binding assay showed that γT3 competed with a fluorescent probe to bind at the hydrophobic groove. Our data suggest that γT3 mimics the action of BH3-only protein by binding to the hydrophobic groove of Bcl-2 and inducing apoptosis via the intrinsic pathway in a Bax- and caspase-9-dependent manner.
Gold nanoparticles (AuNPs) stabilized by new cationic 1‑(3‑(acetylthio)propyl)pyrazin‑1‑ium ligand (PPTA) were synthesized. AuNPs stabilized by PPTA (PPTA-AuNPs) were found to be spherical and polydispersed with the average size of 60 nm. Human neuroblastoma (SHSY-5Y) cells permeability of PPTA-AuNPs was found to be a key feature to study the intracellular quenching of Fe(III) proliferative activity. In vitro MTT assay revealed non-cytotoxicity of PPTA and PPTA-AuNPs at 100 μM concentration, while treatment of 100 μM of Fe(III) with SHSY-5Y cells resulted into higher cells viability. Contrary, a mixture of 1:1 Fe(III) with PPTA-AuNPs showed no change in the viability of cells at same concentration which suggests the intracellular complexation and recognition of Fe(III) by PPTA-AuNPs. AFM morphological analysis of SHSY-5Y cells also supported the MTT assay results, and it is safe to conclude that PPTA-AuNPs can be used as Fe(III) probes in living cells. In addition, Fe(III) caused a significant decrease in the absorbance of surface plasmon resonance (SPR) band of PPTA-AuNPs in a wide range of concentration and pH, with limit of detection 4.3 μM. Moreover, the specific response of PPTA-AuNPs towards Fe(III) was unaffected by the interference of other metals and components of real samples of tap water.
Epigenetics is the study of heritable changes in gene expression that do not involve changes to the underlying DNA sequence. It is a change in phenotype without changing in genotype which in turn affects how cellsread the genes. The epigenetic change is a regular occurrence but can also be influenced by several factors including age, environment, lifestyle, and disease state. It may have damaging effects that result in diseases like cancer. At least three systems including DNA methylation, histone modification and RNA-associated gene silencing are currently considered to initiate and sustain epigenetic change. New and ongoing research is continuously uncovering the role of epigenetics in a variety of diseases including in childhood solid cancer such as Ewing sarcoma, neuroblastoma, Wilms tumours, brain tumours and rhabdomyosarcoma. A better understanding of epigenetic changes in childhood cancers can guide towards future therapy and diagnosis.
Neuroinflammation is associated with neuronal cell death and could lead to chronic neurodegeneration. This study investigated the neuroprotective potential of virgin coconut oil (VCO) against lipopolysaccharide (LPS)-induced cytotoxicity of neuroblastoma SK-N-SH cells. The findings were validated using Wistar rats, which were fed with 1-10 g/kg VCO for 31 days, exposed to LPS (0.25 mg/kg) and subjected to the Morris Water Maze Test. Brain homogenate was subjected to biochemical analyses and gene expression studies. α-Tocopherol (α-T; 150 mg/kg) served as the positive control. VCO (100 µg/mL) significantly (p
Curcumin analogs with excellent biological properties have been synthesized to address and overcome the poor pharmacokinetic profiles of curcumin. This study aims to investigate the cytotoxicity, anti-proliferative, and apoptosis-inducing ability of curcumin analog, MS13 on human glioblastoma U-87 MG, and neuroblastoma SH-SY5Y cells, and to examine the global proteome changes in these cells following treatment. Our current findings showed that MS13 induced potent cytotoxicity and anti-proliferative effects on both cells. Increased caspase-3 activity and decreased bcl-2 concentration upon treatment indicate that MS13 induces apoptosis in these cells in a dose- and time-dependent manner. The label-free shotgun proteomic analysis has defined the protein profiles in both glioblastoma and neuroblastoma cells, whereby a total of nine common DEPs, inclusive of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-enolase (ENO1), heat shock protein HSP 90-alpha (HSP90AA1), Heat shock protein HSP 90-beta (HSP90AB1), Eukaryotic translation initiation factor 5A-1 (EFI5A), heterogenous nuclear ribonucleoprotein K (HNRNPK), tubulin beta chain (TUBB), histone H2AX (H2AFX), and Protein SET were identified. Pathway analysis further elucidated that MS13 may induce its anti-tumor effects in both cells via the common enriched pathways, "Glycolysis" and "Post-translational protein modification." Conclusively, MS13 demonstrates an anti-cancer effect that may indicate its potential use in the management of brain malignancies.
Aromatic L-amino acid decarboxylase deficiency (AADC) is a rare autosomal recessive pediatric neurotransmitter disease. To date it remains poorly understood mainly due to an absence of a disease model. The dopaminergic neuroblastoma cell SH-SY5Y was chosen to develop our AADC deficiency model. These cells are not native dopamine synthesizers. Objective: To develop a dopamine-producing cellular model of AADC deficiency using SH-SY5Y neuroblastoma cells. Methods: Dopamine pathway proteins were identified with Western Blotting. Dopaminergic differentiation was attempted using all-trans retinoic acid (ATRA) with dopamine detection via HPLC-ECD post alumina extraction. Treatment with L-DOPA provided SH-SY5Y with excess precursor. RT-PCR was used to determine the expression of markers of mature neurons. Results: Western Blot screening identified AADC, dopamine β-hydroxylase and tyrosine hyrdoxylase proteins, indicative of a dopaminergic pathway. ATRA was unsuccessful in producing dopamine from the cells. L-DOPA treatment however, generated dopamine first visible as a HPLC-ECD peak 30 minutes post-incubation. Prior to this, SH-SY5Y dopamine synthesis from L-DOPA has never been documented. This de novo synthesis is then inhibited using benserazide to form our AADC deficiency cell model. RT-PCR showed that SH-SY5Y cells express markers of mature neurons in its ‘native’ state and is not affected by L-DOPA and benserazide treatment. This cell model will potentially benefit many areas of AADC deficiency research. Conclusion: SH-SY5Y cells produced HPLC-ECD measureable amounts of dopamine with the addition of L-DOPA. Our model of AADC deficiency is generated by quelling the dopamine production with Benserazide.
Neuroendocrine cervical cancer is a rare subtype of cervical cancer with a highly aggressive malignancy. This study was conducted to analyse the human papillomavirus (HPV) infection and molecular abnormalities in Vietnamese neuroendocrine carcinomas of the uterine cervix. HPV genotyping and p53 mutations were examined using polymerase chain reaction (PCR)-based direct sequencing. Mutations of epidermal growth factor receptor (EGFR), Kirsten rat sarcoma (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS) and v-Raf murine sarcoma viral oncogene homolog B (BRAF) were identified using commercial kits. Four high-risk HPV genotypes were identified in 26 (86.7%) out of a total of 30 tumours. The prevalence of HPV 16, 18, 31 and 45 was 20.0%, 50.0%, 20.0% and 36.7%, respectively. Overexpression of p16INK4a was observed in 93.3% of cases and was significantly correlated with high-risk HPV infections. Furthermore, p53 and NRAS mutations were detected in five (16.7%) and one (3.3%) cases, respectively, whereas no EGFR, KRAS or BRAF mutations were observed. These results demonstrate that high-risk HPV infection may be an important oncogenic factor for the development and progression of cervical neuroendocrine carcinoma.
Neurodegenerative diseases are linked to neuronal cell death and impairment of neurite outgrowth. An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated. The chemical constituents of P. giganteus (linoleic acid, oleic acid, cinnamic acid, caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine) were tested for neurite outgrowth activity. Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells. Uridine which was present in P. giganteus (1.80 ± 0.03 g/100g mushroom extract) increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt). Further, phosphorylation of the mammalian target of rapamycin (mTOR) was also increased. MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells. This study demonstrated that P. giganteus may enhance neurite outgrowth and one of the key bioactive molecules responsible for neurite outgrowth is uridine.
Alzheimer's disease (AD) is the most common cause of dementia. The cardinal neuropathological characteristic of AD is the accumulation of amyloid-β (Aβ) into extracellular plaques that ultimately disrupt neuronal function and lead to neurodegeneration. One possible therapeutic strategy therefore is to prevent Aβ aggregation. Previous studies have suggested that vitamin E analogs slow AD progression in humans. In the present study, we investigated the effects of the tocotrienol-rich fraction (TRF), a mixture of vitamin E analogs from palm oil, on amyloid pathology in vitro and in vivo. TRF treatment dose-dependently inhibited the formation of Aβ fibrils and Aβ oligomers in vitro. Moreover, daily TRF supplementation to AβPPswe/PS1dE9 double transgenic mice for 10 months attenuated Aβ immunoreactive depositions and thioflavin-S-positive fibrillar type plaques in the brain, and eventually improved cognitive function in the novel object recognition test compared with control AβPPswe/PS1dE9 mice. The present result indicates that TRF reduced amyloid pathology and improved cognitive functions, and suggests that TRF is a potential therapeutic agent for AD.
This study investigated the effects of vitamins D and E on an insulin-resistant model and hypothesized that this treatment would reverse the effects of Alzheimer's disease (AD) and improves insulin signalling. An insulin-resistant model was induced in SK-N-SH neuronal cells with a treatment of 250 nM insulin and re-challenged with 100 nM at two different incubation time (16 h and 24 h). The effects of vitamin D (10 and 20 ng/mL), vitamin E in the form of tocotrienol-rich fraction (TRF) (200 ng/mL) and the combination of vitamins D and E on insulin signalling markers (IR, PI3K, GLUT3, GLUT4, and p-AKT), glucose uptake and AD markers (GSK3β and TAU) were determined using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The results demonstrated an improvement of the insulin signalling pathway upon treatment with vitamin D alone, with significant increases in IR, PI3K, GLUT3, GLUT4 expression levels, as well as AKT phosphorylation and glucose uptake, while GSK3β and TAU expression levels was decreased significantly. On the contrary, vitamin E alone, increased p-AKT, reduced the ROS as well as GSK3β and TAU but had no effect on the insulin signalling expression levels. The combination of vitamins D and E only showed significant increase in GLUT4, p-AKT, reduced ROS as well as GSK3β and TAU. Thus, the universal role of vitamin D, E alone and in combinations could be the potential nutritional agents in restoring the sensitivity of neuronal cells towards insulin and delaying the pathophysiological progression of AD.
Neurodegenerative diseases (NDDs) are pathological conditions characterised by progressive damage of neuronal cells leading to eventual loss of structure and function of the cells. Due to implication of multi-systemic complexities of signalling pathways in NDDs, the causes and preventive mechanisms are not clearly delineated. The study was designed to investigate the potential signalling pathways involved in neuroprotective activities of purely isolated glucomoringin isothiocyanate (GMG-ITC) against H2O2-induced cytotoxicity in neuroblastoma (SH-SY5Y) cells. GMG-ITC was isolated from Moringa oleifera seeds, and confirmed with NMR and LC-MS based methods. Gene expression analysis of phase II detoxifying markers revealed significant increase in the expression of all the genes involved, due to GMG-ITC pre-treatment. GMG-ITC also caused significant decreased in the expression of NF-kB, BACE1, APP and increased the expressions of IkB and MAPT tau genes in the differentiated cells as confirmed by multiplex genetic system analysis. The effect was reflected on the expressed proteins in the differentiated cells, where GMG-ITC caused increased in expression level of Nrf2, SOD-1, NQO1, p52 and c-Rel of nuclear factor erythroid factor 2 (Nrf2) and nuclear factor kappa-B (NF-kB) pathways respectively. The findings revealed the potential of GMG-ITC to abrogate oxidative stress-induced neurodegeneration through Nrf2 and NF-kB signalling pathways.
Glutamate is the major mediator of excitatory signals in the mammalian central nervous system. Extreme amounts of glutamate in the extracellular spaces can lead to numerous neurodegenerative diseases. We aimed to clarify the potential of the following vitamin E isomers, tocotrienol-rich fraction (TRF) and α-tocopherol (α-TCP), as potent neuroprotective agents against glutamate-induced injury in neuronal SK-N-SH cells. Cells were treated before and after glutamate injury (pre- and post-treatment, respectively) with 100-300 ng/ml TRF/α-TCP. Exposure to 120 mM glutamate significantly reduced cell viability to 76% and 79% in the pre- and post-treatment studies, respectively; however, pre- and post-treatment with TRF/α-TCP attenuated the cytotoxic effect of glutamate. Compared to the positive control (glutamate-injured cells not treated with TRF/α-TCP), pre-treatment with 100, 200, and 300 ng/ml TRF significantly improved cell viability following glutamate injury to 95.2%, 95.0%, and 95.6%, respectively (p<0.05).The isomers not only conferred neuroprotection by enhancing mitochondrial activity and depleting free radical production, but also increased cell viability and recovery upon glutamate insult. Our results suggest that vitamin E has potent antioxidant potential for protecting against glutamate injury and recovering glutamate-injured neuronal cells. Our findings also indicate that both TRF and α-TCP could play key roles as anti-apoptotic agents with neuroprotective properties.