Tocotrienol-Rich Fraction and Levodopa Regulate Proteins Involved in Parkinson's Disease-Associated Pathways in Differentiated Neuroblastoma Cells: Insights from Quantitative Proteomic Analysis.

Tocotrienol-rich fraction (TRF), a palm oil-derived vitamin E fraction, is reported to possess potent neuroprotective effects. However, the modulation of proteomes in differentiated human neuroblastoma SH-SY5Y cells (diff-neural cells) by TRF has not yet been reported. This study aims to investigate the proteomic changes implicated by TRF in human neural cells using a label-free liquid-chromatography-double mass spectrometry (LC-MS/MS) approach. Levodopa, a drug used in the treatment of Parkinson's disease (PD), was used as a drug control. The human SH-SY5Y neuroblastoma cells were differentiated for six days and treated with TRF or levodopa for 24 h prior to quantitative proteomic analysis. A total of 81 and 57 proteins were differentially expressed in diff-neural cells following treatment with TRF or levodopa, respectively. Among these proteins, 32 similar proteins were detected in both TRF and levodopa-treated neural cells, with 30 of these proteins showing similar expression pattern. The pathway enrichment analysis revealed that most of the proteins regulated by TRF and levodopa are key players in the ubiquitin-proteasome, calcium signalling, protein processing in the endoplasmic reticulum, mitochondrial pathway and axonal transport system. In conclusion, TRF is an essential functional food that affects differential protein expression in human neuronal cells at the cellular and molecular levels.

Immune Biomarkers in Blood from Sarcoma Patients: A Pilot Study.

The main role of the host immune system is to identify and eliminate cancer cells, which is a complex process, but it is not a fail-safe mechanism. Many sarcoma patients succumb to this disease despite treatments rendered. The aim of this pilot study was to compare the levels of CD4+ T-cells, T-regulatory (Treg) cells, and cytokines such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-17A (IL-17A), and transforming growth factor-beta-1 (TGF-ß1) in peripheral blood leukocytes of sarcoma patients and healthy controls. For gene expression studies, total ribonucleic acid (RNA) was extracted from peripheral blood leukocytes and genes that were differentially regulated in peripheral blood leukocytes of sarcoma patients compared with healthy controls were determined using a commercial T-helper cell differentiation quantitative polymerase chain reaction (qPCR) array. Flow cytometer analysis was performed on blood samples from 26 sarcoma patients and 10 healthy controls to identify the levels of CD4+ T-cells and T-reg cells. The level of cytokines in plasma and culture supernatant were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. A marked reduction in the percentage of CD4+ T-cells (p = 0.037) and levels of TNF-α (p = 0.004) and IFN-γ (0.010) was observed in sarcoma patients. Gene expression analysis showed five genes (homeobox A10 (HOXA10), GATA binding protein 3 (GATA3), prostaglandin D2 receptor 2 (PTGDR2), thymocyte selection associated high mobility group box (TOX), and C-C motif chemokine receptor 3 (CCR3)) were dysregulated (p < 0.05) in sarcoma patients. This study suggests that T-helper-1 immune responses are reduced in sarcoma patients.

6-Hydroxydopamine Induces Neurodegeneration in Terminally Differentiated SH-SY5Y Neuroblastoma Cells via Enrichment of the Nucleosomal Degradation Pathway: a Global Proteomics Approach.

The SH-SY5Y human neuroblastoma cells have been used for decades as a cell-based model of dopaminergic neurons to explore the underlying science of cellular and molecular mechanisms of neurodegeneration in Parkinson's disease (PD). However, data revealing the protein expression changes in 6-OHDA induced cytotoxicity in differentiated SH-SY5Y cells remain void. Therefore, we investigated the differentially regulated proteins expressed in terminally differentiated SH-SY5Y cells (differ-SH-SY5Y neural cells) exposed to 6-hydroxydopamine (6-OHDA) using the LC-MS/MS technology and construed the data using the online bioinformatics databases such as PANTHER, STRING, and KEGG. Our studies demonstrated that the neuronal development in differ-SH-SY5Y neural cells was indicated by the overexpression of proteins responsible for neurite formations such as calnexin (CANX) and calreticulin (CALR) besides significant downregulation of ribosomal proteins. The enrichment of the KEGG ribosome pathway was detected with significant downregulation (p < 0.05) of all the 21 ribosomal proteins in differ-SH-SY5Y neural cells compared with undifferentiated cells. Whereas in the PD model, the pathological changes induced by 6-OHDA were indicated by the presence of unfolded and misfolded proteins, which triggered the response of 10 kDa heat shock proteins (HSP), namely HSPE1 and HSPA9. Moreover, the 6-OHDA-induced neurodegeneration in differ-SH-SY5Y neural cells also upregulated the voltage-dependent anion-selective channel protein 1 (VDAC1) protein and enriched the KEGG systemic lupus erythematosus (SLE) pathway that was regulated by 17 histone proteins (p < 0.05) in differ-SH-SY5Y neural cells. These results suggest that the nucleosomal degradation pathway may have regulated the 6-OHDA induced neurodegeneration in PD cell-based model, which is reflected by increased apoptosis and histone release in differ-SH-SY5Y neural cells.

Clinically Relevant Genes and Proteins Modulated by Tocotrienols in Human Colon Cancer Cell Lines: Systematic Scoping Review.

The last decade has witnessed tremendous growth in tocotrienols (T3s) research, especially in the field of oncology, owing to potent anticancer property. Among the many types of cancers, colorectal cancer (CRC) is growing to become a serious global health threat to humans. Chemoprevention strategies in recent days are open to exploring alternative interventions to inhibit or delay carcinogenesis, especially with the use of bioactive natural compounds, such as tocotrienols. This scoping review aims to distil the large bodies of literature from various databases to identify the genes and their encoded modulations by tocotrienols and to explicate important mechanisms via which T3s combat CRC. For this scoping review, research papers published from 2010 to early 2021 related to T3s and human CRC cells were reviewed in compliance with the PRISMA guidelines. The study included research articles published in English, searchable on four literature databases (Ovid MEDLINE, PubMed, Scopus, and Embase) that reported differential expression of genes and proteins in human CRC cell lines following exposure to T3s. A total of 12 articles that fulfilled the inclusion and exclusion criteria of the study were short-listed for data extraction and analysis. The results from the analysis of these 12 articles showed that T3s, especially its γ and δ analogues, modulated the expression of 16 genes and their encoded proteins that are associated with several important CRC pathways (apoptosis, transcriptional dysregulation in cancer, and cancer progression). Further studies and validation work are required to scrutinize the specific role of T3s on these genes and proteins and to propose the use of T3s to develop adjuvant or multi-targeted therapy for CRC.

Reduced infiltration of regulatory T cells in tumours from mice fed daily with gamma-tocotrienol supplementation.

Gamma-tocotrienol (γT3) is an analogue of vitamin E with beneficial effects on the immune system, including immune-modulatory properties. This study reports the immune-modulatory effects of daily supplementation of γT3 on host T helper (Th) and T regulatory cell (Treg ) populations in a syngeneic mouse model of breast cancer. Female BALB/c mice were fed with either γT3 or vehicle (soy oil) for 2 weeks via oral gavage before they were inoculated with syngeneic 4T1 mouse mammary cancer cells (4T1 cells). Supplementation continued until the mice were euthanized. Mice (n = 6) were euthanized at specified time-points for various analysis (blood leucocyte, cytokine production and immunohistochemistry). Tumour volume was measured once every 7 days. Gene expression studies were carried out on tumour-specific T lymphocytes isolated from splenic cultures. Supplementation with γT3 increased CD4+ (p < 0.05), CD8+ (p < 0.05) T-cells and natural killer cells (p < 0.05) but suppressed Treg cells (p < 0.05) in peripheral blood when compared to animals fed with the vehicle. Higher interferon (IFN)-γ and lower transforming growth factor (TGF)-ꞵ levels were noted in the γT3 fed mice. Immunohistochemistry findings revealed higher infiltration of CD4+ cells, increased expression of interleukin-12 receptor-beta-2 (IL-12ꞵ2R), interleukin (IL)-24 and reduced expression of cells that express the forkhead box P3 (FoxP3) in tumours from the γT3-fed animals. Gene expression studies showed the down-regulation of seven prominent genes in splenic CD4+ T cells isolated from γT3-fed mice. Supplementation with γT3 from palm oil-induced T cell-dependent cell-mediated immune responses and suppressed T cells in the tumour microenvironment in a syngeneic mouse model of breast cancer.

Tocotrienols Ameliorate Neurodegeneration and Motor Deficits in the 6-OHDA-Induced Rat Model of Parkinsonism: Behavioural and Immunohistochemistry Analysis.

Parkinson's disease (PD) is a debilitating neurodegenerative disease, which progresses over time, causing pathological depigmentation of the substantia nigra (SN) in the midbrain due to loss of dopaminergic neurons. Emerging studies revealed the promising effects of some nutrient compounds in reducing the risk of PD. One such nutrient compound that possess neuroprotective effects and prevents neurodegeneration is tocotrienol (T3), a vitamin E family member. In the present study, a single dose intracisternal injection of 250 µg 6-hydroxydopamine (6-OHDA) was used to induce parkinsonism in male Sprague Dawley (SD) rats. Forty-eight hours post injection, the SD rats were orally supplemented with alpha (α)- and gamma (γ)-T3 for 28 days. The neuroprotective effects of α- and γ-T3 were evaluated using behavioural studies and immunohistochemistry (IHC). The findings from this study revealed that supplementation of α- and γ-T3 was able to ameliorate the motor deficits induced by 6-OHDA and improve the neuronal functions by reducing inflammation, reversing the neuronal degradation, and preventing further reduction of dopaminergic neurons in the SN and striatum (STR) fibre density.

Gamma-tocotrienol modifies methylation of HOXA10, IRF4 and RORα genes in CD4+ T-lymphocytes: Evidence from a syngeneic mouse model of breast cancer.

DNA methylation plays a crucial role in polarising naïve lymphocytes towards their various sub-populations to fight against many immune challenges including establishment of tumour. Gamma-tocotrienol (γT3) is a natural form of vitamin E, reported to possess anticancer and immunomodulatory effects. This study reports the anticancer effects of γT3 through modulation of DNA methylation in several genes in CD4+ T-lymphocytes using a syngeneic mouse model of breast cancer. Female BALB/c mice were fed with γT3 or vehicle (soy oil) for two-weeks via oral gavage before they were inoculated with 4T1 mouse mammary cancer cells. Supplementation continued until the mice were sacrificed. At autopsy, blood was collected via cardiac puncture and CD4+ T-cells were isolated for DNA extraction. The DNA was analysed using the EpiTech Methyl II mouse T-helper cell differentiation PCR array. γT3 supplementation reduced tumour growth in the tumour-induced animals and modulated host immune system by inducing changes in DNA methylation patterns of the HOXA10, IRF4 and RORα genes, which are involved in differentiation and clonal expansion of CD4+ T-cells. Results suggest that γT3 may enhance cell-mediated immune response in mice with breast cancer by inducing changes in DNA methylation pattern.

Advancing the Role of Gamma-Tocotrienol as Proteasomes Inhibitor: A Quantitative Proteomic Analysis of MDA-MB-231 Human Breast Cancer Cells.

Tocotrienol, an analogue of vitamin E has been known for its numerous health benefits and anti-cancer effects. Of the four isoforms of tocotrienols, gamma-tocotrienol (γT3) has been frequently reported for their superior anti-tumorigenic activity in both in vitro and in vivo studies, when compared to its counterparts. In this study, the effect of γT3 treatment in the cytoplasmic and nuclear fraction of MDA-MB-231 human breast cancer cells were assessed using the label-free quantitative proteomics analysis. The cytoplasmic proteome results revealed the ability of γT3 to inhibit a group of proteasome proteins such as PSMA, PSMB, PSMD, and PSME. The inhibition of proteasome proteins is known to induce apoptosis in cancer cells. As such, the findings from this study suggest γT3 as a potential proteasome inhibitor that can overcome deficiencies in growth-inhibitory or pro-apoptotic molecules in breast cancer cells. The nuclear proteome results revealed the involvement of important nuclear protein complexes which hardwire the anti-tumorigenesis mechanism in breast cancer following γT3 treatment. In conclusion, this study uncovered the advancing roles of γT3 as potential proteasomes inhibitor that can be used for the treatment of breast cancer.

Tocotrienols Modulate Breast Cancer Secretomes and Affect Cancer-Signaling Pathways in MDA-MB-231 Cells: A Label-Free Quantitative Proteomic Analysis.

Tocotrienols (T3), a family of vitamin E, are reported to possess potent anti-cancer effects but the molecular mechanisms behind these effects still remain unclear. The aim of this study was to investigate how T3 exert anti-cancer effects on MDA-MB-231 human breast cancer cells. The MDA-MB-231 cells were chosen for this study as they are triple-negative and highly metastatic cells, which form aggressive tumors in experimental models. The MDA-MB-231 cells were treated with varying concentrations (0-20 µg mL-1) of gamma (γ) or delta (δ) T3 and the secretome profiles of these cells treated with half maximal inhibitory concentration (IC50) of γT3 (5.8 µg mL-1) or δT3 (4.0 µg mL-1) were determined using label-free quantitative proteomic strategy. A total of 103, 174 and 141 proteins were identified with ProteinLynx Global Server (PLGS) score of more than 200 and above 25% sequence coverage in the untreated control and T3-treated cell culture supernatant respectively. A total of 18 proteins were dysregulated between untreated control and T3 (δT3 or γT3) treated conditions. The results showed that T3 treatment downregulated the exogenous Cathepsin D and Serpine1 proteins but upregulated Profilin-1 protein, which play a key role in breast cancer in the MDA-MB-231 cells. These findings strongly suggest that T3 may induce differential expression of secreted proteins involved in the cytoskeletal regulation of RHO GTPase signaling pathway.

Modulatory effects of mesenchymal stem cells on leucocytes and leukemic cells: A double-edged sword?

Mesenchymal stem cells (MSCs) have drawn much attention amongst stem cell researchers in the past few decades. The ability of the MSC to differentiate into cells of mesodermal and non-mesodermal origins has made them an attractive approach for cell-based therapy and regenerative medicine. The MSCs have immunosuppressive activities that may have considerable therapeutic values in autoimmune diseases. However, despite the many beneficial effects reported, there is a growing body of evidence, which suggests that MSCs could be a culprit of enhanced tumour growth, metastasis and drug resistance in leukaemia, via some modulatory effects. Many controversies regarding the interactions between MSCs and leukaemia still exist. Furthermore, the role of MSCs in leukemogenesis and its progression remain largely unknown. Hence it is important to understand how the MSCs modulate leukaemia before these cells could be safely used in the treatment of leukaemia patients.

Quercetin glycosides induced neuroprotection by changes in the gene expression in a cellular model of Parkinson's disease.

Quercetin glycosides, rutin and isoquercitrin, are potent antioxidants that have been found to possess neuroprotective effect in diseases like Parkinson's and Alzheimer's disease. In the present study, we have examined the gene expression changes with rutin and isoquercitrin pretreatment on 6-hydroxydopamine (6-OHDA)-treated toxicity in rat pheochromocytoma (PC12) cells. PC12 cells were pretreated with rutin or isoquercitrin and subsequently exposed to 6-OHDA. Rutin-pretreated PC12 attenuated the Park2, Park5, Park7, Casp3, and Casp7 genes which were expressed significantly in the 6-OHDA-treated PC12 cells. Rutin upregulated the TH gene which is important in dopamine biosynthesis, but isoquercitrin pretreatment did not affect the expression of this gene. Both rutin and isoquercitrin pretreatments upregulated the ion transport and antiapoptotic genes (NSF and Opa1). The qPCR array data were further validated by qRT-PCR using four primers, Park5, Park7, Casp3, and TH. This finding suggests that changes in the expression levels of transcripts encoded by genes that participate in ubiquitin pathway and dopamine biosynthesis may be involved in Parkinson's disease.

Tocotrienol-treated MCF-7 human breast cancer cells show down-regulation of API5 and up-regulation of MIG6 genes.

BACKGROUND: Tocotrienols belong to the vitamin E family and have multiple anticancer effects, such as antiproliferative, antioxidant, pro-apoptosis and antimetastatic. This study aimed to identify the genes that are regulated in human breast cancer cells following exposure to various isomers of vitamin E as these may be potential targets for the treatment of breast cancer. MATERIALS AND METHODS: Gene expression profiling was performed with MCF-7 cells at inhibitory conditions of IC(50) using Illumina's Sentrix Array Human-6 BeadChips. The expression levels of selected differentially expressed genes were verified by quantitative real-time-PCR (qRT-PCR). RESULTS: The treatment with tocotrienol-rich palm oil fraction (TRF), α-tocopherol and isomers of tocotrienols (α, γ, and δ) altered the expression of several genes that code for proteins involved in the regulation of immune response, tumour growth and metastatic suppression, apoptotic signalling, transcription, protein biosynthesis regulation and many others. CONCLUSION: Treatment of human MCF-7 cells with tocotrienol isomers causes the down-regulation of the API5 gene and up-regulation of the MIG6 gene and the differential expression of other genes reported to play a key role in breast cancer biology.