Journal: Cell biology international
To examine cytokine production in response to RSV infection, we assessed the levels of 29 cytokines released from RSV-infected human foetal lung fibroblasts. We also examined the relationships between the effects of fluticasone propionate and various signalling pathways in the cells. Twenty-four hours after infection (1MOI), RSV-infected cells released cytokines, for example proinflammatory cytokines (IL-1β, IL-6 and TNF-α), anti-inflammatory (IL-1ra), Th1 (IFN-γ, IFN-λ1a, IL-2 and IL-12), Th2 (IL-4, IL-5, IL-10 and IL-13), granulopoiesis-inducing (G-CSF and GM-CSF), eosinophil recruitment-inducing (eotaxin and RANTES) and neutrophil recruitment-inducing cytokines (IL-8, IP-10, MCP-1 and MIP-1α). Aberrant release of most was significantly suppressed by fluticasone propionate. Twelve hours after RSV infection, increased phosphorylation of Akt, p38 MAPK, ERK1/2 and IκB-α was noted. Fluticasone propionate suppressed the phosphorylation of Akt, p38 MAPK, and ERK1/2, but not IκB-α, in virus-infected cells. TLR-4 expression was unchanged in control and RSV-infected cells, and TLR-3 and RIG-I expression was not detected. The results indicate that RSV infection induces aberrant production and release of certain cytokines through these signalling pathways in human lung fibroblasts. Overproduction and imbalance of these cytokines may be associated with the pathophysiology of RSV-induced excessive and allergic inflammation.
MicroRNA-455 (miRNA-455), which is downregulated in human cancer, potently mediates the multiple steps of carcinogenesis. However, the role of miR-455 in non-small cell lung cancer (NSCLC) carcinogenesis remains unclear. In present study, we determined the mature miRNA-455 expression in NSCLC tissues and cells by real-time PCR. Follow-up studies examined the effects of a miR-455 mimic (gain of function) on cell proliferation, migration and invasion. Our data indicate that miR-455 was significantly down-regulated in NSCLC cell lines and tissues. In functional assays, overexpression of miR-455 suppressed the proliferation, migration and invasion of NSCLC cell lines. Data from reporter assays showed that miR-455 directly binds to 3'UTR of ZEB1 and suppresses the endogenous level of ZEB1 protein expression. Furthermore, overexpression of ZEB1 reverses miR-455-suppressed malignant phenotype of NSCLC cells. Moreover, we found that upregulation of ZEB1 expression is inversely associated with miR-455 expression in NSCLC tissues. Taken together, miR-455 as an anti-oncogene in non-small cell lung cancer through up-regulation of ZEB1 and serve as a potential therapeutic target in NSCLC.
Parkinson’s disease (PD), the second-most prevalent neurodegenerative disease, is primarily characterized by neurodegeneration in the substantia nigra pars compacta, resulting in motor impairment. Loss-of-function mutations in parkin are the major cause of the early-onset familial form of the disease. Although rodents deficient in parkin (parkin(-/-)) have some dopaminergic system dysfunction associated with central oxidative stress and energy metabolism deficiencies, these animals only display nigrostriatal pathway degeneration under inflammatory conditions. This study investigated the impact of the inflammatory stimulus induced by lypopolisaccharide (LPS) on tetrahydrobiopterin (BH4) synthesizing enzymes (de novo and salvage pathways), since this cofactor is essential for dopamine synthesis. The mitochondrial content and architecture was investigated in the striatum of LPS-exposed parkin(-/-) mice. As expected, the LPS (0.33 mg / kg; i.p.) challenge compromised spontaneous locomotion and social interaction with juvenile parkin(-/-) and WT mice. Moreover, the genotype impacted the kinetics of the investigation of the juvenile. The inflammatory scenario did not induce apparent changes in mitochondrial ultrastructure; however, it increased the quantity of mitochondria, which were of smaller size, and provoked the perinuclear distribution of the organelle. Furthermore, the BH4 de novo biosynthetic pathway failed to be up-regulated in the LPS challenge, a well-known stimulus for its activation. The LPS treatment increased sepiapterin reductase (SPR) expression, suggesting compensation by the salvage pathway. This might indicate that dopamine synthesis is compromised in parkin(-/-) mice under inflammatory conditions. Finally, this scenario impaired the striatal expression of the transcription factor BDNF, possibly favoring cell death.
MicroRNAs have been known to function as important regulators in the vascular system, with various physiopathological effects such as vascular remodeling and hypertension modulation. We aimed to explore whether microRNA-150 (miR-150) regulates endothelial cell function and vascular remodeling in acute coronary syndrome (ACS), and the involvement of PTX3 and NF-κB signaling pathway. Ten normal mice and sixty ApoE-/- mice were chosen, and their coronary artery tissues and endothelial cells were extracted. ApoE-/- mice were injected with a series of inhibitors or mimics for miR-150, or siRNA against PTX3. The miR-150 expression, NF-κB1, RELA, and PTX3 mRNA expressions were assessed by reverse transcription quantitative polymerase chain reaction, and pentraxin-3, p-P50, and p-P65 protein expression by western blot analysis. Cell viability and migration were assessed by MTT assay and scratch test. Matrigel tube formation assay was employed to determine vascular remodeling of endothelial cells. The dual-luciferase reporter assay verified that PTX3 was a target of miR-150. Mice with ACS presented with decreased miR-150 but increased PTX3. It was observed that the miR-150 mimics and siRNA against PTX3 reduced levels of PTX3, NF-κB1 and RELA in mice, and the miR-150 inhibitor reversed the tendency. The in vitro cell experimentation proved that miR-150 might facilitate endothelial cell proliferation, migration, and restrain vascular remodeling via inhibiting PTX3 expression. On the basis of the results of this study, it was hypothesized that miR-150 could possibly maintain endothelial cell function and suppress vascular remodeling by inhibiting PTX3 through the NF-κB signaling pathway in mice with ACS.
Colorectal cancer is one of the global causes of cancer deaths. Cancer stem cells (CSCs) inside the tumour niche responsible for metastasis and relapses, and hence need to be targeted for cancer therapeutics. Although dietary fibre and lifestyle changes have been recommended as measures for colorectal cancer prevention, no such recommendations are available for using water soluble vitamins as prophylaxis measure for colorectal cancers. High dose of Vitamin C has been proven to selectively kill colon cancer cells having BRAF and KRAS mutations by inducing oxidative stress. In this study, we show for the first time the opposing effects of the low and high dose of Vitamin C and vitamin B3 on colon CSCs isolated from HT- 29 and HCT-15 colorectal carcinoma cell lines. At small doses, both of these vitamins exerted a cell proliferative effect only on CSCs, while there was no change in the proliferation status of non-stem cancer cells and wild-type (WT) populations. On the other hand, the death effects induced by high doses of Vitamin C and B3 were of the order of 50-60% and ∼30% on CSCs from HT-29 and HCT15 respectively. Interestingly, the control fibroblast cell line (NIH3T3) was highly refractory all the tested concentrations of Vitamin C and B3, except for the highest dose- 10,000 µg of Vitamin C that induced only 15% of cell death. Hence, these results indicate the future scope of use of therapeutic doses of Vitamin C and B3 especially in patients with advanced colorectal cancer.
Sutherlandia frutescens is a medicinal plant, traditionally used to treat various types of human diseases, including cancer. Previous studies of several botanicals link suppression of prostate cancer growth with inhibition of the Gli/hedgehog (Gli/Hh) signaling pathway. Here we hypothesized the anti-cancer effect of S. frutescens was linked to its inhibition of the Gli/Hh signaling in prostate cancer. We found a dose- and time-dependent growth inhibition in human prostate cancer cells, PC3 and LNCaP, and mouse prostate cancer cell, TRAMP-C2, treated with S. frutescens methanol extract (SLE). We also observed a dose-dependent inhibition of the Gli-reporter activity in Shh Light II and TRAMP-C2QGli cells treated with SLE. In addition, SLE can inhibit Gli/Hh signaling by blocking Gli1 and Ptched1 gene expression in the presence of a Gli/Hh signaling agonist (SAG). A diet supplemented with S. frutescens suppressed the formation of poorly differentiated carcinoma in prostates of TRAMP mice. Finally, we found Sutherlandioside D was the most potent compound in the crude extract that could suppress Gli-reporter in Shh Light II cells. Together, this suggests that the S. frutescens extract may exert anti-cancer effect by targeting Gli/Hh signaling, and Sutherlandioside D is one of the active compounds.
In peripheral nerve injuries, Schwann cells (SC) play pivotal roles in regenerating damaged nerve. However, the use of SC in clinical cell-based therapy is hampered due to its limited availability. In this study, we aim to evaluate the effectiveness of using an established induction protocol for human bone marrow derived-MSC (hBM-MSCs) transdifferentiation into a SC lineage. A relatively homogenous culture of hBM-MSCs was first established after serial passaging (P3), with profiles conforming to the minimal criteria set by International Society for Cellular Therapy (ISCT). The cultures (n = 3) were then subjected to a series of induction media containing β-mercapthoethanol, retinoic acid and growth factors. Quantitative RT-PCR, flow cytometry and immunocytochemistry analyses were performed to quantify the expression of specific SC markers i.e. S100, GFAP, MPZ and p75 NGFR in both undifferentiated and transdifferentiated hBM-MSCs. Based on these analyses, all markers were expressed in undifferentiated hBM-MSCs and MPZ expression (mRNA transcripts) was consistently detected before and after transdifferentiation across all samples. There was upregulation at the transcript level of more than two folds for NGF, MPB, GDNF, p75 NGFR post-transdifferentiation. This study highlights the existence of spontaneous expression of specific SC markers in cultured hBM-MSCs, inter-donor variability and that MSC transdifferentiation is a heterogenous process. These findings strongly oppose the use of a single marker to indicate SC fate. The heterogenous nature of MSC may influence the efficiency of SC transdifferentiation protocols. Therefore, there is an urgent need to re-define the MSC sub-populations and revise the minimal criteria for MSC identification.
Hyperbaric oxygen is a clinical treatment that contributes to wound healing by increasing fibroblasts proliferation, collagen synthesis, and production of growth factors, inducing angiogenesis and inhibiting antimicrobial activity. It also has been shown that hyperbaric oxygen treatment (HBO), through the activation of nitric oxide synthase promotes an increase in the nitric oxide levels that may improve endothelial progenitor cells (EPC) mobilization from bone marrow to the peripheral blood and stimulates the vessel healing process. However, cellular mechanisms involved in cell proliferation and activation of EPC after HBO treatment remain unknown. Therefore, the present work aimed to analyze the effect of HBO on the proliferation of pre-treated bone marrow-derived EPC with TNF-alpha. Also, we investigated the expression of ICAM and eNOS by immunochemistry, the production of reactive species of oxygen and performed an in vitro wound healing. Although 1h of HBO treatment did not alter the rate of in vitro wound closure or cell proliferation, it increased eNOS expression and decreased ICAM expression and reactive oxygen species production in cells pre-treated with TNF-alpha. These results indicate that HBO can decrease the inflammatory response in endothelial cells mediated by TNF-alpha, and thus, promote vascular recovery after injury.
Dental pulp stem cells have emerged as a preferred source of mesenchymal stem cells, because of its easy availability and high stem cell content. Dental pulp is a specific fibrous tissue that contains heterogeneous populations of odontoblasts, fibroblasts, pericytes, progenitors, stem cells, leukocytes and neuronal cells. In this study, we propose sustained explant culture as a simple, economical and efficient process to isolate dental pulp stem cells from human Dental pulp Tissue. Historically explant cultures were used to get fibroblast cells from embryonic chick heart using plasma clot cultures. The subculture was performed by lifting mother explant (original explant) and grafting it in a new plasma clot. We modified this age old technique to suit the modern times. Here we demonstrate for the first time that the mother explant (E0) of human dental pulp tissue could be sub-cultured consecutively seven times (E7) without displacement. This technique is highly reproducible and permits growth and proliferation of dental pulp stem cells yielding an enriched homogeneous mesenchymal stem cells population in the first passage itself as revealed by surface marker expression. These dental pulp stem cells exhibit differentiation into adipogenic, chondrogenic and osteogenic lineage revealing their mesenchymal stem cell nature. We propose that dental pulp stem cells isolated by sustained explant culture are phenotypically and functionally comparable to those obtained by enzymatic method. It is a simple, inexpensive and gentle method, which may be preferred over the conventional techniques for obtaining stem cells from other tissue sources as well especially in cases of limited starting material.
Recent studies have been trying to find out how diet and metabolic changes such as dyslipidaemia, hyperglycaemia and hyperinsulinaemia can stimulate cancer progression. This investigation aimed to evaluate the effect of high concentrations of fatty acids and/or glucose in tumour prostate cells, focusing on the proliferation/migration profile and oxidative stress. PC3 cells were treated with high concentration of saturated fatty acid (palmitate, 100µM), glucose (220 mg/dL) or both for 24 or 48 h. Results demonstrated that PC3 cells showed a significant increase in proliferation after 48 h of treatment with glucose and palmitate + glucose. Cell proliferation was associated with reduced levels of AMPK phosphorylation in glucose group at 24 and 48 h of treatment, while palmitate one presented this result only after 48 h of treatment. Also, there was a significant increase in cell migration between time 0 and 48 h after all treatments, except in the control. Catalase activity was increased by palmitate in the beginning of treatment, while glucose presented a later effect. Also, nitrite production was increased by glucose only after 48 h, and the total antioxidant activity was enhanced by palmitate in the initial hours. Thus, we conclude that the high concentration of the saturated fatty acid palmitate and glucose in vitro influences PC3 cells and stimulates cellular activities related to carcinogenesis such as cell proliferation, migration and oxidative stress in different ways. Palmitate presents a rapid and initial effect, while a glucose environment stimulates cells later on, maintaining high levels of cell proliferation.