Βeta-eudesmol reduces stem cell factor-induced mast cell migration

Previously, we showed the inhibitory effects of β-eudesmol on mast cell-mediated allergic inflammatory responses. Stem cell factor (SCF) participates in allergic reactions through the differentiation and migration of mast cells. However, the effects of β-eudesmol on SCF-mediated allergic reactions are poorly understood. Herein, we showed that a treatment of rat peritoneal mast cells (RPMCs) with β-eudesmol markedly suppressed SCF-induced mast cell migration and morphological alterations in a concentration-dependent manner. β-eudesmol also reduced F-actin formation. The activation of Fyn kinase, Rac1 GTPase, and p38 mitogen-activated protein kinases as increased by SCF was reduced by β-eudesmol. Moreover, β-eudesmol significantly abolished SCF-induced tumor necrosis factor-α and intercellular adhesion molecule-1 production without cytotoxicity. These results demonstrate that β-eudesmol has an anti-allergic inflammatory effect through the inhibition of mast cell migration and inflammatory cytokine production.     

Pharmacology on microfluidics: multimodal analysis for studying cell–cell interaction

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Application of high-content cell imaging system in cell proliferation detection北大核心CSCD

Aim To compare high-content cell imaging system and other methods in detecting cell proliferation, including the traditional thymidine (3H-TdR) incorporation method, methyl thiazolyl tetrazolium (MTT) method and cell counting kit-8 (CCK-8) method. Methods The fibroblast-like synovial cells (FLS) were used as the study object to observe the sensitivity and stability of FLS proliferation in different methods by using the usual proliferative stimulant tumor necrosis factor-a (TNF-a) and the known proliferation inhibitor methotrexate at different concentrations. Results The 3H-TdR method and high-content cell imaging could detect a significant inhibitory effect of 1 nmol L"1 MTX on FLS cell proliferation, MTT assay and CCK-8 method could detect the significant inhibitory effect of 10 nmol L"1 MTX on FLS cell proliferation. 3H-TdR method was found to have a large degree of discretization in the data set, with a standard deviation of 32. 61%-61. 36%, and the MTT method was 11.9%-17.8%, the CCK-8 method was 17.15%-32. 88%, and the high-content cell imaging system method was 12.66%-26.54%. Conclusion The method of high-content cell imaging system is more accurate and stable for detecting cell proliferation. © 2018 Publication Centre of Anhui Medical University. All rights reserved.     

The XS106 cell: a Langerhans’ cell surrogate with a selective type 2 phenotype

Epidermal Langerhans’ cells (LC) play important roles in initiating and regulating cutaneous immune responses. However, LC comprise less than 3% of all epidermal cells and consequently are difficult to study ex vivo. In the current investigations, we have examined the utility of the XS106 cell line, a dendritic cell (DC) line derived from mouse epidermis, as a surrogate for LC. Membrane marker expression, type 1- and type 2-associated chemokine production, and migration patterns have been characterised following treatment of XS106 cells with a range of toll-like receptor (TLR) ligands. Comparisons have been made with mouse bone marrow-derived DC- and LC-derived ex vivo. Like BMDC, XS106 cells expressed generic DC markers, in addition to displaying higher levels of skin DC markers compared with BMDC. XS106 cells and LC-enriched epidermal fractions both displayed higher constitutive expression of type 2-associated chemokines than type 1 chemokines. Furthermore, although treatment with a range of TLR ligands induced cytokine secretion by XS106 cells, only type 2 TLR ligands increased membrane marker expression of major histocompatibility complex class II and co-stimulatory molecules. Moreover, type 1-associated TLR ligands failed to induce selective type 1 chemokine secretion by XS106 cells. XS106 cells also displayed functional similarity to LC, migrating in response to chemokines that are known to induce the migration of LC. On the basis of membrane marker expression and selective type 2 polarisation XS106 cells provide a useful surrogate for LC.     

Antitumor activity of a novel bis-aziridinylnaphthoquinone （AZ4） mediat- ing cell cycle arrest and apoptosis in non-small cell lung cancer cell line NCI-H460

AIM: The cytotoxic activities of a series of bis-aziridinylnaphthoquinone, AZ1 to AZ4, on human lung carcinoma cell lines, H460, and normal lung cells fibroblast cell line, MRC-5, and the mechanisms of H460 cells induced by AZ4 were investigated. METHODS: The MTT assay was used to determine the cell proliferation. Cell cycle was analysed by FACS. The activity of caspase 3, 8 and 9 was determined by cell-permeable fluorogenic detection system. Western blot assay was used to evaluate the regulation of cyclin B, Cdc-2, p53, p21, and the Bcl-2 protein. RESULTS: AZ1 to AZ4 displayed various cytotoxicity activities against H460 and MRC-5 cells. Compared to those compounds, AZ4 was with the most effective agent among the 5 tested analogues at reducing H460 cell viability with an IC(50) value of 1.23 micromol/L; it also exhibited weak cytotoxicity against MRC-5 cells with an IC(50) value of 12.7 micromol/L. The results show that growth arrest on the G2-M phase of H460 cells induced by AZ4 for 24 h was discovered, and this might be altered with the reduced Cdc-2 protein expression of 47% at 2.0 micromol/L AZ4, but not with cyclin B protein expression. The AZ4 treated cells were then led to apoptosis after 48 h. This was associated with the activation of apoptotic enzyme caspase 3 and mediated by caspase 8, but not caspase 9 at various concentrations of AZ4 after being cultured for 48 h and 30 h, respectively. The anti-apoptotic protein (Bcl-2) expression in H460 cells altered by 39% with downregulation, and the p53 protein by 25% with upregulation after being cultured with 2.0 micromol/L AZ4 for 48 h. In a time-dependent manner, the expression of the p53 and p21 proteins were increased to the maximum at 24 h, and then decreased at 48. CONCLUSION: AZ4 represents a novel antitumor aziridinylnaphthoquinone with therapeutic potential against the non-small cell lung cancer cells.     

On-chip investigation of cell–drug interactions

Investigation of cell–drug interaction is of great importance in drug discovery but continues to pose significant challenges to develop robust, fast and high-throughput methods for pharmacologically profiling of potential drugs. Recently, cell chips have emerged as a promising technology for drug discovery/delivery, and their miniaturization and flow-through operation significantly reduce sample consumption while dramatically improving the throughput, reliability, resolution and sensitivity. Herein we review various types of miniaturized cell chips used in investigation of cell–drug interactions. The design and fabrication of cell chips including material selection, surface modification, cell trapping/patterning, concentration gradient generation and mimicking of in vivo environment are presented. Recent advances of on-chip investigations of cell–drug interactions, in particular the high-throughput screening, cell sorting, cytotoxicity testing, drug resistance analysis and pharmacological profiling are examined and discussed. It is expected that this survey can provide thoughtful basics and important applications of on-chip investigations of cell–drug interactions, thus greatly promoting research and development interests in this area.     

Cancer cell death by programmed necrosis?

A recent paper by Zong et al. describes how alkylating agents kill cells by a process they term "programmed necrosis," induced by excessive activation of PARP resulting in degradation of cytosolic NAD(+) and inhibition of glycolysis. We argue that it is not obvious whether chemotherapy in patients can induce sufficient NAD(+) loss to affect glycolysis; that the "programmed" nature of the necrosis requires more evidence; and that there are mechanisms making cancer cells hypersensitive to DNA damage other than their high rate of aerobic glycolysis.     

Can cell systems biology rescue drug discovery?

The focus of innovation in current drug discovery is on new targets, yet compound efficacy and safety in biological models of disease - not target selection - are the criteria that determine which drug candidates enter the clinic. We consider a biology-driven approach to drug discovery that involves screening compounds by automated response profiling in disease models based on complex human-cell systems. Drug discovery through cell systems biology could significantly reduce the time and cost of new drug development.     

Nano-topography: Quicksand for cell cycle progression?

The 3-D spatial and mechanical features of nano-topography can create alternative environments, which influence cellular response. In this paper, murine fibroblast cells were grown on surfaces characterized by protruding nanotubes. Cells cultured on such nano-structured surface exhibit stronger cellular adhesion compared to control groups, but despite the fact that stronger adhesion is generally believed to promote cell cycle progression, the time cells spend in G1 phase is doubled. This apparent contradiction is solved by confocal microscopy analysis, which shows that the nano-topography inhibits actin stress fiber formation. In turn, this impairs RhoA activation, which is required to suppress the inhibition of cell cycle progression imposed by p21/p27. This finding suggests that the generation of stress fibers, required to impose the homeostatic intracellular tension, rather than cell adhesion/spreading is the limiting factor for cell cycle progression. Indeed, nano-topography could represent a unique tool to inhibit proliferation in adherent well-spread cells.     

Epigallocatechin-3-gallate regulates cell growth, cell cycle and phosphorylated nuclear factor-κB in human dermal fibroblasts

Aim:

To investigate the effects of (−)epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, on cell growth, cell cycle and phosphorylated nuclear factor-κB (pNF-κB) expression in neonatal human dermal fibroblasts (nHDFs).

Methods:

The proliferation and cell-cycle of nHDFs were determined using WST-8 cell growth assay and flow cytometry, respectively. The apoptosis was examined using DNA ladder and Annexin V-FITC assays. The expression levels of pNF-κB and cell cycle-related genes and proteins in nHDFs were measured using cDNA microarray analyses and Western blot. The cellular uptake of EGCG was examined using fluorescence (FITC)-labeled EGCG (FITC-EGCG) in combination with confocal microscopy.

Results:

The effect of EGCG on the growth of nHDFs depended on the concentration tested. At a low concentration (200 μmol/L), EGCG resulted in a slight decrease in the proportion of cells in the S and G₂/M phases of cell cycle with a concomitant increase in the proportion of cells in G₀/G₁ phase. At the higher doses (400 and 800 μmol/L), apoptosis was induced. The regulation of EGCG on the expression of pNF-κB was also concentration-dependent, whereas it did not affect the unphosphorylated NF-κB expression. cDNA microarray analysis showed that cell cycle-related genes were down-regulated by EGCG (200 μmol/L). The expression of cyclins A/B and cyclin-dependent kinase 1 was reversibly regulated by EGCG (200 μmol/L). FITC-EGCG was found to be internalized into the cytoplasm and translocated into the nucleus of nHDFs.

Conclusion:

EGCG, through uptake into cytoplasm, reversibly regulated the cell growth and expression of cell cycle-related proteins and genes in normal fibroblasts.     

A role for the cell adhesion molecule CD44 and sulfation in leukocyte-endothelial cell adhesion during an inflammatory response?

CD44 is a widely expressed cell adhesion molecule that has been implicated in a variety of biological processes including lymphopoiesis, angiogenesis, wound healing, leukocyte extravasation at inflammatory sites, and tumor metastasis. The adhesive function of CD44, like other molecules involved in inducible adhesion, is tightly regulated. Post-translational modifications, isoform expression, aggregation state, and protein associations all can affect the ligand binding properties of CD44, and these can vary depending on the cell type and the activation state of the cell. The most extensively characterized ligand for CD44 is hyaluronan, a component of the extracellular matrix. Interactions between CD44 and hyaluronan can mediate both cell-cell and cell-extracellular matrix adhesion. In the immune system, both the selectin molecules and CD44 have been implicated in the initial binding of leukocytes to endothelial cells at an inflammatory site. Sulfation is required for selectin-mediated leukocyte-endothelial cell interactions, and, recently, inducible sulfation also was shown to regulate CD44-mediated leukocyte adhesion to endothelial cells. Sulfation, therefore, may be important in the regulation of cell adhesion at inflammatory sites. In this commentary we have reviewed the molecular aspects of CD44 and the mechanisms that regulate its binding to hyaluronan. In addition, we have summarized the role of CD44 and hyaluronan in mediating leukocyte-endothelial cell interactions and have discussed how this interaction may be regulated. Finally, we examined the potential role of sulfation as an inducible means to regulate CD44-mediated leukocyte adhesion and as a more general mechanism to regulate leukocyte-endothelial cell interactions.     

Cytotoxic activities and effects of atractylodin and β-eudesmol on the cell cycle arrest and apoptosis on cholangiocarcinoma cell line

Cholangiocarcinoma (CCA) is the cancer of bile duct with high mortality rate particularly in Thailand. The clinical efficacy of the standard chemotherapeutics remains unsatisfactory, and therefore, discovery and development of the new alternative drugs with high efficacy and tolerability is needed. The aim of the study was to investigate cytotoxic activity as well as the underlying mechanisms through which atractylodin and β-eudesmol exert their activities on CCA cell growth inhibition, cell cycle arrest, and cell apoptosis. Effects of the compounds on cell cytotoxicity, cell cycle arrest, and cell apoptosis were analyzed using MTT assay, BD Cycletest? Plus DNA kit, and FITC Annexin V Apoptosis Detection Kit I, respectively. The cytotoxic activities of both compounds were concentration- and time-dependent. The IC₅₀ [mean (SD)] of atractylodin and β-eudesmol were 41.66 (2.51) and 39.33 (1.15) μg/ml respectively. Both promoted cell cycle arrest at G1 phase, and induced cell apoptosis through activation of caspase-3/7. The highest activity was observed at 48 h of exposure. Results suggest that these mechanisms are at least in part, explain the cell cytotoxic and anti-CCA activity of atractylodin and β-eudesmol shown in vitro and in vivo models.     

Diversified expression of aryl hydrocarbon receptor dependent genes in human laryngeal squamous cell carcinoma cell lines treated with β-naphthoflavone

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study the effect of administration of β-naphthoflavone (BNF), potent AhR ligand, on the expression of AhR, AhRR, CYP1A1, CYP1A2, CYP1B1, NQO1, GSTA1, ALDH3A1 and UGT1A genes encoding the enzymes controlled by AhR were examined in thirteen laryngeal tumor cell lines and in HepaRG cell line. The analyzed cell lines were derived from patients with squamous laryngeal cancer, with history of cigarette smoking and without signs of human papillomavirus types 16 and 18 infection in investigated cells. Quantitative real-time RT-PCR analysis revealed huge interindividual differences in expression of genes from AhR regulatory network. Our results strongly suggest predominant effect of DNA methylation on induction of CYP1A1 expression by AhR ligands as well. Our results indicate that differentiated HepaRG cell line appeared to be very good substitute for human liver in studies on xenobiotic metabolism by AhR regulated enzymes.     

Interaction of α-synuclein and a cell penetrating fusion peptide with higher eukaryotic cell membranes assessed by ¹⁹F NMR

We show that fluorine NMR can be used to monitor the insertion and change in conformation of a 1?F-labeled cell-penetrating peptide upon interacting with the cellular plasma membrane. α-Synuclein and a construct comprising a cell-penetrating peptide covalently attached to its N-terminus were studied. Important information about the interaction of the proteins with CHO-K1 cells was obtained by monitoring the diminution of 1?F resonances of 3-fluoro-l-tyrosine labeled proteins. For α-synuclein, a decrease in the resonance from position 39 was observed indicating that only the N-terminal third region of the protein interacts with plasma membrane. However, when the fusion construct was incubated with the cells, a decrease in the resonance from the fusion peptide region was noted with no change in the resonances from α-synuclein region. Longer incubation, studied by using confocal fluorescence microscopy, revealed that the fusion construct translocates into the cells, but α-synuclein alone did not cross the membrane in significant amounts.     

Murine macrophage cell lines contain μ3-opiate receptors

Opiate alkaloid-selective, opioid peptide-insensitive μ₃ receptors are present in three murine macrophage cell lines (J774.2; RAW 264.7; BAC1.2F5). The receptor binds morphine, its active metabolite morphine 6-glucuronide and certain other alkaloids, but not morphine 3-glucuronide or any of the opioid peptides tested. The cell lines thus provide valuable model systems for investigation of μ₃-opiate receptors, previously demonstrated to mediate inhibitory effects of morphine on activation of human peripheral blood macrophages (monocytes).     

NFκB inhibitors induce cell death in glioblastomas

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy.     

Modulating effect of mitomycin or cisplatin on lymphokine-activated killer cell proliferation and antitumor activity to bladder cancer cell lines in vitro

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Is cisplatin-induced cell death always produced by apoptosis?

It is generally accepted that DNA damage and subsequent induction of apoptosis may be the primary cytotoxic mechanism of cisplatin and other DNA-binding antitumor drugs (Fisher,1994). Because the final step of apoptosis is characterized by morphological changes in the nucleus, the death signals of the execution phase must be transmitted from the cytoplasm to the nucleus. Thus, the recognition and processing of cisplatin-induced DNA damage through"classic" apoptosis, requires that a nuclear signal, generated at the initiation phase, be transmitted to the cytoplasm to be processed through the effector and execution phases. At the end of the execution phase, the apoptotic signal must come back to the nucleus to produce internucleosomal DNA degradation. Therefore, the induction of apoptosis from detection and subsequent processing of cisplatin-induced DNA damage seems to be a long and complex process of cell death. However, because cisplatin is a nonspecific drug and reacts not only with DNA but also with proteins,we cannot rule out the possibility that in some cases of cisplatin-induced apoptosis, an easier process of initiation, such as damage to cytoplasmic proteins, may take place (Pérez, 1998). Thus, damage to proteins is worth considering as a factor contributing to cisplatin-induced apoptosis. Moreover, it is possible that cisplatin damage to proteins could induce apoptosis at the execution phase level. In fact, initiation of apoptosis at the execution phase (activation of caspases) has been previously reported for the cell killing produced by cytotoxic T lymphocytes (Golstein et al., 1991). Although apoptosis and necrosis are conceptually distinct forms of cell death with very different morphological and biochemical characteristics, these two types of demise may occur simultaneously in tissues or cell cultures exposed to the same insult (Eguchi et al., 1997, Zhan et al., 1999). In fact, both types of cell death have been found in the same population of cisplatin-treated cells (Pestell et al., 2000). Moreover, it has been hypothesized that in a tissue or cell population,apoptosis and necrosis might be two extremes of a continuum of possible types of cell demise. Individual cell death would be decided by factors such as the availability of energy and the metabolic condition of the cell (Leist et al., 1997). Thus, some cells might die as a result of an unfinished apoptotic program. In fact, in L1210 leukemic cells, cisplatin-induced cell death seems to be the result of a defective apoptotic program that lacks some morphological and biochemical characteristics attributed to apoptosis (Segal-Bendirdjian and Jacquemin-Sablon, 1995). In addition, at high doses, cisplatin could damage molecules involved in cellular energy supply (i.e., ATP) and also proteins directly or indirectly involved in the apoptotic process (i.e., p53, Bax, Bcl-2, and caspases), leading to necrotic cell death. In fact, in cisplatin-resistant keratinocytes transformed by H-ras oncogene, a high dose of cisplatin (312 microM) induces characteristic features of necrotic cell death(Pérez et al., 1999). Thus, depending on the level of cellular damage induced by cisplatin, necrosis could take place either directly or as a consequence of an unfinished apoptotic program. In summary, a growing body of evidence suggests that cisplatin-induced cell death does not always come from "classic"apoptosis. Depending on both cisplatin dose and cellular status, cisplatin may also induced cell death by a defective apoptotic program or even by necrosis. Elucidation of the conditions under which the apoptotic program induced by cisplatin as well as other antitumor drugs is totally or partially executed may have important implications for the outcome of cancer chemotherapy.     

Echinacoside stimulates cell proliferation and prevents cell apoptosis in intestinal epithelial MODE-K cells by up-regulation of transforming growth factor-β1 expression

Cistanche deserticola MA (C. deserticola) has been widely used as a laxative herbal in herbal medicine for the treatment of irritable bowel syndrome or constipation, and echinacoside (ECH) is one of the major bioactive ingredients in this herbal. Our aim was to investigate the effect of ECH on intestinal epithelial cell growth and death. MODE-K, an intestinal epithelial cell line, was used as an in vitro model of the intestine. Cell proliferation was measured by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was determined with Annexin-V staining. Here we showed that in cultured MODE-K cells, ECH significantly stimulated cell proliferation and enhanced cell survival by reducing cell apoptosis in the presence of H(2)O(2) or the mixture of pro-inflammatory cytokines, while transforming growth factor (TGF)-β1 expression was up-regulated in a dose-dependent manner. Knockdown of TGF-β1 expression disrupted both the proliferative and cytoprotective activities of ECH, which was further confirmed by neutralization of TGF-β1 activity using anti-TGF-β1 antibody. These data suggest that ECH as one of bioactive ingredients in herbal C. deserticola and others may improve mucosal tissue repair by stimulating intestinal epithelial cell proliferation and preventing cell death via up-regulation of TGF-β.