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.     

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.     

A secreted peptidase involved in T cell β-endorphin metabolism

β-endorphin metabolism by CD4⁺ and CD8⁺ T cells, and the thymoma cell line, EL4, was investigated. In all three cell types, extracellular β-endorphin was metabolized exclusively by a secreted, metal-dependent, thiol peptidase. The enzyme activity is expressed constitutively in EL4 cells and following activation of CD4⁺ and CD8⁺ T cells with anti-CD3 antibody. The enzyme is not one of the proteinases associated with cytolytic T cells and does not appear to be identical with any previously described β-endorphin metabolizing enzyme. The enzyme cleaves β-endorphin at approximately equal rates at either of two sites to yield β-endorphin_1–17 (which is γ-endorphin), β-endorphin_1–18, β-endorphin_18–31 and β-endorphin_19–31. Evidence in the literature indicates that these N- and C-terminal peptides which contain, respectively, the opioid and non-opioid receptor binding domains of β-endorphin, are biologically active. Thus, it is likely that this new T cell peptidase has important immunoregulatory activity.     

Function of HeLa cell cytoplasm in apoptosis induced by cisplatin in a cell-free system

在构建Ｂｃｌ－２高表达及其对照细胞株基础上，用非细胞体系研究了顺铂诱导的ＨｅＬａ细胞凋亡中细胞质的作用．实验结果显示凋亡细胞胞质提取物可引起正常细胞核的固缩及染色质边缘化，类似完整细胞凋亡的改变，而Ｂｃｌ－２高表达株的胞质提取物具有抗凋亡能力．说明引起凋亡的物质及对抗凋亡的物质在细胞质中都存在，细胞质在顺铂诱导的ＨｅＬａ细胞凋亡中起着较重要的作用．     

Does atorvastatin induce aortic smooth muscle cell apoptosis in vivo?

It has been reported that HMG-CoA reductase inhibitors such as atorvastatin induce vascular smooth muscle cell (SMC) apoptosis in vitro. However, this effect remains to be demonstrated in vivo. The present studies were designed to test the ability of atorvastatin to induce SMC apoptosis in vivo, using the spontaneously hypertensive rat (SHR) as a well-known reference model of SMC apoptosis induction in vivo by cardiovascular drugs including the calcium channel blocker amlodipine. Atorvastatin was administered to SHR for 3 or 6 weeks either alone or together with amlodipine, a drug combination clinically available to patients. Primary endpoints included aortic medial hypertrophy and aortic SMC hyperplasia, internucleosomal DNA fragmentation and expression of the apoptosis regulatory proteins Bax and Bcl-2. The SHR aorta showed no evidence of SMC apoptosis induction by atorvastatin, even at the high dose of 50 mg kg(-1) day(-1), although the statin significantly reduced oxidative stress after 3 weeks and blood pressure after 6 weeks of administration. Amlodipine-induced regression of aortic hypertophy and aortic SMC hyperplasia were dose- and time-dependent, but there was no interaction between atorvastatin and amlodipine in modulating the primary endpoints. These results do not support the notion that atorvastatin induces SMC apoptosis in the aortic media in vivo.     

Autophagy in the liver: cell’s cannibalism and beyond

Chronic liver disease and its progression to liver failure are induced by various etiologies including viral infection, alcoholic and nonalcoholic hepatosteatosis. It is anticipated that the prevalence of fatty liver disease will continue to rise due to the growing incidence of obesity and metabolic disorder. Evidence is accumulating to indicate that the onset of fatty liver disease is causatively linked to mitochondrial dysfunction and abnormal lipid accumulation. Current treatment options for this disease are limited. Autophagy is an integral catabolic pathway that maintains cellular homeostasis both selectively and nonselectively. As mitophagy and lipophagy selectively remove dysfunctional mitochondria and excess lipids, respectively, stimulation of autophagy could have therapeutic potential to ameliorate liver function in steatotic patients. This review highlights our up-to-date knowledge on mechanistic roles of autophagy in the pathogenesis of fatty liver disease and its vulnerability to surgical stress, with an emphasis on mitophagy and lipophagy.     

How can we improve prognostic models in renal cell carcinoma?

The therapeutic improvements in renal cell carcinoma brought about by the transition from the ‘cytokine era’ to the ‘targeted agents era’, have not affected the peculiar prognostic heterogeneity of the disease, nor have they diminished the importance of risk group classification based on easily assessable and commonly available laboratory and clinical variables. In the landmark study conducted by Motzer et al. before biological agents were available, the median survival of patients in the good prognosis group was 20 months, while the patients in the poor-risk group had a median survival time of only 4 months. With the introduction of anti-VEGF agents, overall survival has approximately doubled in all risk classes. In a population-based analysis of 670 patients treated with anti-VEGF agents, either in the first-line setting or in the second-line setting after cytokines, stratification according to the Database Consortium model showed that patients in the favorable risk group had a median overall survival of 43.2 months, while patients in the poor-risk group had a median overall survival of 7.8 months.     

Cardiac fibroblasts in cell culture systems: myofibroblasts all along?

The cytoarchitecture of the working myocardium is characterized by densely packed cardiomyocytes that are embedded in a three-dimensional network of numerous fibroblasts. Although the importance of cardiac fibroblasts in maintaining an orderly structured extracellular matrix is well recognized, less is known about their potential paracrine and electrotonic interactions with cardiomyocytes. This is partly the result of the complex intermingling of both cell types in vivo that tends to preclude a direct investigation of heterocellular crosstalk. It is for that reason that most of our present knowledge regarding stromal-parenchymal cell interactions is based on culture systems that permit direct access to either cell type. An often disregarded feature of such studies is that cardiac fibroblasts in standard two-dimensional cell culture have a pronounced tendency to undergo a phenotype switch to myofibroblasts. This cell type typically appears in injured hearts where it contributes importantly to fibrotic remodeling. The present review focuses on recent insights into electrical and paracrine crosstalk between myofibroblasts and cardiomyocytes while acknowledging that a comprehensive understanding of stromal-parenchymal cell interactions will depend on future methodological developments that permit retaining the fibroblast phenotype in cell culture systems and that will, most importantly, allow direct investigations of heterocellular crosstalk in intact tissue.     

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.     

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.     

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.     

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.     

Cladribine enhances apoptotic cell death in lung carcinoma cells over-expressing DNase γ

Worldwide, lung cancer is the most common form of cancer and often has a poor prognosis. Establishment of effective therapies for lung cancer is a major concern in clinical cancer research. We compared the cytotoxic effects of chemotherapeutic agents including cisplatin, 5-fluorouracil, vinorelbine and cladribine, on a human lung cancer cell line, A549, and its derivative transfected with the DNase γ gene. We observed selective cytotoxicity of cladribine on the DNase γ-expressing sub-cell line of A549. Cladribine induces selective apoptosis in DNase γ-expressing A549 cells, which depends on activation of caspases. These results suggest that a combination therapy that includes cladribine along with the introduction of DNase γ has potential as a new therapeutic strategy for lung cancer.     

Nitrofen suppresses cell proliferation and promotes mitochondria-mediated apoptosis in type Ⅱ pneumocytes

AIM: To characterize the molecular mechanisms of nitrofen-induced pulmonary hypoplasia. Methods: After administration of nitrofen to cultured type II A549 pneumocytes, cell proliferation and DNA synthesis were investigated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation assay, flow cytometry and [3H]-thymidine incorporation assay. Apoptosis was measured by terminal transferase-mediated dUTP nick-end-labeling, acridine orange-ethidium bromide staining and flow cytometry. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related genes was assayed by immunofluorescence, RT-PCR and Western blot. RESULTS: Nitrofen inhibited the cell proliferation of A549 cells in a dose- and time-dependent manner, accompanied by downregulation of PCNA. As a result, the DNA synthesis of nitrofentreated A549 cells decreased, while cell cycle was arrested at G0/G1 phase. Moreover, nitrofen induced apoptosis of A549 cells, which was not abolished by Z-Val-Ala- Asp(OCH3)- fluoromethylketone. In addition, nitrofen decreased the expression of Bcl-x( L), but not of Bcl-2, Bax, and Bak, resulting in a loss of mitochondrial membrane potential and the nuclear translocation of apoptosis-inducing factor (AIF). Meanwhile, nitrofen strongly activated the p38 mitogen-activated protein kinase (p38-MAPK). Pretreatment of cells with SB203580 (5 micromol/L) blocked nitrofen-induced phosphorylation of p38-MAPK and abolished nitrofen-induced AIF translocation and apoptosis in A549 cells. CONCLUSION: Nitrofen suppresses the proliferation of cultured type II pneumocytes accompanied by the downregulation of PCNA, and induces mitochondria-mediated apoptosis involving the activation of p38-MAPK.     

Programmed cell death in human pathogenic fungi – a possible therapeutic target

ABSTRACT

Introduction: Diseases caused by pathogenic fungi are increasing because of antibiotic overuse, the rise of immunosuppressive therapies, and climate change. The limited variety of antimycotics and the rapid adaptation of pathogenic fungi to antifungal agents serve to exacerbate this issue. Unfortunately, about 1.6 million people are killed by fungal infections annually.

Areas covered: The discovery of the small antimicrobial proteins produced by microorganisms, animals, humans, and plants will hopefully overcome challenges in the treatment of fungal infections. These small proteins are highly stable and any resistance to them rarely evolves; therefore, they are potentially good candidates for the treatment and prevention of infections caused by pathogenic fungi. Some of these proteins target the programmed cell death machinery of pathogenic fungi; this is potentially a novel approach in antimycotic therapies. In this review, we highlight the elements of apoptosis in human pathogenic fungi and related model organisms and discuss the possible therapeutic potential of the apoptosis-inducing, small, antifungal proteins.

Expert opinion: Small antimicrobial proteins may establish a new class of antimycotics in the future. The rarity of resistance and their synergistic effects with other frequently used antifungal agents may help pave the way for their use in the clinic.     

HPMA copolymer–aminoglutethimide conjugates inhibit aromatase in MCF-7 cell lines

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer–doxorubicin (Dox) has already shown clinical activity in breast cancer patients. Moreover, we have recently found that an HPMA conjugate containing a combination of both Dox and the aromatase inhibitor aminoglutethimide (AGM) shows significantly increased anti-tumour activity in vitro. To better understand the mechanism of action of HPMA copolymer–AGM conjugates several models were used here to investigate their effect on cell growth and aromatase inhibition. Cytotoxicity of HPMA copolymer conjugates containing AGM, Dox and also the combination AGM–Dox was determined by MTT assay in MCF-7 and MCF-7ca cells. Androstenedione (5 × 10^{? 8} M) stimulates the growth of MCF-7ca cells. Both free AGM and polymer-bound AGM (0.2–0.4 mg/ml) were shown to block this mitogenic activity. When MCF-7ca cells were incubated [³H]androstenedione both AGM and HPMA copolymer–GFLG–AGM (0.2 mg/ml AGM-equiv.) showed the ability to inhibit aromatase. Although, free AGM was able to inhibit isolated human placental microsomal aromatase in a concentration dependent manner, polymer-bound AGM was not, suggesting that drug release is essential for activity of the conjugate. HPMA copolymer conjugates containing aromatase inhibitors have potential for the treatment of hormone-dependant cancers, and it would be particularly interesting to explore further as potential therapies in post-menopausal women as components of combination therapy.     

Toxicity of ingredients in artificial tears and ophthalmic drugs in a cell attachment and spreading test†

Abstract

During re-epithelialization of corneal erosions, the epithelial cells are attached and migrate on a fibronectin-containing substratum. In earlier studies we found that topical application of the plasmin inhibitor aprotinin promoted healing of corneal epithelial defects, presumably inhibiting proteolytic degradation of fibronectin and related adhesive proteins. The present study investigated, using an in vitro cell culture test, the action of various ophthalmic preparations on cell attachment and spreading on a fibronectin-coated substratum. The cell culture test was designed to simulate the situation in the wounded corneal epithelium in vivo. Analysis of 16 different ophthalmic preparations indicated that the toxicity of some of them in the test could be assigned to specific ingredients (benzalkonium chloride, thimerosal sodium, Tween 80) used as additives. In contrast, chlorobutanol, used widely as a preservative, was nontoxic in the test, in concentrations used in ophthalmic preparations. It should be noted that the wounded and inflamed cornea may be more vulnerable to the toxicity of topical mediations than an intact corneal epithelium. hoper tests of the vehicle are therefore recommended before clinical trials are undertaken.     

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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