Naringenin (NGEN), a natural flavonoid has growth inhibition and apoptosis‐inducing activities in several cancer cells. However, the cytotoxicity mechanisms of NGEN in cell death of lung cancer cells ...have not been fully defined. In present study, treatment of human lung adenocarcinoma A549 cells with NGEN resulted in time‐ and dose‐dependent decreases in cell viability. Moreover, NGEN significantly induced apoptosis evidenced by morphological changes, DAPI staining, TUNEL assay and sub‐G1 population increase. In NGEN‐treated cells, intensely upregulated Bax and down‐regulated Bcl‐2 proteins were detected and the Bax protein associated with the mitochondrial membrane was analyzed by subcellular fractionation. Knockdown of the Bax expression by the shRNA method dramatically protected A549 cells against NGEN‐induced apoptosis. Treatment with the inhibitors of caspase‐3, ‐8, or ‐9 significantly reduced NGEN‐induced apoptotic deaths. Taken together, our results demonstrate that NGEN‐induced apoptosis may occur via a Bax‐activated mitochondrial pathway in lung adenocarcinoma A549 cells.
Methylprednisolone (MP) is an anti-inflammatory drug approved for the treatment of acute spinal cord injuries (SCIs). However, MP administration for SCIs has become a controversial issue while the ...molecular effects of MP remain unexplored to date. Therefore, delineating the benefits and side effects of MP and determining what MP cannot cure in SCIs at the molecular level are urgent issues. Here, genomic profiles of the spinal cord in rats with and without injury insults, and those with and without MP treatment, were generated at 0, 2, 4, 6, 8, 12, 24, and 48 h post-injury. A comprehensive analysis was applied to obtain three distinct classes: side effect of MP (SEMP), competence of MP (CPMP), and incapability of MP (ICMP). Functional analysis using these genes suggested that MP exerts its greatest effect at 8~12 h, and the CPMP was reflected in the immune response, while SEMP suggested aspects of metabolism, such as glycolysis, and ICMP was on neurological system processes in acute SCIs. For the first time, we are able to precisely reveal responsive functions of MP in SCIs at the molecular level and provide useful solutions to avoid complications of MP in SCIs before better therapeutic drugs are available.
The Golgi apparatus (GA) translocates to the cell leading end during directional migration, thereby determining cell polarity and transporting essential factors to the migration apparatus. The study ...provides mechanistic insights into how GA repositioning (GR) is regulated. We show that the methyltransferase PRMT5 methylates the microtubule regulator HURP at R122. The HURP methylation mimicking mutant 122F impairs GR and cell migration. Mechanistic studies revealed that HURP 122F or endogenous methylated HURP, that is, HURP m122, interacts with acetyl‐tubulin. Overexpression of HURP 122F stabilizes the bundling pattern of acetyl‐tubulin by decreasing the sensitivity of the latter to a microtubule disrupting agent nocodazole. HURP 122F also rigidifies GA via desensitizing the organelle to several GA disrupting chemicals. Similarly, the acetyl‐tubulin mimicking mutant 40Q or tubulin acetyltransferase αTAT1 can rigidify GA, impair GR, and retard cell migration. Reversal of HURP 122F‐induced GA rigidification, by knocking down GA assembly factors such as GRASP65 or GM130, attenuates 122F‐triggered GR and cell migration. Remarkably, PRMT5 is found downregulated and the level of HURP m122 is decreased during the early hours of wound healing‐based cell migration, collectively implying that the PRMT5‐HURP‐acetyl‐tubulin axis plays the role of brake, preventing GR and cell migration before cells reach empty space.
Docetaxel‐based chemotherapy has generally been considered as one of the effective treatments for castration‐resistant prostate cancer (PCa). However, clinical treatment with docetaxel often ...encounters a number of undesirable effects, including drug resistance. Tubulin isoforms have been previously examined for their resistance to docetaxel in many cancers, but their real mechanisms remained unclear. In this study, a series of docetaxel‐resistant PC/DX cell sublines were established by chronically exposing PC3 to progressively increased concentrations of docetaxel. Western blotting results showed significantly higher expression of acetyl‐tubulin, α‐tubulin, β‐tubulin, γ‐tubulin, and βIII‐tubulin in PC/DX25 than in parental PC3 cells. PC/DX25 with greater resistance to docetaxel had higher levels of acetyl‐tubulin and mitotic centromere‐associated kinesin (MCAK) than PC3 cells. This study found that docetaxel induced the expression of acetyl‐tubulin and MCAK in PC3 cells at a dose‐ and time‐dependent manner. Both mRNA and protein levels of histone deacetylase 6 (HDAC6) were significantly decreased in PC/DX25 compared with PC3 cells. PC3 increased the resistance to docetaxel by HDAC6 knockdown and Tubastatin A (HDAC6 inhibitor). Conversely, PC/DX25 reversed the sensitivity to docetaxel by MCAK knockdown. Notably, flow cytometry analysis revealed that MCAK knockdown induced significantly sub G1 fraction in PC/DX cells. Overexpression of polo‐like kinase‐1 increased the cell survival rate and resistance to docetaxel in PC3 cells. Moreover, epidermal growth factor receptor (EGFR) activation induced the upregulation of acetyl‐tubulin in docetaxel‐resistant PCa cells. These findings demonstrated that the EGFR‐mediated upregulated expression of acetyl‐tubulin played an important role in docetaxel‐resistant PCa.
Golgi apparatus (GA) and centrosome reposition toward cell leading end during directional cell migration in a coupling way, thereby determining cell polarity by transporting essential factors to the ...proximal plasma membrane. The study provides mechanistic insights into how GA repositioning (GR) is regulated, and how GR and centrosome repositioning (CR) are coupled. Our previous published works reveals that PRMT5 methylates HURP at R122 and the HURP m122 inhibits GR and cell migration by stabilizing GA‐associated acetyl‐tubulin and then rigidifying GA. The current study further shows that the demethylase JMJD6‐guided demethylation of HURP at R122 promotes GR and cell migration. The HURP methylation mimicking mutant 122 F blocks JMJD6‐induced GR and cell migration, suggesting JMJD6 relays GR stimulating signal to HURP. Mechanistic studies reveal that the HURP methylation deficiency mutant 122 K promotes GR through NF‐κB‐induced CR and subsequently CR‐dependent Cdc42 upregulation, where Cdc42 couples CR to GR. Taken together, HURP methylation statuses provide a unique opportunity to understand how GR is regulated, and the GA intrinsic mechanism controlling Golgi rigidity and the GA extrinsic mechanism involving NF‐κB‐CR‐Cdc42 cascade collectively dictate GR.
Golgi apparatus (GA) is assembled as a crescent-like ribbon in mammalian cells under immunofluorescence microscope without knowing the shaping mechanisms. It is estimated that roughly 1/5 of the ...genes encoding kinases or phosphatases in human genome participate in the assembly of Golgi ribbon, reflecting protein modifications play major roles in building Golgi ribbon.
To explore how Golgi ribbon is shaped as a crescent-like structure under the guidance of protein modifications, we identified a protein complex containing the scaffold proteins Ajuba, two known GA regulators including the protein kinase Aurora-A and the protein arginine methyltransferase PRMT5, and the common substrate of Aurora-A and PRMT5, HURP. Mutual modifications and activation of PRMT5 and Aurora-A in the complex leads to methylation and in turn phosphorylation of HURP, thereby producing HURP p725. The HURP p725 localizes to GA vicinity and its distribution pattern looks like GA morphology. Correlation study of the HURP p725 statuses and GA structure, site-directed mutagenesis and knockdown-rescue experiments were employed to identify the modified HURP as a key regulator assembling GA as a crescent ribbon.
The cells containing no or extended distribution of HURP p725 have dispersed GA membranes or longer GA. Knockdown of HURP fragmentized GA and HURP wild type could, while its phosphorylation deficiency mutant 725A could not, restore crescent Golgi ribbon in HURP depleted cells, collectively indicating a crescent GA-constructing activity of HURP p725. HURP p725 is transported, by GA membrane-associated ARF1, Dynein and its cargo adaptor Golgin-160, to cell center where HURP p725 forms crescent fibers, binds and stabilizes Golgi assembly factors (GAFs) including TRIP11, GRASP65 and GM130, thereby dictating the formation of crescent Golgi ribbon at nuclear periphery.
The Ajuba/PRMT5/Aurora-A complex integrates the signals of protein methylation and phosphorylation to HURP, and the HURP p725 organizes GA by stabilizing and recruiting GAFs to its crescent-like structure, therefore shaping GA as a crescent ribbon. Therefore, the HURP p725 fiber serves a template to construct GA according to its shape. Video Abstract.
Arecoline is the primary alkaloid in betel nuts, which are known as a risk factor for oral submucosal fibrosis and oral cancer. Lung cancer is a severe type of carcinoma with high cell motility that ...is difficult to treat. However, the detailed mechanisms of the correlation between Arecoline and lung cancer are not fully understood. Here, we investigated the effect of Arecoline on migration in lung cancer cell lines and its potential mechanism through the muscarinic acetylcholine receptor 3 (mAChR3)-triggered EGFR/Src/FAK pathway. Our results indicate that different concentrations of Arecoline treatment (10 µM, 20 µM, and 40 µM) significantly increased the cell migration ability in A549 and CL1-0 cells and promoted the formation of the filamentous actin (F-actin) cytoskeleton, which is a crucial element for cell migration. However, migration of H460, CL1-5, and H520 cell lines, which have a higher migration ability, was not affected by Arecoline treatment. The EGFR/c-Src/Fak pathway, which is responsible for cell migration, was activated by Arecoline treatment, and a decreased expression level of E-cadherin, which is an epithelial marker, was observed in Arecoline-treated cell lines. Blockade of the EGFR/c-Src/Fak pathway with the inhibitors of EGFR (Gefitinib) or c-Src (Dasatinib) significantly prevented Arecoline-promoted migration in A549 cells. Gefitinib or Dasatinib treatment significantly disrupted the Arecoline-induced localization of phospho-Y576-Fak during focal adhesion in A549 cells. Interestingly, Arecoline-promoted migration in A549 cells was blocked by a specific mAChR3 inhibitor (4-DAMP) or a neutralizing antibody of matrix metalloproteinase (MMP7 or Matrilysin). Taken together, our findings suggest that mAChR3 might play an essential role in Arecoline-promoted EGFR/c-Src/Fak activation and migration in an A549 lung cancer cell line.
In this study, gallium oxide (Ga2O3) nanorods were deposited onto an indium tin oxide (ITO) glass substrate to develop a real-time living cell viability sensor. Ga2O3 nanorods had characteristics of ...cell population sensing based on cellular metabolism to detect cell viability. The pH sensing characteristics of Ga2O3-based extended-gate field-effect transistors (EGFETs) with and without the annealing process were measured from pH 1–11. Three different sensor heads, ITO/glass, GaOOH/ITO/glass, and Ga2O3/ITO/glass, were produced as EGFETs and immersed into a pH standard buffer and high-glucose Dulbecco’s modified Eagle’s medium (DMEM) to investigate hydrogen ion sensing. Results showed that the Ga2O3/ITO/glass probe had the best hydrogen ion sensing sensitivity and stability in different sample buffers. In contrast, GaOOH/ITO/glass had instability and lower sensitivity due to interference with other components in the medium. The drift characteristic of sensors was analyzed to detect long-term stability. The Ga2O3/ITO/glass probe showed lower drift properties, which can potentially monitor the DMEM situation in real-time for a long-term period. During in vitro detection, the Ga2O3/ITO/glass probe revealed a 70 μA/cells change in IDS-VDS analysis and a 10 μV/cells change in IDS-VGS analysis, which was superior linearly dependent within 10,000 cells. The biocompatibility of the Ga2O3/ITO/glass probe was also detected by 3-(4,5-dimethylthiazol-2-yl)− 2,5-diphenyltetrazolium bromide assay and showed no cytotoxicity. Overall, the Ga2O3/ITO/glass EGFET sensor has potential applications as a living cell viability device in microenvironmental studies in the future.
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•This study creates a device that can be used to monitor changes in living cell viability.•Ga2O3/ITO/glass was connected to 2N7000 MOSFET to synthesize a detection device.•The annealed Ga2O3 film has higher stability and better hydrogen ion concentration response.
ZnO structures were electro-hydrothermally grown on copper and silver wires. To characterize the structures, multiple analyses including field-emission scanning electron microscopy, energy-dispersive ...X-ray spectroscopy, X-ray diffraction and photoluminescence measurements were performed on the ZnO structures on the wires. Results indicate that ZnO nanoflakes were densely deposited on the silver wire but poor ZnO structures were grown on the copper wire.
Furthermore, OD 600 antibacterial tests revealed that ZnO flakes on the silver wire exhibited better antibacterial capability than the silver wire without ZnO nanoflakes. Because of possible integration with silver-related materials and flexibility of wire structures, ZnO nanoflake-coated silver wires have potentials for future antiseptic and biomedical applications.
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