Esophageal squamous cell carcinoma (ESCC) is a common malignancy without effective therapy. Histone deacetylase inhibitors (HDACIs) have been demonstrated as an emerging class of anticancer drugs for ...a range of haematological and solid tumours. However, the effect of HDACIs has not yet been investigated on ESCC cells. In this study, HDACIs were initially considered to have anticancer activity for ESCC, due to the high expression of HDAC genes in ESCC cell lines by analysing expression data of 27 ESCC cell lines from the Broad-Novartis Cancer Cell Line Encyclopedia. Next, we used five ESCC cell lines and one normal immortalized esophageal epithelial cell line to screen three HDACIs, panobinostat (LBH589), vorinostat (SAHA), and trichostatin A (TSA), for the ability to inhibit growth. Here, we report that LBH589 more effectively suppressed cell proliferation of ESCC cell lines, in a dose-dependent manner, than TSA and SAHA, as well as had lower toxicity against the SHEE normal immortalized esophageal epithelial cell line. Further experiments indicated that LBH589 treatment significantly inhibited TP53 (mutated TP53) expression, both at the mRNA and protein level, and simultaneously increased p21 and decreased cyclin D1 expression. Taken together, we propose that LBH589 inhibits ESCC cell proliferation mainly through inducing cell cycle arrest by increasing p21 and decreasing cyclin D1 in a p53-independent manner. SIGNIFICANCE OF THE STUDY: In this study, the antitumor activity of HDACIs LBH589, SAHA, and TSA on ESCC was characterized, with LBH589 displaying the most potent anti-proliferative activity while not harming normal immortalized esophageal epithelial cells. Furthermore, we propose that LBH589 exerts its anti-proliferative effect by inducing cell cycle arrest. The ability to specifically target cancer cells indicates therapeutic potential for use of LBH589 in the treatment of ESCC.
Background
Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post‐translational modifications. However, whether and how Fascin is regulated ...by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis.
Methods
Immunoprecipitation and glutathione‐S‐transferase pull‐down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP‐associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF‐mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live‐cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry.
Results
Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti‐AcK471‐Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin‐K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin‐K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin‐K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471‐acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin‐bundling activity. Clinically, high levels of AcK471‐Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease‐free survival of ESCC patients.
Conclusions
Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin‐K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin‐bundling activity.
Filopodia are dynamic membrane extensions generated by F-actin bundling and are involved in cancer cell migration, invasion and metastasis. Fascin is the crucial actin-bundling protein in filopodia, ...with phosphorylation at fascin serine 39 being well characterized to regulate fascin-mediated actin bundling in filopodia. However, increasing evidence indicates that fascin is phosphorylated at a number of sites. Whether phosphorylation at other sites also regulates fascin function is unknown. In this study, we show that four potential phosphorylation sites in fascin, specifically tyrosine 23, serine 38, serine 39 and serine 274, regulate cell behavior and filopodia formation in esophageal squamous cancer cells. Expression of non-phosphorylatable mutations at each of the four sites promoted anchorage-independent growth, cell motility and filopodia formation, whereas phosphomimetic mutations at each of these sites inhibited these cell behaviors, implying that fascin function in esophageal squamous cancer is regulated by fascin phosphorylation at multiple sites. Furthermore, phosphorylation at S38 and S39 cooperatively regulated cell behavior and filopodia formation, with dual dephosphorylation at both S38 and S39 residues maximally enhancing cell proliferation, migration and filopodia formation, and phosphorylation at any of the two phosphorylatable sites resulting in reduced enhancement. Taken together, our results reveal that phosphorylation at fascin amino acids Y23, S38, S39 and S274, in combination, downregulates the extent of anchorage-independent growth, cell migration and filopodia formation in esophageal squamous cancer cells.
Background For cardiovascular tissue engineering, acellularized biomaterials from pig have been widely investigated. Our purpose was to study mechanical properties and biocompatibility of ...decellularized aorta of fetal pigs （DAFP） to determine its potential as scaffold for small diameter tissue engineered vascular graft. Methods Descending aorta of fetal pigs was removed cells using trypsin, ribonuclease and desoxyribonuclease. Mechanical properties of DAFP were evaluated by tensile stress-strain and burst pressure analysis. Assessment of cell adhesion and compatibility was conducted by seeding porcine aortic endothelial cells. To evaluate biocompatibility in vivo DAFP was implanted subcutaneously into adult male Sprague Dawley rats for 2, 4 and 8 weeks. Results Histochemistry and scanning electron microscopy examination of DAFP revealed well-preserved extracellular matrix proteins and porous three-dimensional structures. Compared with fresh aorta, DAFP had similar ultimate tensile strength, axial compliance and burst pressure. Cell culture studies in vitro showed that porcine aortic endothelial cells adhered and proliferated on the surfaces of DAFP with excellent cell viability. Subdermal implantation demonstrated that the DAFP did not show almost any immunological reaction and exhibited minimal calcification during the whole follow-up period. Conclusion The DAFP has the potential to serve as scaffolds for small diameter tissue engineered vascular graft.
Acute respiratory failure is the most frequent indication for the application of mechanical ventilation. As commonly used in clinical settings, lung protective strategies and recruitment manoeuvres ...are applications of higher than normal airway pressure to open the collapsed alveoli and prevent lung atelectasis caused by minimal vital ventilation. Under those conditions, we pay more attention to the lung injury and circulatory failure,
A new biosensor platform was explored for detection of surfactant based on ?uorescence changes from single strand DNA (ssDNA) and single-walled carbon nanotubes (SWNTs). Thermodynamics assay was ...performed to value the stability of probe. The affinities of SWNT to five common surfactants (SDS, DBS, Triton X-100, Tween-20 and Tween-80) were investigated by real-time fluorescence method. The effects of Mg2+ and pH on the fluorescence intensity of self-assembled quenched sensor were performed. The fluorescent emission spectra were used to measure the responses of self-assembled quenched ?uorescent of ssDNA /SWNTs to different concentration surfactant(Triton X-100). The FAM-DNA wrapped SWNTs probe was stable in a wide temperature range (5 °C to 80 °C). The binding strength of surfactants and single-stranded DNA (ssDNA) on SWNTs surfaces was shown as follows: Triton X-100>DBS>Tween-20>Tween-80>ssDNA>SDS, and the optimized reaction conditions included pH 7.4 and 10 mmol/L Mg2+. The fluorescence of FAM-ssDNA wrapped SWNTs was proportionally recovered as a result of adding different concentrations of Triton X-100, which realizes the quantitative detection of Triton X-100.
Background： In vivo quantification of choroidal neovascularization （CNV） based on noninvasive optical coherence tomography （OCT） examination and in vitro choroidal flatmount immunohistochemistry ...stained of CNV currently were used to evaluate the process and severity of age-related macular degeneration （AMD） both in human and animal studies. This study aimed to investigate the correlation between these two methods in murine CNV models induced by subretinal injection. Methods： CNV was developed in 20 C57BL6/j mice by subretinal injection of adeno-associated viral delivery of a short hairpin RNA targeting sFLT-1 （AAV.shRNA.sFLT- 1）, as reported previously. After 4 weeks, CNV was imaged by OCT and fluorescence angiography. The scaling factors for each dimension, x, y, and z （ktm/pixel） were recorded, and the corneal curvature standard was adjusted from human （7.7） to mice （1 .4）. The volume of each OCT image stack was calculated and then normalized by multiplying the number of voxels by the scaling factors for each dimension in Seg3D software （University of Utah Scientific Computing and Imaging Institute, available at http：//www.sci.utah.edu/cibc-software/seg3d.html）. Eighteen mice were prepared for choroidal flatmounts and stained by CD31. The CNV volumes were calculated using scanning laser confocal microscopy after immunohistochemistry staining. Two mice were stained by Hematoxylin and Eosin for observing the CNV morphology. Results： The CNV volume calculated using OCT was, on average, 2.6 times larger than the volume calculated using the laser confocal microscopy. The correlation statistical analysis showed OCT measuring of CNV correlated significantly with the in vitro method （R： = 0.448, P = 0.001, n = 18）. The correlation coefficient for CNV quantification using OCT and confocal microscopy was 0.693 （n = 18, P = 0.001 ）. Conclusions： There is a fair linear correlation on CNV volumes between in vivo and in vitro methods in CNV models induced by subretinal injection. The result might provide a useful evaluation of CNV both for the studies using CNV models induced by subretinal injection and human AMD studies.
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