Development of small-molecule tropomyosin receptor kinase (TRK) inhibitors for

Tropomyosin receptor kinase A, B and C (TRKA, TRKB and TRKC), which are well-known members of the cell surface receptor tyrosine kinase (RTK) family, are encoded by the neurotrophic receptor tyrosine kinase 1, 2 and 3 (

Expression and functions of long non-coding RNA actin filament-associated protein 1-antisense RNA1 in oral squamous cell carcinoma / 华西口腔医学杂志

OBJECTIVE@#To analyze the expression and clinical significance of long non-coding RNA (lncRNA) actin filament-associated protein 1-antisense RNA1 (AFAP1-AS1) in oral squamous cell carcinoma (OSCC) and its effect on the biobehavior of OSCC cells.@*METHODS@#Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of lncRNA AFAP1-AS1 in the tumor tissue and matching adjacent normal tissue of OSCC patients (n=55), SCC25 cells, and normal oral keratinocyte lines (NOK) cells. The correlation between AFAP1-AS1 expression and the clinicopathological characteristics of OSCC patients was analyzed. The relationship between AFAP1-AS1 and prognosis was also studied with the Kaplan-Meier method. AFAP1-AS1 siRNA was transfected into the SCC25 cells. Cell counting kit-8 (CCK-8) and Trans-well were used to detect cell proliferation, invasion, and migration. The expression of the invasion-associated protein, AFAP1, and Rho GTPase family members, was detected by Western blot. In addition, the immunofluorescence of the cytoskeletal actin filament was observed.@*RESULTS@#The expression of AFAP1-AS1 was higher in the OSCC tissues than in the NOK cells, and the relative expression of AFAP1-AS1 was higher in the SCC25 cells than in the NOK cells (P<0.001). AFAP1-AS1 expression was associated with the degree of diffe-rentiation, TNM stage, lymphatic metastasis, and poor prognosis of OSCC (P<0.05). Patients with a high expression of AFAP1-AS1 had lower survival rates than those with a low expression of AFAP1-AS1 (P<0.05). After transfected by AFAP1-AS1 siRNA, the expression of AFAP1-AS1 was downregulated. The inhibition of AFAP1-AS1 expression consequently suppressed the proliferation, invasion, and migration of SCC25. Moreover, AFAP1-AS1 siRNA upregulated the expression levels of AFAP1, RhoA, Rac2, Rab10, RhoGDI, and Pfn1 but downregulated the expression of RhoC. Immunofluorescence showed that AFAP1-AS1 also reduced the formation of stress filaments in the cytoskeleton and affected the integrity of the actin fila-ment.@*CONCLUSIONS@#The expression of AFAP1-AS1 was high in the OSCC tissues and SCC25 cells and is associated with the development and prognosis of OSCC. The knockdown of AFAP1-AS1 might inhibit the proliferation and invasion of OSCC cells by regulating the integrity of the actin filament.

Actin filaments as the fast pathways for calcium ions involved in auditory processes.

We investigated the polyelectrolyte properties of actin filaments which are in interaction with myosin motors, basic participants in mechano-electrical transduction in the stereocilia of the inner ear. Here, we elaborated a model in which actin filaments play the role of guides or pathways for localized flow of calcium ions. It is well recognized that calcium ions are implicated in tuning of actin-myosin cross-bridge interaction, which controls the mechanical property of hair bundle. Actin filaments enable much more efficient delivery of calcium ions and faster mechanism for their distribution within the stereocilia. With this model we were able to semiquantitatively explain experimental evidences regarding the way of how calcium ions tune the mechanosensitivity of hair cells.

Expression of long non-coding RNA AFAP1-AS1 in human digestive system cancer and its clinical significance / 肿瘤

Objective: To investigate the expressions of actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1), a long non-coding RNA (IncRNA) in four common human digestive system cancers including esophageal cancer, gastric cancer, liver cancer and colorectal cancer, and their clinical significance. Methods: The expression of AFAP1-AS1 was preliminarily detected in several digestive system tumor tissues and their corresponding adjacent normal tissues from 82 cases by multi-tumor tissue microarrays. These 82 patients included 11 with esophageal cancer, 11 with gastric cancer, 26 with liver cancer, and 34 with colorectal cancer. The expression of AFAP1-AS1 which had significant difference in liver tumor tissues was further tested by in situ hybridization (additional 70 cases) and real-time fluorescence quantitative PCR (additional 30 cases). The relationship between the expression of AFAP1-AS1 and the clinicopathological features was analyzed. The role of AFAP1-AS1 in tumor lymph node metastasis was assessed. Results: The expression of AFAP1-AS1 in liver cancer was significantly lower than that in its corresponding adjacent normal liver tissue (P 0.05). Further test also revealed that the expression of AFAP1-AS1 was significantly down-regulated in liver cancer, and this effect was associated with clinical stage and lymph node metastasis (P < 0.05). The sensitivity, specificity, coincidence rate, positive predictive value and negative predictive value of AFAP1-AS1 serving as a molecular marker of metastasis were 68.75%, 65.00%, 65.63%, 28.21% and 91.23%, respectively. Conclusion: The expression of AFAP1-AS1 may play a role in the pathogenesis and progression of liver cancer and esophageal cancer, but this effect is different between these two cancer types. It is suggested that AFAP1-AS1 may become a noval molecular marker for clinical diagnosis of liver cancer. Copyright© 2014 by TUMOR.

Rapid Disruption of Cellular Integrity of Zinc-treated Astroglia Is Regulated by p38 MAPK and Ca(2+)-dependent Mechanisms

Cultured cortical primary astroglia treated with zinc died while rapidly detached from culture plates, a distinct part of zinc-treated astroglia. In the present study, we investigated the mechanism underlying the rapid change in the morphologic integrity of zinc-treated astroglia. Among the early cellular events occurring in zinc-treated astroglia, strong activation of p38 MAPK and JNK was evident. Although inhibitors of p38 (SB203580 and SB202190) or JNK (SP600125) did not protect zinc-insulted astroglia from cell death, the p38 inhibitors, but not the JNK inhibitor, suppressed actin filament and cell morphology disruption. The Ca2+ ionophore, A23187, also suppressed actin filament and cell morphology disruption, but not cell death, of zinc-insulted astroglia. However, A23187 did not inhibit p38 MAPK activation in zinc-treated astroglia. Together these results suggest that zinc influx in astroglia results in rapid loss of the morphologic integrity via mechanisms regulated by p38 kinase and/or Ca2+ signaling.

TGF-?1 induced actin cytoskeletal reorganization in human dental pulp cells / 实用口腔医学杂志

Objective:To determine the effects of TGF-?1 on the cytoskeleton in cultured human dental pulp cells(HPCs).Methods:Human dental pulp cells were cultured from dental pulp tissue explants digested with collagenase I. Semi-confluent cultures of the cells maintained under serum deprivation were treated with 20 ng/ml of TGF-?1 for 30 min, 1, 6 and 24 h respectively.Then cells were processed for BODYPY-phalloidin direct fluorescence examination of the actin filaments,DAPI direct fluorescence of the nucleus and Rhodamine Red(TM) indirect immunofluorescence of tubulin-?. Confocal laser scanning microscopy was used to investigate the changes of actin filaments and microtubules.Results:Disintegration and reorganization of actin filaments were observed in human dental pulp cells treated by TGF-?1 at 20 ng/ml. Actin filaments assembly was found near the cell membrane,specially after 30 min exposure.Disintegration of actin filaments was most obvious after 6 h treatment.Actin filaments were reorganized after 24 h exposure.Microtubules mainly remained intact in the cells during TGF-?1 treatment.Conclusion:TGF-?1 at 20 ng/ml may induce actin cytoskeletal reorganization in human dental pulp cells.

Propofol Decreases Cell Volume and Intracellular Calcium, and Rearranges Actin Filaments in Astroglial Cells / 대한마취과학회지

BACKGROUND: Regulation of cell volume is one of the major physiologic functions in living cells. Intracellular calcium ion and cytoskeletal component of cell membrane play pivotal roles in cell volume regulation. The significance of astroglial cell swelling is in its relation to delayed and permanent neuronal death. The aim of the current study is therefore to elucidate the effect of propofol on the cell volume, [Ca2+]i and actin filaments in astroglial cells. METHODS: Astroglial cell line U1242MG astrocytoma cells were used in vitro. To investigate the alterations of cell volume and [Ca2+]i by propofol, flow cytometry system was used. Actin filaments were determined by tetramethylrhodamine isothiocyanate (TRITC)-phalloidin staining. RESULTS: Treatment with propofol 10 microgram/ml for 15 min or 2 h perfusion reduced significantly both cell volume and [Ca2+]i. Cell volume was reduced 3-4% in either duration of perfusion with propofol. Decreases in [Ca2+]i level in the presence of propofol was duration-dependent. Fifteen min perfusion with propofol caused 5% decrease in [Ca2+]i, but 2 h perfusion decreased [Ca2+]i further to 10%. The changes of actin filaments after propofol treatment for 30 min were apparently observed under fluorescent microscope. CONCLUSIONS: Changes of cell volume, [Ca2+]i and actin filaments are occurred by propofol in astroglial cells. Rearrangement of actin filaments may be associated with volume changes by propofol in astroglial cells.

The Effects of Cytochalasin B and Colchicine on Fine Structure of Bile Canaliculi and Hepatocytes in Mouse Liver / 대한해부학회지

Bile canaliculi is closely related to the cytoskeleton; actin filament web, microtubules and cytokeratin intermediate filaments. To understand how cytoskeletal alteration affects bile canalicular structure, the investigators injected cytochalasin B and colchicine into mice intraperitoneally to inhibit the polymerization of actin filaments and microtubules respectively, and observed the structural changes of bile canaliculi and hepatocytes with transmission and scanning electron microscopes. Bile canaliculi were dilatated and microvilli were decreased in number and length after injection of cytochalasin B and colchicine. Some bile canaliculi branched irregularly after colchicine treatment. Actin filament web in the canalicular ectoplasm was disrupted leaving granular zone after cytochalasin B treatment, but was intact after colchicine treatment. Intermediate filament bundles located at angles to the canalicular membrane appeared after colchicine treatment. Intercellular junctions delimiting bile canaliculi were intact after colchicine treatment, however were disrupted after cytochalsin B treatment. Focal junctions resembling desmosome were formed between microvilli after colchicine treatment. In both cytochalasin B and colchicine treated groups, lumen of rough endoplasmic reticulum were dilated, Golgi apparatus became prominent, and lipid droplets were appeared in the cytoplasm. These results suggest that both intact actin filaments and microtubules are necessary to keep the structural integrity of bile canaliculi.

THE RESPONSE TO CYTOCHALASIN D SEEN IN PRIMARY CULTURED CARDIOCYTES FROM ADULT HUMAN AND ADULT RAT / 解剖学报

10?m cytochalasin D (CD) was used to treat cardiocytes from adult human atrium and adult rat atrium and ventricle in long-term primary cultures. Durations of treatment were 0.5, 1, 6, 12, 24 and 48h. In some cultures, the medium containing CD was removed at the planned time to be replaced with the medium without CD.These dishes were then cultured for an additional 48h. Control cultures were exposed to dimethyl sulfoxide (DMSO), the vehicle used to dissolve CD. All cultured cells were first stained with rhodamine-labelled phalloidin to show F-actin and then stained with fluoreseein-labelled antitubulin to show microtubles. The freshly isolated and rounded cardiocytes did not respond to CD, while the spreading cells responded apparently. The actin filament bundles in the peripheral zone of spreading cells were cut into segments. Most segments were gathered into aggregates and granules. Some aggregates were lodged on the inner aspect of the sarcolemma. Small vacuoles were seen between the myofibrils or somewhere along the course of the myofibrils. The CD response from the atrial spreading cells, especially the human atrial spreading cells, were more obvious than that from the rat ventricular cells. Cells exposed to CD and then cultured in normal medium for 48h did not return to normal. Microtubules were not directly affected by CD, but in places where vacuolization occured they made way for vacuoles. All the control cultures made no response to DMSO. The above-mentioned results suggest that different sensitivity to CD existed in cultured adult cardiocytes between different species and that a difference also existed in the contractile machinery between the atrial culturing cells and ventricular cultureing cells of the same species.