Levels Of Cofilin Research Articles

Inhibition of iron-induced cofilin activation and inflammation in microglia by a novel cofilin inhibitor.

Neuroinflammatory conditions linked to iron dysregulation pose significant challenges in neurodegenerative diseases. Iron-loaded microglia are observed in the brains of patients with various neuroinflammatory conditions, yet how iron overload affects microglial function and contributes to various neuroinflammatory processes is poorly understood. This invitro study elucidates the relationship between excess iron, cofilin activation, and microglial function, shedding light on potential therapeutic avenues. Iron overload was induced in Human Microglial Clone 3 cells using ferrous sulfate, and the expressions of ferritin heavy chain, ferritin light chain, divalent metal transporter 1, cofilin, p-cofilin, nuclear factor-κB (NF-κB), and various inflammatory cytokines were analyzed using real-time quantitative polymerase chain reaction, immunocytochemistry, Western blotting, and enzyme-linked immunosorbent assay. Results revealed a notable increase in cofilin, NF-κB, and inflammatory cytokine expression levels following excess iron exposure. Moreover, treatment with deferoxamine (DFX), a known iron chelator, and a novel cofilin inhibitor (CI) synthesized in our laboratory demonstrate a mitigating effect on iron-induced cofilin expression. Furthermore, both DFX and CI exhibit promising outcomes in mitigating the inflammatory consequences of excess iron, including the expression of pro-inflammatory cytokines and NF-κB activation. These findings suggest that both DFX and CI can potentially alleviate microglia-induced neuroinflammation by targeting both iron dysregulation and cofilin-mediated pathways. Overall, this study provides valuable insights into iron-induced cofilin activation and microglial activation, offering avenues for potential targeted therapies for neuroinflammatory conditions associated with iron and cofilin dysregulation in neurodegenerative diseases.

Serum response factor promoting axonal regeneration by activating the Ras-Raf-Cofilin signaling pathway after the spinal cord injury.

Serum response factor (SRF) is important in muscle development, tissue repair, and neuronal regulation. This research aims to thoroughly examine the effects of SRF on spinal cord injury (SCI) and its ability to significantly impact the recovery and regeneration of neuronal axons. The researchers created rat models of SCI and scratch injury to primary spinal cord neurons to observe the expression of relevant factors after neuronal injury. We found that the SRF, Ras, Raf, and cofilin levels increased after injury and gradually returned to normal levels. Afterward, researchers gave rats with SCI an SRF inhibitor (CCG1423) and studied the effects with nuclear magnetic resonance and transmission electron microscopy. The SRF inhibitor rodents had worse spinal cord recovery and axon regrowth than the control group. And the apoptosis of primary neurons after scratch injury was significantly higher in the SRF inhibitor group. Additionally, the researchers utilized lentiviral transfection to modify the SRF expression in neurons. SRF overexpression increased neuron migration while silencing SRF decreased it. Finally, Western blotting and RT-PCR were conducted to examine the expression changes of related factors upon altering SRF expression. The results revealed SRF overexpression increased Ras, Raf, and cofilin expression. Silencing SRF decreased Ras, Raf, and Cofilin expression. Based on our research, the SRF promotes axonal regeneration by activating the "Ras-Raf-Cofilin" signaling pathway.

Stratifin (SFN) Regulates Cervical Cancer Cell Proliferation, Apoptosis, and Cytoskeletal Remodeling and Metastasis Progression Through LIMK2/Cofilin Signaling

The aberrant expression of Stratifin (SFN) is intricately associated with the initiation and progression of numerous tumors. This study aims to investigate whether SFN regulates the metastasis of cervical cancer cells through the LIMK2/Cofilin signaling pathway. In this study, we compared the expression of SFN in normal cervical tissues and cervical carcinoma tissues. We established SFN overexpression and SFN silencing cellular models to assess the invasive and migratory capabilities of cervical cancer cells using transwell and scratch assays. YO-PRO-1/PI and EdU staining were employed to evaluate apoptotic and proliferative capacities, while Actin-Tracker Green-488 was utilized to investigate cytoskeletal remodeling. The expression levels of SFN, LIMK2, p-LIMK2, Cofilin, and p-Cofilin were examined through Western blotting and immunofluorescence. Our findings revealed elevated expression of SFN in cervical squamous cell carcinoma tissues. SFN overexpression was observed to enhance invasion and migration of cervical cancer cells, induce cytoskeletal remodeling, facilitate cell proliferation, and suppress apoptosis. Furthermore, SFN overexpression upregulated the expression levels of LIMK2, p-LIMK2, Cofilin, and p-Cofilin. Conversely, silencing SFN exerted opposite effects. SFN plays an important role in the diagnosis of cervical cancer. SFN can regulate cervical cancer cell proliferation, apoptosis, cytoskeletal remodeling and metastasis through LIMK2/Cofilin signaling.

Spatiotemporal Cofilin Signaling, Microglial Activation, Neuroinflammation, and Cognitive Impairment Following Hemorrhagic Brain Injury.

Intracerebral hemorrhage (ICH) is a significant health concern associated with high mortality. Cofilin plays a crucial role in stress conditions, but its signaling following ICH in a longitudinal study is yet to be ascertained. In the present study, we examined the cofilin expression in human ICH autopsy brains. Then, the spatiotemporal cofilin signaling, microglia activation, and neurobehavioral outcomes were investigated in a mouse model of ICH. Human autopsy brain sections from ICH patients showed increased intracellular cofilin localization within microglia in the perihematomal area, possibly associated with microglial activation and morphological changes. Various cohorts of mice were subjected to intrastriatal collagenase injection and sacrificed at time points of 1, 3, 7, 14, 21, and 28 days. Mice suffered from severe neurobehavioral deficits after ICH, lasting for 7 days, followed by a gradual improvement. Mice suffered post-stroke cognitive impairment (PSCI) both acutely and in the chronic phase. Hematoma volume increased from day 1 to 3, whereas ventricle size increased from day 21 to 28. Cofilin protein expression increased in the ipsilateral striatum on days 1 and 3 and then decreased from days 7 to 28. An increase in activated microglia was observed around the hematoma on days 1 to 7, followed by a gradual reduction up to day 28. Around the hematoma, activated microglia showed morphological changes from ramified to amoeboid. mRNA levels of inflammatory [tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin-6 (IL-6) and anti-inflammatory markers [interleukin-10 (IL-10), transforming growth factor-β TGF-β, and arginase I (Arg1)] increased during the acute phase and decreased in the chronic phase. Blood cofilin levels increased on day 3 and matched the increase in chemokine levels. slingshot protein phosphatase 1 (SSH1) protein, which activates cofilin, was increased from day 1 to 7. These results suggest that microglial activation might be the sequel of cofilin overactivation following ICH, leading to widespread neuroinflammation and consequent PSCI.

Red CdSe/ZnS QDs' Intracellular Trafficking and Its Impact on Yeast Polarization and Actin Filament.

Quantum dots are nanoparticles (2-10 nm) that emit strong and tunable fluorescence. Quantum dots have been heavily used in high-demand commercialized products, research, and for medical purposes. Emerging concerns have demonstrated the negative impact of quantum dots on living cells; however, the intracellular trafficking of QDs in yeast cells and the effect of this interaction remains unclear. The primary goal of our research is to investigate the trafficking path of red cadmium selenide zinc sulfide quantum dots (CdSe/ZnS QDs) in Saccharomyces cerevisiae and the impact QDs have on yeast cellular dynamics. Using cells with GFP-tagged reference organelle markers and confocal microscopy, we were able to track the internalization of QDs. We found that QDs initially aggregate at the exterior of yeast cells, enter the cell using clathrin-receptor-mediated endocytosis, and distribute at the late Golgi/trans-Golgi network. We also found that the treatment of red CdSe/ZnS QDs resulted in growth rate reduction and loss of polarized growth in yeast cells. Our RNA sequence analysis revealed many altered genes. Particularly, we found an upregulation of DID2, which has previously been associated with cell cycle arrest when overexpressed, and a downregulation of APS2, a gene that codes for a subunit of AP2 protein important for the recruitment of proteins to clathrin-mediated endocytosis vesicle. Furthermore, CdSe/ZnS QDs treatment resulted in a slightly delayed endocytosis and altered the actin dynamics in yeast cells. We found that QDs caused an increased level of F-actin and a significant reduction in profilin protein expression. In addition, there was a significant elevation in the amount of coronin protein expressed, while the level of cofilin was unchanged. Altogether, this suggests that QDs favor the assembly of actin filaments. Overall, this study provides a novel toxicity mechanism of red CdSe/ZnS QDs on yeast actin dynamics and cellular processes, including endocytosis.

Flavonoids from Citrus paradise cv. Changshan-huyou exerts protective effect on ischemia-induced cerebral injury in mice via inhibiting RhoA-ROCK2 signaling pathway.

To investigate the protective effect and mechanism of total flavonoids from Citrus paradise cv. Changshan-huyou extracts (TFC) on oxygen-glucose deprivation (OGD) of primary neurons and chronic ischemia-induced cerebral injury in mice. Primary hippocampal neurons of 18-day fetal rats were isolated and cultured for 1 week, then treated with 0.25, 0.50 and 1.00 mg/mL TFC. After oxygen-glucose deprivation for 1 h, cells were reperfused for 6 and 24 h, respectively. The cytoskeleton was observed by phalloidin staining. In animal study, 6-week ICR male mice were randomly divided into sham operation group, model group, low-dose (10 mg/kg), medium-dose (25 mg/kg) and high-dose (50 mg/kg) TFC treatment groups, with 20 mice in each group. After 3 weeks, chronic cerebral ischemia was induced by unilateral common carotid artery ligation in all groups except sham operation group. Mice were treated with different concentrations of TFC in the three TFC treatment groups for 4 weeks. Open field test, novel object recognition test and Morris water maze test were used to evaluate anxiety, learning and memory of these mice. Nissl, HE and Golgi stainings were used to detect neuronal degeneration and dendritic spine changes in the cortex and the hippocampus. The expression levels of Rho-associated kinase (ROCK) 2, LIM kinase (LIMK) 1, cofilin and its phosphorylation, as well as the expression of globular actin (G-actin) and filamentous actin (F-actin) protein in hippocampus of mice were detected by Western blotting. Neurons subjected to OGD showed that neurites displayed shortening and breakage; while treatment with TFC reversed OGD-induced neurite injury, especially in the 0.50 mg/mL TFC group. Compared with the sham operation group, the mice in the model group showed a significant decline in anxiety and cognitive ability (P<0.01), whereas treatment with TFC significantly reversed anxiety and cognitive deficits (P<0.05). Improvement in the medium-dose TFC group was the most obvious. Histopathological analysis indicated that the number of Nissl bodies and dendritic spines in hippocampus and cortex were decreased in the model group (all P<0.01). However, after treatment with medium dose of TFC, the number of Nissl bodies and dendritic spines (all P<0.05) was significantly recovered. Compared with the sham operation group, the phosphorylation level of ROCK2 in the brain tissue of the model group was significantly increased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly decreased (P<0.05), and the relative content ratio of G-actin/F-actin was significantly increased (P<0.05). After administration of TFC, the phosphorylation level of ROCK2 in brain tissue of each group was significantly decreased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly up-regulated (P<0.05) and the relative content ratio of G-actin/F-actin was significantly decreased (P<0.05). TFC protects from ischemia-induced cytoskeletal damage, reduces neuronal dendritic spine injury and protects mice against chronic cerebral ischemia through RhoA-ROCK2 signaling pathway, indicating that TFC might be a potential candidate for treatment of chronic ischemic cerebral injury.

A novel matrine derivative, WM130, inhibits activation and movement of human hepatic stellate LX-2 cells by targeting cofilin 1.

The online version contains supplementary material available at 10.1007/s10616-022-00548-w.

Abstract 386: Lipocalin 10 Protects Against Sepsis-triggered Vascular Leakage Through Activation Of The Ssh1-cofilin Signaling Pathway

Background: The increased vascular leakage seen in sepsis patients is well-recognized to be responsible for sepsis-triggered organ failure and patient mortality. Prior work suggests that lipocalin 10 (Lcn10), a member of the lipocalin superfamily, is significantly downregulated in the blood of non-survival septic patients when comparing to the survival group. Nonetheless, whether Lcn10 affects endothelial barrier integrity in sepsis remains unknown. Methods and Results: First, we observed that the expression of Lcn10 was remarkably down-regulated in the hearts of both endotoxin LPS- and cecal ligation/puncture (CLP)-induced septic mice, compared to their controls. Interestingly, further analysis of Lcn10 expression in different cell types isolated from LPS- and CLP-hearts showed that reduction of Lcn10 occurred only in cardiac endothelial cells (ECs) but not in cardiomyocytes or fibroblasts. Using a global Lcn10-knockout (KO) mouse model, we found that loss of Lcn10 greatly increased vascular permeability, which correlated with more severe cardiac depression and higher mortality following LPS challenge or CLP surgery, compared to LPS- or CLP-treated wild-type (WT) mice. By contrast, in vitro overexpression of Lcn10 in ECs provided greater resistance to LPS-caused monolayer leak, compared to control cells. A mechanistic analysis by RNA-sequencing and RT-qPCR revealed that both endogenous and exogenous elevation of Lcn10 in ECs could greatly upregulate slingshot homolog 1 (Ssh1) expression. Ssh1 is a phosphatase known to activate Cofilin, a key actin-binding protein that plays an essential role in controlling actin filament dynamics. Accordingly, phosphorylated Cofilin levels were significantly reduced and thereby, reorganized F-actin to cortical actin for stabilizing tight junction molecules in Lcn10-treated ECs, compared to control cells. Finally, knockdown of Ssh1 in ECs by siRNA greatly offsets Lcn10-induced reduction of monolayer leakage upon LPS insult. Conclusions: Endothelial Lcn10 is critical for protecting against sepsis-induced cardiovascular leakage, and its underlying mechanism involves the activation of Ssh1-Cofilin pathway. Our study suggests that Lcn10 could be a novel regulator of vascular barrier integrity.

RHOA inhibits chondrogenic differentiation of mesenchymal stem cells in adolescent idiopathic scoliosis

ABSTRACT Purpose The etiology of adolescent idiopathic scoliosis (AIS) remains unclear. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is important in AIS, and the Ras homolog gene family member A (RHOA) is associated with chondrogenesis. The purpose of this study was to explore the effect of RHOA on the chondrogenic differentiation of MSCs in AIS. Methods We isolated MSCs from patients with AIS (AIS MSCs) and individuals without AIS (control MSCs). The inhibitor Y27632 was used to inhibit the function of RHOA/ROCK signaling, and plasmid-based overexpression and siRNA-mediated knockdown were used to manipulate RHOA expression. CCK-8 was used to detect cell viability. The phosphorylation levels of LIMK1, MLC2 and cofilin were detected by Western blotting. The mRNA expression of aggrecan, SOX9, and COL2A1 were confirmed using RT-PCR. Immunofluorescence was used to analyze F-actin and collagen II. Alcian blue staining was performed to assess the secretion of glycosaminoglycans (GAGs). Results We found that RHOA was significantly upregulated in AIS MSCs, and the phosphorylation levels of LIMK1, MLC2, and cofilin were increased. The mRNA expressions of aggrecan, SOX9, and COL2A1 were notably reduced in AIS MSCs. However, these effects were abolished by Y27632 treatment and RHOA knockdown in AIS MSCs. In addition, RHOA knockdown in AIS MSCs increased the content of collagen II and GAGs. RHOA overexpression in the control MSCs markedly activated the RHOA/ROCK signaling and decreased the expression of aggrecan, SOX9, and COL2A1, F-actin, and GAGs. Conclusion RHOA regulates the chondrogenic differentiation ability of MSCs in AIS via the RHOA/ROCK signaling pathway and this regulation may involve SOX9.

ROCK2 inhibition attenuates profibrogenic immune cell function to reverse thioacetamide-induced liver fibrosis

Background & AimsFibrosis, the primary cause of morbidity in chronic liver disease, is induced by pro-inflammatory cytokines, immune cell infiltrates, and tissue resident cells that drive excessive myofibroblast activation, collagen production, and tissue scarring. Rho-associated kinase 2 (ROCK2) regulates key pro-fibrotic pathways involved in both inflammatory reactions and altered extracellular matrix remodelling, implicating this pathway as a potential therapeutic target.MethodsWe used the thioacetamide-induced liver fibrosis model to examine the efficacy of administration of the selective ROCK2 inhibitor KD025 to prevent or treat liver fibrosis and its impact on immune composition and function.ResultsProphylactic and therapeutic administration of KD025 effectively attenuated thioacetamide-induced liver fibrosis and promoted fibrotic regression. KD025 treatment inhibited liver macrophage tumour necrosis factor production and disrupted the macrophage niche within fibrotic septae. ROCK2 targeting in vitro directly regulated macrophage function through disruption of signal transducer and activator of transcription 3 (STAT3)/cofilin signalling pathways leading to the inhibition of pro-inflammatory cytokine production and macrophage migration. In vivo, KDO25 administration significantly reduced STAT3 phosphorylation and cofilin levels in the liver. Additionally, livers exhibited robust downregulation of immune cell infiltrates and diminished levels of retinoic acid receptor-related orphan receptor gamma (RORγt) and B-cell lymphoma 6 (Bcl6) transcription factors that correlated with a significant reduction in liver IL-17, splenic germinal centre numbers and serum IgG.ConclusionsAs IL-17 and IgG–Fc binding promote pathogenic macrophage differentiation, together our data demonstrate that ROCK2 inhibition prevents and reverses liver fibrosis through direct and indirect effects on macrophage function and highlight the therapeutic potential of ROCK2 inhibition in liver fibrosis.Lay summaryBy using a clinic-ready small-molecule inhibitor, we demonstrate that selective ROCK2 inhibition prevents and reverses hepatic fibrosis through its pleiotropic effects on pro-inflammatory immune cell function. We show that ROCK2 mediates increased IL-17 production, antibody production, and macrophage dysregulation, which together drive fibrogenesis in a model of chemical-induced liver fibrosis. Therefore, in this study, we not only highlight the therapeutic potential of ROCK2 targeting in chronic liver disease but also provide previously undocumented insights into our understanding of cellular and molecular pathways driving the liver fibrosis pathology.

Cofilin 2 Acts as an Inflammatory Linker Between Chronic Periodontitis and Alzheimer's Disease in Amyloid Precursor Protein/Presenilin 1 Mice.

Increasing evidence has shown a correlation between chronic periodontitis (CP) and Alzheimer’s disease (AD). Nevertheless, there is still a lack of direct evidence, and especially key molecules to connect the two diseases. This study aims to investigate potential protein links between CP and AD within the inflammatory aspect. The hippocampus of CP model mice and controls were collected, and changes in protein expression were evaluated using two-dimensional differential in-gel electrophoresis (2D-DIGE) analysis combined with liquid chromatography tandem mass spectrometry. A total of 15 differentially expressed proteins were identified in CP model mice, as compared with the controls. Among them, S100-A9, transthyretin, Cofilin 2, peroxiredoxin 2, and lipocalin-2 were validated by Western blot according to their dual function both in inflammation and AD. Based on 2D-DIGE analysis, CP animal model had higher levels of S100-A9, Cofilin 2, peroxiredoxin 2, and lipocalin-2 compared to controls. The level of Cofilin 2, one of the well-established proteins in the pathology of AD, was strongly correlated with the time course of CP pathology, indicating a specific molecular correlation between CP and AD. Moreover, the in vivo results showed the level of Cofilin 2 increased significantly along with a prominent increase of the phosphorylation of protein phosphatase 2 (PP2A) and tau protein in the cell lysates of Porphyromonas gingivalis (P.g-LPS)-treated SK-N-SH APPwt cells. Cofilin 2 inhibition resulted in a sharp decrease in PP2A dependent of tau phosphorylation. Furthermore, tumor growth factor (TGF)-β1 was one of the most important inflammatory cytokines for the Pg-LPS-induced Cofilin 2 upregulation in SK-N-SH APPwt cells. These results showed inflammation served as the bond between CP and AD, whereas inflammatory related proteins could be the key linkers between the two diseases. Determining the association between CP and AD at the molecular mechanism will not only hold the direct evidence of the association between the two diseases but also provide a new way of preventing and treating AD: the effective prevention and treatment of CP could serve as a useful method to alleviate the development of AD.

Relationship of intracellular proteolysis with CAP1 and cofilin1 in non-small-cell lung cancer

The main cause of death in non-small-cell lung cancer (NSCLC) is tumor progression, in which metastasis and invasion play an important role. The metastatic cascade is marked by a change in morphological, biological, biochemical and functional characteristics, including the acquisition of cellular mobility. The migration activity of tumor cells determines the work of actin-binding proteins that cause their functional partners CAP1 and cofilin. Of interest is the study of the regulation of working tandem CAP1/cofilin in NSCLC. The mechanism that regulates the level of proteins in cells is proteolysis, carried out by proteasomes and calpains. Therefore, the aim of this study was to estimate the expression of CAP1/CFL1 mRNA and their protein level in NSCLC tissues, and to analyze the possible mechanisms of their regulation by the proteasome and calpain systems. Samples of NSCLC and histological unchanged lung tissue were used (n = 42). The CAP1 and CFL1 mRNA expressions were determined by real-time PCR, the contents of proteins encoded by them were determined by Western blotting, and the activity of proteasomes and calpains by the fluorimetric method. There was an increase in the expression of mRNA and protein levels of CAP1 and cofilin in the tumor tissue compared with the unchanged lung tissue. The expression of mRNA and the level of CAP1 in tumor tissue increased during growth of the primary tumor. The cofilin level in the tumor tissue decreases against the background of increased expression of its mRNA. At the same time, during tumor growth, the activity of proteasomes and calpains increased. A negative regression relationships between the activity of proteasomes and the levels of CAP1 and cofilin, as well as the activity of calpains and the level of cofilin, were found. It can be assumed that proteasomes and calpains are involved in the degradation of CAP1 and cofilin. The data obtained suggest the importance of CAP1, cofilin and proteolytic systems in the tumor transformation and lymphogenous metastasis.

Serum levels of cytoskeleton remodeling proteins and their mRNA expression in tumor tissue of metastatic laryngeal and hypopharyngeal cancers

Actin-binding proteins (ABPs) and various signaling systems are involved in the process of squamous cell carcinoma of the larynx and hypopharynx (SCCLH) metastasis. The clinical significance of these proteins has not yet been determined. We analyzed the relationship between the mRNA levels of cofilin 1 (CFL1), profilin 1 (PFN1), adenylyl cyclase-associated protein 1 (CAP1), SNAI1 and RND3 and SCCLH metastasis. The serum levels of the above ABPs were estimated and the relationship between them and their mRNA expressions was analyzed. The expression levels of ABP mRNAs were measured by real-time RT-PCR in paired tissue samples taken from 54 patients with SCCLH (T1-4N0-1M0). Expression analysis was performed using the 2-ΔΔCT method. The levels of ABPs in the blood serum were measured by ELISA. Statistical analysis was carried out using the SPSS Statistica 20.0 software package. No significant difference in the mRNA gene expression in tumor tissue of patients with T1-3N0M0 SCCLH and patients with T2-4N1-2M0 SCCLH was found. High expression of RND3 mRNA was accompanied by an increase in mRNA expression of all studied ABPs. In the blood serum of T2-4N1-2M0 patients, the level of PFN1 was lower by 21% and the level of CAP1 was higher by 75% than those observed in T1-4N0M0 patients. The data obtained showed that RND3 is involved in the regulation of molecular cascades of SCCLH metastasis. PFN1 and CAP1 serum levels can be good classifiers of metastases in patients with SCCLH.

Nujiangexanthone A Inhibits Hepatocellular Carcinoma Metastasis via Down Regulation of Cofilin 1.

Hepatocellular carcinoma (HCC) is one of the malignant tumors with poor prognosis. High expression level of cofilin 1 (CFL1) has been found in many types of cancers. However, the role of CFL1 in HCC hasn’t been known clearly. Here, we found that CFL1 was up regulated in human HCC and significantly associated with both overall survival and disease-free survival in HCC patients. Nujiangexanthone A (NJXA), the caged xanthones, isolated from gamboge plants decreased the expression of CFL1, which also inhibited the migration, invasion and metastasis of HCC cells in vitro and in vivo. Down regulation of CFL1 inhibited aggressiveness of HCC cells, which mimicked the effect of NJXA. Mechanism study indicated that, knockdown of CFL1 or treatment with NJXA increased the level of F-actin and disturbed the balance between F-actin and G-actin. In conclusion, our findings reveal the role of CFL1 in HCC metastasis through the CFL1/F-actin axis, and suggest that CFL1 may be a potential prognostic marker and a new therapeutic target. NJXA can effectively inhibit the metastasis of HCC cells by down regulating the expression of CFL1, which indicates the potential of NJXA for preventing metastasis in HCC.

Electroacupuncture Attenuates Ischemic Brain Injury and Cellular Apoptosis via Mitochondrial Translocation of Cofilin.

To investigate the potential mechanisms of electroacupuncture (EA) to prevent ischemic stroke. The method of middle cerebral artery occlusion (MCAO) was employed to establish a rat model of ischemic stroke. Seventy-eight Sprague-Dawley rats were divided into the sham group, MCAO + EA control (EC) group, and MCAO + EA (EA) group according to a random number table (n=26 per group). EA was applied to the acupoints of Baihui (DU 20) and Shenting (DU 24) 5 min and 6 h, respectively after the onset of MCAO. Rats in the sham and EC groups received only light isoflurane anesthesia for 30 min after MCAO. The neuroprotective effects of EA were evaluated by rota-rod test, neurological deficit scores and infarct volumes. Additionally, Nissl staining and immunostaining were performed to examine brain damage, rod formation, cellular apoptosis, and neuronal loss induced by ischemia. The activities of caspase-3, and expression levels of cofilin and p-cofilin in mitochondria and cytoplasm after ischemic injury were determined by Western blot. Compared with the EC group, EA significantly improved neuromotor function and cognitive ability after ischemic stroke (P<0.05 or P<0.01). Therapeutic use of EA also resulted in a significant decrease of cofilin rod formation and microtubule-associated protein-2 (MAP2) degradation in the cortical penumbra area compared with the EC rats (P<0.01). Furthermore, Western blot analysis showed that EA stimulation significantly inhibited mitochondrial translocation of cofilin and caspase-3 cleavage (P<0.05 or P<0.01). Additionally, brain damage (infarct volume and neuropathy), cellular apoptosis and neuronal loss induced by ischemia were remarkably suppressed by EA in the cortical penumbra of rats (P<0.05 or P<0.01). EA treatment after ischemic stroke may attenuate ischemic brain injury and cellular apoptosis through the regulation of mitochondrial translocation of cofilin, a novel mechanism of EA therapy.

Circulating Actin-Binding Proteins in Laryngeal Cancer: Its Relationship with Circulating Tumor Cells and Cells of the Immune System.

We previously exposed the role of actin-binding proteins (ABPs) in cancer development and progression. In this paper, we studied the relationship between circulating ABPs and the number of ABP-expressing leukocytes and circulating tumor cells (CTCs) in patients with highly aggressive laryngeal squamous cell carcinoma (LSCC). The levels of cofilin (CFL1), profilin (PFN1), ezrin (EZR), fascin (FSCN1), and adenylate cyclase-associated protein 1 (CAP1) were determined using enzyme immunoassay. The ABP expression by the cellular pools was analyzed by flow cytometry. The highest levels of FSCN1 and EZR were found in the blood serum of LSCC patients. There was a difference in ABP expression between the pools of leukocytes and CTCs. Leukocytes were mainly represented by CAP1+ and FSCN1+ pools, and CTCs contained CAP1+, FSCN1+, and EZR+ cells. The serum FSCN1 level correlated with the number of FSCN1-containing and CFL1-containing leukocytes. Thus, the level of circulating EZR is likely related to its expression in CTCs. The levels of CFL1 and PFN1 are likely to be supported by the expression of these proteins by leukocytes. Both CTCs and leukocytes can be a source of FSCN1 and CAP1 in blood serum. The results suggest that serum proteins can be produced by various cells, thus indicating both cancer development and the response of the immune system to this process.

Low-dose X-ray irradiation induces morphological changes and cytoskeleton reorganization in osteoblasts.

Recently, research into the biological effects of low dose X-ray irradiation (LDI) has been a focus of interest. Numerous studies have suggested that cells exhibit different responses and biological effects to LDI compared with high doses. Preliminary studies have demonstrated that LDI may promote osteoblast proliferation and differentiation in vitro, thereby accelerating fracture healing in mice. However, the exact mechanism of action by which LDI exerts its effects remains unclear. Previous studies using microarrays revealed that LDI promoted the expression of genes associated with the cytoskeleton. In the current study, the effect of X-ray irradiation (0.5 and 5 Gy) on the morphology of MC3T3-E1 cells and fiber actin organization was investigated. Osteoblasts were treated with 0, 0.5 and 5 Gy X- ray irradiation, following which changes in the actin cytoskeleton were observed. The levels of RhoA, ROCK, cofilin and phosphorylated-cofilin were measured by reverse transcription-quantitative PCR and western blotting. Subsequently, osteoblasts were pretreated with ROCK specific inhibitor Y27632 to observe the changes of actin skeleton after X-ray irradiation. The results demonstrated that the cellular morphological changes were closely associated with radiation dose and exposure time. Furthermore, the gene expression levels of small GTPase RhoA and its effectors were increased following LDI. These results indicated that the RhoA/Rho-associated kinase pathway may serve a significant role in regulating LDI-induced osteoblast cytoskeleton reorganization.

CELL MOTILITY PROTEINS IN ENDOMETRIUM CARCINOMA AND ENDOMETRIAL HYPERPLASIA: ASSOCIATION WITH CANCER RISK

Introduction. Proteins associated with cellular motility are known to play an important role in invasion and metastasis of cancer, however there is no evidence of their association with the development of malignant tumors including endometrial cancer (EC).The aim of the present study was to investigate the levels of actin-binding proteins, p45-Ser-β-catenin, and calpain activity in endometrial hyperplasia and in EC.Material and Methods. Total calpain activity, p45-Ser β-catenin, Arp3, gelsolin, cofillin and thymosin β-4 levels were evaluated in 43 postmenopausal patients with stage I–II endometrioid EC and 40 endometrial hyperplasia patients. Flow cytometry and Western blotting were used for expression determination of p45 Ser β-catenin and actin-biding proteins. Total calpain activity was estimated by fluorimetric method.Results. Levels of cofilin-1, thymosin β-4 and calpain activity were higher in cancer tissues than in endometrial hyperplasia. Cofilin-1 and thymosin β-4 levels were associated with the depth of myometrial invasion. The thymosin β-4 expression was correlated with the presence of tumor cervical invasion. Revealed correlations between the actin-binding proteins, p45-Ser-β-catenin and total calpain activity in endometrial hyperplasia tissue, but not in the tissue of cancer, is evidence of the involvement of these proteases in regulation of cell migration in endometrial hyperplasia. Levels of thymosin β-4, cofilin and total calpain activity are independent cancer risk factors in patients with endometrial hyperplasia.Conclusion. The level of actin-binding proteins as well as the total calpain activity were enhanced in endometrium carcinoma tissues compared to endometrial hyperplasia. The levels of thymosinβ-4, cofilin and total calpain activity in endometrial hyperplasia tissues are associated with a hyperplasia transition to cancer and may be considered as predictive biomarkers.

Abstract P100: Opioids Cause Sex-Specific Vascular Remodeling Via Cofilin-ERK Signaling: Female Mice Present Higher Risk Of Developing Morphine-Induced Vascular Dysfunction Than Male Mice

Recent studies have shown that chronic use of prescription opioids leads to increased risk of cardiovascular events and pulmonary arterial hypertension. Indices of vascular age and arterial stiffness are increased in opioid dependent patients and the effects are more marked in women. We hypothesized that chronic exposure to exogenous opioids causes sex-specific vascular remodeling via suppression of actin depolymerization. Using culture pressure myographs, we performed continuous intraluminal infusion of morphine (10μM for 24 hours) in pressurized mesenteric resistance arteries (MRA, fifth order) from male and female mice (C57BL/6NTac, 10 weeks old; n=4). Intraluminal pressure curve showed that morphine reduced lumen diameter by 23% (without morphine: 185 ± 10μm vs. morphine: 143 ± 14μm*) in MRA from female mice, whereas little to no change was observed in MRA from male mice (Fig. 1). Cofilin is a key molecule in preventing stress fiber overassembly and is inactivated by extracellular signal-regulated kinases (ERK). Here, we observed that after 24-hour exposure, morphine (10μM) phosphorylated and inactivated cofilin 1.6 fold [AU: control: 0.59 ± 0.14 vs. morphine: 0.95 ± 0.10*, *p&lt;0.05, t-test] and increased ERK phosphorylation 1.8 fold (AU: control: 0.51 ± 0.11 vs. morphine: 0.90 ± 0.13*, *p&lt;0.05) in MRA from female mice. On the other hand, morphine decreased phospho-ERK 1.5 fold (AU: control: 0.91 ± 0.08 vs. morphine: 0.59 ± 0.11*, *p&lt;0.05) but had no effect on the levels of cofilin in MRA from male mice. These results have significant implications for the previously unexplored role of exogenous opioids as a modifiable cardiovascular risk factor, particularly in women.

Superficial and deep zone articular chondrocytes exhibit differences in actin polymerization status and actin-associated molecules in vitro

The actin cytoskeleton regulates cell shape and plays a role in regulating chondrocyte phenotype. Most studies investigating regulation of the chondrocyte phenotype by the actin cytoskeleton use chondrocytes isolated from full-thickness (FT) cartilage, which has a heterogeneous cell population. Superficial zone chondrocytes (SZC) have an elongated morphology and account for 10-20% of chondrocytes, while the remaining chondrocytes in the deeper zones appear more rounded. This study characterizes the actin cytoskeleton and expression of actin-associated molecules in SZC and deep zone (DZ) chondrocytes (DZC) in vitro in order to identify molecules differentially expressed by SZC and DZC that may contribute to the observed differences in zonal chondrocyte shapes. SZ, DZ, and FT chondrocytes isolated from bovine metacarpal-phalangeal joints were cultured in monolayer for 48h. Macroscopic morphology, actin polymerization status, and expression of select actin-associated molecules (adseverin, cofilin, transgelin, vinculin, MRTF-A, and YAP/TAZ) were determined. SZC appeared more elongated and have more filamentous actin compared to DZC, as determined by quantifying cell circularity and G-/F-actin ratio. MRTF-A gene and protein levels were significantly higher in SZC compared to DZC while DZC more highly expressed transgelin and TAZ. Although there was differential gene expression, no significant differences in adseverin, cofilin, vinculin, or YAP protein levels were observed between the two cell populations. This study identifies differences in actin polymerization status and expression of actin-associated molecules in primary SZC and DZC in vitro. These findings further our understanding of candidate actin-related pathways that may be regulating zonal chondrocyte phenotype.