UCH-L1 promotes invasion of breast cancer cells through activating Akt signaling pathway.

As a de-ubiquitin enzyme, ubiquitin C-terminal hydrolase (UCH)-L1 has been shown to be overexpressed in several human cancers. However, the function of UCH-L1 in invasion of breast cancers is still unclear. Here we report that the expression of UCH-L1 is significantly higher in cancer cells with higher invasive ability. While ectopic UCH-L1 expression failed to alter cell proliferation in MCF-7 cells, it caused a significant upregulation of cellular invasion. Furthermore, siRNA mediated knockdown of UCH-L1 led to suppression of invasion in UCH-L1 overexpressing MCF-7 cells. In order to identify molecular mechanisms underlying these observations, a novel in vitro proximity-dependent biotin identification method was developed by fusing UCH-L1 protein with a bacterial biotin ligase (Escherichia coli BirA R118G, BioID). Streptavidin magnetic beads pulldown assay revealed that UCH-L1 can interact with Akt in MCF-7 cells. Pulldown assay with His tagged recombinant UCH-L1 protein and cell lysate from MCF-7 cells further demonstrated that UCH-L1 preferentially binds to Akt2 for Akt activation. Finally, we demonstrated that overexpression of UCH-L1 led to activation of Akt as evidenced by upregulation of phosphorylated Akt. Thus, these findings demonstrated that UCH-L1 promotes invasion of breast cancer cells and might serve as a potential therapeutic target for treatment of human patients with breast cancers.

By Activating Akt/eNOS Bilobalide B Inhibits Autophagy and Promotes Angiogenesis Following Focal Cerebral Ischemia Reperfusion.

BACKGROUND/AIMS: Ischemic stroke is a leading cause of long-term disability. To date, there is no effective treatment for stroke. Previous studies have shown that Ginkgo biloba extract has protective effects against neurodegenerative disorders. In this present study, we sought to test the potential protective role of an active component of Ginkgo biloba extract, bilobalide, in a rat model of middle cerebral artery occlusion (MCAO). METHODS: A rat model of MCAO was used to test the potential protective effects of Bilobalide B on stroke protection. TTC staining was performed to evaluate infarct size of the brains. Neurological deficit score was measured to reveal the effects of the treatments on animal behavior and cognition. Immunohistochemical staining and transmission electronic microscope analysis were performed to measure the cellular responses to drug treatment. Western blotting and ELISA were performed. The expression of Cleaved- Casepase 3, Beclin-1, p62 and LC3I/II were quantified, and the Phosphorylation of eNOS and Akt were evaluated. The ratio of Bcl-2/ Bax was determined to reveal the molecular pathways that are involved in the drug treatment. RESULTS: We found that intraperitoneal delivery of various Bilobalide doses during ischemia can protect against brain injury, as evidenced by reduced infarct size and improved neurological scores after surgery. Histochemical analysis revealed that treatment with bilobalide can significantly reduce apoptosis, autophagy, and promote angiogeneis following ischemia/reperfusion injury to the brain. The performence of increased phosphorylation of eNOS and Akt suggested that bilobalide can activate Akt prosurvival and eNOS pathways to promote cell survival and angiogenesis, respectively. CONCLUSIONS: Our results suggested that bilobalide benefits stroke symptoms by reducing cell death pathways and promoting angiogenesis. As such, bilobalide may be a potential agent for improving self-repair after ischemic stroke.

Modulation of wound healing and scar formation by MG53 protein-mediated cell membrane repair.

Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53(-/-) mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-ß-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-ß signaling may present a potentially effective means for promoting scarless wound healing.

EUS-Guided FNA for Diagnosis of Pancreatic Cystic Lesions: a Meta-Analysis.

BACKGROUND: Preoperative diagnosis of pancreatic cystic lesions (PCLs) must be reliable as the current standard treatment, major or total pancreatectomy, dramatically affects quality of life. Additionally, early diagnosis of malignancy is essential to an improved prognosis. The diagnostic accuracy of fluid analysis using endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) has been demonstrated in pancreatic solid lesions. The utility of this technique in the diagnosis of PCLs is still unknown. METHODS: A comprehensive search was performed in multiple databases. Studies differentiating benign and malignant PCLs via EUS-FNA were included in this meta-analysis. The quality of diagnostic accuracy studies (QUADAS) was adopted to evaluate the selected studies. Pooled sensitivity, specificity, likelihood ratio, diagnostic odds ratio, and summary receiver operating characteristic (sROC) curve analyses were conducted. Two main classification types of malignancy were characterized and analyzed. We also generated a subgroup analysis of available clinical factors. Publication bias was evaluated by Begg's and Egger's tests. RESULTS: Sixteen studies containing 1024 subjects have been published. The pooled sensitivity for malignant cytology according to classification 1 was 0.51 (95% CI, 0.45-0.58), and pooled specificity was 0.94 (95% CI, 0.92-0.96). When the detected PCLs were identified as classification 2, suspicious malignancy or potential malignancy, sensitivity and specificity were similar, 0.52 (95% CI, 0.46-0.57) and 0.97 (95% CI, 0.95-0.98) respectively. CONCLUSION: This meta-analysis demonstrates that EUS-FNA is a reliable clinical tool for the diagnosis of PCLs. However, a more accurate algorithm is needed to reduce various biases and to improve the sensitivity of EUS-FNA in the detection of malignant PCLs.

Store-operated calcium entry is present in HL-1 cardiomyocytes and contributes to resting calcium.

Store-operated Ca(2+) entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca(2+) during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells expressed stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE was tightly coupled to sarcoplasmic reticulum (SR)-Ca(2+) release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca(2+) channels (L-type and T-type channels) or reverse mode Na(+)/Ca(2+) exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca(2+) and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca(2+) homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart.

PTRF suppresses the progression of colorectal cancers.

As a key component of caveolae structure on the plasma membrane, accumulated evidence has suggested that Polymerase I and Transcript Release Factor (PTRF) plays a pivotal role in suppressing the progression of human malignances. However, the function of PTRF in the development of colorectal cancers is still unclear. Here we report that the expression of PTRF is significantly reduced in tumor tissues derived from human patients with colorectal cancers, and that the downregulation of PTRF correlates to the advanced stage of the disease. In addition, we found that the expression of PTRF negatively regulates the tumorigenic activities of colorectal cell lines (Colo320, HT29 and CaCo2). Furthermore, ectopic PTRF expression caused significant suppression of cellular proliferation, and anchorage-independent colony growth of Colo320 cells, which have the lowest expression level of PTRF in the three studied cell lines. Meanwhile, shRNA mediated knockdown of PTRF in CaCo2 cells significantly promoted cellular proliferation and anchorage-independent colony growth. In addition, in vivo assays further revealed that tumor growth was significantly inhibited in xenografts with ectopic PTRF expression as compared to untreated Colo320 cells, but was markedly enhanced in PTRF knockdown CaCo2 cells. Biochemical studies revealed that overexpression of PTRF led to the suppression of the AKT/mTOR pathway, as evidenced by reduced phosphorylation of AKT, mTOR, and downstream MMP-9. Thus, these findings, for the first time, demonstrated that PTRF inhibits the tumorigenesis of colorectal cancers and that it might serve as a potential therapeutic target for human colon cancer patients.

Visualization of MG53-mediated cell membrane repair using in vivo and in vitro systems.

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.