The SLITRK4-CNPY3 axis promotes liver metastasis of gastric cancer by enhancing the endocytosis and recycling of TrkB in tumour cells.

PURPOSE: Gastric cancer (GC) is a malignant tumour with high mortality, and liver metastasis is one of the main causes of poor prognosis. SLIT- and NTRK-like family member 4 (SLITRK4) plays an important role in the nervous system, such as synapse formation. Our study aimed to explore the functional role of SLITRK4 in GC and liver metastasis. METHODS: The mRNA level of SLITRK4 was evaluated using publicly available transcriptome GEO datasets and Renji cohort. The protein level of SLITRK4 in the tissue microarray of GC was observed using immunohistochemistry. Cell Counting Kit-8, colony formation, transwell migration assays in vitro and mouse model of liver metastasis in vivo was performed to investigate the functional roles of SLITRK4 in GC. Bioinformatics predictions and Co-IP experiments were applied to screen and identify SLITRK4-binding proteins. Western blot was performed to detect Tyrosine Kinase receptor B (TrkB)-related signaling molecules. RESULTS: By comparing primary and liver metastases from GC, SLITRK4 was found to be upregulated in tissues of GC with liver metastasis and to be closely related to poor clinical prognosis. SLITRK4 knockdown significantly abrogated the growth, invasion, and metastasis of GC in vitro and in vivo. Further study revealed that SLITRK4 could interact with Canopy FGF Signalling Regulator 3 (CNPY3), thus enhancing TrkB- related signaling by promoting the endocytosis and recycling of the TrkB receptor. CONCLUSION: In conclusion, the CNPY3-SLITRK4 axis contributes to liver metastasis of GC according to the TrkB-related signaling pathway. which may be a therapeutic target for the treatment of GC with liver metastasis.

Visual typing detection of brucellosis with a lateral flow immunoassay based on coloured latex microspheres.

AIMS: Treatment and preventive control strategies for Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) infection differ. A lateral flow immunoassay (LFIA) for the rapid typing and detection of brucellosis by using polychromatic dye-doped latex microspheres (LMs) as a labelling material was developed. METHODS AND RESULTS: This LFIA utilizes a double-antigen sandwich method in which the BP26 protein is used as the diagnostic antigen to detect brucellosis infection and the OMP31 protein is used as the identified antigen to distinguish between bovine and sheep brucellosis. Thus, people and animals infected with brucellosis can be diagnosed according to the different colours of the signals displayed on the detection lines. The results indicated that the accuracy of this assay was found to reach 98%, and the immunochromatographic test strip is highly accurate, shows good sensitivity and can facilitate typing diagnosis, among other features. CONCLUSIONS: The established LFIA can distinguish B. melitensis infection from B. abortus infection and produces results in a short period of time while retaining the advantages of LFIAs. SIGNIFICANCE AND IMPACT OF THE STUDY: This technology lays a foundation for the development of multi-disease test strips and the establishment of methods for rapid, multi-specimen quantitative detection and is thus of great importance for the development of medical diagnostic technologies.

Identification of growth differentiation factor 15 as a pro-fibrotic factor in mouse liver fibrosis progression.

The aim of this study was elucidate the inhibitory role of growth differentiation factor 15 (GDF15) in liver fibrosis and its possible activation mechanism in hepatic stellate cells (HSCs) of mice. We generated a GDF15-neutralizing antibody that can inhibit TGF-ß1-induced activation of the TGF-ß/Smad2/3 pathway in LX-2 cells. All the mice in this study were induced by carbon tetrachloride and thioacetamide. In addition, primary HSCs from mice were isolated from fresh livers using Nycodenz density gradient separation. The severity and extent of liver fibrosis were evaluated by Sirius Red and Masson staining. The effect of GDF15 on the activation of the TGF-ß pathway was detected using dual-luciferase reporter and Western blotting assays. The expression of GDF15 in cirrhotic liver tissue was higher than that in normal liver tissue. Blocking GDF15 with a neutralizing antibody resulted in a delay in primary hepatic stellate cell activation and remission of liver fibrosis induced by carbon tetrachloride or thioacetamide. Meanwhile, TGF-ß pathway activation was partly inhibited by a GDF15-neutralizing antibody in primary HSCs. These results indicated that GDF15 plays an important role in regulating HSC activation and liver fibrosis progression. The inhibition of GDF15 attenuates chemical-inducible liver fibrosis and delays hepatic stellate cell activation, and this effect is probably mainly attributed to its regulatory role in TGF-ß signalling.

Green synthesis and characterization of gold nanoparticles and their application for the rapid detection of glycyrrhizin with immunochromatographic strips.

In this study, a facile and environmentally friendly synthesis process was proposed without regular chemical additives. We successfully synthesized spherical gold nanoparticles (AuNPs) coated with glycyrrhizin (GL) by using GL as both a reductant and a stabilizer to reduce chloroauric acid. The obtained NPs were approximately 35 nm in size. The formation of these GL-AuNPs was verified by the presence of a surface plasmon resonance band at 526 nm. We also experimentally determined that in terms of chemical structure, GL can be used as a reducing agent to obtain colloidal gold. The d-glucuronic acid structure, rather than glycyrrhetinic acid (GA), plays an important reducing role in colloidal gold production. From this, we hypothesized that other compounds with sugar structures in Glycyrrhiza may also have the ability to reduce chloroauric acid. To mitigate the high cost and low efficiency of current GL detection methods, we applied AuNPs to the immunochromatographic system. Then, a colloidal gold immunochromatographic test strip based on the indirect competition method was developed for the rapid detection of GL, and the detection limit of this strip was 25 ng mL-1. The cross-test showed that the strip has high specificity. The test results are consistent with the data obtained by high-performance liquid chromatography (HPLC) with a coincidence rate of up to 100%. The rapid test strip is simple, fast, highly efficient and inexpensive, making it suitable for large-scale, rapid glycyrrhizin content determination.

Role of ubiquitin-conjugating enzyme E2T in the carcinogenesis and progression of pancreatic cancer.

Ubiquitin-conjugating enzyme E2T (UBE2T) is a recently discovered oncogenic protein. Numerous studies reported that UBE2T is highly expressed in various types of human cancer; however, its role in the carcinogenesis and progression of pancreatic cancer remains unknown. The aim of the present study was to investigate the role of UBE2T in pancreatic cancer progression through in vitro experiments in pancreatic cancer tissues and cell lines. The results obtained in the present study demonstrated that UBE2T served an important role in the initiation and progression of pancreatic cancer. Furthermore, increased expression of UBE2T in human pancreatic cancer tissues and pancreatic cancer cells was observed compared with normal tissues and cells. The effect of upregulating and downregulating UBE2T in pancreatic cancer cell lines was investigated using the MTT, wound-healing and migration and invasion assays. The results demonstrated that overexpression of UBE2T significantly promoted pancreatic cancer cell proliferation, migration and invasion compared with controls. However, UBE2T downregulation resulted in the inhibition of pancreatic cancer cell proliferation, migration and invasion. In addition, the results demonstrated that UBE2T may promote the epithelial mesenchymal transition in pancreatic cancer cells.

Blocking follistatin-like 1 attenuates liver fibrosis in mice by regulating transforming growth factor-beta signaling.

AIM: To elucidate the effect of inhibiting follistatin-like 1 on liver fibrosis and activation of hepatic stellate cells in mice. METHODS: We generated a follistatin-like 1 neutralizing antibody that can inhibit TGF-ß 1-induced expression of collagen1α1 in primary mouse liver fibroblasts. All of the mice in our study were induced with carbon tetrachloride and thioacetamide. In addition, primary hepatic stellate cells from mice were isolated from fresh livers using density gradient separation. The degree and extent of fibrosis in mouse livers from the different groups were evaluated by Sirius Red and Masson staining. The effect of the follistatin-like 1 neutralizing antibody on proliferation and migration of hepatic stellate cells was detected using CCK-8 and Transwell assays, respectively. RESULTS: Expression of follistatin-like 1 in human cirrhotic liver tissue was higher than that in normal liver tissue. Blocking follistatin-like 1 resulted in a delay of primary hepatic stellate cell activation and down-regulation of the migratory capacity of hepatic stellate cells. Blocking follistatin-like 1 also down-regulated TGF-beta signaling in primary hepatic stellate cells from mice. Finally, inhibition of follistatin-like 1 attenuated liver fibrosis and liver function damage in vivo. CONCLUSIONS: Inhibiting follistatin-like 1 attenuates liver fibrosis and causes a delay in hepatic stellate cell activation. The effect of follistatin-like 1 on liver fibrosis is mainly attributed to its role in regulating TGF-beta signaling.

Gemcitabine-based polymer-drug conjugate for enhanced anticancer effect in colon cancer.

In this study, we have demonstrated gemcitabine (GEM)-conjugated amphiphilic biodegradable polymeric drug carriers. Our aim was to increase the chemotherapeutic potential of GEM in colon cancer by forming a unique polymer-drug conjugates. The polymer-drug conjugate micelles were nanosized with a typical spherical shape. The GEM-conjugated methoxy poly(ethylene glycol)-poly(lactic acid) (GEM-PL) exhibited a controlled release of drug in both the pH conditions. The developed GEM-PL efficiently killed the HT29 cancers cells in a typical time dependent manner. The clonogenic assay further confirmed the superior anticancer effect of GEM-PL which showed least number of colonies. GEM-PL formulation exhibited a significantly higher apoptosis of cancer cells (∼25%) when stained using Annexin-V/PI kit. Conjugation of GEM to the mPEG-PLA significantly enhanced the blood circulation potential in animal model compared to that of free GEM. GEM-PL could prevent quick elimination of the drug and can provide sufficient time for the greater accumulation of GEM at the tumor sites. GEM-PL showed a remarkable tumor regression effect as evident from the lowest tumor volume in HT-29 containing tumor model. Overall, mPEG-PLA/GEM conjugates showed the potential of polymer-based drug targeting and might hold significant clinical potential in the treatment of colon cancers.

Silencing of MICAL-L2 suppresses malignancy of ovarian cancer by inducing mesenchymal-epithelial transition.

Ovarian cancer remains the disease with the highest associated mortality rate of gynecologic malignancy due to cancer metastasis. Rearrangement of actin cytoskeleton by cytoskeleton protein plays a critical role in tumor cell metastasis. MICAL-L2, a member of MICAL family, can interact with actin-binding proteins, regulate actin cross-linking and coordinate the assembly of adherens junctions and tight junctions. However, the roles of MICAL-L2 in tumors and diseases have not been explored. In this study, we found that MICAL-L2 protein is significantly up-regulated in ovarian cancer tissues along with FIGO stage and associated with histologic subgroups of ovarian cancer. Silencing of MICAL-L2 suppressed ovarian cancer cell proliferation, migration and invasion ability. Moreover, silencing of MICAL-L2 prevented nuclear translocation of ß-catenin, inhibited canonical wnt/ß-catenin signaling and induced the mesenchymal-epithelial transition (MET). Taken together, our data indicated that MICAL-L2 may be an important regulator of epithelial-mesenchymal transition (EMT) in ovarian cancer cells and a new therapeutic target for interventions against ovarian cancer invasion and metastasis.

Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation.

BACKGROUND: A remolded microenvironment in hepatocellular carcinoma (HCC) caused by abnormally expressed matricellular proteins could promote HCC progression. The cell-matrix interactions mediated by integrins play an important role in tumor microenvironment. Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), an extracellular matrix (ECM) protein with angiogenic and anti-inflammatory effects, is abnormally highly expressed in HCC. Here we aim to analyze its expression in liver and HCC tissues, investigate the underlined mechanisms accounted for HCC progression. METHODS: EDIL3 expression level is examined in normal liver, cirrhotic liver and HCC at both mRNA and protein level. The association between EDIL3 and clinical outcomes is analyzed. The pattern of EDIL3 expression and location is examined using Immunofluorescence and ELISA. Overexpression or knock-down of EDIL3 in a panel of cell lines are subjected to assays related to proliferation, invasion, and anoikis to investigate the mechanisms of this matrix protein in HCC progression. Recombinant EDIL3 treatment is applied to confirm the results. RESULTS: Compared with normal liver and cirrhotic liver, EDIL3 is elevated in HCC. High level of EDIL3 protein is much more commonly in patients with larger tumor or portal vein tumor thrombus (PVTT) formation, associated with poor prognosis. EDIL3 is abundantly expressed in HCC cells and secreted by cancer cells. In vitro and in vivo studies indicate that EDIL3, probably in an autocrine manner, inhibits anoikis and promotes anchorage-independent growth of HCC cells. Further mechanistic studies suggest integrin ligation by EDIL3 and thus that the sustained activation of the FAK-Src-AKT signal is responsible for the anoikis resistance and anchorage independence. Both the administration of cilengitide, a RGD-containing integrin antagonist, and silencing of integrin αV, an important RGD-binding integrin, results in the blockade of anoikis-resistance induced by EDIL3. CONCLUSION: Our study suggests that high levels of autocrine EDIL3 may contribute to a receptive microenvironment for the survival of detached HCC cells and may involve in cancer cell spreading. We also highlight the importance of interaction between EDIL3 and integrin αV and suggest disrupting the ligation of EDIL3 to integrins via RGD-blocking in selected patients may bear potential therapeutic value.

PNMA1 promotes cell growth in human pancreatic ductal adenocarcinoma.

Paraneoplastic Ma1 (PNMA1) is a member of an expanding family of 'brain/testis' proteins involved in an autoimmune disorder defined as paraneoplastic neurological syndrome (PNS). Although it is widely studied in PNS, little is known about the underlying clinical significance and biological function of PNMA1 in tumors. Here, we find that elevated PNMA1 expression is more commonly observed in pancreatic ductal adenocarcinoma (PDAC) cell lines, compared with normal pancreatic cell and tissues from pancreatic ductal adenocarcinoma patient. Besides, higher PNMA1 expression is closely correlated with large tumor size. Suppression of endogenous PNMA1 expression decreases cell viability and promotes cell apoptosis. Subsequent studies reveal that the PI3K/AKT, MAPK/ERK pathway and members of the anti-apoptotic Bcl-2 family may be involved in the pro-survival and anti-apoptotic effect of PNMA1 on PDAC. Taken together, this study provides evidence that PNMA1 is involved in tumor growth of pancreatic carcinoma and PNMA1-related pathways might represent a new treatment strategy.

DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3ß1 integrin-Rho GTPase signaling.

Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3ß1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor.

SERPINA3 promotes endometrial cancer cells growth by regulating G2/M cell cycle checkpoint and apoptosis.

Endometrial carcinoma (EC) is the most common gynecologic cancer worldwide and is one of the leading causes of death in women. Therefore, it is urgent to elucidate the pathological mechanisms of EC. SERPINA3 is a member of the serpin super-family of protease inhibitors. Its aberrant expression has been observed in various tumor cells. However, its clinical significance and biological function in endometrial cancer remains unknown. In the present study, we demonstrated that SERPINA3 expression was significantly up-regulated in EC samples and was closely correlated with lower differentiation, higher stage, positive lymph node or vascular thrombosis and negative estrogen receptor (ER), indicating a poor prognosis. We then demonstrated that SERPINA3 promoted EC cells proliferation by regulating G2/M checkpoint in cell cycle and inhibited cells apoptosis, and we further uncovered that the pro-proliferative effect of SERPINA3 on EC was likely ascribed to the activation of MAPK/ERK1/2 and PI3K/AKT signaling. The results of our study may provide insight into the application of SERPINA3 as a novel predictor of clinical outcomes and a potential therapeutic target of EC.

CTHRC1 acts as a prognostic factor and promotes invasiveness of gastrointestinal stromal tumors by activating Wnt/PCP-Rho signaling.

Gastrointestinal stromal tumors (GISTs) are the major gastrointestinal mesenchymal tumors with a variable malignancy ranging from a curable disorder to highly malignant sarcomas. Metastasis and recurrence are the main causes of death in GIST patients. To further explore the mechanism of metastasis and to more accurately estimate the recurrence risk of GISTs after surgery, the clinical significance and functional role of collagen triple helix repeat containing-1 (CTHRC1) in GIST were investigated. We found that CTHRC1 expression was gradually elevated as the risk grade of NIH classification increased, and was closely correlated with disease-free survival and overall survival in 412 GIST patients. In vitro experiments showed that recombinant CTHRC1 protein promoted the migration and invasion capacities of primary GIST cells. A luciferase reporter assay and pull down assay demonstrated that recombinant CTHRC1 protein activated noncanonical Wnt/PCP-Rho signaling but inhibited canonical Wnt signaling. The pro-motility effect of CTHRC1 on GIST cells was reversed by using a Wnt5a neutralizing antibody and inhibitors of Rac1 or ROCK. Taken together, these data indicate that CTHRC1 may serve as a new predictor of recurrence risk and prognosis in post-operative GIST patients and may play an important role in facilitating GIST progression. Furthermore, CTHRC1 promotes GIST cell migration and invasion by activating Wnt/PCP-Rho signaling, suggesting that the CTHRC1-Wnt/PCP-Rho axis may be a new therapeutic target for interventions against GIST invasion and metastasis.