Inhibition of the proliferation, migration, and invasion of human breast cancer cells by leucine aminopeptidase 3 inhibitors derived from natural marine products.

Leucine aminopeptidase 3 is involved in the progression and metastasis of several cancers. This study aimed to screen anti-tumor lead compounds targeting leucine aminopeptidase 3. The compounds' suppression effect on enzyme activity and anti-tumor activity were evaluated through a series of assays. Leucine aminopeptidase 3 overexpression K562 cells were used as an enzyme source to screen 43 natural marine compounds. Compounds 5 and 6 exhibited high suppression effect on leucine aminopeptidase 3 activity. Cell activity tests indicated that both compounds have an anti-proliferative effect on triple-negative breast cancer cells. Wound healing assay and transwell invasion assay showed that both compounds could inhibit the migration and invasion of breast cancer cells. Immunoblot analysis exhibited that both compounds could downregulate the expression of metastasis-related proteins fascin and matrix metalloproteinase-2/9. A molecular dynamic simulation process was applied to discover the key features of compounds 5 and 6 in binding to leucine aminopeptidase 3 active site. This study described the anti-tumor effects of two leucine aminopeptidase 3 small molecule inhibitors. Taken together, compounds 5 and 6 could be used as anti-tumor lead compounds targeting leucine aminopeptidase 3.

Polysialylation promotes neural cell adhesion molecule-mediated cell migration in a fibroblast growth factor receptor-dependent manner, but independent of adhesion capability.

Polysialic acid (PSA), a carbohydrate polymer mainly present in the neural cell adhesion molecule (NCAM), promotes neural plasticity; however, its mode of action in tumor malignancy remains largely unknown. In this study, we investigated the influence of polysialylation on cell migration. PSA consistently promoted cell migration on different extracellular matrices (ECMs) but differentially affected cell adhesion. All of these actions were reversed by endo-N-acetylneuraminidase treatment, and PSA-driven migration was inhibited by the specific fibroblast growth factor receptor (FGFR) inhibitor Su5402. Consistent with this latter observation, PSA-stimulated migration on different ECMs was paralleled by activation of the FGFR and its downstream signaling components, PLC-γ, focal adhesion kinase and extracellular signal-regulated kinase 1/2. In contrast, the pattern of p59(fyn) activation correlated with differential adhesion to different ECMs. Collectively, these results indicate that PSA-conjugated NCAM potentiates signal transduction by the FGFR pathway and thereby enhances cell migration independent of adhesion capability, providing additional insights into the role of PSA in cancer development.

[Inhibitory effect of Astragalus polysaccharides on apoptosis of pancreatic beta-cells mediated by Fas in diabetes mellitus rats].

OBJECTIVE: To observe the effects of Astragalus polysaccharides (APS) on the BG, insulin and C-peptide in serum, ultrastructure and Fas expression of pancreatic beta-cells in diabetes mellitus (DM) rats. METHODS: Thirty DM rats induced by streptozotocin (STZ) were randomly divided into three groups: DM group, APS 200 mg/kg group, APS 400 mg/kg group, another 10 normal rats were taken as the control group. The drug was given by intraperitoneal for 6 weeks. The level of BG was determined by ONE TOUCH II machine. The levels of insulin and C-peptide in serum were measured by radioimmunoassay. The expression of Fas was observed by immunohistochemistry. Moreover, the TEM was used to observe the ultrastructure of pancreatic beta-cells. RESULTS: (1) DM rats showed significant increase in BG compared with control group (P < 0.05). APS could decrease the level of FG (P < 0.05). (2) DM rats showed significant decrease in insulin and C-peptide in serum compared with control group (P < 0.05). APS has no appearance effects on the levels of them (P > 0.05). (3) The expression of Fas of beta-cells was significantly increased in DM rats, which was significantly inhibited by APS treatment. (4) It showed degenerative changes of pancreatic beta-cells ultramicroscopic structure of the DM rats, while APS treatment could significantly improve the damage. CONCLUSION: APS exerts its therapeutic effects on DM, which maybe related to the significant decreasing of the Fas expression and inhibiting the apoptosis of beta-cells.

Ding's herbal enema treats dextran sulfate sodium-induced colitis in mice by regulating the gut microbiota and maintaining the Treg/Th17 cell balance.

Ding's herbal enema (DHEP) is a traditional Chinese medicinal therapy that has been used to treat ulcerative colitis (UC) in China. The present study determined the molecular mechanism of the effect of DHEP in UC treatment. C57BL/6J mice were treated with 3.5% (w/v) dextran sulfate sodium (DSS) for 7 days to establish an animal model of colitis. The mice were divided into five groups (n=5): Control, vehicle, DHEP, mesalazine and ß-sitosterol. After oral administration for 7 days, the body weight, disease activity index, histopathology and inflammatory factors were analyzed. The fractions of CD4+Foxp3+ regulatory T (Treg) cells and CD4+IL-17A+ T helper (Th) cells were determined by flow cytometry. Gut microbiota composition was analyzed by next-generation sequencing. The results revealed that DHEP and ß-sitosterol could significantly alleviate the symptoms of DSS-induced UC. Furthermore, the levels of IL-6, cyclooxygenase-2, TNF-α and p65 were reduced after administration of DHEP. Additionally, the data indicated that DHEP could increase the abundance of seven operational taxonomic units (OTUs) and decrease the abundance of 12 OTUs in the gut microbiota. The content of short-chain fatty acids in the colon remodeled the balance of Treg/Th17 cells in DSS-induced UC in mice. The present study preliminarily defined the mechanism of action of DHEP in UC that may be associated with the regulation of the gut microbiota composition, and maintenance of the balance between Treg and Th17 cells. Furthermore, ß-sitosterol exhibited the same effects with DHEP and it could be a possible substitute for DHEP in UC treatment.

Hsa_circRNA_000166 Promotes Cell Proliferation, Migration and Invasion by Regulating miR-330-5p/ELK1 in Colon Cancer.

INTRODUCTION: Circular RNAs (circRNAs), a novel class of non-coding RNAs, which are widely expressed in human cells, have essential roles in the development and progression of cancers. The aim of this study is to figure out the role of circ_000166 in colon cancer (CC) development and the signaling pathway involved. MATERIALS AND METHODS: HT29 and HCT116 cells were transfected with siRNA of circRNA, miRNA mimics and inhibitors. Cell proliferation, migration and invasion were examined using CCK-8 assay and transwell assay, respectively. Luciferase reporter assay was used to validate the targets of circRNA and miRNA. CC cells were implanted into nude mice subcutaneously to detect tumor growth. RESULTS: hsa_circRNA_000166 was significantly upregulated in the human CC tissue and in the CC cell lines. Knockdown of hsa_circRNA_000166 reduced cell viability, colony formation, migration and invasion in vitro and decreased tumor size and weight in vivo. Luciferase reporter assay revealed that miR-330-5p was the target of circRNA_000166. miR-330-5p could bind to 3' untranslated region (3'UTR) of ELK1 to downregulate both mRNA and protein expression of ELK1. Dual inhibition of circRNA_000166 and miR-330-5p inhibited the suppression of cell proliferation, migration and invasion induced by si-circRNA_000166. CONCLUSION: The data of this study demonstrated that the hsa_circRNA_000166 could upregulated the expression of gene ELK1 by sponging miR-330-5p, which may contribute to a better understanding of the regulatory circRNA/miRNA/mRNA network and CC pathogenesis.

AlCl3 exposure regulates neuronal development by modulating DNA modification.

BACKGROUND: As the third most abundant element, aluminum is widespread in the environment. Previous studies have shown that aluminum has a neurotoxic effect and its exposure can impair neuronal development and cognitive function. AIM: To study the effects of aluminum on epigenetic modification in neural stem cells and neurons. METHODS: Neural stem cells were isolated from the forebrain of adult mice. Neurons were isolated from the hippocampi tissues of embryonic day 16-18 mice. AlCl3 at 100 and 200 µmol/L was applied to stem cells and neurons. RESULTS: Aluminum altered the differentiation of adult neural stem cells and caused apoptosis of newborn neurons while having no significant effects on the proliferation of neural stem cells. Aluminum application also significantly inhibited the dendritic development of hippocampal neurons. Mechanistically, aluminum exposure significantly affected the levels of DNA 5-hydroxy-methylcytosine, 5-methylcytosine, and N6-methyladenine in stem cells and neurons. CONCLUSION: Our findings indicate that aluminum may regulate neuronal development by modulating DNA modifications.

Identification of key genes for predicting colorectal cancer prognosis by integrated bioinformatics analysis.

Colorectal cancer (CRC) is a life-threatening disease with a poor prognosis. Therefore, it is crucial to identify molecular prognostic biomarkers for CRC. The present study aimed to identify potential key genes that could be used to predict the prognosis of patients with CRC. Three CRC microarray datasets (GSE20916, GSE73360 and GSE44861) were downloaded from the Gene Expression Omnibus (GEO) database, and one dataset was obtained from The Cancer Genome Atlas (TCGA) database. The three GEO datasets were analyzed to detect differentially expressed genes (DEGs) using the BRB-ArrayTools software. Functional and pathway enrichment analyses of these DEGs were performed using the Database for Annotation, Visualization and Integrated Discovery tool. A protein-protein interaction (PPI) network of DEGs was constructed, hub genes were extracted, and modules of the PPI network were analyzed. To investigate the prognostic values of the hub genes in CRC, data from the CRC datasets of TCGA were used to perform the survival analyses based on the sample splitting method and Cox regression model. Correlation among the hub genes was evaluated using Spearman's correlation analysis. In the three GEO datasets, a total of 105 common DEGs were identified, including 51 down- and 54 up-regulated genes in CRC compared with normal colorectal tissues. A PPI network consisting of 100 DEGs and 551 edges was constructed, and 44 nodes were identified as hub genes. Among these 44 genes, the four hub genes TIMP metallopeptidase inhibitor 1 (TIMP1), solute carrier family 4 member 4 (SLC4A4), aldo-keto reductase family 1 member B10 (AKR1B10) and ATP binding cassette subfamily E member 1 (ABCE1) were associated with overall survival (OS) in patients with CRC. Three significant modules were extracted from the PPI network. The hub gene TIMP1 was present in Module 1, ABCE1 was involved in Module 2 and SLC4A4 was identified in Module 3. Univariate analysis revealed that TIMP1, SLC4A4, AKR1B10 and ABCE1 were associated with the OS of patients with CRC. Multivariate analysis demonstrated that SLC4A4 may be an independent prognostic factor associated with OS. Furthermore, the results from correlation analysis revealed that there was no correlation between TIMP1, SLC4A4 and ABCE1, whereas AKR1B10 was positively correlated with SLC4A4. In conclusion, the four key genes TIMP1, SLC4A4, AKR1B10 and ABCE1 associated with the OS of patients with CRC were identified by integrated bioinformatics analysis. These key genes may be used as prognostic biomarkers to predict the survival of patients with CRC, and may therefore represent novel therapeutic targets for CRC.

A new method to evaluate the enzyme-suppressing activity of a leucine aminopeptidase 3 inhibitor.

The expression of leucine aminopeptidase 3 (LAP3) is associated with the prognosis for and malignant transformation of many types of tumors. Therefore, a LAP3 inhibitor may represent a new strategy for cancer therapy. Evaluating the suppression of enzyme activity by an LAP3 inhibitor is essential. Right now, leucine aminopeptidases (LAPs) purified from the porcine kidneys are the only enzymes that can be used to evaluate the suppression of enzyme activity by an LAP3 inhibitor. This approach cannot accurately reflect the suppression of human LAP3 by an inhibitor. The current study developed a new method with which to evaluate the suppression of enzyme activity by an LAP3 inhibitor. Total protein from K562 cells seldom catalyzed the LAP3 substrate. A lentivirus was used to induce K562 cells to overexpress LAP3 (K562-LAP3). After puromycin screening, flow cytometry data indicated that 98.8% of cells expressed green fluorescent protein. The expression of LAP3 in K562-LAP3 cells was also assessed using Western blotting. K562-LAP3 cells were lysed with ultrasonication. Total protein was used as an enzyme source and L-leucine p-nitroaniline hydrochloride was used as a substrate to measure enzyme activity. Total protein from K562-LAP3 cells catalyzed the substrate more than that from K562 cells did. The LAP3 inhibitor ubenimex was used as a positive control to evaluate the suppression of LAP3 enzyme activity. Results indicated that ubenimex significantly inhibited the enzyme activity of LAP3. This approach provides a convenient and accurate way to evaluate the suppression of enzyme activity by an LAP3 inhibitor.

[Anticancer effects of tea polyphenols on colorectal cancer with microsatellite instability in nude mice].

OBJECTIVE: To study the anticancer effects of tea polyphenols on colorectal cancer with microsatellite instability (MSI) in nude mice and to explore its mechanism. METHODS: A colostomy was performed on the caecum of nude mice. Tumor fragments collected from the subcutaneous tumor of hMSH2-absence colon carcinoma Lovo cell line were surgically implanted onto the submucosa of the caecum during colostomy to establish the model. Then, the nude mice were divided into untreated group and 50, 75 and 100 mg/kg tea polyphenols groups. The mice in tea polyphenols-treated groups were given intra-abdominal injection of 50, 75 and 100 mg/kg tea polyphenols respectively. The inhibition rates of tumors were calculated, and microsatellite instability (MSI) and the alteration of transforming growth factor-beta1 (TGF-beta1), TGF-beta2 and insulin-like growth factor (IGF) were detected by Genescan method at different times after the injection. RESULTS: The tumor volumes of the three groups began to decrease at the 1st week and decreased most greatly from 2 to 3 weeks after treatment, and then the tumors tended to increase. The study found that tea polyphenols could inhibit the tumor growth. The tumor inhibition rates in the three treated groups were significantly higher than those in untreated group 1, 2, 3 and 4 weeks after treatment (P<0.01). Detection of MSI showed that the colorectal tumor in the untreated group presented with four MSI signs, including BAT-25, D2S123, D5S346 and D17S250, and TGF-beta1, TGF-beta2, IGF expressions. After using the tea polyphenols, the microsatellite tended to become stable. CONCLUSION: Tea polyphenols can inhibit the mismatch-repair-gene deficient colorectal cancer in nude mice by down-regulating the microsatellite instability.

Aminopeptidase N inhibitor 4cc synergizes antitumor effects of 5-fluorouracil on human liver cancer cells through ROS-dependent CD13 inhibition.

Aminopeptidase N (APN, also known as CD13) is involved in cellular processes of various types of tumors and a potential anti-cancer therapeutic target. Here, we report the effect of an APN inhibitor 4cc in enhancing sensitivity of hepatocellular carcinoma (HCC) cell lines and xenograft model in response to 5-fluorouracil (5-FU) in vivo and in vitro. The treatment of the combination of 4cc with 5-FU, compared to the combination of bestain with 5-FU, markedly suppressed cell growth and induced apoptosis of HCC cells, accompanying the increase in the level of reactive oxygen species (ROS) and followed by a decrease in the mitochondrial membrane potential (ΔΨM). Furthermore, the combination of 4cc and 5-FU showed a significant inhibitory effect on the growth of HCC xenograft tumors. In addition, following the treatment of 4cc, APN activity and clonogenic formation and the number of CD13-positive cells in PLC/PRF/5 cells were significantly decreased, suggesting that 4cc may also inhibit liver cancer stem cells by CD13 inhibition. These results showed that the APN inhibitor 4cc synergizes antitumor effects of 5-FU on human liver cancer cells via ROS-mediated drug resistance inhibition and concurrent activation of the mitochondrial pathways of apoptosis.

Intrapulmonary shear stress enhancement: a new therapeutic approach in acute myocardial ischemia.

OBJECTIVE: Ischemic heart disease (IHD) is a leading cause of mortality with insufficient results of current therapies, most probably due to maintained endothelial dysfunction conditions. Alternatively, we propose a new treatment that promotes endothelial shear stress (ESS) enhancement using an intrapulmonary pulsatile catheter. METHODS: Twelve piglets, divided in equal groups of 6: pulsatile (P) and non-pulsatile (NP), underwent permanent left anterior descending coronary artery ligation through sternotomy. After 1 h of ischemia and heparin injection (150 IU/kg): in P group, a pulsatile catheter was introduced into the pulmonary trunk and pulsated intermittently over 1 h, and irrespective of heart rate (110 bpm). In NP group, nitrates were given (7 ± 2 mg/kg/min) for 1 h. RESULTS: In P group all 6 animals survived ischemia for 120 min, but in NP group only 2 animals survived. The 4 animals that died during the experiment in NP group survived for 93 ± 14 min. Hemodynamics and cardiac output (CO) were significantly improved in P group compared with NP group: CO was 0.92 ± 0.15 vs. 0.52 ± 0.08 in NP group (L/min; p < 0.05), respectively. Vascular resistances (dynes.s.cm(-5)/kg) were significantly (p < 0.05) lower in P group versus NP group: pulmonary resistance was 119 ± 13 vs. 400 ± 42 and systemic resistance was 319 ± 43 vs. 1857 ± 326, respectively. Myocardial apoptosis was significantly (p < 0.01) lower in P group (0.66 ± 0.07) vs. (4.18 ± 0.27) in NP group. Myocardial endothelial NO synthase mRNA expression was significantly (p < 0.01) greater in P group (0.90 ± 0.09) vs. (0.25 ± 0.04) in NP group. CONCLUSIONS: Intrapulmonary pulsatile catheter could improve hemodynamics and myocardial contractility in acute myocardial ischemia. This represents a cost-effective method, suitable for emergency setting as a first priority, regardless of classical coronary reperfusion.

Multifunctional nanocomposite based on graphene oxide for in vitro hepatocarcinoma diagnosis and treatment.

Because of its unique chemical and physical properties, graphene oxide (GO) has attracted a large number of researchers to explore its biomedical applications in the past few years. Here, we synthesized a novel multifunctional nanocomposite based on GO and systemically investigated its applications for in vitro hepatocarcinoma diagnosis and treatment. This multifunctional nanocomposite named GO-PEG-FA/Gd/DOX was obtained as the following procedures: gadolinium-diethylenetriamine-pentaacetic acid-poly(diallyl dimethylammonium) chloride (Gd-DTPA-PDDA) as magnetic resonance imaging (MRI) probe was applied to modify GO by simple physical sorption with a loading efficiency of Gd(3+) up to 0.314 mg mg(-1). In order to improve its tumor targeting imaging and treatment efficiency, the obtained intermediate product was further modified with folic acid (FA). Finally, the nanocomposite was allowed to load anticancer drug doxorubicin hydrochloride via π-π stacking and hydrophobic interaction with the loading capacity reaching 1.38 mg mg(-1). MRI test revealed that GO-PEG-FA/Gd/DOX exhibit superior tumor targeting imaging efficiency over free Gd(3+). The in vitro release of DOX from the nanocomposite under tumor relevant condition (pH 5.5) was fast at the initial 10 h and then become relatively slow afterward. Moreover, we experimentally demonstrated that the multifunctional nanocomposite exhibited obviously cytotoxic effect upon cancer cells. Above results are promising for the next in vivo experiment and make it possible to be a potential candidate for malignancy early detection and specific treatment.