Lithium chloride inhibits the migration and invasion of osteosarcoma cells by blocking nuclear translocation of phospho-Erk.

Lithium chloride (LiCl) is an important mood-stabilizing therapeutic agent for bipolar disorders, which has also been shown to inhibit cancer cell metastasis. Investigations of LiCl-induced signaling have focused mainly on extracellular signal regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3 (GSK-3). However, little is known about the differences in cellular activities resulting from specific signaling via each of these pathways. In this study, we investigated the difference in responses between the Wnt/ß-catenin and ERK pathways by LiCl or epidermal growth factor (EGF) treatment of osteosarcoma cells. In particular, we analyzed the mechanisms responsible for differences in cell mobility and cell proliferation when pERK or ß-catenin is activated. In osteosarcoma cells treated with LiCl or EGF, active ß-catenin and p-ERK protein levels were significantly increased compared to those in the control group. However, in wound healing and transwell invasion assays, U2OS and SaOS2 cell migration was significantly reduced by LiCl treatment but increased by EGF treatment. In addition, the proliferation of U2OS cells was reduced by LiCl treatment but increased by EGF treatment. Using immunofluorescence microscopy, we observed nuclear accumulation of phosphorylated ERK (pERK) with EGF treatment, but pERK was restricted to the perinuclear area with LiCl treatment. These results were confirmed using immunoblot assays after subcellular fractionation. Together, these data suggest that LiCl interferes with the translocation of pERK from the cytoplasm to the nucleus.

Oct4 downregulation-induced inflammation increases the migration and invasion rate of oral squamous cell carcinoma.

Inflammatory changes are involved in tumor cell proliferation, migration, and invasion. Tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) play important roles in inflammatory regulation during tumor development. Oct4 acts as a transcription factor that modulates inflammatory changes in mesenchymal stem cells. In this study, we explored the role of Oct4 in the invasion and migration of oral squamous cell carcinoma (OSCC) cells. LPS and TNF-α were used to treat the OSCC cell lines HN4 and CAL27 to induce inflammation. The generation of inflammatory cytokines, including TNF-α, interleukin (IL)-1ß, and IL-6, was evaluated by enzyme-linked immunosorbent assay and real-time quantitative PCR. Western blot analysis was employed to detect the expression and phosphorylation of JNK1, p65, and STAT3, which are key modulators of inflammation. Wound scratch healing and transwell invasion assays were further used to determine the role of inflammation in the invasion and migration of OSCC cells. Robust inflammation was observed in HN4 and CAL27 cells treated with LPS and TNF-α. A marked increase in JNK1, p65, and STAT3 phosphorylation levels in OSCC cells was also detected after LPS and TNF-α treatment. The migration and invasion of HN4 and CAL27 cells were significantly boosted by stimulation with LPS and TNF-α. Furthermore, Oct4 mRNA and protein levels were significantly upregulated by stimulation with LPS and TNF-α. Silencing of Oct4 led to reduced inflammation and decreased levels of phosphorylated JNK1, p65, and STAT3 and impaired invasion and migration in LPS- and TNF-α-stimulated OSCC cells. Overall, LPS- and TNF-α-induced inflammation suppressed the migration and invasion of OSCC cells by upregulating Oct4 expression.

A Dynamic Interbody Cage Improves Bone Formation in Anterior Cervical Surgery: A Porcine Biomechanical Study.

BACKGROUND: Anterior cervical discectomy and fusion (ACDF) with a rigid interbody spacer is commonly used in the treatment of cervical degenerative disc disease. Although ACDF relieves clinical symptoms, it is associated with several complications such as pseudoarthrosis and adjacent segment degeneration. The concept of dynamic fusion has been proposed to enhance fusion and reduce implant subsidence rate and post-fusion stiffness; this pilot preclinical animal study was conducted to begin to compare rigid and dynamic fusion in ACDF. QUESTIONS/PURPOSES: Using a pig model, we asked, is there (1) decreased subsidence, (2) reduced axial stiffness in compression, and (3) improved likelihood of bone growth with a dynamic interbody cage compared with a rigid interbody cage in ACDF? METHODS: ACDF was performed at two levels, C3/4 and C5/6, in 10 pigs weighing 48 to 55 kg at the age of 14 to 18 months (the pigs were skeletally mature). One level was implanted with a conventional rigid interbody cage, and the other level was implanted with a dynamic interbody cage. The conventional rigid interbody cage was implanted in the upper level in the first five pigs and in the lower level in the next five pigs. Both types of interbody cages were implanted with artificial hydroxyapatite and tricalcium phosphate bone grafts. To assess subsidence, we took radiographs at 0, 7, and 14 weeks postoperatively. Subsidence less than 10% of the disc height was considered as no radiologic abnormality. The animals were euthanized at 14 weeks, and each operated-on motion segment was harvested. Five specimens from each group were biomechanically tested under axial compression loading to determine stiffness. The other five specimens from each group were used for microCT evaluation of bone ingrowth and ongrowth and histologic investigation of bone formation. Sample size was determined based on 80% power and an α of 0.05 to detect a between-group difference of successful bone formation of 15%. RESULTS: With the numbers available, there was no difference in subsidence between the two groups. Seven of 10 operated-on levels with rigid cages had subsidence on a follow-up radiograph at 14 weeks, and subsidence occurred in two of 10 operated-on levels with dynamic cages (Fisher exact test; p = 0.07). The stiffness of the unimplanted rigid interbody cages was higher than the unimplanted dynamic interbody cages. After harvesting, the median (range) stiffness of the motion segments fused with dynamic interbody cages (531 N/mm [372 to 802]) was less than that of motion segments fused with rigid interbody cages (1042 N/mm [905 to 1249]; p = 0.002). Via microCT, we observed bone trabecular formation in both groups. The median (range) proportions of specimens showing bone ongrowth (88% [85% to 92%]) and bone volume fraction (87% [72% to 100%]) were higher in the dynamic interbody cage group than bone ongrowth (79% [71% to 81%]; p < 0.001) and bone volume fraction (66% [51% to 78%]; p < 0.001) in the rigid interbody cage group. The percentage of the cage with bone ingrowth was higher in the dynamic interbody cage group (74% [64% to 90%]) than in the rigid interbody cage group (56% [32% to 63%]; p < 0.001), and the residual bone graft percentage was lower (6% [5% to 8%] versus 13% [10% to 20%]; p < 0.001). In the dynamic interbody cage group, more bone formation was qualitatively observed inside the cages than in the rigid interbody cage group, with a smaller area of fibrotic tissue under histologic investigation. CONCLUSION: The dynamic interbody cage provided satisfactory stabilization and percentage of bone ongrowth in this in vivo model of ACDF in pigs, with lower stiffness after bone ongrowth and no difference in subsidence. CLINICAL RELEVANCE: The dynamic interbody cage appears to be worthy of further investigation. An animal study with larger numbers, with longer observation time, with multilevel surgery, and perhaps in the lumbar spine should be considered.

Static Versus Expandable Polyether Ether Ketone (PEEK) Interbody Cages: A Comparison of One-Year Clinical and Radiographic Outcomes for One-Level Transforaminal Lumbar Interbody Fusion.

OBJECTIVE: To examine the effect of static versus expandable polyether ether ketone (PEEK) cages on both clinical and radiographic outcomes. METHODS: A retrospective cohort study was conducted on patients who underwent one-level transforaminal lumbar interbody fusion with either a static or expandable PEEK cage. Patient outcomes were obtained from chart review and radiographic outcomes were measured using standing, lateral radiographs. Recovery ratios and the proportion of patients achieving the minimally clinically important difference were calculated for Oswestry Disability Index (ODI), Physical Component Score-12, Mental Component Score-12, visual analogue scale for back, and visual analogue scale for leg at 1 year and compared between groups. Multivariate linear regression analysis was performed to determine the effect of cage type on the change in patient-reported outcome measures, controlling for demographic factors. RESULTS: A total of 240 patients (137 static, 103 expandable) were included in the final analysis. ΔPhysical Component Score-12 scores at 3 months were significantly greater for the static group (16.0 vs. 10.0, P = 0.043) compared with the expandable group. Multivariate regression demonstrated that use of an expandable cage was associated with greater improvements in ΔODI (ß: -7.82, P = 0.048) at 1 year. No differences were found in the perioperative change in sagittal spinal alignment within or between groups at 1 year. Subsidence rates failed to show any statistically significant difference between the 2 groups. CONCLUSIONS: Transforaminal lumbar interbody fusion with an expandable PEEK cage is an independent predictor of improved ODI scores at 1 year. Our study showed no significant differences in subsidence rates or changes in sagittal spinal alignment between static and expandable PEEK cages.

Tripartite-motif protein 21 knockdown extenuates LPS-triggered neurotoxicity by inhibiting microglial M1 polarization via suppressing NF-κB-mediated NLRP3 inflammasome activation.

Tripartite motif-containing 21 (TRIM21) has been confirmed to mediate the production of inflammatory mediators via NF-κB signaling. However, the function of TRIM21 in microglia-mediated neuroinflammation remains unclear. This study aimed to explore the effect of TRIM21 on LPS-activated BV2 microglia and its underlying mechanism. BV2 cells exposed to lipopolysaccharide (LPS) were used to simulated neuroinflammation in vitro. Loss-of-function and gain-of-function of TRIM21 in BV2 cells were used to assess the effect of TRIM21 on LPS-induced neuroinflammation. BV2 microglia and HT22 cells co-culture system were used to investigate whether TRIM21 regulated neuronal inflammation-mediated neuronal death. TRIM21 knockdown triggered the polarization of BV2 cells from M1 to M2 phenotype. Knockdown of TRIM21 reduced the secretion of TNF-α, IL-6, and IL-1ß, while increased the content of IL-4 in LPS-treated cells. Knockdown of TRIM21 inhibited the expression of p65 and the binding activity of NF-κB-DNA. Additionally, TRIM21 siRNA eliminated the increase in NLRP3 and cleaved caspase-1 proteins expression and caspase-1 activity induced by LPS. TRIM21 knockdown could resist cytotoxicity induced by activated microglia, including increasing the viability of co-cultured HT22 cells and reducing the emancipation of LDH. Moreover, the increased apoptosis and caspase-3 activity of HT22 neurons induced by activated BV2 cells were blocked by TRIM21 siRNA. Blocking of NF-κB abolished the effect of TRIM21 in promoting the expression of M1 phenotype marker genes. Similarly, the blockade of NF-κB pathway eliminated the promotion of TRIM21 on neurotoxicity induced by neuroinflammation. TRIM21 knockdown suppressed the M1 phenotype polarization of microglia and neuroinflammation-mediated neuronal damage via NF-κB/NLRP3 inflammasome pathway, which suggested that TRIM21 might be a potential therapeutic target for the therapy of central nervous system diseases.

A practical toolkit to study aspects of the metastatic cascade in vitro.

While metastasis - the spread of cancer from the primary location to distant sites in the body - remains the principle cause of cancer death, it is incompletely understood. It is a complex process, requiring the metastatically successful cancer cell to negotiate a formidable series of interconnected steps, which are described in this paper. For each step, we review the range of in vitro assays that may be used to study them. We also provide a range of detailed, step-by-step protocols that can be undertaken in most modestly-equipped laboratories, including methods for converting qualitative observations into quantitative data for analysis. Assays include: (1) a gelatin degradation assay to study the ability of endothelial cells to degrade extracellular matrix during tumour angiogenesis; (2) the morphological characterisation of cells undergoing epithelial-mesenchymal transition (EMT) as they acquire motility; (3) a 'scratch' or 'wound-healing' assay to study cancer cell migration; (4) a transwell assay to study cancer cell invasion through extracellular matrix; and (5) a static adhesion assay to examine cancer cell interactions with, and adhesion to, endothelial monolayers. This toolkit of protocols will enable researchers who are interested in metastasis to begin to focus on defined aspects of the process. It is only by further understanding this complex, fascinating and clinically relevant series of events that we may ultimately devise ways of better treating, or even preventing, cancer metastasis. The assays may also be of more broad interest to researchers interested in studying aspects of cellular behaviour in relation to other developmental and disease processes.

Vasoactive Intestinal Peptide Changes the Frequency and Force of Myocardial Contraction in Rats.

We studied the effect of non-selective agonist of VIP receptors of vasoactive intestinal polypeptide in different concentrations on the frequency, force, and duration of isometric contraction of myocardial strips of the right atrium under conditions of spontaneous activity, as well as the force and duration of contractions of the right ventricle in rats. It was found that the agonist produced a positive inotropic and chronotropic effect that depended on its concentration. The maximum effect was observed at vasoactive intestinal peptide concentration of 10-11 M.

Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells.

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air-liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications.

Indirect Decompression of the Neural Elements Utilizing Direct Lateral Interbody Fusion Procedure.

INTRODUCTION: Patients suffering from degenerative scoliosis curves often present with radicular symptoms mainly on the concave side of their curves. Standard treatment includes posterior decompressions, followed by fusions. These procedures carry large morbidity rates. We have observed resolution of radicular and stenotic symptoms with Direct Lateral Interbody Fusions (DLIF). AIM: In this study we radiographically assess indirect decompression effect of DLIF procedure. METHODS: We conducted a case series of four patients with 2-stage procedures. All patients presented with back pain and leg symptoms. Stage one included the insertion of the DLIF polyetheretherketone cages and rh-BMP2. This was followed by a second stage posterior fixation utilizing percutaneous pedicle screws and rods. Plain radiographs were utilized to determine the concave and convex sides of the scoliosis. Pre- and post-DLIF measurements were made from axial and sagittal MRIs. Measurements included central, subarticular, and foraminal areas. Statistical significance was estimated via paired sample t-test. RESULTS: All patients had complete resolution of leg symptoms with remarkable improvement in all areas measured. When both concave and convex sides of the curve are considered, an increase of 49% in the central canal, 82% in the subarticular area, and 71% in the foraminal area was measured. When only the concave levels were measured, there was a 90% increase (0.22 cm2 vs. 0.41 cm2) in the subarticular area and 77% (0.46 cm2 vs. 0.81 cm2) increase in the foraminal area (p < .001). CONCLUSION: The DLIF procedure provides an indirect decompression of the neural elements along with its role in spinal fusion. This negates the need for posterior decompression surgery in degenerative scoliosis associated with spinal stenosis, which might lead to less blood loss and surgical time in these complex surgeries.

Chromogranin A (CGA)-derived polypeptide (CGA47-66) inhibits TNF-α-induced vascular endothelial hyper-permeability through SOC-related Ca2+ signaling.

CGA1-78 (Vasostatin-1, VS-1) a N-terminal Chromogranin A (CGA)-derived peptide, has been shown to have a protective effect against TNF-α-induced impairment of endothelial cell integrity. However, the mechanisms of this effect have not yet been clarified. CGA47-66 (Chromofungin, CHR) is an important bioactive fragment of CGA1-78. The present study aims to explore the protective effects of CHR on the vascular endothelial cell barrier response to TNF-α and its related Ca2+ signaling mechanisms. EA.hy926 cells were used as a vascular endothelial culture model. The synthetic peptides CHR and CGA4-16 were assessed for their ability to suppress TNF-α-induced EA.hy926 cells hyper-permeability through Transwell® and TEER assays. Changes in [Ca2+]i were measured through confocal laser scanning microscopy. SOC channel currents (Isoc) were measured via patch-clamp analysis. RT-PCR and western blot were used to analyze mRNA and protein expression of the transient receptor potential channels TRPC1 and TRPC4, respectively. FITC and rhodamine-phalloidin fluorescence were used to assess cell morphology and the distribution of MyPT-1 and F-actin. Compared to untreated cells, TNF-α increased the permeability of EA.hy926 cells that was inhibited by pre-treatment with CHR (10-1000 nM) in concentration-dependent manner, and the effect was most obvious at 100 nM, but CGA4-16 (100 nM) had no effect. TNF-α treatment increased the phosphorylation of MyPT-1 and stress fiber formation. CHR (10-1000 nM) pretreatment inhibited the cytoskeletal rearrangements and increased [Ca2+]i in response to TNF-α treatment. CHR also reduced TRPC1 expression following TNF-α induction. Similar to SOC inhibitor 2-APB, CHR suppressed IP3 mediated SOC activation. These findings suggest that CHR inhibits TNF-α-induced Ca2+ influx and protects the barrier function of vascular endothelial cells, and that these effects are related to the inhibition of SOC and Ca2+ signaling by CHR.

Broussoflavonol B from Broussonetia kazinoki Siebold Exerts Anti-Pancreatic Cancer Activity through Downregulating FoxM1.

Pancreatic cancer has a high mortality rate due to poor rates of early diagnosis. One tumor suppressor gene in particular, p53, is frequently mutated in pancreatic cancer, and mutations in p53 can inactivate normal wild type p53 activity and increase expression of transcription factor forkhead box M1 (FoxM1). Overexpression of FoxM1 accelerates cellular proliferation and cancer progression. Therefore, inhibition of FoxM1 represents a therapeutic strategy for treating pancreatic cancer. Broussoflavonol B (BF-B), isolated from the stem bark of Broussonetia kazinoki Siebold has previously been shown to inhibit the growth of breast cancer cells. This study aimed to investigate whether BF-B exhibits anti-pancreatic cancer activity and if so, identify the underlying mechanism. BF-B reduced cell proliferation, induced cell cycle arrest, and inhibited cell migration and invasion of human pancreatic cancer PANC-1 cells (p53 mutated). Interestingly, BF-B down-regulated FoxM1 expression at both the mRNA and protein level. It also suppressed the expression of FoxM1 downstream target genes, such as cyclin D1, cyclin B1, and survivin. Cell cycle analysis showed that BF-B induced the arrest of G0/G1 phase. BF-B reduced the phosphorylation of extracellular signal-regulated kinase ½ (ERK½) and expression of ERK½ downstream effector c-Myc, which regulates cell proliferation. Furthermore, BF-B inhibited cell migration and invasion, which are downstream functional properties of FoxM1. These results suggested that BF-B could repress pancreatic cancer cell proliferation by inactivation of the ERK/c-Myc/FoxM1 signaling pathway. Broussoflavonol B from Broussonetia kazinoki Siebold may represent a novel chemo-therapeutic agent for pancreatic cancer.

MIF inhibitor, ISO-1, attenuates human pancreatic cancer cell proliferation, migration and invasion in vitro, and suppresses xenograft tumour growth in vivo.

This study sought to investigate the biological effects of specific MIF inhibitor, ISO-1, on the proliferation, migration and invasion of PANC-1 human pancreatic cells in vitro, and on tumour growth in a xenograft tumour model in vivo. The effect of ISO-1 on PANC-1 cell proliferation was examined using CCK-8 cell proliferation assay. The effect of ISO-1 on collective cell migration and recolonization of PANC-1 cells was evaluated using the cell-wound closure migration assay. The effect of ISO-1 on the migration and invasion of individual PANC-1 cells in a 3-dimensional environment in response to a chemo-attractant was investigated through the use of Transwell migration/invasion assays. Quantitative real time PCR and western blot analyses were employed to investigate the effects of ISO-1 on MIF, NF-κB p65 and TNF-α mRNA and protein expression respectively. Finally, a xenograft tumor model in BALB/c nude mice were used to assess the in vivo effects of ISO-1 on PANC-1-induced tumor growth. We found high expression of MIF in pancreatic cancer tissues. We demonstrated that ISO-1 exerts anti-cancer effects on PANC-1 cell proliferation, migration and invasion in vitro, and inhibited PANC-1 cell-induced tumour growth in xenograft mice in vivo. Our data suggests that ISO-1 and its derivative may have potential therapeutic applications in pancreatic cancer.

Ileo-colonic delivery of conjugated bile acids improves glucose homeostasis via colonic GLP-1-producing enteroendocrine cells in human obesity and diabetes.

BACKGROUND: The bile acid (BA) pathway plays a role in regulation of food intake and glucose metabolism, based mainly on findings in animal models. Our aim was to determine whether the BA pathway is altered and correctable in human obesity and diabetes. METHODS: We conducted 3 investigations: 1) BA receptor pathways were studied in NCI-H716 enteroendocrine cell (EEC) line, whole human colonic mucosal tissue and in human colonic EEC isolated by Fluorescence-activated Cell Sorting (ex vivo) from endoscopically-obtained biopsies colon mucosa; 2) We characterized the BA pathway in 307 participants by measuring during fasting and postprandial levels of FGF19, 7αC4 and serum BA; 3) In a placebo-controlled, double-blind, randomised, 28-day trial, we studied the effect of ileo-colonic delivery of conjugated BAs (IC-CBAS) on glucose metabolism, incretins, and lipids, in participants with obesity and diabetes. FINDINGS: Human colonic GLP-1-producing EECs express TGR5, and upon treatment with bile acids in vitro, human EEC differentially expressed GLP-1 at the protein and mRNA level. In Ussing Chamber, GLP-1 release was stimulated by Taurocholic acid in either the apical or basolateral compartment. FGF19 was decreased in obesity and diabetes compared to controls. When compared to placebo, IC-CBAS significantly decreased postprandial glucose, fructosamine, fasting insulin, fasting LDL, and postprandial FGF19 and increased postprandial GLP-1 and C-peptide. Increase in faecal BA was associated with weight loss and with decreased fructosamine. INTERPRETATIONS: In humans, BA signalling machinery is expressed in colonic EECs, deficient in obesity and diabetes, and when stimulated with IC-CBAS, improved glucose homeostasis. ClinicalTrials.gov number, NCT02871882, NCT02033876. FUNDING: Research support and drug was provided by Satiogen Pharmaceuticals (San Diego, CA). AA, MC, and NFL report grants (AA- C-Sig P30DK84567, K23 DK114460; MC- NIH R01 DK67071; NFL- R01 DK057993) from the NIH. JR was supported by an Early Career Grant from Society for Endocrinology.

DKK1 promotes hepatocellular carcinoma inflammation, migration and invasion: Implication of TGF-ß1.

Dickkopf-1 (DKK1), an inhibitor of the most frequently impaired signaling pathway in hepatocellular carcinoma (HCC), the Wnt/beta-catenin pathway, seems to fulfill contradictory functions in the process of tumorigenesis, acting either as an oncogenic promoter of metastasis or as a tumor suppressor. Elevated serum levels of DKK1 have been reported in HCC; however, little is known about its functional significance. In the current study, we treated HepG2/C3A and PLC/PRF/5 with the recombinant protein DKK1. Cytotoxicity was first determined by the WST-8 assay. AFP expression was measured at both the mRNA and protein levels. Expression of the oncogenes MYC, CCND1, hTERT, and MDM2 and the tumor suppressor genes TP53, P21 and RB was assessed. Western blot analysis of non-phosphorylated ẞ-catenin and Sanger sequencing were performed to explain the functional differences between the two cell lines. Subsequently, inflammation, migration and invasion were evaluated by qPCR, ELISA, the Boyden chamber assay, zymography, and MMP-2 and MMP-9 western blot analysis. Knockdown of DKK1 and TGF-ß1 were also performed. Our results suggest that DKK1 exerts an oncogenic effect on HepG2/C3A cell line by upregulating the expression of oncogenes and downregulating that of tumor suppressor genes, whereas the opposite effect was demonstrated in PLC/PRF/5 cells. This differential impact of DKK1 can be explained by the mutations that affect the canonical Wnt pathway that were detected in exon 3 of the CTNNB1 gene in the HepG2 cell line. We further confirmed that DKK1 promotes inflammation, tumor invasion and migration in both cell types. The canonical pathway was not responsible for the DKK1 proinvasive effect, as indicated by the active ẞ-catenin levels in the two cell lines upon DKK1 treatment. Interestingly, knockdown of TGF-ß1 negatively affected the DKK1 proinvasive effect. Taken together, DKK1 appears to facilitate tumor invasion and migration through TGF- ß1 by remodeling the tumor microenvironment and inducing inflammation. This finding endorses the relevance of TGF-ß1 as a therapeutic target.

THBS4 predicts poor outcomes and promotes proliferation and metastasis in gastric cancer.

Gastric cancer (GC), a common and lethal cancer in the world, has a high risk of metastasis. Our study was to explore the effects of THBS4 on GC progress and metastasis and the underlying mechanisms. The proliferations of MGC-803 and BGC-823 cells were analyzed via cell count, MTT, and soft agar colony formation assay. The migration and invasion of transfected GC cells was investigated via transwell migration and invasion assay. The mRNA abundance of THBS4 and KLF9 was detected by quantitative real-time PCR (qPCR). The analysis of Gene Expression Omnibus (GEO) dataset (GSE26253) suggested that THBS4 was up-regulated in recurrent GC patients and was positively correlated with the increase in pathological stage and poor prognosis in GC. THBS4 stimulated the proliferations of GC cells. Moreover, THBS4 overexpression fostered the migration and invasion of GC cells. Further, the bioinformatics analysis of the cancer genome atlas dataset suggested that there may be a positive correlation between THBS4 and KLF9 expression. QPCR analysis proved that transfected with THBS4 overexpression plasmid enhanced KLF9 expression in GC cells. THBS4 mRNA and protein expression were up-regulated in MGC-803 and BGC-823 cells compared to those in non-tumoral gastric cells. KLF9 overexpression significantly stimulated the proliferation and metastasis of MGC-803 and BGC-823 cells. Besides, KLF9 siRNA inhibited the enhanced viability, migration, and invasion of MGC-803 cells caused by the transfection with THBS4 overexpression plasmid. In conclusion, THBS4 had positive effects on GC proliferation and metastasis via targeting KLF9.

Extent of rescue of F508del-CFTR function by VX-809 and VX-770 in human nasal epithelial cells correlates with SNP rs7512462 in SLC26A9 gene in F508del/F508del Cystic Fibrosis patients.

BACKGROUND: We analyzed the CFTR response to VX-809/VX-770 drugs in conditionally reprogrammed cells (CRC) of human nasal epithelium (HNE) from F508del/F508del patients based on SNP rs7512462 in the Solute Carrier Family 26, Member 9 (SLC26A9; MIM: 608481) gene. METHODS: The Isc-eq measurements of primary nasal epithelial cells from F508del/F508del patients (n = 12) for CFTR function were performed in micro Ussing chambers and compared with non-CF controls (n = 2). Data were analyzed according to the rs7512462 genotype which were determined by real-time PCR. RESULTS: The CRC-HNE cells from F508del/F508del patients evidenced high variability in the basal levels of CFTR function. Also, the rs7512462*C allele showed an increased basal CFTR function and higher responses to VX-809 + VX-770. The rs7512462*CC + CT genotypes together evidenced CFTR function levels of 14.89% relatively to wt/wt (rs7512462*CT alone-15.29%) i.e., almost double of rs7512462*TT (7.13%). Furthermore, sweat [Cl-] and body mass index of patients also evidenced an association with the rs7512462 genotype. CONCLUSION: The CFTR function can be performed in F508del/F508del patient-derived CRC-HNEs and its function and responses to VX-809 + VX-770 combination as well as clinical data, are all associated with the rs7512462 variant, which partially sheds light on the generally inter-individual phenotypic variability and in personalized responses to CFTR modulator drugs.

Interleukin-1ß induces CXCR3-mediated chemotaxis to promote umbilical cord mesenchymal stem cell transendothelial migration.

BACKGROUND: Mesenchymal stem cells (MSCs) are known to home to injured and inflamed regions via the bloodstream to assist in tissue regeneration in response to signals of cellular damage. However, the factors and mechanisms that affect their transendothelial migration are still unclear. In this study, the mechanisms involved in interleukin-1ß (IL-1ß) enhancing the transendothelial migration of MSCs were investigated. METHODS: Immunofluorescence staining and Western blotting were used to observe IL-1ß-induced CXC chemokine receptor 3 (CXCR3) expression on MSCs. Quantitative real-time PCR and ELISA were used to demonstrate IL-1ß upregulated both chemokine (C-X-C motif) ligand 9 (CXCL9) mRNA and CXCL9 ligand secretion in human umbilical vein endothelial cells (HUVECs). Monolayer co-cultivation, agarose drop chemotaxis, and transwell assay were conducted to investigate the chemotaxis invasion and transendothelial migration ability of IL-1ß-induced MSCs in response to CXCL9. RESULTS: In this study, our immunofluorescence staining showed that IL-1ß induces CXCR3 expression on MSCs. This result was confirmed by Western blotting. Following pretreatment with protein synthesis inhibitor cycloheximide, we found that IL-1ß induced CXCR3 on the surface of MSCs via protein synthesis pathway. Quantitative real-time PCR and ELISA validated that IL-1ß upregulated both CXCL9 mRNA and CXCL9 ligand secretion in HUVECs. In response to CXCL9, chemotaxis invasion and transendothelial migration ability were increased in IL-1ß-stimulated MSCs. In addition, we pretreated MSCs with CXCR3 antagonist AMG-487 and p38 MAPK inhibitor SB203580 to confirm CXCR3-CXCL9 interaction and the role of CXCR3 in IL-1ß-induced chemotaxis invasion and transendothelial migration. CONCLUSION: We found that IL-1ß induces the expression of CXCR3 through p38 MAPK signaling and that IL-1ß also enhances CXCL9 ligand secretion in HUVECs. These results indicated that IL-1ß promotes the transendothelial migration of MSCs through CXCR3-CXCL9 axis. The implication of the finding could enhance the efficacy of MSCs homing to target sites.

LincRNA-p21 promotes mesenchymal stem cell migration capacity and survival through hypoxic preconditioning.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from bone marrow have potent stabilizing effects for the treatment of acute respiratory distress syndrome (ARDS). However, low efficiency and survival in MSC homing to injured lung tissue remains to be solved. Therefore, the aim of this study was to assess whether large intergenic noncoding RNA (LincRNA)-p21 promote MSC migration and survival capacity through hypoxic preconditioning in vitro. METHODS: MSCs were cultured and divided into the normoxia culture group (20% O2) and hypoxia culture group (1% O2). To determine roles and mechanisms, lentivirus vector-mediated LincRNA-p21 knockdown of MSCs and hypoxia-inducible factor (HIF-1α) inhibitor KC7F2 were introduced. Additionally, MSC migration was analyzed by scratch test and transwell migration assays. MSC proliferation was tested by cell counting kit-8 and trypan blue dye. Apoptosis was detected by Annexin V-PE/7-AAD stained flow cytometry. Moreover, LincRNA-p21 and HIF-1α mRNA was measured by reverse transcription-polymerase chain reaction, and HIF-1α and CXCR4/7 protein were assayed by western blot (WB) or enzyme-linked immunosorbent assay (ELISA). Apoptosis protein caspase-3 and cleaved-caspase-3 were investigated by WB analysis. Considering interactions between VHL and HIF-1α under LincRNA-p21 effect, co-immunoprecipitation was detected. RESULTS: Hypoxic preconditioning MSC promoted migration capacity and MSC survival than normoxia culture group. MSCs induced by hypoxic preconditioning evoked an increase in expression of LincRNA-p21, HIF-1α, and CXCR4/7(both were chemokine stromal-derived factor-1(SDF-1) receptors). Contrarily, blockade of LincRNA-p21 by shRNA and HIF-1α inhibitor KC7F2 abrogated upregulation of hypoxic preconditioning induced CXCR4/7 in MSCs, cell migration, and survival. Furthermore, co-immunoprecipitation assay revealed that hypoxic preconditioning isolated VHL and HIF-1α protein by increasing HIF-1α expression. CONCLUSIONS: Hypoxic preconditioning was identified as a promoting factor of MSC migration and survival capacity. LincRNA-p21 promotes MSC migration and survival capacity through HIF-1α/CXCR4 and CXCR7 pathway under hypoxic preconditioning in vitro.

Calcium-induced chloride secretion is decreased by Resveratrol in ileal porcine tissue.

OBJECTIVE: Chloride (Cl-) secretion is crucial for intestinal fluid secretion. Therefore, effects of the polyphenol Resveratrol (RSV) on Cl- secretion have been investigated. In a previous study, we observed effects of RSV on forskolin-induced Cl- secretion in the porcine jejunum but not the ileum although RSV itself induced a transepithelial ion current that may represent Cl- secretion in the ileum. The aim of this study was to gain further insights regarding the effects of RSV on characteristics of Cl- secretion in the porcine ileum using the Ussing chamber technique (recording of short circuit currents (Isc) as a measure for epithelial net ion transfer). RESULTS: RSV increased the Isc in the porcine ileum but not in the porcine jejunum as is already known. This increase was absent in a Cl--free buffer system, indicating that RSV indeed induces Cl- secretion. However, the carbachol-induced Isc was significantly inhibited by RSV indicating an inhibition of Ca2+-induced Cl- secretion. The cellular basis for these contradictory, segment specific results of RSV on Cl- secretion has to be subjected to further studies. The results also underline, that is difficult to generalize effects of RSV between different intestinal locations, organs, cell culture models or species.

Tumor-endothelial cell interaction in an experimental model of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a densely vascularized tumor that is highly dependent on angiogenic pathways to direct arterial blood flow to the growing neoplasm, though little is known about how the interaction of tumor and endothelial cells drives these processes and the degree of clinical importance. To this end, we examined the intercellular cross-talk between HepG2 (human HCC) and human endothelial progenitor cells (EPC) in a co-culture system that mimics some aspects of initial tumor parenchyma and stroma interactions. The results showed that the remote cell-to-cell (paracrine) interactions between HepG2 cells and EPC play a critical role in the differentiation and angiogenic activity of endothelial cells, possibly through intercellular signaling function of the exosomes released in the medium by HepG2 cells. The tumor-cell activated phenotype of EPC was associated with increased migration and elevated expression of ephrin-B2, and Delta-like 4 ligand (DLL4). Furthermore, ephrin-B2 was found to be overexpressed in HCC and cholangiocarcinoma tissue samples taken from humans. Overall, our results demonstrate that ephrin-B2 and Dll4 mediated co-dependence of HCC and EPC intercellular crosstalk in the initial stages of HCC establishment and development, a promising target for new clinical strategies.