The aim of this study was to evaluate the diagnostic performance of SonoVue-enhanced ultrasonography in the follow-up of rabbit kidney lesions induced by percutaneous radiofrequency ablation.
Material and methods
New Zealand rabbits (28) underwent percutaneous radiofrequency ablation (RFA) to generate renal lesions. Lesions were evaluated by conventional 2D ultrasound and contrast-enhanced ultrasonography (CEUS) at 1 day, 1 week, 1 month and 3 months after RFA, and the results were compared to gross pathology.
One day and 1 week after RFA, renal lesions were wedge-shaped in the gross dissection cross-sectional profiles. Conventional ultrasound could not indicate the extent of the lesions; however, CEUS could exactly delineate the lesion shape and size. At 1 and 3 months, lesions were observed as hyperechogenic areas on conventional ultrasound, and as small perfusion defects on CEUS. The differences in the lesion measurements obtained by CEUS and in pathological specimens were not statistically significant (p > 0.05).
The conclusion could be deduced from the study that SonoVue-enhanced ultrasonography was effective for the follow-up of normal rabbit kidney percutaneous radiofrequency ablation.
contrast-enhanced ultrasonography; radiofrequency ablation; kidney; intervention
Hepatocellular carcinoma (HCC) is one of the most deadly human cancers, but it is very difficult to establish an animal model by using surgical specimens. In the present experiment, histologically intact fresh surgical specimens of HCC were subcutaneously transplanted in non-obese diabetic/severe combined immunodeficienccy (NOD/SCID) mice. The biological characteristics of the original and the corresponding transplanted tumors and cell lines were investigated. The results showed that 5 new animal models and 2 primary cell lines were successfully established from surgical specimens. Hematoxylin-eosin staining showed that xenografts retained major histological features of the original surgical specimens. The two new cell lines had been cultivated for 3 years and successively passaged for more than 100 passages in vitro. The morphological characteristics and biologic features of the two cell lines were genetically similar to the original tumor. The subcutaneous transplant animal models with histologically intact tumor tissue and primary cell lines could be useful for in vivo and in vitro testing of anti-cancer drugs and be ideal models to study various biologic features of HCC.
Animal model; hepatocellular carcinoma; subcutaneous transplantation; surgical specimens
Hepatocellular carcinoma (HCC) is a prevalent disease worldwide, and the majority of HCC-related deaths occur due to local invasion and distant metastasis. Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have been hypothesized to be responsible for metastatic disease. Recently, we and others have identified a CSC population from human HCC cell lines and xenograft tumors characterized by their expression of CD133. However, the precise molecular mechanisms by which CD133+ cancer stem-like cells mediate HCC metastasis have not been sufficiently analyzed. Here, we have sorted HCC cells using CD133 as a cancer stem cell (CSC) marker by magnetic-activated cell sorting (MACS) and demonstrated that the CD133+ HCC cells not only possess greater migratory and invasive capacity in vitro but are also endowed with enhanced metastatic capacity in vivo and in human HCC specimens when compared to CD133− HCC cells. Gene expression analysis of the CD133+ and CD133− cells of the HCC line SMMC-7721 revealed that G protein-coupled receptor 87 (GPR87) is highly expressed in CD133+ HCC cells. In this study, we explored the role of GPR87 in the regulation of CD133 expression. We demonstrated that the overexpression of GPR87 up-regulated CD133 expression, promoted CSC-associated migratory and invasive properties in vitro, and increased tumor initiation in vivo. Conversely, silencing of GPR87 expression reduced the levels of CD133 expression. Conclusion: GPR87 promotes the growth and metastasis of CD133+ cancer stem-like cells, and our findings may reveal new targets for HCC prevention or therapy.
Perianal infection is a common problem for patients with acute leukemia. However, neutropenia and bleeding tendency are relatively contraindicated to surgical intervention. The epidemiology, microbiology, clinical manifestations and outcomes of perianal infection in leukemic patients are also rarely discussed.
The medical records of 1102 adult patients with acute leukemia at a tertiary medical center in Taiwan between 2001 and 2010 were retrospectively reviewed and analyzed.
The prevalence of perianal infection was 6.7% (74 of 1102) in adult patients with acute leukemia. Twenty-three (31%) of the 74 patients had recurrent episodes of perianal infections. Patients with acute myeloid leukemia had higher recurrent rates than acute lymphoblastic leukemia patients (p = 0.028). More than half (n = 61, 53%) of the perianal infections were caused by gram-negative bacilli, followed by gram-positive cocci (n = 36, 31%), anaerobes (n = 18, 15%) and Candida (n = 1, 1%) from pus culture. Eighteen patients experienced bacteremia (n = 24) or candidemia (n = 1). Overall 41 (68%) of 60 patients had polymicrobial infection. Escherichia coli (25%) was the most common micro-organism isolated, followed by Enterococcus species (22%), Klebsiella pneumoniae (13%), and Bacteroides species (11%). Twenty-five (34%) of 74 patients received surgical intervention. Acute leukemia patients with surgically managed anal fistulas tended to have fewer recurrences (p = 0.067). Four (5%) patients died within 30 days after diagnosis of perianal infection. Univariate analysis of 30-day survival revealed the elderly (≧ 65 years) (p = 0.015) and patients with shock (p<0.001) had worse outcome. Multivariate analysis showed septic shock to be the independent predictive factor of 30-day crude mortality of perianal infections (p = 0.016).
Perianal infections were common and had high recurrence rate in adult patients with acute leukemia. Empirical broad-spectrum antibiotics with anaerobic coverage should be considered. Shock independently predicted 30-day crude mortality. Surgical intervention for perianal infection remains challenging in patients with acute leukemia.
The prognostic implication of immunophenotyping in acute myeloid leukemia (AML) patients with NPM1 mutation remains unclear.
Ninety-four of 543 AML patients diagnosed with NPM1 mutation between 1987 and 2007 were studied. The expression of surface antigens on leukemic cells was evaluated with respect to clinical manifestations and outcomes. In order to validate the prognostic effect of the immunophenotypic cluster, another 36 patients with NPM1 mutation diagnosed between 2008 and 2010 were analyzed.
Ninety-four patients with NPM1 mutations and complete immunophenotyping data were enrolled for a hierarchical cluster analysis and the result was correlated with clinico-laboratory characteristics. Clustering analysis divided the patients with NPM1 mutations into the following two groups: group I, CD34(−)/CD7(−), but with variable expression of HLA-DR; and group II, HLA DR(+)/CD34(+)/CD7(+). With a median follow-up of 53 months, the group II patients had a significantly shorter relapse-free survival (RFS, median: 3 vs. 23 months, p = 0.006) and overall survival (OS, median: 11 vs. 40 months, p = 0.02) than group I patients. Multivariate analysis of variables, including clinico-laboratory data and other gene mutations revealed that the immunophenotypic cluster is an independent prognostic factor (RFS, p = 0.002; OS, p = 0.024). In order to confirm the prognostic effect of the immunophenotypic cluster, another 36 patients with NPM1 mutation diagnosed between 2008 and 2010 were validated. Hierarchical cluster analysis also showed two distinct clusters, group I patient showed significant better RFS (p = 0.021), and OS (p = 0.055). In total, we stratified 130 NPM1-mutant patients, by FLT3-ITD mutation and immunophenotypic cluster into distinct prognostic groups (RFS, p < 0.001 and OS, p = 0.017).
Among NPM1-mutated AML, the antigen expression pattern of HLADR(+) CD34(+) CD7(+) is associated with a poor prognosis, independent to the FLT3-ITD mutation.
Acute myeloid leukemia; NPM1 mutation; Immunophenotype; Prognosis
14-3-3ε is implicated in regulating tumor progression, including hepatocellular carcinoma (HCC). Our earlier study indicated that elevated 14-3-3ε expression is significantly associated with higher risk of metastasis and lower survival rates of HCC patients. However, the molecular mechanisms of how 14-3-3ε regulates HCC tumor metastasis are still unclear.
Methodology and Principal Findings
In this study, we show that increased 14-3-3ε expression induces HCC cell migration and promotes epithelial-mesenchymal transition (EMT), which is determined by the reduction of E-cadherin expression and induction of N-cadherin and vimentin expression. Knockdown with specific siRNA abolished 14-3-3ε-induced cell migration and EMT. Furthermore, 14-3-3ε selectively induced Zeb-1 and Snail expression, and 14-3-3ε-induced cell migration was abrogated by Zeb-1 or Snail siRNA. In addition, the effect of 14-3-3ε-reduced E-cadherin was specifically restored by Zeb-1 siRNA. Positive 14-3-3ε expression was significantly correlated with negative E-cadherin expression, as determined by immunohistochemistry analysis in HCC tumors. Analysis of 14-3-3ε/E-cadherin expression associated with clinicopathological characteristics revealed that the combination of positive 14-3-3ε and negative E-cadherin expression is significantly correlated with higher incidence of HCC metastasis and poor 5-year overall survival. In contrast, patients with positive 14-3-3ε and positive E-cadherin expression had better prognostic outcomes than did those with negative E-cadherin expression.
Our findings show for the first time that E-cadherin is one of the downstream targets of 14-3-3ε in modulating HCC tumor progression. Thus, 14-3-3ε may act as an important regulator in modulating tumor metastasis by promoting EMT as well as cell migration, and it may serve as a novel prognostic biomarker or therapeutic target for HCC.
To explore the key regulatory genes associated with lung cancer in order to reduce its occurrence and progress through silencing these key genes.
To identify the key regulatory genes involved in lung cancer, we performed a combination of gene array and bioinformatics analyses to compare gene transcription profiles in 3 monoclonal cell strains with high, medium or low metastatic abilities, which were separated from the SPC-A-1sci and SPC-A-1 cell lines by limiting dilution monoclone assay. We then analyzed those genes’ biological activities by knocking down their expression in SPC-A-1sci cells using siRNA and lenti-viral shRNA vectors, followed by determinations of the invasion and migration capabilities of the resulting cell lines in vitro as well as their potential for inducing occurrence and metastasis of lung cancer in vivo. To examine the clinical relevance of these findings, we analyzed the expression levels of the identified genes in human lung cancer tissues (n = 135) and matched adjacent normal tissues by immunohistochemical (IHC) staining.
Three monoclonal cell strains characterized with high, medium or low metastatic abilities were successfully selected. Gene array and bioinformatics analyses implied that osteopontin, LAMB3 and ITGB1 were key genes involved in lung cancer. Knockdown of these genes suppressed human lung cancer cell invasion and metastasis in vitro and in vivo. Clinical sample analyses indicated that osteopontin, LAMB3 and ITGB1 protein expression levels were higher in lung cancer patients, compared to non-cancerous adjacent tissues, and correlated with lymphatic metastasis.
We confirmed that osteopontin, LAMB3 and ITGB1 played important roles in the occurrence and metastasis of lung cancer, thus provided important clues to understanding the molecular mechanism of metastasis and contributing to the therapeutic treatment of lung cancer.
It has been previously reported that IL-22, one of the cytokines secreted by Th17 cells, demonstrates both a protective and inflammatory promotion effect in inflammatory bowel disease (IBD) through STAT3 signaling activation. We sought to investigate the role of IL-22 expression in colon cancer (CC).
The expression of IL-22 and related molecules were detected in human CC, the detail function and mechanism of IL-22 were investigated by in vivo and in vitro model.
Our results demonstrated significant upregulation of IL-22 in human CC tumor infiltrated leukocytes (TILs) compared to peripheral lymphocytes. Moreover, our findings demonstrated that IL-22 expression was significantly higher in ulcerative colitis (UC) tissues versus normal colon tissues. Both IL-22 receptor α1 (IL-22RA1) and IL-23 were highly expressed in CC and UC tissues compared to normal controls. TILs exhibiting various IL-22 expression levels isolated from CC patients were demonstrated to enhance tumor growth and metastasis co-transplanted with Hct-116 cells underwent subcutaneous transplantation in mice model. Tumor growth and metastasis was promoted by STAT3 phosphorylation and upregulation of its downstream genes such as Bcl-xl, CyclinD1, and VEGF. In vitro studies confirmed the anti-apoptotic and pro-proliferation effect of IL-22 according to the BrdU cooperation assay and peroxide induced apoptosis analysis with or without the presence of IL-22.
In this study we demonstrated that excessive IL-22 in the CC and UC microenvironment leads to tumor growth, inhibition of apoptosis, and promotion of metastasis depend on STAT3 activation.
Colon cancer; Ulcerative colitis; IL-22; TILs; STAT3
Partitioning defective 3 (Par-3), a crucial component of partitioning-defective complex proteins, controls cell polarity and contributes to cell migration and cancer cell epithelial-to-mesenchymal transition. However, the clinical relevance of Par-3 in tumor progression and metastasis has not been well elucidated. In this study, we investigated the impact and association of Par-3 expression and clinical outcomes with hepatocellular carcinoma (HCC). We first confirmed that Par-3 was abundantly expressed in HCC cell lines by Western blot analysis. We used immunohistochemistry to analyze the association of Par-3 expression and clinicopathological characteristics in primary and subsequent metastatic tumors of patients with HCC. Par-3 was overexpressed in 47 of 111 (42.3%) primary tumors. Increased expression of Par-3 in primary tumors predicted an increased five-year cumulative incidence of extrahepatic metastasis. In addition, multivariate analysis revealed that Par-3 overexpression was an independent risk factor of extrahepatic metastasis. Increased Par-3 expression in primary tumors was associated with poor five-year overall survival rates and was an independent prognostic factor on Cox regression analysis. In conclusion, we show for the first time that increased Par-3 expression is associated with distant metastasis and poor survival rates in patients with HCC. Par-3 may be a novel prognostic biomarker and therapeutic target for HCC.
hepatocellular carcinoma; metastasis; Par-3; survival
The Maasai are a pastoral people in Kenya and Tanzania, whose traditional diet of milk, blood and meat is rich in lactose, fat and cholesterol. In spite of this, they have low levels of blood cholesterol, and seldom suffer from gallstones or cardiac diseases. Field studies in the 1970s suggested that the Maasai have a genetic adaptation for cholesterol homeostasis. Analysis of HapMap 3 data using Fixation Index (Fst) and two metrics of haplotype diversity: the integrated Haplotype Score (iHS) and the Cross Population Extended Haplotype Homozygosity (XP-EHH), identified genomic regions and single nucleotide polymorphisms (SNPs) as strong candidates for recent selection for lactase persistence and cholesterol regulation in 143–156 founder individuals from the Maasai population in Kinyawa, Kenya (MKK). The non-synonmous SNP with the highest genome-wide Fst was the TC polymorphism at rs2241883 in Fatty Acid Binding Protein 1(FABP1), known to reduce low density lipoprotein and tri-glyceride levels in Europeans. The strongest signal identified by all three metrics was a 1.7 Mb region on Chr2q21. This region contains the genes LCT (Lactase) and MCM6 (Minichromosome Maintenance Complex Component) involved in lactase persistence, and the gene Rab3GAP1 (Rab3 GTPase-activating Protein Catalytic Subunit), which contains polymorphisms associated with total cholesterol levels in a genome-wide association study of >100,000 individuals of European ancestry. Sanger sequencing of DNA from six MKK samples showed that the GC-14010 polymorphism in the MCM6 gene, known to be associated with lactase persistence in Africans, is segregating in MKK at high frequency (∼58%). The Cytochrome P450 Family 3 Subfamily A (CYP3A) cluster of genes, involved in cholesterol metabolism, was identified by Fst and iHS as candidate loci under selection. Overall, our study identified several specific genomic regions under selection in the Maasai which contain polymorphisms in genes associated with lactase persistence and cholesterol regulation.
Clinical characteristics and outcomes of intracranial hemorrhage (ICH) among adult patients with various hematological malignancies are limited.
A total of 2,574 adult patients diagnosed with hematological malignancies admitted to a single university hospital were enrolled into this study between 2001 and 2010. The clinical characteristics, image reports and outcomes were retrospectively analyzed.
A total of 72 patients (48 men and 24 women) with a median age of 56 (range 18 to 86) had an ICH. The overall ICH incidence was 2.8% among adult patients with hematological malignancies. The incidence of ICH was higher in acute myeloid leukemia (AML) patients than in patients with other hematological malignancies (6.3% vs 1.1%, P = 0.001). ICH was more common among patients with central nervous system (CNS) involvement of lymphoma than among patients with CNS involved acute leukemia (P <0.001). Sites of ICH occurrence included the cerebral cortex (60 patients, 83%), basal ganglia (13 patients, 18%), cerebellum (10 patients, 14%), and brainstem (5 patients, 7%). A total of 33 patients (46%) had multifocal hemorrhages. In all, 56 patients (77%) had intraparenchymal hemorrhage, 22 patients (31%) had subdural hemorrhage, 15 patients (21%) had subarachnoid hemorrhage (SAH), and 3 patients (4%) had epidural hemorrhage. A total of 22 patients had 2 or more types of ICH. In all, 46 (64%) patients died of ICH within 30 days of diagnosis, irrespective of the type of hematological malignancy. Multivariate analysis revealed three independent prognostic factors: prolonged prothrombin time (P = 0.008), SAH (P = 0.021), and multifocal cerebral hemorrhage (P = 0.026).
The incidence of ICH in patients with AML is higher than patients with other hematological malignancies. But in those with intracranial malignant disease, patients with CNS involved lymphoma were more prone to ICH than patients with CNS involved acute leukemia. Mortality was similar regardless of the type of hematological malignancy. Neuroimaging studies of the location and type of ICH could assist with prognosis prediction for patients with hematological malignancies.
central nervous system (CNS) involvement; cerebral hemorrhage; hematological malignancy; prognosis; neuroimage
Isocorydine (ICD), an anticancer agent under current evaluation, decreased the percentage of side population (SP) cells significantly in hepatocellular carcinoma (HCC) cell lines. ICD treatment sensitized cancer cells to doxorubicin (DXR), a conventional clinical chemotherapeutic drug for HCC. We found that ICD decreased the percentage of SP cells in HCC cell lines by preferentially killing SP cells. In the early stage of treatment, ICD inhibited SP cell growth by arresting cells in G2/M; later, it induced apoptosis. Our xenograft model confirmed that ICD selectively reduced the size and weight of SP-induced tumor masses in vivo. Furthermore, it was found that programmed cell death 4 (PDCD4), a tumor suppressor gene, was relatively low when expressed in SP cells compared with non-SP cells, and its expression level was remarkably elevated when cells were treated with ICD. Taken together, these data suggest that ICD is a drug that may target the SP cells of HCC.
The multikinase inhibitor sorafenib is the first oral agent to show activity against human hepatocellular carcinoma (HCC). Although the clinical application of sorafenib has shown good tolerability in the studied populations, it also causes multiple human dose-limiting toxicities. Thus, there is a strong need to reduce the overall dose of sorafenib. We have reported that the epidermal growth factor receptor variant III (EGFRvIII) expression can decrease the sensitivity of HCC cells to chemotherapeutic drugs. Therefore, we sought to explore whether EGFRvIII can affect the sensitivity of HCC cells to sorafenib. In this study, we observed that EGFRvIII expression significantly decreased the sensitivity of HCC cells to sorafenib. To enhance the antitumor effect and reduce the overall dose of sorafenib, we evaluated the combined effects of CH12, a monoclonal antibody against EGFRvIII, and sorafenib on the growth of HCC cells expressing EGFRvIII in vitro and in vivo. The results showed that, when CH12 was combined with sorafenib, the tumor growth suppression effect was significantly increased, and the concentration of sorafenib required for growth inhibition was substantially reduced. Mechanistically, the combination could more noticeably downregulate the phosphorylation of constitutively active extracellular signal-regulated kinase (ERK), Akt (Thr308), and signal transducer and activator of transcription 3 (STAT3) than sorafenib alone. Collectively, these findings demonstrate that CH12 interacts additively with sorafenib to strongly inhibit the tumor growth of HCC xenografts expressing EGFRvIII by enhancing the sorafenib-mediated inhibition of the MEK/ERK, phosphoinositide 3-kinase/AKT, and STAT3 pathways.
The treatment of human hepatocellular carcinoma (HCC) cell lines with (+)-isocorydine, which was isolated and purified from Papaveraceae sp. plants, resulted in a growth inhibitory effect caused by the induction of G2/M phase cell cycle arrest and apoptosis. We report that isocorydine induces G2/M phase arrest by increasing cyclin B1 and p-CDK1 expression levels, which was caused by decreasing the expression and inhibiting the activation of Cdc25C. The phosphorylation levels of Chk1 and Chk2 were increased after ICD treatment. Furthermore, G2/M arrest induced by ICD can be disrupted by Chk1 siRNA but not by Chk2 siRNA. In addition, isocorydine treatment led to a decrease in the percentage of CD133+ PLC/PRF/5 cells. Interestingly, isocorydine treatment dramatically decreased the tumorigenicity of SMMC-7721 and Huh7 cells. These findings indicate that isocorydine might be a potential therapeutic drug for the chemotherapeutic treatment of HCC.
Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene (PPP2R2D) remain largely unknown. To explore genetic variations in the 5′-flanking region of PPP2R2D gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5′-flanking region of PPP2R2D. Four genetic variants were identified in the 1836 bp 5′-flanking region of PPP2R2D. Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human PPP2R2D promoter luciferase constructs, we found that a 601 bp (−540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5′-flanking fragment revealed the existence of three haplotypes in the distal promoter of PPP2R2D. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the −462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1). In vitro reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of PPP2R2D proximal promoter. Introduction of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of PPP2R2D gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5′-flanking region of PPP2R2D are critical for transcriptional regulation of PP2A-Bδ.
In this study, a series of manganese [Mn]-doped zinc oxide [ZnO] hierarchical microspheres [HMSs] are prepared by hydrothermal method only using zinc acetate and manganese acetate as precursors and ethylene glycol as solvent. X-ray diffraction indicates that all of the as-obtained samples including the highest Mn (7 mol%) in the crystal lattice of ZnO have a pure phase (hexagonal wurtzite structure). A broad Raman spectrum from as-synthesized doping samples ranges from 500 to 600 cm-1, revealing the successful doping of paramagnetic Mn2+ ions in the host ZnO. Optical absorption analysis of the samples exhibits a blueshift in the absorption band edge with increasing dopant concentration, and corresponding photoluminescence spectra show that Mn doping suppresses both near-band edge UV emission and defect-related blue emission. In particular, magnetic measurements confirm robust room-temperature ferromagnetic behavior with a high Curie temperature exceeding 400 K, signifying that the as-formed Mn-doped ZnO HMSs will have immense potential in spintronic devices and spin-based electronic technologies.
Mn-doped ZnO; hierarchical microspheres; optical properties; magnetic properties
Lysosomal exocytosis and fusion to cellular membrane is critical in the oxidative stress formation of endothelium under apoptotic stimulus. We investigated the role therein of it in hyperglycaemia-induced endothelial dysfunction. The lysosome-membrane fusion was shown by the expression of lamp1, the lysosomal membrane marker, on cellular membrane and the transportation of lysosomal symbolic enzymes into cultural medium. We also examined the ceramide production, lipid rafts (LRs) clustering, colocalization of gp91phox, a NADPH oxidase subunit (NOX) to LRs clusters, superoxide (O2.-) formation and nitric oxide (NO) content in human umbilical vein endothelial cells (HUVEC) and the endothelium-dependent NO-mediated vasodilation in isolated rat aorta. As compared to normal glucose (5.6 mmol/l, Ctrl) incubation, high glucose (22 mmol/l, HG) exposure facilitated the lysosome-membrane fusion in HUVEC shown by significantly increased quantity of lamp1 protein on cellular membrane and enhanced activity of lysosomal symbolized enzymes in cultural medium. HG incubation also elicited ceramide generation, LRs clustering and gp91phox colocalization to LRs clusters which were proved to mediate the HG induced O2.- formation and NO depletion in HUVEC. Functionally, the endothelium-dependent NO-mediated vasodilation in aorta was blunted substantially after HG incubation. Moreover, the HG-induced effect including ceramide production, LRs clustering, gp91phox colocalization to LRs clusters, O2.- formation and endothelial dysfunction could be blocked significantly by the inhibition of lysosome-membrane fusion. We propose that hyperglycaemia-induced endothelial impairment is closely related to the lysosome-membrane fusion and the following LRs clustering, LRs-NOX platforms formation and O2.- production.
The sequential order of secondary structural elements in proteins affects the folding and activity to an unknown extent. To test the dependence on sequential connectivity, secondary structural elements were reconnected by their solvent-exposed ends, permuting their sequential order, called “re-wiring.” This new protein design strategy changes the topology of the backbone without changing the core sidechain packing arrangement. While circular and non-circular permutations have been observed in protein structures that are not related by sequence homology, to date no one has attempted to rationally design and construct a protein whose sequence is non-circularly permuted while conserving three-dimensional structure. Herein we show that Green Fluorescent Protein (GFP) can be rewired, still functionally fold, and exhibit wild-type fluorescence excitation and emission spectra.
Protein engineering; GFP; re-wire; topology; protein folding; loop design
DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G2/M. These data support a crucial role for BMI1 in the cellular response to DNA damage.
Previous studies have demonstrates that, after nerve injury, extracellular signal-regulated protein kinase (ERK) activation in the spinal cord-initially in neurons, then microglia, and finally astrocytes. In addition, phosphorylation of ERK (p-ERK) contributes to nociceptive responses following inflammation and/or nerve injury. However, the role of spinal cells and the ERK/MAPK pathway in cancer-induced bone pain (CIBP) remains poorly understood. The present study analyzed activation of spinal cells and the ERK/MAPK pathway in a rat model of bone cancer pain.
A Sprague Dawley rat model of bone cancer pain was established and the model was evaluated by a series of tests. Moreover, fluorocitrate (reversible glial metabolic inhibitor) and U0126 (a MEK inhibitor) was administered intrathecally. Western blots and double immunofluorescence were used to detect the expression and location of phosphorylation of ERK (p-ERK). Our studies on pain behavior show that the time between day 6 and day 18 is a reasonable period ("time window" as the remaining stages) to investigate bone cancer pain mechanisms and to research analgesic drugs. Double-labeling immunofluorescence revealed that p-ERK was sequentially expressed in neurons, microglia, and astrocytes in the L4-5 superficial spinal cord following inoculation of Walker 256 cells. Phosphorylation of ERK (p-ERK) and the transcription factor cAMP response element-binding protein (p-CREB) increased in the spinal cord of CIBP rats, which was attenuated by intrathecal injection of fluorocitrate or U0126.
The ERK inhibitors could have a useful role in CIBP management, because the same target is expressed in various cells at different times.
bone cancer pain; hyperalgesia; spinal cord; extracellular signal-regulated protein kinase (ERK); cAMP response element-binding protein (CREB); rat
The differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca2+ determined by the alterations in the functions of plasma membrane CaL channels and ryanodine-sensitive Ca2+ releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively.
Sprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of CaL channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca2+ releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of CaL channels and ryanodine-sensitive Ca2+ releases to their control levels in cerebral and small mesenteric VSMCs, respectively.
The differential regulation of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca2+, which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.
Several types of epidermal growth factor receptor (EGFR) gene alternations have been observed in human tumors. Here we present a novel EGFR variant with aberrant splicing of exon 4 (named as de4 EGFR). Variant-specific polymerase chain reaction showed that de4 EGFR was expressed in some glioma (4/40), prostate cancer (3/11), and ovarian cancer (3/9) tissues but not in tissues adjacent to tumors or normal tissues. de4 EGFR displayed an enhanced transformation and a higher metastasis-promoting capacity in comparison to wild-type EGFR. With minimal EGF-binding activity, de4 EGFR underwent ligand-independent autophosphorylation and self-dimerization. Moreover, in serum-starved condition, de4 EGFR expression in U87 MG cells significantly upregulated the extracellular signal-regulated kinase and AKT phosphorylation and expression of JUN and Src. Importantly, E-cadherin expression was barely detectable in the U87 MG cells expressing de4 EGFR and restored expression of E-cadherin in these cells inhibited their metastatic behaviors. Taken together, we identified a novel EGFR variant with increased metastasis-promoting activity that may become a promising new target for cancer therapy.
ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma in situ (DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel.
Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts.
ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest.
ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest.
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) in the tumor microenvironment play important roles in tumor invasion and metastasis. Recently, ROS have been reported to cause a significant increase in the production and expression of matrix metalloproteinase (MMP)-7, which is closely correlated with metastatic colorectal cancer. The present study was undertaken to evaluate the scavenging activity of dimerumic acid (DMA) for H2O2 isolated from Monascus-fermented rice to investigate the inhibitory effects of DMA on the invasive potential of SW620 human colon cancer cells, and to explore the mechanisms underlying both these phenomena. Our results showed that increased MMP-7 expression due to H2O2 exposure was mediated by activation of mitogen-activated protein kinases (MAPKs) such as Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and p38 kinase. DMA pretreatment suppressed activation of H2O2-mediated MAPK pathways and cell invasion. Moreover, H2O2-triggered MMP-7 production was demonstrated via JNK/c-Jun and ERK/c-Fos activation in an activating protein 1 (AP-1)-dependent manner. Taken together, these results suggest that DMA suppresses H2O2-induced cell invasion by inhibiting AP-1-mediated MMP-7 gene transcription via the JNK/c-Jun and ERK/c-Fos signaling pathways in SW620 human colon cancer cells. Our data suggest that DMA may be useful in minimizing the development of colorectal metastasis. In the future, DMA supplementation may be a beneficial antioxidant to enhance surgical outcomes.
dimerumic acid; H2O2; MAPK; metastasis; MMP-7; ROS.