The high-resolution crystal structures of apo and peptide-bound XIAP BIR2 are presented and compared with BIR3 structures to understand their selectivity. This crystal system can be used to determine the structures of BIR2–inhibitor complexes.
XIAP, a member of the inhibitor of apoptosis family of proteins, is a critical regulator of apoptosis. Inhibition of the BIR domain–caspase interaction is a promising approach towards treating cancer. Previous work has been directed towards inhibiting the BIR3–caspase-9 interaction, which blocks the intrinsic apoptotic pathway; selectively inhibiting the BIR2–caspase-3 interaction would also block the extrinsic pathway. The BIR2 domain of XIAP has successfully been crystallized; peptides and small-molecule inhibitors can be soaked into these crystals, which diffract to high resolution. Here, the BIR2 apo crystal structure and the structures of five BIR2–tetrapeptide complexes are described. The structural flexibility observed on comparing these structures, along with a comparison with XIAP BIR3, affords an understanding of the structural elements that drive selectivity between BIR2 and BIR3 and which can be used to design BIR2-selective inhibitors.
apoptosis; XIAP; BIR domains; caspases; extrinsic pathway; inhibitor of apoptosis; peptide complex; SMAC; AVPI
The identification of deregulated modules (such as induced by oncogenes) is a crucial step for exploring the pathogenic process of complex diseases. Most of the existing methods focus on deregulation of genes rather than the links of the path among them. In this study, we emphasize on the detection of deregulated links, and develop a novel and effective regulatory path-based approach in finding deregulated modules. Observing that a regulatory pathway between two genes might involve in multiple rather than a single path, we identify condition-specific core regulatory path (CCRP) to detect the significant deregulation of regulatory links. Using time-series gene expression, we define the regulatory strength within each gene pair based on statistical dependence analysis. The CCRPs in regulatory networks can then be identified using the shortest path algorithm. Finally, we derive the deregulated modules by integrating the differential edges (as deregulated links) of the CCRPs between the case and the control group. To demonstrate the effectiveness of our approach, we apply the method to expression data associated with different states of Human Epidermal Growth Factor Receptor 2 (HER2). The experimental results show that the genes as well as the links in the deregulated modules are significantly enriched in multiple KEGG pathways and GO biological processes, most of which can be validated to suffer from impact of this oncogene based on previous studies. Additionally, we find the regulatory mechanism associated with the crucial gene SNAI1 significantly deregulated resulting from the activation of HER2. Hence, our method provides not only a strategy for detecting the deregulated links in regulatory networks, but also a way to identify concerning deregulated modules, thus contributing to the target selection of edgetic drugs.
Iron deficiency is a common comorbidity in chronic heart failure (CHF) which may exacerbate CHF. The c-kit+ cardiac stem cells (CSCs) play a vital role in cardiac function repair. However, much is unknown regarding the role of iron deficiency in regulating c-kit+ CSCs function. In this study, we investigated whether iron deficiency regulates c-kit+ CSCs proliferation, migration, apoptosis, and differentiation in vitro.
All c-kit+ CSCs were isolated from adult C57BL/6 mice. The c-kit+ CSCs were cultured with deferoxamine (DFO, an iron chelator), mimosine (MIM, another iron chelator), or a complex of DFO and iron (Fe(III)), respectively. Cell migration was assayed using a 48-well chamber system. Proliferation, cell cycle, and apoptosis of c-kit+ CSCs were analyzed with BrdU labeling, population doubling time assay, CCK-8 assay, and flow cytometry. Caspase-3 protein level and activity were examined with Western blotting and spectrophotometric detection. The changes in the expression of cardiac-specific proteins (GATA-4,TNI, and β-MHC) and cell cycle-related proteins (cyclin D1, RB, and pRB) were detected with Western blotting.
DFO and MIM suppressed c-kit+ CSCs proliferation and differentiation. They also modulated cell cycle and cardiac-specific protein expression. Iron chelators down-regulated the expression and phosphorylation of cell cycle-related proteins. Iron reversed those suppressive effects of DFO. DFO and MIM didn’t affect c-kit+ CSCs migration and apoptosis.
Iron deficiency suppressed proliferation and differentiation of c-kit+ CSCs. This may partly explain how iron deficiency affects CHF prognosis.
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely reported to display strong efficacy for cancer chemoprevention, although their mechanism of action is poorly understood. The most well documented effects of NSAIDs include inhibition of tumor cell proliferation and induction of apoptosis, but their effect on tumor cell invasion has not been well studied. Here we show that the NSAID, sulindac sulfide (SS) can potently inhibit the invasion of human MDA-MB-231 breast and HCT116 colon tumor cells in vitro at concentrations less than those required to inhibit tumor cell growth. To study the molecular basis for this activity, we investigated the involvement of microRNA (miRNA). A total of 132 miRNAs were found to be altered in response to SS treatment including miR-10b, miR-17, miR-21, and miR-9, which have been previously implicated in tumor invasion and metastasis. We confirmed that these miRNA can stimulate tumor cell invasion and show that SS can attenuate their invasive effects by down-regulating their expression. Employing luciferase and chromatin immunoprecipitation assays, NF-κB was found to bind the promoters of all four miRNAs to suppress their expression at the transcriptional level. We show that SS can inhibit the translocation of NF-κB to the nucleus by decreasing the phosphorylation of IKKβ and IκB. Analysis of the promoter sequences of the miRNAs suppressed by SS revealed that 81 of 115 sequences contained NF-κB binding sites. These results show that SS can inhibit tumor cell invasion by suppressing NF-κB mediated transcription of miRNAs.
sulindac; invasion; microRNA; NF-κB
Herein, we report the rational design of a DEVD-based heptapeptide hydrogelator 1 which is susceptible to caspase-3 (CASP3), and its isomeric control hydrogelator 2 with a DEDV-based heptapeptide sequence. Self-assembly of 1 in water results in flexuous, long nanofibers to form supramolecular hydrogel I with higher mechanical strength than that of hydrogel II which is composed of rigid, short nanofibers of 2. In vitro enzymatic analysis indicated that 1 is susceptive to CASP3 while 2 is not. 3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyl tetrazolium bromide (MTT) and Western blot analyses indicated that DEDV-based hydrogelator 2 induces cell death via apoptotic pathway while the DEVD-based hydrogelator 1 minimizes cellular apoptosis induction.
Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMP-induced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation.
Herein we developed a new “smart” Gd-based MR contrast agent (i.e., 1) which is susceptive to furin, a protease overexpressed in tumor. Under the action of furin, 1 condenses to form dimers (1-Ds) and the latter self-assemble into gadolinium nanparticles (Gd-NPs). Relaxivity of 1-D is more than 2 folds of those of 1 and magnevist at 1.5 T, and 1.4 folds of that of 1 at 3 T. Intracellular condensation of 1 in furin-overexpressed MDA-MB-468 cells was proven with direct two-photon laser microscopy (TPLM) fluorescence imaging of the cells incubated with the europium analog of 1 (i.e., 2). Intracellular Gd-NPs of 1 were uncovered and characterized for the first time. MRI of MDA-MB-468 tumors showed that 1 has enhanced MR contrast within the tumors than that of its scrambled control 1-Scr.
More and more evidences demonstrate that the long non-coding RNAs (lncRNAs) play many key
roles in diverse biological processes. There is a critical need to annotate the functions
of increasing available lncRNAs. In this article, we try to apply a global network-based
strategy to tackle this issue for the first time. We develop a bi-colored network based
global function predictor, long non-coding RNA global function predictor
(‘lnc-GFP’), to predict probable functions for lncRNAs at large scale by
integrating gene expression data and protein interaction data. The performance of lnc-GFP
is evaluated on protein-coding and lncRNA genes. Cross-validation tests on protein-coding
genes with known function annotations indicate that our method can achieve a precision up
to 95%, with a suitable parameter setting. Among the 1713 lncRNAs in the bi-colored
network, the 1625 (94.9%) lncRNAs in the maximum connected component are all
functionally characterized. For the lncRNAs expressed in mouse embryo stem cells and
neuronal cells, the inferred putative functions by our method highly match those in the
Studying protein complexes is very important in biological processes since it helps reveal the structure-functionality relationships in biological networks and much attention has been paid to accurately predict protein complexes from the increasing amount of protein-protein interaction (PPI) data. Most of the available algorithms are based on the assumption that dense subgraphs correspond to complexes, failing to take into account the inherence organization within protein complex and the roles of edges. Thus, there is a critical need to investigate the possibility of discovering protein complexes using the topological information hidden in edges.
To provide an investigation of the roles of edges in PPI networks, we show that the edges connecting less similar vertices in topology are more significant in maintaining the global connectivity, indicating the weak ties phenomenon in PPI networks. We further demonstrate that there is a negative relation between the weak tie strength and the topological similarity. By using the bridges, a reliable virtual network is constructed, in which each maximal clique corresponds to the core of a complex. By this notion, the detection of the protein complexes is transformed into a classic all-clique problem. A novel core-attachment based method is developed, which detects the cores and attachments, respectively. A comprehensive comparison among the existing algorithms and our algorithm has been made by comparing the predicted complexes against benchmark complexes.
We proved that the weak tie effect exists in the PPI network and demonstrated that the density is insufficient to characterize the topological structure of protein complexes. Furthermore, the experimental results on the yeast PPI network show that the proposed method outperforms the state-of-the-art algorithms. The analysis of detected modules by the present algorithm suggests that most of these modules have well biological significance in context of complexes, suggesting that the roles of edges are critical in discovering protein complexes.
Network alignment is one of the most common biological network comparison methods. Aligning protein-protein interaction (PPI) networks of different species is of great important to detect evolutionary conserved pathways or protein complexes across species through the identification of conserved interactions, and to improve our insight into biological systems. Global network alignment (GNA) problem is NP-complete, for which only heuristic methods have been proposed so far. Generally, the current GNA methods fall into global heuristic seed-and-extend approaches. These methods can not get the best overall consistent alignment between networks for the opinionated local seed. Furthermore These methods are lost in maximizing the number of aligned edges between two networks without considering the original structures of functional modules.
We present a novel seed selection strategy for global network alignment by constructing the pairs of hub nodes of networks to be aligned into multiple seeds. Beginning from every hub seed and using the membership similarity of nodes to quantify to what extent the nodes can participate in functional modules associated with current seed topologically we align the networks by modules. By this way we can maintain the functional modules are not damaged during the heuristic alignment process. And our method is efficient in resolving the fatal problem of most conventional algorithms that the initialization selected seeds have a direct influence on the alignment result. The similarity measures between network nodes (e.g., proteins) include sequence similarity, centrality similarity, and dynamic membership similarity and our algorithm can be called Multiple Hubs-based Alignment (MHA).
When applying our seed selection strategy to several pairs of real PPI networks, it is observed that our method is working to strike a balance, extending the conserved interactions while maintaining the functional modules unchanged. In the case study, we assess the effectiveness of MHA on the alignment of the yeast and fly PPI networks. Our method outperforms state-of-the-art algorithms at detecting conserved functional modules and retrieves in particular 86% more conserved interactions than IsoRank.
We believe that our seed selection strategy will lead us to obtain more topologically and biologically similar alignment result. And it can be used as the reference and complement of other heuristic methods to seek more meaningful alignment results.
Over 350 million people worldwide are infected with hepatitis B virus (HBV), a major cause of liver failure and hepatocellular carcinoma. Current therapeutic agents are highly effective, but are also associated with development of viral resistance. Therefore, strategies for identifying other anti-HBV agents with specific, but distinctive mechanisms of action are needed. The human La (hLa) protein, which forms a stabilizing complex with HBV RNA ribonucleoprotein to promote HBV replication, is a promising target of molecular therapy.
This study aimed to discover novel inhibitors of hLa that could inhibit HBV replication and expression.
A multistage molecular docking approach was used to screen a Specs database and an in-house library against hLa binding sites. Sequential in vitro evaluations were performed to detect potential compounds with high scores in HepG2.2.15 cells.
Of the 26 potential compounds with high scores chosen for experimental verification, 12 had HBV DNA inhibition ratios of less than 50% with P<0.05. Six had significant inhibition of HBV e antigen (HBeAg) levels, and 13 had significant inhibition of HBV surface antigen (HBsAg) levels by in vitro assays. Compounds HBSC-11, HBSC-15 and HBSC-34 (HBSC is system prefix for active compounds screened by the library) were selected for evaluation. HBSC-11 was found to have an obvious inhibitory effect on hLa transcription and expression.
Our findings suggest that anti-HBV activity of HBSC-11 may be mediated by a reduction in hLa levels. In addition, our data suggest the potential clinical use of hLa inhibitors, such as HBSC-11, for treating HBV infection.
Antiangiogenic therapy is one of the most significant advances in anticancer treatment. The benefits of antiangiogenic therapies of late-stage cancers have been investigated but are still too limited.
We used an ovarian cancer model to test the effect of short-term bevacizumab treatment on metastasis as measured by bioluminescence. Western blotting and CD34-PAS dual staining were performed to assess hypoxia-inducible transcription factor-1α (HIF-1α) expression and vasculogenic mimicry(VM) formation. Cell viability was examined by a CCK8 assay.
Bevacizumab demonstrated antitumor effects in models of ovarian cancer, but also accelerated metastasis together, with marked hypoxia and VM formation in mice receiving short-term therapy. Bevacizumab treatment did not affect SKOV3 cell viability and the amount of VM in three-dimensional culture.
These results suggest that antiangiogenic therapy may potentially influence the progression of metastatic disease, which has been linked to the hypoxic response and VM formation.
Antiangiogenic therapies; Metastasis; Hypoxia; Vasculogenic mimicry
Auto-fluorescence bronchoscopy (AFB) has been used for the identification and localization of intra-epithelial pre-neoplastic and neoplastic lesions within the bronchus.
To determine the applicability of AFB for the detection and localization of precancerous and cancerous lesions, in addition to analyzing the morphologic presentation, their association to histological type and the variation between genders.
A five-year study involving 4983 patients, who underwent routine bronchoscopy [B] examination in a local tertiary teaching hospital, was done. The B examination was performed under intratracheal lidocaine, and samples were obtained using suitable approach. One thousand four hundred and eighty-five pathologically confirmed lung cancer patients were included in the study. The following parameters were studied: Morphological presentation, biopsy sites, histology. Differences between the groups were analyzed using Chi square test.
One thousand four hundred and eighty-five patients who had hyperplasia or neoplastic lesions were further confirmed as lung cancer pathologically. Lung cancer was more commonly found in the right lung (51.58% vs. 42.82%). The lesion occurred more frequently in the upper lobe than the lower lobe (44.17% vs. 22.42%). Male patients with squamous cell carcinoma showed upper lobe involvement more commonly, while the left main bronchus was more commonly involved in female patients. Adenocarcinoma mostly involved lesion of the upper lobe. Squamous cell carcinoma and small cell carcinoma were the major proliferative types (80.15% and 76.16% respectively).
AFB is efficient in the detection of pre-invasive and invasive lung lesions. The morphological presentation is associated to the histological type. There is variation in the presentation and histology of cancerous lung lesions between genders.
Auto-fluorescence bronchoscopy; gender; invasive lesion; lung cancer; screening
AIM: To investigate the anti-tumor effects of Paris chinensis dioscin (PCD) and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells.
METHODS: Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope (LSCM) using Annexin-V/propidium iodide (PI) staining, and the cell cycle was evaluated using PI staining with flow cytometry. Intracellular calcium ions were detected under fluorescence microscope. The expression of cell cycle and apoptosis-related proteins cyclin B1, CDK1, cytochrome C and caspase-3 was measured by immunohistochemical staining.
RESULTS: PCD had an anti-proliferation effect on human gastric cancer SGC-7901 cells in a dose- and time-dependent manner. After treatment of SGC-7901 cells with PCD, apoptosis appeared in SGC-7901 cells. Morphological changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining, and the cell number of the G0/G1 phase was decreased, while the number of cells in the G2/M phase was increased. Cell cycle-related proteins, such as cyclin B1 and CDK1, were all down-regulated, but caspase-3 and cytochrome C were up-regulated. Moreover, intracellular calcium accumulation occurred in PCD-treated cells.
CONCLUSION: G2/M phase arrest and apoptosis induced by PCD are associated with the inhibition of CDK-activating kinase activity and the activation of Ca2+-related mitochondrion pathway in SGC-7901 cells.
CyclinB1/CDK1; Cell cycle arrest; Caspase-3, Ca2+; Cytochrome C
The identification of disease-causing genes is a fundamental challenge in human health and of great importance in improving medical care, and provides a better understanding of gene functions. Recent computational approaches based on the interactions among human proteins and disease similarities have shown their power in tackling the issue. In this paper, a novel systematic and global method that integrates two heterogeneous networks for prioritizing candidate disease-causing genes is provided, based on the observation that genes causing the same or similar diseases tend to lie close to one another in a network of protein-protein interactions. In this method, the association score function between a query disease and a candidate gene is defined as the weighted sum of all the association scores between similar diseases and neighbouring genes. Moreover, the topological correlation of these two heterogeneous networks can be incorporated into the definition of the score function, and finally an iterative algorithm is designed for this issue. This method was tested with 10-fold cross-validation on all 1,126 diseases that have at least a known causal gene, and it ranked the correct gene as one of the top ten in 622 of all the 1,428 cases, significantly outperforming a state-of-the-art method called PRINCE. The results brought about by this method were applied to study three multi-factorial disorders: breast cancer, Alzheimer disease and diabetes mellitus type 2, and some suggestions of novel causal genes and candidate disease-causing subnetworks were provided for further investigation.
To investigate the biological significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and ATF-2 in the TGF-β signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation.
Joint disease in Smad3 knockout (Smad3−/−) mice was examined by micro-CT and histology. Numerous in vitro methods including immunostaining, real-time PCR, Western blotting, an ATF-2 DNA-binding assay and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3−/− mice.
Smad3−/− mice gradually developed an end-stage OA phenotype. TGF-β-induced TAK1-ATF-2 signaling was disrupted in Smad3−/− chondrocytes at the level of p38 MAP kinase activation resulting in reduced ATF-2 phosphorylation and transcriptional activity. Re-introduction of Smad3 into the Smad3−/− cells restored the normal p38 response to TGF-β. Phospho-p38 formed a complex with Smad3 by binding to the Smad3 MH1-linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAP kinase phosphatase-1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild type and Smad3−/− chondrocytes. p38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms α, β and γ, but not δ.
Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1-ATF-2 signaling, most likely by disrupting the Smad3-phospho-p38 complex and, thereby, promoting p38 dephosphorylation and inactivation by MKP-1. p38 and ATF-2 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA.
Kallistatin is a plasma protein with anti-inflammatory properties. In this study, we investigated the role and mechanisms of kallistatin in inhibiting endothelial inflammation through its heparin-binding domain. We showed that recombinant wild-type kallistatin dose-dependently competed with tumor necrosis factor (TNF)-α binding to TNF-α receptor in endothelial cells, whereas kallistatin mutant at the heparin-binding domain had no effect. Kallistatin, but not kallistatin mutant at the heparin-binding domain, abrogated TNF-α-induced endothelial cell activation as evidenced by inhibition of TNF-α receptor/TRADD activation, IκB-α degradation, nuclear factor-κB translocation and p38MAPK phosphorylation, as well as cell adhesion molecule and cytokine expression. Moreover, kallistatin, but not kallistatin mutant at the heparin-binding domain, inhibited TNF-α-induced THP-1 monocyte adhesion to endothelial cells, and prevented vascular endothelial growth factor-induced endothelial permeability. In mice, kallistatin gene delivery prevented vascular leakage provoked by complement factor C5a, whereas delivery of kallistatin heparin mutant gene had no effect. Similarly, gene transfer of kallistatin, but not the kallistatin heparin mutant, inhibited collagen/adjuvant-induced arthritis in rats. These results indicate that kallistatin's heparin–binding site plays an essential role in preventing TNF-α–mediated endothelial activation and reducing vascular endothelial growth factor-induced vascular permeability, resulting in attenuation of vascular inflammation in cultured endothelial cells and animal models. This study identifies a protective role of kallistatin in vascular injury, thereby implicating therapeutic potential of kallistatin for vascular and inflammatory diseases.
kallistatin; tumor necrosis factor-α; vascular endothelial growth factor; nuclear factor κB; inflammation; vascular permeability
Background and Objectives
Glutathione S-transferases (GSTs) are multifunctional enzymes that play a key role in the detoxification of varieties of both endogenous products of oxidative stress and exogenous carcinogens.
In this meta-analysis, twenty-five studies were identified by searching PubMed, EMBASE, ISI Web of Science and CBM databases: 23 evaluated GSTM1 and 19 evaluated GSTT1. Crude odds ratios with corresponding 95% confidence intervals were used to estimate the association between GSTM1 and GSTT1 polymorphisms and risk of cervical neoplasia. Subgroup analyses were conducted by pathological history, ethnicity, source of DNA for genotyping, quality score, and matching variable.
The null genotypes of GSTM1 and GSTT1 polymorphisms were associated with a significantly increased risk of cervical neoplasia (for GSTM1: OR = 1.40; 95%CI, 1.19–1.65; for GSTT1: OR = 1.30; 95%CI, 1.05–1.62, respectively). Subgroup analyses showed that the null genotype of GSTM1 increased the risk of cervical neoplasia in Asians, studies with DNA isolation from white blood cells and tissue samples, both high and low quality studies, and matched studies. In GSTM1-GSTT1 interaction analysis, individuals with dual null genotype were associated with a significantly increased risk of cervical neoplasia (OR = 1.72; 95%CI, 1.18–2.51).
These findings indicate that GSTM1 and GSTT1 polymorphisms, particularly GSTM1-GSTT1 interaction, may play critical roles in the development of cervical neoplasia. A conservative manner should be adopted to interpret these results because of obvious heterogeneity between-study, unadjusted data, and relatively small sample size in this meta-analysis. Well designed studies with larger sample size are of great value to confirm these results.
Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR1), and by PAR1 inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK-antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR1-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.
tissue kallikrein; keratinocyte; migration; proteinase-activated receptor 1; epidermal growth factor receptor
Since genes associated with similar diseases/disorders show an increased tendency for their protein products to interact with each other through protein-protein interactions (PPI), clustering analysis obviously as an efficient technique can be easily used to predict human disease-related gene clusters/subnetworks. Firstly, we used clustering algorithms, Markov cluster algorithm (MCL), Molecular complex detection (MCODE) and Clique percolation method (CPM) to decompose human PPI network into dense clusters as the candidates of disease-related clusters, and then a log likelihood model that integrates multiple biological evidences was proposed to score these dense clusters. Finally, we identified disease-related clusters using these dense clusters if they had higher scores. The efficiency was evaluated by a leave-one-out cross validation procedure. Our method achieved a success rate with 98.59% and recovered the hidden disease-related clusters in 34.04% cases when removed one known disease gene and all its gene-disease associations. We found that the clusters decomposed by CPM outperformed MCL and MCODE as the candidates of disease-related clusters with well-supported biological significance in biological process, molecular function and cellular component of Gene Ontology (GO) and expression of human tissues. We also found that most of the disease-related clusters consisted of tissue-specific genes that were highly expressed only in one or several tissues, and a few of those were composed of housekeeping genes (maintenance genes) that were ubiquitously expressed in most of all the tissues.
Disease-related gene cluster; Clustering analysis; PPI network; Gene expression data
Saikosaponin-a and -d, two naturally occurring compounds derived from Bupleurum radix, have been shown to exert anti-cancer activity in several cancer cell lines. However, the effect of combination of saikosaponins with chemotherapeutic drugs has never been addressed. Thus, we investigated whether these two saikosaponins have chemosensitization effect on cisplatin-induced cancer cell cytotoxicity.
Two cervical cancer cell lines, HeLa and Siha, an ovarian cancer cell line, SKOV3, and a non-small cell lung cancer cell line, A549, were treated with saikosaponins or cisplatin individually or in combination. Cell death was quantitatively detected by the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit. Cellular ROS was analyzed by flow cytometry. Apoptosis was evaluated by AO/EB staining, flow cytometry after Anexin V and PI staining, and Western blot for caspase activation. ROS scavengers and caspase inhibitor were used to determine the roles of ROS and apoptosis in the effects of saikosaponins on cisplatin-induced cell death.
Both saikosaponin-a and -d sensitized cancer cells to cisplatin-induced cell death in a dose-dependent manner, which was accompanied with induction of reactive oxygen species (ROS) accumulation. The dead cells showed typical apoptotic morphologies. Both early apoptotic and late apoptotic cells detected by flow cytometry were increased in saikosaponins and cisplatin cotreated cells, accompanied by activation of the caspase pathway. The pan-caspase inhibitor z-VAD and ROS scanvengers butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) dramatically suppressed the potentiated cytotoxicity achieved by combination of saikosaponin-a or -d and cisplatin.
These results suggest that saikosaponins sensitize cancer cells to cisplatin through ROS-mediated apoptosis, and the combination of saikosaponins with cisplatin could be an effective therapeutic strategy.
Emerging evidence suggests that ataxia telangiectasia-mutated (ATM) is involved in numerous damage repair signaling pathways and cell-cycle checkpoints. Heterozygous carriers of ATM-mutations have an increased risk for the development of breast cancer. The purpose of this study is to evaluate the association between ATM exon39 5557G > A (D1853N, rs1801516) polymorphism and breast cancer susceptibility with the use of a meta-analysis.
By searching PubMed and Embase databases, a total of 9 epidemiological studies with 4,191 cases and 3,780 controls were identified. Crude odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) for ATM D1853N polymorphism and breast cancer risk were calculated using fixed- or random-effects model based on the degree of heterogeneity among studies.
No significant association between the ATM D1853N polymorphism and breast cancer risk was observed in overall analysis (GA versus GG: OR = 1.18; 95% CI, 0.90-1.53; AA versus GG: OR = 0.77; 95% CI, 0.58-1.03; dominant model: OR = 1.16; 95% CI, 0.89-1.51; and recessive model: OR = 0.78; 95% CI, 0.59-1.04, respectively).
Our results indicate that ATM D1853N polymorphism is not a risk factor for developing breast cancer.
Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into many cell types. Chondrogenesis is induced in hMSCs cultured as a micromass pellet to mimic cellular condensation during cartilage development, and exposed to transforming growth factor β (TGFβ). Interestingly, TGFβ also can induce hMSC differentiation to smooth-muscle-like cell types, but it remains unclear what directs commitment between these two lineages. Our previous work revealed that cell shape regulates hMSC commitment between osteoblasts and adipocytes through RhoA signaling. Here we show that cell shape also confers a switch between chondrogenic and smooth muscle cell (SMC) fates. Adherent and well-spread hMSCs stimulated with TGFβ3 upregulated SMC genes, while cells allowed to attach onto micropatterned substrates, but prevented from spreading and flattening, upregulated chondrogenic genes. Interestingly, cells undergoing SMC differentiation exhibited little change in RhoA but significantly higher Rac1 activity than chondrogenic cells. Rac1 activation inhibited chondrogenesis, and was necessary and sufficient for inducing SMC differentiation. Furthermore, TGFβ3 and Rac1 signaling upregulated N-cadherin, which was required for SMC differentiation. These results demonstrate a chondrogenic-SMC fate decision mediated by cell shape, Rac1, and N-cadherin, and highlight the tight coupling between lineage commitment and the many changes in cell shape, cell-matrix adhesion, and cell-cell adhesion that occur during morphogenesis.
Mesenchymal stem cells; Cell shape; Chondrogenesis; Smooth muscle cells; Rac1; N-cadherin
We investigated the role of the Akt-glycogen synthase kinase (GSK)-3β signalling pathway in mediating the protective effects of tissue kallikrein on myocardial injury by promoting angiogenesis and blood flow in rats after myocardial infarction (MI).
Methods and results
Human tissue kallikrein gene in an adenoviral vector, with or without co-administration of dominant-negative Akt (Ad.DN-Akt) or constitutively active GSK-3β (Ad.GSK-3βS9A), was injected into rat myocardium after MI. The expression of recombinant human kallikrein in rat heart significantly improved cardiac function and reduced infarct size 10 days after gene delivery. Kallikrein administration significantly increased myocardial blood flow as well as capillary and arteriole densities in the infarcted myocardium. Kallikrein increased cardiac Akt and GSK-3β phosphorylation in conjunction with decreased GSK-3β activity and the upregulation of vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2). All of kallikrein’s effects on the myocardium were abrogated by Ad.DN-Akt and Ad.GSK-3βS9A. Moreover, in cultured human aortic endothelial cells, tissue kallikrein stimulated capillary tube formation and promoted cell migration; however, these effects were blocked by Ad.DN-Akt, Ad.GSK-3βS9A, icatibant (a kinin B2 receptor antagonist), Tki (a VEGF receptor tyrosine kinase inhibitor), and a neutralizing VEGF antibody. In addition, tissue kallikrein decreased GSK-3β activity via the phosphatidylinositol 3-kinase-Akt pathway and enhanced VEGF and VEGFR-2 expression in endothelial cells.
These data provide the first direct evidence that tissue kallikrein protects against acute-phase MI by promoting neovascularization, restoring regional blood flow and improving cardiac function through the kinin B2 receptor-Akt-GSK-3β and VEGF signalling pathways.
Tissue kallikrein; Angiogenesis; Myocardial infarction; GSK-3β; VEGF
Tissue kallikrein exerts various biological functions through kinin formation with subsequent kinin B2 receptor activation. Recent studies showed that tissue kallikrein directly activates kinin B2 receptor in cultured cells expressing human kinin B2 receptor. In the present study, we investigated the role of tissue kallikrein in protection against cardiac injury through direct kinin B2 receptor activation using kininogen-deficient Brown Norway Katholiek (BNK) rats after acute myocardial infarction (MI). Tissue kallikrein was injected locally into the myocardium of BNK rats after coronary artery ligation, with and without co-injection of icatibant (a kinin B2 receptor antagonist) and L-NAME (a nitric oxide synthase inhibitor). One day after MI, tissue kallikrein treatment significantly improved cardiac contractility, reduced myocardial infarct size and left ventricle-end-diastolic pressure in BNK rats. Kallikrein attenuated ischemia-induced apoptosis and monocyte/macrophage accumulation in the ischemic myocardium in conjunction with increased nitric oxide (NO) levels and reduced myeloperoxidase activity. Icatibant and L-NAME abolished kallikrein’s effects, indicating a kinin B2 receptor-NO-mediated event. Moreover, inactive kallikrein had no beneficial effects in cardiac function, myocardial infarction, apoptosis or inflammatory cell infiltration after MI. In primary cardiomyocytes derived from BNK rats under serum-free conditions, active, but not inactive, kallikrein reduced hypoxia/reoxygenation-induced apoptosis and caspase-3 activity, and the effects were mediated by kinin B2 receptor/NO formation. This is the first study to demonstrate that tissue kallikrein directly activates kinin B2 receptor in the absence of kininogen to reduce infarct size, apoptosis and inflammation and improve cardiac performance of infarcted hearts.
tissue kallikrein; kinin B2 receptor; cardiac function; infarct size; apoptosis