metastatic cell line of recent origin designated MIM/8. We found a good correlation between the metastatic potentials of the melanoma cells as measured in nude mice and their ability to adhere to cryostat sections of human lymph nodes. When adhesion to immobilized extracellular matrix proteins was measured, a significant increase in adhesion, which correlated with increased metastasis, was seen mainly on vitronectin and to a lesser extent on fibronectin. The adhesion to vitronectin and to the frozen sections were specifically blocked by an RGD-containing peptide, mAb 661 to vitronectin and mAb LM609 to integrin alphavbeta3. FACS analysis revealed a significant and specific increase in cell surface expression of alphavbeta3 on the metastatic cells as compared to the parent line. Together these results suggest that the adhesion of melanoma cells to lymph node vitronectin via the alphavbeta3 receptor plays a role in the process of lymphatic dissemination.
As we showed that the proteins involved in ECM cell adhesion were downregulated in shENO1 cells, we evalu- ated the adhesive ability of shCTRL or shENO1 PDA cells on fibronectin (FN), collagen I (Col-I), collagen IV (Col- IV), and vitronectin (VN). Compared to shCTRL cells, shENO1 PDA cells (CFPAC-1, PT45 and T3M4) showed a significantly lower adhesion to FN, Col-I, and Col-IV, and a greater adhesion to VN (Fig. 3 and Additional file 1: Figure S1a–b). We then evaluated the expression levels of alpha 5/ beta 1 and alphav/beta3 complexes, the integrins most involved in binding on these extracellular matrices, in shCTRL and shENO1 cells. Alpha 5 integrin was strongly upregulated by shENO1, both at the mRNA (Fig. 4a) and protein levels (Fig. 4b), while the mRNA, protein, and cell surface levels of beta 1 were unchanged (Fig. 4a–c). mRNA and protein levels of alphav and beta3 integrins were de- creased by shENO1 (Fig. 1, Fig. 4a and b, respectively). Consistently, the surface expression of the complex alphav/beta3, evaluated by a specific Ab that recognized the whole complex, was decreased on shENO1 cells compared to control cells (Fig. 4c). To identify the mechanism involved in the increased VN adhesion mediated by ENO1 silencing, we also analyzed the expression of the other VN receptor alpha IIb/beta3 integrin. However, no surface ex- pression of this complex was detected in either shENO1 or control cells (Fig. 4c). To understand the involvement of beta3 integrin in the adhesion to VN, a specific anti-beta3 Ab was employed. In beta3-expressing shCTRL cells, the anti-beta3 Ab significantly decreased the adhesion to VN, while in shENO1 cells that did not express beta3, it had no effect (Fig. 4d). The increase in adhesion to VN in shENO1
Glioblastoma multiforme, the most malignant astroglial-derived tumor, grows as an adherent mass and locally invades normal brain. An examination of adult cerebral glioblastoma biopsy material for the expression of adhesive proteins that might potentiate adhesion and invasion demonstrated tumor cell-associated vitronectin (5/5). In contrast, vitronectin was not detected associated with glial cells in low grade astroglial tumors (0/4), reactive astrogliosis (0/4), or in normal adult cortex and cerebral white matter (0/5). Also, a wide variety of other adhesive ligands were absent from the glioblastoma tumor parenchyma. The alphavbeta3 integrin was the only vitronectin receptor identified in glioblastoma tumors in situ, and was also not expressed on low grade astroglial-derived tumors, reactive
α v β 3 expression on the surface of eleven different cell lines was performed by FCM using antibody (ab93513; Abcam), which was monocloned based on the RGD recognizing mode (as per Abcam’s product description); however, in the current research, T7 or T7-6H bound to α v β 3 integrin in a different way, which may provide an explanation for the discrepancy between FCM results and Kd values. It also indicated that non-RGD binding mode may have a stronger correlation, specifically between α v β 3 integrin and the tracer, than RGD binding mode. At this point, mathematical approaches should be used to assess this phenomenon.
Figure 1 The figure describes the process by which αVβ3 mediates prostate cancer cell metastasis. Macroscopically, after cancer cells destroy the ECM and leave their primary site, cancer cells depend on αVβ3, which destroys vascular endothelial cells, to enter blood vessels. In blood vessels, tumor cells under the protection of BSP escape the attack of the immune system and then pass through the blood vessels to reach the metastatic site. Here, bone tissue is used as an example. Under the induction of VEGF, cancer cells recognize SPARC and HA and interact with them on the surface of bone with the help of αVβ3; at the same time, VEGF induces neovascularization to supply blood to the cell mass system. At the microscopic level, IL-8, IGF-I, CCN2 and other ligands activate different signaling pathways to promote the expression of αVβ3 in cancer cells. In addition, CCN2 transmits signals into the cell through αVβ3 to increase the acetylation of the transcription factor RUNX2, and RUNX2 promotes RANKL expression in tumor cells; these tumor cells then secrete RANKL to induce osteoblast differentiation into osteoclasts, and osteoclast-mediated bone tissue remodeling can expose HA and SPARC on the bone surface. BKCa and CXCL16 promote the aggregation and activation of αVβ3 and trigger the activation of downstream FAK. In addition to enhancing the activity of the ERK or PI3K pathway, activated FAK triggers cytoskeletal protein remodeling with the help of adaptor proteins, further enhancing tumor cell invasion ability. Cell information: The PC-3 cell line was initiated from the bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old Caucasian male; DU145 was initiated from a 69-year-old Caucasian male with brain metastasis of prostatic adenocarcinoma; LNCAP was isolated from the left supraclavicular lymph node of a 50-year-old prostate cancer patient. LNCAP is sensitive to hormones; C4-2B is a subtype derived from the LNCAP cell line. Abbreviations: BSP, bone sialoprotein; VEGF, vascular endothelial growth factor; HA, hydroxyapatite; SPARC, osteonectin; FAK, focal adhesion kinase; IGF-I, insulin-like growth factor; IL-8, interleukin8; OPN, osteopontin; MMP (2 or 9), matrix metalloproteinase (2 or 9); CCN2, connective tissue growth factor; CCL2, chemokines2; CXCL16, chemokines16; P, phosphorylated; AC, acetylated.
thyroid hormone. Covalently bound to a 200 nm nanoparticle that limits its activity to the cell exterior, tetrac reformulated as Nanotetrac has additional effects mediated by αvβ3 beyond the inhibition of binding of T 4 and T 3 to the integrin. These actions of Nanotetrac include disruption of transcription of cell survival pathway genes, promotion of apoptosis by multiple mechanisms, and interruption of repair of double-strand deoxyribonucleic acid breaks caused by irradiation of cells. Among the genes whose expression is suppressed by Nanotetrac are EGFR, VEGFA, multiple cyclins, catenins, and multiple cytokines. Nanotetrac has been effective as a chemotherapeutic agent in preclinical studies of human cancer xenografts. The low concentrations of αvβ3 on the surface of quiescent nonmalignant cells have minimized toxicity of the agent in animal studies.
uptake of RGD-SPIONs for both internalization through the cell membrane and accumulation within endosomes. More- over, the uptake was reduced after inhibiting the cells with free RGD peptides, which verified the specificity of RGD- SPIONs for integrin α v β 3. In line with the in vitro observa- tions, after intravenous injection of RGD-SPIONs into mice bearing squamous carcinoma (HaCaT-ras-A-5RT3 or A341), T2*-weighted MR imaging identified the heterogeneous distribution of α v β 3-positive tumor vessels by an irregular signal intensity decrease in HaCaT-ras-A-5RT3 xenografts, whereas the signal intensity decreased more homogeneously in the control tumors (A431) with predominantly small and uniformly distributed vessels (Figure 1). This study indicated that integrin α v β 3-targeted SPIONs were able to noninva- sively differentiate tumors with high and lower area fractions of α v β 3-positive tumor vessels. In an extended study, the same RGD-SPIONs were applied for the detection of tumor angiogenesis with different α v β 3 expression profiles by a 3T MRI scanner. 50 R2* pseudo-color images revealed that A549
an important role in cell-mediated immunity and also serve as host cells for a number of important viral and bacterial patho- gens, the ability to deliver genes or antiviral agents into these cells via adenovirus is of potential value. Human monocyte- derived and alveolar macrophages have been reported to be permissive for adenovirus cell entry; however, freshly isolated human monocytes are unable to support virus internalization (3, 10). Adenovirus has also been reported to have limited capacity for binding to human lymphocytes (12, 19), although these cells are clearly capable of being infected (11). Since the expression of integrins a v b 3 and a v b 5 can be differentially induced on human monocytes by the hematopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M- CSF) (7), this suggested that freshly isolated human monocytes were susceptible to adenovirus infection following upregula- tion of these cell surface receptors. In these studies, we dem- onstrate that induction of a v integrin expression on human monocytes/macrophages as well as human T lymphocytes by hematopoietic growth factors or cell-activating agents facili- tates adenovirus-mediated gene delivery. These studies further substantiate the role of a v integrins in adenovirus infection and also suggest a mechanism by which the host cell range of adenovirus is extended to monocytes and lymphoid cells.
maintain the nano-class diameter of the particles. Gener- ally, the smaller the diameter of the probe, the better the pharmacokinetics. In addition, such a probe usually has better extravasation and binding ability with the target after coupling with specific ligands. 19,20 The RGD polypeptide Figure 3 Transmission electron microscopy images of hUVecs co-cultured with RgD-UsPIO for 10 min A) and 30 min B) or UsPIO for 30 min C) at the iron concentration of 0.03 µmol/mL. A certain amount of RgD-UsPIO probe was ready internalized by the cells 10 min after co-culture and even more after 30 min co-culture. In contrast, the plain UsPIO probe was distributed mainly outside endothelial cells, with only a small number of particles attached to the cell membrane.
survival of rat Walker W256 cells cultured in the absence of serum. In this study, we confirmed the effects of 12-LOX overexpression in both cell lines by treating wild-type PC3 or A431 cells with 12(S)- HETE to examine whether this provided a survival advantage in both cell lines. As expected, treatment with 12(S)-HETE, and not other LOX metabolites, i.e., 5(S)-HETE, 15(S)-HETE, or 13(S)-HODE, resulted in significantly more viable cells compared with controls. In addition, 12(S)-HETE increased the surface expression of ␣ v ␤ 5 in wild-type cells, as illustrated by an integrin-mediated adhesion assay, an effect that was not observed when cells were treated with the other LOX metabolites. Also, treatment of PC3 or A431 cells overexpress- ing platelet-type 12-LOX with inhibitors of 12-LOX (Baicalein or BHPP) resulted in a significant reduction in the survival of either cell line, whereas the selective 5-LOX inhibitor Rev-5901 had no effect. We have demonstrated previously (10) that inhibition of 12-LOX with either of these structurally different inhibitors induces cell cycle arrest and apoptosis in two prostate cancer cell lines. Our results suggest that a more general phenomenon may be observed because survival and apoptosis of an epidermal tumor cell line, A431, was also regulated by 12-LOX and its end product, 12(S)-HETE.
breast cancer cases of different stages from the institutional hospital catchment area. Patients were offered treatment according to the stage of the disease, either surgery followed by chemotherapy and ± Radiotherapy or Neo-adjuvant chemotherapy followed by surgery then completion chemotherapy and radiation depending on the mode of the surgery, the patient’s menopausal status, and the stage of the disease, according to the national guidelines. In brief, postoperative radiotherapy was given to all patients treated with breast-conservation surgery. Hormonal therapy was offered to receptor ER or PR positive patients with axillary node positive (pN+) or T3 and T4 tumors irrespective of the mode of surgery. All pre-menopausal patients were treated with Tablet- Tamoxifen for 5 years. Post menopausal patients were either treated for 2 - 3 years with tamoxifen followed by 2 - 3 years of aromatase inhibitor or with aromatase inhibitor for 5 years. Patients with pN+ status and some with axillary node negative (pN−) status presenting with other adverse prognostic factors such as estrogen re- ceptor (ER)/progesterone receptor (PR) negative or poorly differentiated tumor, were given adjuvant chemothe- rapy (FEC/FAC cyclophosphamide, anthracyclin, taxens, methotrexate and 5-flurouracil) for six cycles. Stage was assessed by using the TNM classification. Patients with noninvasive carcinomas, a previous history of breast cancer, metastatic disease (stage-IV), or insufficient tumor material was excluded from the present study. Thus 81 patients with sufficient primary tumors and complete clinical histories were available for the present study. The mean age of the patients was 59.2 years (median 56.8 years; range, 23.3 - 91.6 years). The mean fol- low up time was 55.0 months (median 57.5 months; range, 1.2 - 115.1 months). The clinicopathological data of the patients are summarized in Table 1.
with similar particle concentrations ( ∼ 40 fmol) of untargeted and targeted NSs. The targeted NSs treatment group had more profound hemorrhage during treatment than the untargeted NSs treatment group. Greater amounts of hemorrhagic and necrotic debris were evident within the tumor core upon sectioning of the tumors from the targeted treatment group than the untargeted treatment group. As noted in Figure 5, the lower magnification images (A and C, 4 × ) demonstrate greater necrosis in the vascular-targeted group, suggestive of more significant vascular disruption. The results suggest that greater tumor and tumor vascular s pecificity via the active targeting technique improves the efficacy of thermal therapy. The observed enhancement in therapeutic efficacy is attributed to the higher tumor concentrations of targeted NSs as well as the possibly more intense focal temperature hot spots generated in the vicinity of v ascular-targeted NSs, resulting in more vascular disruption.
Single-cell RNAseq data for the mouse the T helper cells have previously been described  and we downloaded data from the ArrayExpress  under accession num- ber E-MTAB-2512. The RNAseq data generated for three replicate human Jurkat cell lines was downloaded from GEO  under accession number GSE45428. Simulated RNAseq reads for the in silico testing was generated as follows: GemSim , a General Error- Model based SIMulator, capable of generating paired- end reads generated using a reference genome and a given error model was used on the hg19 human refer- ence genome to generate read pairs of read lengths 25 bp, 50 bp, 75 bp, and 100 bp with a mean length of fragments of 300 bp with standard deviation of 30 bp. Noise and errors were added according to the ill100v4_p error model: llumina GA IIx with Illumina Sequencing Kit v4 chemistry, paired reads. TCR alpha and beta chain sequences were generated for 30 pairs by randomly con- catenating variable (diversity), junction, and constant re- gions (V-J-C for alpha and V-D-J-C for beta) selected at random from the IMGT reference human databases for each gene region . Random bases were then inserted at the junction segments of lengths 0 bp, 3 bp, 6 bp, and 9 bp to model the junctional diversity of VDJ recombin- ation that occurs in TCRs. Empirically we calculated from the Jurkat cell line RNAseq data that approxi- mately 0.3 % of all mapped reads mapped to the recon- structed TCR loci; therefore, for each simulated alpha and beta chain sequence we then again applied GemSim to the synthetic TCR alpha and beta sequences to
Recently, many investigators have focused their attention on the molecular mechanisms involved in the anti-IFN effect me- diated by paramyxovirus (4–7, 14, 36, 37). Sendai virus, hPIV-3, SV5, and mumps virus have been found to block both IFN-␣/␤ and IFN-␥ signaling, whereas hPIV-2 blocks IFN-␣/␤ signaling (37). There was a specific reduction in the level of the serine 727-phosphorylated form of Stat1␣ in Sendai virus- and hPIV-3-infected cells (37). The C protein of Sendai virus is responsible for preventing the induction of an IFN-induced antiviral state (6, 7). In contrast, the V protein of SV5 targets Stat1 for proteasome-mediated degradation and is thus re- sponsible for the observed block in IFN signaling in SV5- infected human cells, although SV5 does not inhibit the IFN- ␣/␤-responsive promoter in murine cells (5). A specific loss of Stat2 in hPIV-2-infected cells was reported by Young et al. (37). In various cells persistently infected with mumps virus, Stat1␣, but not Stat2, disappeared, and no difference between the levels of Stat1␣ mRNA transcript in the persistently in- fected cells and uninfected control cells was observed (36). Unexpectedly, the level of Stat1␣ apparently could not be improved by treating the cells with proteasome inhibitors (36). HeLa-CA cells and HeLa-V cells showed complete resis- tance to hIFN-␣ and hIFN-␤ irrespective of whether VSV or Sindbis virus was used as a challenge virus. In addition, when VSV was used, these cells were about 10 3 times less susceptible
healing as in normal epidermis with very little change in the relative intensity or distribution of staining at the leading edge of the migrating epidermis. However, at the time of wound closure, when the epidermis is still hyperproliferative, alpha 2, alpha3, alpha 6, and beta 1 were no longer confined to the basal layer, as in normal epidermis, but were also found in all the living suprabasal cell layers, coexpressed with the terminal differentiation markers involucrin, keratin 10, and keratin 16. Strong suprabasal staining for alphav was also found in one specimen. beta 4, which normally forms a heterodimer with alpha 6, and alpha 5 remained predominantly basal. Three of the integrin ligands, fibronectin, type IV collagen, and laminin, remained largely confined to the basement membrane zone and dermis. By 14 d after wounding, the integrins were once more restricted to the basal layer. Suprabasal integrin expression was also observed in involved psoriatic lesions. Thus, in two situations in which the epidermis is hyperproliferative, there is a failure […]
Objective: Insilico study using molecular docking with Hex had already surpassed all of the other previous approach in assessment of molecular binding to scaffold. The combination of insilico study to investigate the molecular binding to scaffold and electron microscope to assess the population density of adipocyte derivative stem cells by measuring the number of cluster cells could show the inter cell communication between adipocyte derived stem cell and scaffold. The aim of this study is to prove the attachment of mesenchymal stem cell (MSC) from stromal vascular fraction (SVF) to scaffold. Methods: The research is true experimental research using molecular docking with HEX version 8.0. Electron microscope used to measure the density and number of cluster cells of adipocyte derivative stem cells. Results: The strongest bound to scaffold is between MSC from SVF to hydroxyapatite in both receptor which was Integrin AlphaV to Hydroxyapatite which need a total energy of -89.24 (J/Mol) and Integrin Beta 2 to Hydroxyapatite which need a total energy of -177.8 (J/Mol). The highest impact obtained from hydroxyapatite-calcium phosphate with an average value of 12.66 cluster cells counting per 100 µm 2
After P. falciparum infection, many immune response- related genes of the TH1 immunological pathway were up-regulated (Table 1). Although interferon alpha/beta was not detected in this study, the major transcription factor of interferon alpha/beta synthesis, IRF7, was up- regulated after malaria. Many interferon alpha/beta in- ducible genes were up-regulated during the early stage of malaria including: tryptophenyl-tRNA (IFI53), inter- feron alpha inducible protein 27 (IFI27), interferon- induced protein 44 (IFI44), interferon-stimulated protein 15KD (G1P2), interferon regulatory factor 1 (IRF1), 2′- 5′-oligoadenylate synthetase 3 (OAS3), signal transducer and activator of transcription 1 (STAT1), interferon stimulated protein 35 (IFI35), Myxovirus resistance 1 (MX1), interferon-induced protein with tetratricopeptide repeat 4 (IFIT4), interferon-induced protein with tetra- tricopeptide repeat 1 (IFIT1), and interferon-induced protein with tetratricopeptide repeat 2 (IFIT2). The ex- pression levels of the above genes were greater than two-fold in early malaria, as compared to un-infection baseline. In acute febrile malaria, the expression levels of most of the alpha/beta interferon genes tended to de- cline and all returned to baseline levels of expression during the recovery period (Table 1). Summary of all four immunological pathways in malarial infection is shown in Figure 1.
In R statistical programming language (version 3.1.1 ), the MTrackJ results and background values were imported and relevant tracks were selected based on starting time point and minimum time point length. Baseline correction was performed by normalizing the tracks for baseline intensity. To identify the two different unloading profiles, tracks were hierarchically clustered in sub-populations based on the decrease of signal im- mediately after stimulation. The threshold criteria for clustering were (1) a decrease of the slope after stimula- tion of 25 %, (2) a relative amount of variation based on a ratio stdev/mean intensity lower than 0.5 and (3) stability of the baseline slope. Statistical analysis was per- formed using a t test with permutations to look for differences in the amount of synaptic boutons and ana- lysis of variance (ANOVA) to compare the regression lines of the synaptic bouton dye release.
Objective: To study a comprehensive proteomic analysis of celecoxib in oseteoarthritis (OA) chon- drocytes. Methods: OA chondrocytes were stimulated with celecoxib, IL-1β and IL-1β together with celecoxib. Proteins were extracted from the cells and subjected to 2-dimensional differential im- age gel electrophoresis (2D-DIGE). Proteins of interest were identified by mass spectrometry. Re- sults: Eighty-six protein spots showed significantly different intensities with each reagent or rea- gent combination. AAA+ protein, HSP47/Serpin, cAMP-dependent protein kinase type II-beta reg- ulatory subunit, alpha-actin-4 and tubulin decreased with the addition of celecoxib, while apoli- poprotein A-V, glutamate carboxipeptide 2, mitochondrial stress-70 protein, sorting nexin-9 and GRP78 increased with the addition of celecoxib. GRP78 is a stress protein and may be chondro- protective. Celecoxib modulated IL-1β stimulated chondrocytes, and CD200R and moesin were identified as such resulting proteins. Conclusion: Protein profiles of OA chondrocytes changed af- ter administration of celecoxib. Further investigation is needed to elucidate the function of each protein in OA chondrocytes.