Osteo mineralization of fibrin-decorated graphene oxide

Surface functionalized graphene oxide (GO) was synthesized by coating fibrin on its surface. The fibrin coated graphene oxide (FGO) was characterized for its physiochemical properties and its potential as an osteoinductive material was evaluated using osteoblast like cell line MG-63 and normal cell line NIH 3T3. The scanning electron microscopy (SEM) has confirmed the coating of fibrin on GO and the transmission electron microscopy (TEM) revealed both spherical and cubical nature of GO nanoparticles and FGO. In in vitro studies, FGO exhibited higher levels of alkaline phosphatase and calcium ion release which confirmed the osteoinductive nature of FGO. The 3-(4,5-dimethylthiazol-2-Y)-2,5-diphenyltetrazolium bromide (MTT) assay proved FGO as a biocompatible material. The results have suggested that FGO might be a promising scaffold for bone tissue engineering applications.     

聚氨酯-羧甲基壳聚糖席夫碱水凝胶的制备与性能

将2,4-二羟基苯甲醛（DDBA）作为扩链剂，丙烯酸羟乙酯(HEA)作为封端剂制备了含有醛基的水性聚氨酯（DPU），在温和的条件下引入羧甲基化壳聚糖（CMCh）合成了聚氨酯-CMCh席夫碱，再由自由基聚合法引入聚丙酰胺合成了聚氨酯-羧甲基壳聚糖席夫碱水凝胶（DPU-Ch）。通过FTIR、SEM、力学性能、溶胀保水试验、抗菌试验和血液相容性测试等对水凝胶结构和性能进行表征。结果表明，水凝胶的机械性能随着CMCh质量分数的增加而提升，同时水凝胶也显示出良好的溶胀能力和保水能力；当CMCh添加量为2%时，水凝胶对革兰氏阳性和阴性细菌菌株均显示出良好的抗菌性能；水凝胶溶血率均低于5%表明其具有良好的细胞相容性，NIH3T3细胞存活率在90%以上证明其没有细胞毒性，因此在生物医疗领域中具有潜在的应用前景。     

Anticancer and Antioxidant Activity of Water-Soluble Polysaccharides from Ganoderma aff. australe against Human Osteosarcoma Cells

Wild mushrooms have gained great importance for being a source of biologically active compounds. In this work, we evaluate the anticancer and antioxidant activity of a water-soluble crude polysaccharide extract isolated from the fruiting bodies of the Ganoderma aff. australe (GACP). This mushroom was collected in San Mateo (Boyacá, Colombia) and identified based on macroscopic and microscopic characterization. GACP was characterized by UV–Vis spectroscopy, Fourier-transform infrared spectroscopy, high-performance liquid chromatography–diode array detector, and nuclear magnetic resonance. The antiradical and antioxidant activity were evaluated by different methods and its anticancer activity was verified in the osteosarcoma MG-63 human cell line. Chemical and spectroscopic analysis indicated that GACP consisted of β-D-Glcp-(1→, →3)-β-D-Glcp-(1→ and α-D-Glcp-(1→ residues. The results of the biological activity showed that GACP exhibited high antioxidant activity in the different methods and models studied. Moreover, the results showed that GACP impaired cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay) and cell proliferation (clonogenic assay) in a dose–response manner on MG-63 cells. The findings of this work promote the use of mushroom-derived compounds as anticancer and antioxidant agents for potential use in the pharmaceutical and food industries.     

Optimized Zn-doped hydroxyapatite/doxorubicin bioceramics system for efficient drug delivery and tissue engineering application

The synthesis and fabrication of multifunctional nanostructures with enhanced biocompatibility are the most important characteristics for biomedical research. The goal of our present research is to study the optimum zinc (Zn)-loading on pure hydroxyapatite (HAp) bioceramics and its potential advantages in biomedical application. In this study, different mole concentrations (1, 2, 5 mol%) of Zn doped HAp (Zn-HAp) nanoparticles were synthesized through a facile co-precipitation technique using zinc nitrate as a source for Zn metal. The synthesized Zn-HAp nanoparticles were critically characterized for their structural and morphological changes by different spectroscopy and electron microscopy analysis. The potential advances of Zn-HAp nanoparticles in biological application was studied by using MG-63 cell line, drug model experiment and scaffold cell attachment, proliferation study. The cell cytotoxicity test (MTT assay and trypan blue) was first conducted to confirm the nontoxic characteristics of Zn-HAp with enhanced MG-63 cell proliferation activity. The drug loading experiment of Zn-HAp nanoparticles was then confirmed with 1 mol% Zn-HAp (which had the maximum drug loading efficiency with pH responsive drug interaction). Furthermore, the optimized 1 mol% Zn-HAp constructed biomimetic scaffold shows excellent cell attachment and proliferation behavior with MG-63 cells. The result suggests that the biomimetic 1 mol% Zn-HAp scaffolds may be of enormous potential in bone repair and regeneration. This research distinguishes from other research by showing an advanced analysis of the Zn-HAp and its enhanced physicochemical properties for tissue engineering and pH responsive drug delivery application.     

Potential Anti-Metastatic Role of the Novel miR-CT3 in Tumor Angiogenesis and Osteosarcoma Invasion

Osteosarcoma (OS) is the most common primary bone tumor mainly occurring in young adults and derived from primitive bone-forming mesenchyme. OS develops in an intricate tumor microenvironment (TME) where cellular function regulated by microRNAs (miRNAs) may affect communication between OS cells and the surrounding TME. Therefore, miRNAs are considered potential therapeutic targets in cancer and one of the goals of research is to accurately define a specific signature of a miRNAs, which could reflect the phenotype of a particular tumor, such as OS. Through NGS approach, we previously found a specific molecular profile of miRNAs in OS and discovered 8 novel miRNAs. Among these, we deepen our knowledge on the fifth candidate renamed now miR-CT3. MiR-CT3 expression was low in OS cells when compared with human primary osteoblasts and healthy bone. Through TargetScan, VEGF-A was predicted as a potential biological target of miR-CT3 and luciferase assay confirmed it. We showed that enforced expression of miR-CT3 in two OS cell lines, SAOS-2 and MG-63, reduced expression of VEGF-A mRNA and protein, inhibiting tumor angiogenesis. Enforced expression of miR-CT3 also reduced OS cell migration and invasion as confirmed by soft agar colony formation assay. Interestingly, we found that miR-CT3 behaves inducing the activation of p38 MAP kinase pathway and modulating the epithelial-mesenchymal transition (EMT) proteins, in particular reducing Vimentin expression. Overall, our study highlights the novel role of miR-CT3 in regulating tumor angiogenesis and progression in OS cells, linking also to the modulation of EMT proteins.     

Biobanked Glioblastoma Patient-Derived Organoids as a Precision Medicine Model to Study Inhibition of Invasion

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.     

Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line

CD19 is the most promising target for developing chimeric-antigen receptor (CAR) T cells against B-cell leukemic cancer. Currently, two CAR-T-cell products, Kymriah and Yescarta, are approved for leukemia patients, and various anti-CD19 CAR T cells are undergoing clinical trial. Most of these anti-CD19 CAR T cells use FMC63 single-chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti-CD19 CAR T cells. We used the KHYG-1 NK/T-cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti-CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti-CD19 CAR KHYG-1 cells. Interestingly, only FMC63 CAR KHYG-1 and 4G7 CAR KHYG-1 efficiently lysed CD19-positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL-2 when co-cultured with CD19-positive cell line, NALM-6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19-positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM-6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B-cell leukemia patients.     

Different Cell Responses to Hinokitiol Treatment Result in Senescence or Apoptosis in Human Osteosarcoma Cell Lines

Hinokitiol is a tropolone-related compound isolated from the heartwood of cupressaceous plants. It is known to exhibit various biological functions including antibacterial, antifungal, and antioxidant activities. In the study, we investigated the antitumor activities of hinokitiol against human osteosarcoma cells. The results revealed that hinokitiol treatment inhibited cell viability of human osteosarcoma U-2 OS and MG-63 cells in the MTT assay. Further study revealed that hinokitiol exposure caused cell cycle arrest at the S phase and a DNA damage response with the induction of γ-H2AX foci in both osteosarcoma cell lines. In U-2 OS cells with wild-type tumor suppressor p53, we found that hinokitiol exposure induced p53 expression and cellular senescence, and knockdown of p53 suppressed the senescence. However, in MG-63 cells with mutated p53, a high percentage of cells underwent apoptosis with cleaved-PARP expression and Annexin V staining after hinokitiol treatment. In addition, up-regulated autophagy was observed both in hinokitiol-exposed U-2 OS and MG-63 cells. As the autophagy was suppressed through the autophagy inhibitor chloroquine, hinokitiol-induced senescence in U-2 OS cells was significantly enhanced accompanying more abundant p53 expression. In MG-63 cells, co-treatment of chloroquine increased hinokitiol-induced apoptosis and decreased cell viability of the treated cells. Our data revealed that hinokitiol treatment could result in different cell responses, senescence or apoptosis in osteosarcoma cell lines, and suppression of autophagy could promote these effects. We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors might provide more precise and efficacious therapies in hinokitiol-related trials for treating osteosarcoma.     

Amino Alcohol Acrylonitriles as Activators of the Aryl Hydrocarbon Receptor Pathway: An Unexpected MTT Phenotypic Screening Outcome

Lead (Z)-N-(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide, 1 showed MCF-7 GI₅₀=30 nM and 400-fold selective c.f. MCF10A (normal breast tissue). Acetamide moiety modification ( 13 a - g ) to introduce additional hydrophobicity was favoured with MCF-7 breast cancer cell activity enhanced at 1.3 nM. Other analogues were potent against the HT29 colon cancer cell line at 23 nM. Textbook SAR data was observed in the MCF-7 cell line, in an MTT assay, via the ortho ( 17 a ), meta ( 17 b ) and para ( 13 f ). The amino alcohol -OH moiety was pivotal, but no stereochemical preference noted. But, these data did not fit our homology modelling expectations. Aberrant MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) screening results and metabolic interference confirmed by sulforhodamine B (SRB) screening. Interfering analogues resulted in 120 and 80-fold CYP1A1 and CYP1A2 amplification, with no upregulation of SULT1A1. This is consistent with activation of the AhR pathway. Piperidine per-deuteration reduced metabolic inactivation. 3-OH / 4-OH piperidine analogues showed differential MTT and SRB activity supporting MTT assay metabolic inactivation. Data supports piperidine 3-OH, but not the 4-OH, as a CYP substrate. This family of β-amino alcohol substituted 3,4-dichlorophenylacetonitriles show broad activity modulated via the AhR pathway. By SRB analysis the most potent analogue was 23 b , (Z)-3-(4-(3-(4-phenylpiperidin-1-yl)-2-hydroxypropoxy)phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile.     

Exploiting the Lactose–GM3 Interaction for Drug Delivery

Protein–protein and protein–carbohydrate interactions as a means to target the cell surface for therapeutic applications have been extensively investigated. However, carbohydrate–carbohydrate interactions (CCIs) have largely been overlooked. Here, we investigate the concept of CCI‐mediated drug delivery. Lactose‐functionalized β‐cyclodextrin (L‐β‐CD) hosting doxorubicin (Dox) was evaluated for site‐specific delivery to cancer cells via interaction with GM₃, a cell‐surface carbohydrate. The host–guest complex was evaluated in B16 melanoma cells, which express exceptionally high levels of GM₃, and acute monocytic leukemia (THP‐1) and mouse fibroblast (NIH‐3T3) cells, which lack GM₃ on the cell surface. Doxorubicin (Dox) was delivered more efficiently into B16 cells compared with NIH‐3T3 and THP‐1 cells. In B16 cells pretreated with sialidase or sodium periodate, thus preventing CCI formation, drug uptake was significantly decreased. Taken together, the results of these studies strongly support CCI‐mediated uptake via the GM₃–lactose interaction as the mechanism of controlled drug delivery.     

The Role of TKS5 in Chromosome Stability and Bladder Cancer Progression

TKS5 promotes invasion and migration through the formation of invadopodia in some tumour cells, and it also has an important physiological function in cell migration through podosome formation in various nontumour cells. To date, the role of TKS5 in urothelial cells, and its potential role in BC initiation and progression, has not yet been addressed. Moreover, the contribution of TKS5 to ploidy control and chromosome stability has not been reported in previous studies. Therefore, in the present study, we wished to address the following questions: (i) Is TKS5 involved in the ploidy control of urothelial cells? (ii) What is the mechanism that leads to aneuploidy in response to TKS5 knockdown? (iii) Is TKS5 an oncogene or tumour-suppressor gene in the context of BC? (iv) Does TKS5 affect the proliferation, migration and invasion of BC cells? We assessed the gene and protein expressions via qPCR and Western blot analyses in a set of nontumour cell strains (Y235T, HBLAK and UROtsa) and a set of BC cell lines (RT4, T24, UMUC3 and J82). Following the shRNA knockdown in the TKS5-proficient cells and the ectopic TKS5 expression in the cell lines with low/absent TKS5 expression, we performed functional experiments, such as metaphase, invadopodia and gelatine degradation assays. Moreover, we determined the invasion and migration abilities of these genetically modified cells by using the Boyden chamber and wound-healing assays. The TKS5 expression was lower in the bladder cancer cell lines with higher invasive capacities (T24, UMUC3 and J82) compared to the nontumour cell lines from human ureter (Y235T, HBLAK and UROtsa) and the noninvasive BC cell line RT4. The reduced TKS5 expression in the Y235T cells resulted in augmented aneuploidy and impaired cell division. According to the Boyden chamber and wound-healing assays, TKS5 promotes the invasion and migration of bladder cancer cells. According to the present study, TKS5 regulates the migration and invasion processes of bladder cancer (BC) cell lines and plays an important role in genome stability.     

N6-Isopentenyladenosine Hinders the Vasculogenic Mimicry in Human Glioblastoma Cells through Src-120 Catenin Pathway Modulation and RhoA Activity Inhibition

Background: Vasculogenic mimicry (VM) is a functional microcirculation pattern formed by aggressive tumor cells. Thus far, no effective drugs have been developed to target VM. Glioblastoma (GBM) is the most malignant form of brain cancer and is a highly vascularized tumor. Vasculogenic mimicry represents a means whereby GBM can escape anti-angiogenic therapies. Methods: Here, using an in vitro tube formation assay on Matrigel, we evaluated the ability of N6-isopentenyladenosine (iPA) to interfere with vasculogenic mimicry (VM). RhoA activity was assessed using a pull-down assay, while the modulation of the adherens junctions proteins was analyzed by Western blot analysis. Results: We found that iPA at sublethal doses inhibited the formation of capillary-like structures suppressing cell migration and invasion of U87MG, U343MG, and U251MG cells, of patient-derived human GBM cells and GBM stem cells. iPA reduces the vascular endothelial cadherin (VE-cadherin) expression levels in a dose-dependent manner, impairs the vasculogenic mimicry network by modulation of the Src/p120-catenin pathway and inhibition of RhoA-GTPase activity. Conclusions: Taken together, our results revealed iPA as a promising novel anti-VM drug in GBM clinical therapeutics.     

Claudin 4 Is Differentially Expressed between Ovarian Cancer Subtypes and Plays a Role in Spheroid Formation

Claudin 4 is a cellular adhesion molecule that is frequently overexpressed in ovarian cancer and other epithelial cancers. In this study, we sought to determine whether the expression of claudin 4 is associated with outcome in ovarian cancer patients and may be involved in tumor progression. We examined claudin 4 expression in ovarian cancer tissues and cell lines, as well as by immunohistochemical staining of tissue microarrays (TMAs; n = 500), spheroids present in patients' ascites, and spheroids formed in vitro. Claudin 4 was expressed in nearly 70% of the ovarian cancer tissues examined and was differentially expressed across ovarian cancer subtypes, with the lowest expression in clear cell subtype. No association was found between claudin 4 expression and disease-specific survival in any subtype. Claudin 4 expression was also observed in multicellular spheroids obtained from patients' ascites. Using an in vitro spheroid formation assay, we found that NIH:OVCAR5 cells treated with shRNA against claudin 4 required a longer time to form compact spheroids compared to control NIH:OVCAR5 cells that expressed high levels of claudin 4. The inability of the NIH:OVCAR5 cells treated with claudin 4 shRNA to form compact spheroids was verified by FITC-dextran exclusion. These results demonstrate a role for claudin 4 and tight junctions in spheroid formation and integrity.     

Ionising Radiation Promotes Invasive Potential of Breast Cancer Cells: The Role of Exosomes in the Process

Along with the cells that are exposed to radiation, non-irradiated cells can unveil radiation effects as a result of intercellular communication, which are collectively defined as radiation induced bystander effects (RIBE). Exosome-mediated signalling is one of the core mechanisms responsible for multidirectional communication of tumor cells and their associated microenvironment, which may result in enhancement of malignant tumor phenotypes. Recent studies show that exosomes and exosome-mediated signalling also play a dynamic role in RIBE in cancer cell lines, many of which focused on altered exosome cargo or their effects on DNA damage. However, there is a lack of knowledge regarding how these changes in exosome cargo are reflected in other functional characteristics of cancer cells from the aspects of invasiveness and metastasis. Therefore, in the current study, we aimed to investigate exosome-mediated bystander effects of 2 Gy X-ray therapeutic dose of ionizing radiation on the invasive potential of MCF-7 breast cancer cells in vitro via assessing Matrigel invasion potential, epithelial mesenchymal transition (EMT) characteristics and the extent of glycosylation, as well as underlying plausible molecular mechanisms. The findings show that exosomes derived from irradiated MCF-7 cells enhance invasiveness of bystander MCF-7 cells, possibly through altered miRNA and protein content carried in exosomes.     

Surface modification of poly (ethylene terephthalate) fabric by soy protein isolate hydrogel for wound dressing application

In this study, a composite of poly (ethylene terephthalate) (PET) fabric and soy protein isolate (SPI) hydrogel loaded with gabapentin was developed. For covalent attachment of SPI on the surface of PET fabric, graft polymerization of acrylic acid (AA) on the surface of PET fabric was performed and then carboxyl groups available in the structure of AA were activated using EDAC and then SPI was coated on the surface of PET fabric. The results revealed appropriate connection between hydrogel and modified fabric. The hydrogel was characterized by swelling test and the drug release behavior was investigated. It was found that the casting temperature affects the swelling ratio of the hydrogel and an appropriate release profile of the drug was observed. The surface of fabric was characterized by contact angle measurement, electron microscopy, and infrared spectroscopy. In vitro cell culture study was performed using NIH 3T3 mouse fibroblasts to investigate the biocompatibility of final composite and MTS results along with morphology of cells on the surface of PET fabric coated with SPI revealed the biocompatibility of final product and no cell cytotoxicity was observed in modified PET fabric.     

Multifunctional heteropolysaccharide hydrogel under photobiomodulation for accelerated wound regeneration

The present study evaluated the effect of fucoidan/alginate-polyethylene glycol-gellan gum (Fu/AL-PEG@GGH) hydrogel using low-level laser therapy (LLLT) on facilitating wound healing for wound care management. The hydrogel was fabricated and characterized to evaluate the wound healing potential. Cytotoxicity and apoptotic effects were evaluated with L929 and NIH3T3 cells. Uniform spherical sheets were observed with high thermal stability caused by porous matrixes with the increased cell viability and fast cell migration. Scar tissue was reduced by larger wound contraction with faster healing effects from the hydrogel + LLLT-treated group at day 14. The polysaccharides may promote wound healing due to the strong bonds by the physical cross-linking in hydrogel preparation. The results from hydrogel + LLLT-treated group confirmed an effective wound healing potential from the presence of high fibroblast and collagen deposition. Therefore, the combined practice of the hydrogel with LLLT may enhance a wound healing process for effective wound care applications.     

Solid lipid nanoparticles loaded thermoresponsive pluronic–xanthan gum hydrogel as a transdermal delivery system

The aim of this study was to develop and investigate thermoresponsive hydrogel incorporating curcumin (Cur) for application as a transdermal delivery system. Cur was encapsulated within solid lipid nanoparticles via ultrasonic homogenization, and these were introduced into a thermoresponsive hydrogel composed of pluronic F68 (PF68) and F127 (PF127). The hydrogel composed of PF68 and PF127 in 10:90 ratio transformed from sol to gel at 29.3 °C close to skin temperature. The skin adhesiveness and adhesive strength of the hydrogel with 0.2% (w/w) of XG was 1.64 and 1.24 times higher than those of the hydrogel without XG, respectively. The physiochemical characteristics of prepared formulations were investigated via observation of particle size, polydispersity index, transmission electron microscopy, and scanning electron microscopy. Moreover, Fourier transform infrared spectroscopy analysis was performed at physiological temperature, which revealed lower hydrogen bonding intensity at gel phase than at sol phase. The cumulative amount of Cur that penetrated significantly increased compared with the Cur ethanol solution. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46004.