技术资料

The identification and targeted therapy of cells involved in hepatocellular carcinoma (HCC) recurrence remain challenging. Here,we generated a monoclonal antibody against recurrent HCC,1B50-1,that bound the isoform 5 of the α2δ1 subunit of voltage-gated calcium channels and identified a subset of tumor-initiating cells (TICs) with stem cell-like properties. A surgical margin with cells detected by 1B50-1 predicted rapid recurrence. Furthermore,1B50-1 had a therapeutic effect on HCC engraftments by eliminating TICs. Finally,α2δ1 knockdown reduced self-renewal and tumor formation capacities and induced apoptosis of TICs,whereas its overexpression led to enhanced sphere formation,which is regulated by calcium influx. Thus,α2δ1 is a functional liver TIC marker,and its inhibitors may serve as potential anti-HCC drugs. View Publication

Novel derivatives of 1,2,4-triazoles are described as potent ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds display VEGFR-2 inhibitory activity comparable to that of Vatalanib and Vandetanib in both homogenous time-resolved fluorescence enzymatic and cellular assays. Several active molecules feature high intrinsic permeability (textgreater30 x 10(-5) cm/min) across Caco-2 cell monolayer. View Publication

Embryonic stem cells (ESC) are derived from the inner cell mass (ICM) of preimplantation blastocysts. To date,it has been challenging to establish pluripotent ESC lines for domestic animals,which could be important for biotechnological applications,such as genetic engineering and SCNT,and biomedical research. The aim of this work was to derive and characterise bovine embryonic stem-like cells (bESC) from in vitro-produced bovine blastocysts. Embryos were produced by in vitro fertilization of in vitro-matured oocytes aspirated from abattoir ovaries and cultured in groups of 25 in 50-μL drops of KSOM (Evolve,Zenith Biotech) with 4mgmL(-1) BSA for 7 days until they reached the blastocyst stage (Ross et al.,2009 Reproduction 137,427-437). At that point,the zona pellucida (ZP) was removed using 1mgmL(-1) Pronase (Sigma,St. Louis,MO),and ZP-free blastocysts were washed 6 times in SOF-HEPES. Three derivation approaches were tested: ZP-free whole blastocysts,mechanically isolated ICM,and immunosurgery-derived ICM. In each case,individual blastocysts/ICM were placed in 1 well of a 12-well dish seeded with a monolayer of mouse embryo fibroblasts (MEF) and cultured in mTeSR1 basal medium (without growth factors) supplemented with 20ngmL(-1) FGF2 and 2.5μM IWR1 (CTFR) (Wu et al. 2015 Nature 521,316-321). After 48h,blastocysts/ICM that failed to adhere were physically pressed against the bottom of the culture dish with a 22-gauge needle under a stereoscope to aid attachment. Thereafter,the media was changed daily. Outgrowths (after 6-7 days in culture) were dissociated and passaged using TrypLE and re-seeded in the presence of ROCK inhibitor (Y-27632,10μM) onto newly prepared wells containing MEF. Established bESC lines were cultured on MEF and passaged every 4 to 5 days at a 1:10 split ratio. The bESC lines were characterised by immunofluorescence (IF),RNA-seq,and teratoma formation. The efficiency of cell line derivation (evaluated at passage 3) was similar for the 3 approaches: whole blastocysts (9/16,56.3%),mechanical ICM isolation (7/12,58.3%),and immunosurgical ICM isolation (7/16,43.8%). The bESC were passaged and cultured long-term (more than 15 passages) and were subjected to several rounds of freezing and thawing while retaining their morphology and characteristics. IF analysis showed that long-term cultured bESC expressed the markers SOX2 and OCT4 (pluripotency),but did not express CDX2 (trophectoderm) or GATA6 (primitive endoderm). RNAseq analysis of 2 bESC lines showed that ICM markers (POU5F1,NANOG,SOX2,LIN28B,DNAMT3B,UTF1,SALL4) were expressed (RPKMtextgreater0.4),while trophectoderm markers (CDX2,GATA2,GATA3,FGF4,TFAP2A) and primitive endoderm markers (GATA6,HNF4A) were not expressed (RPKMtextless0.4). Finally,bESC lines (n=2) were able to form teratomas in immunodeficient mice. The teratomas contained tissues representative of the 3 germ lineages and expressed lineage-specific markers (ectoderm: TUJ1,endoderm: FOXA2,and mesoderm: ASM). In conclusion,the culture condition used in this work (CTFR) enables robust derivation and long-term in vitro propagation of pluripotent bESC. View Publication

2-Hydroxyglutarate (2HG) exists as two enantiomers,(R)-2HG and (S)-2HG,and both are implicated in tumor progression via their inhibitory effects on $\alpha$-ketoglutarate ($\alpha$KG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations,whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme and conversely it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH rather than attempting to block 2HG production by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore our results uncover an unexpected link between oncometabolites altered DNA repair and genetic instability." View Publication

Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study,we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single,living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique,it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research. View Publication

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells,it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system,aerobic glycolysis,and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells,while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass,increased proton leak,and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference,and highlight the importance of further research concerning energy metabolism of stem cells. View Publication

AbstractThe landscape of cancer treatment has been transformed by immune checkpoint inhibitors; however,the failure to benefit a large number of patients with cancer has underlined the need to identify promising targets for more effective interventions. In this study,we leverage 23andMe,Inc.’s large-scale human germline genetic and health database to uncover the previously unknown role of UL16-binding protein 6 (ULBP6),a high-affinity NK group 2D (NKG2D) ligand,in cancer and its promise as an immuno-oncology therapeutic target. We confirm ULBP6 expression in human tumors and demonstrate that soluble ULBP6 shed from tumors circumvents NKG2D activation provided by membrane-anchored NKG2D ligands to inhibit immune cell activation and tumor cell killing. Based on these findings,we developed 23ME-01473,a humanized Fc effector–enhanced antibody that binds to ULBP6 and its closely related family members,ULBP2 and ULBP5. 23ME-01473 effectively blocks soluble ULBP6-mediated immunosuppression to restore the NKG2D axis on NK and T cells to elicit tumor growth control. Moreover,the Fc effector–enhanced design of 23ME-01473 increases its binding affinity to fragment crystallizable gamma receptor IIIa,which,together with 23ME-01473’s binding to membrane-anchored ULBP6/2/5 on cancer cells,allows for augmented antibody-dependent cellular cytotoxicity induction,providing a second activation node for NK cells. Our studies demonstrate the therapeutic potential of an Fc effector–enhanced anti-ULBP6/2/5 antibody to reinvigorate NK cell and T-cell activation and cytotoxicity for the treatment of cancer.Significance:This study emphasizes the utility of population-based genome-wide assessments for discovering naturally occurring genetic variants associated with lifetime risks for cancer or immune diseases as novel drug targets. We identify ULBP6 as a potential keystone member of the NKG2D pathway,which is important for antitumor immunity. Targeting ULBP6 may hold therapeutic promise for patients with cancer. View Publication

Two types of acquired loss of heterozygosity are possible in cancer: deletions and copy-neutral uniparental disomy (UPD). Conventionally,copy number losses are identified using metaphase cytogenetics,whereas detection of UPD is accomplished by microsatellite and copy number analysis and as such,is not often used clinically. Recently,introduction of single nucleotide polymorphism (SNP) microarrays has allowed for the systematic and sensitive detection of UPD in hematologic malignancies and other cancers. In this study,we have applied 250K SNP array technology to detect previously cryptic chromosomal changes,particularly UPD,in a cohort of 301 patients with myelodysplastic syndromes (MDS),overlap MDS/myeloproliferative disorders (MPD),MPD,and acute myeloid leukemia. We show that UPD is a common chromosomal defect in myeloid malignancies,particularly in chronic myelomonocytic leukemia (CMML; 48%) and MDS/MPD-unclassifiable (38%). Furthermore,we show that mapping minimally overlapping segmental UPD regions can help target the search for both known and unknown pathogenic mutations,including newly identified missense mutations in the proto-oncogene c-Cbl in 7 of 12 patients with UPD11q. Acquired mutations of c-Cbl E3 ubiquitin ligase may explain the pathogenesis of a clonal process in a subset of MDS/MPD,including CMML. View Publication

Overexpression of high mobility group AT-hook 2 (HMGA2) is found in a number of benign and malignant tumors,including the clonal PIGA(-) cells in 2 cases of paroxysmal nocturnal hemoglobinuria (PNH) and some myeloproliferative neoplasms (MPNs),and recently in hematopoietic cell clones resulting from gene therapy procedures. In nearly all these cases overexpression is because of deletions or translocations that remove the 3' untranslated region (UTR) which contains binding sites for the regulatory micro RNA let-7. We were therefore interested in the effect of HMGA2 overexpression in hematopoietic tissues in transgenic mice (ΔHmga2 mice) carrying a 3'UTR-truncated Hmga2 cDNA. ΔHmga2 mice expressed increased levels of HMGA2 protein in various tissues including hematopoietic cells and showed proliferative hematopoiesis with increased numbers in all lineages of peripheral blood cells,hypercellular bone marrow (BM),splenomegaly with extramedullary erythropoiesis and erythropoietin-independent erythroid colony formation. ΔHmga2-derived BM cells had a growth advantage over wild-type cells in competitive repopulation and serial transplantation experiments. Thus overexpression of HMGA2 leads to proliferative hematopoiesis with clonal expansion at the stem cell and progenitor levels and may account for the clonal expansion in PNH and MPNs and in gene therapy patients after vector insertion disrupts the HMGA2 locus. View Publication

The pancreatic adenocarcinoma is an aggressive and devastating disease,which is characterized by invasiveness,rapid progression,and profound resistance to actual treatments,including chemotherapy and radiotherapy. At the moment,surgical resection provides the best possibility for long-term survival,but is feasible only in the minority of patients,when advanced disease chemotherapy is considered,although the effects are modest. Several studies have shown that thyroid hormone,3,3',5-triiodo-l-thyronine (T(3)) is able to promote or inhibit cell proliferation in a cell type-dependent manner. The aim of the present study is to investigate the ability of T(3) to reduce the cell growth of the human pancreatic duct cell lines chosen,and to increase the effect of chemotherapeutic drugs at conventional concentrations. Three human cell lines hPANC-1,Capan1,and HPAC have been used as experimental models to investigate the T(3) effects on pancreatic adenocarcinoma cell proliferation. The hPANC-1 and Capan1 cell proliferation was significantly reduced,while the hormone treatment was ineffective for HPAC cells. The T(3)-dependent cell growth inhibition was also confirmed by fluorescent activated cell sorting analysis and by cell cycle-related molecule analysis. A synergic effect of T(3) and chemotherapy was demonstrated by cell kinetic experiments performed at different times and by the traditional isobologram method. We have showed that thyroid hormone T(3) and its combination with low doses of gemcitabine (dFdCyd) and cisplatin (DDP) is able to potentiate the cytotoxic action of these chemotherapic drugs. Treatment with 5-fluorouracil was,instead,largely ineffective. In conclusion,our data support the hypothesis that T(3) and its combination with dFdCyd and DDP may act in a synergic way on adenopancreatic ductal cells. View Publication

A novel series of 3,4-dihydroxychalcones was synthesized to evaluate their effects against 5-lipoxygenase and cyclooxygenase. Almost all compounds exhibited potent inhibitory effects on 5-lipoxygenase with antioxidative effects,and some also inhibited cyclooxygenase. The 2',5'-disubstituted 3,4-dihydroxychalcones with hydroxy or alkoxy groups exhibited optimal inhibition of cyclooxygenase. We found that 2',5'-dimethoxy-3,4-dihydroxychalcone (37; HX-0836) inhibited cyclooxygenase to the same degree as flufenamic acid and 5-lipoxygenase,more than quercetin. Finally,these active inhibitors of 5-lipoxygenase inhibited arachidonic acid-induced mouse ear edema more than phenidone. View Publication

The thyroid hormone,3,5,3'-Triiodo-L-thyronine (T3),is essential for growth,differentiation,and regulation of metabolic functions in multicellular organisms,although the specific mechanisms of this control are still unknown. In this study,treatment of a human pancreatic duct cell line (hPANC-1) with T3 blocks cell growth by an increase of cells in G(0)/G(1) cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a beta-cell-like phenotype,we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion,our results suggest,for the first time,that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC-1) into pancreatic-beta-cell-like cells. These findings have important implications in cell-therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes. View Publication