技术资料

Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood,although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First,we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness,serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA),an activator of myosin that increases cell contractility,increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling,PP2,was found to significantly inhibit LPA-induced increases in cortical stiffness,as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue,we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine,which also increases myosin activation and contractility,increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins,including FAK,CAS,and paxillin. Thus,in the present study we identify,for the first time,the FA of the VSMC,in particular the FAK-Src signaling complex,as a significant subcellular regulator of aortic stiffness and stress. View Publication

The receptor tyrosine kinase c-MET is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-MET axis is often dysregulated in tumors. c-MET activation can be caused by MET gene amplification,activating mutations,and auto- or paracrine mechanisms. Thus,c-MET inhibitors are under development as anticancer drugs. Tivantinib (ARQ 197) was reported as a small-molecule c-MET inhibitor and early clinical studies suggest antitumor activity. To assess whether the antitumor activity of tivantinib was due to inhibition of c-MET,we compared the activity of tivantinib with other c-MET inhibitors in both c-MET-addicted and nonaddicted cancer cells. As expected,other c-MET inhibitors,crizotinib and PHA-665752,suppressed the growth of c-MET-addicted cancers,but not the growth of cancers that are not addicted to c-MET. In contrast,tivantinib inhibited cell viability with similar potency in both c-MET-addicted and nonaddicted cells. These results suggest that tivantinib exhibits its antitumor activity in a manner independent of c-MET status. Tivantinib treatment induced a G(2)-M cell-cycle arrest in EBC1 cells similarly to vincristine treatment,whereas PHA-665752 or crizotinib treatment markedly induced G(0)-G(1) cell-cycle arrest. To identify the additional molecular target of tivantinib,we conducted COMPARE analysis,an in silico screening of a database of drug sensitivities across 39 cancer cell lines (JFCR39),and identified microtubule as a target of tivantinib. Tivantinib-treated cells showed typical microtubule disruption similar to vincristine and inhibited microtubule assembly in vitro. These results suggest that tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET. View Publication

PURPOSE: MET,the high-affinity receptor for hepatocyte growth factor,is frequently deregulated in human cancer. Tivantinib (ARQ197; Arqule),a staurosporine derivative that binds to the dephosphorylated MET kinase in vitro,is being tested clinically as a highly selective MET inhibitor. However,the mechanism of action of tivantinib is still unclear. EXPERIMENTAL DESIGN: The activity of tivantinib was analyzed in multiple cellular models,including: cells displaying c-MET gene amplification,strictly 'addicted' to MET signaling; cells with normal c-MET gene copy number,not dependent on MET for growth; cells not expressing MET; somatic knockout cells in which the ATP-binding cleft of MET,where tivantinib binds,was deleted by homologous recombination; and a cell system 'poisoned' by MET kinase hyperactivation,where cells die unless cultured in the presence of a specific MET inhibitor. RESULTS: Tivantinib displayed cytotoxic activity independently of c-MET gene copy number and regardless of the presence or absence of MET. In both wild-type and isogenic knockout cells,tivantinib perturbed microtubule dynamics,induced G2/M arrest,and promoted apoptosis. Tivantinib did not rescue survival of cells 'poisoned' by MET kinase hyperactivation,but further incremented cell death. In all cell models analyzed,tivantinib did not inhibit HGF-dependent or -independent MET tyrosine autophosphorylation. CONCLUSIONS: We conclude that tivantinib displays cytotoxic activity via molecular mechanisms that are independent from its ability to bind MET. This notion has a relevant impact on the interpretation of clinical results,on the design of future clinical trials,and on the selection of patients receiving tivantinib treatment. View Publication

New small molecules that regulate the step-wise differentiation of human pluripotent stem cells into dopaminergic neurons have been identified. The steroid,guggulsterone,was found to be the most effective inducer of neural stem cells into dopaminergic neurons. These neurons are extensively characterized and shown to be functional. We believe this new approach offers a practical route to creating neurons of sufficient quality to be used to treat Parkinson's disease patients. View Publication

Bone remodeling,a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs),is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature,osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence,anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca²?/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones,along with significantly more OBs and fewer multinuclear OCs. In vitro,although Camkk2?/? mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs,bone marrow cells from Camkk2?/? mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective,cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further,we find phospho-protein kinase A (PKA) and Ser¹³³ phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand,genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target,nuclear factor of activated T cells,cytoplasmic (NFATc1). Moreover,in vivo administration of STO-609 results in increased OBs and diminished OCs,conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall,our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis. View Publication

We have previously synthesized a series of 1,8-naphthyridine-3-carboxamide derivatives to identify potential anti-cancer/anti-inflammatory compounds. Three derivatives,7-chloro-N-(3-(cyclopentylamino)-3-oxo-1-phenylpropyl)-6-fluoro-4-oxo-1-(prop-2-yn-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (C-22),7-chloro-N-(2-hydroxy-3-oxo-1-phenyl-3-(phenylamino)propyl)-4-oxo-1-(prop-2-yn-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (C-31) and 7-chloro-6-fluoro-N-(2-hydroxy-3-oxo-1-phenyl-3-(phenylamino)propyl)-4-oxo-1-(prop-2-yn-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (C-34) demonstrated high cytotoxicity against a number of cancer cell lines and inhibited secretion of IL-1-β and IL-6. In the present study,C-22,C-31 and C-34 were assessed for modulation of pro-inflammatory cytokines,TNF-α and IL-8,chemokine RANTES and NO produced by lipopolysaccharide (LPS)-treated mouse Dendritic cells (DCs). Among the 3 compounds,C-34 showed the most potent inhibition of inflammatory markers in DC model at 0.2 and 2 μM. C-34 also significantly downregulated the secretion of TNF-α,IL-1-β and IL-6 by murine splenocytes and THP-1 cells against LPS induced levels. In vitro effects of C-34 on bone marrow toxicity were assessed in CFU-GM assay. Human CFU-GM population was comparatively more sensitive to C-34 (0.1-10 μM) than murine CFU-GM. IC50 values for murine and human CFU-GM were not attained. C-34 was further examined for in vivo suppression of LPS induced cytokines in a mice model. At doses ranging from 1.25 to 5 mg/kg,C-34 led to significant inhibition of TNF-α,IL-1-β,IL-6 and MIP-1-α. At the highest dose of 5 mg/kg,C-34 also protected LPS-treated mice against endotoxin-induced lethality. In conclusion,C-34 demonstrates anti-inflammatory activity in vitro and in vivo in addition to cytotoxic properties. This finding suggests its potential for further development as a synthetic naphthyridine derivative with dual anti-cancer and anti-inflammatory (cytokine inhibition) properties. View Publication

The comparative evaluation of different 3D matrices-Matrigel,Puramatrix,and inverted colloidal crystal (ICC) scaffolds-provides a perspective for studying the pathology and potential cures for many blood and bone marrow diseases,and further proves the significance of 3D cultures with direct cell-cell contacts for in vitro mimicry of the human stem cell niche. View Publication

A small molecule compound,JTP-74057/GSK1120212/trametinib,had been discovered as a very potent antiproliferative agent able to induce the accumulation of CDK inhibitor p15INK4b. To conduct its drug development rationally as an anticancer agent,molecular targets of this compound were identified as MEK1/2 using compound-affinity chromatography. It was shown that JTP-74057 directly bound to MEK1 and MEK2 and allosterically inhibited their kinase activities,and that its inhibitory characteristics were similar to those of the known and different chemotype of MEK inhibitors PD0325901 and U0126. It was further shown that JTP-74057 induced rapid and sustained dephosphorylation of phosphorylated MEK in HT-29 colon and other cancer cell lines,while this decrease in phosphorylated MEK was not observed in PD0325901-treated cancer cells. Physicochemical analyses revealed that JTP-74057 preferentially binds to unphosphorylated MEK (u-MEK) in unique characteristics of both high affinity based on extremely low dissociation rates and ability stabilizing u-MEK with high thermal shift,which were markedly different from PD0325901. These findings indicate that JTP-74057 is a novel MEK inhibitor able to sustain MEK to be an unphosphorylated form resulting in pronounced suppression of the downstream signaling pathways involved in cellular proliferation. View Publication

Human embryonic stem cells are derived from the inner cell mass of preimplantation embryo at the blastocyst stage and their differentiation occurs through an intermediate step involving the formation of embryoid bodies (EBs),which are aggregates of embryonic stem cells. The EBs seem to be a powerful tool for investigating the development of embryos,as they can mimic the initial stages of embryonic development. In this study,we aimed to investigate the effect of estrogen compounds on the proliferation and differentiation of short-term and long-term cultured EBs in vitro. For this study,10-day-old (short-term cultured) and 30-day-old (long-term cultured) EBs were subjected to estradiol (E2),estriol (E3),selective estrogen receptor modulator (raloxifene [RLX]),bisphenol A,and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole for 7 days. To confirm the effects of estrogen treatment,ICI-182780 was added to the respective EBs for additional 7 days following estrogen treatment. Quantitative reverse transcription-polymerase chain reaction was performed to analyze the relative expression of differentiation marker genes representing the 3 germ layers. The expression of 7 marker genes,which included α-fetoprotein,hepatocyte nuclear factor (HNF)-3β,HNF-4α (endoderm),brachyury,cardiac actin ([cACT]; mesoderm),nestin (ectoderm),and Oct-4 (undifferentiated),was measured. Significantly,lower expression of HNF-4α in both short-term and long-term cultured EBs was observed after treatment of estrogen compounds compared to control. The expression of HNF-3β in short-term cultured EBs has been positively affected by E2,E3,and RLX. Regarding cACT,higher expression was observed after treatment of E2 (10(-7) mol/L) and E3 (10(-9) mol/L) in short-term cultured EBs,but opposite effects were demonstrated in long-term cultured EBs. The lower expressions of HNF-4α by E2 and RLX were negated by ICI-182780 treatment,although these findings were not statistically significant in E3-treated group. These findings suggest that estrogen compounds have effects on endodermal and mesodermal differentiation of human EBs. View Publication

Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet,it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study,we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment,investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore,their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together,our findings revealed that AML exosome trafficking alters the proliferative,angiogenic,and migratory responses of cocultured stromal and hematopoietic progenitor cell lines,helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML. View Publication

This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol,passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization,centrifugation or drug treatment. It also allows for higher throughput,requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation,colony expansion,cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion,and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages,this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium. View Publication

We isolated neural stem cells/neural progenitors (NSC) from 1-day-old rat pups born to mothers fed diets that were deficient or supplemented with n-3 polyunsaturated fatty acids (PUFAs) and compared their proliferation and differentiation in vitro. The cells isolated from the n-3PUFA-deficient pups consistently proliferated more slowly than cells that were isolated from n-3PUFA-supplemented pups,despite the fact that both were cultured under the same conditions. The differences in the proliferation rates were evaluated up until 40 days of culture and were highly significant. When the cells were allowed to differentiate,the deficient cells exhibited a higher degree of neuronal maturation in response to the addition of PUFAs in the medium,as demonstrated by an increase in neurite length,whereas the neurons derived from the supplemented pups showed no change. This result was consistent,regardless of the age of the culture. The properties of the NSC were durably modified throughout the length of the culture,although the membrane phospholipid compositions were similar. We examined the differential expression of selected mRNAs and micro RNAs. We found significant differences in the gene expression of proliferating and differentiating cells,and a group of genes involved in neurogenesis was specifically modified by n-3 PUFA treatment. We conclude that n-3 PUFA levels in the maternal diet can induce persistent modifications of the proliferation and differentiation of NSCs and of their transcriptome. Therefore,the n-3 supply received in utero may condition on a long-term basis cell renewal in the brain. View Publication