技术资料

Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed,at least in part,to the presence of oleuropein and hydroxytyrosol. In this study,oleuropein and hydroxytyrosol,major phenolic compound of olive oil,was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay),cell viability (Guava ViaCount assay),cell apoptosis,cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability,inhibited cell proliferation,and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 mug/mL of oleuropein or 50 mug/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G(1) to S phase transition manifested by the increase of cell number in G(0)/G(1) phase. View Publication

BACKGROUND Asthma is a heterogenous disease characterized by chronic inflammation and airway remodeling. An increase in the severity of airway remodeling is associated with a more severe form of asthma. There is increasing interest in the epithelial to mesenchymal transition process and mechanisms involved in the differentiation and repair of the airway epithelium,especially as they apply to severe asthma. Growing evidence suggests that Epithelial-Mesenchymal transition (EMT) could contribute to airway remodeling and fibrosis in asthma. Severe asthmatic patients with remodeled airways have a neutrophil driven inflammation. Neutrophils are an important source of TGF-$\beta$1,which plays a role in recruitment and activation of inflammatory cells,extracellular matrix (ECM) production and fibrosis development,and is a potent inducer of EMT. OBJECTIVE As there is little data examining the contribution of neutrophils and/or their mediators to the induction of EMT in airway epithelial cells,the objective of this study was to better understand the potential role of neutrophils in severe asthma in regards to EMT. METHODS We used an in vitro system to investigate the neutrophil-epithelial cell interaction. We obtained peripheral blood neutrophils from severe asthmatic patients and control subjects and examined for their ability to induce EMT in primary airway epithelial cells. RESULTS Our data indicate that neutrophils from severe asthmatic patients induce changes in morphology and EMT marker expression in bronchial epithelial cells consistent with the EMT process when co-cultured. TGF-$\beta$1 levels in the culture medium of severe asthmatic patients were increased compared to that from co-cultures of non-asthmatic neutrophils and epithelial cells. CONCLUSIONS AND CLINICAL RELEVANCE As an inducer of EMT and an important source of TGF-$\beta$1,neutrophils may play a significant role in the development of airway remodeling and fibrosis in severe asthmatic airways. View Publication

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Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties,yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME,we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway,possibly by modulating availability of hyaluronic acid (HA),the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships,and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions,respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction,whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo,targeting CD44 decreased PI3K-4EBP1-SOX2 signaling,tumor growth,and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally,a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes,respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44,TAM,and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery. View Publication

BACKGROUND Taking into consideration a recent surge of a lung injury condition associated with electronic cigarette use,we devised an in vitro model of sub-chronic exposure of human bronchial epithelial cells (HBECs) in air-liquid interface,to determine deterioration of epithelial cell barrier from sub-chronic exposure to cigarette smoke (CS),e-cigarette aerosol (EC),and tobacco waterpipe exposures (TW). METHODS Products analyzed include commercially available e-liquid,with 0{\%} or 1.2{\%} concentration of nicotine,tobacco blend (shisha),and reference-grade cigarette (3R4F). In one set of experiments,HBECs were exposed to EC (0 and 1.2{\%}),CS or control air for 10 days using 1 cigarette/day. In the second set of experiments,exposure of pseudostratified primary epithelial tissue to TW or control air exposure was performed 1-h/day,every other day,until 3 exposures were performed. After 16-18 h of last exposure,we investigated barrier function/structural integrity of the epithelial monolayer with fluorescein isothiocyanate-dextran flux assay (FITC-Dextran),measurements of trans-electrical epithelial resistance (TEER),assessment of the percentage of moving cilia,cilia beat frequency (CBF),cell motion,and quantification of E-cadherin gene expression by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS When compared to air control,CS increased fluorescence (FITC-Dextran assay) by 5.6 times,whereby CS and EC (1.2{\%}) reduced TEER to 49 and 60{\%} respectively. CS and EC (1.2{\%}) exposure reduced CBF to 62 and 59{\%},and cilia moving to 47 and 52{\%},respectively,when compared to control air. CS and EC (1.2{\%}) increased cell velocity compared to air control by 2.5 and 2.6 times,respectively. The expression of E-cadherin reduced to 39{\%} of control air levels by CS exposure shows an insight into a plausible molecular mechanism. Altogether,EC (0{\%}) and TW exposures resulted in more moderate decreases in epithelial integrity,while EC (1.2{\%}) substantially decreased airway epithelial barrier function comparable with CS exposure. CONCLUSIONS The results support a toxic effect of sub-chronic exposure to EC (1.2{\%}) as evident by disruption of the bronchial epithelial cell barrier integrity,whereas further research is needed to address the molecular mechanism of this observation as well as TW and EC (0{\%}) toxicity in chronic exposures. View Publication

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO,Ac-DEVD-CHO,Ac-YVAD-CHO,t-butoxycarbonyl-IETD-CHO,and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes,with dissociation constants ranging from 75 pM to {\textgreater}10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor,with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki {\textless} 20 nM) and selective inhibitor of Group I caspases (caspase-1,-4,and -5) and most Group III caspases (caspase-8,-9,and -10),suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response. View Publication

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PURPOSE A major clinical problem in the treatment of breast cancer is the inherent and acquired resistance to antiestrogen therapy. In this study,we sought to determine whether antiprogestin treatment,used as a monotherapy or in combination with antiestrogen therapy,induced growth arrest and active cell death in antiestrogen-resistant breast cancer cells. EXPERIMENTAL DESIGN MCF-7 sublines were established from independent clonal isolations performed in the absence of drug selection and tested for their response to the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (fulvestrant),and the antiprogestin mifepristone (MIF). The cytostatic (growth arrest) effects of the hormones were assessed with proliferation assays,cell counting,flow cytometry,and a determination of the phosphorylation status of the retinoblastoma protein. The cytotoxic (apoptotic) effects were analyzed by assessing increases in caspase activity and cleavage of poly(ADP-ribose) polymerase. RESULTS All of the clonally derived MCF-7 sublines expressed estrogen receptor and progesterone receptor but showed a wide range of antiestrogen sensitivity,including resistance to physiological levels of 4-OHT. Importantly,all of the clones were sensitive to the antiprogestin MIF,whether used as a monotherapy or in combination with 4-OHT. MIF induced retinoblastoma activation,G(1) arrest,and apoptosis preceded by caspase activation. CONCLUSIONS We demonstrate that: (a) estrogen receptor(+)progesterone receptor(+),4-OHT-resistant clonal variants can be isolated from an MCF-7 cell line in the absence of antiestrogen selection; and (b) MIF and MIF plus 4-OHT combination therapy induces growth arrest and active cell death of the antiestrogen-resistant breast cancer cells. These preclinical findings show potential for a combined hormonal regimen of an antiestrogen and an antiprogestin to combat the emergence of antiestrogen-resistant breast cancer cells and,ultimately,improve the therapeutic index of antiestrogen therapy. View Publication

Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2),a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2-CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently,myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca,but not Na,currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw,a model of postoperative pain. These effects are compared with those of tat-CBD3-the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex,superior suppression of calcium influx,and better antinociceptive potential. View Publication

Many tumors become addicted to autophagy for survival,suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation,how ULK1 promotes autophagy remains poorly understood. Here,we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered 15 phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells,regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells,providing a strong rationale for their combined use in the clinic. View Publication