ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of ...the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate (125)Iiodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ingenol-3-angelate (Ing3A), extracted from Euphorbia peplus, is currently in clinical trials for eradicating basal cell carcinoma, actinic keratosis, and squamous cell carcinoma (SCC) in situ by ...topical application. Although structurally related to phorbol esters and a protein kinase C activator, topical Ing3A, but not phorbol 12-myristate 13-acetate (PMA), inhibited the growth of subcutaneous tumors derived from PAM212 (mouse SCC) and B16 (mouse melanoma). Ing3A and PMA both induced acute neutrophilic inflammation on mouse skin, but only Ing3A caused subcutaneous hemorrhage and vascular damage. Both Ing3A and PMA activated extracellular signal-regulated kinase 1/2 (ERK1/2) in epidermis, but Ing3A also activated ERK1/2 in skin dermal fibroblasts and endothelial cells. Pretreatment with topical cyclosporin A (CsA), verapamil, or XR9576, modulators of P-glycoprotein (P-gp), prevented Ing3A-induced hemorrhage but not neutrophil infiltration. CsA also impaired the anticancer activity of Ing3A, whereas the anti-inflammatory dexamethasone did not. Ing3A, but not PMA, blocked photoaffinity labeling of human P-gp with (125)Iiodoaryazidoprazosin and inhibited P-gp-mediated drug resistance to HCT-15 cells. The intracellular levels of Ing3A were significantly lower in P-gp-expressing cells, and treatment with XR9576 increased the levels to those of cells that do not express P-gp, showing that Ing3A binds to and is transported by P-gp. Taken together, our results suggest that P-gp-mediated absorptive transport, dermal penetration, and vascular damage contribute to the anticancer activity of Ing3A in vivo.
Nuclear translocation of cytosolic CLIC4 is an essential feature of its proapoptotic and prodifferentiation functions. Here we demonstrate that CLIC4 is induced concurrently with inducible nitric ...oxide synthase (iNOS) and S-nitrosylated in proinflammatory peritoneal macrophages. Chemical inhibition or genetic ablation of iNOS inhibits S-nitrosylation and nuclear translocation of CLIC4. In macrophages, iNOS-induced nuclear CLIC4 coincides with the pro- to anti-inflammatory transition of the cells because IL-1β and CXCL1 mRNA remain elevated in CLIC4 and iNOS knockout macrophages at late time points, whereas TNFα mRNA is elevated only in the iNOS knockout macrophages. Active IL-1β remains elevated in CLIC4 knockout macrophages and in macrophages in which CLIC4 nuclear translocation is prevented by the NOS inhibitor L-NAME. Moreover, overexpression of nuclear-targeted CLIC4 down-regulates IL-1β in stimulated macrophages. In mice, genetically null for CLIC4, the number of phagocytosing macrophages stimulated by LPS is reduced. Thus, iNOS-induced nuclear CLIC4 is an essential part of the macrophage deactivation program.
Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway ...protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCδ and PKCϵ from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCδ and PKCϵ were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCα enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCδ and PKCϵ provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.
RasGRP3 mediates the activation of the Ras signaling pathway that is present in many human cancers. Here, we explored the involvement of RasGRP3 in the formation and maintenance of the prostate ...cancer phenotype. RasGRP3 expression was elevated in multiple human prostate tumor tissue samples and in the human androgen-independent prostate cancer cell lines PC-3 and DU 145 compared with the androgen-dependent prostate cancer cell line LNCaP. Downregulation of endogenous RasGRP3 in PC-3 and DU 145 cells reduced Ras-GTP formation, inhibited cell proliferation, impeded cell migration, and induced apoptosis. Anchorage-independent growth of the PC-3 cells and tumor formation in mouse xenografts of both cell lines were likewise inhibited. Inhibition of RasGRP3 expression reduced AKT and extracellular signal-regulated kinase 1/2 phosphorylation and sensitized the cells to killing by carboplatin. Conversely, exogenous RasGRP3 elevated Ras-GTP, stimulated proliferation, and provided resistance to phorbol 12-myristate 13-acetate-induced apoptosis in LNCaP cells. RasGRP3-overexpressing LNCaP cells displayed a markedly enhanced rate of xenograft tumor formation in both male and female mice compared with the parental line. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses, caused the cells to resume the LNCaP morphology, and suppressed growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. We conclude that RasGRP3 contributes to the malignant phenotype of the prostate cancer cells and may constitute a novel therapeutic target for human prostate cancer.
Keratinocyte differentiation requires integrating signaling among intracellular ionic changes, kinase cascades, sequential gene expression, cell cycle arrest, and programmed cell death. We now show ...that Cl⁻ intracellular channel 4 (CLIC4) expression is increased in both mouse and human keratinocytes undergoing differentiation induced by Ca²⁺, serum and the protein kinase C (PKC)-activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Elevation of CLIC4 is associated with signaling by PKCδ, and knockdown of CLIC4 protein by antisense or shRNA prevents Ca²⁺-induced keratin 1, keratin 10 and filaggrin expression and cell cycle arrest in differentiating keratinocytes. CLIC4 is cytoplasmic in actively proliferating keratinocytes in vitro, but the cytoplasmic CLIC4 translocates to the nucleus in keratinocytes undergoing growth arrest by differentiation, senescence or transforming growth factor β (TGFβ) treatment. Targeting CLIC4 to the nucleus of keratinocytes via adenoviral transduction increases nuclear Cl⁻ content and enhances expression of differentiation markers in the absence of elevated Ca²⁺. In vivo, CLIC4 is localized to the epidermis in mouse and human skin, where it is predominantly nuclear in quiescent cells. These results suggest that CLIC4 participates in epidermal homeostasis through both alterations in the level of expression and subcellular localization. Nuclear CLIC4, possibly by altering the Cl⁻ and pH of the nucleus, contributes to cell cycle arrest and the specific gene expression program associated with keratinocyte terminal differentiation.
The diagram vividly depicts the reduction of CO2 to CH4 on the active sites of TM@N3-VS2.
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•The novel SACs (TM@N3-VS2) were designed based on the properties of VS2 nanosheets with ...unique electronic structure, and the outstanding performance of nitrogen adjusting the coordination environment of transition metal for CO2RR.•The catalytic performance of TM@N3-VS2 and TM@VS2 for CO2RR was systematically compared and investigated.•TM@N3-VS2 possess excellent performance for reduction of CO2 to CH4, especially for Cr@N3-VS2 with the lowest limiting potential of 0.2 eV.•The reason why N-doping leads to the excellent electrocatalytic CO2 reduction activity was explained.
The electrocatalytic reduction of CO2 using single-atom catalysts (SACs) has significant potential to make a substantial impact on energy regeneration and greenhouse effect control in the future. The novel SACs were designed based on the properties of vanadium disulfide (VS2) nanosheets with large surface area and unique electronic structure, and the outstanding catalytic performance of transition metal atoms bonding with nitrogen atoms for CO2 reduction reaction (CO2RR). In this work, the electrocatalytic performance of N-doped vanadium disulfide nanosheets loading a series of transition metal single atoms for CO2RR was systemically evaluated by density functional theory (DFT). The results showed that TM@N3-VS2 (TM = Ti, V, Cr, Mn, Zr, Nb, Mo, Ru, Rh) possess excellent performance for reduction of CO2 to CH4, with low limiting potential of 0.36 eV, 0.28 eV, 0.2 eV, 0.34 eV, 0.54 eV, 0.33 eV, 0.55 eV, 0.38 eV, and 0.25 eV, respectively. The calculation results indicated that the nitrogen atoms could adjust the coordination environment of transition metal atoms and modulate the ability of transition metal atoms and VS2 nanosheets accepting and donating electrons, which makes CO2 adsorb on TM@N3-VS2 with moderate adsorption energy, and significantly reduces the reaction energy barrier.
Polo-like kinase 1 (Plk1) plays a pivotal role in the regulation of cellular proliferation. Plk1 is overexpressed in almost equal to80% of human tumors of diverse origins, and overexpression of Plk1 ...promotes neoplastic transformation of human cells. A growing body of evidence suggests that deregulation of Plk1 closely correlates with prognosis of various cancers in humans. Thus, accurate assessment of Plk1 deregulation would provide clear clinical advantages. However, because of the limited amount of cancer tissues available, quantification of the Plk1 activity has not been feasible. Here, we report the development of a rapid, highly sensitive, and specific ELISA-based Plk1 assay that can quantify the level of Plk1 activity with a small amount (2-20 μg) of total cellular proteins. Unlike the conventional immunocomplex kinase assay, this assay directly utilizes total cellular lysates and does not require a Plk1 enrichment step such as immunoprecipitation or affinity purification. Using this assay, we demonstrated that Plk1 activity is elevated in tumors but not in the surrounding normal tissues and that the level of Plk1 activity significantly diminishes after an antiproliferative chemotherapy. The method described here may provide an innovative tool for assessing the predisposition for cancer development, monitoring early tumor response after therapy, and estimating the prognosis of patients with cancers from multiple organ sites.
Abstract
In this paper, polyacrylate (PA) was modified by waterborne polyurethane (WPU) to synthesize waterborne polyurethane-acrylate coating material. The structural changes of each component ...before and after modification, emulsion performance, film performance and printing performance were studied. The effects of raw material ratio and process conditions on application performance of printing emulsion were explored. The results showed that under the conditions of drying temperature and time of 150 °C and 3 min, the optimal formula was resin coating (WPU : PA=4 : 6), mixed solvent, deionized water and pigment content of 25 %, 15 %, 45 % and 15 %, respectively. The obtained emulsion was stable, the water absorption and solvent resistance of the film were good, and the rubbing fastness of printing could reach 4–5 grade. The structural analysis showed that the polyacrylate emulsion was synthesized, and the modified emulsion had the composite structure of WPU/PA.
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