Electrochemistry and Electrogenerated Chemiluminescence of a Novel Donor−Acceptor FPhSPFN Red Fluorophore

We report here the electrogenerated chemiluminescence (ECL) of a red fluorophore diphenylaminospirobifluorenylfumaronitrile (FPhSPFN). FPhSPFN contains two diphenylamino groups as strong electron donors at the ends linked through a nonplanar bulky spirobifluorene linker to a common fumaronitrile group as a strong electron acceptor. The cyclic voltammetry of this compound shows two one-electron transfer reduction waves and one single overall two-electron oxidation wave consisting of closely spaced waves with a peak separation of 63 mV. Chronoamperometry with an ultramicroelectrode (UME) confirmed that each reduction wave involves a 1e transfer and that the oxidation wave contains an overall 2e transfer. The first reduction wave is Nernstian, Eo1,red = −1.09 V versus SCE, and the second reduction wave, Eo2,red = −1.50 V versus SCE, produces a film on the electrode. By comparison to a digital simulation, the oxidation is assigned to two reversible, closely spaced, one-electron processes for oxidation with Eo1,ox = 1.05 V and Eo2,ox = 1.11 V versus SCE. A large wavelength shift (∼35 nm) was observed in ECL as compared to photoluminescence (PL). The ECL spectrum showed a maximum emission at 708 nm with a red-shifted shoulder at around 750 nm. ECL was studied in mixtures of benzene/acetonitrile at different composition ratios; an increase in the ratio of the ECL intensity of the shoulder with respect to the main peak emission was observed with increasing solvent polarity. ECL generated with BPO as a coreactant also showed the red-shifted shoulder at the same ratio as that with annihilation

Utilization of Multi-Heterodonors in Thermally Activated Delayed Fluorescence Molecules and Their High Performance Bluish-Green Organic Light-Emitting Diodes

We report a series of pentacarbazolyl-benzonitrile derivatives such as 2,4,6-tri­(9H-carbazol-9-yl)-3,5-bis­(3,6-di­(pyridin-3-yl)-9H-carbazol-9-yl)­benzonitrile (mPyBN), 3,5-bis­(3,6-bis­(4-(trifluoromethyl)­phenyl)-9H-carbazol-9-yl)-2,4,6-tri­(9H-carbazol-9-yl)­benzonitrile (pCF3BN), 2,4,6-tri­(9H-carbazol-9-yl)-3-(3,6-di­(pyridin-3-yl)-9H-carbazol-9-yl)-5-(3,6-diphenyl-9H-carbazol-9-yl)­benzonitrile (PyPhBN), 3-(3,6-bis­(4-(trifluoromethyl)­phenyl)-9H-carbazol-9-yl)-2,4,6-tri­(9H-carbazol-9-yl)-5-(3,6-di­(pyridin-3-yl)-9H-carbazol-9-yl)­benzonitrile (PyCF3BN), and 3-(3,6-bis­(4-(trifluoromethyl)­phenyl)-9H-carbazol-9-yl)-2,6-di­(9H-carbazol-9-yl)-5-(3,6-di­(pyridin-3-yl)-9H-carbazol-9-yl)-4-(9H-pyrido­[3,4-b]­indol-9-yl)­benzonitrile (CbPyCF3BN) in which some of the carbazoles are substituted with modified 3,5-diphenyl carbazoles, exhibiting thermally activated delayed fluorescence (TADF) properties. These emitters comprised two, three, and four different types of donors, capable of bluish-green emission of around 480 nm with relatively high photoluminescence quantum yields over 90% in solution. Emitters, namely, PyPhBN, PyCF3BN, and CbPyCF3BN, composed of three and four different types of donors endowed a rather short delayed lifetime (τd) of 4.25, 5.01, and 3.65 μs in their film state, respectively. Bluish-green organic light-emitting diodes based on PyPhBN, PyCF3BN, and CbPyCF3BN exhibit a high external quantum efficiency of 20.6, 19.5, and 19.6%, respectively, with unsurpassed efficiency roll-off behavior. These results indicate that the TADF properties of multidonor type molecules can be manipulated by controlling the types and number of electron donor units

Decrease in PP2A activity increases phosphorylation of p38MAPK, pMAPKAPK2 and Hsp27 in CD133+ cells.

<p>(A upper panel) Immunoblot analysis and (A lower panel) PP2A activity analysis for HT-29 CD133+ and CD133− cells after MACS separation at day 4 of exposure to hypoxia and serum depletion. (B upper panel) Immunoblot analysis and (B lower panel) PP2A activity of CCS cells cultured under serum depletion in the presence of EGF (10 ng/mL) and FGF2 (10 ng/mL) (EGF+FGF2) for indicated time periods. The fold values are relative to the band intensity of the 5 day treatment.</p

Survival of Cancer Stem Cells under Hypoxia and Serum Depletion via Decrease in PP2A Activity and Activation of p38-MAPKAPK2-Hsp27

<div><p>Hypoxia and serum depletion are common features of solid tumors that occur upon antiangiogenesis, irradiation and chemotherapy across a wide variety of malignancies. Here we show that tumor cells expressing CD133, a marker for colorectal cancer initiating or stem cells, are enriched and survive under hypoxia and serum depletion conditions, whereas CD133− cells undergo apoptosis. CD133+ tumor cells increase cancer stem cell and epithelial-mesenchymal transition properties. Moreover, via screening a panel of tyrosine and serine/threonine kinase pathways, we identified Hsp27 is constitutively activated in CD133+ cells rather than CD133− cell under hypoxia and serum depletion conditions. However, there was no difference in Hsp27 activation between CD133+ and CD133− cells under normal growth condition. Hsp27 activation, which was mediated by the p38MAPK-MAPKAPK2-Hsp27 pathway, is required for CD133+ cells to inhibit caspase 9 and 3 cleavage. In addition, inhibition of Hsp27 signaling sensitizes CD133+ cells to hypoxia and serum depletion -induced apoptosis. Moreover, the antiapoptotic pathway is also activated in spheroid culture-enriched CD133+ cancer stem cells from a variety of solid tumor cells including lung, brain and oral cancer, suggesting it is a common pathway activated in cancer stem cells from multiple tumor types. Thus, activation of PP2A or inactivation of the p38MAPK-MAPKAPK2-Hsp27 pathway may develop new strategies for cancer therapy by suppression of their TIC population.</p> </div

CD133+ cells resist hypoxia and serum depletion-induced apoptosis via activation of Hsp27.

<p>(A) TUNEL staining for apoptosis was performed after MACS separation of CD133− and CD133+ cells under hypoxia and serum depletion for 4 days. (B) CD133− and CD133+ cells under hypoxia and serum depletion for 3 days were harvested by MACS separation, reseeded under hypoxia and serum depletion, and APOPercentage assay was performed 16 h later. (C) Quantitative RT-PCR for mRNA levels. (D-F) Cell lysates of CD133+ and CD133− cells after MACS separation at day 4 of exposure to hypoxia and serum depletion were prepared and used for (D, F) immunoblot analysis for protein levels, and (E) MAPK phospho-antibody array for analyzing the levels of indicated signaling pathways. Graphs show each signaling after normalized with control. CD133+ cells decreased in the expression and the activation of caspase 9 and 3 and increased in the expression of phospho-Hsp27. Error bars represent standard deviations. (*p<0.05 and **p<0.01 as determined by the Student’s t test.) Data of HT-29 cells are shown as representative results.</p

The involvement of Hsp27 activation in the anti-apoptosis pathway of CD133+ cells.

<p>Cells were lentivirally transfected with Hsp27 shRNA (shR1 and shR2) or scrambled shRNA, then exposed to hypoxia and serum depletion. CD133+ and CD133− cells were isolated using MACS separation 4 days later. (A) Immunoblot analysis for HT-29 protein levels of CD133+ and CD133− cells after MACS separation. (B) The percentage of CD133+ cells were analyzed by flow cytometry and the increased CD133+ percentage compared to CD133+ percentage in normoxia and growth medium was calculated. (C) Cells were re-exposed to hypoxia and serum depletion for 1 day, followed by TUNEL staining. (D-F) HT-29 cells were exposed to serum depletion and hypoxia in the presence of Quercetin, KRIBB3, or DMSO (vehicle control) for 4 days. (D) CD133+ cell were isolated and the protein levels were analyzed by western blotting. (E) The increased percentage of CD133+ cells and (F) apoptosis of CD133+ cells were analyzed by flow cytometry and TUNEL staining, respectively. Error bars represent standard deviations. (**p<0.01 compared with the scrambled as determined by the Student’s t test.)</p

CD133+ cells form undifferentiated spheres and increase in embryonic gene expression and markers for EMT.

<p>(A)Sphere formation capacity of CD133+ and CD133− cells. CD133+ and CD133− cells, separated by MACS after 4 days cultured under hypoxia and serum depletion, were cultured under serum free conditions in the presence of EGF (10 ng/mL) and FGF2 (10 ng/mL). Sphere formation was calculated at 2 weeks (>25 or <25 indicates the cell number of each sphere.). Error bars represent standard deviation. (*p<0.05 compared with CD133−cells as determined by the Student’s t test.) (B) CD133+ cells after MACS separation at day 4 of exposure to hypoxia and serum depletion, cultured under serum free condition in the presence of EGF (10 ng/mL) and FGF2 (10 ng/mL), formed spheres at 2 weeks. Immunofluorescence shows that these spheres are undifferentiated spheres (Undifferentiated), which are positive for CD133, and negative for nuclear β-catenin, CDX2 and CK20. The spheres were then seeded into matrigel and exposed to serum-containing condition for another 2 weeks. Immunofluorescence shows that these spheres are differentiated (Differentiated), which decrease in CD133 expression, increase in the expression of nuclear β-catenin, CDX2 and CK20. Scale bar, 20 µm (C) Percentages of cells positive for CD133, nuclear β-catenin, CDX2 and CK20 were counted. (D) and (E upper two panels) Quantitative RT-PCR for mRNA levels, (E lower panel) immunoblot analysis. (F) cell migration and invasion assay for CD133+ and CD133− cells after MACS separation at day 4 of exposure to hypoxia and serum depletion. Data of HT-29 cells are shown as representative results.</p

CD133+ cells increase in percentage under hypoxia and serum depletion.

<p>(A) HT-29, (B) CCS and HCW cells were seeded with normal growth medium (FBS) under normoxia (Nor). Cells were exposed to serum depletion (SF), hypoxia (Hyp), hypoxia and serum depletion, or, as a control, under the same condition, and 2, 4 or 6 days later, the cells were harvested for assaying CD133 percentage by flow cytometry. (C left panel) Total cell numbers for each condition and (C right panel) cell number of CD133− and CD133+ of HT-29 cells at indicated period under hypoxia and serum depletion were measured. (D left panel) Total cell number and (D right panel) numbers of CD133+ and CD133− cells of CCS and HCW cells at 4 days under each condition. (E and F) Flow cytometry for surface protein profiles of cells cultured under control, or under hypoxia and serum depletion for 4 days.</p