27 research outputs found
Visible Light-Controlled Nitric Oxide Release from Hindered Nitrobenzene Derivatives for Specific Modulation of Mitochondrial Dynamics
Nitric oxide (NO) is a physiological
signaling molecule, whose
biological production is precisely regulated at the subcellular level.
Here, we describe the design, synthesis, and evaluation of novel mitochondria-targeted
NO releasers, Rol-DNB-mor and Rol-DNB-pyr, that are photocontrollable
not only in the UV wavelength range but also in the biologically favorable
visible wavelength range (530–590 nm). These caged NO compounds
consist of a hindered nitrobenzene as the NO-releasing moiety and
a rhodamine chromophore. Their NO-release properties were characterized
by an electron spin resonance (ESR) spin trapping method and fluorometric
analysis using NO probes, and their mitochondrial localization in
live cells was confirmed by costaining. Furthermore, we demonstrated
visible light control of mitochondrial fragmentation <i>via</i> activation of dynamin-related protein 1 (Drp1) by means of precisely
controlled NO delivery into mitochondria of cultured HEK293 cells,
utilizing Rol-DNB-pyr
A Reductant-Resistant and Metal-Free Fluorescent Probe for Nitroxyl Applicable to Living Cells
Nitroxyl
(HNO) is a one-electron reduced and protonated derivative
of nitric oxide (NO) and has characteristic biological and pharmacological
effects distinct from those of NO. However, studies of its biosynthesis
and activities are restricted by the lack of versatile HNO detection
methods applicable to living cells. Here, we report the first metal-free
and reductant-resistant HNO imaging probe available for use in living
cells, P-Rhod. It consists of a rhodol derivative moiety as the fluorophore,
linked via an ester moiety to a diphenylphosphinobenzoyl group, which
forms an aza-ylide upon reaction with HNO. Intramolecular attack of
the aza-ylide on the ester carbonyl group releases a fluorescent rhodol
derivative. P-Rhod showed high selectivity for HNO in the presence
of various biologically relevant reductants, such as glutathione and
ascorbate, in comparison with previous HNO probes. We show that P-Rhod
can detect not only HNO enzymatically generated in the horseradish
peroxidase-hydroxylamine system <i>in vitro</i> but also
intracellular HNO release from Angeli’s salt in living cells.
These results suggest that P-Rhod is suitable for detection of HNO
in living cells
Photocontrollable Peroxynitrite Generator Based on <i>N</i>-Methyl-<i>N</i>-nitrosoaminophenol for Cellular Application
We designed and synthesized a photocontrollable peroxynitrite
(ONOO<sup>–</sup>) generator, P-NAP, which has <i>N</i>-methyl-<i>N</i>-nitrosoaminophenol structure with four
methyl groups introduced
onto the benzene ring to block reaction of the photodecomposition
product with ONOO<sup>–</sup> and to lower the semiquinoneimine’s
redox potential. The semiquinoneimine intermediate generated by photoinduced
release of nitric oxide (NO) reduces dissolved molecular oxygen to
generate superoxide radical anion (O<sub>2</sub><sup>•–</sup>), which reacts with NO to afford ONOO<sup>–</sup> under diffusion
control (<i>k</i> = 6.7 × 10<sup>9</sup> M<sup>–1</sup> s<sup>–1</sup>). NO release from P-NAP under UV-A (330–380
nm) irradiation was confirmed by ESR spin trapping. Tyrosine nitration,
characteristic of ONOO<sup>–</sup>, was demonstrated by HPLC
analysis of a photoirradiated aqueous solution of P-NAP and <i>N</i>-acetyl-l-tyrosine ethyl ester. ONOO<sup>–</sup> formation was confirmed with a ONOO<sup>–</sup>-specific
fluorogenic probe, HKGreen-3, and compared with that from 3-(4-morpholinyl)Âsydnonimine
hydrochloride (SIN-1), which is the most widely used ONOO<sup>–</sup> generator at present. The photoreaction of P-NAP was influenced
by superoxide dismutase, indicating that generation of O<sub>2</sub><sup>•–</sup> occurs before ONOO<sup>–</sup> formation. The quantum yield for formation of duroquinone, the main
P-NAP photodecomposition product, was measured as 0.86 ± 0.07
at 334 nm with a potassium ferrioxalate actinometer. Generation of
ONOO<sup>–</sup> from P-NAP in HCT-116 cells upon photoirradiation
was successfully imaged with HKGreen-3A. This is the first example
of a photocontrollable ONOO<sup>–</sup> donor applicable to
cultured cells
Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets
<div><p>Medulloblastoma is a fatal brain tumor in children, primarily due to the presence of treatment-resistant medulloblastoma stem cells. The energy metabolic pathway is a potential target of cancer therapy because it is often different between cancer cells and normal cells. However, the metabolic properties of medulloblastoma stem cells, and whether specific metabolic pathways are essential for sustaining their stem cell-like phenotype and radioresistance, remain unclear. We have established radioresistant medulloblastoma stem-like clones (rMSLCs) by irradiation of the human medulloblastoma cell line ONS-76. Here, we assessed reactive oxygen species (ROS) production, mitochondria function, oxygen consumption rate (OCR), energy state, and metabolites of glycolysis and tricarboxylic acid cycle in rMSLCs and parental cells. rMSLCs showed higher lactate production and lower oxygen consumption rate than parental cells. Additionally, rMSLCs had low mitochondria mass, low endogenous ROS production, and existed in a low-energy state. Treatment with the metabolic modifier dichloroacetate (DCA) resulted in mitochondria dysfunction, glycolysis inhibition, elongated mitochondria morphology, and increased ROS production. DCA also increased radiosensitivity by suppression of the DNA repair capacity through nuclear oxidization and accelerated the generation of acetyl CoA to compensate for the lack of ATP. Moreover, treatment with DCA decreased cancer stem cell-like characters (e.g., CD133 positivity and sphere-forming ability) in rMSLCs. Together, our findings provide insights into the specific metabolism of rMSLCs and illuminate potential metabolic targets that might be exploited for therapeutic benefit in medulloblastoma.</p></div
Diminution of mitochondrial respiration in ONS-F8 and -B11.
<p>(A) Mitochondria mass, (B) mitochondria membrane potential, (C) OCR (using MitoXpress) in ONS-76, -F8 and -B11 cells. All quantitative data are means ± S.D. *P<0.05, Welch’s t-test.</p
HDAC-Inhibitory Activity of vorinostat (3), compound 1, T247, and T326 <sup>a</sup>.
a<p>Values are means of at least three experiments.</p
ONS-76, -F8 and DCA-treated cells exhibit different metabolic profiles.
<p>Schematic diagram of the metabolic pathway. The red and blue arrows represent increased and decreased metabolite concentrations in ONS-F8 compared with ONS-76 (A), and in DCA-treated cells compared with non-DCA-treated cells (B).</p
Relative glycolysis and TCA cycle related metabolites in ONS-76 and -F8 cells with and without DCA.
<p>Relative glycolysis and TCA cycle related metabolites in ONS-76 and -F8 cells with and without DCA.</p
Design of triazole-containing HDAC inhibitor candidates.
<p>Design of triazole-containing HDAC inhibitor candidates.</p
Total HDACs activity in the presence of 48 hydroxamates (1 µM).
<p>Total HDACs activity in the presence of 48 hydroxamates (1 µM).</p