Valery V. Khramtsov

Macrophage-Colony-Stimulating Factor-Induced Activation of Extracellular-Regulated Kinase Involves Phosphatidylinositol 3-Kinase and Reactive Oxygen Species in Human Monocytes

We previously reported that activation of the phosphatidylinositol (PI) 3-kinase pathway was important in M-CSF-induced monocyte survival. Because M-CSF also induces activation of the mitogen-activated protein (MAP) kinase extracellular-regulated kinase (Erk), we focused on dissecting the mechanism used by M-CSF to induce Erk activation in human monocytes. We found that, in addition to the MAP/Erk kinase inhibitor PD098059, the PI 3-kinase inhibitors LY294002 and wortmannin both suppressed Erk activation in M-CSF-treated monocytes, suggesting that 3-phosphorylated products of PI 3-kinase played a role in Erk activation. Investigating the biochemical pathways regulated by PI 3-kinase to activate Erk, we found that, in response to M-CSF, normal human monocytes induced reactive oxygen species (ROS), which were suppressed by the PI 3-kinase inhibitor wortmannin but not by the solvent control DMSO or the MAP/Erk kinase inhibitor PD098059. We next found that, in the absence of M-CSF, ROS could induce Erk activation in human monocytes. Exogenous H2O2 induced Erk activation in human monocytes, which was suppressed by exogenous catalase. To determine whether ROS induced by M-CSF played a role in Erk activation, we found that N-acetylcysteine and diphenyleneiodonium both suppressed Erk activation in M-CSF-treated monocytes. Erk activation by M-CSF also seemed to play a role in cellular survival in monocytes. These data suggest that, in M-CSF-stimulated human monocytes, PI 3-kinase products and ROS production play a role in Erk activation and monocyte survival.

Synthesis of the tetraethyl substituted pH-sensitive nitroxides of imidazole series with enhanced stability towards reduction

The synthesis of 2,2,5,5-tetraethylimidazole nitroxides from 3-ethylpent-2-ene is described. The newly synthesized nitroxides, namely 4-methyl-2,2,5,5-tetraethyl-2,5-dihydro-1H-imidazol-1-yloxy (1), 3,4-dimethyl-2,2,5,5-tetraethylperhydroimidazol-1-yloxy (2) and 2,2,5,5-tetraethyl-4-pyrrolidin-1-yl-2,5-dihydro-1H-imidazol-1-oxyl (3), were found to be pH sensitive spin probes, with pK values of 1.2, 4.95 and 7.4, respectively. The most important finding was the fact that these new nitroxides were 20-30 times more stable in the presence of ascorbate and had significantly longer halflifes in rat blood as compared to 2,2,5,5-tetramethyl analogs. The latter observation provides a unique advantage for the application of tetraethyl substituted imidazole nitroxides as functional EPR probes.

In vivo monitoring of pH, redox status, and glutathione using L-band EPR for assessment of therapeutic effectiveness in solid tumors

Approach for in vivo real‐time assessment of tumor tissue extracellular pH (pHe), redox, and intracellular glutathione based on L‐band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony‐stimulating factor. It was observed that tumor pHe is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony‐stimulating factor decreased the value of pHe by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony‐stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pHe mapping was performed using recently proposed variable frequency proton–electron double‐resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pHe and a difference of about 0.4 pH units between average pHe values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pHe, extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors. Magn Reson Med, 2011.

Synthesis and Characterization of Amino Derivatives of Persistent Trityl Radicals as Dual Function pH and Oxygen Paramagnetic Probes

Triarylmethyl radicals, TAMs, are useful soluble paramagnetic probes for EPR spectroscopic and imaging applications because of their extraordinary stability in living tissues, narrow line width, high analytical resolution at micromolar concentrations and enhanced sensitivity to molecular oxygen. Recently we proposed the concept of dual function pH and oxygen TAM probes based on the incorporation of ionizable groups into the TAM structure (J. Am. Chem. Soc. 2007, 129 (23), 7240-7241). In this paper we report the synthesis of TAM derivatives containing amino groups. The synthesized TAMs combine stability with oxygen and pH sensitivity, in the range of pH from 6.8 to 9.0. To decrease the number of spectral components and improve probe solubility at physiological pH, asymmetric TAM derivatives containing both carboxyl and amino functions were synthesized. The presence of nitrogen and hydrogen atoms in direct proximity to protonatable amino groups resulted in strong pH-induced changes to the corresponding hyperfine splittings, Delta hfs approximately (300-1000) mG, comparable to the values of hfs themselves. Large pH-dependent line shifts of individual spectral components, with narrow linewidths of (160-280) mG, allow for easy discrimination between the pH effect and the observed oxygen-dependent line broadening of about (6 +/- 0.5) mG per % oxygen. The synthesized TAM derivatives represent the first dual function pH and oxygen paramagnetic probes with reasonably valuable properties for biomedical research.

NMR spin trapping: detection of free radical reactions using a phosphorus-containing nitrone spin trap.

This study employs 31P‐nuclear magnetic resonance (NMR) to probe for changes in molecular structure arising from reactions between free radicals and a phosphorus‐containing nitrone spin trap, 5‐diethoxyphosphoryl‐5‐methyl‐1‐pyrroline‐N‐oxide (DEPMPO). A number of biologically relevant free radical reactions were detected: a) reactions of DEPMPO with ⋅OH resulted in a new 31P‐NMR resonance at 27.05 ppm (shifted from the parent compound at 23.67 ppm); evidence suggests that this species is a diamagnetic hydroxy‐pyrrolidone reduction product; b) 31P‐NMR spectra of DEPMPO/⋅CH3 reactions resulted in peaks at 24.54, 30.83, and 32.31 ppm, while DEPMPO/⋅CH2OH produced peaks at 24.05, 30.80 and 32.52 ppm; in the presence of excess ascorbate, only resonances between 30 and 32 ppm were evident, which we have tentatively assigned to the hydroxylamine isomers of their respective adducts; and c) reaction of DEPMPO with O2⋅−, produced by xanthine/xanthine oxidase or stimulated neutrophils, resulted in a single line, indistinguishable from DEPMPO/⋅OH reaction products. We conclude that NMR spin trapping is a useful approach for detecting free radical reaction pathways. It may have future applications for human free radical biology and imaging. Magn Reson Med 42:228–234, 1999.

Nitroxides with two pK values—useful spin probes for pH monitoring within a broad range

A series of 4-dialkylamino-2,5-dihydroimidazole nitroxides with pyridine-4-yl, 4-dimethylaminophenyl or 4-hydroxyphenyl groups in position 2 of the imidazole ring were prepared using the reaction of RMgBr with corresponding 5-dialkylamino-4,4-dimethyl-4H-imidazole 3-oxides. The EPR spectra of the nitroxides were shown to be pH-sensitive due to consecutive protonation of the amidino moiety and the basic group(s) at position 2 of the imidazole ring. The 5,5-dimethyl-4-(dimethylamino)-2-ethyl-2-pyridine-4-yl-2,5-dihydro-1H-imidazol-1-oxyl showed a monotonic increase in the isotropic nitrogen hyperfine (hfi) coupling constant alpha(N) of 1 .4 G over a pH range from 2 to 6.5. Such a broad range of pH-sensitivity could be useful for many biophysical and biomedical applications, including pH-monitoring in the stomach.

NCX-4040, a nitric oxide-releasing aspirin, sensitizes drug-resistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols

BackgroundOvarian carcinoma is the leading cause of mortality among gynecological cancers in the world. The high mortality rate is associated with lack of early diagnosis and development of drug resistance. The antitumor efficacy and mechanism of NCX-4040, a nitric oxide-releasing aspirin derivative, against ovarian cancer is studied.MethodsNCX-4040, alone or in combination with cisplatin (cis-diamminedichloroplatinum, cDDP), was studied in cisplatin-sensitive (A2780 WT) and cisplatin-resistant (A2780 cDDP) cell lines as well as xenograft tumors grown in nude mice. Electron paramagnetic resonance (EPR) was used for measurements of nitric oxide and redox state. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice was used for mechanistic studies.ResultsCells treated with NCX-4040 (25 μM) showed a significant reduction of cell viability (A2780 WT, 34.9 ± 8.7%; A2780 cDDP, 41.7 ± 7.6%; p < 0.05). Further, NCX-4040 significantly enhanced the sensitivity of A2780 cDDP cells (cisplatin alone, 80.6 ± 11.8% versus NCX-4040+cisplatin, 26.4 ± 7.6%; p < 0.01) and xenograft tumors (cisplatin alone, 74.0 ± 4.4% versus NCX-4040+cisplatin, 56.4 ± 7.8%; p < 0.05), to cisplatin treatment. EPR imaging of tissue redox and thiol measurements showed a 5.5-fold reduction (p < 0.01) of glutathione in NCX-4040-treated A2780 cDDP tumors when compared to untreated controls. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice treated with NCX-4040 and cisplatin revealed significant downregulation of pEGFR (Tyr845 and Tyr992) and pSTAT3 (Tyr705 and Ser727) expression.ConclusionThe results suggested that NCX-4040 could resensitize drug-resistant ovarian cancer cells to cisplatin possibly by depletion of cellular thiols. Thus NCX-4040 appears to be a potential therapeutic agent for the treatment of human ovarian carcinoma and cisplatin-resistant malignancies.

Trityl-based EPR probe with enhanced sensitivity to oxygen

An asymmetric derivative of the triarylmethyl radical, TAM-H, containing one aldehyde and two carboxyl groups, was synthesized. The electron paramagnetic resonance (EPR) spectrum of TAM-H is characterized by a doublet of narrow lines with a linewidth of 105 mG under anoxic conditions and hyperfine interaction constant of 245 mG. The partial overlap of the components of the doublet results in enhanced sensitivity of the spectral amplitudes ratio to oxygen compared with oxygen-induced linewidth broadening of a single line. Application of the TAM-H probe allows for EPR measurements in an extended range of oxygen pressures from atmospheric to 1 mm Hg, whereas the EPR spectrum linewidth of the popular TAM-based oxygen sensor Oxo63 is practically insensitive to oxygen partial pressures below 20 mm Hg. Enhanced sensitivity of the TAM-H probe relative to Oxo63 was demonstrated in the detection of oxygen consumption by Met-1 cancer cells. The TAM-H probe allowed prolonged measurements of oxygen depletion during the hypoxia stage and down to true anoxia (<or=1.5 mm Hg).

In Vitro and In Vivo Measurement of pH and Thiols by EPR-Based Techniques

In vitro and in vivo measurements of pH and thiols provide critical information on physiology and pathophysiology of living organisms, particularly related to oxidative stress. Stable nitroxides of imidazoline and imidazolidine types provide the unique possibility of measuring local values of pH and glutathione content in various biological systems, including in vivo studies. The basis for these applications is the observation of specific chemical reactions of these nitroxides with protons or thiols, followed by significant changes in the electron paramagnetic resonance (EPR) spectra of these probes, measured by low-frequency EPR techniques. The applications of some newly developed pH and SH probes in model systems of pharmacological interest, biological fluids, tissues, and cells as well as in vivo studies in isolated hearts and in the gut of living animals are discussed.

In vivo detection of a pH‐sensitive nitroxide in the rat stomach by low‐field ESR‐based techniques

A study was made of the in vivo detectability of a pH‐sensitive, imidazolidine spin probe, and the efficacy of low‐frequency electron spin resonance (ESR)‐based techniques for pH measurement in vitro and in vivo in rats. The techniques used were longitudinally‐detected ESR (LODESR) and field‐cycled dynamic nuclear polarization (FC‐DNP) for in vitro and in vivo measurements, and radiofrequency (RF)‐ and X‐band ESR for comparisons in vitro. The spin probe was hexamethyl imidazolidine (HMI) with a pK of 4.6. All techniques detected HMI. Detection by FC‐DNP implies coupling between the free radical and solvent water spins. Separations between the three spectral lines of the nitroxide radical, relative to measurement frequency, were consistent with theory. The overall spectrum width from unprotonated HMI (pH > pK) was greater than that from protonated agent (pH < pK). This was observed in vitro and in vivo. Longer‐term studies showed that HMI is detectable and has the same spectral width (i.e., is at the same pH) up to 2 hr after gavage into the stomach, although the magnitude of the signal decreases rapidly during the first hour. These findings demonstrate the suitability of LODESR and FC‐DNP for monitoring HMI and measuring pH in vivo. These techniques would be useful for monitoring disease and drug pharmacology in the living system. Magn Reson Med 49:558–567, 2003.