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Stéphane Bouchonnet - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the ultraviolet–visible photoproducts of thiophanate-methyl using high performance liquid chromatography coupled with high resolution tandem mass spectrometry—Detection in grapes and tomatoes
Journal of Chromatography A, 2016Co-Authors: Houda Chayata, Yannick Lassalle, Edith Nicol, Sophia Maria Manolikakes, Yasmine Souissi, Sophie Bourcier, Corinne Gosmini, Stéphane BouchonnetAbstract:UV-visible irradiation of thiophanate-methyl (TM) led to the formation of nine photoproducts that were characterized by high performance liquid chromatography coupled with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Although carbendazime has been reported in the literature to be the major metabolite and photoproduct of thiophanate-methyl, it was not detected in this study. However, an isomer of carbendazime referred as PP2, which was unambiguously characterized owing to CID experiments, was found in great abundance. Grape berries and cherry tomatoes treated with aqueous solutions of thiophanate-methyl were submitted to irradiation under laboratory conditions. TM and PP2 were detected in both peel and flesh of berries. The ability of TM and PP2 to pass through the fruit skin has been shown to be highly compound and matrix dependent. In vitro bioassays on Vibrio fischeri bacteria showed that the global ecotoxicity of the TM solution increases significantly with the irradiation time. PP2 should likely contribute to this ecotoxicity enhancement since in silico estimations for Daphnia magna provide a LC50 value seven times lower for PP2 than for the parent molecule.
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Characterization of the ultraviolet-visible photoproducts of thiophanate-methyl using high performance liquid chromatography coupled with high resolution tandem mass spectrometry-Detection in grapes and tomatoes.
Journal of chromatography. A, 2016Co-Authors: Houda Chayata, Yannick Lassalle, Edith Nicol, Sophia Maria Manolikakes, Yasmine Souissi, Sophie Bourcier, Corinne Gosmini, Stéphane BouchonnetAbstract:UV-visible irradiation of thiophanate-methyl (TM) led to the formation of nine photoproducts that were characterized by high performance liquid chromatography coupled with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Although carbendazime has been reported in the literature to be the major metabolite and photoproduct of thiophanate-methyl, it was not detected in this study. However, an isomer of carbendazime referred as PP2, which was unambiguously characterized owing to CID experiments, was found in great abundance. Grape berries and cherry tomatoes treated with aqueous solutions of thiophanate-methyl were submitted to irradiation under laboratory conditions. TM and PP2 were detected in both peel and flesh of berries. The ability of TM and PP2 to pass through the fruit skin has been shown to be highly compound and matrix dependent. In vitro bioassays on Vibrio fischeri bacteria showed that the global ecotoxicity of the TM solution increases significantly with the irradiation time. PP2 should likely contribute to this ecotoxicity enhancement since in silico estimations for Daphnia magna provide a LC50 value seven times lower for PP2 than for the parent molecule.
Michael Lee - One of the best experts on this subject based on the ideXlab platform.
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Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2
Journal of cellular physiology, 2017Co-Authors: Sung-hee Hwang, Byeal-i Han, Michael LeeAbstract:Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/Cas9 system. In ATG5 KO cells, RT-PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild-type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto-ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G2 /M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G2 /M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.
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Src Family Kinase Inhibitor PP2 Induces LC3 Conversion in a Manner That is Uncoupled from Autophagy and Increases Apoptosis in Multidrug-Resistant Cells.
Biomolecules & therapeutics, 2012Co-Authors: Yun-ki Kim, Jun-ho Ahn, Michael LeeAbstract:Recently, we reported that defective autophagy may contribute to the inhibition of the growth in response to PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a selective SFK inhibitor, in multidrug-resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr). In this study, we demonstrated that PP2 induces LC3 conversion via a mechanism that is uncoupled from autophagy and increases apoptosis in Ras-NIH 3T3/Mdr cells. PP2 preferentially induced autophagy in Ras-NIH 3T3 cells rather than in Ras-NIH 3T3/Mdr cells as determined by LC3-I to LC3-II conversion and GFP-LC3 fluorescence microscopy. Beclin 1 knockdown experiments showed that, regardless of drug resistance, PP2 induces autophagy via a Beclin 1-dependent mechanism. PP2 induced a conformational change in Beclin 1, resulting in the enhancement of the pro-autophagic activity of Beclin 1, in Ras-NIH 3T3 cells. Further, PI3K inhibition induced by wortmannin caused a significant increase in apoptosis in Ras-NIH 3T3 cells, as demonstrated by flow cytometric analysis of Annexin V staining, implying that autophagy inhibition through PI3K increases apoptosis in response to PP2 in Ras-NIH 3T3 cells. However, despite the fact that wortmannin abrogates PP2-induced GFP-LC3 punctae formation, some LC3 conversion remains in Ras-NIH 3T3/Mdr cells, suggesting that LC3 conversion may occur in an autophagy-independent manner. Taken together, these results suggest that PP2 induces LC3 conversion independent of PI3K, concomitant with the uncoupling of LC3 conversion from autophagy, in multidrug-resistant cells.
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Suppression of autophagy sensitizes multidrug resistant cells towards Src tyrosine kinase specific inhibitor PP2.
Cancer letters, 2011Co-Authors: Jun-ho Ahn, Michael LeeAbstract:Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we employed 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a selective SFK inhibitor, to determine the possible involvement of tyrosine phosphorylation in the modulation of autophagy, for overcoming multidrug resistance. We found that multidrug-resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) were more susceptible to PP2 treatment than were their parental cells (Ras-NIH 3T3). The antiproliferative activity of PP2 appeared to be due to cell-cycle arrest at G1/S without induction of apoptosis. Interestingly, PP2 preferentially induced autophagy in Ras-NIH 3T3 cells but not in Ras-NIH 3T3/Mdr cells, which implies that a high level of autophagy may protect PP2-treated cells from undergoing cell death. PP2-induced autophagy in Ras-NIH 3T3 cells is accompanied by an inhibition of the mTOR signaling pathway. However, we found that in Ras-NIH 3T3/Mdr cells, PP2-induced mTOR inhibition was uncoupled from the induction of autophagy-likely due to the hyperactivation of AMPK by delayed Raf activation. We also found that PP2-induced dissociation of Beclin 1 from Bcl-2 leads to autophagy in Ras-NIH 3T3 cells. Taken together, these results suggest that functional autophagy in response to PP2 may lead to cell survival in Ras-NIH 3T3 cells, while defective autophagy may contribute to inhibition of growth in Ras-NIH 3T3/Mdr cells. Thus, modulators of autophagy may be used beneficially as adjunctive therapeutic agents during the treatment of cancers with SFK inhibitors.
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raf 1 kinase activation is uncoupled from downstream mek erk pathway in cells treated with src tyrosine kinase inhibitor PP2
Biochemical and Biophysical Research Communications, 2006Co-Authors: Michael LeeAbstract:Abstract Recently we demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, markedly enhanced Ras-independent activation of Raf-1 by the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H2O2). We report here that Raf-1 knockdown cells were significantly more sensitive to treatment of PP2 than control cells. This PP2-induced growth inhibition was found to be linked to decreased ERK and p38 activity. Interestingly, the growth of Sprouty knockdown cells appeared to be inhibited at earlier time points of PP2 treatment when compared with control cells. Unexpectedly, siRNA-mediated knockdown of Spry2, which is known to modulate the Ras/Raf/MAPK signal through feedback regulation, resulted in decreased Raf-1 kinase activity. PP2 had limited effect on the ability of PMA/H2O2 to induce significant phosphorylation of MEK/ERK proteins in both Spry2 knockdown and control cells, indicating that PP2-mediated activation of Raf-1 did not potentiate signaling through the downstream MEK/ERK pathway. Taken together our results suggest that Raf-1 signaling may be bypassed in PP2-treated cells by uncoupling from downstream MEK/ERK pathway.
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Raf-1 kinase activation is uncoupled from downstream MEK/ERK pathway in cells treated with Src tyrosine kinase inhibitor PP2.
Biochemical and biophysical research communications, 2006Co-Authors: Michael LeeAbstract:Abstract Recently we demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, markedly enhanced Ras-independent activation of Raf-1 by the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H2O2). We report here that Raf-1 knockdown cells were significantly more sensitive to treatment of PP2 than control cells. This PP2-induced growth inhibition was found to be linked to decreased ERK and p38 activity. Interestingly, the growth of Sprouty knockdown cells appeared to be inhibited at earlier time points of PP2 treatment when compared with control cells. Unexpectedly, siRNA-mediated knockdown of Spry2, which is known to modulate the Ras/Raf/MAPK signal through feedback regulation, resulted in decreased Raf-1 kinase activity. PP2 had limited effect on the ability of PMA/H2O2 to induce significant phosphorylation of MEK/ERK proteins in both Spry2 knockdown and control cells, indicating that PP2-mediated activation of Raf-1 did not potentiate signaling through the downstream MEK/ERK pathway. Taken together our results suggest that Raf-1 signaling may be bypassed in PP2-treated cells by uncoupling from downstream MEK/ERK pathway.
Houda Chayata - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the ultraviolet–visible photoproducts of thiophanate-methyl using high performance liquid chromatography coupled with high resolution tandem mass spectrometry—Detection in grapes and tomatoes
Journal of Chromatography A, 2016Co-Authors: Houda Chayata, Yannick Lassalle, Edith Nicol, Sophia Maria Manolikakes, Yasmine Souissi, Sophie Bourcier, Corinne Gosmini, Stéphane BouchonnetAbstract:UV-visible irradiation of thiophanate-methyl (TM) led to the formation of nine photoproducts that were characterized by high performance liquid chromatography coupled with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Although carbendazime has been reported in the literature to be the major metabolite and photoproduct of thiophanate-methyl, it was not detected in this study. However, an isomer of carbendazime referred as PP2, which was unambiguously characterized owing to CID experiments, was found in great abundance. Grape berries and cherry tomatoes treated with aqueous solutions of thiophanate-methyl were submitted to irradiation under laboratory conditions. TM and PP2 were detected in both peel and flesh of berries. The ability of TM and PP2 to pass through the fruit skin has been shown to be highly compound and matrix dependent. In vitro bioassays on Vibrio fischeri bacteria showed that the global ecotoxicity of the TM solution increases significantly with the irradiation time. PP2 should likely contribute to this ecotoxicity enhancement since in silico estimations for Daphnia magna provide a LC50 value seven times lower for PP2 than for the parent molecule.
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Characterization of the ultraviolet-visible photoproducts of thiophanate-methyl using high performance liquid chromatography coupled with high resolution tandem mass spectrometry-Detection in grapes and tomatoes.
Journal of chromatography. A, 2016Co-Authors: Houda Chayata, Yannick Lassalle, Edith Nicol, Sophia Maria Manolikakes, Yasmine Souissi, Sophie Bourcier, Corinne Gosmini, Stéphane BouchonnetAbstract:UV-visible irradiation of thiophanate-methyl (TM) led to the formation of nine photoproducts that were characterized by high performance liquid chromatography coupled with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Although carbendazime has been reported in the literature to be the major metabolite and photoproduct of thiophanate-methyl, it was not detected in this study. However, an isomer of carbendazime referred as PP2, which was unambiguously characterized owing to CID experiments, was found in great abundance. Grape berries and cherry tomatoes treated with aqueous solutions of thiophanate-methyl were submitted to irradiation under laboratory conditions. TM and PP2 were detected in both peel and flesh of berries. The ability of TM and PP2 to pass through the fruit skin has been shown to be highly compound and matrix dependent. In vitro bioassays on Vibrio fischeri bacteria showed that the global ecotoxicity of the TM solution increases significantly with the irradiation time. PP2 should likely contribute to this ecotoxicity enhancement since in silico estimations for Daphnia magna provide a LC50 value seven times lower for PP2 than for the parent molecule.
Sophie Bourcier - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the ultraviolet–visible photoproducts of thiophanate-methyl using high performance liquid chromatography coupled with high resolution tandem mass spectrometry—Detection in grapes and tomatoes
Journal of Chromatography A, 2016Co-Authors: Houda Chayata, Yannick Lassalle, Edith Nicol, Sophia Maria Manolikakes, Yasmine Souissi, Sophie Bourcier, Corinne Gosmini, Stéphane BouchonnetAbstract:UV-visible irradiation of thiophanate-methyl (TM) led to the formation of nine photoproducts that were characterized by high performance liquid chromatography coupled with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Although carbendazime has been reported in the literature to be the major metabolite and photoproduct of thiophanate-methyl, it was not detected in this study. However, an isomer of carbendazime referred as PP2, which was unambiguously characterized owing to CID experiments, was found in great abundance. Grape berries and cherry tomatoes treated with aqueous solutions of thiophanate-methyl were submitted to irradiation under laboratory conditions. TM and PP2 were detected in both peel and flesh of berries. The ability of TM and PP2 to pass through the fruit skin has been shown to be highly compound and matrix dependent. In vitro bioassays on Vibrio fischeri bacteria showed that the global ecotoxicity of the TM solution increases significantly with the irradiation time. PP2 should likely contribute to this ecotoxicity enhancement since in silico estimations for Daphnia magna provide a LC50 value seven times lower for PP2 than for the parent molecule.
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Characterization of the ultraviolet-visible photoproducts of thiophanate-methyl using high performance liquid chromatography coupled with high resolution tandem mass spectrometry-Detection in grapes and tomatoes.
Journal of chromatography. A, 2016Co-Authors: Houda Chayata, Yannick Lassalle, Edith Nicol, Sophia Maria Manolikakes, Yasmine Souissi, Sophie Bourcier, Corinne Gosmini, Stéphane BouchonnetAbstract:UV-visible irradiation of thiophanate-methyl (TM) led to the formation of nine photoproducts that were characterized by high performance liquid chromatography coupled with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Although carbendazime has been reported in the literature to be the major metabolite and photoproduct of thiophanate-methyl, it was not detected in this study. However, an isomer of carbendazime referred as PP2, which was unambiguously characterized owing to CID experiments, was found in great abundance. Grape berries and cherry tomatoes treated with aqueous solutions of thiophanate-methyl were submitted to irradiation under laboratory conditions. TM and PP2 were detected in both peel and flesh of berries. The ability of TM and PP2 to pass through the fruit skin has been shown to be highly compound and matrix dependent. In vitro bioassays on Vibrio fischeri bacteria showed that the global ecotoxicity of the TM solution increases significantly with the irradiation time. PP2 should likely contribute to this ecotoxicity enhancement since in silico estimations for Daphnia magna provide a LC50 value seven times lower for PP2 than for the parent molecule.
Yasmine Souissi - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the ultraviolet–visible photoproducts of thiophanate-methyl using high performance liquid chromatography coupled with high resolution tandem mass spectrometry—Detection in grapes and tomatoes
Journal of Chromatography A, 2016Co-Authors: Houda Chayata, Yannick Lassalle, Edith Nicol, Sophia Maria Manolikakes, Yasmine Souissi, Sophie Bourcier, Corinne Gosmini, Stéphane BouchonnetAbstract:UV-visible irradiation of thiophanate-methyl (TM) led to the formation of nine photoproducts that were characterized by high performance liquid chromatography coupled with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Although carbendazime has been reported in the literature to be the major metabolite and photoproduct of thiophanate-methyl, it was not detected in this study. However, an isomer of carbendazime referred as PP2, which was unambiguously characterized owing to CID experiments, was found in great abundance. Grape berries and cherry tomatoes treated with aqueous solutions of thiophanate-methyl were submitted to irradiation under laboratory conditions. TM and PP2 were detected in both peel and flesh of berries. The ability of TM and PP2 to pass through the fruit skin has been shown to be highly compound and matrix dependent. In vitro bioassays on Vibrio fischeri bacteria showed that the global ecotoxicity of the TM solution increases significantly with the irradiation time. PP2 should likely contribute to this ecotoxicity enhancement since in silico estimations for Daphnia magna provide a LC50 value seven times lower for PP2 than for the parent molecule.
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Characterization of the ultraviolet-visible photoproducts of thiophanate-methyl using high performance liquid chromatography coupled with high resolution tandem mass spectrometry-Detection in grapes and tomatoes.
Journal of chromatography. A, 2016Co-Authors: Houda Chayata, Yannick Lassalle, Edith Nicol, Sophia Maria Manolikakes, Yasmine Souissi, Sophie Bourcier, Corinne Gosmini, Stéphane BouchonnetAbstract:UV-visible irradiation of thiophanate-methyl (TM) led to the formation of nine photoproducts that were characterized by high performance liquid chromatography coupled with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Although carbendazime has been reported in the literature to be the major metabolite and photoproduct of thiophanate-methyl, it was not detected in this study. However, an isomer of carbendazime referred as PP2, which was unambiguously characterized owing to CID experiments, was found in great abundance. Grape berries and cherry tomatoes treated with aqueous solutions of thiophanate-methyl were submitted to irradiation under laboratory conditions. TM and PP2 were detected in both peel and flesh of berries. The ability of TM and PP2 to pass through the fruit skin has been shown to be highly compound and matrix dependent. In vitro bioassays on Vibrio fischeri bacteria showed that the global ecotoxicity of the TM solution increases significantly with the irradiation time. PP2 should likely contribute to this ecotoxicity enhancement since in silico estimations for Daphnia magna provide a LC50 value seven times lower for PP2 than for the parent molecule.