Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the... more Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the estrogen receptor (ER) subtypes, ERα and ERβ, and subtype-specific agonist/antagonist properties were determined. Cyclopentane-and cyclohexane-fused pyrazoles with p-...
Recent reports have indicated that in cells ectopically expressing only ERα or the full-length ho... more Recent reports have indicated that in cells ectopically expressing only ERα or the full-length hormone-binding isoform of ERβ (ERβ1), the receptors interact with chromatin with different efficacies and that antibodies capable of probing such interactions by chromatin immunoprecipitation (ChIP) are scarce. We therefore produced nine subtype and isoform-specific antibodies to ERα or ERβ and validated their performance in receptor probing in cell lines and tissue biopsies by various immunochemical methods, including ChIP. We also produced clones of HEK-293 cells stably transfected with an estrogen response element (ERE)-dependent luciferase reporter and ERα or ERβ1, in order to comparatively study their interaction with reporter ERE. We show that ERα was located in the nucleus and ERβ1 in the cytoplasm as well as the nucleus of the stably transfected cells, while both receptors were found predominantly in the nucleus in transiently transfected cells and in all estrogen target tissues examined using the same antibodies. The cells displayed wild-type transcriptional activity and canonical regulation of ERE-dependent luciferase expression by estrogen agonists and antagonists. However, unlike ERα, ERβ1 recruitment to the reporter ERE could be probed only by sequential ChIP with antibodies to receptor N- and C-terminus. These data suggest that in HEK-293 cells stably expressing ERα or ERβ1, ER subtype-specific constraints apply to ERβ1 nuclear entry; and that in cells displaying cytoplasmic as well as nuclear localization of ERβ1, sequential ChIP with different antibodies to the receptor is the method of choice for probing its interaction with chromatin.
Recent reports have indicated that in cells ectopically expressing only ERα or the full-length ho... more Recent reports have indicated that in cells ectopically expressing only ERα or the full-length hormone-binding isoform of ERβ (ERβ1), the receptors interact with chromatin with different efficacies and that antibodies capable of probing such interactions by chromatin immunoprecipitation (ChIP) are scarce. We therefore produced nine subtype and isoform-specific antibodies to ERα or ERβ and validated their performance in receptor probing in cell lines and tissue biopsies by various immunochemical methods, including ChIP. We also produced clones of HEK-293 cells stably transfected with an estrogen response element (ERE)-dependent luciferase reporter and ERα or ERβ1, in order to comparatively study their interaction with reporter ERE. We show that ERα was located in the nucleus and ERβ1 in the cytoplasm as well as the nucleus of the stably transfected cells, while both receptors were found predominantly in the nucleus in transiently transfected cells and in all estrogen target tissues examined using the same antibodies. The cells displayed wild-type transcriptional activity and canonical regulation of ERE-dependent luciferase expression by estrogen agonists and antagonists. However, unlike ERα, ERβ1 recruitment to the reporter ERE could be probed only by sequential ChIP with antibodies to receptor N- and C-terminus. These data suggest that in HEK-293 cells stably expressing ERα or ERβ1, ER subtype-specific constraints apply to ERβ1 nuclear entry; and that in cells displaying cytoplasmic as well as nuclear localization of ERβ1, sequential ChIP with different antibodies to the receptor is the method of choice for probing its interaction with chromatin.
Phytochemical investigation of the dichloromethane extract of the leaves of Peltophorum pterocarp... more Phytochemical investigation of the dichloromethane extract of the leaves of Peltophorum pterocarpum, a tropical ornamental tree, led to the isolation of twelve compounds (1-12). One new derivative of peltogynoid ophioglonin (1) and a new 2-phenoxychromone (2) with its 3'-O-β-D-glucoside derivative (3) are described here for the first time. In addition, nine flavonoid derivatives, including peltogynoid ophioglonin (4), were isolated for the first time from this plant. The structures were determined by spectroscopic and chemical methods. Evaluation of the estrogenic activities of 1, 2, and 4 using different model cell systems revealed that 4 was estrogenic and that 2 was largely inactive. Interestingly, 1 was unable to stimulate the proliferation of breast and endometrial cancer cells but exhibited substantial estrogen receptor α-mediated activation of gene expression. This observation indicates that 1 can be further evaluated for its cancer chemopreventive potential.
The Journal of steroid biochemistry and molecular biology, 2009
Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the... more Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the estrogen receptor (ER) subtypes, ERalpha and ERbeta, and subtype-specific agonist/antagonist properties were determined. Cyclopentane- and cyclohexane-fused pyrazoles with p-hydroxyphenyl rings at positions 1 and 3 displayed modest ERbeta-binding selectivity and variable agonism through ERalpha, while behaving as full estrogen antagonists through ERbeta in estrogen-responsive element (ERE)-dependent gene expression assays. By contrast, the 2,3-diphenolic derivatives were non-selective and considerably less effective ERbeta antagonists compared to 1,3-diphenolic ones. The cyclohexane-fused 1,3-diphenolic pyrazole 8, in particular, behaved as full ERalpha agonist/ERbeta antagonist in these assays. Molecular modelling revealed the structural determinants possibly accounting for the differential regulation of transcription through the two ERs exhibited by 8. The data also shows that the ER ...
Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the... more Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the estrogen receptor (ER) subtypes, ERα and ERβ, and subtype-specific agonist/antagonist properties were determined. Cyclopentane-and cyclohexane-fused pyrazoles with p-...
Recent reports have indicated that in cells ectopically expressing only ERα or the full-length ho... more Recent reports have indicated that in cells ectopically expressing only ERα or the full-length hormone-binding isoform of ERβ (ERβ1), the receptors interact with chromatin with different efficacies and that antibodies capable of probing such interactions by chromatin immunoprecipitation (ChIP) are scarce. We therefore produced nine subtype and isoform-specific antibodies to ERα or ERβ and validated their performance in receptor probing in cell lines and tissue biopsies by various immunochemical methods, including ChIP. We also produced clones of HEK-293 cells stably transfected with an estrogen response element (ERE)-dependent luciferase reporter and ERα or ERβ1, in order to comparatively study their interaction with reporter ERE. We show that ERα was located in the nucleus and ERβ1 in the cytoplasm as well as the nucleus of the stably transfected cells, while both receptors were found predominantly in the nucleus in transiently transfected cells and in all estrogen target tissues examined using the same antibodies. The cells displayed wild-type transcriptional activity and canonical regulation of ERE-dependent luciferase expression by estrogen agonists and antagonists. However, unlike ERα, ERβ1 recruitment to the reporter ERE could be probed only by sequential ChIP with antibodies to receptor N- and C-terminus. These data suggest that in HEK-293 cells stably expressing ERα or ERβ1, ER subtype-specific constraints apply to ERβ1 nuclear entry; and that in cells displaying cytoplasmic as well as nuclear localization of ERβ1, sequential ChIP with different antibodies to the receptor is the method of choice for probing its interaction with chromatin.
Recent reports have indicated that in cells ectopically expressing only ERα or the full-length ho... more Recent reports have indicated that in cells ectopically expressing only ERα or the full-length hormone-binding isoform of ERβ (ERβ1), the receptors interact with chromatin with different efficacies and that antibodies capable of probing such interactions by chromatin immunoprecipitation (ChIP) are scarce. We therefore produced nine subtype and isoform-specific antibodies to ERα or ERβ and validated their performance in receptor probing in cell lines and tissue biopsies by various immunochemical methods, including ChIP. We also produced clones of HEK-293 cells stably transfected with an estrogen response element (ERE)-dependent luciferase reporter and ERα or ERβ1, in order to comparatively study their interaction with reporter ERE. We show that ERα was located in the nucleus and ERβ1 in the cytoplasm as well as the nucleus of the stably transfected cells, while both receptors were found predominantly in the nucleus in transiently transfected cells and in all estrogen target tissues examined using the same antibodies. The cells displayed wild-type transcriptional activity and canonical regulation of ERE-dependent luciferase expression by estrogen agonists and antagonists. However, unlike ERα, ERβ1 recruitment to the reporter ERE could be probed only by sequential ChIP with antibodies to receptor N- and C-terminus. These data suggest that in HEK-293 cells stably expressing ERα or ERβ1, ER subtype-specific constraints apply to ERβ1 nuclear entry; and that in cells displaying cytoplasmic as well as nuclear localization of ERβ1, sequential ChIP with different antibodies to the receptor is the method of choice for probing its interaction with chromatin.
Phytochemical investigation of the dichloromethane extract of the leaves of Peltophorum pterocarp... more Phytochemical investigation of the dichloromethane extract of the leaves of Peltophorum pterocarpum, a tropical ornamental tree, led to the isolation of twelve compounds (1-12). One new derivative of peltogynoid ophioglonin (1) and a new 2-phenoxychromone (2) with its 3'-O-β-D-glucoside derivative (3) are described here for the first time. In addition, nine flavonoid derivatives, including peltogynoid ophioglonin (4), were isolated for the first time from this plant. The structures were determined by spectroscopic and chemical methods. Evaluation of the estrogenic activities of 1, 2, and 4 using different model cell systems revealed that 4 was estrogenic and that 2 was largely inactive. Interestingly, 1 was unable to stimulate the proliferation of breast and endometrial cancer cells but exhibited substantial estrogen receptor α-mediated activation of gene expression. This observation indicates that 1 can be further evaluated for its cancer chemopreventive potential.
The Journal of steroid biochemistry and molecular biology, 2009
Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the... more Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the estrogen receptor (ER) subtypes, ERalpha and ERbeta, and subtype-specific agonist/antagonist properties were determined. Cyclopentane- and cyclohexane-fused pyrazoles with p-hydroxyphenyl rings at positions 1 and 3 displayed modest ERbeta-binding selectivity and variable agonism through ERalpha, while behaving as full estrogen antagonists through ERbeta in estrogen-responsive element (ERE)-dependent gene expression assays. By contrast, the 2,3-diphenolic derivatives were non-selective and considerably less effective ERbeta antagonists compared to 1,3-diphenolic ones. The cyclohexane-fused 1,3-diphenolic pyrazole 8, in particular, behaved as full ERalpha agonist/ERbeta antagonist in these assays. Molecular modelling revealed the structural determinants possibly accounting for the differential regulation of transcription through the two ERs exhibited by 8. The data also shows that the ER ...
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