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American Journal of Physiology. Heart... Jul 2004The objective of this work was to evaluate the effects of testosterone (T) and 17beta-estradiol (E(2)) on coronary microvascular endothelial cells (CMECs) of male and...
The objective of this work was to evaluate the effects of testosterone (T) and 17beta-estradiol (E(2)) on coronary microvascular endothelial cells (CMECs) of male and female rats. To analyze the short-term effects of such sex steroid hormones on intracellular Ca(2+) concentration ([Ca(2+)](i)) kinetics, we used the chelating agent fura-2 acetoxymethyl ester. We also explored the possibility of testosterone aromatization by using selective inhibitors of the aromatase enzyme cytochrome P-450 aromatase (P450(arom)), aminoglutethimide (4 microM), and 4-hydroxyandrostenedione (4 microM). The presence of P450(arom) was investigated by immunocytochemical and immunoblot assays using peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of rat P450(arom) and by in situ hybridization to locate the aromatase mRNA in such cells. The activity of P450(arom) was demonstrated by the stereospecific loss of the tritium atom of [1beta-(3)H]androstenedione. Our results indicate that both T and E(2) induced a rapid increase in [Ca(2+)](i). The fact that the effects of E(2) and T were carried out within milliseconds suggests that they were exerted at the membrane level and not through intracellular receptors. The possibility of involvement of PLC-beta in these effects is suggested because U-73122 (a PLC inhibitor) blocked the effects of both T and E(2). Immunocytochemical assays indicated the expression of androgenic and estrogenic receptors in these cells. The effects of T were blocked by the selective aromatase inhibitors. We also demonstrated membrane association of P450(arom), expression of the ovary-specific mRNA after in situ hybridization, and E(2) formation resulting from a significant activity of P450(arom) in CMECs. There were no gender-based differences.
Topics: Animals; Aromatase; Calcium; Coronary Vessels; Endothelium, Vascular; Estradiol; Estrenes; Estrogen Receptor alpha; Estrogens; Female; Immunoblotting; Immunohistochemistry; In Situ Hybridization; Intracellular Membranes; Kinetics; Male; Microcirculation; Pyrrolidinones; Rats; Rats, Wistar; Receptors, Androgen; Receptors, Estrogen; Testosterone; Time Factors; Tritium; Type C Phospholipases; Water
PubMed: 14726302
DOI: 10.1152/ajpheart.00784.2003 -
Annals of Oncology : Official Journal... Nov 2004Vinorelbine (VRL) has been shown to be active in hormone-refractory prostate cancer (HRPC) in phase II studies, alone or in combination. Its moderate toxicity profile is... (Clinical Trial)
Clinical Trial Comparative Study Randomized Controlled Trial
BACKGROUND
Vinorelbine (VRL) has been shown to be active in hormone-refractory prostate cancer (HRPC) in phase II studies, alone or in combination. Its moderate toxicity profile is well tolerated in elderly patients.
PATIENTS AND METHODS
Patients with metastatic prostate cancer, progressive after primary hormonal therapy, were randomised to receive intravenous VRL 30 mg/m2 on days 1 and 8 every 3 weeks, and hydrocortisone 40 mg/day or hydrocortisone alone until disease progression. Centres could choose to add aminoglutethimide 1000 mg/day to hydrocortisone as second-line hormone therapy (HT) for all their patients. Randomisation was stratified by centre. Further chemotherapy was allowed after progression. The primary end point was progression-free survival (PFS). The final analysis was performed on a total of 414 patients. Reported results were all based on intention-to-treat analyses. All progressions and responses were reviewed by an independent panel.
RESULTS
PFS was significantly prolonged in the VRL plus HT arm compared with the HT alone arm, according to the statistical hypothesis of the protocol (P=0.055 in the two-sided log-rank test with a pre-specified significance level of 10%). The 6-month PFS rates were 33.2% versus 22.8%, and the median durations of PFS were 3.7 versus 2.8 months. In the multivariate Cox analysis, which included age, Karnofsky performance status (PS), haemoglobin, alkaline phosphatase at study entry and number of prior hormonal treatments, the P value was decreased to 0.005. The prostate-specific antigen (PSA) response rate (> or =50% decline sustained for at least 6 weeks) was significantly higher for VRL plus HT compared with HT (30.1% versus 19.2%; P=0.01). Clinical benefit, defined as a decrease in pain intensity or analgesic consumption or an improvement of Karnofsky PS for at least 9 weeks, and at least stable assessment in the other two, was also more frequently observed in patients who received VRL plus HT versus HT alone (30.6% and 19.2%; P=0.008). There was no statistical difference in overall survival. Forty-three per cent of patients in the HT arm received at least one line of further chemotherapy after progression, compared with 28% of patients in the VRL-based arm. Aminoglutethimide did not seem to result in better efficacy for either arm. VRL plus HT was well tolerated, with a median administered relative dose intensity of 90%; grade 4 neutropenia occurred in 6.5% of patients and non-haematological toxicity was rare.
CONCLUSIONS
The combination of VRL and hydrocortisone compared with hydrocortisone alone resulted in improved clinical benefit, PFS and PSA response rate. This therapeutic gain is similar to that previously reported with mitoxantrone in combination with low-dose corticosteroids. There was no gain in survival; however, the combination is well tolerated in this elderly group of patients, who often present cardiac co-morbidities, and therefore offers an active and safe therapeutic option for patients with hormone-refractory prostate cancer.
Topics: Aged; Aged, 80 and over; Anemia; Antineoplastic Combined Chemotherapy Protocols; Hormones; Humans; Infusions, Intravenous; Male; Middle Aged; Neutropenia; Proportional Hazards Models; Prostatic Neoplasms; Survival Analysis; Survival Rate; Treatment Outcome; Vinblastine; Vinorelbine
PubMed: 15520061
DOI: 10.1093/annonc/mdh429 -
The Journal of Biological Chemistry Sep 1984We examined the subcellular localization of ACTH (adrenocorticotropic hormone)-induced changes in adrenal phospholipids using dexamethasone-treated rats. In adrenal...
We examined the subcellular localization of ACTH (adrenocorticotropic hormone)-induced changes in adrenal phospholipids using dexamethasone-treated rats. In adrenal mitochondrial fraction, ACTH significantly enhanced both concentrations and contents of phosphatidylinositol (37%), phosphatidylcholine (22%), and phosphatidylethanolamine (20%). Other mitochondrial phospholipids including cardiolipin did not change upon administration of ACTH. In adrenal plasma membrane, endoplasmic reticulum, and peroxisomes, no increase in phospholipids was observed. The ACTH-induced increases in mitochondrial phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine were specific to adrenal among tissues tested. These changes were observed specifically in cortical cells rather than medulla. Nonsteroidogenic ACTH fragments and related peptides were unable to induce the change in adrenal mitochondrial phospholipids. From the dose-response profile with ACTH, the changes in mitochondrial phospholipids were closely related to ACTH-dependent stimulation of steroidogenesis. Furthermore, in vitro treatment with cyclic AMP enhanced both concentrations and contents of mitochondrial phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine similar to those by the in vivo administration of ACTH. Both in vivo and in vitro experiments revealed that the hormone-induced changes in mitochondrial phospholipids were sensitive to a protein-synthesis inhibitor, cycloheximide. However, aminoglutethimide and cytochalasin B, which strongly inhibited the hormone-induced formation of corticosterone, did not affect the increases in mitochondrial phospholipids. These results suggest that the hormone-induced increases in these phospholipids occur between ACTH-mediated ribosomal protein synthesis and corticosterone formation.
Topics: Adrenal Glands; Adrenocorticotropic Hormone; Animals; Cell Fractionation; Centrifugation, Density Gradient; Cycloheximide; Dexamethasone; Male; Mitochondria; Organ Specificity; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Phospholipids; Rats; Rats, Inbred Strains
PubMed: 6088515
DOI: No ID Found -
Frontiers in Pharmacology 2019Research indicates that neurosteroids are locally synthesized in the central nervous system and play an important modulatory role in nociception. While the...
Inhibition of Cytochrome P450 Side-Chain Cleavage Attenuates the Development of Mechanical Allodynia by Reducing Spinal D-Serine Production in a Murine Model of Neuropathic Pain.
Research indicates that neurosteroids are locally synthesized in the central nervous system and play an important modulatory role in nociception. While the neurosteroidogenic enzyme, cytochrome P450 side-chain cleavage enzyme (P450scc), is the initiating enzyme of steroidogenesis, P450scc has not been examined under the pathophysiological conditions associated with peripheral neuropathy. Thus, we investigated whether chronic constriction injury (CCI) of the sciatic nerve increases the expression of P450scc in the spinal cord and whether this increase modulates serine racemase (Srr) expression and D-serine production contributing to the development of neuropathic pain. CCI increased the immunoreactivity of P450scc in astrocytes of the ipsilateral lumbar spinal cord dorsal horn. Intrathecal administration of the P450scc inhibitor, aminoglutethimide, during the induction phase of neuropathic pain (days 0 to 3 post-surgery) significantly suppressed the CCI-induced development of mechanical allodynia and thermal hyperalgesia, the increased expression of astrocyte Srr in both the total and cytosol levels, and the increases in D-serine immunoreactivity at day 3 post-surgery. By contrast, intrathecal administration of aminoglutethimide during the maintenance phase of pain (days 14 to 17 post-surgery) had no effect on the developed neuropathic pain nor the expression of spinal Srr and D-serine immunoreactivity at day 17 post-surgery. Intrathecal administration of exogenous D-serine during the induction phase of neuropathic pain (days 0 to 3 post-surgery) restored the development of mechanical allodynia, but not the thermal hyperalgesia, that were suppressed by aminoglutethimide administration. Collectively, these results demonstrate that spinal P450scc increases the expression of astrocyte Srr and D-serine production, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury.
PubMed: 31866864
DOI: 10.3389/fphar.2019.01439 -
Annals of Oncology : Official Journal... Nov 2002To evaluate the efficacy and safety of the combination of cisplatin and vinorelbine in metastatic breast cancer. (Clinical Trial)
Clinical Trial Comparative Study
BACKGROUND
To evaluate the efficacy and safety of the combination of cisplatin and vinorelbine in metastatic breast cancer.
PATIENTS AND METHODS
Cisplatin (80 mg/m(2) day 1) and vinorelbine (25 mg/m(2) days 1 and 8) were administrated every 3 weeks to 52 patients (mean age 57 years; range 35-75 years) with metastatic breast cancer. Thirty-two patients were previously untreated for metastatic disease. Treatment was repeated for a maximum of six cycles.
RESULTS
Objective responses were obtained in 27 patients (52.9%; complete response 9.8%). The response rate was similar in pretreated and untreated patients (50% and 54.7%, respectively; P = 0.7). ECOG performance status was good (grade 0 or 1) in 55.7% of patients at baseline assessment and in 90.3% at the end of treatment (P = 0.0001). Median time to progression was 8.5 months (8.5 months in first-line and 8.7 months in second-line patients). Median survival was 16.6 months (21.2 months in first-line and 16.1 months in second-line patients). Grade 3/4 toxicity included neutropenia (44% in first-line, 60% in second-line patients), nausea (17.3%), anemia (17%), asthenia (3.8%) and thrombocytopenia (1.9%). There were no cases of febrile neutropenia or treatment-related deaths. Alopecia did not develop in any of the patients.
CONCLUSIONS
Cisplatin plus vinorelbine is active and tolerable in metastatic breast cancer, in untreated and pretreated patients.
Topics: Adult; Aged; Aminoglutethimide; Antineoplastic Combined Chemotherapy Protocols; Biopsy, Needle; Bone Neoplasms; Breast Neoplasms; Cisplatin; Confidence Intervals; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Resistance, Neoplasm; Female; Follow-Up Studies; Humans; Infusions, Intravenous; Liver Neoplasms; Lung Neoplasms; Megestrol Acetate; Middle Aged; Neoplasm Staging; Probability; Risk Assessment; Soft Tissue Neoplasms; Survival Analysis; Tamoxifen; Vinblastine; Vinorelbine
PubMed: 12419744
DOI: 10.1093/annonc/mdf290 -
European Journal of Biochemistry Jan 2001Aromatase (CYP19) catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, five human aromatase...
Aromatase (CYP19) catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, five human aromatase mutants (E302D, S478A, S478T, H480K, and H480Q) were prepared using a mammalian cell expression system. These mutants were evaluated by enzyme kinetic analysis, inhibitory profile studies, and reaction intermediate measurements. Three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1,4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)] were used in the inhibitory profile studies. Our computer model of aromatase suggests that Glu302 is situated in the conserved I-helix region and located near the C-19 position of the steroid substrate. The model was supported by significant changes in kinetic parameters and a sevenfold increase in the Ki value of MDL 101,003 for the mutant E302D. As S478A was found to have kinetic properties similar to the wild-type enzyme and a much higher activity than S478T, Ser478 is thought to be situated in a rather restricted environment. There was a 10-fold increase in the Ki value of 7alpha-APTADD for S478T over that for the wild-type enzyme, suggesting that Ser478 might be near the C-7 position of the substrate. The reaction intermediate analysis revealed that significantly more 19-ol intermediate was generated by both S478A and S478T than the wild-type enzyme. These results would support a hypothesis that Ser478 plays a role in the first and second hydroxylation reactions. A positive charged amino acid is preferred at position 480 as shown by the fact that H480K has a significantly higher activity than H480Q. The Ki value of 4-OHA for H480Q was found to be three times that of the wild-type enzyme. In addition, significantly more 19-ol and 19-al intermediates were detected for both mutants H480K and H480Q than for the wild-type enzyme. Evaluation of the two mutations at His480 allows us to propose that this residue may participate in the aromatization reaction (the third step) by acting as a hydrogen bond donor for the C-3 keto group of the substrate. Furthermore, new products were generated when the enzyme was mutated at Ser478 and His480. Thus, these two residues must play an important role in the catalysis and are likely closer to the substrate binding site than previously predicted.
Topics: Androgens; Aromatase; Aromatase Inhibitors; Catalytic Domain; Computer Simulation; Estrogens; Humans; Hydroxylation; Kinetics; Microsomes; Models, Molecular; Mutagenesis, Site-Directed; Point Mutation; Recombinant Proteins
PubMed: 11168357
DOI: 10.1046/j.1432-1033.2001.01886.x -
The Journal of Cell Biology May 1978The cytochalasins stimulate steroid secretion of Y-1 adrenal tumor cells two-to threefold. The order of potencies is cytochalasin E is greater than D is greater than B,...
The cytochalasins stimulate steroid secretion of Y-1 adrenal tumor cells two-to threefold. The order of potencies is cytochalasin E is greater than D is greater than B, but the maximum response is the the same and always less than with ACTH. Like that with ACTH, the stimulation has a rapid onset, is easily reversible, is inhibited by cucloheximide and aminoglutethimide, and occurs at a stage before pregnenolone. Although the cytochalasin, like ACTH, produce cell rounding, it is shown that this morphological change is not necessarily coupled to steridogenesis. Unlike ACTH, cytochalasin B does not measurably increase cellular levels of cAMP at concentrations that lead to maximal steroidogenesis. The cytochalasin B-induced stimulation of steroidogenesis, unlike the short-term ACTH effect, fails to occur in the absence of serum. This lack of response can be corrected by even low concentrations of human high density lipoproteins (HDL) but not by low density lipoproteins (LDL). We, therefore, propose that cytochalasin B enhances the availability of cholesterol bound to HDL for steroidogenesis by Y-1 adrenal cells.
Topics: 20-alpha-Dihydroprogesterone; Adrenal Gland Neoplasms; Adrenocorticotropic Hormone; Aminoglutethimide; Cell Line; Cycloheximide; Cytochalasin B; Cytochalasins; Drug Antagonism; Lipoproteins, HDL; Progesterone; Stimulation, Chemical
PubMed: 206563
DOI: 10.1083/jcb.77.2.507 -
The Journal of Biological Chemistry Aug 1983Two-dimensional electrophoretic techniques were used to identify and characterize a protein that is not produced in quiescent isolated rat adrenal cells but is produced...
Two-dimensional electrophoretic techniques were used to identify and characterize a protein that is not produced in quiescent isolated rat adrenal cells but is produced in response to acute stimulation by adrenocorticotropic hormone (ACTH) or dibutyryl cAMP. The molecular weight of this protein is 28,000 (sodium dodecyl sulfate electrophoresis), and its isoelectric point is 6.5 (isoelectric focusing). Mapping of proteolytic peptides suggests that this induced protein (i) is quite similar in primary structure to another protein (p), which is produced only in nonstimulated adrenal cells. The time course of formation of protein i and its ACTH dose response closely parallel the increase of corticosteroid production in stimulated cells. The possibility that protein i is produced in response to increased levels of some steroid of the glucocorticoid pathway is precluded by the observation that inhibition of corticosteroid synthesis by aminoglutethimide does not alter the rate of production of i. Addition of cycloheximide before ACTH, which prevents stimulation of corticosteroidogenesis, also prevents formation of protein i implying that the production of protein i depends on continuing protein synthesis. [35S/32S]Methionine pulse-chase experiments, i.e. addition of excess [32S] methionine and ACTH after prelabeling with [35S]methionine, show that protein i is not produced from pre-existing protein p or other pre-existing proteins even if protein synthesis (and increased steroid production) is not inhibited. These findings exclude post-translational modification as a mechanism for the production of i but are consistent with p and i being related by cotranslational modification. Addition of cycloheximide after stimulation causes the formation of protein i to cease, but the amount of the protein does not decrease with the same kinetics as the return of corticosteroid production to its unstimulated level. [35S/32S]Methionine pulse-chase experiments imply protein i, even under conditions of ongoing ACTH stimulation and protein synthesis, is degraded with approximately the same kinetics as after cycloheximide inhibition. The close concurrence under a wide variety of experimental conditions between the appearance of protein i and the increase in adrenal corticosteroid production, coupled with the fact that the former does not occur as a result of the latter, make protein i a likely candidate for the postulated corticosteroidogenic stimulatory protein (Ferguson, J.J. (1962) Biochim. Biophys. Acta 57, 616-617). The fact that stimulation of steroidogenesis may occur via co-translational modification of a regulatory protein is an intriguing possibility which readily explains both the observed rapid and protein synthesis-dependent stimulation and the lack of dependence of stimulation on transcription (Schulster, D. (1974) Mol. Cell. Endocr. 1, 55-64).
Topics: Adrenal Glands; Adrenocorticotropic Hormone; Aminoglutethimide; Animals; Bucladesine; Corticosterone; Cycloheximide; Dose-Response Relationship, Drug; Female; Molecular Weight; Protein Biosynthesis; Rats; Time Factors
PubMed: 6309771
DOI: No ID Found -
Drug Metabolism and Disposition: the... Feb 2011Human pregnane X receptor (hPXR) plays a key role in regulating metabolism and clearance of endogenous and exogenous substances. Identification of novel hPXR activators...
Human pregnane X receptor (hPXR) plays a key role in regulating metabolism and clearance of endogenous and exogenous substances. Identification of novel hPXR activators among commercial drugs may aid in avoiding drug-drug interactions during coadministration. We applied ligand-based computational approaches for virtual screening of a commonly prescribed drug database (SCUT). Bayesian classification models were generated with a training set comprising 177 compounds using Fingerprints and 117 structural descriptors. A cell-based luciferase reporter assay was used for evaluation of chemical-mediated hPXR activation in HepG2 cells. All compounds were tested at 10 μM concentration with rifampicin and dimethyl sulfoxide as positive and negative controls, respectively. The Bayesian models showed specificity and overall prediction accuracy up to 0.92 and 0.69 for test set compounds. Screening the SCUT database with this model retrieved 105 hits and 17 compounds from the top 25 hits were chosen for in vitro testing. The reporter assay confirmed that nine drugs, i.e., fluticasone, nimodipine, nisoldipine, beclomethasone, finasteride, flunisolide, megestrol, secobarbital, and aminoglutethimide, were previously unidentified hPXR activators. Thus, the present study demonstrates that novel hPXR activators can be efficiently identified among U.S. Food and Drug Administration-approved and commonly prescribed drugs, which should lead to detection and prevention of potential drug-drug interactions.
Topics: Bayes Theorem; Computational Biology; Databases, Factual; Drug Evaluation, Preclinical; Drug Interactions; Hep G2 Cells; Humans; Ligands; Luciferases; Models, Biological; Predictive Value of Tests; Pregnane X Receptor; Prescription Drugs; Principal Component Analysis; Receptors, Steroid; Reproducibility of Results
PubMed: 21068194
DOI: 10.1124/dmd.110.035808 -
British Journal of Cancer Sep 1984By analogy with combination chemotherapy, endocrine agents with different mechanisms of action have been combined in the treatment of patients with advanced breast...
By analogy with combination chemotherapy, endocrine agents with different mechanisms of action have been combined in the treatment of patients with advanced breast cancer. The clinical use of tamoxifen+aminoglutethimide+hydrocortisone showed no clinical benefit over the individual use of tamoxifen or aminoglutethimide+hydrocortisone. The endocrine changes occurring in postmenopausal patients as a consequence of their treatment with tamoxifen+aminoglutethimide+hydrocortisone have been examined. Suppression of gonadotrophin and oestrogen levels and increased levels of sex hormone binding globulin were observed. These changes might be expected to be of benefit in the treatment of advanced breast cancer, and do not explain the lack of clinical benefit in combining the treatments. Non-responders to this combination therapy had higher levels of oestrone and dehydroepiandrosterone sulphate whilst on treatment than responders, confirming previous observations in patients treated with aminoglutethimide+hydrocortisone.
Topics: Aminoglutethimide; Androgens; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Estrogens; Female; Gonadotropins, Pituitary; Humans; Hydrocortisone; Menopause; Sex Hormone-Binding Globulin; Tamoxifen
PubMed: 6540595
DOI: 10.1038/bjc.1984.183