Progesterone Metabolites Regulate Breast Cell Tumors
Progesterone Metabolites Regulate Breast Cell Tumors
Progesterone, 5αP, cell-culture media, insulin, penicillin, and streptomycin were obtained from Sigma Chemical Co. (Oakville, ON, Canada). 3αHP was obtained from Steraloids (Newport, RI, USA). Serum was purchased from Invitrogen (Burlington, ON, Canada). [1,2,6,7-H]Progesterone and [9,11,12-H]5α-pregnan-3α-ol-20-one were purchased from Perkin-Elmer (Woodbridge, ON, Canada). Other chemicals and solvents were of appropriate analytic grade and were purchased from Sigma Chemical Co., BDH Inc., (Toronto, ON, Canada), VWR (Mississauga, ON, Canada), or Fisher Scientific Ltd. (Toronto, ON, Canada). Ethanol was double (glass) distilled.
The human breast cell line MDA-MB-231 was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and cells were grown in a 1:1 Ham F12 Medium and Dulbecco Modified Eagle Medium with supplements and 10% calf serum as described. Cells were grown in T-75 flasks (Sarstedt) and were harvested at approximately 80% confluence. Cell-proliferation and -detachment responses to 5αP and 3αHP were tested before harvesting for inoculation into animals, and cell viability was determined with the trypan blue exclusion test. Cells intended for inoculation into mice were harvested, washed, and then suspended in serum-free medium (about 5 × 10 cells per 100 μl).
Severe combined immunodeficiency (SCID) female mice with impaired T- and B-cell lymphocyte development (NOD SCID) were obtained from Charles River Laboratories (Saint-Constant, Quebec, Canada) at 5 to 6 weeks of age and maintained under specific pathogen-free conditions with food and water ad libitum. All animal handling and procedures were approved by Western University Institutional Care and Use Committee. After acclimation (8 to 27 days), cells were implanted on day 0. Each mouse was anesthetized with a mixture of isofluorane and oxygen, and about 5 × 10 cells, suspended in 100 μl cold (0°C to 4°C) serum-free medium, were injected into the right thoracic mammary fat pad through a 5-mm incision at the sternum region, by using a 1.0-ml syringe with 26-gauge needle. The wound was closed in one layer with metal wound clips or with tissue adhesive (3M Vetbond; St. Paul, MN, USA). The surgical, injection, and handling procedures were conducted in approved laminar-flow sterility hoods. At termination (asphyxiation by CO2), blood was collected, tumors were excised and weighed, necropsies were conducted, and tissues were fixed in 10% formalin for histopathologic observation (5-μm sections, hematoxylin and eosin). Some tumors were stored in methanol for steroid extraction.
Suspensions of 5αP and 3αHP were prepared under sterile conditions in sterile-filtered vehicle (0.9% NaCl in double-distilled H2O, containing 0.1% double-distilled ethanol and 0.05% Tween 80) at 4 to 5 mg/150 μl. The suspensions were stored at 4°C before use and were administered SC (150 μl/injection) by using a 1.0-ml syringe with a 23-gauge needle in the nape of the neck.
The growth of tumors was monitored at regular intervals (weekly at first, and after appearance of palpable tumors, every second day or every day). Tumor volumes were determined from digital caliper measurements of length and width. The formula (length × (width) × 0.6) was determined to be a good approximation of tumor volumes (mm) as calculated empirically by water-displacement measurements of various irregular tumorlike shapes and sizes of modeling clay.
[9,11,12-H]5αP was prepared by oxidation of [9,11,12-H]5α-pregnan-3α-ol-20-one, as described. Purification of [H]-labeled 5αP was by high-pressure liquid chromatography (HPLC; C18 column and methanol/water, 3:1) and TLC (Fisherbrand silica gel GF; three runs in hexane:ethyl acetate, 5:2). Preparation of 5α-pregnane-3,20-dione-11α-hemisuccinate-BSA (5αP-BSA conjugate) was by previously described procedures, and purity of the conjugate was confirmed with HPLC in the solvent system acetonitrile:H2O:trifluoroacetic acid (45:55:0.1) by using a Vydac C4 column (4.6 × 250 mm) for protein with particle size, 5 μm, and pore diameter, 300 A.
Tritiated 3αHP ([1,2,6,7-H]3αHP) was prepared from freshly TLC-cleaned [1,2,6,7-H]progesterone by using potassium trisamylborohydride (KS-Selectride; Aldrich) as reducing agent, as described, with some modifications. In brief, [H]progesterone (50 to 100 μCi) was transferred to a dry siliconized reaction tube; the ethanol was evaporated under N2, and the tube was dried overnight in a vacuum desiccator over gypsum (Drierite). Dry molecular sieves (3 to 4) were added to the tube under an N2 cone, and the tube was sealed with a rubber septum. Dry tetrahydrofuran (THF; 200 μl) was added, and the N2-purged tube was cooled to -80°C. The reaction was initiated at -80°C with slow dropwise addition of cold KS-Selectride (100 μl) under an N2-purged atmosphere and with gentle agitation. After 1 hour, the reaction was continued in an ice bath (0°C) for another 3 hours and terminated with the addition of 1.0 ml THF and 1.5 ml cold (0°C) 0.1N NaOH. The reaction mixture was extracted 3 times with 5 ml ethyl ether or ether:CH2Cl2 (5:1) and cleaned by backwashing and C18 bonded silica gel columns. The reaction products were separated and purified with TLC and HPLC, as described earlier under Synthesis of [H]-5αP, and [H]3αHP was stored in double-distilled ethanol (purged under N2), at -20°C.
The 3αHP antiserum (lyophilized) was from our stock originally generated in rabbits by using a 3αHP-carboxymethyloxime-BSA conjugate. For the preparation of 5αP and progesterone antiserum, two male SPF New Zealand white rabbits were immunized with 5αP-BSA, and titer was determined. The serum was stored at -80°C. The antiserum from Rabbit 1 showed high specificity for 5αP, and low cross reaction (percentage relative to 5αP at 100%) with progesterone (2.2%), 3αHP (1.3%), estradiol (2.2%), 4-pregnen-20α-ol-3-one (0.9%), and other 5α-pregnanes and testosterone (<0.1%). The serum from Rabbit 2 had lower specificity for 5αP but acceptable specificity for progesterone and was therefore used for the progesterone radioimmunoassay (RIA).
Sera (100 to 300 μl) from 29 mice from different experiments were extracted 3 times with 2.0 ml ether/chloroform (6:1). The water and organic solvent phases were separated by freezing (-80°C), the combined solvent portions were dried down under a stream of N2, and the residue was brought up in 0.5 ml of methanol/CH2Cl2 (5:1), purged with N2, and stored at -20°C until chromatography was performed. Tumors were weighed, cut into pieces, and homogenized in 5-ml methanol by using a Polytron and extracted with methanol and 3 times with ether/CH2Cl2 (5:1). The combined solvent was evaporated under N2, and the samples brought up in methanol/CH2Cl2 (5:1) were cleaned by solid-phase extraction (C18-bonded silica gel columns) by using methanol/CH2Cl2 (20:1) as eluant at a flow rate of about 0.8 ml/min. The fractions containing the steroids were combined, evaporated under N2, and brought up in 0.5 ml methanol/CH2Cl2 (5:1), purged with N2, and stored at -20°C until chromatography.
Thin-layer chromatography (TLC) of serum and tumor extracts was performed on 20 × 20-cm silica gel G(F) TLC plates (250 μm; Fisher Scientific, Pittsburgh, PA, USA). Extracts and standards (5αP, 3αHP, progesterone) were run in separate lanes (2×) in a solvent system consisting of hexane/chloroform/ethyl acetate (60:60:30). The standards were located by UV absorption and exposure to iodine vapors and, on average, were located at Rf of 0.62 (5αP), 0.44 (progesterone), and 0.27 (3αHP). Regions in the sample lanes coinciding with the standards were extracted with ether/chloroform (6:1); the extracts were evaporated under N2 and brought up in 0.4-ml double-distilled ethanol.
The RIAs for 5αP, 3αHP, and progesterone were basically as described, with the generated antisera (see earlier) and scintillation spectrometry (Beckman-Coulter LS 6500 Scintillation Counter). For purposes of comparison, concentrations were standardized to nanograms per milliliter for serum and nanograms per gram for tumors, and it was assumed that these two measures represent a reasonable equivalence.
For verification of RIA measurements of 5αP, 3αHP, and progesterone, portions of TLC-separated extracts from four tumor tissues were tested with both RIA and GC/MS (Hewlett-Packard GC-Mass Spectrometer, model 5790A/5970A, used in the selected ion mode (SIM) with a DB-1MS 12-m × 0.2 mm × 0.33 μm cross-linked methyl silicone capillary column). The conditions were similar to those described previously: splitless mode, 0.7 kg/cm helium, 230°C injection temperature, column temperature at 150°C (initial) to 230°C at 20°/min, and scan speed of 690 amu/sec at an electron multiplier setting of 2,200 V. Underivatized authentic standards at various concentrations as well as samples were brought up in 10 μl MeOH or CH2Cl2, and 5 μl was injected for each GC/MS analysis. The authentic steroids were first run in standard mode, which showed that, under the condition employed, 5αP and progesterone each eluted as single peaks (at about 12.5 and 14.2 minutes, respectively), and 3αHP eluted as three separate peaks consisting of two isomeric dehydrated moieties and whole 3αHP (at 7.6, 8.3, and 11.1 minutes, respectively) (see Additional file 1). The runs in standard mode also showed that the major ions (m/e) were 246, 283, 298, and 316, for 3αHP and its two dehydrated fragments; 231, 258, 298, and 316 for 5αP; and 124, 229, 272, and 314 for progesterone (Additional file 1 through E). Authentic steroids and sample extracts were then run in SIM set for the respective major ions. Quantification of samples was calculated by comparison with concentration curves developed from standards (range of 0.04 to 20 ng), and the limit of detection was about 50 pg.
Statistical analyses were carried out with GraphPad Instat software (Graph-Pad Software, Inc., San Diego, CA, USA). Results are presented as mean ± SEM and were analyzed with the unpaired Student t test, with P < 0.05 considered statistically significant.
Methods and Materials
Chemicals and Reagents
Progesterone, 5αP, cell-culture media, insulin, penicillin, and streptomycin were obtained from Sigma Chemical Co. (Oakville, ON, Canada). 3αHP was obtained from Steraloids (Newport, RI, USA). Serum was purchased from Invitrogen (Burlington, ON, Canada). [1,2,6,7-H]Progesterone and [9,11,12-H]5α-pregnan-3α-ol-20-one were purchased from Perkin-Elmer (Woodbridge, ON, Canada). Other chemicals and solvents were of appropriate analytic grade and were purchased from Sigma Chemical Co., BDH Inc., (Toronto, ON, Canada), VWR (Mississauga, ON, Canada), or Fisher Scientific Ltd. (Toronto, ON, Canada). Ethanol was double (glass) distilled.
Cells
The human breast cell line MDA-MB-231 was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and cells were grown in a 1:1 Ham F12 Medium and Dulbecco Modified Eagle Medium with supplements and 10% calf serum as described. Cells were grown in T-75 flasks (Sarstedt) and were harvested at approximately 80% confluence. Cell-proliferation and -detachment responses to 5αP and 3αHP were tested before harvesting for inoculation into animals, and cell viability was determined with the trypan blue exclusion test. Cells intended for inoculation into mice were harvested, washed, and then suspended in serum-free medium (about 5 × 10 cells per 100 μl).
Animals
Severe combined immunodeficiency (SCID) female mice with impaired T- and B-cell lymphocyte development (NOD SCID) were obtained from Charles River Laboratories (Saint-Constant, Quebec, Canada) at 5 to 6 weeks of age and maintained under specific pathogen-free conditions with food and water ad libitum. All animal handling and procedures were approved by Western University Institutional Care and Use Committee. After acclimation (8 to 27 days), cells were implanted on day 0. Each mouse was anesthetized with a mixture of isofluorane and oxygen, and about 5 × 10 cells, suspended in 100 μl cold (0°C to 4°C) serum-free medium, were injected into the right thoracic mammary fat pad through a 5-mm incision at the sternum region, by using a 1.0-ml syringe with 26-gauge needle. The wound was closed in one layer with metal wound clips or with tissue adhesive (3M Vetbond; St. Paul, MN, USA). The surgical, injection, and handling procedures were conducted in approved laminar-flow sterility hoods. At termination (asphyxiation by CO2), blood was collected, tumors were excised and weighed, necropsies were conducted, and tissues were fixed in 10% formalin for histopathologic observation (5-μm sections, hematoxylin and eosin). Some tumors were stored in methanol for steroid extraction.
Treatments
Suspensions of 5αP and 3αHP were prepared under sterile conditions in sterile-filtered vehicle (0.9% NaCl in double-distilled H2O, containing 0.1% double-distilled ethanol and 0.05% Tween 80) at 4 to 5 mg/150 μl. The suspensions were stored at 4°C before use and were administered SC (150 μl/injection) by using a 1.0-ml syringe with a 23-gauge needle in the nape of the neck.
Tumor Growth Monitoring
The growth of tumors was monitored at regular intervals (weekly at first, and after appearance of palpable tumors, every second day or every day). Tumor volumes were determined from digital caliper measurements of length and width. The formula (length × (width) × 0.6) was determined to be a good approximation of tumor volumes (mm) as calculated empirically by water-displacement measurements of various irregular tumorlike shapes and sizes of modeling clay.
Synthesis of [H]-5αP and 5αP-BSA Conjugate
[9,11,12-H]5αP was prepared by oxidation of [9,11,12-H]5α-pregnan-3α-ol-20-one, as described. Purification of [H]-labeled 5αP was by high-pressure liquid chromatography (HPLC; C18 column and methanol/water, 3:1) and TLC (Fisherbrand silica gel GF; three runs in hexane:ethyl acetate, 5:2). Preparation of 5α-pregnane-3,20-dione-11α-hemisuccinate-BSA (5αP-BSA conjugate) was by previously described procedures, and purity of the conjugate was confirmed with HPLC in the solvent system acetonitrile:H2O:trifluoroacetic acid (45:55:0.1) by using a Vydac C4 column (4.6 × 250 mm) for protein with particle size, 5 μm, and pore diameter, 300 A.
Synthesis of [H]3αHP
Tritiated 3αHP ([1,2,6,7-H]3αHP) was prepared from freshly TLC-cleaned [1,2,6,7-H]progesterone by using potassium trisamylborohydride (KS-Selectride; Aldrich) as reducing agent, as described, with some modifications. In brief, [H]progesterone (50 to 100 μCi) was transferred to a dry siliconized reaction tube; the ethanol was evaporated under N2, and the tube was dried overnight in a vacuum desiccator over gypsum (Drierite). Dry molecular sieves (3 to 4) were added to the tube under an N2 cone, and the tube was sealed with a rubber septum. Dry tetrahydrofuran (THF; 200 μl) was added, and the N2-purged tube was cooled to -80°C. The reaction was initiated at -80°C with slow dropwise addition of cold KS-Selectride (100 μl) under an N2-purged atmosphere and with gentle agitation. After 1 hour, the reaction was continued in an ice bath (0°C) for another 3 hours and terminated with the addition of 1.0 ml THF and 1.5 ml cold (0°C) 0.1N NaOH. The reaction mixture was extracted 3 times with 5 ml ethyl ether or ether:CH2Cl2 (5:1) and cleaned by backwashing and C18 bonded silica gel columns. The reaction products were separated and purified with TLC and HPLC, as described earlier under Synthesis of [H]-5αP, and [H]3αHP was stored in double-distilled ethanol (purged under N2), at -20°C.
Preparation of Antisera
The 3αHP antiserum (lyophilized) was from our stock originally generated in rabbits by using a 3αHP-carboxymethyloxime-BSA conjugate. For the preparation of 5αP and progesterone antiserum, two male SPF New Zealand white rabbits were immunized with 5αP-BSA, and titer was determined. The serum was stored at -80°C. The antiserum from Rabbit 1 showed high specificity for 5αP, and low cross reaction (percentage relative to 5αP at 100%) with progesterone (2.2%), 3αHP (1.3%), estradiol (2.2%), 4-pregnen-20α-ol-3-one (0.9%), and other 5α-pregnanes and testosterone (<0.1%). The serum from Rabbit 2 had lower specificity for 5αP but acceptable specificity for progesterone and was therefore used for the progesterone radioimmunoassay (RIA).
Steroid Extractions From Serum and Tumor Tissues
Sera (100 to 300 μl) from 29 mice from different experiments were extracted 3 times with 2.0 ml ether/chloroform (6:1). The water and organic solvent phases were separated by freezing (-80°C), the combined solvent portions were dried down under a stream of N2, and the residue was brought up in 0.5 ml of methanol/CH2Cl2 (5:1), purged with N2, and stored at -20°C until chromatography was performed. Tumors were weighed, cut into pieces, and homogenized in 5-ml methanol by using a Polytron and extracted with methanol and 3 times with ether/CH2Cl2 (5:1). The combined solvent was evaporated under N2, and the samples brought up in methanol/CH2Cl2 (5:1) were cleaned by solid-phase extraction (C18-bonded silica gel columns) by using methanol/CH2Cl2 (20:1) as eluant at a flow rate of about 0.8 ml/min. The fractions containing the steroids were combined, evaporated under N2, and brought up in 0.5 ml methanol/CH2Cl2 (5:1), purged with N2, and stored at -20°C until chromatography.
Chromatographic Separation of Progesterone, 5αP, and 3αHP
Thin-layer chromatography (TLC) of serum and tumor extracts was performed on 20 × 20-cm silica gel G(F) TLC plates (250 μm; Fisher Scientific, Pittsburgh, PA, USA). Extracts and standards (5αP, 3αHP, progesterone) were run in separate lanes (2×) in a solvent system consisting of hexane/chloroform/ethyl acetate (60:60:30). The standards were located by UV absorption and exposure to iodine vapors and, on average, were located at Rf of 0.62 (5αP), 0.44 (progesterone), and 0.27 (3αHP). Regions in the sample lanes coinciding with the standards were extracted with ether/chloroform (6:1); the extracts were evaporated under N2 and brought up in 0.4-ml double-distilled ethanol.
Radioimmunoassays
The RIAs for 5αP, 3αHP, and progesterone were basically as described, with the generated antisera (see earlier) and scintillation spectrometry (Beckman-Coulter LS 6500 Scintillation Counter). For purposes of comparison, concentrations were standardized to nanograms per milliliter for serum and nanograms per gram for tumors, and it was assumed that these two measures represent a reasonable equivalence.
Mass Spectrometry
For verification of RIA measurements of 5αP, 3αHP, and progesterone, portions of TLC-separated extracts from four tumor tissues were tested with both RIA and GC/MS (Hewlett-Packard GC-Mass Spectrometer, model 5790A/5970A, used in the selected ion mode (SIM) with a DB-1MS 12-m × 0.2 mm × 0.33 μm cross-linked methyl silicone capillary column). The conditions were similar to those described previously: splitless mode, 0.7 kg/cm helium, 230°C injection temperature, column temperature at 150°C (initial) to 230°C at 20°/min, and scan speed of 690 amu/sec at an electron multiplier setting of 2,200 V. Underivatized authentic standards at various concentrations as well as samples were brought up in 10 μl MeOH or CH2Cl2, and 5 μl was injected for each GC/MS analysis. The authentic steroids were first run in standard mode, which showed that, under the condition employed, 5αP and progesterone each eluted as single peaks (at about 12.5 and 14.2 minutes, respectively), and 3αHP eluted as three separate peaks consisting of two isomeric dehydrated moieties and whole 3αHP (at 7.6, 8.3, and 11.1 minutes, respectively) (see Additional file 1). The runs in standard mode also showed that the major ions (m/e) were 246, 283, 298, and 316, for 3αHP and its two dehydrated fragments; 231, 258, 298, and 316 for 5αP; and 124, 229, 272, and 314 for progesterone (Additional file 1 through E). Authentic steroids and sample extracts were then run in SIM set for the respective major ions. Quantification of samples was calculated by comparison with concentration curves developed from standards (range of 0.04 to 20 ng), and the limit of detection was about 50 pg.
Statistical Analyses
Statistical analyses were carried out with GraphPad Instat software (Graph-Pad Software, Inc., San Diego, CA, USA). Results are presented as mean ± SEM and were analyzed with the unpaired Student t test, with P < 0.05 considered statistically significant.
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