Progestins and breast cancer

In the last years there has been an extraordinary development in the synthesis of new progestins. These compounds are classified, in agreement with their structure, in various groups which include progesterone, retroprogesterones, 17-hydroxyprogesterones, 19-norprogesterones, 17-hydroxyprogesterone derivatives, androstane and estrane derivatives. The action of progestins is a function of many factors: its structure, affinity to the progesterone receptor or to other steroid receptors, the target tissue considered, the biological response, the experimental conditions, dose, and metabolic transformation. The information on the action of progestins in breast cancer patients is very limited. Positive response with the progestins: medroxyprogesterone acetate and megestrol acetate was obtained in post-menopausal patients with advanced breast cancer. However, extensive information on the effect of progestins was obtained in in vitro studies using hormone-dependent and hormone-independent human mammary cancer cell lines. It was demonstrated that in the hormone-dependent breast cancer cells, various progestins (nomegestrol acetate, tibolone, medrogestone, promegestone) are potent sulfatase inhibitory agents. The progestins can also involve the inhibition of mRNA of this enzyme. In another series of studies it was also demonstrated that various progestins are very active in inhibiting the 17β-hydroxysteroid dehydrogenase for the conversion of estrone to estradiol. More recently it was observed that the progestins promegestone or medrogestone stimulate the sulfotransferase for the formation of estrogen sulfates. Consequently, the blockage in the formation of estradiol via sulfatase, or the stimulatory effect on sulfotransferase activity, by progestins can open interesting and new possibilities in clinical applications in breast cancer.     

Effect of some progestational steroids on lactation in Egyptian women. I. Milk yield during the first year of lactation.

The effect of monthly injectios of 300 mg Depo-Provera or 150 mg Deladroxone, and of daily oral administration of .5 mg chlormadinone acetate or .3 mg quingestanol acetate on lactation was studied in Egyptian women during the 1st year of lactation. Women receiving Depo-Provera had the highest milk yield, followed by those taking Deladroxone and quingestanol acetate. The milk yields while taking these hormonal preparations were higher than in untreated controls. However, those women taking chlormadinone acetate had lower milk yields than untreated controls. The increased milk yield is probably due to the progestagenic activity, and minimal estrogenicity, of these drugs.     

Aromatase activity of human adipose tissue stromal cells: effects of thyroid hormones and progestogens

In order to determine the direct effects of thyroid hormones and progestogens on extraglandular aromatization, human adipose stromal cells in monolayer culture were used as a model system for this study on the regulation of aromatase enzyme activity. It was found that 1-thyroxine at 2- and 4-fold normal concentrations (220 and 440 nM, respectively) and triiodothyronine at 4-fold normal concentration (7.4 nM) had no effect on basal, dibutyryl cyclic AMP ((Bu)2 cAMP)-induced, or dexamethasone-induced aromatization. Medroxyprogesterone acetate at a concentration of 25.9 nM, but not progesterone, 47.7 nM, stimulated basal aromatization slightly but not significantly. In contrast, both medroxyprogesterone acetate and progesterone potentiated the effect of (Bu)2 cAMP on aromatase activity (P less than 0.05 and P less than 0.01, respectively) but had no effect on dexamethasone-stimulated aromatase activity. We concluded that (i) the increased peripheral aromatization associated with hyperthyroidism is not due to the direct effect of thyroid hormones on aromatase activity, and (ii) neither progesterone nor medroxyprogesterone acetate inhibit aromatase activity of adipose tissue stromal cells, but may stimulate this activity under certain conditions.     

Steroid hormones and the fertilizing capacity of spermatozoa

The effects of eleven different steroid hormones on $\text{in vitro}$ development of fertilizing capacity by hamster sperm were examined. Capacitation of epididymal sperm occurred only in the presence of female genital tract secretions. Fertilizing ability of sperm was poor if estradiol-17β, cortisol, chlormadinone acetate, medroxyprogesterone acetate, or megestrol acetate were present in the incubation medium at 10^?5M, whereas similar concentrations of estradiol-17α, progesterone, norethisterone acetate, ethynodiol diacetate, or norgestrel had little effect. Testosterone was a weak inhibitor of capacitation. Capacitation activity of female uterus and oviduct washings was higher at estrus than diestrus. This activity was reduced by treating intact animals with progesterone, cortisol, or testosterone, but increased by estradiol-17β or HCG. Estradiol-17α has no effect. Activity was low in pregnant or ovariectomized hamsters. Treatment of ovariectomized animals with estradiol-17β increased capacitation activity, but estradiol-17α, HCG or progesterone treatment was ineffective.     

A new assay and solubilization procedure for steroid 17,20-lyase from rat testes

We report an assay for testicular 17, 20-lyase which depends on the use of [21?¹⁴C]progesterone as a substrate. The method is made possible by a simplified procedure for the synthesis of [21?¹⁴C]progesterone. A chromatographic separation of the unreacted substrate and the 2-carbon by-product on mini silica gel colums permitted a quantitative assay of the lyase activity.The lyase complex from rat testes has been solubilized by treatment with Triton CF-54 detergent. The solubilized enzyme complex catalyzes the formation of androstenedione (4-androstene-3,17-dione) from progesterone without equilibrium with added 17-hydroxyprogesterone and the solubilized enzyme complex responds to the presence of cytosol activator. Both of these characteristics are similar to the properties of the intact microsomes. Thus, solubilization with this detergent preserves the special properties of the microsome bound enzyme complex.     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U     

Subcellular distribution of phospholipid-sensitive calcium-dependent protein kinase in guinea pig heart,spleen and cerebral cortex,and inhibition of the enzyme by Triton X-100

The subcellular distribution of phospholipid-sensitive Ca²⁺-dependent protein kinase in guinea pig heart was found to be: cytosol, 73%; microsome, 18%; plasma membrane, 9%; nuclei and mitochondria, < 0.1%. The enzyme in spleen and cerebral cortex was distributed nearly equally in the cytosolic and total (unfractionated) particulate fractions. The particulate enzyme in heart was released by EGTA (2.5 mM) alone but not by Triton X-100 (0.3%) alone, although a combination of the two was most effective. On the other hand, the particulate enzyme in spleen and cerebral cortex was released only by a combination of Triton X-100 and EGTA. Triton X-100 inhibited the enzyme, and this inhibition was reversed by phosphatidylserine (a phospholipid cofactor for the enzyme). The detergent, however, was without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases.     

Steroidal oxathiazine inhibitors of estrone sulfatase

The presence of estrone sulfatase in breast tumors and the high levels of circulating estrone sulfate may contribute the major portion of estrogen synthesized locally in breast tissues through conversion of estrone sulfate to estrone by the enzyme. Using inhibitors of estrone sulfatase for the treatment of estrogen-dependent (estrogen receptor positive, ER(+)) breast cancer could be a very effective therapeutic strategy for the treatment of estrogen-dependent breast tumors in postmenopausal women. Therefore, we designed and synthesized several steroidal 2',3'-oxathiazines that inhibit estrone sulfatase and have greatly reduced estrogenic side effects. Our in vitro studies indicate that the oxathiazine compounds have inhibitory activity on estrone sulfatase in MCF-7 human breast cancer cells. These estrone sulfatase inhibitors (ESIs) also inhibit the growth of MCF-7 cells induced by estrone sulfate. In addition, our in vivo experiments demonstrate that our ESIs have moderate antitumor activity against MCF-7 breast cancer xenografts in Balb/c athymic nude mice. The synthesis and biological activity of a number of these unique steroidal ESIs are described.     

The metabolism of 4-14C-pregnenolone in tissue sections of human ovaries and their modification by chlormadinone acetate]

In vitro incubations with slices of two normal human ovaries and 4-14C-pregnenolone as precursor were carried out to study the possibility of a direct influence of chlormadinone acetate on the metabolism of pregnenolone. In agreement with our previous studies the incubations of the ovary from the follicle phase of the cycle yields a profile of steroids different from that of the ovary from the corpus luteum phase of the cycle. Under the experimental condition chosen, the presence of enzymes of the steroidogenic pathway responsible for the synthesis of 17alpha-hydroxy-pregnenolone, DHA, androstenediol (basic metabolites) and androstenedione represents a characteristic profile of steroids of the ovaries from the follicle phase. After the addition of chlormadinone acetate to the incubation medium, the formation of androstenedione was inhibited, whereas the basic metabolites increased. The biosynthesis of progesterone, 17alpha-hydroxyprogesterone, estrone and estradiol represents a characteristic profile of steroids of the ovaries from the corpus luteum phase. After a addition of chlormadinone acetate to the incubation medium, the formation of this characteristic profile of steroids was inhibited. The influence of chlormadinone acetate on the two different profiles of steroids indicated, that chlormadinone acetate exerts an inhibitory effect on the 3beta-hydroxysteroid-dehydrogenase-delta5-4-isome     

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin activity of S. aureus cells under nongrowing conditions, and this lytic response was markedly reduced in energy-poisoned cells. In contrast, the detergent had no effect on the activity of autolysins in cell-free systems, and growth in the presence of Triton X-100 did not alter either the cellular autolysin activity or the susceptibility of cell walls to exogenous lytic enzymes. Treatment with either Triton X-100 or penicillin G in the growth medium stimulated release of predominantly acylated intracellular lipoteichoic acid and sensitized staphylococci to Triton X-100-induced autolysis. There was no significant difference in the cell wall and membrane compositions or Triton X-100 binding between the parental strains and the resistant mutants. The resistant mutant TXR1, derived from S. aureus H, had a higher level of L-alpha-glycerophosphate dehydrogenase activity, and its oxygen uptake was more resistant to inhibition by a submicellar concentration (0.008%) of Triton X-100. Growth in the presence of subinhibitory concentrations of Triton X-100 rendered S. aureus H cells phenotypically resistant to the detergent and greatly stimulated the level of oxygen uptake. Membranes isolated from such cells exhibited enhanced activity of the respiratory enzymes succinic dehydrogenase and L-alpha-glycerophosphate dehydrogenase.     

Affinities of progestogen and estrogen receptors in rabbit uterus for synthetic progestogens

The affinities of progestogen and estrogen receptors of rabbit uterus for a number of synthetic progestogens in clinical use and some analogues have been measured. Progesterone, 17-hydroxyprogesterone caproate, the chlormadinone, megestrol and cyproterone acetates had similar affinities for the progestogen receptor. Medroxyprogesterone acetate and particularly DU-41164 possessed much higher affinities. 17-hydroxyprogesterone or other agents with a free 17-hydroxyl had much lower receptor affinities than the corresponding 17-esters.Of the nortestosterone derivatives tested, norethisterone equalled progesterone in affinity for the progestogen receptor while d-norgestrel and Wy-4355 were more active. Norethynodrel and ethynodiol diacetate had much lower receptor affinities than progesterone. These results are discussed in relation to possible metabolic bioactivation.Only norethynodrel and ethynodiol (free alcohol) showed marked affinity for the estrogen receptor.     

Development of novel steroid sulfatase inhibitors; II. TZS-8478 potently inhibits the growth of breast tumors in postmenopausal breast cancer model rats

In postmenopausal breast cancer tissue, steroid sulfatase (STS) activity is high and much estrone sulfate also exists; these facts reveal that estrone sulfate may be involved in the growth of breast cancer as an estrogen source. Steroid sulfatase is an enzyme, which catalyzes hydrolysis from estrone sulfate to estrone, and the development of steroid sulfatase inhibitors is expected as novel therapeutic drugs for postmenopausal breast cancer. We have developed a novel compound 2',4'-dicyanobiphenyl-4-O-sulfamate (TZS-8478), which has potent steroid sulfatase-inhibitory activity and exhibits no estrogenicity in vitro and in vivo. To elucidate its usefulness as a therapeutic drug for postmenopausal breast cancer, we examined the breast cancer cell proliferation- and breast tumor growth-inhibitory activity of TZS-8478 in postmenopausal breast cancer model rats. TZS-8478 dose-dependently suppressed the estrone sulfate-stimulated proliferation of MCF-7 cells. Regarding nitrosomethylurea (NMU)-induced postmenopausal breast cancer models, furthermore, TZS-8478 (0.5 mg/kg per day) markedly inhibited the estrone sulfate-stimulated growth of breast tumors similarly to estrone sulfate-depletion. TZS-8478 completely inhibited steroid sulfatase activity in tumor, uterus and liver, and also markedly lowered plasma concentrations of estrone and estradiol. The above mentioned results suggested that TZS-8478 may be useful as a therapeutic drug for estrogen-dependent postmenopausal breast cancer.     

Characterization of the stimulatory effect of medroxyprogesterone acetate and chlormadinone acetate on growth factor treated normal human breast epithelial cells

OBJECTIVE: Evidence is increasing that adding progestogens to hormone replacement therapy may be more harmful than beneficial, however it is debatable whether all progestogens act equally on breast cells. Mitogenic growth factors from stromal breast tissue are important in growth-regulation of breast cells, and may modify responses to progestogens. We investigated the effect of two C-21 derivatives, medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) on growth-factor treated normal breast epithelial cells and tried to explore the underlying mechanisms of proliferation. METHOD: MCF10A (human epithelial, estrogen- and progesterone-receptor negative normal breast cells) were incubated with MPA or CMA at 0.1 and 1 microM for 7 days with the growth factors (GFs) EGF, bFGF and IGF-I at 1pM. The same combinations, as well as growth factors alone, were also incubated with the proliferation inhibitors PD98059 and LY294002 at 1 microM for 4 days. Cell proliferation rate was measured by the ATP-assay. RESULTS: MPA 0.1 and 1 microM, and CMA 1 microM in combination with GFs both significantly increased cell proliferation rate, with MPA having the greatest effect. MPA- and CMA-induced proliferation of GF stimulated cells was blocked by both PD98059 (selective inhibitor of MAP kinases) and LY294002 (phosphatidylinositol 3-kinase inhibitor); GF stimulated cells could not be significantly reduced by any of the inhibitors used. CONCLUSION: MPA and CMA have a stimulatory effect on benign growth factor stimulated MCF10A cells, possibly via activation of MAP kinase and subsequent substrates and activation of PI3-kinase. GF induced proliferation appear to be mediated by pathways other than those investigated here. Growth factors and progestogens therefore have an additive, synergistic effect on cell proliferation, eliciting their effects via different pathways.     

Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by Triton X-100 for efficient production of d-malate

Pseudomonas pseudoalcaligenes can only form d-malate from maleate after incubation of the cells with a solvent or a detergent. The effect of the detergent Triton X-100 on d-malate production was studied in more detail. The longer the cells were incubated with Triton X-100, the higher was the d-malate production activity, until the maximal malease activity was reached. Incubation of P. pseudoalcaligenes cells with Triton X-100 also resulted in an increase in the protein concentration of the supernatant, indicating that cell lysis had occurred. The rate at which the d-malate production activity increased was dependent on the Triton X-100 concentration and on the cell density. Also the rate at which lysis occurred depended on the Triton X-100 concentration.     

Optimum Conditions for Indoleacetic Acid Oxidase Extraction from Betula Leaves

This report deals with total extraction and activation of soluble indoleacetic acid oxidase from Betula alleghaniensis leaves as affected by different buffers, varying pH, phenol binder, detergent, plus volume and time parameters. For all buffers and pH levels tested, only tris pH 8 gave a high activity. This result was not a pH effect, since a wide-range, citrate-phosphate buffer at pH 8 gave a very low activity. Addition of a neutral detergent, Triton X-100, to all buffers gave considerable activity in every case. Most activity with Triton X-100 occurred at pH 6 and least at pH 8 regardless of buffer composition. A phenol binder, polyvinylpyrollidone, increased activity also, but less than the detergent Triton X-100. Both of these compounds in combination gave an additive effect and the highest measure of enzyme activity. Further increases in measurable indoleacetic acid oxidase activity were obtained by using the best combination of these factors to determine the optium tissue: buffer ratio and optimum soaking time. Increases in activity of 70 and 60%, respectively, were achieved.     

Norelgestromin as selective estrogen enzyme modulator in human breast cancer cell lines. Effect on sulfatase activity in comparison to medroxyprogesterone acetate

Human breast cancer tissue contains enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol (E(2)) formation. In this tissue, E(2) can be synthesized by two main pathways: (1) sulfatase-transforms estrogen sulfates into bioactive E(2), and the (2) aromatase-converts androgens into estrogens. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization.In the present study, we demonstrated in T-47D and MCF-7 human breast cancer cells that norelgestromin (NGMN) (a metabolite of norgestimate) is a potent inhibitory agent of the estrone sulfatase activity. After 24h incubation of physiological concentrations of E(1)S (5 x 10(-9)mol/l) the inhibitory effect of NGMN at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 43+/-7, 74+/-4 and 97+/-2%, respectively, in T-47D cells; 25+/-4, 57+/-5 and 96+/-2% respectively, in MCF-7 cells. Comparative studies using medroxyprogesterone acetate (MPA) showed that this progestin also has an inhibitory effect on sulfatase activity, but significantly less intense than that of NGMN. The inhibition for MPA at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 31+/-5, 47+/-3 and 61+/-3%, respectively, for T-47D cells; 6+/-3, 20+/-3 and 63+/-4%, respectively, for MCF-7 cells.In conclusion, the present data show that NGMN is a very potent inhibitory agent for sulfatase activity in the hormone-dependent breast cancer cells, resulting in decreased tissue concentration of E(2). The clinical significance of this finding remains to be elucidated.     

Detergent activation and solubilization of 2'':3''-cyclic nucleotide 3''-phosphodiesterase from isolated myelin and c6 cells.

Several detergents were investigated for their ability to increase activity of 2':3'-cyclic nucleotide 3'-phosphodiesterase in isolated myelin. The ability of Triton X-100 and Sulfobetaine DLH to solubilize the enzyme was also examined. Solubilization with Triton X-100 was only effective in the presence of salt, for example with NaCl 51% of the activity was solubilized. A single extraction with Sulfobetaine DLH yielded slightly more solubilized enzyme and did not require added salt. Both activation and solubilization of 2':3'-cyclic nucleotide 3'-phosphodiesterase appeared to be similarly dependent on detergent concentration, suggesting a common action of the detergent in the two processes. Myelin basic protein was solubilized more readily than the enzyme. In contrast with the enzyme in myelin, 2':3'-cyclic nucleotide 3'-phosphodiesterase activity in C6 cells was not increased in the presence of Triton X-100, and was partially solubilized by either Triton X-100 or NaCl alone. No myelin basic protein could be detected in C6 cells by radioimmunoassay.     

Interaction of Triton X-100 with the pigment-protein complexes of photosynthetic membranes.

The interaction of the non-ionic detergent Triton X-100 with photosynthetic membrane components of Pisum sativum (pea) is described. The detergent affected both the wavelength and the intensity of the 77K fluorescence-emission peaks of both Photosystem I and Photosystem II preparations, in addition to the effects on whole thylakoids recently described by Murphy & Woodrow [(1984) Biochem. J. 224, 989-993]. Below its critical micellar concentration, Triton X-100 had no effect on 77K fluorescence emissions even after prolonged incubations of up to 30 min. Above the critical micellar concentration of about 0.16 mg X ml-1, Triton X-100 caused a dramatic increase in the intensity of the 680 nm emission. The intensity of the 680 nm fluorescence emission continued to increase as more Triton X-100 was added, until limiting concentrations of detergent were reached. These limiting concentrations were proportional to the amount of membrane present and generally occurred at Triton X-100/chlorophyll (w/w) ratios of 100-200:1. In all cases the detergent effect was seen within 10 min, and is often considerably faster, with longer detergent treatments causing no further effects. The data are discussed in terms of a three-stage mechanism for detergent solubilization of membrane components.     

Lack of inhibition of placental estrone sulfatase and aromatase enzymes by vitamin D3 and its analogs

The aromatase and estrone sulfatase enzymes are important sources of biologically active estrogens in postmenopausal women with breast cancer. Promising initial results in the treatment of endocrine-responsive breast cancer have been exhibited by 125-dihydroxyvitamin D₃ and the synthetic vitamin D analogues MC903 and EB1089. However, these compounds together with vitamin D₃ and vitamin D₃ sulfate did not inhibit the human placental aromatase enzyme when assayed up to 20 μm. Only vitamin D₃ sulfate and 125-dihydroxyvitamin D inhibited the estrone sulfatase activity in human placental microsomes, albeit at high concentration (32 and 37% inhibition, respectively with 50 μm each inhibitor). It is unlikely that inhibition of aromatase or estrone sulfatase enzymes contribute to the inhibitory effect of this group of compounds on breast cancer cells in vivo.