In vitro characterization of trimegestone: a new potent and selective progestin

Trimegestone (TMG) is a novel 19-norpregnane progestin under development for hormone replacement therapy and oral contraception. The objective of the current study was to characterize the potency and steroid receptor selectivity of TMG in several in vitro assays and to compare its activity to that of medroxyprogesterone acetate (MPA). TMG and MPA had a similar competitive binding affinity for human and rabbit progesterone receptor (PR). However, TMG had a significantly higher affinity for rat PR (IC(50) = 3.3 nM) than MPA (IC(50) = 53.3 nM). In T47D cells, both compounds increased alkaline phosphatase activity and cell proliferation with comparable potencies (EC(50s) of 0.1 nM and of 0.02 nM, respectively). To further characterize the progestational activity and steroid receptor selectivity, we established an immortalized human endometrial stromal cell line (HESC-T). This cell line lacks endogenous estrogen receptor (ER) and PR but does have functional glucocorticoid receptors (GR). When ER is transiently expressed in the cells, 17beta-estradiol (E(2)) induces PR, allowing the study of PR-regulated genes. In HESC-T cells expressing exogenous ER, and therefore PR, both TMG and MPA increased HRE-tk-luciferase activity tenfold with an EC(50) of 0.2 nM. In HESC-T cells without exogenous ER, and therefore no PR, TMG did not induce HRE-tk-luciferase activity, whereas MPA induced the reporter activity with an EC(50) of about 10 nM. This MPA-induced reporter activity is believed to be mediated through GR. The steroid receptor selectivity of TMG was further evaluated using the HRE-tk-luciferase assay in the human lung carcinoma cell line A549, which contains GR but no PR. In these cells TMG had no effect on luciferase activity, whereas MPA increased the reporter activity in a dose-dependent manner with an EC(50) of approximately 30 nM. Furthermore, HRE-tk-luciferase assay in mouse fibroblast cell line L929, which expresses androgen receptor (AR) but no PR, showed that TMG had weak antiandrogenic activity whereas MPA had androgenic activity. In summary, data from several in vitro assays demonstrate that TMG is a potent progestin with a better receptor selectivity profile than MPA.     

The effect of medroxyprogesterone acetate on aromatase and DNA polymerase alpha activities in breast tumours

The effect of treatment with the progestogen medroxyprogesterone acetate (MPA) on the peripheral conversion of androstenedione to oestrone and tumour aromatase activity has been examined in post-menopausal women with advanced breast cancer. In addition to being a potent progestational compound, MPA also possesses glucocorticoid properties and glucocorticoids have been shown to stimulate in vitro aromatase activity. While some evidence was obtained of an increase in aromatase activity in tumour tissue after treatment with MPA, peripheral conversion of androstenedione to oestrone was similar when measured before (2.12 +/- 0.67%) and after (1.89 +/- 0.16%) treatment. DNA polymerase alpha activity, measured as a marker of cellular proliferation, decreased from 331 +/- 145 to 156 +/- 93 pmol thymidine triphosphate (TTP)/mg protein per h (P less than 0.02) in tumour samples examined before and after treatment. It is concluded that treatment with high doses of MPA has no effect on the peripheral conversion of androstenedione to oestrone but results in a significant reduction in tumour DNA polymerase alpha activity.     

Effects in vitro of medroxyprogesterone acetate on steroid metabolizing enzymes in the rat: selective inhibition of 3 alpha-hydroxysteroid oxidoreductase activity

The effects of 6 alpha-methyl-17 alpha-acetoxy-4-pregnene 3,20-dione (MPA) on the activity of different steroid metabolizing enzymes in vitro were investigated in several organs in the rat. MPA seems to be a potent inhibitor of 3 alpha-reduction of 17 beta-hydroxy-5 alpha-androstan-3-one (Dht) in homogenates of the testis, ovary, epididymis, prostate, kidney and the adrenal glands. In testicular homogenates MPA acts like a competitive inhibitor of the 3 alpha-reduction of Dht, with Ki of 0.42 [microM]. MPA seems to be a selective inhibitor of 3 alpha-hydroxysteroid oxidoreductase in numerous organs. Steroid metabolizing enzymes like 5 alpha-reductase, 7 alpha-hydroxylase, 3 beta-hydroxysteroid oxidoreductase and 17 beta-hydroxysteroid oxidoreductase were not inhibited by MPA under the conditions of incubation employed in these studies.     

Novel 6-aryl-1,4-dihydrobenzo[d]oxazine-2-thiones as potent,selective, and orally active nonsteroidal progesterone receptor agonists

The functional activity of 6-aryl benzoxazinone-based progesterone (PR) antagonists changed to PR agonism when the 2-carbonyl group was replaced by a 2-thiocarbonyl moiety. Based on this finding novel 6-aryl benzoxazine-2-thiones were synthesized and evaluated as PR agonists in various in vitro and in vivo assays. Several analogues had sub-nanomolar in vitro potency and showed excellent oral activities in rats. Compounds 15 and 29 had similar potencies to medroxyprogesterone acetate (MPA) in the in vitro T47D alkaline phosphatase assay and in vivo rat decidualization model. In contrast to MPA, 29 was highly selective (>500-fold) for PR over glucocorticoid and androgen receptors.     

beta]-Glucan Synthesis in the Cotton Fiber (IV. In Vitro Assembly of the Cellulose I Allomorph)

In vitro assembly of cellulose from plasma membrane extracts of the cotton (Gossypium hirsutum) fiber was enriched by a combination of 3-(N-morpholino)propanesulfonic acid extraction buffer and two independent digitonin solubilization steps consisting of 0.05% digitonin (SE1) followed by 1% digitonin (SE2). Glucan synthase activity assays revealed that, although the SE2 fraction possessed higher activity, only 8.6% of the in vitro product survived acetic/nitric acid treatment. On the other hand, the SE1 fraction was less active, but 32.1% of the total glucan in vitro product was resistant to acetic/nitric acid. In vitro products synthesized from the SE1 fraction contained [beta]-1,3-glucan and fibrillar cellulose I, whereas the SE2 fraction produced [beta]-1,3-glucan and cellulose II. Both celluloses assembled in vitro were labeled with cellobiohydrolase I-gold complex, and the electron diffraction patterns of both products from SE1 and SE2 revealed cellulose I and cellulose II, respectively. Contamination of native cellulose was ruled out by extensive evidence from autoradiography of the ethanol-insoluble and acetic/nitric acid-insoluble materials, including three different controls.     

Comparison of two synthetic progesterones on ventilation in normal males: CMA vs. MPA

We administered chlormadinone acetate (CMA), medroxyprogesterone acetate (MPA), and placebo to 16 normal male subjects using a randomized double-blind crossover study. After CMA administration, minute alveolar ventilation increased by +1.04 +/- 0.22 (SE) 1/min (P less than 0.05) accompanied by decrements of arterial PCO2 (-4.0 +/- 1.0 Torr) (P less than 0.01) and [HCO3-] (-2.1 +/- 0.05 mM/l) (P less than 0.01). On the other hand, in the MPA runs the corresponding changes of the above parameters were +0.71 +/- 0.21 l/min (P less than 0.05), -2.9 +/- 0.6 Torr (P less than 0.01), and -1.3 +/- 0.3 mM/l (P less than 0.01), respectively. The slopes of hypoxic ventilatory and occlusion pressure response lines remained unchanged in hypocapnia after CMA or MPA ingestion, but they increased when entidal PCO2 was adjusted to the predrug level. The hypercapnic ventilatory and occlusion pressure response lines merely shifted to the left without changing their slopes with these agents. No significant differences in all the above parameters were found between CMA and MPA runs. We concluded that in the normal males the effect of CMA on ventilation was similar to that of MPA, despite the fact that the luteinizing activity of CMA was reported to be approximately 10 times higher than the latter.     

The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase

It is not yet clear to what extent depletion of intracellular GTP pools contributes to the antiviral activity of ribavirin. Therefore, the antiviral activities of (i) ribavirin, (ii) its 5-ethynyl analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and (iii) mycophenolic acid (MPA) (a compound that inhibits only cellular IMP dehydrogenase activity) were studied on the replication of flaviviruses and paramyxoviruses. In addition, the effects of these three compounds on intracellular GTP pools were assessed. A linear correlation was observed over a broad concentration range between the antiviral activities of ribavirin, EICAR, and MPA and the effects of these compounds on GTP pool depletion. When the 50% effective concentrations (EC50s) for the antiviral activities of ribavirin, EICAR, and MPA were plotted against the respective EC50 values for GTP pool depletion, a linear correlation was calculated. These data provide compelling evidence that the predominant mechanism of action of ribavirin in vitro against flavi- and paramyxoviruses is based on inhibition of cellular IMP dehydrogenase activity.     

Structure-activity relationships for inhibition of inosine monophosphate dehydrogenase and differentiation induction of K562 cells among the mycophenolic acid derivatives

Inosine monophosphate dehydrogenases (IMPDHs) are the committed step in de novo guanine nucleotide biosynthesis. There are two separate, but very closely related IMPDH isoenzymes, termed type I and type II. IMPDHs are widely believed to be major targets for cancer and transplantation therapy. Mycophenolic acid (MPA) is a potent inhibitor of IMPDHs. Previously, we found that MPA acted as a latent agonist of this nuclear hormone receptor in U2OS cells, and 6'-hydroxamic acid derivatives of MPA inhibited tubulin-specific histone deacetylase[s] (HDAC[s]) in HeLa cells. Although MPA is a promising lead compound, structure-activity relationships (SARs) for inhibition of IMPDH, and the mechanism action of MPA derivatives have not well been understood. We therefore synthesized, evaluated MPA derivatives as IMPDH inhibitor in vitro and cellular level, and explored their biological function and mechanism in cultured cells. This paper exhibits that (i) functional groups at C-5, C-7, and C-6' positions in MPA are important for inhibitory activity against IMPDH, (ii) it is difficult to improve specificity against IMPDH II by modification of 5-, 7-, and 6'-group, (iii) demethylation of 5-OMe results in increasing hydrophilicity, and lowering cell permeability, (iv) ester bonds of protective groups at C-7 and C-6' positions are hydrolyzed to give MPA in cultures, (v) the effects of a tubulin-specific HDAC[s] inhibitor on proliferation and differentiation are weaker than its inhibitory activity against IMPDH. The present work may provide insight into the development of a new class of drug lead for treating cancer and transplantation.     

Bioactivity of nacre water-soluble organic matrix from the bivalve mollusk Pinctada maxima in three mammalian cell types: fibroblasts, bone marrow stromal cells and osteoblasts

In vivo and in vitro studies provide strong evidence of the osteogenic activity of nacre obtained from Pinctada maxima. The in vitro studies indicate that diffusible factors from nacre are involved in cell stimulation. The water-soluble matrix (WSM) was extracted from nacre by a non-decalcifying process, and four fractions (SE(1)-SE(4)) were separated by SE-HPLC. Those fractions were tested in vitro on MRC5 fibroblasts. Alkaline phosphatase (ALP) activity was measured as a marker of osteoblastic differentiation. The anti-apoptotic protein Bcl-2 was also immunodetected in cultured osteoblasts from rat calvaria. WSM and fraction SE(4) increased ALP activity. BMP-2 had the same effect on the cells as WSM and SE(4). WSM greatly increased the amount of Bcl-2 in the cytoplasm and nucleus of osteoblasts. These in vitro studies support our initial hypothesis that nacre organic matrix (WSM) of a bivalve mollusk contains signal-molecules that can stimulate the osteogenic pathway in mammalian cells that are targets for bone induction.     

Preliminary studies of plasma growth hormone releasing activity during medical therapy of acromegaly.

The in vitro growth hormone releasing activity of plasma obtained from six acromegalic subjects was measured before and during therapy. In five subjects, plasmas were obtained before and during successful medical therapy with medroxyprogesterone acetate (MPA). The sixth subject was sampled before and after transphenoidal Sr90-induced hypopituitarism. All subjects had a decrement in fasting growth hormone levels with respective therapies (29-88%). The in vitro growth hormone released from Rhesus monkey anterior pituitaries was assessed after incubating one lateral half in control plasma (pre-therapy) and the contralateral pituitary half in plasma obtained during or after therapy. Studies with plasmas obtained from the five patients successfully treated with MPA showed a decreased in growth hormone releasing activity during therapy in all (18-57%). Plasma obtained after Sr90 pituitary ablation in the sixth subject had 35% more growth hormone releasing activity than obtained before therapy. These results suggest that active acromegalics who respond to MPA with significantly lowered growth hormone levels may actually achieve this response because of a decrease in growth hormone releasing factor measured peripherally. The opposite response in one acromegalic subject, following Sr90 pituitary ablation and hypopituitarism, suggests that growth hormone releasing factor secretion may increase when growth hormone levels are lowered by ablative therapy.     

The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC), we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment.     

Role of monoamine oxidase-B in medroxyprogesterone acetate (17-acetoxy-6 alpha-methyl-4-pregnene-3, 20-dione) induced changes in brain dopamine levels of rats

The effect of medroxyprogesterone acetate (MPA) on brain monoamine levels and monoamine oxidase (MAO) activity was studied in adult, healthy, non-pregnant female rats. MpA was injected in a single dose of 100 mg/kg i.m. Dopamine (DA), noradrenaline (NA), 5-hydroxytryptamine (5-HT) levels and MAO activity were estimated fluorometrically in rat brian. No change in DA, NA, 5-HT or MAO activity was observed after 7 days of MPA treatment while a significant decrease in DA levels along with a significant increase in MAO activity was observed after 21 days of MPA treatment. However, there was no change in NA and 5-HT levels after 21 days of MPA administration. The selective reduction of DA by MPA could be due to an increase in MAO-B activity. MPA does not appear to increase MAO-A activity because neither of the specific substrates (NA and 5-HT) of MAO-A was found to be decreased inspite of the increase in MAO activity as estimated by the kynuramine method. These findings suggest the importance of MAO-B also in DA metabolism in rat brain.     

Ontogeny of dexamethasone binding and sodium potassium ATPase activity in experimental murine polycystic kidney disease.

The induction of polycystic kidney disease (PKD) by glucocorticoids in newborn mice behaves as a "threshold" trait, with prevalence of PKD varying in different inbred strains after exposure to an inducing steroid. C3H mice (low threshold for PKD) demonstrated greater specific dexamethasone binding than DBA mice (high threshold) on the second day of life. Treatment with methylprednisolone acetate (MPA), a cyst-inducing steroid, down regulated dexamethasone binding earlier than in DBA mice. C3H mice demonstrated greater whole kidney homogenate Na-K ATPase activity than DBA mice within 24h of MPA injection. Specific renal glucocorticoid binding may be a regulator of threshold for murine glucocorticoid induced PKD. Our findings support in vitro evidence that glucocorticoid induced Na-K ATPase activity during critical periods of nephron development is an important regulatory point of this model.     

Effect of water-soluble matrix fraction extracted from the nacre of Pinctada maxima on the alkaline phosphatase activity of cultured fibroblasts

A new approach to the isolation of the water-soluble factors from nacre without any demineralization is described and examined their effect on fibroblast cells in culture. The soluble matrix in pure water from the nacre of Pinctada maxima was analysed by size-exclusion HPLC. Four fractions (SE1-SE4) of the water-soluble matrix (WSM) were further analysed by anion-exchange HPLC. The amino acid composition of the WSM showed that it is mainly composed of glycine and alanine. SE1 and SE4 had different amino acid compositions from the whole WSM. The WSM and SE4 tested on a culture of human foetus lung tissue fibroblasts increased the alkaline phosphatase (ALP) activity. SE1 caused a decrease in ALP activity. Our results support the hypothesis that WSM promotes the differentiation of cells in vitro.     

Inhibitory Effect of Silkworm-Extract(SE) on Monoamine Oxidase Activity in Vitro and in Vivo

In vitro.MAO‐A activity was inhibited 16‐25%, and MAO‐B activity was inhibited 20‐50% by SE treatment (12.5, 25 and 50 μg), In vivo.male C57BL/6 mice Received intraperitoneal injection of SE (20 mg/kg/day) for 14 days. The results showed that MAO‐A activity of pre‐SE‐treatment mice brain was inhibited in whole brain, cerebral cortex, substantia nigra. MAO‐B activity of pre‐SE‐treatment mice brain was inhibited in substantia nigra and cerebellum than saline‐treated control group. These results suggest that SE inhibits MAO activity in vivo.which would be expected to results in anti‐depressive and neuroprotective effects.     

Issue information

The effect of 3‐mercaptopropionic acid (MPA)‐capped CdTe quantum dots (QDs) on lysozyme was systematically investigated by spectroscopic methods, enzyme activity assay, and calorimetry techniques. Results show that the MPA‐capped CdTe QDs binded to lysozyme through van der Walls forces and hydrogen bondings, causing the decrement of α‐helical content (～7%) and increment of β‐sheet content (～11%) of lysozyme. The binding caused static quenching of the fluorescence, while the microenvironment of aromatic amino acid residues did not show any significant alteration. The lysozyme activity was affected by the increasing exposure of QDs, it was inhibited to 53.77% under a 6 × 10^?7 M exposure compared with the control group. This work will provide direct evidence about enzyme toxicity of QDs to lysozyme in vitro .     

Expansion of CD11b+Ly6G+Ly6Cint cells driven by medroxyprogesterone acetate in mice bearing breast tumors restrains NK cell effector functions

The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b⁺Gr-1⁺, mostly CD11b⁺Ly6G⁺Ly6C^int and CD11b⁺Ly6G^?Ly6C^high cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b⁺Ly6G⁺Ly6C^int cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b⁺Ly6G⁺Ly6C^int cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b⁺Gr-1⁺ cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b⁺Gr-1⁺ cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b⁺Ly6G⁺Ly6C^int cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.     

Medroxyprogesterone acetate attenuates estrogen-induced nitric oxide production in human umbilical vein endothelial cells

We report the novel observation that medroxyprogesterone acetate (MPA) attenuates the induction by 17beta estradiol (E2) of both nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells. Although MPA had no effect on basal NO production or basal eNOS phosphorylation or activity, it attenuated the E2-induced NO production and eNOS phosphorylation and activity. Moreover, we examined the mechanism by which MPA attenuated the E2-induced NO production and eNOS phosphorylation. MPA attenuated the E2-induced phosphorylation of Akt, a kinase that phosphorylates eNOS. Treatment with pure progesterone receptor (PR) antagonist RU486 completely abolished the inhibitory effect of MPA on E2-induced Akt phosphorylation and eNOS phosphorylation. In addition, the effects of actinomycin D were tested to rule out the influence of genomic events mediated by nuclear PRs. Actinomycin D did not affect the inhibitory effect of MPA on E2-induced Akt phosphorylation. Furthermore, the potential roles of PRA and PRB were evaluated. In COS cells transfected with either PRA or PRB, MPA attenuated E2-induced Akt phosphorylation. These results indicate that MPA attenuated E2-induced NO production via an Akt cascade through PRA or PRB in a non-genomic manner.     

In vitro processing of the 3'-overhanging DNA in the postcleavage complex involved in V(D)J joining

The postcleavage complex involved in V(D)J joining is known to possess a transpositional strand transfer activity, whose physiological role is yet to be clarified. Here we report that RAG1 and RAG2 proteins in the signal end (SE) complex cleave the 3'-overhanging structure of the synthetic coding-end (CE) DNA in two successive steps in vitro. The 3'-overhanging structure is attacked by the SE complex imprecisely, near the double-stranded/single-stranded (ds/ss) junction, and transferred to the SE. The transferred overhang is then resolved and cleaved precisely at the ds/ss junction, generating either the linear or the circular cleavage products. Thus, the blunt-end structure is restored for the SE and variably processed ends are generated for the synthetic CE. This 3'-processing activity is observed not only with the core RAG2 but also with the full-length protein.