Stimulation of apolipoprotein D secretion by steroids coincides with inhibition of cell proliferation in human LNCaP prostate cancer cells

Although steroid hormones are known to play a predominant role in the regulation of cell growth in hormone-sensitive cancers, their mechanisms of action, especially their interaction with growth factors and/or growth inhibitors, is poorly understood. We have recently observed that the effects of androgens and estrogens on the expression of the major protein found in human breast gross cystic disease fluid, protein-24, are opposite to their respective action on cell proliferation in human breast cancer cell lines. Somewhat surprisingly, the recent elucidation of the amino acid sequence of this progesterone binding protein reveals that this tumor marker is apolipoprotein D (apo D), a member of a superfamily of lipophilic ligand carrier proteins. The present study was designed to determine whether apo D is secreted by human prostate cancer cells and could thus be a new marker of steroid action in these cancer cells, and whether the sex steroid-induced stimulation of apo D secretion coincides with inhibition of cell proliferation. We took advantage of the biphasic pattern of the effect of steroids on the proliferation of the human prostate cancer LNCaP cell line, which offers the opportunity to discriminate between positive and negative steroid receptor-regulated cell growth processes. A 10-day exposure to low concentrations of dihydrotestosterone and testosterone caused a potent stimulation of LNCaP cell proliferation, whereas incubation with higher concentrations of these androgens led to a progressive decrease in cell proliferation towards basal levels. The biphasic action of androgens was also observed on apo D secretion, the effects on apo D secretion being inversely related to their action on LNCaP cell proliferation. Similar opposite biphasic effects were also observed with 9 other steroids, thus indicating that the stimulation of secretion of this new biochemical marker coincides with inhibition of cell proliferation in LNCaP human prostatic cancer cells.

Estradiol and insulin cross -talk in breast cancer

Approximately 33% of clinical breast carcinomas require estrogens to proliferate. Epidemiological data show that insulin resistance and diabetes mellitus is 2–3 times more prevalent in women with breast cancer than those with benign breast lesions, suggesting a clinical link between insulin and estradiol. Insulin and estradiol have a synergistic effect on the growth of MCF7 breast cancer cells, and long-term estradiol treatment upregulates the expression of the key insulin signaling protein IRS-1. The goal of this study was to further define the mechanism(s) of cross-talk between insulin and estradiol in regulating the growth of breast cancer. Using MCF7 cells, acute treatment with insulin or estradiol alone was found to stimulate two activities associated with growth: Erk MAP kinase and PI 3-kinase. However, combined acute treatment had an antagonistic effect on both activities. Acute estradiol treatment inhibited the insulin-stimulated tyrosine phosphorylation of IRS-1 while increasing its serine phosphorylation; the serine phosphorylation was attenuated by the PI 3-kinase inhibitor wortmannin. The acute antagonism observed with combined estradiol and insulin are not consistent with the long-term synergistic effect on growth. In contrast, chronic estradiol treatment enhanced the insulin-sensitivity of breast cancer cells as measured by increases in total cellular insulin-stimulated tyrosine phosphorylation of IRS-1 and activation of PI 3-kinase. Estradiol stimulation of gene transcription was found to require PI 3-kinase activity but not MAP kinase activity. Insulin alone had no effect on ER transcriptional activity, but chronic treatment in combination with estradiol resulted in synergism of ER transcription. The synergistic effect of insulin and estradiol on MCF7 cell growth was also found to require PI 3-kinase but not MAP kinase activity. Therefore, chronic estradiol treatment increases insulin stimulation of PI 3-kinase, and PI 3-kinase is required for estradiol stimulation of gene transcription alone and in combined synergy with insulin. These data demonstrate that PI 3-kinase is the locus for the cross-talk between insulin and estradiol which results in enhanced breast cancer growth with long-term exposure to both hormones. This may have important clinical implications for women with high risk for breast cancer and/or diabetes mellitus. ^

The low-affinity site of the β1-adrenoceptor and its relevance to cardiovascular pharmacology

β-Adrenoceptor blocking agents (β-blockers) that at low concentrations antagonize cardiostimulant effects of catecholamines, but at high concentrations also cause cardiostimulation, have been appearing since the late 1960s. These cardiostimulant β-blockers, coined non-conventional partial agonists, antagonize the effects of catecholamines through a high-affinity site (β1HAR), but cause cardiostimulation mainly through a low-affinity site (β1LAR) of the myocardial β1-adrenoceptor. The experimental non-conventional partial agonist (−)-CGP12177 increases cardiac L-type Ca2+ current density and Ca2+ transients, shortens action potential duration but augments action potential plateau, increases heart rate and force, as well as causes arrhythmic Ca2+ transients and arrhythmic cardiocyte contractions. Other β-blockers, which do not cause cardiostimulation, consistently have lower affinity for β1LAR than β1HAR. These sites were verified and the cardiac pharmacology of non-conventional partial agonists confirmed on recombinant β1-adrenoceptors and on β1-adrenoceptors overexpressed into the heart. A targeted mutation of Asp138 to Glu138 virtually abolished the pharmacology of β1HAR but left intact the pharmacology of β1LAR. Non-conventional partial agonists may be beneficial for the treatment of peripheral autonomic neuropathy but probably due to their arrhythmic propensities, may be harmful for the treatment of chronic heart failure.

Transport of IgG across the blood-luminal barrier of the male reproductive tract of the rat and the effect of estradiol administration on reabsorption of fluid and IgG by the epididymal ducts

In rats immunized systemically with tetanus toxoid the concentration of specific anti-tetanus-toxoid-specific IgG in fluid from the rete testis and cauda epididymidis were respectively 0.6% and 1.4% the concentration in blood serum. The extratesticular duct system reabsorbed 97% of the IgG and 99% of the fluid leaving the rete, but estradiol administration affected the site of reabsorption. In untreated rats, the ductuli efferentes reabsorbed 94% of the IgG and 96% of the fluid leaving the rete, whereas estradiol-treated rats reabsorbed 83% of the IgG and 86% of the fluid, and the ductus epididymidis fully compensated for these different effects of estradiol on the ductuli efferentes. The concentrations of IgG in secretions of the seminal vesicles and prostate gland were lower (0.1% and 0.3% respectively of the titers in blood serum) than in fluids from the extratesticular ducts, and were not affected by the administration of estradiol. RT-PCR showed that Fcgrt (neonatal Fc receptor, also known as FcRn) is expressed in the reproductive ducts, where IgG is probably transported across epithelium, being particularly strong in the ductuli efferentes (where most IgG was reabsorbed) and distal caput epididymidis. It is concluded that IgG enters the rete testis and is concentrated only 2.5-fold along the extratesticular duct system, unlike spermatozoa, which are concentrated 95-fold. Further, the ductus epididymidis can recognize and compensate for changes in function of the ductuli efferentes.

Modulation of the Chlamydia trachomatis In vitro transcriptome response by the sex hormones estradiol and progesterone

BACKGROUND:Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment.RESULTS:Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response.CONCLUSION:Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.

Surviving Bioscience and Pharmacology – an eBook for accelerated students in Nursing

An eBook to support accelerated nursing students is being developed at QUT. The first component of this is a formative activity comprising key bioscience and pharmacology concepts and self-help quizzes. This initiative has been reviewed favourably by the students. The eBook will also cover requisite academic skills and revision bioscience material.

Do nursing students have the recall of bioscience necessary to study pharmacology?

Background: For medical and allied health students, bioscience knowledge underpins the successful scaffolding of learning in their developmental and advanced level units. Many of these students complete theory-based Bioscience units, followed by a unit in Pharmacology, which specifically requires knowledge of anatomy, physiology and microbiology. In general, studies of recall report relatively large losses over short retention intervals (months), which accumulate, but level off, for longer retention intervals (years) (Custers, 2010). However, there are no studies that specifically test the recall of bioscience knowledge by allied health students. Methods: We are tracking the recall of bioscience in nursing students prior to, and during, their Pharmacology unit. In each semester, students complete short, basic, knowledge-based MCQ quizzes on concepts from (i) the gastrointestinal system and (ii) fundamental microbiology. Students were given 5 days warning about the microbiology quizzes but were given no warning prior to the gastrointestinal system quiz. Performance in these quizzes was compared to individual student’s results in the final examination on these topics in the first semester of their degree. Results: At the start of the study, the nursing students performed better in the exam MCQs on the gastrointestinal system than on microbiology. In the exam, the students’ mean marks for the gastrointestinal system ranged from 69–83%, and this was successively reduced to 63%, 53% and 49% after 4, 9 and 16 months, respectively. The mean exam marks for microbiology was 48–58%, and this did not change significantly after 4 (63%), 9 (59%) or 16 months (47%). This suggests that warning the nursing students that they were to be quizzed on microbiology may have helped their recall. However, after 16 months regardless of the subject, the nursing students undertaking the Pharmacology unit recalled less than half of the bioscience quiz answers. Conclusions: Nursing students may not have the recall of bioscience necessary to study pharmacology, and this may limit their success in pharmacology. Reference: Custers, E. J. F. M. (2010). Long-term retention of basic science knowledge: a review study. Advances in Health Science Education, 15, 109–128.

Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

Although cytosolic glutathione S-transferase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes, GSTT1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GSTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals, e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestive tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

Pharmacology in one semester [Version 3, Semester 1, 2014]

- For use in Introductory Units/Courses to Biomedical/Science Students - For use with Allied Health Students who are taking pharmacology as a Unit/Course or a part Unit/Course

Infection and cellular defense dynamics in a novel 17beta-estradiol murine model of chronic human group B streptococcus genital tract colonization reveal a role for hemolysin in persistence and neutrophil accumulation

Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17β-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (106–107 CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

Identification of actin as an estradiol 17-beta-stimulated protein in the human placenta

Addition of estradiol 17-beta to first trimester human placental minces resulted in an increased synthesis of a protein of apparent molecular weight 45 kDa. The specific involvement of estrogen in the stimulation of this protein was established by demonstrating a reduction in the level of this protein by the addition of CCS 16949 A, an inhibitor of aromatase, a key enzyme in the biosynthesis of estradiol 17-beta and ICI 182,780, an estrogen receptor antagonist. The protein was purified to homogeneity and N-terminal sequencing of two of the internal peptides obtained by enzymatic digestion of the protein, as well as the absence of a free N-terminal indicated that it could be actin. This was confirmed by Western blotting using commercially available actin antiserum. The role of estradiol 17-beta in the stimulation of actin synthesis in human placenta was also established by monitoring the quantitative inhibition of DNase I by actin.