Annexin 1 (ANXA1) has a well-demonstrated role in early delayed inhibitory feedback of glucocorticoids in the pituitary. ANXA1 is located in folliculo-stellate (FS) cells, and glucocorticoids act on these cells to externalize and stimulate the synthesis of ANXA1. However, ANXA1 lacks a signal sequence so the mechanism by which ANXA1 is externalized from FS cells was unknown and has been investigated. The ATP-binding cassette (ABC) transporters are a large group of transporters with varied roles that include the externalization of proteins. Glucocorticoid-induced externalization of ANXA1 from an FS cell line (TtT/GF) and rat anterior pituitary was blocked by glyburide, which inhibits ABC transporters. Glyburide also blocked the glucocorticoid inhibition of forskolin-stimulated ACTH release from pituitary tissue in vitro. RT-PCR revealed mRNA and Western blotting demonstrated protein for the ATP binding cassette A1 (ABCA1) transporter in mouse FS, TtT/GF, and A549 lung adenocarcinoma cells from which glucocorticoids also induce externalization of ANXA1. In TtT/GF cells, immunofluorescence labeling revealed a near total colocalization of cell surface ANXA1 and ABCA1. We conclude that ANXA1, which mediates the early delayed feedback of glucocorticoids in the anterior pituitary, is externalized from FS cells by an ABC transporter and that the ABCA1 transporter is a likely candidate.

Our recent studies on rat pituitary tissue suggest that the annexin I-dependent inhibitory actions of glucocorticoids may not be exerted directly on endocrine cells but indirectly via folliculo-stellate (FS) cells. FS cells contain glucocorticoid receptors and abundant annexin I. We have studied the localization of annexin I in FS cells and the ability of dexamethasone to induce annexin I secretion by an FS (TtT/GF) cell line, using Western blotting and immunofluorescence microscopy. Exposure of TtT/GF cells to dexamethasone (0.1 micro M, 3 h) caused an increase in the amount of annexin I protein in the intracellular compartment and attached to the surface of the cells. In nonpermeabilized cells, immunofluorescence labeling revealed that annexin I immunoreactivity was associated with the cell surface and concentrated in focal patches on the ends of cytoplasmic processes; dexamethasone (0.1 micro M, 3 h) increased both the number and intensity of these foci. Immunogold electron microscopy confirmed in anterior pituitary tissue the presence of immunoreactive-annexin at the surface of FS cell processes contacting endocrine cells. These data support our hypothesis that annexin I is released by FS cells in response to glucocorticoids to mediate glucocorticoid inhibitory actions on pituitary hormone release via a juxtacrine mechanism.

In the male rat anterior pituitary, three morphological subtypes of cells secreting primarily prolactin (PRL) (lactotrophs) have been described. Type I contain predominantly large irregularly shaped granules, whereas type II and type III lactotrophs contain smaller spherical granules. We have previously shown that oestradiol and testosterone exert a rapid stimulatory effect selectively on type II lactotrophs but it is not known how the lactotroph subtypes respond to peptide secretagogues. We have therefore examined which cell subtype(s) release PRL in response to vasoactive intestinal peptide (VIP), thyrotrophin-releasing hormone (TRH) and prolactin-releasing peptide (PrRP-31). Pituitary segments were incubated in medium containing tannic acid (to capture exocytosis of secretory granules), either alone or with secretagogue peptide. VIP (1-10 nM), TRH (10 nM) and PrRP-31 (10 nM) all caused a significant increase (P< 0.05) in the amount of PRL granule exocytosis from type II and III lactotrophs, but had no effect on PRL exocytosis from type I. Dopamine (100 nM) inhibited basal exocytosis of immunoreactive (ir)-PRL from type I, II and III lactotrophs and PrRP-31-stimulated ir-PRL granule exocytosis from II and III lactotrophs. Treatment of lactating female rats with the dopamine D(2) receptor antagonist sulpiride (40 microg/kg) produced a significant increase (P < 0.05) in PRL granule exocytosis from type I and type III lactotrophs and a significant increase (P < 0.05) in the proportion of type I and II cells undergoing exocytosis of PRL. In conclusion, VIP, TRH and PrRP-31 selectively stimulate exocytosis from type II and III lactotrophs in the male rat, whereas all three lactotroph types are sensitive to dopamine inhibition of exocytosis in male and female rats.

The transcription factor STAT6 is strongly expressed in various tumours and is most highly expressed in human malignant lymphomas and pancreatic, colorectal, prostate and breast cancers. STAT6 is associated with cancer cell proliferation, an increased malignancy and poor prognosis. Thus, techniques aimed at reducing or blocking STAT6 expression may be useful in treating STAT6 high cancers. Among these cancers, colorectal and breast cancers represent two of the most common worldwide and their incidence is increasing every year. In 2018, colorectal and breast cancers represented 10.2% and 11.6% of all new cases of cancer diagnosed, respectively. In this study, four proprietary STAT6 specific small interfering RNA (siRNA) sequences were tested in vitro using the human colon adenocarcinoma cell line, HT-29, and the breast/duct carcinoma cell line, ZR-75-1. Decreases in STAT6 mRNA and protein levels were analysed to confirm the transfection was successful and STAT6 knockdown effects were measured by analysing cell proliferation and apoptosis. Results showed that 100nM siRNA concentration was the most effective and, although all individual sequences were capable of significantly inhibiting cell proliferation, STAT6 siRNA sequences 1 and 4 had the largest effects. STAT6 silencing also significantly induced apoptotic events. In conclusion, these results demonstrate that STAT6 siRNA sequences are capable of inhibiting proliferation of and inducing apoptosis of HT-29 colorectal cancer cells and ZR-75-1 breast cancer cells, halving the number of cancer cells in a short period of time. These experiments will be repeated in other STAT6 high cancers in vitro , and animal studies in immunocompromised mice have been planned using xenografts of STAT6-expressing human colorectal and breast cancer cells. The STAT6 siRNA sequences therefore represent a potential treatment for STAT6 high colorectal and breast cancers and a wide variety of other STAT6-expressing cancers.

Prolactin secretion is controlled by the hypothalamus, and by circulating steroids; oestrogens stimulate, but glucocorticoids inhibit prolactin release. Lactotrophs express intracellular receptors for oestrogens, but apparently not glucocorticoids. Therefore, a genomic effect of oestrogens could be direct, but that of glucocorticoids appears to be indirect. Lactotrophs are not a homogeneous cell population: some have large irregular dense-cored vesicles, others have small round vesicles, but the functional significance of this inhomogeneity is far from clear. Oestradiol and testosterone can stimulate rapid release of prolactin selectively from type II lactotrophs characterised by small round vesicles. Progesterone and other steroids do not exert this effect, which results from a non-genomic action of oestradiol and testosterone. Glucocorticoid inhibition of secretagogue-induced prolactin secretion is mimicked by annexin 1 (lipocortin 1), a protein induced by glucocorticoids in the pituitary and many other tissues, and can be blocked by annexin 1 immunoneutralisation and antisense. Glucocorticoid inhibition of ACTH and growth hormone secretion also involves annexin 1. Pituitary annexin 1 is located in folliculo-stellate cells; these express glucocorticoid receptors, and glucocorticoids induce annexin-1 synthesis. Annexin 1 is externalised from folliculo-stellate cells in response to glucocorticoids, despite the fact that it lacks a secretory signal sequence and is not packaged in vesicles. Inhibition of annexin 1 externalisation by glyburide suggests involvement of an ABC (ATP-binding cassette) transporter in externalisation. Both oestradiol and glucocorticoids therefore influence the secretion of prolactin by novel direct and indirect mechanisms, in addition to their much better understood effects on transcription via classical intracellular steroid receptors.

Glucocorticoid feedback on the pituitary inhibits the secretion of many pituitary hormones, including ACTH, growth hormone and prolactin. This inhibition involves not only the relatively slow genomic inhibition of hormone synthesis via classical intracellular glucocorticoid receptors but also much a more rapid inhibition which is independent of transcription. Annexin 1 is a protein produced by the folliculo‐stellate cells of the pituitary. Antibodies and antisense to annexin 1 block this more rapid inhibition. We now show that, despite the fact that annexin 1 lacks a signal sequence and is not packaged in secretory granules, glucocorticoids cause the externalisation of annexin 1 to a membrane‐associated pool (calcium‐dependent and bound to phospholipid) from which annexin 1 can be released. Annexin 1 is externalised at specific points on the membrane of a folliculo‐stellate cell line; these may correspond to the points at which folliculo‐stellate cells are attached to endocrine cells in situ. The externalisation of annexin 1 can be blocked by the sulphonylurea glyburide, an inhibitor of ATP‐binding cassette (ABC) transporters which can translocate other large proteins across membranes. ABC1 transporter protein and mRNA are both present in pituitary folliculo‐stellate cells. Annexin 1 is not restricted to the pituitary, but is widely distributed in macrophages and other tissues in which it is involved in the suppression of inflammation. It therefore appears that paracrine regulation can be exerted by a protein that is not packed in secretory vesicles and which is externalised from cells by a mechanism very different to that which controls the secretion of most peptides and proteins.

The transcription factor STAT6 is strongly expressed in various tumours and is most highly expressed in malignant lymphomas and pancreatic, colorectal, prostate and breast cancers. STAT6 expression in colorectal cancer is associated with an increased malignancy, poor prognosis and poor survival rates. Colorectal cancer has an incidence of approximately 1,361,000 patients per annum worldwide and approximately 60% of those cancers show STAT6 expression. Techniques aimed at reducing or blocking STAT6 expression may be useful in treating colorectal cancers. Celixir’s four proprietary STAT6 specific small interfering RNA (siRNA) sequences were tested in vitro using the human colon adenocarcinoma cell line, HT-29. The four sequences were introduced individually and in combination into HT-29 cells at different concentrations (10 to 200 nM). Decreases in STAT6 mRNA and protein levels were analysed to confirm the transfection was successful. STAT6 knockdown effects were measured by analysing cell proliferation and apoptosis. Results showed that 100nM siRNA concentration was the most effective and all four individual sequences knocked-down STAT6 mRNA and protein by more than 50%. Although all individual sequences were capable of significantly inhibiting cell proliferation, STAT6.1 and STAT6.4 were the best. STAT6 silencing also significantly induced late and total apoptotic events. In conclusion, these results demonstrate that STAT6 siRNA sequences are capable of inhibiting the proliferation, and inducing late apoptosis, of HT-29 colon cancer cells and, in some instances, halving the number of cancer cells. These experiments will be repeated using xenografts of STAT6-expressing colon cancer cells in immunocompromised mice and the STAT6 siRNA sequences will be tested in other cancers in which STAT6 is expressed. The STAT6 siRNA sequences therefore represent a potential treatment for the most serious colorectal cancers and a wide variety of STAT6-expressing cancers.

proteins.Xenografts in nude mice were used to evaluate the effect of ZQT on lung cancer cell in vivo.ELISA assay was test the liver and kidney function post ZQT treatment. Results and discussions Lung cancer cells were significantly killed by ZQT,and it inhibited the proliferation of lung cancer cells and induced cell cycle arrest at S phase and mitochondrial-related apoptosis. Moreover, ZQT induced a sustained activation of the phosphorylation of ERK and JNK. Moreover, ZQT provoked the generation of reactive oxygen species (ROS) in lung cancer cells.In vivo, ZQT suppressed tumour growth in mouse xenograft models.Besides, ZQT was safe, showing minimum toxicity in liver and kidney. Conclusion These findings suggest Traditional Chinese medicine Ze-Qi-Tang formula is promising to be a novel, potent and safe anti tumour drug candidate for lung cancer. Introduction The transcription factor STAT6 is strongly expressed in various tumours with highest expression in malignant lymphomas and pancreatic, colorectal, prostate and breast cancers. STAT6 expression in colorectal cancer is associated with an increased malignancy and a poor prognosis. Incidence of colorectal cancer is approximately 1,361,000 patients per annum worldwide and approximately 60% show STAT6 expression. Patients with colorectal cancer expressing STAT6 also show poor survival rates. The 5 year relative survival rate for patients with stage IIIC and IV colon cancer is about 53% and 11% respectively. Techniques aimed at reducing or blocking STAT6 expression may be useful in treating those cancers with STAT6 expression. Material and methods Celixir owns a patent covering four STAT6 specific small interfering RNA (siRNA) sequences. We tested these four sequences in vitro using the human colon adenocarcinoma cell line HT-29. The four sequences were introduced individually and in combination into HT-29 cells by cationic lipid-mediated transfer at different concentrations (10 to 200 nM). STAT6 mRNA and protein levels were analysed to confirm the transfection was successful. The proliferation of cells was analysed using cell counting at various times (using a NucleoCounter) and the apoptosis of cells was analysed using Annexin V and propidium iodide (PI) staining. Results and discussions 100 nM siRNA concentration was the most effective and all four individual sequences knocked-down STAT6 mRNA and protein by more than 50%. All individual sequences were capable of significantly inhibiting cell proliferation, with sequences #1 and #4 reducing the number of cells to~50% compared with control 7 days after treatment. STAT6 silencing also significantly induced late apoptotic events. Despite using several combinations of all four sequences at the same time and serially, the best results were obtained with sequences #1 and #4 individually. Introduction Albeit the advancement in breast cancer (BC) treatment, BC remains as the second leading cause of cancer death worldwide. Unlike the other BC subtypes, the lack of hormone receptor (HR) rendering the aggressive triple ne...