Circulating tumour cell RNA characterisation from colorectal cancer patient blood after inertial microfluidic enrichment.

The detection and molecular analysis of circulating tumour cells (CTCs) potentially provides a significant insight to the characterisation of disease, stage of progression and therapeutic options for cancer patients. Following on from the protocol by Warkiani et al. 2016, which describes a method of enriching CTCs from cancer patient blood with inertial microfluidics, we describe a method to measure the CTC RNA expression in the enriched fraction using droplet digital PCR and compare transcript detection with and without RNA pre-amplification.

The Combination of Metformin and Valproic Acid Has a Greater Anti-tumoral Effect on Prostate Cancer Growth than Either Drug Alone.

Background/aim: The hypoglycemic drug metformin (MET) and the anti-epileptic drug valproic acid (VPA) have individually shown anti-tumor effects in prostate cancer in vitro. The present study intended to investigate the efficacy of the combination of MET and VPA in prostate cancer treatment in a pre-clinical xenograft model.

Materials And Methods: Prostate cancer cell lines (LNCaP and PC-3) were inoculated under the skin of BALB/c nude mice.

Phase I study of BNC105P, carboplatin and gemcitabine in partially platinum-sensitive ovarian cancer patients in first or second relapse (ANZGOG-1103).

Purpose: The primary objective of this study was to determine the recommended dose of the vascular disrupting agent, BNC105P, in combination with gemcitabine and carboplatin in patients with ovarian cancer in first or second relapse with a minimum 4 month progression-free interval after last platinum.

Methods: Patients received carboplatin AUC4 on day 1 in combination with escalating doses of 800 or 1000 mg/m gemcitabine on days 1 and 8 and escalating doses of 12 or 16 mg/m BNC105P on days 2 and 9 every 21 days for a maximum for six cycles. Maintenance treatment with 16 mg/m BNC105P treatment continued for a maximum of six additional cycles.

The vascular disrupting agent BNC105 potentiates the efficacy of VEGF and mTOR inhibitors in renal and breast cancer.

BNC105 is a tubulin targeting compound that selectively disrupts vasculature within solid tumors. The severe tumor hypoxia and necrosis that ensues translates to short term tumor growth inhibition. We sought to identify the molecular and cellular events activated following BNC105 treatment that drives tumor recovery.

Discovery of 7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]furan (BNC105), a tubulin polymerization inhibitor with potent antiproliferative and tumor vascular disrupting properties.

A structure-activity relationship (SAR) guided design of novel tubulin polymerization inhibitors has resulted in a series of benzo[b]furans with exceptional potency toward cancer cells and activated endothelial cells. The potency of early lead compounds has been substantially improved through the synergistic effect of introducing a conformational bias and additional hydrogen bond donor to the pharmacophore. Screening of a focused library of potent tubulin polymerization inhibitors for selectivity against cancer cells and activated endothelial cells over quiescent endothelial cells has afforded 7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]furan (BNC105, 8) as a potent and selective antiproliferative.

BNC105: a novel tubulin polymerization inhibitor that selectively disrupts tumor vasculature and displays single-agent antitumor efficacy.

Vascular disruption agents (VDA) cause occlusion of tumor vasculature, resulting in hypoxia-driven tumor cell necrosis. Tumor vascular disruption is a therapeutic strategy of great potential; however, VDAs currently under development display a narrow therapeutic margin, with cardiovascular toxicity posing a dose-limiting obstacle. Discovery of new VDAs, which display a wider therapeutic margin, may allow attainment of improved clinical outcomes.

Effects of the insecticide amitraz, an alpha2-adrenergic receptor agonist, on human luteinized granulosa cells.

Background: Amitraz, an insecticide used to prevent tick and mite infestation of cattle, crops and dogs, is an alpha2-adrenergic receptor agonist that inhibits GnRH release and the ovulatory LH surge in rats. Noradrenalin, the physiological ligand for adrenergic receptors, inhibits progesterone production by IVF-derived granulosa cells, but the effects of amitraz are unknown.

Methods: Luteinized granulosa cells obtained from women undergoing ovarian stimulation were exposed to amitraz (1, 10, 50, 100 microg/ml) for 2-72 h, and to amitraz (50 microg/ml) +/- hCG or the specific alpha2-adrenergic receptor antagonist yohimbine, for 6 h.

Discovery and validation of drug targets for tumour angiogenesis.

The formation of blood vessels is a key process in the progression of solid tumours, providing the means for tumour growth and metastasis. A number of drugs are currently being developed to exploit inhibition of angiogenesis in the therapy of cancer. An even greater number of genes that are regulated in models of in vitro angiogenesis have been identified.

The effect of obesity on the ratio of type 3 17beta-hydroxysteroid dehydrogenase mRNA to cytochrome P450 aromatase mRNA in subcutaneous abdominal and intra-abdominal adipose tissue of women.

Objectives: To investigate (1) whether type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD), the enzyme which catalyzes the conversion of androstenedione to testosterone in the testis, is co-expressed with P450aromatase in the preadipocytes of women, and (2) whether the relative expression of type 3 17beta-HSD and aromatase varies in subcutaneous abdominal vs intra-abdominal adipose tissue of women.

Subjects: Subcutaneous abdominal and intra-abdominal adipose tissue was obtained from women undergoing elective abdominal surgery (age 22-78 y, body mass index (BMI) 22.4-52.

Dynamics of the membrana granulosa during expansion of the ovarian follicular antrum.

As an endocrine organ, the ovary has some unique characteristics. The formation, the maturation and the regression of the hormone producing cells really determine the timing, the amount and the type of hormone secreted. Here, we focus on the granulosa cells of ovarian follicles which express 17beta-hydroxysteroid dehydrogenase type 1 and cytochrome P450 aromatase.

Roles of extracellular matrix in follicular development.

The cellular biology and changes in the extracellular matrix of ovarian follicles during their development are reviewed. During growth of the bovine ovarian follicle the follicular basal lamina doubles 19 times in surface area. It changes in composition, having collagen IV alpha 1-26 and laminin alpha 1, beta 2 and gamma 1 at the primordial stage, and collagen IV alpha 1 and alpha 2, reduced amounts of alpha 3-alpha 5, and a higher content of laminin alpha 1, beta 2 and gamma 1 at the antral stage.

Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division.

Development of the ovarian follicular epithelium.

A lot is known about the endocrine control of the development of ovarian follicles, but a key question now facing researchers is which molecular and cellular processes take part in control of follicular growth and development. The growth and development of ovarian follicles occurs postnatally and throughout adult life. In this review, we focus on the follicular epithelium (membrana granulosa) and its basal lamina.

Evidence for alternative pathways of granulosa cell death in healthy and slightly atretic bovine antral follicles.

Granulosa cell death is an early feature of atresia; however, there are many apparent contradictions in the literature concerning the mode of granulosa cell death. We have therefore examined this process in bovine healthy and atretic antral follicles, using a variety of established techniques. Light and electron microscopic observations indicated the presence of pyknotic or shrunken nuclei in both the membrana granulosa and the antrum.

Distribution of the alpha1 to alpha6 chains of type IV collagen in bovine follicles.

During follicular development the proliferative and differentiated state of the epithelioid granulosa cells changes, and the movement of fluid across the follicular basal lamina enables the formation of an antrum. Type IV collagen is an important component of many basal laminae. Each molecule is composed of three alpha chains; however, six different type IV collagen chains have been identified.

Effects of insulin-like growth factors and binding protein 1 on bovine granulosa cell division in anchorage-independent culture.

Granulosa cells can exhibit the properties of stem cells and tumour cells. Contact with neighbouring cells does not inhibit their replication in vivo and they can divide in vitro while embedded in agar and thus without anchorage on a substratum. By culturing granulosa cells without anchorage, those cells that do not require anchorage, and thus exhibit at least one property of stem cells, divide.

Production of extracellular matrix, fibronectin and steroidogenic enzymes, and growth of bovine granulosa cells in anchorage-independent culture.

A proportion of the granulosa cells from bovine antral follicles will survive, like stem cells, in anchorage-independent culture. To study these cells, bovine granulosa cells were isolated from medium-sized follicles (3-5 mm), plated out (in aliquots of 2.5 x 10(4) viable cells) onto a 1 mL agar base, and overlaid with 1 mL of methycellulose solution in culture medium (control).

Basal lamina and other extracellular matrix produced by bovine granulosa cells in anchorage-independent culture.

Bovine granulosa cells from 3-7 mm follicles were cultured without anchorage in soft agar/methylcellulose solution for 14 days, with or without 50 ng/ml basic fibroblast growth factor. The granulosa cells divided to form colonies of cells. These were analysed by light and electron microscopy, immunohistochemistry and Western immunoblotting.

Trophic effects of myeloid leukaemia inhibitory factor (LIF) on mouse embryos.

Myeloid leukaemia inhibitory factor (LIF) is expressed at highest concentrations in the maternal endometrial glands at about the stage of blastocyst implantation. LIF is also expressed by the extraembryonic membranes of the early mouse embryo. Embryos of different ages were cultured with, or without, LIF, and embryo growth in vivo and in vitro was examined to determine whether LIF is important for embryo development.

The physiology of the ovary: maturation of ovarian granulosa cells and a novel role for antioxidants in the corpus luteum.

During folliculogenesis the granulosa cells divide whilst in contact with each other, and so exhibit some of the characteristics of stem cells. In vitro we have shown that bovine granulosa cells from 3-7 mm follicles, like stem cells, divide without the need for a substratum, and produce colonies of cells. Growth factors, bFGF and IGF's, stimulate their division.

Anchorage-independent culture of bovine granulosa cells: the effects of basic fibroblast growth factor and dibutyryl cAMP on cell division and differentiation.

During ovarian folliculogenesis granulosa cells divide while in contact with stromal cells and other granulosa cells. Following ovulation, however, they cease dividing and differentiate into large luteal cells. When cultured in monolayer, granulosa cells spontaneously differentiate into luteal cells, thus confounding the study of the follicular functions of granulosa cells in vitro, such as cell division.

Distribution of extraneuronal uptake1 in reproductive tissues: studies on cells in culture.

Cultures of stromal cells from pregnant mouse uterus, and an FL cell line derived from human amnion, displayed significant capacities to O-methylate noradrenaline. O-methylation was inhibited in the stromal cells by uptake1-inhibitors, and in the FL cell line by uptake2 inhibitors. These findings are discussed in terms of the distribution and possible functional importance of catecholamine metabolising systems in the female reproductive system.

Addition of steroids to embryo-uterine monolayer co-culture enhances embryo survival and implantation in vitro.

Co-culture with a mixed cell monolayer established from trypsinized uterine tissue increased the numbers of embryos developing in a minimum essential medium beyond the hatching blastocyst stage in the 5-day period of culture to 56.0% (343/613) compared with 30.2% (93/308) for embryos cultured in medium alone.