Vitamin D and docosahexaenoic acid inhibit proliferation of the ovarian cancer cell line OVCAR4.

BACKGROUND: Ovarian cancer is one of the deadliest cancers in women. Improved preventative, diagnostic, and therapeutic strategies are needed. Certain dietary patterns and nutrients such as vitamin D and omega-3 fatty acids are associated with reduced cancer risk, but their effects on ovarian cancer remain to be fully elucidated, and their combined effects have not been explored. AIM: To determine the individual and combined effects of the active vitamin D metabolite, calcitriol, and the omega-3 fatty acid, docosahexaenoic acid, on cell growth, and the abundance of the vitamin D receptor (VDR), proteins that modulate cell cycle progression, and apoptotic markers. METHODS: OVCAR4 cells, a model of ovarian cancer, were treated with calcitriol, and docosahexaenoic acid, either alone or in combination. Effects on cell growth were determined by the sulforhodamine B assay. Changes in VDR, the cell cycle promotor c-Myc, the cell cycle inhibitor p27 and cleaved PARP, were determined by Western blotting. RESULTS: While OVCAR4 cell growth was inhibited by individual treatment with either calcitriol or docosahexaenoic acid, the combined treatment revealed enhanced growth inhibition as compared to either treatment alone. Furthermore, long-term treatment (12 days) yielded stronger growth inhibition at lower concentrations as compared to short-term treatments (3 days). Accompanying this growth inhibition was a decrease in c-Myc, and an increase in p27. CONCLUSIONS: The observed reduction in cell growth mediated by calcitriol and docosahexaenoic acid highlights the need for further research utilizing these nutrients, alone and especially in combination, to support ovarian cancer prevention and treatment.

Water-Soluble Nanoconjugate for Enhanced Cellular Delivery of Receptor-Targeted Magnetic Resonance Contrast Agents.

ProGlo is an efficient steroid receptor-targeted magnetic resonance (MR) imaging contrast agent (CA). It has been shown to bind to the progesterone receptor (PR) and produce enhanced image contrast in PR-positive cells and tissues in vitro and in vivo. However, the hydrophobicity of the steroid targeting domain of ProGlo (logP = 1.4) limits its formulation and delivery at clinically relevant doses. In this work, a hydrophobic moiety was utilized to drive efficient adsorption onto nanodiamond (ND) clusters to form a water-soluble nanoconstruct (logP = -2.4) with 80% release in 8 h under biological conditions. In cell culture, the ND-ProGlo construct delivered increased concentrations of ProGlo to target cells compared to ProGlo alone. Importantly, these results were accomplished without the use of solvents such as DMSO, providing a significant advance toward formulating ProGlo for translational applications. Biodistribution studies confirm the delivery of ProGlo to PR(+) tissues with enhanced efficacy over untargeted controls. These results demonstrate the potential for a noncovalent ND-CA construct as a general strategy for solubilizing and delivering hydrophobic targeted MR CAs.

Microphysiologic systems in female reproductive biology.

Microphysiologic systems (MPS), including new organ-on-a-chip technologies, recapitulate tissue microenvironments by employing specially designed tissue or cell culturing techniques and microfluidic flow. Such systems are designed to incorporate physiologic factors that conventional 2D or even 3D systems cannot, such as the multicellular dynamics of a tissue-tissue interface or physical forces like fluid sheer stress. The female reproductive system is a series of interconnected organs that are necessary to produce eggs, support embryo development and female health, and impact the functioning of non-reproductive tissues throughout the body. Despite its importance, the human reproductive tract has received less attention than other organ systems, such as the liver and kidney, in terms of modeling with MPS. In this review, we discuss current gaps in the field and areas for technological advancement through the application of MPS. We explore current MPS research in female reproductive biology, including fertilization, pregnancy, and female reproductive tract diseases, with a focus on their clinical applications. Impact statement This review discusses existing microphysiologic systems technology that may be applied to study of the female reproductive tract, and those currently in development to specifically investigate gametes, fertilization, embryo development, pregnancy, and diseases of the female reproductive tract. We focus on the clinical applicability of these new technologies in fields such as assisted reproductive technologies, drug testing, disease diagnostics, and personalized medicine.

Genome-wide transcriptional regulation of estrogen receptor targets in fallopian tube cells and the role of selective estrogen receptor modulators.

BACKGROUND: The fallopian tube epithelium is one of the potential sources of high-grade serous ovarian cancer (HGSC). The use of estrogen only hormone replacement therapy increases ovarian cancer (OVCA) risk. Despite estrogen's influence in OVCA, selective estrogen receptor modulators (SERMs) typically demonstrate only a 20 % response rate. This low response could be due to a variety of factors including the loss of estrogen receptor signaling or the role of estrogen in different potential cell types of origin. The response of fallopian tube epithelium to SERMs is not known, and would be useful when determining therapeutic options for tumors arising from this cell type, such as HGSC. RESULTS: Using normal murine derived oviductal epithelial cells (mouse equivalent to the fallopian tube) estrogen receptor expression was confirmed and interaction with its ligand, estradiol, triggered mRNA and protein induction of progesterone receptor (PR). The SERMs 4-hydroxytamoxifen, raloxifene and desmethylarzoxifene, functioned as estrogen receptor antagonists in oviductal cells. Cellular proliferation and migration assays suggested that estradiol does not significantly impact cellular migration and increased proliferation. Further, using RNAseq, the oviduct specific transcriptional genes targets of ER when stimulated by estradiol and 4-hydroxytamoxifen signaling were determined and validated. The RNA-seq revealed enrichment in proliferation, anti-apoptosis, calcium signaling and steroid signaling processes. Finally, the ER and PR receptor status of a panel of HGSC cell lines was investigated including Kuramochi, OVSAHO, OVKATE, OVCAR3, and OVCAR4. OVSAHO demonstrated receptor expression and response, which highlights the need for additional models of ovarian cancer that are estrogen responsive. CONCLUSIONS: Overall, the fallopian tube has specific gene targets of estrogen receptor and demonstrates a tissue specific response to SERMs consistent with antagonistic action.

Spontaneous Transformation of Murine Oviductal Epithelial Cells: A Model System to Investigate the Onset of Fallopian-Derived Tumors.

High-grade serous carcinoma (HGSC) is the most lethal ovarian cancer histotype. The fallopian tube secretory epithelial cells (FTSECs) are a proposed progenitor cell type. Genetically altered FTSECs form tumors in mice; however, a spontaneous HGSC model has not been described. Apart from a subpopulation of genetically predisposed women, most women develop ovarian cancer spontaneously, which is associated with aging and lifetime ovulations. A murine oviductal cell line (MOE(LOW)) was developed and continuously passaged in culture to mimic cellular aging (MOE(HIGH)). The MOE(HIGH) cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOE(HIGH) cells proliferated significantly faster than MOE(LOW), and the MOE(HIGH) cells produced more 2D foci and 3D soft agar colonies as compared to MOE(LOW) MOE(HIGH) were xenografted into athymic female nude mice both in the subcutaneous and the intraperitoneal compartments. Only the subcutaneous grafts formed tumors that were negative for cytokeratin, but positive for oviductal markers, such as oviductal glycoprotein 1 and Pax8. These tumors were considered to be poorly differentiated carcinoma. The differential molecular profiles between MOE(HIGH) and MOE(LOW) were determined using RNA-Seq and confirmed by protein expression to uncover pathways important in transformation, like the p53 pathway, the FOXM1 pathway, WNT signaling, and splicing. MOE(HIGH) had enhanced protein expression of c-myc, Cyclin E, p53, and FOXM1 with reduced expression of p21. MOE(HIGH) were also less sensitive to cisplatin and DMBA, which induce lesions typically repaired by base-excision repair. A model of spontaneous tumorogenesis was generated starting with normal oviductal cells. Their transition to cancer involved alterations in pathways associated with high-grade serous cancer in humans.

Tumorigenesis and peritoneal colonization from fallopian tube epithelium.

Ovarian cancer is the most lethal gynecological malignancy, primarily because its origin and initiation factors are unknown. A secretory murine oviductal epithelial (MOE) model was generated to address the hypothesis that the fallopian tube is an origin for high-grade serous cancer. MOE cells were stably altered to express mutation in p53, silence PTEN, activate AKT, and amplify KRAS alone and in combination, to define if this cell type gives rise to tumors and what genetic alterations are required to drive malignancy. Cell lines were characterized in vitro and allografted into mice. Silencing PTEN formed high-grade carcinoma with wide spread tumor explants including metastasis into the ovary. Addition of p53 mutation to PTEN silencing did not enhance this phenotype, whereas addition of KRAS mutation reduced survival. Interestingly, PTEN silencing and KRAS mutation originating from ovarian surface epithelium generated endometrioid carcinoma, suggesting that different cellular origins with identical genetic manipulations can give rise to distinct cancer histotypes. Defining the roles of specific signaling modifications in tumorigenesis from the fallopian tube/oviduct is essential for early detection and development of targeted therapeutics. Further, syngeneic MOE allografts provide an ideal model for pre-clinical testing in an in vivo environment with an intact immune system.

Progesterone-targeted magnetic resonance imaging probes.

Determination of progesterone receptor (PR) status in hormone-dependent diseases is essential in ascertaining disease prognosis and monitoring treatment response. The development of a noninvasive means of monitoring these processes would have significant impact on early detection, cost, repeated measurements, and personalized treatment options. Magnetic resonance imaging (MRI) is widely recognized as a technique that can produce longitudinal studies, and PR-targeted MR probes may address a clinical problem by providing contrast enhancement that reports on PR status without biopsy. Commercially available MR contrast agents are typically delivered via intravenous injection, whereas steroids are administered subcutaneously. Whether the route of delivery is important for tissue accumulation of steroid-modified MRI contrast agents to PR-rich tissues is not known. To address this question, modification of the chemistry linking progesterone with the gadolinium chelate led to MR probes with increased water solubility and lower cellular toxicity and enabled administration through the blood. This attribute came at a cost through lower affinity for PR and decreased ability to cross the cell membrane, and ultimately it did not improve delivery of the PR-targeted MR probe to PR-rich tissues or tumors in vivo. Overall, these studies are important, as they demonstrate that targeted contrast agents require optimization of delivery and receptor binding of the steroid and the gadolinium chelate for optimal translation in vivo.

Chemical map of Schizosaccharomyces pombe reveals species-specific features in nucleosome positioning.

Using a recently developed chemical approach, we have generated a genome-wide map of nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S. pombe is due to a preponderance of nucleosomes separated by ∼4/5 bp, placing nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position nucleosomes is equally strong in exons as in introns, demonstrating that nucleosome positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe nucleosomes, particularly at ±20 bp around the dyad. This difference in nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete nucleosomes in S. pombe, and they prefer special rotational positions within the nucleosome, with longer tracts enriched in the 10- to 30-bp region from the dyad. S. pombe does not have a well-defined nucleosome-depleted region immediately upstream of most transcription start sites; instead, the -1 nucleosome is positioned with the expected spacing relative to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher nucleosome occupancy on DNA with high A/T content.

Two-step mechanism for modifier of transcription 1 (Mot1) enzyme-catalyzed displacement of TATA-binding protein (TBP) from DNA.

The TATA box binding protein (TBP) is a central component of the transcription preinitiation complex, and its occupancy at a promoter is correlated with transcription levels. The TBP-promoter DNA complex contains sharply bent DNA and its interaction lifetime is limited by the ATP-dependent TBP displacement activity of the Snf2/Swi2 ATPase Mot1. Several mechanisms for Mot1 action have been proposed, but how it catalyzes TBP removal from DNA is unknown. To better understand the Mot1 mechanism, native gel electrophoresis and FRET were used to determine how Mot1 affects the trajectory of DNA in the TBP-DNA complex. Strikingly, in the absence of ATP, Mot1 acts to unbend DNA, whereas TBP remains closely associated with the DNA in a stable Mot1-TBP-DNA ternary complex. Interestingly, and in contrast to full-length Mot1, the isolated Mot1 ATPase domain binds DNA, and its affinity for DNA is nucleotide-dependent, suggesting parallels between the Mot1 mechanism and DNA translocation-based mechanisms of chromatin remodeling enzymes. Based on these findings, a model is presented for Mot1 that links a DNA conformational change with ATP-induced DNA translocation.

Structural constraints in collaborative competition of transcription factors against the nucleosome.

Cooperativity in transcription factor (TF) binding is essential in eukaryotic gene regulation and arises through diverse mechanisms. Here, we focus on one mechanism, collaborative competition, which is of interest because it arises both automatically (with no requirement for TF coevolution) and spontaneously (with no requirement for ATP-dependent nucleosome remodeling factors). Previous experimental studies of collaborative competition analyzed cases in which target sites for pairs of cooperating TFs were contained within the same side of the nucleosome. Here, we utilize new assays to measure cooperativity in protein binding to pairs of nucleosomal DNA target sites. We focus on the cases that are of greatest in vivo relevance, in which one binding site is located close to the end of a nucleosome and the other binding site is located at diverse positions throughout the nucleosome. Our results reveal energetically significant positive (favorable) cooperativity for pairs of sites on the same side of the nucleosome but, for the cases examined, energetically insignificant cooperativity between sites on opposite sides of the nucleosome. These findings imply a special significance for TF binding sites that are spaced within one-half nucleosome length (74 bp) or less along the genome and may prove useful for prediction of cooperatively acting TFs genome wide.