Jessica G. Drenzek

Notch 1 signaling is active in ovarian cancer.

OBJECTIVE.: Despite advances in chemotherapy and radical surgery, most advanced stage ovarian cancer patients die from their disease, highlighting the need for the development of novel treatment strategies. The Notch signaling pathway plays an important role in cellular differentiation, proliferation and apoptosis. We hypothesized that the active form of Notch 1, the Notch 1 intracellular domain (NICD), would be overexpressed in ovarian cancer cells and that depletion of NICD would lead to growth reduction. METHODS.: Following institutional review board approval, NICD expression was analyzed in human ovarian cancer specimens as well as the ovarian cancer cell lines OVCAR3, SKOV3, and CaOV3. In addition, the effects of Notch 1 depletion on ovarian cancer cell growth were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay for 6 days following transfection with siRNA against Notch 1. RESULTS.: Western blot analysis revealed abundant NICD expression in all 3 ovarian cancer cell lines, as well as in 16 of 21 (76%) human ovarian cancer samples. Following treatment with Notch 1 siRNA, expression of NICD was greatly reduced in all three cell lines. Furthermore, this depletion of NICD was associated with significant growth inhibition of all three ovarian cancer cell lines. CONCLUSIONS.: NICD was frequently expressed in ovarian cancer cell lines and human ovarian cancer specimens. Importantly, depletion of Notch 1 led to growth inhibition of ovarian cancer cells. These findings support the hypothesis that Notch 1 plays a role in ovarian cancer proliferation, encouraging the investigation of this pathway as a therapeutic target.

Xanthohumol decreases Notch1 expression and cell growth by cell cycle arrest and induction of apoptosis in epithelial ovarian cancer cell lines

OBJECTIVE Notch1 signaling is active in ovarian cancer and is a promising pathway for new therapies in ovarian cancer. We have previously detected high Notch1 expression in ovarian tumors. Xanthohumol has been shown to inhibit cancer cell growth and invasion, including Kaposis sarcoma, which also highly expresses Notch1. We hypothesized that the Notch1 signaling pathway is targeted by xanthohumol leading to decreased ovarian cancer cell growth. METHODS SKOV3 and OVCAR3 cells were utilized. MTT growth assays were conducted following treatment with xanthohumol. Quantitative RT-PCR and Western blot analyses were conducted to assess Notch1 down-regulation. Luciferase reporter assays were performed to assess functional down-regulation of Notch1. Cell cycle analysis was performed by flow cytometry. RESULTS Significant growth inhibition and down-regulation of Notch1 transcription and protein expression were found following xanthohumol treatment. In addition, xanthohumol increased Hes6 transcription and decreased Hes1 transcription, known downstream targets of Notch 1. These observations were associated with cell cycle inhibition as demonstrated by an increase in p21 expression and S and G2/M cell cycle arrest confirmed by an increase in phosphorylated cdc2. Furthermore, an increase in the apoptotic markers, cleaved caspase-3 and cleaved PARP were observed. CONCLUSION Xanthohumol was a potent inhibitor of ovarian cancer cell growth, and our results suggest that xanthohumol may be influencing the Notch1 pathway. These findings suggest that xanthohumol could be useful as a therapeutic agent in ovarian cancer.

Modulation of Cytokine and Chemokine Secretions in Rhesus Monkey Trophoblast Co-Culture With Decidual but not Peripheral Blood Monocyte–Derived Macrophages

Citation 
Rozner AE, Dambaeva SV, Drenzek JG, Durning M, Golos TG. Modulation of cytokine and chemokine secretions in rhesus monkey trophoblast co‐culture with decidual but not peripheral blood monocyte–derived macrophages. Am J Reprod Immunol 2011; 66: 115–127

Expression of indoleamine 2,3-dioxygenase in the rhesus monkey and common marmoset

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step of tryptophan degradation along the kynurenine pathway, and is hypothesized to limit tryptophan availability at embryo implantation and prevent maternal T cell activation at the maternal-fetal interface. To determine if nonhuman primates are suitable models for investigating the role of IDO during pregnancy, we defined the expression of IDO in the rhesus monkey and common marmoset with particular attention to the female reproductive tract and placenta. IDO mRNA was detected by RT-PCR in the rhesus monkey term placenta, lung, small intestine, spleen, lymph node and nonpregnant uterus, and also in the common marmoset placenta. Immunohistochemical analysis of rhesus monkey tissues localized IDO to glandular epithelium of nonpregnant endometrium and first trimester decidua, vessel endothelium of nonpregnant myometrium, first trimester decidua and term decidua, and villous vessel endothelium and syncytiotrophoblast of term placenta. Western blot analysis confirmed IDO in rhesus monkey term placenta. In the common marmoset, IDO was detected in glandular epithelium of the nonpregnant uterus and in the decidua at day 60 and day 128 of gestation. IDO activity was higher in rhesus monkey and common marmoset decidua and placentas than in other tissues. Confirmation of IDO expression in rhesus monkey and common marmoset uterine and placental tissues supports the hypothesis that this enzyme regulates immune activation at the maternal-fetal interface and demonstrates that nonhuman primates may provide models with distinct similarities to human placentation to study the role of IDO in maternal-fetal immune dialogue.

Generation of macrophages from peripheral blood monocytes in the rhesus monkey

Macrophages are found in tissues throughout the body and are important immune cells, however, these tissue macrophages are difficult to collect and study. Therefore, the ability to differentiate macrophages from peripheral blood precursors is an important research tool. Macrophage differentiation has been well studied in humans, but differentiation in the non-human primate is poorly characterized. Using human models is not always feasible for invasive experimental studies and, therefore, developing reliable protocols for the non-human primate model is important. We describe a method to differentiate macrophages in vitro in the rhesus monkey by culturing adherent peripheral blood mononuclear cells for five days in RPMI-1640 supplemented with 1% human serum, M-CSF, and IL-1beta. The resulting cells had a distinct macrophage phenotype, the ability to secrete cytokines in response to LPS, and antigen uptake and processing capabilities.

Suppression of Mamu-AG by RNA interference.

Problem  The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu‐AG, the rhesus monkey homology of human leukocyte antigen (HLA)‐G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function.

Trophoblast differentiation, invasion and hormone secretion in a three-dimensional in vitro implantation model with rhesus monkey embryos

BackgroundThe initiation of primate embryo invasion into the endometrium and the formation of the placenta from trophoblasts, fetal mesenchyme, and vascular components are essential for the establishment of a successful pregnancy. The mechanisms which direct morphogenesis of the chorionic villi, and the interactions between trophectoderm-derived trophoblasts and the fetal mesenchyme to direct these processes during placentation are not well understood due to a dearth of systems to examine and manipulate real-time primate implantation. Here we describe an in vitro three-dimensional (3-D) model to study implantation which utilized IVF-generated rhesus monkey embryos cultured in a Matrigel explant system.MethodsBlastocyst stage embryos were embedded in a 3-D microenvironment of a Matrigel carrier and co-cultured with a feeder layer of cells generating conditioned medium. Throughout the course of embryo co-culture embryo growth and secretions were monitored. Embedded embryos were then sectioned and stained for markers of trophoblast function and differentiation.ResultsSigns of implantation were observed including enlargement of the embryo mass, and invasion and proliferation of trophoblast outgrowths. Expression of chorionic gonadotropin defined by immunohistochemical staining, and secretion of chorionic gonadotropin and progesterone coincident with the appearance of trophoblast outgrowths, supported the conclusion that a trophoblast cell lineage formed from implanted embryos. Positive staining for selected markers including Ki67, MHC class I, NeuN, CD31, vonWillebrand Factor and Vimentin, suggest growth and differentiation of the embryo following embedding.ConclusionsThis 3-D in vitro system will facilitate further study of primate embryo biology, with potential to provide a platform for study of genes related to implantation defects and trophoblast differentiation.

Abstract 4142: The effects of SBHA on Notch 1 expression and platinum sensitivity in ovarian cancer cells

Objective: Despite advances in radical surgical debulking and intraperitoneal chemotherapy, ovarian cancer remains the most deadly gynecologic malignancy. The Notch signaling pathway plays an important role in cellular differentiation, proliferation, and apoptosis. We have previously found Notch 1 intracellular domain (NICD) to be highly expressed in the most platinum resistant ovarian cancer cell lines. Recent evidence has shown that histone deacetylase inhibitors (HDACi9s) decrease platinum resistance in oral squamous cell cancers. In addition, the HDACi suberoyl bis-hydroxamic acid (SBHA) has been found to modify Notch 1 expression in other cancers. We hypothesized that SBHA would reduce ovarian cancer cell proliferation through inhibition of the Notch 1 signaling pathway and help sensitize ovarian cancer cells to platinum. Methods: Following treatment with SBHA, NICD expression in the ovarian carcinoma cell lines OVCAR3, SKOV3, and CaOV3 was analyzed by Western blot. The effects of SBHA and cisplatin on ovarian cancer cell growth were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay. Results: NICD expression by Western blot was consistently reduced in all 3 cell lines following treatment with SBHA. Dose-dependent growth inhibition of ovarian cancer cells was demonstrated with SBHA treatment. Furthermore, the addition of SBHA to cisplatin resulted in additional reduction of NICD and further growth suppression in SKOV3, the most platinum resistant cell line tested. Conclusions: SBHA treatment of ovarian cancer cells led to decreased NICD expression and dose dependent growth reduction in ovarian cancer cells. In addition, we found that SBHA in combination with platinum further reduced NICD expression and growth in SKOV3 cells. These findings provide a compelling argument for further study into the role that Notch 1 signaling may be playing in platinum resistance and ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4142.

Abstract 4147: Xanthohumol decreases Notch1 expression and cell growth in human epithelial ovarian cancer cell lines

Epithelial ovarian cancer is the most lethal of all gynecologic malignancies in the United States with the majority of cases detected with advanced disease. Therefore, it is critical that novel therapies be developed that can effectively treat ovarian cancer. Notch1 is a transmembrane receptor that upon activation is cleaved by gamma-secretase resulting in Notch1 intracellular domain (NICD) translocation to the nucleus where it heterodimerizes with the transcription factor CBF-1 converting CBF-1 from a transcriptional repressor to a transcriptional activator via recruitment of coactivators. Deregulation of the Notch pathway has been observed in cancers such as T cell lymphoblastic leukemia, pancreatic, and breast. In addition, it has been shown that NICD is expressed in ovarian cancer cell lines and ovarian tumors. Xanthohumol, a prenylated chalcone derived from hops used in brewing beer, has been shown to exhibit anti-cancer properties. Since xanthohumol has been shown to inhibit cancer cell growth and invasion, we hypothesized that the Notch1 signaling pathway is suppressed by xanthohumol leading to decreased cell growth. OVCAR3 and SKOV3 are human epithelial ovarian cancer cell lines that express high levels of NICD. These cells were treated with increasing doses of xanthohumol, and cell growth was measured by 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay on days 0, 2, 4, and 6. In addition, protein lysates were isolated on days 2 and 4 of treatment, detected by western blot using a NICD primary antibody, and protein expression was evaluated by densitometry. These treatments resulted in decreased cell growth when measured by MTT assay. In addition, when analyzed by western blot, there was a decrease in NICD protein expression. In conclusion, xanthohumol was a potent inhibitor of ovarian cancer cell growth, and our results suggest that xanthohumol is affecting the Notch pathway. Therefore, it is essential that further investigation into the role that xanthohumol has on the Notch pathway be pursued. These outcomes could prompt the use of xanthohumol as a potential chemotherapeutic agent in ovarian cancer patients who do not respond long term to standard chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4147.

ORIGINAL ARTICLE: Suppression of Mamu-AG by RNA Interference: SUPPRESSION OF MAMU-AG BY RNA INTERFERENCE

Problem  The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu‐AG, the rhesus monkey homology of human leukocyte antigen (HLA)‐G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function.