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 (MOELOW) was developed and continuously passaged in culture to mimic cellular aging (MOEHIGH). The MOEHIGH cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOEHIGH cells proliferated significantly faster than MOELOW, and the MOEHIGH cells produced more 2D foci and 3D soft agar colonies as compared to MOELOW. MOEHIGH were xenografted into athymic female nude mice both in the subcutaneous and the intraperiteonal 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 MOEHIGH and MOELOW 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. MOEHIGH had enhanced protein expression of c-myc, Cyclin E, p53 and FOXM1 with reduced expression of p21. MOEHIGH 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

Kaempferol Exhibits Progestogenic Effects in Ovariectomized Rats

OBJECTIVE: Progesterone (P4) plays a central role in women's health. Synthetic progestins are used clinically in hormone replacement therapy (HRT), oral contraceptives, and for the treatment of endometriosis and infertility. Unfortunately, synthetic progestins are associated with side effects, including cardiovascular disease and breast cancer. Botanical dietary supplements are widely consumed for the alleviation of a variety of gynecological issues, but very few studies have characterized natural compounds in terms of their ability to bind to and activate progesterone receptors (PR). Kaempferol is a flavonoid that functions as a non-steroidal selective progesterone receptor modulator (SPRM) in vitro. This study investigated the molecular and physiological effects of kaempferol in the ovariectomized rat uteri.METHODS: Since genistein is a phytoestrogen that was previously demonstrated to increase uterine weight and proliferation, the ability of kaempferol to block genistein action in the uterus was investigated. Analyses of proliferation, steroid receptor expression, and induction of well-established PR-regulated targets Areg and Hand2 were completed using histological analysis and qPCR gene induction experiments. In addition, kaempferol in silico binding analysis was completed for PR. The activation of estrogen and androgen receptor signalling was determined in vitro.RESULTS: Molecular docking analysis confirmed that kaempferol adopts poses that are consistent with occupying the ligand-binding pocket of PRA. Kaempferol induced expression of PR regulated transcriptional targets in the ovariectomized rat uteri, including Hand2 and Areg. Consistent with progesterone-l ke activity, kaempferol attenuated genistein-induced uterine luminal epithelial proliferation without increasing uterine weight. Kaempferol signalled without down regulating PR expression in vitro and in vivo and without activating estrogen and androgen receptors.CONCLUSION: Taken together, these data suggest that kaempferol is a unique natural PR modulator that activates PR signaling in vitro and in vivo without triggering PR degradation.</p

In vivo tumor growth of high-grade serous ovarian cancer cell lines

OBJECTIVE: Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown. METHODS: To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119 and UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression. RESULTS: Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic. CONCLUSIONS: Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community

Conditional Inactivation of <i>p53</i> in Mouse Ovarian Surface Epithelium Does Not Alter MIS Driven Smad2-Dominant Negative Epithelium-Lined Inclusion Cysts or Teratomas

<div><p>Epithelial ovarian cancer is the most lethal gynecological malignancy among US women. The etiology of this disease, although poorly understood, may involve the ovarian surface epithelium or the epithelium of the fallopian tube fimbriae as the progenitor cell. Disruptions in the transforming growth factor beta (TGFβ) pathway and p53 are frequently found in chemotherapy-resistant serous ovarian tumors. Transgenic mice expressing a dominant negative form of Smad2 (Smad2DN), a downstream transcription factor of the TGFβ signaling pathway, targeted to tissues of the reproductive tract were created on a FVB background. These mice developed epithelium-lined inclusion cysts, a potential precursor lesion to ovarian cancer, which morphologically resembled oviductal epithelium but exhibited protein expression more closely resembling the ovarian surface epithelium. An additional genetic “hit” of <i>p53</i> deletion was predicted to result in ovarian tumors. Tissue specific deletion of <i>p53</i> in the ovaries and oviducts alone was attempted through intrabursal or intraoviductal injection of Cre-recombinase expressing adenovirus (AdCreGFP) into <i>p53</i>^flox/flox mice. Ovarian bursal cysts were detected in some mice 6 months after intrabursal injection. No pathological abnormalities were detected in mice with intraoviductal injections, which may be related to decreased infectivity of the oviductal epithelium with adenovirus as compared to the ovarian surface epithelium. Bitransgenic mice, expressing both the Smad2DN transgene and <i>p53</i>^flox/flox, were then exposed to AdCreGFP in the bursa and oviductal lumen. These mice did not develop any additional phenotypes. Exposure to AdCreGFP is not an effective methodology for conditional deletion of floxed genes in oviductal epithelium and tissue specific promoters should be employed in future mouse models of the disease. In addition, a novel phenotype was observed in mice with high expression of the Smad2DN transgene as validated through qPCR analysis, characterized by teratoma-like lesions implicating Smad signaling in teratoma development.</p></div

Intrabursal injection of AdCreGFP, but not intraoviductal injection, modifies ovarian phenotype.

<p>Fluid-filled bursal cysts and degenerate ovaries were observed in <i>p53</i>^flox/flox mice 6 months after intrabursal injection of AdCreGFP (A-C). Black arrow indicates ovarian tissue and blue arrow indicates oviductal tissue. The TEC showed normal expression of CK8 (D), PAX8 (E), and OVGP1 (F) both three and six months after intraoviductal viral injection. Scale bar represents 100 µm.</p

Summary of Tumor Incidence.

*<p>Intraoviductal injection with leakage into the bursa.</p

Intrabursal or intraoviductal injection of adenovirus results in OSE or TEC specific infection.

<p>Immunohistochemical and immunofluorescent detection of GFP and Cre-recombinase indicates tissue specific infection. Intrabursal injection shows staining of the OSE with limited leakage to adjacent tissue including the bursa. Intraoviductal injection results in limited infection of the TEC. Scale bars represent 100 µm.</p

Synthesis of a zinc oxide/graphene hybrid material by the direct thermal decomposition of oxalate

Hybrid materials of zinc (II) oxide (ZnO) nanocrystals and graphene are of current interest due to their cheap, Earth-abundant composition, low toxicity, and varied applications in photocatalysis, sensing, and electronics among others. We have developed a novel methodology for the synthesis of such materials utilizing the thermal decomposition of zinc (II) oxalate in solid-state solution with graphene nanoplatelets. Although the procedure simply involves precursor mixing and heating, electronic interaction between the ZnO and graphitic phases is spectroscopically observed in the hybrid material—beyond that of a homogeneous mixture of ZnO and graphene—via powder XRD, XPS, and ATR-IR spectroscopy. The synthetic method employed can be easily tuned for the desired hybrid product stoichiometry, and is easily industrially scalable with minimal chemical waste products

High Smad2DN transgene expression on a FVB background results in teratoma formation.

<p>The large teratomas contained many diverse structures including squamous cysts filled with layers of keratin (A), nervous tissue (B), bone formation (C), undifferentiated cells invading into an area of nervous tissue-like cells (D), thyroid follicle-like glands (E), and ciliated cells lining fluid-filled cysts (F). Immunohistochemical staining for CK8 was strongly positive in epithelial cells lining inclusion cysts (G) and infiltrating tumor cells (H). The OSE had an altered, papillary morphology and stained positive for epithelial marker CK8 (I). Acetylated tubulin marked the ciliated cells of the inclusion cysts as well as ovarian stroma (J). Abnormal OVGP1 expression was noted in the ovarian cortex obtained from an eight-week-old mouse (K). The teratomas lacked staining for phopsho-Smad2/3 (L). Scale bar represent 100 µm<b>.</b> Amplification of the Smad2DN by real time qPCR revealed significantly higher transgene expression in mice with the teratoma phenotype as compared to the mice with only the ovarian inclusion cysts phenotype (M).</p