Ovulation provides excessive coagulation and hepatocyte growth factor signals to cause postoperative intraabdominal adhesions.

Postoperative adhesions show a higher occurrence in females aged 16-60, especially after pelvic surgeries. This study explores the role of ovulation in adhesion formation in mice. Ovarian surgery in mice with normal- or super-ovulation led to pronounced adhesions, whereas ovulation-defective Pgr-KO mice showed minimal adhesions. Specifically, exposure to ovulatory follicular fluid (FF) markedly increased the adhesion. The hazardous exposure time window was one day before to 2.5 days after the surgery. Mechanistically, early FF exposure triggered adhesions via the blood coagulation cascade, while later exposure relied on the HGF/cMET signaling pathway. Prophylactic administration of a thrombin inhibitor pre-operatively or a cMET inhibitor postoperatively effectively mitigated FF-induced adhesions, while COX inhibitor treatment exhibited no discernible effect. These findings underscore ovulation as a pivotal factor in the development of pelvic wound adhesions and advocate for targeted preventive strategies such as c-MET inhibition, scheduling surgeries outside the ovulatory period, or employing oral contraceptive measures.

Cellular PSMB4 Protein Suppresses Influenza A Virus Replication through Targeting NS1 Protein.

The nonstructural protein 1 (NS1) of influenza A virus (IAV) possesses multiple functions, such as the inhibition of the host antiviral immune responses, to facilitate viral infection. To search for cellular proteins interacting with the IAV NS1 protein, the yeast two-hybrid system was adopted. Proteasome family member PSMB4 (proteasome subunit beta type 4) was found to interact with the NS1 protein in this screening experiment. The binding domains of these two proteins were also determined using this system. The physical interactions between the NS1 and cellular PSMB4 proteins were further confirmed by co-immunoprecipitation assay and confocal microscopy in mammalian cells. Neither transiently nor stably expressed NS1 protein affected the PSMB4 expression in cells. In contrast, PSMB4 reduced the NS1 protein expression level, especially in the presence of MG132. As expected, the functions of the NS1 protein, such as inhibition of interferon activity and enhancement of transient gene expression, were suppressed by PSMB4. PSMB4 knockdown enhances IAV replication, while its overexpression attenuates IAV replication. Thus, the results of this study suggest that the cellular PSMB4 protein interacts with and possibly facilitates the degradation of the NS1 protein, which in turn suppresses IAV replication.

The Double Engines and Single Checkpoint Theory of Endometriosis.

Endometriosis is a chronic disease characterized by the ectopic localization of the endometrial tissue in the peritoneal cavity. Consequently, it causes local pathological changes and systemic symptoms, affecting at least one in every ten women. This disease is difficult to diagnose early, it is prone to dissemination, is difficult to eradicate, tends to recur, and is regarded as "a cancer of no kill". Indeed, the development of endometriosis closely resembles that of cancer in the way of mutagenesis, pelvic spreading, and immunological adaptation. While retrograde menstruation has been regarded as the primary cause of endometriosis, the role of ovulation and menstrual stimuli in the development of endometriosis has long been overlooked. The development of ovarian and peritoneal endometrioses, similar to the development of high-grade serous carcinoma in the fallopian tube fimbriae with intraperitoneal metastasis, depends highly on the carcinogens released during ovulation. Moreover, endometriosis carries an extremely hypermutated genome, which is non-inferior to the ultra-mutated endometrial cancer. The hypermutation would lead to an overproduction of new proteins or neoantigens. Because of this, the developing endometriosis may have to turn on the PD-1/PDL-1 "self-tolerance" checkpoint to evade immune surveillance, leaving an Achilles tendon for an immune checkpoint blockade. In this review, we present the double engines and single checkpoint theory of the genesis of endometriosis, provide the current pieces of evidence supporting the hypothesis, and discuss the new directions of prevention and treatment.

Ovulation Enhances Intraperitoneal and Ovarian Seedings of High-Grade Serous Carcinoma Cells Originating from the Fallopian Tube: Confirmation in a Bursa-Free Mouse Xenograft Model.

Background: Recently, new paradigms for the etiology and origin of ovarian high-grade serous carcinoma (HGSC) have emerged. The carcinogens released during ovulation transform fallopian tube epithelial cells, exfoliating and metastasizing to the peritoneal organs, including the ovaries. Solid in vivo evidence of the paradigms in a mouse model is urgently needed but is hampered by the differing tubo-ovarian structures. In mice, there is a bursa structure surrounding the distal oviduct and ovary. This, on one hand, prevents the direct influence of ovulatory follicular fluid (FF) on the exfoliated tumor cells. On the other hand, it hinders the seeding of exfoliated tumor cells into the ovary. Methods: In this study, we created a bursa-free mouse xenograft model to examine the effect of superovulation on peritoneal and ovarian metastases of transformed human tubal epithelial cells after intraperitoneal injection in NSG mice. Results: The bursa-free mouse model showed a better effect of ovulation on peritoneal metastasis. In this model, superovulation increased the number of transformed human tubal epithelial cell seedlings after intraperitoneal injection. Compared to the bursa-intact state, bursa-free ovaries were more vulnerable to external tumor seeding in either normal ovulation or superovulation state. Conclusions: This study provides the first in vivo evidence that intraperitoneal spreading of tubal HGSC cells is enhanced by ovulation. This study also demonstrated a mouse model for studying ovary-peritoneum interaction in cancer development.

Cellular models of development of ovarian high-grade serous carcinoma: A review of cell of origin and mechanisms of carcinogenesis.

High-grade serous carcinoma (HGSC) is the most common and malignant histological type of epithelial ovarian cancer, the origin of which remains controversial. Currently, the secretory epithelial cells of the fallopian tube are regarded as the main origin and the ovarian surface epithelial cells as a minor origin. In tubal epithelium, these cells acquire TP53 mutations and expand to a morphologically normal 'p53 signature' lesion, transform to serous tubal intraepithelial carcinoma and metastasize to the ovaries and peritoneum where they develop into HGSC. This shifting paradigm of the main cell of origin has revolutionarily changed the focus of HGSC research. Various cell lines have been derived from the two cellular origins by acquiring immortalization via overexpression of hTERT plus disruption of TP53 and the CDK4/RB pathway. Malignant transformation was achieved by adding canonical driver mutations (such as gain of CCNE1) revealed by The Cancer Genome Atlas or by noncanonical gain of YAP and miR181a. Alternatively, because of the extreme chromosomal instability, spontaneous transformation can be achieved by long passage of murine immortalized cells, whereas in humans, it requires ovulatory follicular fluid, containing regenerating growth factors to facilitate spontaneous transformation. These artificially and spontaneously transformed cell systems in both humans and mice have been widely used to discover carcinogens, oncogenic pathways and malignant behaviours in the development of HGSC. Here, we review the origin, aetiology and carcinogenic mechanism of HGSC and comprehensively summarize the cell models used to study this fatal cancer having multiple cells of origin and overt genomic instability.

Ovulatory Follicular Fluid Facilitates the Full Transformation Process for the Development of High-Grade Serous Carcinoma.

Background: High-grade serous carcinoma (HGSC) is mainly derived from the stepwise accumulation of driver mutations in the fallopian tube epithelium (FTE), and it subsequently metastasizes to the ovary and peritoneum that develops into a clinically evident ovarian carcinoma. The developmental process involves cell proliferation/clonal expansion, cell migration, anoikis resistance, anchorage-independent growth (AIG), peritoneum attachment, and cell invasion. Previously, we discovered FTE could be transformed by follicular fluid (FF) released from ovulation, the most crucial risk factor of ovarian cancer, and IGF axis proteins in FF confers stemness activation and clonal expansion via IGF-1R/AKT pathway. However, whether other phenotypes in advanced cancer development are involved is unknown. Methods: A panel of FTE and ovarian HGSC cell lines with different severity of transformation were treated with FF with or without IGF-1R and AKT inhibitors and analyzed for the transformation phenotypes in vitro, ex vivo, and in vivo. Results: FF largely promotes (by order of magnitude) cell migration, AIG, cell invasion, peritoneum attachment, anoikis resistance, and cell proliferation. Most of these activities worked in the full panel of cell lines. The AIG activity largely depends on IGF-1R/AKT phosphorylation, and the proliferation activity depends on an AKT phosphorylation not mediated by IGF-1R. In contrast, both AKT- and non-AKT-mediated signals are responsible for the other transformation activities. Conclusions: Our data demonstrate an extensive transformation activity of FF in the full journey of carcinogenesis, and endorsed ovulation-inhibition for the prevention and AKT-inhibition for the treatment of ovarian HGSC.

Natural compounds as potential adjuvants to cancer therapy: Preclinical evidence.

Traditional chemotherapy is being considered due to hindrances caused by systemic toxicity. Currently, the administration of multiple chemotherapeutic drugs with different biochemical/molecular targets, known as combination chemotherapy, has attained numerous benefits like efficacy enhancement and amelioration of adverse effects that has been broadly applied to various cancer types. Additionally, seeking natural-based alternatives with less toxicity has become more important. Experimental evidence suggests that herbal extracts such as Solanum nigrum and Claviceps purpurea and isolated herbal compounds (e.g., curcumin, resveratrol, and matairesinol) combined with antitumoral drugs have the potential to attenuate resistance against cancer therapy and to exert chemoprotective actions. Plant products are not free of risks: Herb adverse effects, including herb-drug interactions, should be carefully considered. LINKED ARTICLES: This article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc.

IGF-axis confers transformation and regeneration of fallopian tube fimbria epithelium upon ovulation.

BACKGROUND: The fallopian tube fimbria is regarded as the main tissue of origin and incessant ovulation as the main risk factor of ovarian high-grade serous carcinoma. Previously, we discovered the tumorigenesis activity of human ovulatory follicular fluid (FF) upon injection to the mammary fat pad of Trp53-null mice. We also found a mutagenesis activity of FF-ROS and a apoptosis-rescuing activity of Hb from retrograde menstruation. However, neither of them can explain the tumorigenesis activities of FF. METHODS: From two cohorts of ovulatory FF retrieved from IVF patients, the main growth factor responsible for the transformation of human fimbrial epithelial cells was identified. Mechanism of activation, ways of signal transduction of the growth factor, as well as the cellular and genetic phenotypes of the malignant transformation was characterized. FINDINGS: In this study, we showed that insulin-like growth factor (IGF)-axis proteins, including IGFBP-bound IGF2 as well as the IGFBP-lytic enzyme PAPP-A, are abundantly present in FF. Upon engaging with glycosaminoglycans on the membrane of fimbrial epithelial cells, PAPP-A cleaves IGFBPs and releases IGF2 to bind with IGF-1R. Through the IGF-1R/AKT/mTOR and IGF-1R/AKT/NANOG pathways, FF-IGF leads to stemness and survival, and in the case of TP53/Rb or TP53/CCNE1 loss, to clonal expansion and malignant transformation of fimbrial epithelial cells. By depleting each IGF axis component from FF, we proved that IGF2, IGFBP2/6, and PAPP-A are all essential and confer the majority of the transformation and regeneration activities. INTERPRETATION: This study revealed that the FF-IGF axis functions to regenerate tissue damage after ovulation and promote the transformation of fimbrial epithelial cells that have been initiated by p53- and Rb-pathway disruptions. FUND: The study was supported by grants of the Ministry of Science and Technology, Taiwan (MOST 106-2314-B-303-001-MY2; MOST 105-2314-B-303-017-MY2; MOST 107-2314-B-303-013-MY3), and Buddhist Tzu Chi General Hospital, Taiwan (TCMMP104-04-01).

Haemoglobin in pelvic fluid rescues Fallopian tube epithelial cells from reactive oxygen species stress and apoptosis.

Fallopian tube fimbrial epithelium is considered to be the major site of origin of ovarian high-grade serous carcinoma, with p53 loss being the earliest and universal change. We previously reported that reactive oxygen species (ROS) in the ovulatory follicular fluids (FFs) are mutagenic and cytotoxic to fimbrial epithelial cells, which are bathed in the peritoneal fluid mixed with FFs. Here, we observed that ferryl haemoglobin (Hb), which was abundantly present in ovulatory FFs and pelvic peritoneal fluids, could rescue p53-deficient immortalized fimbrial epithelial (FE25) cells and oviduct epithelial cells from Trp53-null mice from lethal ovulatory ROS stress. Ferryl Hb and FF containing high Hb levels protected FE25 cells from apoptosis, mainly by consuming extracellular ROS and reducing NADPH oxidase-mediated cell death. The remaining extracellular ROS could still induce DNA double-strand breaks in the fimbrial epithelial cells. Our study revealed that ferryl Hb in peritoneal fluid rescued ROS-stressed, DNA-damaged fimbrial epithelial cells from death, and suggested that peritoneal blood from various sources may contribute to the ovulation-induced transformation of Fallopian tube epithelium. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Platelet-Derived Growth Factor in the Ovarian Follicle Attracts the Stromal Cells of the Fallopian Tube Fimbriae.

During human ovulation, the fallopian tube fimbriae must move to the ovulation site to catch the oocyte. As the tissue-of-origin of the majority of ovarian high-grade serous carcinoma (HGSC), the fallopian tube fimbriae carrying a precursor cancer lesion may also approach the ovulatory site for metastasis. We hypothesize that platelet-derived growth factor (PDGF) in mature follicle fluid (FF) attracts the migration of PDGFR-expressing fimbriae toward the ovulating follicle. We observed that more PDGFR-ß was expressed in the distal part than in the proximal parts of the fallopian tube, particularly in stromal cells in the lamina propria. The stromal cells, but not the epithelial cells, from normal fimbriae and fallopian tube HGSC were highly chemotactic to mature FF. The chemotactic activities were positively correlated with PDGF-BB and estradiol levels in FF and were abolished by a blocking antibody of PDGFR-ß and by tyrosine kinase inhibitor imatinib. When PDGF-BB/AB was depleted from the FF, more than 80% of chemotaxis activities were diminished. This study suggests an ovarian follicle-directed and PDGF-dependent attraction of fallopian tube fimbriae before ovulation. The same mechanism may also be crucial for the ovarian homing of HGSC, which largely originates in the fimbriae.

Mutagenic, surviving and tumorigenic effects of follicular fluid in the context of p53 loss: initiation of fimbria carcinogenesis.

Ovulation is the strongest risk factor for ovarian high-grade serous carcinoma (HGSC) that largely originates from the fallopian tube fimbriae and always carries loss-of-function mutations of TP53 in both early and late lesions. Mature ovarian follicle contains high level of reactive oxygen species (ROS). When released from ovulation, follicular fluid (FF) bathes the fimbriae and may lead to DNA double-strand break (DSB) and neoplastic transformation. In this study, we examined the mutagenic and tumorigenic activities of human pre-ovulatory FFs. A subset (6/11) of FFs was found with high levels of ROS whereas the antioxidant capacities were indifferent. These ROS(high) FFs induced intracellular ROS and DSBs in the secretory cell population of fimbriae epithelium. When p53 and Rb were turned down, the FF-exposed secretory cells overcame apoptosis and expanded the population carrying ROS and DSB. The cancer initiation and promotion effects of FF were further recapitulated in Trp53 (-/-) mice. When introduced into the mammary fat pad, ROS(high) but not ROS(low) FFs induced early-onset B-cell lymphoma. Cotreatment with physiological concentration of melatonin, a potent antioxidant, ameliorated the mutagenic and tumorigenic effect of ROS(high) FF in vitro and in vivo. The study revealed ROS and mitogens in mature ovarian follicles could initiate the transformation of fimbria epithelium in the context of p53 loss and melatonin is a potent preventive agent.

Enterovirus type 71 2A protease functions as a transcriptional activator in yeast.

Enterovirus type 71 (EV71) 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME) in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

Increased ATP generation in the host cell is required for efficient vaccinia virus production.

To search for cellular genes up-regulated by vaccinia virus (VV) infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR) assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 microM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.