Podocalyxin promotes an impermeable epithelium and inhibits pro-implantation factors to negatively regulate endometrial receptivity

Embryo implantation is a key step in establishing pregnancy and a major limiting factor in IVF. Implantation requires a receptive endometrium but the mechanisms governing receptivity are not well understood. We have recently discovered that podocalyxin (PCX or PODXL) is a key negative regulator of human endometrial receptivity. PCX is expressed in all endometrial epithelial cells in the non-receptive endometrium but selectively down-regulated in the luminal epithelium at receptivity. We have further demonstrated that this down-regulation is essential for implantation because PCX inhibits embryo attachment and penetration. However, how PCX confers this role is unknown. In this study, through RNAseq analysis of Ishikawa cell line stably overexpressing PCX, we discovered that PCX suppresses expression of genes controlling cell adhesion and communication, but increases those governing epithelial barrier functions, especially the adherens and tight junctions. Moreover, PCX suppresses multiple factors such as LIF and signaling pathways including Wnt and calcium signaling that support receptivity but stimulates anti-implantation genes such as LEFTY2. Functional studies confirmed that PCX promotes epithelial barrier functions by increasing key epithelial junction proteins such as E-cadherin and claudin 4. PCX thus promotes an anti-adhesive and impermeable epithelium while impedes pro-implantation factors to negatively control endometrial receptivity for implantation.

DJ-1 Activates the Noncanonical NF-κB Pathway via Interaction with Cezanne to Inhibit the Apoptosis and Promote the Proliferation of Ishikawa Cells

Endometrial cancer is generally one of the most evident malignant tumours of the female reproductive system, and the mechanisms underlying its cell proliferation and apoptosis are key to research in gynaecological oncology. In the paper, the in-depth molecular mechanism by which DJ-1 protein regulates the proliferation and apoptosis of Ishikawa cells was investigated. DJ-1 knockdown and overexpressing Ishikawa stable cell lines were established by lentiviral transduction. The levels of DJ-1 and noncanonical NF-κB signaling proteins were evaluated by Western blotting. Cell counting kit-8 (CCK-8) and flow cytometry were applied to analyze the cell viability and apoptosis. Co-immunoprecipitation experiment was utilized to assess the DJ-1-Cezanne interaction. The results showed that DJ-1 overexpression conferred apoptosis resistance and high proliferation on Ishikawa cells, while DJ-1 knockdown in Ishikawa cells produced the opposite results. These findings again suggested that DJ-1 inhibits the apoptosis and promotes the proliferation of Ishikawa cells. More crucially, further data showed that the noncanonical NF-κB activation was required for the regulation of Ishikawa cell proliferation and apoptosis by DJ-1. Meanwhile, it was found that noncanonical NF-κB pathway may be activated by DJ-1 interacting with and negatively regulating Cezanne in Ishikawa cells. Overall, this work revealed that DJ-1 associates with and negatively regulates Cezanne and consequently triggers the noncanonical NF-κB activation, thereby regulating Ishikawa cell proliferation and apoptosis.

Synthesis, Cytoxicity Evaluation and Molecular Docking of Fluorine Containing Hexahydroquinoline-3-Carbonitrile Derivatives

Background: Fluorine containing Hexahydroquinoline-3-carbonitrile derivatives were found to have potent cytotoxicity. Further, Fluorine can modulate pharmacokinetic and pharmacodynamic profile of drugs. Hence, new derivatives containing fluorine were explored as potential cytotoxic agents. Objective: Difluoro substituted compounds containing aromatic/heteroaromatic rings were designed, synthesized and screened for in vitro cytotoxicity on cancer cell lines. The active compounds were subjected to docking on Mcl-1 and ADME/T prediction. Methods: The synthesized compounds were characterized using various spectral techniques like FT-IR, 1 H NMR, 13C NMR and Mass spectra. Compounds were screened for cytotoxicity on NCI-60 cell lines at National Cancer Institute. The active compounds were evaluated additionally by MTT and SRB assay. Results: Compounds (6l and 6o) showed maximum cytotoxicity with (% GI) of 69 and 63.7 at 10 µM drug concentration respectively. Compound 6i showed potent cytotoxicity with GI50 of 7.2 µM against Ishikawa cell line. Compound 6o was nearly as active as reference with IC50 of 9.39 µM and 13.54 µM against HT-29 and HCT-116 respectively and compound 6l also showed equal potency to that of reference with IC50 of 9.66 µM against Caco-2. Compounds 6i, 6o and 6l showed high docking score suggesting their cytotoxicity. Further, ADME/T prediction revealed that all the compounds have drug likeness properties. Conclusion: Enhanced lipophilic interaction of compounds due to presence of fluorine in compounds 6i and 6l was revealed during docking study. Compound 6i can be explored as a lead molecule against other endometrial cancer in futuristic drug development.

Brain-Derived Neurotrophic Factor Regulates Ishikawa Cell Proliferation through the TrkB-ERK1/2 Signaling Pathway

(1) Background: Endometrial regulation is a necessary condition for maintaining normal uterine physiology, which is driven by many growth factors. Growth factors produced in the endometrium are thought to be related to the proliferation of endometrial cells induced by estradiol-17β (E2). In this study, we found that E2 can induce the secretion of brain-derived neurotrophic factor (BDNF) in Ishikawa cells (the cells of an endometrial cell line). Furthermore, Ishikawa cells were used in exploring the regulatory role of BDNF in endometrial cells and to clarify the potential mechanism. (2) Methods: Ishikawa cells were treated with different concentrations of BDNF (100, 200, 300, 400, and 500 ng/mL). The mRNA expression levels of various proliferation-related genes were detected through quantitative reverse transcription polymerase chain reaction, and the expression of various proliferation-related genes was detected by knocking out BDNF or inhibiting the binding of BDNF to its receptor TrkB. The expression levels of various proliferation-related genes were detected by performing Western blotting on the TrkB-ERK1/2 signaling pathway. (3) Results: Exogenous BDNF promoted the growth of the Ishikawa cells, but the knocking down of BDNF or the inhibition of TrkB reduced their growth. Meanwhile, BDNF enhanced cell viability and increased the expression of proliferation-related genes, including cyclin D1 and cyclin E2. More importantly, the BDNF-induced proliferation of the Ishikawa cells involved the ERK1/2 signaling pathway. (4) Conclusions: The stimulating effect of exogenous E2 on the expression of BDNF in the uterus and the action of BDNF promoted the proliferation of the Ishikawa cells through the TrkB-ERK1/2 signal pathway.

Catecholestradiol Activation of Adrenergic Receptors Induces Endometrial Cell Survival via p38 MAPK Signaling

Context Enhanced levels of catecholestradiols, 2-hydroxyestradiol (2-OHE2) or 4-hydroxyestradiol (4-OHE2), are reported in endometriosis. During gestation, catecholestradiol activation of adrenergic receptors (AR) elevates estrogen receptor (ER)-independent proliferation of uterine arterial endothelial cells. Objective To investigate β-AR-mediated catecholestradiol effects on human endometrial stromal cell (HESC) and epithelial cell survival in endometriosis. Design β-AR immunostaining of eutopic and ectopic endometria (n = 9). Assays for cell viability, 5-bromo-2′-deoxyuridine proliferation, apoptosis, quantitative PCR, and estrogenicity (alkaline phosphatase activity), as well as siRNA β-AR silencing and immunoblot analyses of cultured HESCs or Ishikawa cells treated with control or 2-OHE2 or 4-OHE2 ±β-AR antagonist or ±p38 MAPK inhibitor. Setting University research institution. Patients Women with or without endometriosis. Interventions None. Main Outcome Measures β-AR expression in eutopic vs ectopic endometria and regulation of HESC survival by 2-OHE2 and 4-OHE2. Results Eutopic and ectopic endometrial stromal and epithelial cells displayed β2-AR immunoreactivity with increased staining in the functionalis vs basalis layer (P < 0.05). Both 2-OHE2 and 4-OHE2 enhanced HESC and Ishikawa cell survival (P < 0.05), an effect abrogated by β-AR antagonist propranolol, but not ER antagonist ICI182,780. 2-OHE2 or 4-OHE2 failed to induce cell survival and estrogenic activity in ADRB2-silenced HESCs and in Ishikawa cells, respectively. Although 2-OHE2 inhibited apoptosis and BAX mRNA expression, 4-OHE2 induced proliferation and decreased apoptosis (P < 0.05). Both catecholestradiols elevated phospho-p38 MAPK levels (P < 0.05), which was blocked by propranolol, and p38 MAPK inhibitor reversed catecholestradiol-enhanced HESC survival. Conclusions Catecholestradiols increase endometrial cell survival by an ER-independent β-AR-mediated p38 MAPK activation, suggesting that agents blocking β-AR (e.g., propranolol) or inhibiting 2-OHE2- or 4-OHE2-generating enzymes (i.e., CYP1A1/B1) could treat endometriosis.

Cisplatin Changes Expression of SEMA3B in Endometrial Cancer

Background: Semaphorin 3B (SEMA3B) is characterized as a strong suppressing factor of the proliferation of cancerous cells and also by its anti-angiogenic effect. However, the knowledge on the changes in the expression profile of SEMA3B under the influence of cisplatin in endometrial cancer remains fragmented. The aim of this work was to note the changes in expression of SEMA3B when under the influence of cisplatin in the endometrial cancer cell line. Methods: Ishikawa cell line cells were exposed to three different concentrations of cisplatin: 2.5μM; 5μM; 10μM for 12, 24 and 48 hours and were compared to cells untreated by the drug. Changes in the expression profile of SEMA3B were determined based upon RtqPCR (mRNA) alongside the ELISA assay (protein). The Statistica 13.0 PL program was used for statistical analysis (p<0.05). Results: Changes on the transcriptome level seem to be more dynamic than on the proteome level. Regardless of the concentration given or the exposition period, the expression of semaphorin 3B was, in fact, higher in cells exposed to cisplatin. Statistically substantial differences (p<0.05) in the expression of SEMA3B mRNA and protein were seen for all incubation periods at the given cisplatin level when compared to the control. Conclusions: Cisplatin causes a growth in the expression of SEMA3B in an endometrial cancer cell culture, this results in the restoration in the state of cell homeostasis and shows the effectiveness of pharmacotherapy, including a low risk of drug resistance.

Evaluation of Changes in the Expression Profile of mRNA and Proteinencoding Adiponectin in Ishikawa Cell Line under the Influence of Cisplatin – Preliminary Report

Background: A reduced concentration of adiponectin is considered as an independent factor of the risk of inducing endometrial cancer. Cisplatin is a drug used in the therapy of this type of neoplasm. However, knowledge of the effects of cisplatin on the adiponectin level is still limited. Objective: he purpose of this study was to assess the impact of cisplatin depending on the concentration and time of exposition of the cells to the drug on the adiponectin level in the endometrial cancer cell line. Methods: : Cells of endometrial cancer cell line Ishikawa were exposed for 12,24 and 48 hour periods to cisplatin with the following concentrations: 2.5μM, 5μM, 10μM. The changes in the expression profile of adiponectin were compared to the RtqPCR reaction and ELISA test. The STATISTICA 13.0 PL program was used for statistical analysis (p<0.05). Results: : In the culture without the drug, the concentration of adiponectin was statistically lower than in the cell culture incubated with the drug. Changes on the mRNA level seem to be more specific than on the protein level, although in both cases, the same trend in the expression changes was noted. Discussion: The longer the time of exposition of the cells to the drug, the expression of mRNA, and the adiponectin protein increased. Changes in the expression profile were characterized statistically (p<0.05). Conclusion: Cisplatin, in a noticeable way, changes the expression profile of adiponectin. Molecular analysis indicated that in the case of endometrial cancer therapy should be implemented with a concentration of no less than 5 μM.

Stigmasterol sensitizes endometrial cancer cells to chemotherapy by repressing Nrf2 signal pathway

Background Chemoresistance reduces the 5-year survival rate of endometrial cancer patient, which is the current major obstacle for cancer therapy. Increasing evidence state that Nrf2 contributes to chemoresistance in several kinds of cancer. However, its role in endometrial cancer cells remains unclarified. Methods Immunohistochemistry staining was used to detect the expression of Nrf2 in normal patient and endometrial cancer patient. Stable transfection Ishikawa cell line with high level of Nrf2 was established to evaluate its role in chemoresistance. Dot blot assays were used to assess global hydroxymethylation level after stigmasterol treatment. Cellular growth profile was detected by CCK8 assay. Western blot was used to evaluate the changes of the target molecules after various treatments. Results Nrf2 is overexpressed in endometrial cancer tissues compared with the normal endometrium. Overexpression of Nrf2 resulted in decrease sensitivity to cisplatin. In addition, stigmasterol has been identified as a novel Nrf2 inhibitor. It enhanced the sensitivity of endometrial cancer cells to cisplatin, and the underlying mechanism is that stigmasterol declines the Nrf2 protein level. Conclusions Our findings identified stigmasterol as a new potential inhibitor of Nrf2 and highlight a critical role of stigmasterol in overcoming chemoresistance in endometrial cancer therapy.

miR-23a-3p increases endometrial receptivity via CUL3 during embryo implantation

A receptive endometrium is required in a successful embryo implantation. The ubiquitination-induced β-catenin degradation is related to the implantation failure.This study aimed to elucidate whether miR-23a-3p regulates endometrial receptivity via the modulation of β-catenin ubiquitination.The expressions of miR-23a-3p and CUL3 were detected in endometrial epithelial cells (EECs) isolated from pregnant mice and in hormone-induced EEC-like Ishikawa cells. The ubiquitination experiment was performed to explore the effect of CUL3 and miR-23a-3p on β-catenin ubiquitination level. The trophoblast attachment was detected by co-culturing JAR (choriocarcinoma cell line) spheroids with Ishikawa cell monolayers. miR-23a-3p was upregulated while CUL3 was downregulated in EECs at day 4 after pregnancy compared with day 1, as well as in hormone-induced Ishikawa cells. miR-23a-3p positively regulated the protein level of β-catenin without affecting the mRNA level. The ubiquitination and degradation of β-catenin was suppressed by miR-23a-3p, while it was promoted by CUL3. Immunoprecipitation confirmed the binding between CUL3 and β-catenin. Luciferase reporter assay confirmed the target relationship between miR-23a-3p and CUL3. The ubiquitination of β-catenin was modulated by the miR-23a-3p/CUL3 pathway. The overexpression of miR-23a-3p promoted JAR spheroid attachments in Ishikawa cells. miR-23a-3p is beneficial for the endometrial receptivity and embryo implantation, whose mechanism is partly through the modulation of CUL3/β-catenin.