HEC-1A Cells Research Articles

Long non‑coding RNA LINC01224 promotes cell proliferation and inhibits apoptosis by regulating AKT3 expression via targeting miR‑485‑5p in endometrial carcinoma.

Endometrial carcinoma (EC) is the most common cancer in women worldwide, yet little is known about the underlying molecular basis of EC development. LINC01224, a novel long non-coding (lnc)RNA, was recently identified as an oncogene in various types of cancer. However, the function and underlying mechanism of LINC01224 in EC is still unclear. A total of 50 pairs of tumor and adjacent normal tissue from patients with EC, three EC cell lines and one human normal endometrial stromal cell (ESC) line were subjected to reverse transcription-quantitative PCR assay to evaluate the expression levels of LINC01224. Cell Counting Kit-8, colony formation and flow cytometry assays were used to assess cell proliferation and apoptosis. Western blotting was used to measure expression levels of apoptosis- and proliferation-associated proteins and AKT3 protein. A xenograft model of HEC1A cells was established to validate the in vivo function of LINC01224 in EC tumor growth. Starbase 3.0 database prediction and luciferase reporter and RNA pull-down assays were performed to verify the binding sites between LINC01224 and microRNA (miR)-485-5p and miR-485-5p and AKT3. LINC01224 expression was significantly upregulated in both EC tumor tissue and cell lines. The upregulation of LINC01224 was negatively associated with survival of patients with EC. Functionally, LINC01224 promoted proliferation and inhibited apoptosis of EC cells; LINC01224 directly bound to and downregulated miR-485-5p to elevate the expression levels of AKT3, thereby promoting EC progression. LINC01224 depletion in EC cells hindered tumor growth in a xenograft model. The tumor suppressing effect of LINC01224-knockdown on EC progression was partly rescued by treatment with miR-485-5p inhibitor. The present data demonstrated the expression levels, clinical relevance and functional mechanism of LINC01224 in EC. LINC01224 promoted EC development via sponging miR-485-5p to elevate AKT3 expression levels; this may provide a promising therapeutic target pathway for EC treatment.

Synergistic effects of a combined treatment of PI3K/mTOR dual inhibitor LY3023414 and carboplatin on human endometrial carcinoma

ObjectivesOur study aims to investigate whether PI3K/mTOR dual inhibitor LY3023414 has synergistic effects with carboplatin in suppressing endometrial cancer (EC), and explore the mechanisms and toxicity of combined therapy. MethodsThe effects of combined therapy of LY3023414 and carboplatin on cell viability, long-term survival and cell apoptosis were studied in vitro, and on subcutaneous xenograft model of HEC-1A cells and patient derived xenograft (PDX) models with different PI3K pathway mutational patterns in vivo. The synergistic mechanisms were explored on ATM/Chk2 and PI3K signaling pathway. The toxicity of combined therapy was also observed. ResultsCombined treatment of LY3023414 and carboplatin synergistically inhibited proliferation, colony formation, promoted apoptosis of EC cells, and significantly activated ATM/Chk2 signaling pathway. LY3023414 had synergistic anti-tumor effects with carboplatin in HEC-1A subcutaneous xenograft which harbors PIK3CA mutation. The sensitivity to LY3023414 and carboplatin differed in PDX of EC cases with different mutational patterns of PI3K pathway, and combined therapy exhibited distinct synergistic anti-tumor effects in those harboring different PI3K pathway mutations. No increased drug toxicity in nude mice was seen in combined groups. ConclusionsCombined therapy of PI3K/mTOR dual inhibitor LY3023414 and carboplatin had synergistic anti-tumor effects in EC cell line and some of the PDX EC models, without increasing the toxicity of single drug. Enhanced carboplatin-induced DNA damage response (DDR) and cell apoptosis may be the mechanisms of synergistic effects. The anti-tumor effects may correlate with the mutational pattern of PI3K pathway, which provides experimental basis of individual treatments of ECs in the future.

CircZNF124 regulates cell proliferation, leucine uptake, migration and invasion by miR-199b-5p/SLC7A5 pathway in endometrial cancer.

BackgroundRecent studies have revealed that circular RNA participates in endometrial carcinoma (EC) progression. Here we investigated the role of circRNA zinc finger protein 124 (circZNF124) in EC genesis and underlying mechanism.MethodsThe expression levels of circZNF124, microRNA‐199b‐5p (miR‐199b‐5p) and solute carrier family 7 member 5 (SLC7A5) were detected by quantitative real‐time polymerase chain reaction. The expression of SLC7A5 and other indicated marker proteins was determined by western blot analysis. For functional assay, cell proliferation, leucine uptake and metastasis were investigated by total cell number, cell counting kit‐8, cell colony formation, leucine uptake or transwell assay. The interaction between miR‐199b‐5p and circZNF124 or SLC7A5 was predicted by starbase online database, and identified by mechanism assays. The impact of circZNF124 absence on tumor growth in vivo was revealed by xenograft mouse model assay. Immunohistochemistry assay was implemented to detect the positive expression rate of nuclear proliferation marker (Ki67).ResultsCircZNF124 and SLC7A5 expression were significantly increased, while miR‐199b‐5p was decreased in EC tissues and cells compared with normal endometrial tissues or cells. CircZNF124 expression was closely associated with EC severity and lymph node metastasis. Additionally, circZNF124 depletion repressed cell proliferation, leucine uptake, migration and invasion in both HEC1A and Ishikawa cells. CircZNF124 regulated SLC7A5 expression by binding to miR‐199b‐5p. MiR‐199b‐5p inhibitors or SLC7A5 overexpression attenuated circZNF124 silencing‐mediated EC malignant progression. Furthermore, SLC7A5 absence inhibited tumor growth in vivo.ConclusionCircZNF124 depletion inhibited EC cell malignancy by miR‐199b‐5p/SLC7A5 pathway, which demonstrated that circZNF124 had the potential as a therapeutic target for EC.

Nitric oxide positively affects endometrial receptivity via FAAH and NAPE-PLD in vitro.

SummaryObjectiveTo determine if models of human 'receptive' and 'non-receptive endometrium' differ in their responses to nitric oxide (NO) supplementation by measuring the levels of the enzymes of the endocannabinoid system (ECS) (fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD)), which control the 'anandamide tone' essential for successful pregnancy.DesignA study of FAAH and NAPE-PLD expression (using human endometrium) through the menstrual cycle and an in vitro using a model of 'receptive' (Ishikawa) and 'non-receptive' (HEC-1A) human endometrial cell lines treated with the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP).ResultsImmunoreactivity measured by optimised H-score for both FAAH and NAPE-PLD was reduced in secretory (receptive) endometrium compared to proliferative (non-receptive) endometrium (P = 0.0009 and <0.0001, respectively). FAAH and NAPE transcript levels were significantly higher in untreated Ishikawa cells than in HEC-1A cells (P = 0.0228 and 0.0001, respectively). Treatment of cultures with SNAP resulted in an increase in the amount of FAAH mRNA produced by Ishikawa cells and a decrease in NAPE-PLD mRNA. No effect of SNAP was observed in HEC-1A cells. Similarly, FAAH protein was significantly decreased in endometria representative of the receptive endometrium.ConclusionThese data suggest that NO most likely affects the expression of ECS enzymes in the implantation site of a receptive endometrium; a phenomenon not seen in a non-receptive endometrium. These effects are most marked with FAAH expression, suggesting that FAAH may play the more critical role in ensuring the correct 'anandamide tone' for successful embryo implantation than NAPE-PLD.Lay summaryEmbryo implantation into the wall of the uterus is only successful when the inner wall of the uterus (the endometrium) is ‘receptive’, because if it is ‘non-receptive’, implantation will fail. Previous work showed that enzymes of the 'endocannabinoid system' are critical for implantation by maintaining the correct level of a fat called anandamide. This is by balancing its synthesis (by N-acylphosphatidylethanolamine specific phospholipase D, NAPE-PLD) and degradation (by fatty acid amide hydrolase, FAAH). Using immortalised cell lines as models of ‘receptive’ and ‘non-receptive’ human endometrium, we demonstrate a key stimulator of implantation, nitric oxide, has a positive effect on implantation by both increasing the mRNA levels of the degrading enzyme (FAAH) and decreasing the expression of the synthesising enzyme (NAPE-PLD). These effects are most marked with the degrading enzyme, suggesting that FAAH plays a more critical role than NAPE-PLD in ensuring the correct 'anandamide tone' for successful embryo implantation.

OSU-03012 Disrupts Akt Signaling and Prevents Endometrial Carcinoma Progression in vitro and in vivo.

PurposeOSU-03012 is a celecoxib derivative lacking cyclooxygenase-2 inhibitory activity and a potent PDK1 inhibitor which has been shown to inhibit tumor growth in various ways. However, the role of OSU-03012 in endometrial carcinoma (EC) in which the PI3K/Akt signaling pathway highly activated has not been studied. Here, we determined the potency of OSU-03012 in suppressing EC progression in vitro and in vivo, and studied the underlined mechanisms.MethodsThe human EC Ishikawa and HEC-1A cells were used as the in vitro models. CCK8 assay and flow cytometry were conducted to evaluate cell proliferation, cell cycle progression, and apoptosis. The metastatic ability was evaluated using the transwell migration assay. The Ishikawa xenograft tumor model was used to study the inhibitory effects of OSU-03012 on EC growth in vivo. Western blot analysis was performed to evaluate expressions of the cell cycle and apoptosis associated proteins.ResultsOSU-03012 could inhibit the progression of EC both in vitro and in vivo by disrupting Akt signaling. It reduced the metastatic ability of EC, led to G2/M cell cycle arrest and induced apoptosis via the mitochondrial apoptosis pathway.ConclusionOur data indicated that OSU-03012 could inhibit the progression of EC in vitro and in vivo. It can potentially be used as the targeted drug for the treatment of EC by inhibiting Akt signaling.

Fractalkine enhances endometrial receptivity and activates iron transport towards trophoblast cells in an in vitro co-culture system of HEC-1A and JEG-3 cells

Endometrium receptivity and successful implantation require a complex network of regulatory factors whom production is strictly controlled especially at the implantation window. Many regulators like steroid hormones, prostaglandins, cytokines, extracellular matrix proteins and downstream cell signalling pathways are involved in the process of embryo-endometrium interaction. Our work reveals the effect of fractalkine (FKN), a unique chemokine on progesterone receptor, SOX-17 and NRF2 expressions in HEC-1A endometrial cell line. FKN activates fractalkine receptor signalling and the expression of SOX-17 through progesterone receptor in HEC-1A endometrial cells, and as a consequence it increases endometrial receptivity. Fractalkine also activates the NRF2-Keap-1 signal transduction pathway regulating the IL-6 and IL-1β cytokine productions, which increase endometrial receptivity, as well. The NRF2 transcription factor increases the expression of the iron exporter ferroportin in HEC-1A cells activating iron release towards JEG-3 trophoblast cells. The iron measurements show that iron content of endometrial cells decreases while heme concentration increases at FKN treatment. At the same time, the trophoblast cells show increased iron uptake and total iron content. Based on our results it seems that FKN enhances the establishment of endometrial receptivity and meanwhile it regulates the iron homeostasis of endometrium contributing to the iron availability of the trophoblast cells and the embryo.

Par3/integrin β1 regulates embryo adhesion via changing endometrial luminal epithelium polarity†.

The objective is to investigate the pathophysiological significance of Par3 and integrin β1 with regard to the functionality of the endometrial luminal epithelium (LE). Design: laboratory study; setting: university research laboratory. Analysis involved endometrial aspirates and endometrial adenocarcinoma cells (HEC-1A) and endometrial carcinoma cells (RL95-2). We first examined the expression and localization of Par3 and integrin β1 in HEC-1A cells and RL95-2 cells. Then we knocked down Par3 and integrin β1 in HEC-1A cells and RL95-2 cells, respectively, and found that Par3/integrin β1 affected embryo adhesion by regulating the intercellular tight junctions' (TJs') structure and thus the polarity of the endometrial LE. These findings were also confirmed in the endometrium specimens from human and mice. The main outcome measures were the expression and localization of Par3 and integrin β1 in the endometrial epithelial cell lines and endometrium specimens and the regulations of Par3 and integrin β1 on TJs, polarity, and embryo adhesion. Following the knockdown of Par3 in HEC-1A cells, there was a reduction in the complexity of the TJs and cell polarity, and the adhered blastocysts number was significantly increased. However, the reduction of integrin β1 in RL95-2 cells resulted in effects that directly opposed those following the knockdown of Par3 in HEC-1A cells. Estrogen and progesterone reduced the expression of Par3 and promoted the expression of integrin β1 in HEC-1A cells. Par3/integrin β1 regulates embryo adhesion by regulating intercellular TJs' structure and polarity of endometrial LE under the action of ovarian hormones.

Abstract PO049: Compartment-specific profiling for evaluation of disrupted signaling in early stage, low-grade endometrial cancer

Abstract Understanding tumor microenvironment is critical to improving conservative management (non-surgical) strategies for early stage, low grade endometrial cancer (EC). This remains an unmet clinical need for women who wish to maintain fertility and those who cannot undergo surgery due to medical co-morbidities. Current regimens rely on progestin treatment, but resistance and early relapse require improved therapeutic strategies. We sought to evaluate alterations in gene expression in the epithelial and stromal compartment of EC compared to normal endometrium to identify targets for combination therapies. RNA was extracted from microdissected epithelial and stromal compartments of formalin fixed paraffin embedded early stage, low grade EC and normal endometrium (NE). Comprehensive transcriptome profiling was performed with Affymetrix Clariom D assay and pathway analysis was performed. Immunohistochemistry (IHC) was used for validation. Functional studies of differentially expressed genes were performed in Hec1A EC cell lines. Among differentially expressed genes in EC vs NE, multiple altered signaling pathways were identified. EIF2 signaling, eIF4 and p70S6K signaling, and PI3K/AKT/mTOR were the most significant pathways activated in both epithelial and stromal compartments. In the epithelia compartment, the most significant gene expression changes included reduced expression of RBPMS (RNA Binding Protein with Multiple Splicing), SORD (Sorbitol Dehydrogenase), DDX17 (DEAD-Box Helicase 17), and TPT1 (Translationally-Controlled Tumor Protein) in EC, while MTDH (Metadherin) was significantly increased in EC. Similar changes were observed in the stroma compartment, but with lower fold changes than that observed in gland. Together with decreased RBPMS expression, decreased RBPMS-interacting molecules IGF2, TCF7L2 (Transcription Factor 7 Like 2), FOS (Proto-Oncogene C-Fos) and increased GPS2 (G protein Pathway Suppressor 2) were also observed. Differential expression of proteins in gland and stroma was also confirmed by IHC. An inverse correlation of RBPMS expression and EC proliferation was also observed in EC at the protein level. Enhanced AKT and S6 protein phosphorylation were observed by RBPMS silencing in Hec1A endometrial cells. Reverse Phase Protein Array demonstrated activation of Caveolin 1, FOXO3 (Forkhead Box O3), MT-CO1 (Mitochondrially Encoded Cytochrome C Oxidase I), L1CAM (L1 Cell Adhesion Molecule) with RBPMS silencing in Hec1A cell. These molecules are involved in pathways such as molecular mechanisms of cancer, estrogen receptor signaling, and senescence pathway. While estrogen-regulated changes were prominent in early stage low-grade EC, the identification of additional signaling changes provide support for an ongoing trial of levonorgestrel IUD + everolimus (mTOR inhibitor) and can be probed further to identify new targets for combination conservative management strategies. Additionally, biomarkers from these pathways, such as RBPMS, can be studied to guide the selection of progestin-only therapy versus combination therapy. Citation Format: Qian Zhang, Xiaoping Su, Joseph Celestino, Ying Yuan, Samuel C. Mok, Karen H. Lu, Melinda S. Yates. Compartment-specific profiling for evaluation of disrupted signaling in early stage, low-grade endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO049.

Progesterone induces apoptosis by activation of caspase-8 and calcitriol via activation of caspase-9 pathways in ovarian and endometrial cancer cells in vitro

Previously we have shown inhibition of endometrial cancer cell growth with progesterone and calcitriol. However, the mechanisms by which the two agents attenuate proliferation have not been well characterized yet. Herein, we investigated how progesterone and calcitriol induce apoptosis in cancer cells. DNA fragmentation was upregulated by progesterone and calcitriol in ovarian and endometrial cancer cells. Time-dependent treatment of ovarian cancer cells, ES-2, and TOV-21G with progesterone enhanced caspase -8 activity after 12h, whereas OV-90, TOV-112D, HEC-1A, and HEC-59 cells showed increased activity after 24h. Caspase 9 activity was increased in all cell lines after 24h treatment with calcitriol. Pretreatment of cancer cells with a caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (Z-LEHD-fmk) significantly attenuated progesterone and calcitriol induced caspase-8 and caspase-9 expression, respectively. The expression of FasL, Fas, FAD, and pro-caspase-8, which constitute the death-inducing signaling complex (DISC), was upregulated in progesterone treated cancer cells. Knockdown of FAS or FADD with specific siRNAs significantly blocked progesterone-induced caspase-8. Cleavage of the BID was not affected by caspase-8 activation suggesting the absence of cross-talk between caspase-8 and caspase-9 pathways. Calcitriol treatment decreased mitochondrial membrane potential and increased the release of cancer cytochrome C. These findings indicate that progesterone induces apoptosis through activation of caspase-8 and calcitriol through caspase-9 activation in cancer cells. A combination of progesterone-calcitriol activates both extrinsic and intrinsic apoptotic pathways in cancer cells.

Long-Term Metformin Effect on Endometrial Cancer Development Depending on Glucose Environment &amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt;

Objectives: Hyperinsulinemia as well as prolonged and elevated estrogen exposure are considered as risk factors for endometrial cancer (EC) development. Metformin, an anti-hyperglycemic and insulin-sensitizing biguanide, displayed anti-proliferative effects in recent studies. In the present study, the effects of long-term exposure of endometrial cancer cells to low and moderate concentrations of metformin on cell viability, proliferation, clonogenicity and migration were investigated under different metabolic conditions. Study Design: EC cell lines HEC-1A and Ishikawa were cultured under normo- (NG, 5.5 mM) or hyperglycemic (HG, 17.0 mM) conditions and treated with metformin (0.01 - 5.0 mM) in the presence of β-estradiol (E2) for 7 d. Results: A concentration-dependent decrease of cellular viability was observed in the MTT and ATP assays after metformin treatment. IC50 values were between 0.7 - 3.7 mM (NG) and 3.0 - 18.3 mM (HG), respectively. A protective effect of glucose on cellular viability was detected only in the ATP assay. Furthermore, metformin (0.5 - 5.0 mM) led to a significant decrease in proliferation by 12% - 55% (NG). However, a decreased proliferation rate was only induced at 5.0 mM metformin (40%) in the presence of high glucose levels in HEC-1A cells, indicating a glucose-related resistance to anti-proliferative metformin effects, which—to a lesser extent—was also observed in Ishikawa cells. Metformin treatment also caused concentration-dependent effects on clonogenicity and decreased the number and size of colonies. In HEC-1A cells, metformin (0.5 - 5.0 mM) reduced the colony formation by 44% - 80% (NG) and 29% - 81% (HG), respectively. Slightly higher metformin concentrations (1.0 - 5.0 mM) were necessary in Ishikawa cells to reduce clonogenicity by 36% - 86% independent of glucose levels. An investigation of migration in the wound healing assay revealed that the % wound closure decreased with increasing metformin concentrations, but independent of glucose levels. After treatment with 5.0 mM metformin, migration was significantly reduced in both cell lines. Conclusion: Our in vitro findings support the hypothesis that metformin has a direct effect on endometrial cancer cell lines and reflects the importance of the local glucose environment, suggesting that metformin may be considered as a potential adjuvant agent in endometrial cancer therapy due to its direct and indirect effects on endometrial development. However, further studies are necessary that confirm the relevance of our data for clinical applications.

LncRNA RHPN1-AS1 promotes the progression of endometrial cancer through the activation of ERK/MAPK pathway.

This study aimed to investigate the function of long noncoding RNA RHPN1 antisense RNA 1 (lncRNA RHPN1-AS1) in the progression of endometrial cancer (EC) and its underlying molecular mechanisms. The RHPN1-AS1 expression was measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in EC tissues and cells. The cell clones, proliferation, cell cycle, apoptosis, migration and invasion in Ishikawa and HEC-1A cells were respectively measured by colony formation assay, cell counting kit-8 assay (CCK-8) assay, flow cytometry, wound healing assay and transwell assay. In addition, the protein expressions in Ishikawa and HEC-1A cells were measured using western blot and Immunofluorescence assay. Our data showed the RHPN1-AS1 expression was significantly upregulated in both EC tissues and cells. The expression of RHPN1-AS1 was significantly correlated with FIGO stage, histological grade, and lymph node metastasis. Additionally, silencing RHPN1-AS1 could inhibit proliferation, cell cycle progression, migration and invasion, and also promote apoptosis in Ishikawa and HEC-1A cells. Moreover, silencing RHPN1-AS1 could markedly elevate the expressions of caspase-3 and Bax, but reduce the Bcl-2 expression in Ishikawa and HEC-1A cells. We also found that silencing RHPN1-AS1 could significantly inhibit the phosphorylation of MEK and ERK in Ishikawa and HEC-1A cells. After U0126 pretreatment, the inhibition effect of silencing RHPN1-AS1 on the phosphorylation of MEK and ERK was strengthened. Our study demonstrated that RHPN1-AS1 could facilitate cell proliferation, migration and invasion, as well as inhibit apoptosis via activating ERK/MAPK pathway in EC.

Curcusone C induces apoptosis in endometrial cancer cells via mitochondria-dependent apoptotic and ERK pathway.

Jatropha curcashas been used in traditional medicine in Africa to treat cancer for thousands of years. This study aimed to examine the anti-endometrial cancer effect of Curcusone C, a naturally occurring rhamnofolane diterpene, isolated from J. curcasand reveal its molecular mechanism of action. Curcusone C treatment caused significant anti-proliferative and apoptotic effects in human endometrial cancer (EC) Ishikawa and HEC-1A cells in a dose-dependent manner. Exposure of EC cells to Curcusone C resulted in apoptosis, which was associated with cytochrome c release, caspase-3 and caspase-9 activation, Bcl-xL/Bax dysregulation, and decreased expression of inhibitors of apoptosis proteins, such as XIAP and survivin. The inhibitory effect induced by Curcusone C was greatly impaired by the overexpression of survivin or Bax-/- MEFs or the knockdown of Bim expression. Moreover, Curcusone C activated mitogen-activated protein kinases, and the ERK inhibitor U0126 significantly attenuated the growth-inhibitory and apoptotic effects of Curcusone C in Ishikawa cells. Taken together, the results demonstrate the anti-endometrial cancer potential of Curcusone C for the treatment of endometrial cancer.

CD47 Enhances Cell Viability and Migration Ability but Inhibits Apoptosis in Endometrial Carcinoma Cells via the PI3K/Akt/mTOR Signaling Pathway.

Purposes: To measure expression levels of CD47 during endometrial carcinoma development, and to determine specific modulatory effects.Methods: CD47 expression levels in endometrial carcinoma tissues and adjacent tissues were analyzed using qRT-PCR. CD47-overexpressed or downregulated cell models were established using CD47 plasmid or CD47 shRNA. The effects of CD47 on HEC-1A and Ishikawa cell growth were evaluated using CCK-8 assays. Migration ability of transfected HEC-1A and Ishikawa cells were examined using wound healing assays. Flow cytometry was used to measure the effects of CD47 on apoptosis and the cell cycle in HEC-1A and Ishikawa cells. Western blot was used to analyze the correlation between CD47 expression level and PI3K/Akt/mTOR signaling pathway.Results: Highly expressed CD47 was observed in endometrial carcinoma tissues, with higher levels in more advanced tissues than in early tissues. Upregulation of CD47 enhanced cell viability and migration ability in HEC-1A and Ishikawa cells, while silencing CD47 caused the opposite results. CD47 overexpression suppressed apoptosis and inhibited cell cycle arrest in HEC-1A and Ishikawa cells. CD47 upregulation contributes to the activation of PI3K/Akt/mTOR signaling pathway in endometrial carcinoma cells.Conclusion: CD47 exerts oncogenic functions in endometrial carcinoma by activating PI3K/Akt/mTOR signaling, suggesting it may be a novel immunotherapeutic target for therapeutic interventions.

Abstract 4143: Identification of molecular targets for conservative management of early stage, low-grade endometrial cancer through compartment-specific transcriptome profiling

Abstract Understanding tumor microenvironment is critical to improving conservative management (non-surgical) strategies for early stage, low grade endometrial cancer (EC). This remains an unmet clinical need for women who wish to maintain fertility and those who cannot undergo surgery due to medical co-morbidities. Current regimens rely on progestin treatment, but resistance and early relapse require improved therapeutic strategies. We sought to evaluate alterations in gene expression in the epithelial and stromal compartment of EC compared to normal endometrium to identify targets for combination therapies. RNA was extracted from microdissected epithelial and stromal compartments of formalin fixed paraffin embedded early stage, low grade EC and normal endometrium (NE). Comprehensive transcriptome profiling was performed with Affymetrix Clariom D assay and pathway analysis was performed. Immunohistochemistry (IHC) was used for validation. Functional studies of differentially expressed genes were performed in EC cell lines, ECC-1 and Hec1A. Focusing on differentially expressed genes in the epithelial compartment of EC vs NE, multiple activated signaling pathways were identified, including eIF2 signaling, eIF4 and p70S6K signaling, PI3K/AKT/mTOR, and hormone signaling. Top gene expression changes with significantly lower expression in EC included RBPMS (RNA-binding protein with multiple splicing), SORD (sorbitol dehydrogenase), DDX17 (DEAD-box helicase 17), and TPT1 (translationally-controlled tumor protein) gene expression, while MTDH (metadherin) was significantly increased in EC. Differential expression of proteins coded by these genes was confirmed by IHC. Additionally, RBPMS silencing enhanced AKT and S6 protein phosphorylation in Hec1A cells and forced expression of RBPMS in ECC-1 cells attenuated Erk1/2 MAP kinase activation. Expression of RBPMS inhibited cell proliferation and knockdown of RBPMS increased proliferation. Treatment of EC cells with everolimus (mTOR inhibitor) resulted in differential inhibition of proliferation based on RBPMS expression. Communication between tumor and stroma is also being evaluated. While estrogen-regulated changes were prominent in early stage low-grade EC, the discovery of alterations in other signaling pathways suggest additional targets for conservative management of the disease. The identification of signaling nodes shared between hormone-regulated pathways and mTOR signaling provides further justification for the ongoing combination therapy trial (progestin plus everolimus). Additionally, biomarkers from these pathways, such as RBPMS, can be studied to guide the selection of progestin-only therapy versus combination therapy. Citation Format: Qian Zhang, Xiaoping Su, Joseph Celestino, Ying Yuan, Samuel C. Mok, Karen H. Lu, Melinda S. Yates. Identification of molecular targets for conservative management of early stage, low-grade endometrial cancer through compartment-specific transcriptome profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4143.

Lanthanide(III) complexes with N-(2,6-dimethylphenyl)oxamate and 1,10-phenanthroline: Synthesis, characterisation and cytotoxicity against MCF-7, HEC-1A and THP-1 cell lines

Seven novel mixed-ligand complexes, [Ln(Hpma)2(phen)2(OAc)]·2H2O (Ln = La (1), Ce (2) and Nd (3)), [Er(Hpma)2(phen)(OAc)(H2O)]·EtOH·H2O (4), [Yb(Hpma)3(phen)]·2EtOH (5) and [Ln(Hpma)3(phen)(H2O)]·4H2O (Ln = Nd (6) and Gd (7)) (Hpma = N-(2,6-dimethylphenyl)oxamate; phen = 1,10-phenanthroline) were prepared in the presence of NaOH. Complexes 1–3 are isostructural and each metal ion is ten-coordinate, adopting a distorted sphenocorona geometry, with continuous shape measures of 2.702, 2.677 and 2.592, respectively. In contrast, the nona-coordinate complexes 4, 6 and 7 displayed spherical capped square antiprism molecular geometry, while the Yb(III) complex is bonded to three bidentate Hpma– ligands and one phen, forming a square antiprism structure (coordination number = 8). Besides the strong Ln(III)-ligand bonds as the main driving force for coordination, the relatively weak hydrogen bonds (OH∙∙∙O and NH∙∙∙O), CH∙∙∙O contacts, CH∙∙∙π, NH∙∙∙π and offset π-π interactions between the aromatic rings, play a key role in crystal packing. The UV–Vis-NIR absorption spectra of the metal complexes are associated with the π → π* transitions of phen and the Laporte-forbidden f-f transitions of the Nd(III), Er(III) and Gd(III) ions from the 4I9/2, 4I15/2 and 8S7/2 ground states, respectively, to the various excited J-levels. Cytotoxicity studies revealed antiproliferative activity in MCF-7 cells (p < 0.05) after treatment with phen and coordination compounds 1–7. Statistically significant cytotoxic effects were also exerted by Nd(NO3)3·6H2O, phen, 3, 6 and 7 in HEC-1A cells (p < 0.05). No cytotoxic effects were observed in THP-1 cells (p > 0.05) for all the compounds, except for the positive control curcumin; indicating that the target compounds do not exert adverse effects on the immune system monocytes.

Effects of Cordycepin, Gold Nanostar, and Their Combination on Endometrial Cancer Cells

The incidence rate of (EC) has increased significantly in the past 2 decades. The current chemotherapy for this disease can cause intolerable side effects. Recently, both cordycepin and gold nanostars have demonstrated the ability to inhibit the growth and differentiation of cancers. This study aimed to investigate the antiEC effects of cordycepin, gold nanostars (Au NS), and the combination of the two. The gold nanostars were made by seed-mediated reduction, and their morphology was confirmed by laser particle size analysis, spectrophotometry, and electron microscopy. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assays, clonogenic assays, and flow cytometry were employed to evaluate the inhibition rate, survival fraction, and apoptosis in HEC-1A cells under different concentrations of cordycepin, Au NS, and their combination. This study revealed that both cordycepin and Au NS with 808 nm light exposure could inhibit cell proliferation, cause cell apoptosis, and inhibit the clone ability of EC cells. This study supports further investigations into Au NS-cordycepin in the development of new treatments for EC.

Ginsenoside 20(S)-protopanaxadiol induces cell death in human endometrial cancer cells via apoptosis

Background20(S)-protopanaxadiol (20(S)-PPD), one of the aglycone derivatives of major ginsenosides, has been shown to have an anticancer activity toward a variety of cancers. This study was initiated with an attempt to evaluate its anti-cancer activity toward human endometrial cancer by cell and xenograft mouse models. MethodsHuman endometrial cancer (HEC)-1A cells were incubated with different 20(S)-PPD concentrations. 20(S)-PPD cytotoxicity was evaluated using MTT assay. Apoptosis was detected using the annexin V binding assay and cell cycle analysis. Cleaved poly (ADP-ribose) polymerase (PARP) and activated caspase-9 were assessed using western blotting. HEC-1A cell tumor xenografts in athymic mice were generated by inoculating HEC-1A cells into the flank of BALB/c female mice and explored to validate 20(S)-PPD anti-endometrial cancer toxicity. Results20(S)-PPD inhibited HEC-1A cell proliferation in a dose-dependent manner with an IC50 value of 3.5 μM at 24 h. HEC-1A cells morphologically changed after 20(S)-PPD treatment, bearing resemblance to Taxol-treated cells. Annexin V-positive cell percentages were 0%, 10.8%, and 58.1% in HEC-1A cells when treated with 0, 2.5, and 5 μM of 20(S)-PPD, respectively, for 24 h. 20(S)-PPD subcutaneously injected into the HEC-1A cell xenograft-bearing mice three times a week for 17 days manifested tumor growth inhibition by as much as 18% at a dose of 80 mg/kg, which sharply contrasted to controls that showed an approximately 2.4-fold tumor volume increase. These events paralleled caspase-9 activation and PARP cleavage. Conclusion20(S)-PPD inhibits endometrial cancer cell proliferation by inducing cell death via a caspase-mediated apoptosis pathway. Therefore, the 20(S)-PPD-like ginsenosides are endowed with ample structural information that could be utilized to develop other ginsenoside-based anticancer agents.

Novel 3D embryo implantation model within macroporous alginate scaffolds

BackgroundImplantation failure remains an unsolved obstacle in reproductive medicine. Previous studies have indicated that estrogen responsiveness, specifically by estrogen receptor alpha (ERα), is crucial for proper implantation. There is an utmost need for a reliable in vitro model that mimics the events in the uterine wall during the implantation process for studying the regulatory mechanisms governing the process. The current two-dimensional and hydrogel-based in vitro models provide only short-term endometrial cell culture with partial functionality.ResultsEndometrial biopsies showed an increase in E-cadherin expression on the typical window of implantation of fertile women, compared to negligible expression in recurrent implantation failure (RIF) patients. These clinical results indicated E-cadherin as a marker for receptivity. Three-dimensional (3D) macroporous alginate scaffolds were the base for epithelial endometrial cell-seeding and long-term culture under hormone treatment that mimicked a typical menstrual cycle. The RL95–2 epithelial cell culture in macroporous scaffolds was viable for 3 weeks and showed increased E-cadherin levels in response to estrogen. Human choriocarcinoma (JAR) spheroids were used as embryo models, seeded onto cell constructs and successfully adhered to the RL95–2 cell culture. Moreover, a second model of HEC-1A with low ERα levels, showed lower E-cadherin expression and no JAR attachment. E-cadherin expression and JAR attachment were recovered in HEC-1A cells that were transfected with ERα plasmid.ConclusionsWe present a novel model that enables culturing endometrial cells on a 3D matrix for 3 weeks under hormonal treatment. It confirmed the importance of ERα function and E-cadherin for proper implantation. This platform may serve to elucidate the regulatory mechanisms controlling the implantation process, and for screening and evaluating potential novel therapeutic strategies for RIF.

Identification of early stage recurrence endometrial cancer biomarkers using bioinformatics tools.

Endometrial cancer (EC) is the sixth most common cancer in women worldwide. Early diagnosis is critical in recurrent EC management. The present study aimed to identify biomarkers of EC early recurrence using a workflow that combined text and data mining databases (DisGeNET, Gene Expression Omnibus), a prioritization algorithm to select a set of putative candidates (ToppGene), protein-protein interaction network analyses (Search Tool for the Retrieval of Interacting Genes, cytoHubba), association analysis of selected genes with clinicopathological parameters, and survival analysis (Kaplan-Meier and Cox proportional hazard ratio analyses) using a The Cancer Genome Atlas cohort. A total of 10 genes were identified, among which the targeting protein for Xklp2 (TPX2) was the most promising independent prognostic biomarker in stage I EC. TPX2 expression (mRNA and protein) was higher (P<0.0001 and P<0.001, respectively) in ETS variant transcription factor 5-overexpressing Hec1a and Ishikawa cells, a previously reported cell model of aggressive stage I EC. In EC biopsies, TPX2 mRNA expression levels were higher (P<0.05) in high grade tumors (grade 3) compared with grade 1–2 tumors (P<0.05), in tumors with deep myometrial invasion (>50% compared with <50%; P<0.01), and in intermediate-high recurrence risk tumors compared with low-risk tumors (P<0.05). Further validation studies in larger and independent EC cohorts will contribute to confirm the prognostic value of TPX2.

Efavirenz nanomicelles loaded vaginal film (EZ film) for preexposure prophylaxis (PrEP) of HIV

Preexposure prophylaxis (PrEP) using oral or vaginal microbicide is an emerging and effective strategy to prevent HIV transmission. Vaginal film is becoming more acceptable and a convenient dosage form compared to cream, gel and suppository. Extremely poor aqueous solubility of efavirenz (EFV) limits its use as vaginal microbicide. The aim of this study was to develop and evaluate a monomeric surfactant free, rapidly soluble vaginal film of EFV (EZ film). EZ film was prepared using a tetrafunctional block polymer (Tetronic 1107), carrageenan and polyvinyl alcohol (PVA) by solvent evaporation method. First, different solubilizers were screened for EFV solubility, in vitro cytotoxicity and cell membrane integrity assay on HeLa cells. Optimized film was characterized for solid state, mechanical strength, epithelial integrity, in vitro drug release in simulated vaginal fluid (SVF), simulated seminal fluid (SSF) and in vitro anti-HIV activity. Optimized EZ film showed a particle size of 48 ± 3.8 nm with PDI of 0.299. Differential scanning colorimetry (DSC) thermogram suggested the complete amorphization of EFV within the film. EZ film rapidly disintegrated (30 s) with complete release of EFV in SVF and SSF. The film was found to be non-toxic to HeLa cells and showed similar anti-HIV-1 activity as that of EFV in DMSO. EZ film did not show any significant change in the TEER value in HEC 1A cell line. Hence, the findings from the current study strongly suggest that the EZ film could be a cost-effective and convenient dosage form for PrEP of HIV.