Ishikawa Cells Research Articles

Genome-wide CRISPR screening reveals ADCK3 as a key regulator in sensitizing endometrial carcinoma cells to MPA therapy

BackgroundThe effectiveness of conservative treatment of endometrial carcinoma (EC) with oral progesterone therapy, such as medroxyprogesterone acetate (MPA), can be blunted due to primary or acquired resistance, but the underlying mechanisms remain incompletely defined.MethodsGenome-wide CRISPR screening was performed to identify potential regulators in response to MPA in Ishikawa cells. Crystal violet staining, RT–qPCR, western blotting, ChIP–qPCR and luciferase assays were employed to elucidate the p53-AarF domain-containing kinase 3 (ADCK3) regulatory axis and its roles in sensitizing EC cells to MPA treatment.ResultsADCK3 is identified as a previously unrecognized regulator in response to MPA in EC cells. Loss of ADCK3 in EC cells markedly alleviated MPA-induced cell death. Mechanistically, loss of ADCK3 primarily suppresses MPA-mediated ferroptosis by abrogating arachidonate 15-lipoxygenase (ALOX15) transcriptional activation. Moreover, we validated ADCK3 as a direct downstream target of the tumor suppressor p53 in EC cells. By stimulating the p53-ADCK3 axis, the small-molecule compound Nutlin3A synergized with MPA to efficiently inhibit EC cell growth.ConclusionsOur findings reveal ADCK3 as a key regulator of EC cells in response to MPA and shed light on a potential strategy for conservative EC treatment by activating the p53-ADCK3 axis to sensitize MPA-mediated cell death.

Talin1 regulates glucose metabolism and endometrial receptivity via GLUT-4 in patients with polycystic ovary syndrome and insulin resistance

Objectives: Talin1 is a cytoskeletal protein and is localized between cells and the extracellular matrix. This study aimed to investigate the mechanism by which Talin1 affects glucose metabolism and endometrial receptivity via glucose transporter proteins-4 (GLUT-4) in patients with polycystic ovary syndrome (PCOS) and insulin resistance (IR). Methods: We examined the expression of Talin1 and GLUT4 in the receptive endometrium of PCOS-IR and control patients. GLUT4 expression was examined after silencing and overexpression of Talin1 in Ishikawa cells. We validated the interaction between Talin1 and GLUT-4 proteins using a co-immunoprecipitation (Co-IP) assay. After successfully establishing the C57BL/6j mouse model of PCOS-IR, the expression of Talin1 and GLUT-4 were examined in PCOS-IR and control mice. The effect of Talin1 on embryo implantation and the number of live births in mice were examined. Results: Our study found low expression of Talin1 and GLUT-4 in the receptive endometrium of PCOS-IR patients compared to that in control patients (p < 0.01). The level of GLUT-4 expression decreased after silencing Talin1 in Ishikawa cells and increased after overexpression of Talin1. Co-IP results showed that Talin1 interacts with GLUT-4 protein. We successfully established a PCOS-IR C57BL/6j mouse model and found that Talin1 and GLUT-4 expression in the receptive endometrium of PCOS-IR mice were lower than that in control mice (p < 0.05). In vivo experiments confirmed that the knockdown of Talin1 affects embryo implantation (p < 0.05) and live birth rate in mice (p < 0.01). Conclusions: Talin1 and GLUT-4 expression were decreased in the endometrium of PCOS-IR patients, and Talin1 may affect glucose metabolism and endometrial receptivity through GLUT4.

Saikosaponin D exerts cytotoxicity on human endometrial cancer ishikawa cells by inducing apoptosis and inhibiting metastasis through MAPK pathways.

Saikosaponin D (SSD) is one of plant secondary metabolic active substance with effective anti-tumor ability; however, the toxicity of Saikosaponin D on human endometrial cancer Ishikawa cells is still unclear. Our results revealed that SSD displayed cytotoxicity on the Ishikawa cell with an IC50 = 15.69 μM, but was non-toxic to the human normal cell line HEK293. SSD could upregulate p21 and Cyclin B to keep cells in the G2/M stage. In addition, it activated the death receptor and mitochondrion routes to induce apoptosis in Ishikawa cells. The transwell chamber and wound healing results showed that SSD inhibited the cell migration and invasion. In addition, we found that it was closely related to the MAPK cascade pathway, and it could mediate the three classical MAPK pathways to block cell metastasis. In conclusion, SSD could be potentially beneficial as a natural secondary metabolite in preventing and treating endometrial carcinoma.

Endometriyal Hücrelerde Spirulina platensis Ekstresinin Kimyasal Bileşiminin Seçici Sitotoksisitesi: İn Vitro ve İn Siliko Yaklaşım

Microalgae are considered as an ample source of bioactive compounds. These wide variety of chemicals possess promising potential to translate into drug candidates. Hence, the aim of this study is to determine potential anti-cancer effects of Spirulina platensis microalgae extract against endometrial cancer cells. The selective cytotoxic effect and cytotoxicity index of S. platensis microalgae extract on the Ishikawa endometrial cancer cell line, the determination of DNA fragmentation and the change in gene expression levels of apoptosis-related Bad, Poly-ADP Ribose Polymerase-1 (PARP1), p53, Akt1 and caspase-3 signaling proteins were determined. ECV304 human umbilical vein endothelial cell line was used for a control cell line. Cytotoxic effects of 500 μg mL-1 of S. platensis microalgae extract on Ishikawa and ECV304 cells were determined as 43±4.87 % and 22±1.87 % respectively. In order to delineate specific compounds in the extract, GC-MS analysis was further conducted and seven major compounds revealed to be abundantly present. Since DNA fragmentation induced by S. platensis extract might be related interaction of GC-MS identified compounds with PARP1, an in silico analysis was further implemented. Results indicated the presence of a possible PARP1 inhibitory mechanism contributing to the apoptotic response. In conclusion, a possible link between a specific compound found in S. platensis microalgae extract and the cytotoxicity in endometrial cells was formed through combining in vitro and in silico approaches. The result of these approaches has contributed to the identification of potential anti-cancer compound candidates found in S. platensis microalgae extract.

Casein kinase 1 controls the shuttling of epidermal growth factor receptor and estrogen receptor in endometrial carcinoma induced by breast cancer hormonal therapy: Relevance of GPER1/Src

Casein kinase 1 plays a crucial role in carcinogenesis. 4-Hydroxytamoxifen (4-OHT), which is widely used to treat breast cancer, often leads to the development of endometrial carcinoma with poor prognosis, particularly among women who receiving long-term treatment. This study was performed to elucidate whether specific inhibition of casein kinase 1 (CK1) controls 4-OHT-mediated Ishikawa cell carcinogenesis. 4-OHT significantly stimulated the activity of estrogen receptor alpha (ERα) and nuclear translocation and expression of epidermal growth factor receptor (EGFR) from the plasma membrane to perinuclear or nuclear regions, as well as the activities of G-protein-coupled estrogen receptor 1 (GPER1) and Src in Ishikawa cells. However, inhibition of EGFR by Gefitinib blocked all these events, and inhibition of GPER1 or Src produced a partial block. GPER1 and Src controlled Ishikawa cell carcinogenesis in different manners: GPER1 accelerated EGFR mobility without affecting ERα activity, while Src activated ERα and EGFR without any change in GPER1 expression. EGFR and GPER1 performed reciprocal regulation in endometrial cell carcinogenesis via direct interaction in 4-OHT-treated Ishikawa cells, implying a possible key role of GPER1 in these events. Inhibition of CK1 by CKI-7 and IC261, however, impeded all changes beginning with EGFR translocation and activity in 4-OHT-treated Ishikawa cells. These findings indicate that inhibition of CK1 could control 4-OHT-mediated activation and translocation of ER/EGFR and GPER1/Src expression, inhibiting 4-OHT-triggered endometrial carcinogenesis. Therefore, targeting of CK1 by CKI-7 and IC261 could be a prospective adjuvant therapy for breast cancer patients taking tamoxifen.

The transcriptional foundations of interferon-λ-mediated endometrial cell to uterine receptivity.

Interferon-λ (IFN-λ) is a novel non-redundant regulator that participates in the fetal-maternal immune interaction, including immune regulation, uterine receptivity, cell migration and adhesion, and endometrium apoptosis. However, the exact transcriptional foundation for endometrial signaling of IFN-λ is not completely understood, and studies regarding IFN-λ to implantation failure in vivo are limited. The gene expression profile of human endometrial Ishikawa cell line treated with IFN-λ or IFN-α (100ng/mL) for 6h was analyzed using RNA-sequencing. Real-time qPCR, western blotting, and enzyme-linked immunosorbent assay (ELISA) tests were used to validate these sequencing data. An in vivo IFN-λ knock-down mouse pregnancy model was performed, and the phenotype analysis and the intrauterine biomarkers detection were applied with the uterus samples. High levels of messenger RNA (mRNA) were detected for genes previously associated with endometrial receptivity, including LIF, AXL, CRYAB, EPHB2, CCL5, and DDX58, following IFN-λ treatment. Moreover, the data indicated IFN-λ reduced pro-inflammatory gene activity compared with IFN-α, including members of the ISG, TNF, SP100 and interleukin genes. The in vivo mouse pregnancy model showed that inhibition of intrauterine IFN-λ results in aberrant epithelial phenotype and significantly decreases the embryo implantation rates and derails normal uterine receptivity. These findings demonstrate the antagonistic and agonistic roles of IFNs in the endometrial cell, suggesting a selective role of IFN-λ in endometrial receptivity and immunological tolerance regulation. Moreover, the findings provide valuable insight into potential biomarkers related to endometrial receptivity and facilitate an understanding of the molecular changes observed during infertility treatment and contraception usage.

Transcriptional response of endometrial cells to Insulin, cultured using microfluidics.

Obesity is a rapidly growing public health issue among women of reproductive age associated with decreased reproductive function including implantation failure. This can result from a myriad of factors including impaired gametes and endometrial dysfunction. The mechanisms of how obesity-related hyperinsulinaemia disrupts endometrial function are poorly understood. We investigated potential mechanisms by which insulin alters endometrial transcript expression. Ishikawa cells were seeded into a microfluidics device attached to a syringe pump to deliver a constant flow rate of 1uL/min of the following: 1) control 2) vehicle control (acetic acid) or, 3) Insulin (10 ng/ml) for 24 hours (n=3 biological replicates). Insulin-induced transcriptomic response of endometrial epithelial cells was determined via RNA sequencing, and DAVID and Webgestalt to identify Gene Ontology (GO) terms and signalling pathways. A Total of 29 transcripts showed differential expression levels across two comparison groups (control v vehicle control; vehicle control v insulin). Nine transcripts were differentially expressed in vehicle control v insulin comparison (p<0.05). Functional annotation analysis of transcripts altered by insulin (n=9) identified three significantly enriched GO terms: SRP-dependent cotranslational protein targeting to membrane, poly(A) binding, and RNA binding (p<0.05). Over-representation analysis found three significantly enriched signalling pathways relating to insulin-induced transcriptomic response: protein export, glutathione metabolism, and ribosome pathways (p<0.05). Transfection of siRNA for RASPN successfully knocked down expression (p<0.05) but this did not have any effect on cellular morphology. Insulin-induced dysregulation of biological functions and pathways highlight potential mechanisms by which high insulin concentrations within maternal circulation may perturb endometrial receptivity.

Bisphenol S Increases Cell Number and Stimulates Migration of Endometrial Epithelial Cells.

To determine whether bisphenol S (BPS), a common substitute for bisphenol A (BPA), induces cell proliferation and migration in human endometrial epithelial cells (Ishikawa) and adult mouse uterine tissues. Human endometrial Ishikawa cells were exposed to low doses of BPS (1 nM and 100 nM) for 72 hours. Cell proliferation was assessed through the viability assays MTT and CellTiter-Glo®. Wound healing assays were also used to evaluate the migration potential of the cell line. The expression of genes related to proliferation and migration was also determined. Similarly, adult mice were exposed to BPS at a dose of 30 mg/kg body weight/day for 21 days, after which, the uterus was sent for histopathologic assessment. BPS increased cell number and stimulated migration in Ishikawa cells, in association with the upregulation of estrogen receptor beta (ESR2) and vimentin (VIM). In addition, mice exposed to BPS showed a significantly higher mean number of endometrial glands within the endometrium. Overall, in vitro and in vivo results obtained in this study showed that BPS could significantly promote endometrial epithelial cell proliferation and migration, a phenotype also observed with BPA exposure. Hence, the use of BPS in BPA-free products must be reassessed, as it may pose adverse reproductive health effects to humans.

TMT-based quantitative proteomic analysis of spheroid cells of endometrial cancer possessing cancer stem cell properties

BackgroundCancer stem cells (CSCs) play an important role in endometrial cancer progression and it is potential to isolate CSCs from spheroid cells. Further understanding of spheroid cells at protein level would help find novel CSC markers.MethodsSpheroid cells from endometrial cancer cell lines, Ishikawa and HEC1A, exhibited increased colony forming, subsphere forming, chemo-drug resistance, migration, invasion ability and tumorigenicity, verifying their cancer stem-like cell properties. The up-regulated CD90, CD117, CD133 and W5C5 expression also indicated stemness of spheroid cells. TMT-based quantitative proteomic analysis was performed to explore the potential alterations between parent cells and cancer stem-like spheroid cells. HK2-siRNA was transfected to Ishikawa and HEC1A cells to explore the roles and molecular mechanism of HK2 in endometrial cancer.ResultsWe identified and quantified a total of 5735 proteins and 167 overlapped differentially expressed proteins of two cell types, 43 proteins were up-regulated and 124 were down-regulated in spheroid cells comparing with parent cells. KEGG pathway revealed a significant role of HIF-1 pathway in spheroid cells. qRT-PCR and western blot results of GPRC5A, PFKFB3 and HK2 of HIF-1 pathway confirmed their elevated expressions in spheroid cells which were consistent with proteomic results. HK2 promoted cancer stemness in endometrial cancer.ConclusionThese findings indicate that spheroid cells from endometrial cancer cell lines possess cancer stem-like cell properties and enrich CSCs. HIF-1 pathway is activated in endometrial cancer stem-like spheroid cells.

Phytochemical Profiling and Biological Effects of Glycyrrhiza glabra L.

Glycyrrhiza glabra L. commonly known as licorice belonging to the family Leguminosae is an ayurvedic medicinal plant having substantial pharmacological properties such as anti-inflammatory and anticancer effects. Analysed ethnopharmacological studies show phytochemical and biological activities of the plant extract and serve as a resource for studying anti- endometriosis. According to WHO, 176 million women in the world are affected with endometriosis condition and up to 30-50% of women may experience infertility. Our study aims to evaluate in vitro activity of ethanolic extract of Glycyrrhiza glabra in different cell lines, DPPH assay and HPLC studies in order to propose it to be an effective remedy in endometriosis treatment. In vitro cytotoxic assessment of the extract was determined in estrogenic (MCF 7), non-estrogenic (MDAMB 231) and endometrial cancer cell lines (Ishikawa) along with its safety assessment in normal cell lines (L929). The extract of G. glabra exhibited DPPH radical scavenging activity with 77.09% inhibition in 100 μg concentration. Cytotoxic activity of extract in MCF 7, MDAMB 231, endometrial cancer cells- Ishikawa and significant cell viability in normal L929 cell line shows promising effect of the extract and suggests it as a possible therapeutic adjuvant for treating endometriosis.

YBX2 modulates mRNA stability via interaction with YTHDF2 in endometrial cancer cells

RNA-binding proteins (RBPs) fine-tune gene expression by modulating RNA stability, translation, and degradation. RBPs are involved in the development of endometrial cancer. In particular, Y-box-binding protein 2 (YBX2), a germ cell-specific member of the YBX family, has been reported to maintain cancer stem cell-like phenotypes in endometrial cancer. However, the mechanism by which YBX2 modulates mRNA stability in endometrial cancer cells remains unknown. In this study, we examined the effects of the ectopic expression of YBX2 in endometrial adenocarcinoma-derived Ishikawa cells. We found that elevated levels of YBX2 delayed cell proliferation, without increasing cell apoptosis. Transcriptomic analysis revealed disturbances in gene expression caused by YBX2. Interestingly, heat shock protein family A (Hsp70) member 6 (HSPA6) levels were downregulated due to the reduced mRNA stability after YBX2 binding. YBX2 facilitated the formation of relatively stable cytoplasmic granules in tumor cells via its mRNA binding domain. Moreover, N6-methyladenosine (m6A) reader proteins are recruited by YBX2 granules via the cold-shock domain. Notably, knockdown of YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2), an m6A reader, ameliorated the reduction in HSPA6 mRNA levels induced by YBX2, indicating the synergistic effects of YBX2 and YTHDF2 on mRNA stability. Therefore, YBX2 regulates RNA stability by interacting with the m6A reader proteins.

CCND1-Induced Autophagy Contributes to Lymph Node Metastasis in Endometrial Cancer.

Endometrial cancer with lymph node metastasis shows poor prognosis, while the biomarker to predict the metastasis is lacking. The relative mRNA or protein expression of cyclin D1 (CCND1) and autophagy-related molecules were detected in real-time PCR and Western blot. Correlation analysis was applied to identify any significant patterns, and receiver operating characteristics (ROC) was performed to assess the prediction value. CCND1 vector was transfected in Ishikawa (ISK) cells, and the relative expression of autophagy-related molecules was detected with Western blot. CCND1 was overexpressed in endometrial cancer and correlated with lymph node metastasis. ROC analysis found that CCND1 had a predictive value to discriminate tumors from normal tissues (cut off=1.455; sensitivity, 71%; specificity, 84%; area under curve (AUC) 0.82; p<0.001) and had a predictive value to indicate metastasis (cut off=1.871; sensitivity, 54.17%; specificity, 75%; AUC 0.674; p=0.003). Increased BECLIN1 (r=0.39, p<0.001) and ATG5 (r=0.41, p<0.001) expression were positively correlated to CCND1. On the other hand, the relative protein expression of CCND1, BECLIN1, ATG5, ATG7, and LC3 I/II were also increased in tumor tissues. CCND1 overexpressed ISK cells showed upregulated BECLIN1, ATG5, ATG7, and LC3 I/II expression. CCND1 promoted autophagy may contribute to lymph node metastasis in endometrial cancer.

Apigenin, a natural flavonoid, promotes autophagy and ferroptosis in human endometrial carcinoma Ishikawa cells in vitro and in vivo

Apigenin, a natural flavonoid has been reported against a variety of cancer types. However, it is unclear whether apigenin can promote autophagy and ferroptosis in Ishikawa cells. There are few reports on the mechanism of apigenin on autophagy and ferroptosis of endometrial cancer Ishikawa cells. We found that iron accumulation, lipid peroxidation, glutathione consumption, p62, HMOX1, and ferritin were increased, while, solute carrier family 7 member 11 and glutathione peroxidase 4 were decreased. Ferrostatin-1, an iron-death inhibitor could reverse the effects of apigenin in Ishikawa cells. On the other hand, apigenin could promote autophagy via up-regulating Beclin 1, ULK1, ATG5, ATG13, and LC3B and down-regulating AMPK, mTOR, P70S6K, and ATG4. Furthermore, apigenin could inhibit tumor tissue proliferation and restrict tumor growth via ferroptosis in vivo.

Abstract 4877: Mutant or overexpressed SF3B1 facilitates endometrial cancer tumorigenesis

Abstract Aberrant expression or mutations in core splicing factors including U2AF1, SRSF2, ZRSR2, and SF3B1 found to drive tumorigenesis of various malignant tumors e.g., myelodysplastic syndromes, chronic lymphocytic leukemia, uveal melanoma, and breast cancer. Importantly, the amplifications and mutations in SF3B1, the pioneer splicing factor, are reportedly far more common than those in other splicing factors. However, the functional relevance and underlying mechanisms of dysregulated SF3B1 in endometrial cancer are not investigated. Endometrial cancer is the fourth most common cancer in women in the United States and there will be about 65,950 new endometrial cancer cases in the current year alone with 12,550 estimated cancer deaths in the United States. In this study, we thus investigated the role of SF3B1 in endometrial adenocarcinoma. We found SF3B1 is one of the most frequently amplified or mutated splicing factors in endometrial cancer. Specifically, analysis of The Cancer Genome Atlas (TCGA) uterine pan-cancer dataset revealed that up to 9% of endometrial cancers have SF3B1 mutations or amplification. Additionally, we showed that the SF3B1 protein is overexpressed in human as well as mouse endometrial tumor samples. Further analysis found elevated SF3B1 protein in multiple endometrial cancer cell lines even in the absence of SF3B1 mutations. However, we did not observe any altered expression at the transcript level. Further, we identified a correlation between the over-expressed SF3B1 levels and the tumorigenicity/invasive potential of cancer cell lines. For instance, endometrial cancer cell lines (Ishikawa, AN3CA, KLE) with over-expressed SF3B1 levels were much more tumorigenic and invasive than those with low endogenous SF3B1 levels (normal human endometrial epithelial cells, RL-95-2 cells). Moreover, cell viability assays revealed that Ishikawa, AN3CA, and KLE cells were more susceptible to the loss of SF3B1 or treatment with the SF3B1 inhibitor Pladienolide-B (PLAD-B) than RL-95-2 cells. To further ensure that overexpressed or mutated SF3B1 drives endometrial tumorigenesis, we transiently transfected an empty vector or a vector expressing FLAG-tagged wild-type SF3B1 or vector with mutated SF3B1 construct R957Q into RL-95-2 cells which have minimal endogenous SF3B1. Analysis of proliferation assays revealed that cells overexpressing SF3B1 proliferated significantly more than cells expressing empty vector. Intriguingly, we observed that RL-95-2 cells over-expressing R957Q mutants were even more proliferative than the cells expressing wild-type SF3B1. Together, this data suggest that SF3B1 over-expression and mutation play a crucial oncogenic role in endometrial cancer, and these findings may support the development of SF3B1 inhibitors to treat this disease. Citation Format: Pooja Popli, Xinchao Deng, Premal Thaker, Ramakrishna Kommagani. Mutant or overexpressed SF3B1 facilitates endometrial cancer tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4877.

Abstract 3102: Synthesis of N-benzoylamino-tetrahydropyridine analogues as selective COX-2 inhibitors and anti-breast cancer agents

Abstract The World Health Organization confirms breast cancer as the most common cancer globally, as of 2021, tallying 12% of new annual cancer cases. Female breast cancer is the second leading cancer in incidence rates among women after lung cancer. It is also more deadly amongst Black and Hispanic women as mortality rates increase by 40% compared to their caucasian counterparts. Hence, there is a dire need to design and develop novel molecules as effective anticancer agents. Rational design-based drug discovery has led to advancements in developing novel small molecules as effective anticancer agents. Chronic inflammation is directly related to cancer. Literature testifies to the presence and overexpression of cyclooxygenase-2 (COX-2) in various inflammation-based ailments, diseases, and many cancers. Therefore, it is imperative to investigate the relevance of cyclooxygenase-2 inhibition in cancer therapy. The tetrahydropyridine structure has been documented in several natural products. They display biological properties including antioxidant, anti-inflammatory, and chemotherapeutic. Substituted benzoylamino-5-ethyl-1,2,3,6-tetrahydropyridine analogues have been designed as COX-2 inhibitors. This project aims to synthesize thirteen novel THP analogues using a 4-step synthesis. The synthesis of these 1,2,3,6-THP analogues primarily involves the hydrolysis of ethyl-o-(mesitylenesulfonyl) acethydroxymate to render O-(mesitylsulfonyl)hydroxylamine (MSH). MSH then acts as an aminating agent as it reacts with 3-ethyl pyridine. This amination reaction affords the mesitylene salt, 1-amino-3-ethylpyridin-1-ium. Next, the salt product undergoes acylation with various substituted benzoyl chlorides to generate stable N-ylide products. Finally, the partial reduction of the ylides yields the desired final tetrahydropyridine products. Purification was completed by flash chromatography. The novel THP analogues were characterized using nuclear magnetic resonance, infrared spectroscopy, and elemental analysis. Computer-aided drug design studies provided the preliminary data constituting that the likelihood of the THP moiety generating more effective COX-2 inhibitors is dependent on suitable modifications to the structure. Further studies include testing the novel THP analogues as cyclooxygenase-2 inhibitors using COX-2 inhibition assays, and lastly, determining their anti-breast cancer activity in MDA-MD-231, MCF-7 and Ishikawa cell lines to assess their antiproliferative effects. This research was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number U54 MD007582. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Citation Format: Shasline Gedeon, Madhavi Gangapuram, Kinfe K. Redda, Tiffany Ardley. Synthesis of N-benzoylamino-tetrahydropyridine analogues as selective COX-2 inhibitors and anti-breast cancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3102.

Anticancer Activity of Methanol extract of Limnophila repens and Argyeia cymosa by Using SRB Assay

Introduction: Plants have a special place in the treatment of cancer. It is estimated that plant-derived compounds constitute more than 50% of anticancer agents. In this present study I attempted an experiment to find out the anti-cancer activity of selected plants Limnophila repens and Argyeia cymosa by using SRB Assay. Material method: I have collected the plants, dried very well and extracted with methanol crude methanol extract of the both plants tested for its anti-cancer activity. Results and disscussionThe anticancer activity of methanolic extracts of Argyreia cymosa and Limnophila repens was determined by sulforhodamine B colorimetric assay. The results of the cytotoxicity of extracts from both plant extracts analysed and The Methanol extract of Limnophila repens extract showed comparable activity to the standard compound, i.e., Adriamycin on Ishikawa and SCC-29B cell lines, respectively. This extract showed TGI, and GI50 was 38.9 and &lt;10 µg/ ml on Ishikawa cell lines and 58.12, &lt;10 and &lt;10 µg/ ml of LC50, TGI and GI50 activity on SCC-29B cell lines respectively. The Methanol extract of Argyeia cymosa showed GI50 was &gt;80 µg/ ml on Ishikawa cell lines and &lt;10 and &lt;10 µg/ ml of, TGI and GI50 activity on SCC-29B cell lines .Estimations based on GI50 values shows that MELR was more active against Ishikawa and SCC-29B cell lines than MEAC on Ishikawa (human endometrial adenocarcinoma) and SCC-29B (human oral cancer) cell lines.

Vitexin suppresses the proliferation, angiogenesis and stemness of endometrial cancer through the PI3K/AKT pathway

Context Endometrial cancer is a common gynecologic malignancy. Vitexin is an active flavonoid compound with an antitumor function. Objective This study elucidated the role of vitexin in endometrial cancer development and clarified the potential mechanism. Materials and methods The toxicity of vitexin (0–80 μM) treatment for 24 h on HEC-1B and Ishikawa cells was tested utilizing the CCK-8 assay. Endometrial cancer cells were divided into vitexin 0, 5, 10, and 20 μM groups. Cell proliferation, angiogenesis and stemness in vitro after treatment with vitexin (0, 5, 10, 20 μM) for 24 h were evaluated using the EdU staining assay, tube formation assay and sphere formation assay, respectively. Twelve BALB/c mice were grouped into control and vitexin (80 mg/kg) groups to monitor tumour growth for 30 days. Results Vitexin suppressed cell viability of HEC-1B (IC50 = 9.89 μM) and Ishikawa (IC50 = 12.35 μM) cells. The proliferation (55.3% and 80% for HEC-1B; 44.7% and 75% for Ishikawa), angiogenesis (54.3% and 78.4% for HEC-1B; 47.1% and 68.2% for Ishikawa) and stemness capacity (57.2% and 87.3% for HEC-1B; 53.4% and 78.4% for Ishikawa) of endometrial cancer cells were inhibited by 10 and 20 μM vitexin. Furthermore, the inhibitory effects of vitexin on endometrial cancer were reversed by PI3K/AKT agonist 740Y-P (20 μM). Moreover, the xenograft tumour experiment lasting for 30 days proved that vitexin (80 mg/kg) blocked tumour growth of endometrial cancer in vivo. Discussion and conclusions Vitexin has therapeutic potential on endometrial cancer, which supports further clinical trials.

Downregulation of SEPTIN11 inhibits endometrial epithelial cell adhesive function in patients with elevated peripheral blood natural killer cell counts

Research questionWhat is the underlying mechanism of IVF and embryo transfer (IVF-ET) failure in patients with elevated peripheral blood natural killer cell (pNK) counts? DesignPatients undergoing IVF-ET cycles for tubal obstruction or pelvic adhesion (n = 486) were assigned to three groups: high (CD56+CD16+pNK >30% [n = 49]); medium (15< CD56+CD16+pNK ≤30% [n = 211]); and normal pNK groups (5≤ CD56+CD16+pNK ≤15% [n = 226]). Their general condition, previous pregnancy history and IVF outcomes were compared. Uterine fluid and endometrial tissue from patients in the high and normal pNK groups were collected during the mid-secretory phase and studied to elucidate the molecular mechanism underlying impaired endometrial receptivity. ResultsThe highest incidence of IVF-ET cycles (P < 0.0001) and biochemical pregnancy losses (P < 0.0001), and lowest implantation and clinical pregnancy rates (both P < 0.0001), were observed in patients with pNK over 30%. No significant difference was found in the number of previous miscarriages and rate of spontaneous miscarriage in IVF outcomes. Lower Septin11 (SEPT11) expression in the uterine fluid and endometrial epithelial cells (EEC), and higher endometrial IFN-γ, was observed in patients with high pNK. Ishikawa cell and human endometrial epithelial cell (HEEC) adhesion was inhibited after SEPT11 knock-down. Elevated IFN-γ decreased the SEPT11 protein levels in Ishikawa cells and HEECs. ConclusionsCD56+CD16+pNK above 30% may be a threshold for adverse IVF-ET outcomes. Low SEPT11 expression in EEC inhibits cell adhesion, which may cause impaired endometrial receptivity in patients with elevated pNK. The level of SEPT11 in mid-secretory uterine fluid could serve as a non-invasive marker to assess endometrial receptivity in these patients.

Infectious Recombinant Senecavirus A Expressing p16INK4A Protein.

Senecavirus A (SVA) is an oncolytic RNA virus, and it is the ideal oncolytic virus that can be genetically engineered for editing. However, there has not been much exploration into creating SVA viruses that carry antitumor genes to increase their oncolytic potential. The construction of SVA viruses carrying antitumor genes that enhance oncolytic potential has not been fully explored. In this study, a recombinant SVA-CH-01-2015 virus (p15A-SVA-clone) expressing the human p16INK4A protein, also known as cell cycle-dependent protein kinase inhibitor 2A (CDKN2A), was successfully rescued and characterized. The recombinant virus, called SVA-p16, exhibited similar viral replication kinetics to the parent virus, was genetically stable, and demonstrated enhanced antitumor effects in Ishikawa cells. Additionally, another recombinant SVA virus carrying a reporter gene (iLOV), SVA-iLOV, was constructed and identified using the same construction method as an auxiliary validation. Collectively, this study successfully created a new recombinant virus, SVA-p16, that showed increased antitumor effects and could serve as a model for further exploring the antitumor potential of SVA as an oncolytic virus.

The Inactivation of Hippo Signaling Pathway Promotes the Development of Adenomyosis by Regulating EMT, Proliferation, and Apoptosis of Cells.

Adenomyosis is a benign gynecological disease. The pathogenesis of adenomyosis is still unclear. The Hippo signaling pathway is highly conserved in vivo and associated with endometriosis and various cancers. Our objective was to study the expression of Hippo signaling pathway-related proteins in the uterus of mice with and without adenomyosis. We also sought to determine the relationship between the Hippo signaling pathway and cell migration, invasion, proliferation, and apoptosis in adenomyosis. The inactivation of Hippo signaling pathway and abnormal expression of EMT-related proteins were observed in mice with adenomyosis. In vitro, the YAP inhibitor verteporfin can inhibit the proliferation and migration of Ishikawa cells and promote apoptosis, while inhibiting the EMT process. In addition, intraperitoneal injection of verteporfin inhibits EMT process and proliferation and promotes apoptosis of cells in the uterus of adenomyosis mice. It suggests that the Hippo signaling pathway participates in the EMT, proliferation, and apoptosis of cells in adenomyosis. In conclusion, these results suggest that Hippo signaling pathway may be involved in the development of adenomyosis by regulating EMT, proliferation, and apoptosis of cells, which provide a potential target for the treatment of adenomyosis.