Ovarian Cancer Cell Lines OVCAR3 Research Articles

The effects of miR-140-5p on the biological characteristics of ovarian cancer cells through the Wnt signaling pathway.

Ovarian cancer is usually not diagnosed until the late stage, and it is resistant to platinum and other standard chemotherapy drugs, resulting in high mortality. To investigate the effects of miR-140-5p on cell proliferation, apoptosis, invasion, and migration capability in the SKOV3 and OVCAR3 ovarian cancer cell lines through Wnt signaling pathway. Expression levels of miR-140-5p were checked using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of miR-140-5p was upregulated by transfecting cells with a miR-140-5p mimic or a mimic negative control (NC). Cell proliferation was assessed using a CCK8 assay, and cell cycle distribution and apoptosis percentage were detected with flow cytometry. A transwell invasion assay was employed to evaluate cell migration and invasion ability. The target complementary relationship between miR-140-5p and WNT1 was verified using a dual-luciferase reporter assay while â-catenin in the nuclei was observed using immunofluorescence. The expression of Wnt signaling pathway-related proteins was examined using western blot and qRT-PCR. The relative expression level of miR-140-5p in SKOV3 and OVCAR3 cells was obviously decreased compared with that in the IOSE80 cells (p < 0.05). Besides, upregulated miR-140-5p effectively suppressed cell proliferation and increased the apoptosis ratio of SKOV3 and OVCAR3 cells (p < 0.05). In addition, the invasion and migration capability of SKOV3 and OVCAR3 cells in miR-140-5p mimic group was largely suppressed compared with the NC group (p < 0.05). What is more, the target complementary relationship between miR-140-5p and the WNT1 gene was revealed; upregulated miR-140-5p suppressed the expression of Wnt signaling-related genes, and restrained nuclear transfer of â-catenin (p < 0.05). The overexpression of miR-140-5p restricted the proliferation, migration and invasion abilities, and accelerated cell apoptosis in ovarian cancer cell lines SKOV3 and OVCAR3 through the Wnt signaling pathway.

Abstract A16: A novel death receptor ligand fabclavine inhibits cancer and cancer stem cell proliferation by extrinsic apoptosis

Abstract Many antibiotics produced by bacteria also have antineoplastic activity. Examples of such bacterial compounds include actinomycin D, doxorubicin, mitoxantrone, bleomycin, mitomycin, etoposide, and others. Recently, it was identified that Xenorhabdus budapestnesis (Xbu) produces a secondary metabolite, fabclavine, that has antibiotic property. Here, for the first time we demonstrate that fabclavine exhibits potent cytotoxic activity against ovarian cancer cells and ovarian cancer stem cells and demonstrate its mechanism of action, Fabclavine was isolated and purified from cell-free supernatants of Xbu cultures and analyzed by mass spectrometry. Proliferation assays conducted on several ovarian cancer cell lines Ovcar3, OVCA433, ID8, and breast cancer cell lines MCF7 and 4T1 demonstrate that fabclavine inhibits the cancer cell proliferation at nanomolar (250-400nM) concentration. Fabclavine is ten times more potent than cisplatin in inhibiting cell proliferation of ovarian cancer cell line OVCA33 and breast cancer cell line MCF7. We also tested the effect of fabclavine in 3D culture of ovarian cancer cell lines. The results indicate, even in 3D culture, fabclavine killed 40-50% of the cells at 300nM concentration. In addition, fabclavine was also tested against ovarian cancer stem cells isolated from five ovarian cancer patients and grown in 3D culture. Fabclavine inhibited the growth of stem cells by 70-85%. In cancer cell lines fabclavine induced apoptosis in 20-30% cells after 24-hour treatment. Caspase activity assay for caspase3, caspase8, and caspase9 shows a significant increase in caspase3 and caspse8 activity with no change in caspase9 activity, suggesting an activation of the extrinsic apoptosis pathway in the fabclavine-treated cells. This observation was supported by the increase in phospho-FADD upon exposure to fabclavine. When conjugated to agarose beads, fabclavine efficiently precipitated two death receptors, DR4 and DR5, from ovarian and breast cancer cell extracts. These experiments indicate that fabclavine is a novel DR4/5 ligand and should therefore be considered as a death receptor-targeting agent for the treatment of ovarian and other tumors. We will present data on the comparison of DR4/5 binding to fabclavine versus its natural ligand, TRAIL, and will also present data from PDex and mouse xenograft models on the ex vivo and in vivo effects of fabclavine on the proliferation of ovarian tumors and cancer stem cells. Citation Format: Arvinder Kapur, Mayur Kajla, Susan Paskewitz, Allegra Cappuccini, Pooja Mehta, Geeta Mehta, Manish Patankar. A novel death receptor ligand fabclavine inhibits cancer and cancer stem cell proliferation by extrinsic apoptosis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A16.

FTO accelerates ovarian cancer cell growth by promoting proliferation, inhibiting apoptosis, and activating autophagy

ObjectiveFat mass and obesity-associated protein (FTO) is identified as a critical demethylase involved in various physiological processes. Despite efforts have been made to study the biological functions of FTO in certain cancers, the role of FTO in ovarian cancer is largely unknown. In this study, we sought to investigate the function of FTO on proliferation, apoptosis and autophagy of ovarian cancer cells. MethodsQuantitative real-time PCR was performed to detect FTO expression in ovarian tumor tissues and ovarian cancer cell lines OVCAR-3, SKOV-3, COC1, HO-8910 and A2780. SKOV-3 cells were constructed with FTO overexpression and A2780 cells were constructed with FTO knockdown. CCK-8 assay was used to examine cell viability and flow cytometry was used to detect cell apoptosis. Activity assay kits were applied to detect caspase-3 and caspase-9 levels. Western blot was performed to measure the expressions of FTO, PCNA, Bax, Bcl-2, LC3, ATG5, P62, p-AKT and AKT. Stable FTO-overexpression SKOV-3 cells or FTO-depletion A2780 cells were injected subcutaneously into male Balb/c-nu mice. Xenografted tumors were assayed by H&E staining. Immunohistochemistry was subjected to measure FTO and Ki67 expressions. ResultsFTO was up-regulated in ovarian tumor tissues compared with non-cancerous ovarian tissues. FTO overexpression markedly increased viability and autophagy function, but decreased apoptosis of ovarian cancer cells. In addition, FTO overexpression promoted AKT phosphorylation. In contrast, FTO silence showed the opposite effect. ConclusionFTO accelerated ovarian cancer cell growth by promoting proliferation, inhibiting apoptosis, and activating autophagy.

Cytokine mRNA and protein expression by cell cultures of epithelial ovarian cancer-Methodological considerations on the choice of analytical method for cytokine analyses.

To get a comprehensive picture of cytokine expression in health and disease is difficult, cytokines are transiently and locally expressed, and protein analyses are burdened by biological modifications, technical issues, and sensitivity to handling of samples. Thus, alternative methods, based on molecular techniques for cytokine mRNA analyses, are often used. We compared cytokine mRNA and protein expression to evaluate whether cytokine mRNA profiles can be used instead of protein analyses. In kinetic experiments, cytokine mRNA and protein expression of IL-1β, IL-6, IL-8, TNF-α, and TNF-β/LTA were studied using real-time RT-qPCR and Luminex® microarrays in the ovarian cancer cell lines OVCAR-3, SKOV-3 and the T-cell line Jurkat, after activation of transcription by thermal stress. In addition, we analyzed IL-6 and IL-8 mRNA and protein in a small number of ovarian cancer patients. Ovarian cancer cells can express cytokines on both mRNA and protein level, with 1-4hours' time delay between the mRNA and protein peak and a negative Spearman correlation. The mRNA and protein expression in patient samples was poorly correlated, reflecting previous studies. Cytokine mRNA and protein expression levels show diverging results, depending on the material analyzed and the method used. Considering the high sensitivity and reproducibility of real-time RT-qPCR, we suggest that cytokine mRNA profiles could be used as a proxy for protein expression for some specific purposes, such as comparisons between different patient groups, and in defining mechanistic pathways involved in the pathogenesis of cancer and other pathological conditions.

Effects of Wnt/β-Catenin Signal Pathway Regulated by miR-342-5p Targeting CBX2 on Proliferation, Metastasis and Invasion of Ovarian Cancer Cells.

ObjectiveThis study aimed to investigate the effect of Wnt/β-catenin signal pathway mediated by miR-342-5p targeting CBX2 gene on the proliferation, metastasis, invasion and apoptosis of ovarian cancer cells, and to explore its related regulatory mechanism.MethodsHuman normal ovarian epithelial cell line IOSE80, human ovarian cancer cell line SKOV3 and OVCAR3 were the subjects. Software were used to predict the binding site of miR-342-5p targeting CBX2 gene. The proliferation rate of ovarian cancer cells was detected by MTT method; the cell viability of each group was observed by colony formation test; the apoptosis of cells in each group was detected by flow cytometry; the invasive ability of cells was determined by transwell test, and the migration ability of cells was detected by scratch test. The mRNA expression levels of miR-342-5p, CBX2, Wnt1, β-catenin, C-myc and Cyclin D1 were measured by qRT-PCR. Also, Western blot was used to determine the protein expression levels of CBX2, Wnt1, β-catenin, C-myc and Cyclin D1.ResultsCBX2 was identified as the target gene of miR-342-5p. MTT test results showed that miR-342-5p could significantly inhibit the proliferation of SKOV3 and OVCAR3 cells, colony formation assay results indicated that the viability of SKOV3 and OVCAR3 cells transfected with miR-342-5p decreased significantly, and flow cytometry results suggested that miR-342-5p could promote the apoptosis of SKOV3 and OVCAR3 cells. Also, the results of transwell showed that miR-342-5p could significantly inhibit the invasive ability of SKOV3 and OVCAR3 cells, and the results of scratch assay suggested that miR-342-5p could significantly inhibit the migration of SKOV3 and OVCAR3 cells. Moreover, qRT-PCR and Western blot results indicated that the mRNA and protein expression levels of CBX2, Wnt1, β-catenin, C-myc and Cyclin D1 decreased in SKOV3 and OVCAR3 cells transfected with miR-342-5p, while the mRNA expression levels of miR-342-5p increased significantly (P<0.05).ConclusionMiR-342-5p targeted gene is CBX2, which can significantly reduce the proliferation, invasion, migration and viability of ovarian cancer cell lines SKOV3 and OVCAR3, and promote their apoptosis. The mechanism may be related to the mediation of Wnt/β-catenin signal pathway and down-regulation of the related genes expression.

Exendin-4 exhibits a tumour suppressor effect in SKOVR-3 and OVACR-3 ovarian cancer cells lines by the activation of SIRT1 and inhibition of NF-κB.

This study investigated if EX-527 has an anti-tumour effect in SKOV-3 and OVCAR-3 ovarian cancer (OC) cell lines and if this effect involves the SIRT1/NF-κB axis. Cells were cultured in the presence or absence of EX-527, a selective SIRT-1 inhibitor. Exendin-4 significantly induced cell death in both cell lines and inhibited cell migration and invasion. Also, it decreased protein levels of Bcl-2, MMP-9, and ICAM-1 and increased those of Bax, cyclin D1 and cleaved caspase-3. Mechanistically, Exendin-4 increased the activity and nuclear accumulation of SIRT1 and decreased nuclear levels of NF-κB p65; acetylated levels of NF-κB p65, and cytoplasmic levels of p-IKKα and p-IκBα. EX-527 partially ameliorated the effect of Exendin-4 on cell death, migration, and invasion, as well as on the expression of Bcl-2, MMP-9, Bax, cleaved caspase-3 and ICAM-1. In addition, EX-527 did not affect the levels of nuclear p65 and p-p65 (Ser536); p-IκBα (Ser32) and p-IKKαβ. In conclusion, Exendin-4 can suppress OC by inhibiting NF-kB through SIRT1 dependent and independent mechanisms.

METTL3 serves an oncogenic role in human ovarian cancer cells partially via the AKT signaling pathway.

Methyltransferase-like 3 (METTL3) has been identified as a methyltransferase responsible for N6-methyla-denosine (m6A) modification of mRNA. METTL3 functions in tumorigenesis and tumor development by promoting the translation of oncoproteins; however, the role of METTL3 in ovarian cancer has not been extensively studied. The present study performed immunohistochemistry to detect METTL3 expression levels in 52 samples of ovarian cancer tissue paired with corresponding paracancerous tissue. RNA interference was conducted to downregulate the expression levels of METTL3 in the SKOV3 and OVCAR3 ovarian cancer cell lines. Reverse transcription-quantitative PCR and western blot analysis demonstrated the effects of METTL3 knockdown on mRNA and protein levels, respectively. CCK-8, colony formation, apoptosis and Transwell assays were also performed. The results demonstrated that METTL3 exhibited significantly higher expression levels in ovarian cancer tissues compared with corresponding paracancerous tissue. High METTL3 expression levels were associated with large tumors, lymph node metastasis and high pathological grade. Cell proliferation analysis revealed that METTL3 knockdown reduced the proliferation and clonogenic ability of SKOV3 and OVCAR3 cells. Apoptotic rates were increased in METTL3-silenced ovarian cancer cells, which may have been mediated by the activation of the mitochondrial apoptosis pathway, and METTL3 knockdown reduced cell invasion. METTL3 knockdown downregulated the phosphorylation levels of AKT and the expression of the downstream effector Cyclin D1. These results suggested that METTL3 may serve an oncogenic function in the progression of human ovarian cancer cells partially through the AKT signaling pathway, indicating that METTL3 may be a potentially novel therapeutic target for the treatment of ovarian cancer.

Regulatory mechanism of lncRNA NORAD on proliferation and invasion of ovarian cancer cells through miR-199a-3p.

To explore the regulatory mechanism of lncRNA NORAD on proliferation and invasion of ovarian cancer cells through miR-199a-3p. Eighty-six ovarian cancer tissues and 86 tissues adjacent to cancer, human ovarian cancer cell lines SKOV3, HO-8910, A2780, OVCAR-3, and human normal ovarian epithelial cell line IOSE80 were collected. MiR-199a-3p-mimics, miR-199a-3p-inhibitor, miR-NC, si-NORAD, Sh-NORAD, and NC were transfected into HO-8910 and A2780 cells, the expression levels of lncRNA NORAD and miR-199a-3p in ovarian cancer tissues and cells were detected by qRT-PCR, and the expression levels of N-cadherin, E-cadherin, and vimentin in cells were detected by WB. Cell Counting Kit-8 (CCK-8), transwell, and cell scratch tests were used to detect proliferation, invasion, and migration of cells, and the relationship between lncRNA NORAD and miR-199a-3p was confirmed by the Dual-Luciferase reporter assay. LncRNA NORAD was highly expressed and miR-199a-3p was lowly expressed in ovarian cancer, and the expression levels of LNCRNARAD and miR-199a-3p were negatively correlated. Cell experiments showed that inhibiting the expression of lncRNA NORAD or up-regulating the expression of miR-199a-3p could inhibit the proliferation, invasion, migration, and EMT of ovarian cancer cells, while up-regulating the expression of lncRNA NORAD or inhibiting the expression of miR-199a-3p could promote their proliferation, invasion, migration, and EMT. Dual-Luciferase reporter assay confirmed that there was a regulatory relationship between lncRNA NORAD and miR-199a-3p. LncRNA NORAD was highly expressed in ovarian cancer tissues, while silencing lncRNA NORAD expression could inhibit the proliferation, invasion, migration, and EMT of ovarian cancer cells by regulating miR-199a-3p, which might be a new target for the diagnosis and treatment of ovarian cancer.

LPS and IL-8 activated umbilical cord blood-derived neutrophils inhibit the progression of ovarian cancer.

Background: Immunotherapy including immune checkpoint blockade, cancer vaccines, and adoptive cell therapy. However, no immune therapies support ovarian cancer. It is not clear whether the neutrophils, the component of the immune system derived from umbilical cord blood play a role in inhibiting the progression of ovarian cancer.Methods: We investigate the impact of LPS and IL-8 activated neutrophils derived from umbilical cord blood(UCB)on ovarian cancer progression. After co-culture LPS and IL-8 activated UCB-derived neutrophils with ovarian cancer cell line SKOV3 and OVCAR3, CCK8, Transwell assay, and Flow Cytometry was performed to detect cell proliferation, migration, invasion, and apoptosis of ovarian cancer cell lines SKOV3 and OVCAR3. Furthermore, RT-PCR and western blotting assay were used to analyze the mechanism of metastasis and apoptosis of ovarian cancer cell lines respectively to support previous function experiments.Results: We demonstrate LPS and IL-8 activated neutrophils derived from umbilical cord blood inhibit proliferation, invasion migration and promote apoptosis of SKOV3 and OVCAR3. Meanwhile, LPS and IL-8 activated UCB-derived neutrophils significantly decreased BAX and increased BCL2 expression in SKOV3 and OVCAR3 which account for the mechanism of apoptosis. Moreover, LPS and IL-8 activated UCB derived neutrophils significantly up-regulated E-cadherin and downregulated N-cadherin, MMP2 expression in SKOV3 and OVCAR3.Conclusion: Taken together, these results approved that LPS and IL-8 activated neutrophils from UCB may be the novel strategy in immune therapy for ovarian cancer.

SPOP Regulates The Biological Mechanism Of Ovarian Cancer Cells Through The Hh Signaling Pathway.

BackgroundOvarian cancer is characterized by high metastatic potential and high mortality. More than 80% of primary ovarian malignancies are epithelial ovarian cancers. There is increasing evidence that Speckle-type POZ protein (SPOP) is highly correlated with the development of various types of cancer. However, the effects of SPOP on epithelial ovarian cancer and the associated molecular mechanisms remain unclear.Materials and methodsWe compared SPOP expression between epithelial ovarian cancer tissues and normal ovarian tissues by using immunohistochemical staining. To determine the role of SPOP in epithelial ovarian cancer cells, we overexpressed or knocked down SPOP in the epithelial ovarian cancer cell line OVCAR-3 using lentiviral vectors.ResultsOur results from the present study indicated that SPOP expression was significantly downregulated in human epithelial ovarian cancer and was associated with the FIGO stage and the histopathologic grading of the tumor. The overexpression and knockdown experiments revealed that SPOP inhibited proliferation while promoting apoptosis in ovarian cancer cells. Inhibition of SPOP mis-activated the Hedgehog (Hh) signaling pathway, thereby inhibiting apoptosis in ovarian cancer cells.ConclusionSPOP suppresses proliferation and promotes apoptosis in human ovarian cancer cells by inhibiting the Hh signaling pathway, offering the possibility of new approaches for the treatment of ovarian cancer.

Asparagine Synthetase and Filamin A Have Different Roles in Ovarian Cancer.

Early-stage ovarian serous carcinoma is usually difficult to detect in clinical practice. The profiling of protein expression in high-grade serous carcinoma (HGSC) and low-grade serous carcinoma (LGSC) would provide important information for diagnoses and chemotherapy. Here, we performed proteomic profiling of specimens from 13 HGSC and 7 LGSC patients by iTRAQ. A total of 323 proteins that were differentially expressed were identified. After immunohistochemical confirmation of expressed proteins in 166 clinical tissues, asparagine synthetase (ASNS) and filamin A (FLNA) were selected for further functional study. Cisplatin-sensitive (CS; ASNShigh and FLNAlow) and cisplatin-resistant (CR; ASNSlow and FLNAhigh) SKOV3 and OVCAR3 ovarian cancer cell lines were used for subsequent in vitro and in vivo experiments. Notably, ASNS overexpression (ASNS+) or FLNA knockdown (shFLNA) enabled cisplatin-induced apoptosis and autophagy in CR cells. However, ASNS+ and shFLNA promoted and attenuated tumor growth, respectively. In CS cells, ASNS knockdown (shASNS) attenuated clonogenicity, cell proliferation, and the epithelial–mesenchymal transition, whereas FLNA overexpression (FLNA+) protected cells from cisplatin. In vivo, cisplatin resistance was attenuated in mice xenografted with ASNS+, shFLNA, or ASNS+-shFLNA CR cells, whereas xenografts of shASNS or FLNA+ CS cells exhibited resistance to cisplatin. Clinically, all HGSC patients (83/83) responded to cisplatin, while 6 in 41 LGSC patients exhibited cisplatin resistance. These findings identify ASNS and FLNA as distinct biomarkers for HGSC and LGSC, which may have potential value in the prognosis and clinical treatment of serous carcinoma.

1866 The Role of Microrna-424/503-Wee1 Axis in Ovarian Cancer Stem Like Cells

Study Objective We investigated the role of miR-424/503-WEE1 axis in ovarian cancer and its potential utility as a therapeutic target. Design To determine whether miR 424/503-WEE1 axis is associated with the generation of CSCs, we up/down-regulated miR 424/503-WEE1 axis in ovarian cancer sphere cells. Setting This study was performed in Pusan National University Hospital, Busan, Korea Patients or Participants The human epithelial ovarian cancer cell lines OVCAR3, SKOV3, and OVCAR429 were obtained from the Korean Cell Line Bank. Ovarian cancer cells were maintained in MEM (Life Technologies, Inc., Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 100 μg/ml streptomycin in a humidified 5% CO2 incubator. Interventions In order to assess spheroid formation, approximately 5 × 103 cells were suspended in 10 mL of serum-free DMEM-F12, supplemented with 10 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor and plated in an ultra-low attachment plate to prevent adherence. Spheres were counted at 14 days after plating. All experiments were performed in triplicate. Measurements and Main Results Firstly, we discovered that miR-424 and miR-503 are lowly expressed in ovarian cancer stem-like cells (CSCs) compared with non-CSCs. Over-expression of miR-424 and miR-503 resulted in decreased cancer colony formation, migration in ovarian cancer cell lines. Moreover, miR-424 and miR-503 over-expression caused inhibition of spheroid formation and the DNA damage repair pathway, while over-expression of Wee1 caused increased spheroid formation and cell survival in ovarian CSCs. Secondly, we found that the stem cell marker NANOG decreased miR-424 and miR-503 transcription in ovarian CSCs. In addition, miR-424 and miR-503 bind to the 3’ UTR of WEE1 to decrease its expression. Finally, we found that atorvastatin treatment increased miR-424 and miR-503 expression while it significantly decreased NANOG and WEE1 expression in ovarian CSCs. Conclusion Our findings indicate that increased WEE1 is an oncogenic marker for ovarian CSCs and is a potential target for CSC-specific therapy.

PDMS-PMOXA-Nanoparticles Featuring a Cathepsin B-Triggered Release Mechanism.

Background: It was our intention to develop cathepsin B-sensitive nanoparticles for tumor-site-directed release. These nanoparticles should be able to release their payload as close to the tumor site with a decrease of off-target effects in mind. Cathepsin B, a lysosomal cysteine protease, is associated with premalignant lesions and invasive stages of cancer. Previous studies have shown cathepsin B in lysosomes and in the extracellular matrix. Therefore, this enzyme qualifies as a trigger for such an approach. Methods: Poly(dimethylsiloxane)-b-poly(methyloxazoline) (PDMS-PMOXA) nanoparticles loaded with paclitaxel were formed by a thin-film technique and standard coupling reactions were used for surface modifications. Despite the controlled release mechanism, the physical properties of the herein created nanoparticles were described. To characterize potential in vitro model systems, quantitative polymerase chain reaction and common bioanalytical methods were employed. Conclusions: Stable paclitaxel-loaded nanoparticles with cathepsin B digestible peptide were formed and tested on the ovarian cancer cell line OVCAR-3. These nanoparticles exerted a pharmacological effect on the tumor cells suggesting a release of the payload.

Europium Nanoparticle-Based Sialyl-Tn Monoclonal Antibody Discriminates Epithelial Ovarian Cancer-Associated CA125 from Benign Sources.

The Sialyl-Thomsen-nouveau antigen (STn) is abundantly produced on many types of human epithelial cancers including epithelial ovarian cancer (EOC). We previously developed an EOC-specific lectin sandwich immunoassay (CA125MGL) using a human macrophage galactose-binding lectin coated on fluorescent europium nanoparticles (Eu+3-NPs) as a tracer and an anti-CA125-specific mAb for capture. Here we have identified a novel STn-mAb that efficiently recognizes the EOC-associated STn antigen on CA125 when coated on Eu+3-NPs. CA125 from the ovarian cancer cell line OVCAR-3, placental homogenate, and ascites fluid from patients with liver cirrhosis was captured by anti-CA125 antibody immobilized on microtitration wells and traced with anti-STn-mAb-Eu+3-NPs. Samples from EOC or patients with endometriosis with marginally increased conventional CA125 immunoassay (CA125IA; 35-200 U/mL) and healthy controls were analyzed. An analytically sensitive CA125STn assay that specifically recognized the CA125 isoform produced by OVCAR-3 was achieved. Serum CA125STn concentration was significantly higher in EOC patients than in those with endometriosis (P < 0.001). Furthermore, the sensitivity for detection of EOC with CA125STn assay was 73.3% when 95% of endometriosis cases were undetectable. Our findings suggest that Eu+3-NPs-based CA125STn assay could help reduce the false-positive rates of CA125IA to improve differential diagnosis. The results encourage studying further the potential use of CA125STn to detect EOC at earlier clinical stages. This approach warrants further investigation in other cancers as well.

Oncogene miR-934 promotes ovarian cancer cell proliferation and inhibits cell apoptosis through targeting BRMS1L.

Ovarian cancer is a common malignant cancer among women. Increasing studies have demonstrated that microRNAs function as important regulation factors in the progression of ovarian cancer. Human ovarian cancer cell lines HO8910 and OVCAR-3 were transfected with miR-934 inhibitor and corresponding negative control (inhibitor control). Cell proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) and TUNEL assay, respectively. The expression levels of proliferation/apoptosis-related genes and BRMS1L were measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blotting. Furthermore, the association between miR-934 and BRMS1L was investigated through luciferase reporter assays. MiR-934 was significantly increased in ovarian cancer cell lines, whereas BRMS1L was significantly decreased. Downregulated miR-934 remarkably inhibited cell proliferation and induced cell apoptosis. Meanwhile, miR-934 could influence the expression levels of Ki67, Cyclin D1, Caspase3, and Bcl-2. In addition, BRMS1L was identified as a target gene of miR-934. Oncogene miR-934 promotes ovarian cancer cell proliferation and inhibits cell apoptosis through targeting BRMS1L. MiR-934 and BRMS1L may be novel biomarkers or therapeutic targets for ovarian cancer in the future.

CEMIP promotes ovarian cancer development and progression via the PI3K/AKT signaling pathway

CEMIP is a cell migration-inducing protein that is closely associated with carcinogenesis. However, the function of CEMIP has not been reported in ovarian cancer. Here we show that CEMIP expression level in ovarian cancer tissues was higher than that in normal ovaries. Silencing of CEMIP in ovarian cancer cell lines A2780 and OVCAR3 significantly decreased cell proliferation, cell migration and invasion capacity, while the proportion of apoptotic cells was increased. In addition, Western blotting demonstrated that knockdown of CEMIP inhibited activation of the PI3K/AKT signaling pathway. Altogether, these data suggest that CEMIP may promote the development of ovarian cancer by regulating PI3K/AKT signaling.

Interaction of ERα and NRF2 Impacts Survival in Ovarian Cancer Patients.

Nuclear factor erythroid 2-related factor 2 (NRF2) regulates cytoprotective antioxidant processes. In this study, the prognostic potential of NRF2 and its interactions with the estrogen receptor α (ERα) in ovarian cancer cells was investigated. NRF2 and ERα protein expression in ovarian cancer tissue was analyzed as well as mRNA expression of NRF2 (NFE2L2) and ERα (ESR1) in four ovarian cancer and one benign cell line. NFE2L2 silencing was carried out to evaluate a potential interplay between NRF2 and ERα. Cytoplasmic NRF2 expression as inactive form had significantly higher expression in patients with low-grade histology (p = 0.03). In the serous cancer subtype, high cytoplasmic NRF2 expression (overall survival (OS), median 50.6 vs. 29.3 months; p = 0.04) and high ERα expression (OS, median 74.5 vs. 27.1 months; p = 0.002) was associated with longer overall survival as well as combined expression of both inactive cytoplasmic NRF2 and ERα in the whole cohort (median 74.5 vs. 37.7 months; p = 0.04). Cytoplasmic NRF2 expression showed a positive correlation with ERα expression (p = 0.004). NFE2L2 was found to be highly expressed in the ovarian cancer cell lines OVCAR3, UWB1.289, and TOV112D. Compared with the benign cell line HOSEpiC, ESR1 expression was reduced in all ovary cancer cell lines (all p < 0.001). Silencing of NFE2L2 induced a higher mRNA expression of ESR1 in the NFE2L2 downregulated cancer cell lines OVCAR3 (p = 0.003) and ES2 (p < 0.001), confirming genetic interactions of NRF2 and ERα. In this study, both inactive cytoplasmic NRF2 and high ERα expression were demonstrated to be associated with improved survival in ovarian cancer patients. Further understanding of interactions within the estradiol–ERα–NRF2 pathway could better predict the impact of endocrine therapy in ovarian cancer.

Human Wharton's Jelly Stem Cell (hWJSC) Extracts Inhibit Ovarian Cancer Cell Lines OVCAR3 and SKOV3 in vitro by Inducing Cell Cycle Arrest and Apoptosis.

Ovarian cancer is a highly lethal and the second highest in mortality among gynecological cancers. Stem cells either naïve or engineered are reported to inhibit various human cancers in both in-vitro and in-vivo. Herein we report the cancer inhibitory properties of human Wharton's jelly stem cell (hWJSC) extracts, namely its conditioned medium (hWJSC-CM) and cell lysate (hWJSC-CL) against two ovarian cancer cell lines (OVCAR3 and SKOV3) in-vitro. Cell metabolic activity assay of OVCAR3 and SKOV3 cells treated with hWJSC-CM (12.5, 25, 50, 75, 100%) and hWJSC-CL (5, 10, 15, 30, and 50 μg/ml) demonstrated concentration dependent inhibition at 24–72 h. Morphological analysis of OVCAR3 and SKOV3 cells treated with hWJSC-CM (50, 75, 100%) and hWJSC-CL (15, 30, and 50 μg/ml) for 24–72 h showed cell shrinkage, membrane damage/blebbings and cell death. Cell cycle assay demonstrated an increase in the sub-G1 and G2M phases of cell cycle following treatment with hWJSC-CM (50, 75, 100%) and hWJSC-CL (10, 15, and 30 μg/ml) at 48 h. Both OVCAR3 and SKOV3 cells demonstrated mild positive expression of activated caspase 3 following treatment with hWJSC-CM (50%) and hWJSC-CL (15 μg/ml) for 24 h. Cell migration of OVCAR3 and SKOV3 cells were inhibited following treatment with hWJSC-CM (50%) and hWJSC-CL (15 μg/ml) for 48 h. Tumor spheres (TS) of OVCAR3 and SKOV3 treated with hWJSC-CM (50, 75, 100%) and hWJSC-CL (10, 15, 30 μg/ml) for 48 h showed altered surface changes including vacuolations and reduction in size of TS. TS of OVCAR3 and SKOV3 also showed the presence of few ovarian cancer stem cells (CSCs) in minimal numbers following treatment with hWJSC-CM (50%) or hWJSC-CL (15 μg/ml) for 48 h. Real-time gene expression analysis of OVCAR3 and SKOV3 treated with hWJSC-CM (50%) or hWJSC-CL (15 μg/ml) for 48 h demonstrated decreased expression of cell cycle regulatory genes (cyclin A2, Cyclin E1), prostaglandin receptor signaling genes (EP2, EP4) and the pro-inflmmatory genes (IL-6, TNF-α) compared to untreated controls. The results indicate that hWJSC-CM and hWJSC-CL inhibit ovarian cancer cells at mild to moderate levels by inducing cellular changes, cell cycle arrest, apoptosis, decreasing the expression of CSC markers and related genes regulation. Therefore, the stem cell factors in hWJSCs extracts can be useful in cancer management.

Overexpression of the lncRNA FER1L4 inhibits paclitaxel tolerance of ovarian cancer cells via the regulation of the MAPK signaling pathway.

To determine how the lncRNA FER1L4 in ovarian cancer cells influences paclitaxel (PTX) resistance, we examined the expression level of FER1L4 in human ovarian epithelial cell lines IOSE80 and HOSEpiC and human ovarian cancer cell lines OVCAR-3, Caov-3, and SKOV3 through RNA isolation and quantitative polymerase chain reaction (qRT-PCR). SKOV3 cell lines were treated with PTX. The cell survival rate and apoptosis rate of SKOV3 and SKOV3-PR at different PTX dose levels were evaluated. Next, qRT-PCR was performed to detect the expression of FER1L4 in SKOV3 and SKOV3-PR cell lines. SKOV3-PR cell lines were transfected with pcDNA3.1 as the control group (SKOV3-PR/pcDNA3.1) or pcDNA3.1-FER1L4 to upregulate the expression level of FER1L4 (SKOV3-PR/pcDNA3.1-FER1L4). The level of cell survival, apoptosis, and colony formation were compared between the two groups using MTT, flow cytometry analysis, and colony formation assay. To reveal the molecular mechanism, we measured the relative protein phosphorylation level of ERK and MAPK in SKOV3, SKOV3-PR, SKOV3-PR/pcDNA3.1, and SKOV3-PR/pcDNA3.1-FER1L4 groups using an enzyme-linked immunosorbent assay. The effects of SB203580 (a p38 MAPK inhibitor) on PTX were also investigated to reveal the function of the MAPK pathway on the PTX tolerance of SKOV3. In comparison with normal ovarian epithelial cells, FER1L4 was downregulated. The FER1L4 level was decreased in human ovarian cancer cells with drug resistance than in common ovarian cancer cells. The upregulation of FER1L4 could promote the PTX sensitivity of ovarian cancer cells. The increased level of FER1L4 could suppress the PTX resistance of ovarian cancer cells through the inhibition of the MAPK signaling pathway.

Celastrol strongly inhibits proliferation, migration and cancer stem cell properties through suppression of Pin1 in ovarian cancer cells

Ovarian cancer is one of the most serious diseases worldwide and the fifth-most common cancer among women. Celastrol, extracted from Thunder God Vine, exerts anti-cancer effects on various cancers; however, the mechanism underlying these anti-cancer effects in ovarian cancer needs further investigation. Herein, we investigated the anti-cancer efficacy of celastrol and its underlying mechanism in human ovarian cancer cell lines A2780, OVCAR3, and SKOV3. Celastrol significantly suppressed cell proliferation and migration in a dose-dependent manner. Celastrol resulted in a G2/M cell cycle arrest, accompanied with the down-regulation of Cyclin D1, CDK2, and CDK4. Celastrol induced apoptosis primarily via up-regulation of caspase-3, caspase-8, and Bax, and down-regulation of Bcl-2. Celastrol treatment inhibited the expression of stem cell marker CD44, Nanog, Klf4, and Oct4, and reduced a portion of the CD44highCD24low cell population. To further understand the cancer therapeutic target, we assessed the effect of celastrol on expression of Pin1, which is reportedly overexpressed in many human cancers and activates more than 40 oncogenes or inactivates more than 20 tumor suppressor genes. We report that celastrol particularly suppressed Pin1 expression, thereby inhibiting Akt, STAT3, P38, JNK, P65, and IL-6 expression. Taken together, these findings indicate that celastrol is a potential therapeutic agent for ovarian cancer in humans via inhibition of Pin1 expression.