Ovarian Cancer Cell Lines Caov-3 Research Articles

Integrated transcriptome and cell phenotype analysis suggest involvement of PARP1 cleavage, Hippo/Wnt, TGF-β and MAPK signaling pathways in ovarian cancer cells response to cannabis and PARP1 inhibitor treatment.

Introduction: Cannabis sativa is utilized mainly for palliative care worldwide. Ovarian cancer (OC) is a lethal gynecologic cancer. A particular cannabis extract fraction ('F7') and the Poly(ADP-Ribose) Polymerase 1 (PARP1) inhibitor niraparib act synergistically to promote OC cell apoptosis. Here we identified genetic pathways that are altered by the synergistic treatment in OC cell lines Caov3 and OVCAR3. Materials and methods: Gene expression profiles were determined by RNA sequencing and quantitative PCR. Microscopy was used to determine actin arrangement, a scratch assay to determine cell migration and flow cytometry to determine apoptosis, cell cycle and aldehyde dehydrogenase (ALDH) activity. Western blotting was used to determine protein levels. Results: Gene expression results suggested variations in gene expression between the two cell lines examined. Multiple genetic pathways, including Hippo/Wnt, TGF-β/Activin and MAPK were enriched with genes differentially expressed by niraparib and/or F7 treatments in both cell lines. Niraparib + F7 treatment led to cell cycle arrest and endoplasmic reticulum (ER) stress, inhibited cell migration, reduced the % of ALDH positive cells in the population and enhanced PARP1 cleavage. Conclusion: The synergistic effect of the niraparib + F7 may result from the treatment affecting multiple genetic pathways involving cell death and reducing mesenchymal characteristics.

Proteomic analysis of exosomes secreted during the epithelial-mesenchymal transition and potential biomarkers of mesenchymal high-grade serous ovarian carcinoma

BackgroundThe epithelial-mesenchymal transition (EMT) promotes cell signaling and morphology alterations, contributing to cancer progression. Exosomes, extracellular vesicles containing proteins involved in cell-cell communication, have emerged as a potential source of biomarkers for several diseases.MethodsOur aim was to assess the proteome content of exosomes secreted after EMT-induction to identify potential biomarkers for ovarian cancer classification. EMT was induced in the ovarian cancer cell line CAOV3 by treating it with EGF (10 ng/mL) for 96 h following 24 h of serum deprivation. Subsequently, exosomes were isolated from the supernatant using selective centrifugation after decellularization, and their characteristics were determined. The proteins present in the exosomes were extracted, identified, and quantified using Label-Free-Quantification (LFQ) via Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). To identify potential biomarkers, the obtained proteomic data was integrated with the TGGA database for mRNA expression using principal component analysis and a conditional inference tree.ResultsThe exosomes derived from CAOV3 cells exhibited similar diameter and morphology, measuring approximately 150 nm, regardless of whether they were subjected to EMT stimulation or not. The proteomic analysis of proteins from CAOV3-derived exosomes revealed significant differential regulation of 157 proteins, with 100 showing upregulation and 57 downregulation upon EMT induction. Further comparison of the upregulated proteins with the TCGA transcriptomic data identified PLAU, LAMB1, COL6A1, and TGFB1 as potential biomarkers of the mesenchymal HGSOC subtype.ConclusionsThe induction of EMT, the isolation of exosomes, and the subsequent proteomic analysis highlight potential biomarkers for an aggressive ovarian cancer subtype. Further investigation into the role of these proteins is warranted to enhance our understanding of ovarian cancer outcomes.

The Activity of Vitamin C Against Ovarian Cancer Cells Is Enhanced by Hyperthermia.

Vitamin C is essential for the proper functioning of the human body and plays a crucial role in many biological processes as a cofactor for enzymes. The anticancer activity of vitamin C has been indicated for years. Hyperthermia used in clinics allows increasing the effectiveness of anticancer therapies and may also be useful in enhancing the action of other substances. The purpose of this study was to enhance the anticancer activity of vitamin C through hyperthermia against ovarian cancer cells. The ovarian cancer cell lines Caov-3, NIH:OVCAR-3, and human fibroblasts CCD-1064Sk were tested in the present study. Vitamin C was used in the following concentrations: 0.24, 2.50 and 5.25 mM. Each of the selected concentrations was combined with the different temperatures (37°C, 40°C and 43°C). Cell survival, adhesion and changes at the molecular level were assessed. The obtained results revealed that hyperthermia enhances the anticancer activity of vitamin C. Ovarian cancer cells showed greater sensitivity to vitamin C at elevated temperatures. Cells may have different sensitivity to vitamin C due to the activation of different gene signatures associated with redox reactions and apoptosis, therefore we examined the following genes: BCAP31, BCL2L13, BID, CASP7, FADD and HTRA2. The increase in expression of these genes in cancer cells generated a stronger proapoptotic response. The present study showed that hyperthermia enhanced the anticancer activity of vitamin C in vitro.

Decitabine-Mediated Upregulation of CSPG4 in Ovarian Carcinoma Cells Enables Targeting by CSPG4-Specific CAR-T Cells.

Simple SummaryCancer therapy by specifically redirected T cells has revolutionized the field of oncology. However, the repertoire of targetable antigens is small. Here, we use the FDA-approved drug decitabine to upregulate the surface antigen CSPG4 on CSPG4-negative ovarian carcinoma cells. By optimizing decitabine dosing, we converted more than 50% of treated ovarian carcinoma cells to CSPG4-positive cells. Importantly, CSPG4 is a very well-established target antigen in melanoma, and we could previously demonstrate that T cells engineered to target CSPG4 could reliably kill CSPG4-positive melanoma cells. Using CSPG4-specific T cells, we demonstrate CSPG4-directed killing of decitabine-treated ovarian carcinoma cells, thereby adding CSPG4 to the repertoire of target antigens for ovarian cancer.The addition of CAR-T cells to the armamentarium of immunotherapy revigorated the field of oncology by inducing long-lasting remissions in patients with relapsing/refractory hematological malignancies. Nevertheless, in the lion’s share of patients diagnosed with solid tumors, CAR-T-cell therapy so far failed to demonstrate satisfactory anti-tumor activity. A crucial cause of resistance against the antigen-specific attack of CAR-T cells is predicated on the primary or secondary absence of suitable target antigens. Thus, the necessity to create a broad repertoire of different target antigens is vital. We aimed to evaluate the potential of the well-established melanoma antigen chondroitin sulfate proteoglycan 4 (CSPG4) as an inducible antigen in ovarian cancer cells, using CSPG4-negative SKOV-3 ovarian cancer cells as a model. Based on the hypomethylating activity of the FDA-approved drug decitabine, we refined a protocol to upregulate CSPG4 in the majority of decitabine-treated SKOV-3 cells. CSPG4-specific CAR-T cells generated by mRNA-electroporation showed CSPG4-directed cytokine secretion and cytotoxicity towards decitabine-treated SKOV-3. Another ovarian cancer cell line (Caov-3) and the neoplastic cell line 293T behaved similar. In aggregate, we generated proof-of-concept data paving the way for the further exploration of CSPG4 as an inducible antigen for CAR-T cells in ovarian cancer.

IL-1β inhibitor sensitizes to olaparib in homologous recombination deficiency proficient ovarian cancer cells

Objective: To investigate the inhibitory effect of combined strategy of poly adenosine diphosphate ribose polymerase (PARP) inhibitor and interleukin-1β (IL-1β) inhibitor on homologous recombination deficiency (HRD)-proficient ovarian cancer cells. Methods: (1) HRD-proficient ovarian cancer cell lines OVCAR3 and CAOV3 were treated with PARP inhibitor olaparib. Screening by RNA sequencing analysis, the expression level of IL-1β was validated by enzyme-linked immunosorbent assay (ELISA) and western blot. (2) The dose-response curves of IL-1β inhibitor diacerein were evaluated by cell counting kit-8 (CCK-8) assays in OVCAR3 and CAOV3 cells. CCK-8 assays were further applied to determine the viabilities of OVCAR3 and CAOV3 cells. (3) To evaluate the synergistic effects of olaparib and IL-1β inhibitor in vivo, the transplanted ovarian cancer model was constructed. BALB/c-nude mice (n=16) were injected intraperitoneally with 1×107 OVACR3 cells labelled with luciferase (OVCAR3-Luc). Immunohistochemistry (IHC) assay was performed to determine nuclear antigen associated with cell proliferation (Ki-67) expression. (4) Blood routine tests, kidney and liver function tests were performed to analyze the toxic reaction of different drug treatments. The potential drug-induced injuries of vital organs including heart, liver, spleen, lungs and kidneys of nude mice were determined by hematoxylin-eosin (HE) staining. Results: (1) The RNA sequencing results showed that the mRNA level of IL-1β was the most significantly increased among the 25 differentially expressed genes in OVCAR3 cells treated with olaparib, compared to the negative control group. Olaparib treatment significantly promoted the secretion and expression of IL-1β protein in both OVACR3 and CAOV3 cells by ELISA [(36.2±3.5) and (49.5±3.5) pg/ml, respectively; all P<0.001] and western bolt (2.87±0.37 and 2.05±0.08, respectively; all P<0.01). (2) The half maximal inhibitory concentration (IC50) value of IL-1β inhibitor was determined as follows: 75 μmol/L for OVACR3 cells and 100 μmol/L for CAOV3 cells. The treatments were divided into four groups including control group, olaparib monotherapy group, IL-1β inhibitor monotherapy group and the combination therapy group. The cell viabilities of each group in OVCAR3 and CAOV3 were determined by CCK-8 assay. The data in each group were showed as follows for OVCAR3 and CAOV3 cells: (100.0±0.4)% and (100.0±3.5)% in control group; (63.1±6.2)% and (63.3±3.8)% in olaparib monotherapy group; (61.6±4.7)% and (63.8±3.5)% in IL-1β inhibitor monotherapy group; and (32.9±5.2)% and (30.0±1.3)% in the combination therapy group. The viability assay showed that the combined strategy exhibited a significant inhibition effect on OVACR3 and CAOV3 cells, compared to the monotherapy group and the control group (all P<0.01). (3) All mice with transplanted tumors of HRD-proficient ovarian cancer cells were randomly divided into four groups, and treated with four different treatments as mentioned above, respectively. After 4 weeks (on day 29), the vivo fluorescence imaging were determined. The results showed that the amount of fluorescence of transplanted tumors was mostly decreased in the combination therapy group [(0.5±0.4)×1010 p/s], compared to the control group [(4.2±1.0)×1010 p/s] or the groups treated with any single drug [(3.1±0.9)×1010, (2.2±0.9)×1010 p/s; all P<0.05]. Mice were then sacrificed under anesthesia, and all transplanted tumors detached and weighed for further investigation. The weight of transplanted tumors was significantly decreased in the combination therapy group [(0.09±0.03) g], compared to that in control group [(0.25±0.05) g] or groups treated with any single drug [(0.17±0.03), (0.19±0.04) g; all P<0.05]. The measurement of the expression of Ki-67 showed that it was significantly decreased in the combination therapy group (0.33±0.10), compared to that in the control group (1.00±0.20) or monotherapy groups (0.76±0.07, 0.77±0.12; all P<0.05). (4) There were no significant differences of body weights, blood routine test, renal and liver function tests among mice with different treatments (all P>0.05). Moreover, no significant injuries were observed in the vital organs among the four groups. Conclusions: The combination of olaparib and IL-1β inhibitor synergistically exhibits significant cytotoxicity in HRD-proficient ovarian cancer cells. Moreover, the blood routine and blood biochemistry results confirmed the biosafety of the combination of olaparib and IL-1β inhibitor.

MiR-4324 inhibits ovarian cancer progression by targeting FEN1

BackgroundOvarian cancer is one of the most lethal malignancies, with a 1.9% mortality rate worldwide. The dysregulation of the FEN1 gene and miR-4324 has been associated with cancer progression. However, the relationship between miR-4324 and-FEN1 requires further investigation.MethodsmiR-4324 and FEN1 expressions in ovarian cancer tissues and cell lines were measured via RT-qPCR. The interaction between miR-4324 and FEN1 was assessed using luciferase and RNA pull-down assays. The effects of miR-4324 and FEN1 on cell proliferation, adhesion and apoptosis were determined by CCK-8, BrdU, colony formation, cell adhesion, Caspase-3 and western blot assays in ovarian cancer cell lines CaOV3 and OVCAR3, respectively.ResultsThe results showed that miR-4324 expression was significantly decreased and FEN1 expression was enhanced in ovarian cancer tissues and cell lines. miR-4324 inhibitor promoted cell proliferation, adhesion and migration, and prevented apoptosis. Furthermore, the downregulation of FEN1 inhibited ovarian cancer cell growth and increased apoptosis. miR-4324 inhibited FEN1 expression and repressed ovarian cancer progression.ConclusionOur study found that miR-4324 inhibited FEN1 expression, suppressed cell growth, and increased apoptosis in ovarian cancer cells. Therefore, we identified miR-4324 and FEN1 as potential therapeutic targets for ovarian cancer treatment.

CircCRIM1 promotes ovarian cancer progression by working as ceRNAs of CRIM1 and targeting miR-383-5p/ZEB2 axis

BackgroundOvarian cancer is the leading cause of death in patients with gynecologic cancer, and circular RNAs (circRNAs) are involved in cancer progression. However, there are limited studies on the roles of circRNAs in ovarian cancer.MethodsWe designed divergent and convergent primers, used sanger sequencing and RNase R digestion to verify the source of circCRIM1. We detected the expression of circCRIM1 and its parental gene cysteine rich transmembrane BMP regulator 1 (CRIM1) in ovarian cancer and normal ovarian samples via qRT-PCR. MTT viability assay, apoptosis assay, wound healing assay and invasion assay were used to investigate the function of circCRIM1 and CRIM1 in ovarian cancer cell lines OVCAR3 and CAOV3. Mice xenografts experiment was performed. Bioinformatics predicted the microRNAs that bond with circCRIM1 and CRIM1, and dual luciferase reporter system confirmed it. Rescue experiments of microRNAs mimics transfection on the basis of circCRIM1 over-expression were carried out to uncover the mechanism by which circCRIM1 played cancer-promoting roles in ovarian cancer.ResultsCircCRIM1 was derived from CRIM1 by back-splicing. CircCRIM1 and CRIM1 had higher expression in ovarian cancer than in normal ovarian tissues, and both of them promoted ovarian cancer progression in vitro. In vivo circCRIM1 promoted the growth of tumors. CircCRIM1 and CRIM1 had a positive correlation relationship in the same cohort of ovarian cancer tissues. Bioinformatics predicted and dual luciferase assay confirmed circCRIM1 and CRIM1 bond with miR-145-5p, and circCRIM1 bond with miR-383-5p additionally. CircCRIM1 positively affected the expression of CRIM1. After circCRIM1 was over-expressed, miR-145-5p mimics transfection reversed the expression of CRIM1. Western blot discovered circCRIM1 positively affected the expression of zinc finger E-box binding homeobox 2 (ZEB2). Rescue experiments found miR-383-5p mimics reversed ZEB2 expression and the cancer-promoting effects of circCRIM1.ConclusionsCircCRIM1 bond with miR-145-5p to work as competing endogenous RNA (ceRNA) of CRIM1, and circCRIM1 bond with miR-383-5p to improve the expression of ZEB2 in ovarian cancer. CircCRIM1 and CRIM1 promoted the ovarian cancer progression and supplied a novel insight into the researches of ovarian cancer.

Arsenic compound sensitizes homologous recombination proficient ovarian cancer to PARP inhibitors

The poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors show survival benefits in ovarian cancer patients with BRCA1/2 mutation or homologous recombination (HR) deficiency, but only limited efficacy in HR-proficient ones. Another drug, arsenic trioxide (ATO) or arsenic drug (RIF), exerts antitumor effects via inducing DNA damage. Here, we investigated the combined therapeutic effects of the PARP inhibitors and the arsenic compound in HR-proficient ovarian cancer. The combined treatment of niraparib, olaparib, or fluazolepali with ATO showed a significant suppression in tumor cell viability and colony formation. The drug treatment also induced synergistic inhibition of cell proliferation and DNA damage, and acceleration of cell apoptosis in two HR-proficient ovarian cancer cell lines SKOV3 and CAOV3, either by simultaneous or sequential administration. The mechanism underlying these synergistic effects were reflected by the significantly increased ratio of cleaved-PARP/total PARP and decreased ratio of p-AKT/total AKT. Consistently, the combination of olaparib with RIF synergistically reduced the tumor growth in mouse xenograft models. In conclusion, the arsenic compound greatly sensitizes HR-proficient ovarian cancer cells to the PARP inhibitors, and our findings provide an evidence for the clinical treatment development of this combination in HR-proficient ovarian cancer patients.

Evaluating the Inhibition of the Na + ‐H + exchanger Isoform 1 (NHE1) and Kinases that Activate NHE1 as a Potential Combination Therapies for the Treatment of Ovarian Cancer with Paclitaxel

Every year in the United States there are over 22,000 newly diagnosed cases of ovarian cancer leading to over 14,000 deaths. Ovarian Cancer is the fifth leading cause of cancerous deaths among women. Due to that advanced stage of cancer progression at the time of diagnosis treatment is typically challenging and frequently accompanied by drug resistance and disease recurrence. Increased expression and activation of NHE1 has been shown to regulate cell proliferation in cancer cells from ovarian cancer patients. NHE1 is an 815 amino acid transmembrane protein that regulates intracellular pH. NHE1 hyperactivity is involved in the pH inversion which creates an optimal environment for cancer cell proliferation and invasion. In an effort to combat this disease recurrence and drug resistance, it is hypothesized that combining the chemotherapeutic paclitaxel with inhibitors that alter NHE1 function there will be a synergistic effect that slows ovarian cancer progression. Paclitaxel is a secondary treatment for ovarian cancer. It is a taxane based chemotherapeutic that acts as a microtubule stabilizer that affects the cells in the G2 and M phases of cell growth. In these experiments a direct inhibitor of NHE1, Ethylisopropylamiloride, is used. Additionally, we evaluate BI-D1870, the inhibitor of Ribosomal S6 Kinase (Rsk). Rsk is activated in ovarian cancer and has the ability to stimulate NHE1 activity. The ability to develop a synergistic effect was assessed using a proliferation assay in two ovarian cancer cell lines CAOV-3 and SKOV-3. Proliferation assays were run with paclitaxel alone and then with both paclitaxel and one of the inhibitors. The IC50 values were then determined for paclitaxel alone and in combination with the inhibitor. For example, in SKOV-3 Cells, the IC50 for paclitaxel was 0.086 µM. When combined with BI-D 1870 the IC50 for paclitaxel decreased to 0.12 µM, an 86% reduction. These data indicate that inhibitors of NHE1 or NHE1 activation may be combined with paclitaxel to improve the efficacy of ovarian cancer treatment.

Analysis of ovarian cancer cell secretome during epithelial to mesenchymal transition reveals a protein signature associated with advanced stages of ovarian tumors

Ovarian cancer (OvCA) is the most lethal neoplasia among gynecologic malignancies and faces high rates of new cases particularly in South America. In special, the High Grade Serous Ovarian Carcinoma (HGSC) presents very poor prognosis with deaths caused mainly by metastasis. Among several mechanisms involved in metastasis, the Epithelial to Mesenchymal Transition (EMT) molecular reprogramming represents a model for latest stages of cancer progression. EMT promotes important cellular changes in cellular adhesion and cell-cell communication, which particularly depends on the paracrine signaling from neighbor cells. Considering the importance of cellular communication during EMT and metastasis, here we analyzed the changes in the secretome of the ovarian cancer cell line Caov-3 induced to EMT by Epidermal Growth Factor (EGF). Using a combination of GEL-LC-MS/MS and stable isotopic metabolic labelling (SILAC), we identified up-regulated candidates during EMT as a starting point to identify relevant proteins for HGSC. Based on public databases, our candidate proteins were validated and prioritized for further analysis. Importantly, several of the protein candidates were associated with cellular vesicles, which are important to the cell-cell communication and metastasis. Furthermore, the association of candidate proteins with gene expression data uncovered a subset of proteins correlated with the mesenchymal subtype of ovarian cancer. Based on this relevant molecular signature for aggressive ovarian cancer, supported by protein and gene expression data, we developed a targeted proteomic method to evaluate individual OvCA clinical samples. The quantitative information obtained for 33 peptides, representative of 18 proteins, was able to segregate HGSC from other tumor types. Our study highlighted the richness of the secretome and EMT to reveal relevant proteins for HGSC, which could be used in further studies and larger patient cohorts as a potential stratification signature for ovarian cancer tumor that could guide clinical conduct for patient treatment.

The oncogenic and prognostic role of PDK1 in the progression and metastasis of ovarian cancer

Ovarian cancer (OC) is the most lethal of gynecological tumors in women. Tumor metabolism has become a new opportunity in the treatment of tumors. Pyruvate dehydrogenase kinase 1 (PDK1), as a key regulatory enzyme implicated in metabolic reprogramming of tumors, abnormally high expressed in various tumors and involved in the regulation of tumor cell biological behavior. However, the role of PDK1 in the occurrence and development of ovarian cancer remains unclear. Our team identified the expression of PDK1 in ovarian cancer cell lines and tissues through RT-PCR and immunohistochemical staining and evaluated the correlation of PDK1 expression with clinicopathologic features of patients and survival analyses. We used a variety of in vitro experiments to explore the influence of PDK1 on proliferation, invasion, migration, colony formation, apoptosis and the cell cycle of ovarian cancer cell lines CAOV3 and SKOV3. PDK1 was highly expressed in ovarian cancer cell lines and OC tissues. High expression of PDK1 was closely correlated to tumor size, FIGO stage, extraovarian metastases status and distribution. Univariate and multivariate Cox regression analysis identified that PDK1 was an independent prognostic factor for overall survival. Moreover, PDK1 was a superior predictor in prognosis of ovarian cancer and the incorporation of CA125 into PDK1 generated a predictive combination that displayed better predictive accuracy for overall survival. Downregulation of PDK1 suppressed the biological behavior of ovarian cancer cells due to S phase arrest and cellular apoptosis. PDK1 may serve as a novel prognostic biomarker, even a promising antineoplastic target of ovarian cancer.

MicroRNA miR-502-5p inhibits ovarian cancer genesis by downregulation of GINS complex subunit 2

ABSTRACT Ovarian cancer (OC) is one of the most common malignancies with high incidence and mortality and the eighth most common cancer-associated mortality in women worldwide. Aberrant expression of the GINS complex subunit 2 (GINS2) gene and miR-502-5p has been associated with cancer progression. This study aims to investigate the specific molecular mechanism of the miR-502-5p-GINS2 axis in OC. GINS2 and miR-502-5p expression in OC tissues and cell lines was measured using RT-qPCR. Next, we investigated the interaction between miR-502-5p and GINS2 using a luciferase assay. The role of the miR-502-5p-GINS2 axis was detected by assessing cell proliferation, migration, and apoptosis levels, such as caspase-3 activity and caspase-3 protein expression, in the OC cell lines CaOV3 and SKOV3, respectively. MiR-502-5p expression was decreased, and GINS2 expression was dramatically elevated in OC tissues and cells. Upregulation of miR-502-5p expression repressed cellular proliferation and migration levels but increased the cellular apoptosis level. GINS2 overexpression enhanced the proliferation and migration levels but hampered OC cell apoptosis. Moreover, miR-502-5p inhibited GINS2 expression and suppressed OC tumorigenesis. miR-502-5p targeting GINS2 suppressed OC progression by inhibiting cell growth and promoting cell apoptosis. Hence, we provide a comprehensive understanding of OC involving both miR-502-5p and GINS2, which might be effective therapeutic targets for OC patients.

Vinculin presents unfavorable prediction in ovarian cancer and prevents proliferation and migration of ovarian cancer cells.

The influences of Vinculin on many cancers were blurry, including ovarian cancer. Thus, we concentrated on the efficient role of Vinculin in ovarian cancer and explored the potential mechanism(s). Expression of Vinculin in ovarian cancer tissues and cell lines was investigated by real-time polymerase chain reaction, immunohistochemistry, and Western blot. The Kaplan-Meier manner with the logrank was performed to assess overall survival. We further evaluated the relations between Vinculin expression and clinicopathological features of ovarian cancer. Moreover, Vinculin was overexpressed or silenced by respectively transfection with pcDNA-Vinculin or small interfering (si-Vinculin) into human ovarian cancer cell line Caov3 or human ovarian epithelial cell line (HOEpiC). Thereafter, cell viability, cell apoptosis, and migration were checked by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, flow cytometer, and scratch assay, respectively. Likewise, the apoptosis- and migration-related proteins were distinguished by Western blot. Compared to the nontumor tissues or HOEpiC cells, Vinculin was significantly lower expressed in the ovarian cancer tissues and cells. Furthermore, we found out that Vinculin was primarily distributed at the cell membrane and cytoplasm. Moreover, Vinculin was negatively associated with International Federation of Gynecology and Obstetrics stage, grade, and distant metastasis. Overexpression of Vinculin dramatically weakened cell viability and migration and stimulated apoptosis. Conversely, suppression of Vinculin showed opposite results. Vinculin presents unfavorable prediction in ovarian cancer and inhibits ovarian cancer proliferation and migration.

MiR-6086 inhibits ovarian cancer angiogenesis by downregulating the OC2/VEGFA/EGFL6 axis

miRNAs have emerged as a pivotal component of gene regulatory networks, mediating cytokines secretion, cell cycle, and differentiation regulation. However, how miRNAs collaborate with transcription factors and downstream effector proteins that determine the fate of ovarian cancer cells remains to be understood, especially regarding to mechanism of tumor angiogenesis regulation. Based on the qRT-PCR and IHC analysis, we found that miR-6086 was maintained a very low level both in ovarian cancer cell lines and tissues. Further, we identified OC2 and EGFL6 as the direct targets of miR-6086 by luciferase assay and we observed an inverse relationship between the expression of miR-6086 and the OC2/VEGFA/EGFL6 axis. The Western blotting analysis suggested that OC2 could directly upregulate VEGFA and indirectly up-regulate EGFL6 through VEGFA. Moreover, miR-6086 could indirectly downregulate VEGFA through OC2. In addition, miR-6086, siOC2 and siEGFL6 could negatively regulate the tumor growth and angiogenesis of ovarian cancer (Skov3) in the animal studies, with the inhibition rates of 77.07%, 69.89%, and 73.62%, respectively (**p < 0.01). Moreover, the tumor cell proliferation, migration, and invasion of ovarian cancer cell lines (Caov3 and Skov3) and vascular formation (HUVECs) were significantly suppressed in vitro, by decreasing the AKT/MAPK pathways (*p < 0.05). Taken together, our results reveal that miR-6086 can suppress the angiogenesis networks in ovarian cancer by down-regulating the OC2/VEGFA/EGFL6 axis, directly or indirectly, which may provide potential targets for tumor therapeutics.

Use of Epigenetic Therapy Shortens Duration of Standard Chemotherapy for Ovarian Cancer with Minimal Toxicity to Normal Tissue

Objective: (1) Determine if sequential administration of standard chemotherapy (paclitaxel and cisplatin, P/C) with epigenetic drugs effectively targets ovarian cancer and limits toxicity to normal cells. (2) Define whether epigenetic treatment can shorten the exposure to P/C. Methods: Normal cells—adipocyte-derived stem cells (ASC), primary fibroblasts (PF), and human intestinal epithelial cells (HIEC-6)—were treated with 48 h IC50 values of P/C and epigenetic drugs, 5-azacytidine (AZA) and or suberoylanilide hydroxamic acid (SAHA), in combination and sequentially. The least toxic regimens to normal cells were administered to the ovarian cancer cell lines Caov-3, SKOV-3, OVCAR-3. Cell viability after treatments were assessed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and cell count assays. Secretome analysis of conditioned medium collected from the treated ovarian cancer cells was performed using ELISA. Results: P/C with AZA and SAHA targeted all ovarian cancer cell lines (82-99% cell death), but also caused significant normal cell death (66-100%). In contrast, P/C followed by AZA or SAHA is less toxic to ASC and PF (25-96% viability) when compared to a four-drug combination therapy (1% viability, p<0.0001). P/C followed by SAHA was least toxic to HIEC-6 (100% viability, p=0.0356). P/C followed by epigenetic drugs targets ovarian cancer cells more efficiently than two rounds of P/C (p<0.001). Levels of VEGF and IL-6 were downregulated after treatment with P/C followed by SAHA in SKOV-3 and OVCAR-3. Conclusions: Sequential treatments of P/C with epigenetic drugs, specifically SAHA, preserves the viability of normal cells, efficiently targets ovarian cancer, and minimizes exposure to P/C.

LncRNA PCAT6 promotes occurrence and development of ovarian cancer by inhibiting PTEN.

The purpose of this study was to explore the expression and function of long non-coding RNA (lncRNA) PCAT6 in ovarian cancer. Quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of lncRNA PCAT6 in 42 pairs of ovarian cancer tissues and adjacent normal tissues. Then, the relationship between PCAT6 expression and pathological indicators of ovarian cancer was analyzed. Subsequently, the transfection efficiency of PCAT6 in ovarian cancer cells was verified, and the PCAT6 knockdown model was constructed using lentiviruses in SKOV3 and CAOV3 ovarian cancer cell lines. In addition, Cell Counting Kit-8 (CCK-8) test, wound healing assay and transwell invasion and migration experiments were performed to estimate the effect of PCAT6 on the biological function of ovarian cancer cells, to further explore the possible potential mechanisms. QRT-PCR results showed that the expression level of PCAT6 in ovarian cancer was higher than that in the adjacent normal tissues. The incidence of distant metastasis and lymph node metastasis in patients with high expression of PCAT6 was higher than those with low PCAT6 expression. Compared with the NC group, the proliferation, metastasis and invasion ability of ovarian cancer cells in si-PCAT6 group decreased significantly. QRT-PCR results demonstrated that the PTEN expression was increased after the knockdown of PCAT6. In addition, the recovery experiment also revealed that PCAT6 and PTEN have a mutual regulation, which can jointly regulate the development of ovarian cancer. LncRNA PCAT6 was up-regulated in ovarian cancer tissues and was closely related to distant metastasis or lymph node metastasis. Additionally, lncRNA PCAT6 might promote the proliferation, migration and invasion of ovarian tumor cells by inhibiting PTEN.

Knockdown of long noncoding RNA-taurine-upregulated gene 1 inhibits tumor angiogenesis in ovarian cancer by regulating leucine-rich α-2-glycoprotein-1.

To investigate the role of long noncoding RNA taurine-upregulated gene 1 (TUG1) on ovarian cancer-induced angiogenesis and to explore possible signaling pathways. Ovarian cancer cell line SKOV3 or CAOV3 was transfected with short hairpin-TUG1 to suppress TUG1 expression. Supernatant from cultured cancer cells was used as a condition medium to incubate endothelial cell line human umbilical vein endothelial cells, whose proliferation rate was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration and invasion of endothelial cells were examined by wound healing and Transwell assays, followed by in-vitro angiogenesis assay. One of the secretory factors mediating angiogenesis, leucine-rich α-2-glycoprotein-1 (LRG1), was measured in ovarian cancer cells. Signaling pathway mediating angiogenesis was further detected by western blotting. TUG1 was down-regulated in ovarian cancer cells by short hairpin RNA. Conditional medium originating from TUG1-knockdown cancer cells led to suppressed proliferation, migration, or invasion of endothelial cell line human umbilical vein endothelial cells. LRG1 expression and secretion was suppressed in ovarian cancer cells after TUG1 knockdown. Moreover, recombinant LRG1 rescued TUG1 knockdown-induced angiogenesis inhibition, and LRG1 probably mediated angiogenesis by tumor growth factor-β signaling pathway in endothelial cells. Long noncoding RNA-TUG1 mediates angiogenesis of endothelial cells by regulating LRG1 secretion from ovarian cancer cells partially through tumor growth factor-β pathway. Our results indicate the potency of TUG1 as a biomarker and therapeutic target for tumor-induced angiogenesis.

GPR30 signaling to regulate epithelial-mesenchymal transition and predict survival in ovarian cancer.

e17041 Background: G protein-coupled receptor 30 (GPR30) is a 7-transmembrane estrogen receptor that functions alongside traditional estrogen receptors to regulate the cellular responses to estrogen. Recent studies suggest that the high expression of GPR30 is associated with a poor prognosis in breast cancer or endometrial cancer. Although the role of GPR30 in ovarian cancer was unclear, we revealed that GPR30 is associated with poor prognosis in ovarian cancer. On the other hand, Epithelial-to-Mesenchymal Transition (EMT) is involved in cancer metastasis. The purpose of this study is to reveal how GPR30 was associated with poor prognosis and whether associated with EMT in ovarian cancer. Methods: We examined whether GPR30 signaling activates the EGFR-Akt pathway in an ovarian cancer cell line (Caov-3) by a Western blotting analysis. We also examined the effect of GPR30 on EMT were evaluated in Caov-3, which were cultured both in two-dimensional (2D) culture and three-dimensional (3D) culture model. GPR30 agonist, G1, was used to confirm the regulatory effects of GPR30 on the change of phenotypic modulation and EMT markers expression. Results: The phosphorylation of the EGFR and Akt could be significantly enhanced by G1 (p &lt; 0.05) and inhibited by a Src family kinase inhibitor. In 3D culture, the stimulation of GPR30 leads the floating and sphere formation in Caov-3. G1-induced EMT was observed with related regulation of EMT markers expression at both mRNA and protein level. G1 induced the decrease of E-cadherin level and the increase of Snail and Vimentin in RT-PCR and Western blotting. Knockdown of GPR30, using siRNA, blocked G1-induced EMT. Conclusions: GPR30 increases the phosphorylation of Akt via the EGFR in ovarian cancer cells and changes ovarian cancer cells to the EMT state.GPR30 might be an important molecule related to metastasis process in ovarian cancer.

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.

Poly r(C) Binding Protein 1 Regulates Posttranscriptional Expression of the Ubiquitin Ligase TRIM56 in Ovarian Cancer.

Our earlier work has shown that the E3 ligase TRIM56 messenger RNA (mRNA) level and vimentin protein expression followed an inverse correlation in ovarian carcinoma patients; however, the regulatory mechanisms underlying TRIM56 expression is unclear. Steady state expression of TRIM56 mRNA expression in the normal ovarian cell line Moody and ovarian cancer cell lines SKOV-3, A2780, and Caov-3 were not significantly different; however, TRIM56 protein expression was significantly lower in the ovarian cancer cell lines compared to the Moody cell line. Polysome profiling showed significant increase in translation of TRIM56 messenger RNA in the Moody cells compared to the SKOV-3 cells. We performed RNA-affinity pulldown using biotinylated TRIM56 5 'and 3'-UTR and postnuclear extracts from Moody and SKOV-3 cells. Whereas no notable difference was observed in affinity pull-down obtained with the 5'-UTR, there was obvious difference in protein binding patterns with the 3'-UTR. Mass spectrometry was used to determine the most differentially binding protein as poly r (c) binding protein 1 (PCBP1). PCBP1 expression and binding to the 3'-UTR was both higher in SKOV-3 cells compared to the Moody cells. Silencing of TRIM56 in Moody cells cause an increase in in vitro migration and invasion, and a similar effect was mimicked by overexpression of PCBP1. Conversely, silencing of PCBP1 or overexpression of TRIM56 in SKOV-3 cells significantly decreased in vitro migration and invasion. In xenograft assays, SKOV-3 cells stably overexpressing shRNA targeting PCBP1 decreased metastasis, whereas shRNA-targeting TRIM56 potentiated detection of metastatic lesions, compared to the parental SKOV-3 cells themselves. Taken together our results reveal a yet undefined posttranscriptional regulatory mechanism underlying low expression of TRIM56 in ovarian cancer. © 2018 IUBMB Life, 71(1):177-182, 2019.