SKOV3 Ovarian Cancer Cells Research Articles

Abstract 4880: Parsortix™ system: Analytic performance evaluation using ovarian cancer cell lines

Abstract Background: Ovarian cancer has the worst prognosis among gynecologic malignancies, in part due to presentation with later stage disease in a majority of women. Much effort has been expended towards early detection, but it remains a significant issue and challenge. One approach which may have potential in improving the detection of ovarian cancer, particularly in women who present with an adnexal or pelvic mass, is the isolation and interrogation of circulating tumor cells (CTCs) from peripheral blood. The microfluidic Parsortix™ system isolates rare cells from biological fluids, particularly blood, on the basis of cell size and deformability. Objectives: The primary aim of the present study was to evaluate the performance of a modified Parsortix™ system for the isolation of ovarian cancer cell lines spiked into blood drawn from healthy volunteers (HVs). Parameters such as sensitivity, determined by dilution linearity, reproducibility and stability (time after blood draw) were assessed. In addition, the ability to molecularly interrogate Parsortix™ harvests by RT-qPCR for the CTC-specific genes Epithelial Cell Adhesion Molecule (EpCAM) and CytoKeratin (CK) was assessed. Methods: Two ovarian cancer cell lines, CaOV3 and SKOV3, were used as model CTCs. Live cells were spiked into HV blood samples collected into K2EDTA vacutainers and stored for up to 120h. The spiked blood samples were then processed using modified Parsortix™ instruments (optimized to speed-up blood processing) and separation cassettes with a 6.5µm critical gap size. Harvests were evaluated for reproducibility, linearity, and stability by counting harvested cells and/or by using an optimized RT-qPCR assay. For experiments where cell counting and percentage recovery were assessed, cancer cells (CaOV3 or SKOV3) labelled with CellTracker™ Green were utilized and counted via fluorescence microscopy. When RT-qPCR was used to assess the recovered cells, unlabeled Parsortix™-isolated cells were lysed in a LiDS buffer, mRNA isolated using Dynabeads™, and cDNA synthesis performed followed by RT-qPCR for selected gene products. Results: The sensitivity of the Parsortix™ system for the isolation/harvest of CaOV3 and SKOV3 cells was approximately 1 cell/mL blood. There was no appreciable effect of storage of the spiked K2EDTA blood samples on recovery of the pre-labelled CaOV3 cells for up to 120h hours after collection/spiking, with either room temperature or refrigerated (4oC) blood storage. The isolation/harvest of model CTCs was reproducible with a CV of ≤14%. RT-qPCR, while gene dependent, was shown to be linear with model CTC input and showed a sensitivity of approximately 1 cell for EpCAM. Conclusions: These data demonstrate both the robustness and sensitivity of the Parsortix™ system for the isolation and interrogation of CTCs, and further studies, specifically in the area of ovarian cancer, are ongoing. Citation Format: Arianna Hustler, Gabrielle Wishart, Amy Templeman, Erica Bravo Sales, Sara Arocas Torrecillas, Kyra Mumford, Daniel J. O’Shannessy. Parsortix™ system: Analytic performance evaluation using ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4880.

Abstract 3501: Entinostat enhances the efficacy of olaparib in preclinical models of ovarian cancer

Abstract Introduction: Ovarian cancer is a rare but often fatal disease and a leading cause of gynecologic cancer death, in the United States. Poly ADP ribose polymerase inhibitors (PARPi) are promising new drugs that are most effective in patients with tumors with altered homologous recombination (HR) DNA repair genes, particularly BRCA1/2. To expand their clinical utility, PARPi are being tested in combination with other drugs. Our group has published that histone deacetylase inhibitors (HDACi) sensitize HR proficient BRCA wild-type ovarian cancer cells to PARPi. We have developed an investigator-initiated phase 1/2 clinical trial of the PARPi olaparib combined with the HDACi entinostat in patients with recurrent, HR proficient/BRCA wild-type ovarian cancer. Objective: To test the effects of a novel combination of olaparib and entinostat in preclinical models of BRCA wild-type ovarian cancer. Methods: HR proficient BRCA wild-type SKOV3 and ID8 ovarian cells were treated with Entinostat followed by Olaparib alone or the combination of Entinostat and Olaparib. Treated and control cells were analyzed for cell proliferation by sulforhodamine B assays, clonogenicity, and DNA damage by Comet assays. SKOV-3-luciferase cells were injected intraperitoneally into NOD-SCID mice. Mice were randomized into 4 treatment groups and treated with vehicle, entinostat, olaparib, or the combination. The mice were monitored for toxicity and by bioluminescence imaging (BLI). At sacrifice, tumor burden was quantified. Results: In cell culture assays, entinostat significantly enhanced the sensitivity of SKOV3 and ID8 cells to Olaparib. Entinostat combined with olaparib led to synergistic reductions in cell proliferation and clonogenicity in SKOV-3 and ID8 ovarian cancer cells, compared to each drug alone. Evidence of DNA damage was enhanced by the drug combination. In the SKOV3-luc xenograft mouse model, a significant reduction in tumor burden in the group of mice treated with the combination of entinostat and olaparib, compared to each drug alone. There was no significant toxicity with drug treatment. Conclusion: Entinostat sensitizes ovarian cancer cells to Olaparib in vitro and in vivo. Experiments in ID-luciferase and patient-derived xenograft models are ongoing. These preclinical models are powerful tools that will be used to discover markers and mechanisms of sensitivity and resistance to inform the parallel clinical trial. Citation Format: Vijayalaxmi G. Gupta, Yoskaly Lazo Fernandez, Katherine Roby, Harsh Pathak, Jeff Hirst, Andrew J. Wilson, Andrew Godwin, Dineo Khabele. Entinostat enhances the efficacy of olaparib in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3501.

Abstract 3809: Modulation of MRP1 activity reverses chemotherapy resistance in adult cancer cells

Abstract Multidrug resistance protein 1 (MRP1) is an ATP-dependent efflux pump that extrudes chemotherapeutic drugs from cancer cells, preventing their intracellular accumulation and thus their efficacy. MRP1 also regulates intracellular levels of glutathione (GSH), an antioxidant that when elevated in cancer provides resistance against chemotherapy- and radiotherapy-induced oxidative damage. The different binding sites for drugs and GSH on MRP1 has allowed the development of small molecule MRP1 modulators that block chemotherapy efflux whilst simultaneously enhancing GSH efflux to deplete GSH. As no therapeutic options are available to combat MRP1-mediated multidrug resistance, we have been investigating MRP1 modulators for their efficacy in sensitizing MRP1-expressing cancers to treatment. MRP1 immunohistochemistry staining in ovarian and non-small cell lung cancer (NSCLC) patient samples was assessed to determine the frequency of high MRP1 expression. High MRP1 staining was frequently observed in the subcellular compartment (37%, 47%) and, to a lesser extent, plasma membrane (7%, 16%) in ovarian and NSCLC patient tumour samples respectively. In NSCLC, subcellular MRP1 was associated with poorer overall survival in late stage disease (p=0.0095) and membrane MRP1 with poorer disease-free survival in Stage 4 cancer (p=0.01449). Analysis for ovarian cancer is ongoing. As MRP1 is therefore an attractive target for inhibition, we used membrane vesicle uptake assays to compare the ability of modulators to inhibit [3H]-labelled MRP1 substrate transport. 7-(difluoromethyl)-N-4-morpholinyl-5-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7914321) was identified as an analogue of the MRP1 inhibitor Reversan with improved potency. Interestingly, this inhibition was GSH dependent. Exploring further this relationship with GSH, the high MRP1-expressing A549 lung and SKOV3 ovarian cancer cell lines were treated with 7914321 alone or in combination with the GSH synthesis inhibitor buthionine sulfoximine (BSO), then GSH levels were determined by glutathione recycling assay. 7914321 stimulated GSH efflux in both cell lines in an MRP1-dependent manner and strongly synergised with BSO to cause near complete GSH depletion. To determine the potential of this synergistic GSH depletion to chemosensitize cells, the viability of A549 and SKOV3 cells treated with chemotherapy, 7914321, and BSO was assessed by clonogenic and resazurin-based cytotoxicity assays. 7914321/BSO significantly diminished clonogenicity in high but not low MRP1-expressing cells, and further sensitized cancer cell lines to MRP1-substrate chemotherapeutics compared to the single agents. Together, these findings provide preliminary evidence that 7914321 is a potent MRP1 modulator whose dual effects on MRP1 place it in a unique position to potentially enhance the therapeutic window to treat chemoresistant MRP1-overexpressing cancers. Citation Format: Kimberley M. Hanssen, Christine C. Gana, Madeleine S. Wheatley, Denise M. Yu, Claudia L. Flemming, Richard Young, Gavin M. Wright, Gwenaelle Conseil, Catherine J. Kennedy, Anna deFazio, Ben Solomon, Susan P. Cole, Murray D. Norris, Michelle Haber, Jamie I. Fletcher. Modulation of MRP1 activity reverses chemotherapy resistance in adult cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3809.

Targeting tumor cells and neovascularization using RGD-functionalized magnetoliposomes.

PurposeMagnetoliposomes (MLs) have shown great potential as magnetic resonance imaging contrast agents and as delivery vehicles for cancer therapy. Targeting the MLs towards the tumor cells or neovascularization could ensure delivery of drugs at the tumor site. In this study, we evaluated the potential of MLs targeting the αvβ3 integrin overexpressed on tumor neovascularization and different tumor cell types, including glioma and ovarian cancer.MethodsMLs functionalized with a Texas Red fluorophore (anionic MLs), and with the fluorophore and the cyclic Arginine-Glycine-Aspartate (cRGD; cRGD-MLs) targeting the αvβ3 integrin, were produced in-house. Swiss nude mice were subcutaneously injected with 107 human ovarian cancer SKOV-3 cells. Tumors were allowed to grow for 3 weeks before injection of anionic or cRGD-MLs. Biodistribution of MLs was followed up with a 7T preclinical magnetic resonance imaging (MRI) scanner and fluorescence imaging (FLI) right after injection, 2h, 4h, 24h and 48h post injection. Ex vivo intratumoral ML uptake was confirmed using FLI, electron paramagnetic resonance spectroscopy (EPR) and histology at different time points post injection.ResultsIn vivo, we visualized a higher uptake of cRGD-MLs in SKOV-3 xenografts compared to control, anionic MLs with both MRI and FLI. Highest ML uptake was seen after 4h using MRI, but only after 24h using FLI indicating the lower sensitivity of this technique. Furthermore, ex vivo EPR and FLI confirmed the highest tumoral ML uptake at 4 h. Last, a Perl’s stain supported the presence of our iron-based particles in SKOV-3 xenografts.ConclusionUptake of cRGD-MLs can be visualized using both MRI and FLI, even though the latter was less sensitive due to lower depth penetration. Furthermore, our results indicate that cRGD-MLs can be used to target SKOV-3 xenograft in Swiss nude mice. Therefore, the further development of this particles into theranostics would be of interest.

Abstract 3641: Biodegradable polysaccharides based paclitaxel formation for ovarian cancer therapy

Abstract Over 90% of pipeline therapeutic molecules have low solubility and lack of suitable delivery vehicle, which results the treatment potential could be limited. To address this challenge, ZY Therapeutics developed a tunable biodegradable polysaccharide-based nanoparticle drug delivery platform. The first candidate ZY-010-PNP is a nanoformulation using folate-modified polysaccharide encapsulating paclitaxel (PTX). ZY-010-PNP is supplied as a lyophilized powder that is readily reconstituted in saline to form a transparent suspension. The reconstituted formulation has an average particle size < 150 nm and remains stable for 24 hours at room. Drug release profile of ZY-010-PNP in human plasma was evaluated from 20 min to 4 hrs at different concentrations, using the stable isotope tracer ultrafiltration assay developed at NCI. The ZY-010-PNP formulation released 20-40% of the encapsulated PTX in human plasma within 20 min, and 60-100% was released over the subsequent 4-hour period. The data clearly showed that the drug release profile of ZY-010-PNP was time / concentrations dependent in serum, in contrast with burst release of all other marketed Taxane formulations. The in vitro efficacy was evaluated in human ovarian cancer SKOV3 cells. ZY-010-PNP induced dose- and time-dependent cell death and showed a similar potency as the benchmark drug in terms of cytotoxicity. However, the vehicle showed very low toxicity on SKOV3 and THLE-2 epithelial cells. The PK properties of ZY-010-PNP were also studied. ZY-010-PNP administrated on rats yielded the highest total plasma PTX level (Cmax), followed by a rapid declining and relatively longer elimination phase (t1/2) in comparison with the benchmark drug. The in vivo efficacy of ZY-010-PNP was demonstrated in two xenograft models, Triple Negative Breast Cancer (4T1) and Ovarian Cancer (SKOV3). In the SKOV3 study, multiple doses (10, 30 and 45 mg/kg) of ZY-010-PNP were tested in comparison to the benchmark drug. ZY-010-PNP at doses of over 30 mg/kg demonstrated complete tumor growth inhibition. Body weight (group means up to 15%) dropped during 30 and 45 mg/kg dosing period, but it was regained quickly post administration. In summary, a lyophilized dosage form of PTX formulation was developed with time and concentration dependent drug release profile. The in vitro and in vivo efficacy studies showed a similar tumor growth inhibition effect compared to the benchmark formulation. The PK profile demonstrated that ZY formulation yielded the highest total plasma Taxane level and relatively longer elimination phase. ZY’s vitamin-modified-polysacchride drug delivery platform showed advantages on improved stability, easy reconstitution, no foaming and no immunogenicity in compare to other benchmark delivery vehicle. The proprietary tunable platform can be applied to other high potency with low solubility molecules to increase the tolerance and reduce the toxicity of these drugs. Citation Format: Jian Bao, Hui Zhang, Aili Rong, Bajin Han, Nazar Filonov, Jun Li. Biodegradable polysaccharides based paclitaxel formation for ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3641.

Tetramethylpyrazine Enhances the Antitumor Effect of Paclitaxel by Inhibiting Angiogenesis and Inducing Apoptosis.

Recent published findings have demonstrated the effectiveness of combining molecules from traditional Chinese medicine with chemotherapeutic drugs to treat cancer. Combined administration of these agents can overcome drug-mitigating responses as well as reduce adverse side effects, thereby enhancing the efficacy of the therapy. Tetramethylpyrazine (TMP), an alkaloid monomer from the medicinal herb Ligusticum chuanxiong hort, is known to exert a variety of antitumor effects including inhibition of tumor cell proliferation, metastasis, and drug resistance. In this research, we investigated antitumor effects of TMP combined with paclitaxel (PTX), a frontline chemotherapeutic drug, in vitro and in vivo. Our results indicate that TMP enhances the antitumor effects of PTX in ovarian cancer A2780 and SKOV3 cells. Furthermore, we found that combined treatment of TMP and PTX suppressed angiogenesis by inhibiting both ERK1/2 and Akt pathways and promoted apoptosis of tumor cells compared to TMP or PTX treatment alone. Moreover, TMP augmented the antitumor effects of PTX in ovarian cancer A2780 xenograft mouse models by significantly decreasing tumor burden and partially decreasing the toxicity of PTX, as evidenced by the decreased expression of proliferation and angiogenesis markers as well as the hematoxylin and eosin (H&E) staining and biochemical indexes assay. Overall, our findings provide novel mechanistic insight into the efficacy of combining of potent molecules present in traditional Chinese medicine with chemotherapeutic drugs for therapeutic intervention in cancer.

Anticancer Effects of Cordyceps Militaris Extract in Human Ovarian Cancer Cells via ADORA2B (P05-012-19)

ObjectivesThe study was aimed to determine anticancer effects of Cordyceps militaris extract (CME) and its major bioactive compound, cordycepin, in human ovarian cancer cells, and to identify their putative molecular mechanism mediated by adenosine receptors (ADORAs). MethodsCME was prepared in 50% ethanol solution. LC-MS was used for quantification and Q-TOF MS for qualifying bioactive compounds in CME. MTT assay was performed for cell viability in A2780, SKOV-3, TOV112D, and OVCAR-3 human ovarian cancer cell lines. cAMP response element (CRE)-luciferase reporter gene assays were used to determine whether antitumorigenic effect of CME/cordycepin is based on adenosine derivatives. Additionally, the involvement of ADORA signaling pathway was measured using with ADORA2A antagonist SCH 58261 and ADORA2B antagonist PSB 603. ResultsCordycepin concentrations of CME was 21.8%. CME was effective to reduce cell viability in A2780 and OVCAR-3 with IC50 115.2 μg/ml and 155.94 μg/ml respectively, while SKOV-3 and TOV112D were relatively resistant to CME. cAMP production was significantly increased by treatment with cordycepin and, lesser extent, with CME. Among the four types of ADORAs, ADORA2A and 2B showed relatively higher expression levels in ovarian cancer cells. The cAMP production by CME was ameliorated by PSB 603, not SCH 58261, treatment. ConclusionsCME and cordycepin have anticancer effects in human ovarian cancer cells via ADORA2B-cAMP pathway. Funding SourcesNRF of Korea (2017R1D1A1B03034936 & 22A20130012143) and Health Fellowship Foundation.

BDNF activates TrkB/PLCγ1 signaling pathway to promote proliferation and invasion of ovarian cancer cells through inhibition of apoptosis.

Abnormal expression and activation of tropomyosin-related kinase receptor B (TrkB) are observed in many pathological conditions, including many types of cancer. We try to explore the relationship between ovarian cancer and Brain-derived neurotrophic factor (BDNF), a ligand of TrkB. Human ovarian cancer cell line SKOV-3 was used in this study. qPCR, immunohistochemistry, and immunoblot were used to assay BDNF and TrkB expression level. Scratch assay was used to test the cell motility, and transwell assay was used to test the cell migration ability. We found that BDNF promotes the proliferation and invasion of human ovarian cancer SKOV-3 cells depend on the activation of TrkB. To illuminate the downstream pathway of BDNF/TrkB, we silenced AKT1 and PLCγ1 by siRNA. The functional assay showed that activated PLCγ1 signaling pathway is necessary for the proliferation and invasion of cancer cells other than the AKT pathway. Further study showed that PLCγ1 could inhibit the apoptosis of cancer cells. BDNF triggers TrkB/PLCγ1 signaling pathway to promote proliferation and invasion of ovarian cancer cells through inhibition of apoptosis.

The Effect of Triptolide-Loaded Exosomes on the Proliferation and Apoptosis of Human Ovarian Cancer SKOV3 Cells.

Triptolide has been proven to possess anticancer efficacy; however, its application in the clinical practice was limited by poor water solubility, hepatotoxicity, and nephrotoxicity. In this study, a triptolide-loaded exosomes delivery system (TP-Exos) was constructed and its effects on the proliferation and apoptosis of SKOV3 cells in vitro and in vivo were observed. SKOV3-exosomes (SK-Exos) were collected by ultracentrifugation and ultrafiltration centrifugation. TP-Exos was constructed by sonication and ultrafiltration centrifugation. SK-Exos and TP-Exos were characterized by transmission electron microscopy, western blotting, nanoparticle-tracking analysis, and high-performance liquid chromatography. Cellular uptake of exosomes, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) cell proliferation assay, and cell apoptosis experiment were used to study the effect of TP-Exos on ovarian cancer in vitro. Tumor-targeting study of exosomes, monitoring the tumor volume of mice, and TdT-mediated dUTP Nick-End labeling (TUNEL) assay were used to evaluate the effect of TP-Exos on ovarian cancer in vivo. The toxicity of TP-Exos in vivo was evaluated by liver and kidney function and histopathology of major organs (heart, liver, spleen, lung, kidney, and ovary). The results revealed that TP-Exos not only have the general characteristics of exosomes but also have high drug encapsulation efficiency. Besides, PKH26 labeled exosomes (PKH26-Exos) could be uptaken by SKOV3 cells, and Dir labeled exosomes (Dir-Exos) could be enriched to the tumor site of tumor bearing mice. Furthermore, the cytotoxic and apoptotic effects on SKOV3 cells of TP-Exos were weaker than those of free TP, and tumor cell proliferation inhibition and tumor growth inhibition were stronger than that of free TP. Moreover, TP-Exos have toxic effect on liver and spleen. In conclusion, the TP-Exos could be a promising strategy for ovarian cancer, but they need to be further optimized to attenuate the damage to liver and spleen.

Increasing potency and safety of anticancer drugs through sortilin receptor-mediated cancer therapy: A new targeted approach for the treatment of ovarian cancer.

e17068 Background: The development of personalized therapies against ovarian cancer remains highly challenging in current modern oncology. One strategy to achieve greater selectivity and better anticancer drug delivery into cancer cells is to conjugate cytotoxic agents to specific peptide ligands that selectively target receptors abundantly and/or exclusively expressed on these cells. Increased expression of Sortilin, a scavenging receptor, has been clinically observed in invasive ovarian cancer biopsies, and correlated with tumor grades. In light of this, we developed a peptide conjugation platform and a new Sortilin receptor-mediated vectorization strategy to increase cell targeting selectivity and cell delivery efficacy of anticancer agents. Methods: As a proof-of-concept, Doxorubicin was conjugated to a Sortilin binding peptide (KA-peptide). In vitro, intracellular delivery of the Doxorubicin-KA-peptide conjugate (DoxKA) was assessed in ES-2 and SKOV-3 ovarian cancer cell line models using flow cytometry and fluorescent microscopy. Sortilin gene silencing was performed with specific siRNA. DoxKA efficacy and safety were evaluated in vivo using ES-2 (CD1 nude mice) and SKOV-3 (athymic mice) subcutaneous xenograft models. Results: Uptake of DoxKA was observed in both Sortilin-positive ovarian cancer cell lines tested and was reduced when Sortilin expression was specifically silenced or upon competition with the Sortilin ligands Neurotensin and Progranulin. Results indicate that the uptake of DoxKA occurs via Sortilin-mediated endocytosis in contrast to simple diffusion for Doxorubicin. DoxKA was found to bypass the P-glycoprotein (P-gp) efflux pump in MDCK-MDR1 cells overexpressing P-gp as the uptake of DoxKA was unaffected by the P-gp inhibitor Cyclosporin A. In vivo, DoxKA showed lower potential side effects than Doxorubicin alone did, with decreased accumulation in healthy tissues such as heart and ovary. DoxKA caused a more potent inhibition of human ovarian tumor xenografts growth in mice and was better tolerated (absence of leukopenia and neutropenia) than the unconjugated Doxorubicin at an equivalent dose. Conclusions: These results strongly support the future clinical use of this platform to generate novel personalized therapeutics with specific targeting of Sortilin-positive tumors in the next stage of development in phase 1 clinical trial.

The human amniotic fluid mesenchymal stem cells therapy on, SKOV3, ovarian cancer cell line.

PurposeOne of the most common malignancies peculiar to female health with few symptoms, low response to therapy, difficult diagnosis, frequent relapse, and high mortality, is ovarian cancer. Thus, our experiment, using Human amniotic fluid mesenchymal stem cells (hAFMSCs) as a therapeutic tool, aims to find an efficient treatment approach for patients suffering from SKOV3 ovarian cancer.Material & MethodsIn this study, we obtained 5 ml amniotic fluid from 16–20 week pregnant women who underwent amniocentesis for routine prenatal diagnosis by karyotyping in Al‐Zahra Hospital of Tabriz University of Medical Sciences, Iran. Using trans wells in 24 wells plate, hAFMSCs were isolated from all samples, co‐cultured with SKOV3 ovarian cancer cell line, and characterized via flow cytometry and RT‐PCR. Human skin fibroblast cells (HSFCs) were isolated and used as a negative control. SKOV3 and HSFCs' viability after 5 days was evaluated by MTT assay. Cell cycle and apoptotic genes were analyzed by real‐time PCR.ResultsWe successfully isolated and characterized hAFMSCs through it positivity for CD44 and CD90 specific mesenchymal stem cell markers and negativity for CD31 and CD45. Oct4 and NANOG were evaluated by RT‐PCR as pluripotency markers, and visualized on 2% gel electrophoresis. We established hAFMS cell lines after 5 days of co‐culturing the SKOV3 cells, viability was decreased; however, HSFCs did not show toxicity by MTT assay. The genes indicated upregulation and high expression by a real‐time PCR.ConclusionsOur findings showed that hAFMSCs have natural tumor tropism, and can release soluble factors in a cell culture, which cause an efficient anticancer effect. Thus, we can use hAFMSCs for complete anticancer therapy on SKOV3 cell line at cell culture condition and possibly in vivo in the near future.

HypoxamiRs Profiling Identify miR-765 as a Regulator of the Early Stages of Vasculogenic Mimicry in SKOV3 Ovarian Cancer Cells

Vasculogenic mimicry (VM) is a novel cancer hallmark in which malignant cells develop matrix-associated 3D tubular networks with a lumen under hypoxia to supply nutrients needed for tumor growth. Recent studies showed that microRNAs (miRNAs) may have a role in VM regulation. In this study, we examined the relevance of hypoxia-regulated miRNAs (hypoxamiRs) in the early stages of VM formation. Data showed that after 48 h hypoxia and 12 h incubation on matrigel SKOV3 ovarian cancer cells undergo the formation of matrix-associated intercellular connections referred hereafter as 3D channels-like structures, which arose previous to the apparition of canonical tubular structures representative of VM. Comprehensive profiling of 754 mature miRNAs at the onset of hypoxia-induced 3D channels-like structures showed that 11 hypoxamiRs were modulated (FC>1.5; p < 0.05) in SKOV3 cells (9 downregulated and 2 upregulated). Bioinformatic analysis of the set of regulated miRNAs showed that they might impact cellular pathways related with tumorigenesis. Moreover, overall survival analysis in a cohort of ovarian cancer patients (n = 485) indicated that low miR-765, miR-193b, miR-148a and high miR-138 levels were associated with worst patients outcome. In particular, miR-765 was severely downregulated after hypoxia (FC < 32.02; p < 0.05), and predicted to target a number of protein-encoding genes involved in angiogenesis and VM. Functional assays showed that ectopic restoration of miR-765 in SKOV3 cells resulted in a significant inhibition of hypoxia-induced 3D channels-like formation that was associated with a reduced number of branch points and patterned tubular-like structures. Mechanistic studies confirmed that miR-765 decreased the levels of VEGFA, AKT1 and SRC-α transducers and exerted a negative regulation of VEGFA by specific binding to its 3‘UTR. Finally, overall survival analysis of a cohort of ovarian cancer patients (n = 1435) indicates that high levels of VEGFA, AKT1 and SRC-α and low miR-765 expression were associated with worst patients outcome. In conclusion, here we reported a novel hypoxamiRs signature which constitutes a molecular guide for further clinical and functional studies on the early stages of VM. Our data also suggested that miR-765 coordinates the formation of 3D channels-like structures through modulation of VEGFA/AKT1/SRC-α axis in SKOV3 ovarian cancer cells.

Convergence of Wnt and Notch signaling controls ovarian cancer cell survival.

In the last 40 years ovarian cancer mortality rates have slightly declined and, consequently, it continues to be the fifth cause of cancer death in women. In the present study, we showed that β-catenin signaling is involved in the functions of ovarian cancer cells and interacts with the Notch system. Wnt and Notch systems showed to be prosurvival for ovarian cancer cells and their inhibition impaired cell proliferation and migration. We also demonstrated that the inhibition of β-catenin by means of two molecules, XAV939 and ICG-001, decreased the proliferation of the IGROV1 and SKOV3 ovarian cancer cell lines and that ICG-001 increased the percentage of IGROV1 cells undergoing apoptosis. The simultaneous inhibition of β-catenin and Notch signaling, by using the DAPT inhibitor, decreased ovarian cancer cell proliferation to the same extent as targeting only the Wnt/β-catenin pathway. A similar effect was observed in IGROV1 cell migration with ICG-001 and DAPT. ICG-001 increased the Notch target genes Hes-1 and Hey-1 and increased Jagged1 expression. However, no changes were observed in Dll4 or Notch 1 and 4 expressions. Our results suggest that Notch and β-catenin signaling co-operate in ovarian cancer to ensure the proliferation and migration of cells and that this could be achieved, at least partly, by the upregulation of Notch Jagged1 ligand in the absence of Wnt signaling. We showed that the Wnt pathway crosstalks with Notch in ovarian cancer cell functions, which may have implications in ovarian cancer therapeutics.

Inhibition of JAK2/STAT3 and activation of caspase‑9/3 are involved in KYS05090S‑induced apoptosis in ovarian cancer cells.

To overcome the poor prognosis of patients with ovarian cancer, attempting to target ovarian cancer with effective antitumor compounds has been conducted for numerous years. Although the 3,4‑dihydroquinazoline derivative KYS05090S was known to exert antitumor effects in A549 and ovarian cancer cells by inhibition of T‑type Ca2+ channels, the complete underlying antitumor mechanism of this compound remains unclear. Thus, in the present study, the potential apoptotic mechanism of KYS05090S was elucidated in SKOV3 and OVCAR3 ovarian cancer cells. KYS05090S exerted significant cytotoxicity in SKOV3 and OVCAR3 ovarian cancer cells, and also increased the number of apoptotic bodies, and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells and the sub‑G1 population as a feature of apoptosis. Consistently, KYS05090S induced cleavage of poly(ADP‑ribose) polymerase and caspase‑9/3 in ovarian cancer cells. Notably, KYS05090S attenuated the expression of anti‑apoptotic proteins, including cyclinD1 and B‑cell lymphoma‑2(Bcl‑2), and reduced the phosphorylation of Janus kinase2(JAK2) and signal transducer and activator of transcription3 (STAT3) in ovarian cancer cells. Additionally, KYS05090S blocked the nuclear translocation of STAT3 and suppressed the signaling of JAK2/STAT3 in interleukin‑6‑treated SKOV3 cells, as a STAT3 activator. Overall, these observations indicated that inhibition of JAK2/STAT3 signaling and activation of caspase‑9/3 are critically involved in the effects of KYS05090S on apoptosis in ovarian cancer types, and the compound may be beneficial as a potent antitumor agent.

Effects of arsenic trioxide on migration and invasion of human ovarian cancer SKOV3 cells and study on related molecular mechanism

Effects of arsenic trioxide on migration and invasion of human ovarian cancer SKOV3 cells and study on related molecular mechanism

\u03b1-Mangostin and Apigenin Induced Cell Cycle Arrest and Programmed Cell Death in SKOV-3 Ovarian Cancer Cells

Ovarian cancer is the fifth main cause of pre-senescent death in women. Although chemotherapy is generally an efficient treatment, its side effects and the occurrence of chemotherapeutic resistance have prompted the need for alternative treatments. In this study, α-mangostin and apigenin were evaluated as possible anticancer alternatives to the chemotherapeutic drug doxorubicin, used herein as a positive control. The ovarian adenocarcinoma cell line SKOV-3 (ATCC No. HTB77) was used as model ovarian cancer cells, whereas the skin fibroblast line CCD-986Sk (ATCC No. CRL-1947) and lung fibroblast line WI-38 (ATCC No. CCL-75) were used as model untransformed cells. Apigenin and doxorubicin inhibited the growth of SKOV-3 cells in a dose- and time-dependent manner. After 72 hr exposure, doxorubicin was mostly toxic to SKOV-3 cells, whereas apigenin was toxic to SKOV-3 cells but not CCD-986Sk and WI-38 cells. α-Mangostin was more toxic to SKOV-3 cells than to CCD-986Sk cells. A lower cell density, cell shrinkage, and more unattached (floating round) cells were observed in all treated SKOV-3 cells, but the greatest effects were observed with α-mangostin. With regard to programmed cell death, apigenin caused early apoptosis within 24 hr, whereas α-mangostin and doxorubicin caused late apoptosis and necrosis after 72 hr of exposure. Caspase-3 activity was significantly increased in α-mangostin-treated SKOV-3 cells after 12 hr of exposure, whereas only caspase-9 activity was significantly increased in apigenin-treated SKOV-3 cells at 24 hr. Both α-mangostin and apigenin arrested the cell cycle at the G2/M phase, but after 24 and 48 hr, respectively. Significant upregulation of BCL2 (apoptosis-associated gene) and COX2 (inflammation-associated gene) transcripts was observed in apigenin- and α-mangostin-treated SKOV-3 cells, respectively. α-Mangostin and apigenin are therefore alternative options for SKOV-3 cell inhibition, with apigenin causing rapid early apoptosis related to the intrinsic apoptotic pathway, and α-mangostin likely being involved with inflammation.

MicroRNA-663 facilitates the growth, migration and invasion of ovarian cancer cell by inhibiting TUSC2

BackgroundMicroRNAs (miRNAs) have emerged as the critical modulators of the tumorigenesis and tumor progression.MethodsThe levels of miR-663 in ovarian cancer cell lines and clinical tissues were detected using qRT-PCR assays. The Transwell invasion and wound healing assay were conducted to assess the roles of miR-663 in the migration and invasion of ovarian cancer cell in vitro. Rescue assays were carried out to confirm the contribution of tumor suppressor candidate 2 (TUSC2) in the aggressiveness of cancer cell which was regulated by miR-663.ResultsThe levels of miR-663 were up-regulated in ovarian cancer tissues in comparison with the corresponding normal tissues. Up-regulation of miR-663 increased the proliferation, colony formation, migration and invasion of ovarian cancer SKOV3 cell. Additional, over-expression of miR-663 increased the tumor growth of SKOV3 in xenograft model. Bioinformatics analysis and luciferase reporter assay identified that miR-663 decreased the level of TUSC2 via binding to the 3′-UTR of TUSC2 gene. Finally, the expression of TUSC2 was inversely associated with the level of miR-663 in ovarian carcinoma tissue and over-expression of TUSC2 inhibited the migration and invasion abilities of SKOV3 that was promoted by miR-663.ConclusionAltogether, these results indicate that miR-663 acts as a potential tumor-promoting miRNA through targeting TUSC2 in ovarian cancer.

Interferon Induces Expression of the Immune Checkpoint PD‐L1 via Bcl3 Upregulation in Ovarian Cancer Cells

Interferon‐g (IFN) induces PD‐L1 expression in ovarian cancer (OC) cells, thus promoting OC growth. In addition to inhibiting anti‐tumor T responses, the tumor‐expressed PD‐L1 has tumor‐intrinsic effects that include increased cancer cell survival and proliferation. However, the mechanisms by which IFN induces PD‐L1 expression in OC cells are unknown. Here, we show that IFN increases gene and protein levels of the proto‐oncogene Bcl3 in ovarian cancer SKOV3 and OVCAR3 cells, resulting in their increased survival, proliferation, and migration. The IFN‐induced Bcl3 expression then promotes IFN‐mediated PD‐L1 transcription in OC cells. The mechanism consists of an IFN‐induced, Bcl3‐ and p300‐dependent PD‐L1 promoter occupancy by K314/315 acetylated p65 NFkB. Since blocking PD‐L1 by neutralizing antibody reduces proliferation of OC cells overexpressing Bcl3, these results indicate that the pro‐proliferative effect of Bcl3 in OC cells is mediated by PD‐L1. Together, our work demonstrates that IFN induces Bcl3 expression in OC cells, identifies PD‐L1 as a novel target of Bcl3, and links Bcl3 to IFN signaling and PD‐L1‐mediated immune escape.Support or Funding InformationSponsor: Dr. Ivana Vancurova (ASBMB member ID: 32743).Funding: NIH CA202775 grant (to Dr. Ivana Vancurova)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phyto-fabrication of silver nanoparticles by Acacia nilotica leaves: Investigating their antineoplastic, free radical scavenging potential and application in H2O2 sensing

Abstract In this study, a green approach by utilizing Acacia nilotica leaves (ANL) extract to produce silver nanoparticles (AgNPs) has been made. The biosynthesized AgNPs displayed surface plasmon resonance (SPR) centered at 430 nm. Fourier transform infrared spectroscopy (FTIR) and energy-dispersive spectra (EDS) results showed the contribution of phyto-constituents of ANL in the reduction, capping and stabilizing matrix for AgNPs. The AgNPs were characterized by X-ray diffraction (XRD), and high resolution transmission electron microscopy (HR-TEM) revealed the synthesized ANL-AgNPs were crystalline and spherical forms having about ∼20 nm average particle size. ANL-AgNPs exhibited significant antineoplastic efficacy towards SKOV3 ovarian cancer cells (LD50; 66.5 µg/mL) thus could be useful to treat the cancer and to trounce limitations of conventional chemotherapy treatment. The synthesized AgNPs unveiled substantial antioxidant activity represented by common enzyme markers 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (IC50; 48.4 µg/mL) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (IC50; 55.2 µg/mL) activity. The ANL-AgNPs displayed substantial anti-bacterial activity against human pathogenic bacterial strains and also studied their minimum inhibitory and minimum bactericidal concentration (MIC and MBC) for individual strain. Additionally, the synthesized ANL-AgNPs exhibited colorimetric detection of hydrogen peroxide (H2O2) with better selectivity which showed its viability in determining reactive oxygen species in biological and environmental samples. Overall, the ANL-AgNPs could be used in multitasking biomedical applications, including anticancer, antioxidant, and antibacterial potential and also in sensing application.

Sustained release paclitaxel-loaded core-shell-structured solid lipid microparticles for intraperitoneal chemotherapy of ovarian cancer

The current clinical paradigm for ovarian cancer treatment has a poor prognosis, partially due to the efficacy and toxicity concerns associated with the available chemotherapeutic formulations. To overcome these limitations, we have designed core-shell-structured paclitaxel (PTX) laden solid lipid microparticles (PTX-SLMPs) for intraperitoneal treatment of ovarian cancer. A single-step coaxial electro hydrodynamic atomization (CEHDA) process has been explored to synthesize core-shell structure of PTX-SLMPs with the particle size of 1.76 ± 0.37 µm. Core-shell PTX-SLMPs have high encapsulation efficiency of 94.73% with sustained drug release profile. In vitro evaluation of PTX-SLMPs in SKOV-3 ovarian cancer cells yield significant enhancement in cytotoxicity when compared with Taxol®. In vivo pharmacokinetic study demonstrated slower absorption of PTX into the systemic circulation after intraperitoneal (i.p.) administration of PTX-SLMPs in Wistar rats implying the PTX-SLMPs remained in the peritoneal cavity and released the PTX for prolonged period of time. Through these studies, we have demonstrated the technical potential of core-shell structured PTX-SLMPs, which can enhance passive targeting of PTX to the tumor in the treatment of not only ovarian cancer but also in other peritoneal cancer.