ES-2 Ovarian Cancer Cell Lines Research Articles

Abstract 1061: Increasing potency of anticancer drugs through Sortilin receptor-mediated cancer therapy: A new targeted approach for the treatment of ovarian cancer

Abstract Rational : The development of personalized therapies against ovarian cancer remains highly challenging even in current modern oncology. One strategy to achieve greater selectivity and better anticancer drug delivery into cancer cells is to conjugate cytotoxic agents to peptide ligands that selectively target receptors abundantly and/or exclusively expressed in these tumor cells. Increased expression of Sortilin (SORT1), a scavenging receptor also known as neurotensin receptor-3, has been observed in invasive ovarian cancer biopsies. In addition, increased SORT1 expression correlated with breast tumor grades. Procedures : In light of these observations, we developed a peptide conjugation platform to target SORT1 and to increase cell selectivity and internalization of anticancer agents. As a proof-of-concept, doxorubicin and docetaxel were conjugated to a Sortilin-binding proprietary peptide (TH19P01). Results : In vitro, significant intracellular delivery of the doxorubicin-TH19P01 conjugate (TH1904) and docetaxel-TH19P01 conjugate (TH1902) were observed in a ES-2 ovarian cancer cell line model while preserving efficient doxorubicin or docetaxel cytotoxic mechanism. TH19P01 and drug conjugate uptake in ovarian cancer cells assays were reduced when SORT1 expression was specifically silenced using siRNA or upon competition with the SORT1 ligands neurotensin and progranulin. Importantly, drug-TH19P01 conjugates were found to bypass the P-glycoprotein (P-gp) efflux pump in MDCK-MDR1 cells overexpressing P-gp as the uptake of conjugates was unaffected by the P-gp inhibitor Cyclosporin A. In vivo, TH1904 and TH1902 caused a more potent inhibition of human ovarian tumor xenografts grown in mice and were better tolerated than their unconjugated parent drugs. Conclusion : These results strongly support future clinical development of this targeted technology platform to generate novel personalized therapeutics with specific indications in Sortilin-positive cancers. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Larocque, Alain Zgheib, Sophie Kozelko, Richard Béliveau, Christian Marsolais, Borhane Annabi. Increasing potency of anticancer drugs through Sortilin receptor-mediated cancer therapy: A new targeted approach for the treatment of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1061.

Diet-Derived Gallated Catechins Prevent TGF-β-Mediated Epithelial-Mesenchymal Transition, Cell Migration and Vasculogenic Mimicry in Chemosensitive ES-2 Ovarian Cancer Cells

Background: Transforming growth factor (TGF)-β triggers ovarian cancer metastasis through epithelial-mesenchymal transition (EMT). Whereas drug design strategies targeting the TGF-β signaling pathway have been envisioned, the anti-TGF structure:function aspect of chemopreventive diet-derived catechins remains unexplored.Aim: We assessed the effects of eight catechins on TGF-β-mediated cell migration and induction of EMT biomarkers, as well as on In Vitro vasculogenic mimicry (VM), a process partly regulated by EMT-related transcription factors.Results: TGF-β-mediated phosphorylation of Smad-3 and p38 signaling intermediates was more effective in a chemosensitive ES-2 ovarian cancer cell line but was inoperative in cis-platinum- and adriamycin-chemoresistant SKOV-3 ovarian cancer cells. Increases in cell migration and in gene/protein expression of EMT biomarkers Fibronectin, Snail, and Slug were observed in ES-2 cells. When VM was assessed in ES-2 cells, 3D capillary-like structures were formed and increases in EMT biomarkers found. Catechins bearing the galloyl moiety (CG, ECG, GCG, and EGCG) exerted potent inhibition of TGF-β-induced cell migration as well as EMT, and inhibited VM, in part through inhibition of Snail and matrix metalloproteinase-2 secretion.Conclusions: Our data suggest that diet-derived catechins exhibit chemopreventive properties that circumvent the TGF-β-mediated signaling which contributes to the ovarian cancer metastatic phenotype.

Abstract 5146: Increasing penetration of anticancer drugs through sortilin receptor-mediated cancer therapy: A new targeted and personalized Approach in the treatment of ovarian cancer

Abstract The development of personalized therapies against ovarian cancer remains highly challenging in current modern oncology. One way to achieve greater selectivity and better anticancer drug delivery into cancer cells is to conjugate cytotoxic agents to specific peptide ligands that will selectively target receptors abundantly and/or exclusively expressed on these cells. Increased expression of Sortilin, a scavenging receptor, has been clinically observed in several human cancers including breast, prostate, colon, pancreas, skin, and pituitary. In particular, Sortilin has also been reported to be overexpressed in ovarian cancers as compared to non-malignant ovarian tissue. In light of this, we developed a peptide conjugation platform using a new Sortilin receptor-mediated vectorization strategy to increase cell targeting selectivity and cell delivery efficacy of anticancer agents. As a proof-of-concept, Doxorubicin was conjugated to peptide sequences, termed Katana peptides (KA). In vitro, significantly increased Sortilin mediated intracellular delivery of the Doxorubicin-Katana peptide conjugate (DoxKA) was observed in SKOV3 and ES-2 ovarian cancer cell line models, with conserved efficient Doxorubicin cytotoxic mechanism. Uptake of DoxKA in ES-2 ovarian cancer cells was reduced when Sortilin expression was specifically silenced by using siRNA or upon competition with the Sortilin ligands neurotensin and progranulin. In addition, DoxKA was found to bypass the P-glycoprotein (P-gp) efflux pump since, in contrast to Doxorubicin uptake in MDCK-MDR1 cells overexpressing the P-gp efflux pump, the uptake of DoxKA was unaffected by the P-gp inhibitor Cyclosporin A. In vivo, DoxKA was better tolerated and caused a more potent inhibition of human ovarian tumor xenografts growth than did Doxorubicin alone. These results provide strong evidence for the potential of this drug development platform in the generation of novel personalized therapeutics with specific targeting of SORT1-positive cancer cells. Citation Format: Michel Demeule, Jean-Christophe Curie, Alain Larocque, Cyndia Charfi, Richard Béliveau, Claude Vezeau, Borhane Annabi. Increasing penetration of anticancer drugs through sortilin receptor-mediated cancer therapy: A new targeted and personalized Approach in the treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5146. doi:10.1158/1538-7445.AM2017-5146

Inhibition of leucine aminopeptidase 3 suppresses invasion of ovarian cancer cells through down-regulation of fascin and MMP-2/9

Leucine aminopeptidase 3 (LAP3) is a cell surface aminopeptidase that catalyzes the hydrolysis of leucine residues from the amino termini of protein or peptide substrates. The over-expression of LAP3 correlates with prognosis and malignant development of several human cell carcinomas. However, the molecular mechanism remains unknown. In this study, we used ES-2 ovarian cancer cell line as a model system to explore the role of LAP3 in regulation of cancer cell invasion by employing a natural LAP3 inhibitor bestatin and LAP3 siRNA. Bestatin inhibited tumor cell migration and invasion in a dose-dependent manner. More interestingly, bestatin down-regulated expression of fascin protein and inhibited activity of fascin promoter luciferase reporter. Both proteome profiler array and Western blot assay showed that bestatin up-regulated the phosphorylation of Hsp27. Furthermore, LAP3 siRNA could up-regulate the phosphorylation of Hsp27 and down-regulate the expression of fascin. Meanwhile, LAP3 siRNA could also down-regulate the phosphorylation of Akt and the expression of MMP-2/9. Taken together, LAP3 could affect the expression of fascin and MMP-2/9 and may act as a potential anti-metastasis therapeutic target.

BRCA1 as a nicotinamide adenine dinucleotide (NAD)-dependent metabolic switch in ovarian cancer

Both hereditary factors (e.g., BRCA1) and nicotinamide adenine dinucleotide (NAD)-dependent metabolic pathways are implicated in the initiation and progression of ovarian cancer. However, whether crosstalk exists between BRCA1 and NAD metabolism remains largely unknown. Here, we showed that: (i) BRCA1 inactivation events (mutation and promoter methylation) were accompanied by elevated levels of NAD; (ii) the knockdown or overexpression of BRCA1 was an effective way to induce an increase or decrease of nicotinamide phosphoribosyltransferase (Nampt)-related NAD synthesis, respectively; and (iii) BRCA1 expression patterns were inversely correlated with NAD levels in human ovarian cancer specimens. In addition, it is worth noting that: (i) NAD incubation induced increased levels of BRCA1 in a concentration-dependent manner; (ii) Nampt knockdown-mediated reduction in NAD levels was effective at inhibiting BRCA1 expression; and (iii) the overexpression of Nampt led to higher NAD levels and a subsequent increase in BRCA1 levels in primary ovarian cancer cells and A2780, HO-8910 and ES2 ovarian cancer cell lines. These results highlight a novel link between BRCA1 and NAD. Our findings imply that genetic (e.g., BRCA1 inactivation) and NAD-dependent metabolic pathways are jointly involved in the malignant progression of ovarian cancer.

Clinical importance and therapeutic implication of E-cadherin gene methylation in human ovarian cancer

E-cadherin (E-cad) is widely expressed in epithelial cells and acts as a pivotal tumor suppressor. The promoter methylation of E-cad has been reported to closely relate to its downregulation in many kinds of cancers. E-cad expression and methylation status were detected by immunohistochemistry (IHC) and methylation-specific polymerase chain reaction (MS-PCR) in 50 ovarian cancer tissues. 5-Aza-2'-deoxycytidine (5-Aza-dC) was used to demethylate E-cad in SKOV3 and ES2 ovarian cancer cell lines, of which the effect was verified by Western blot and MS-PCR. Then MTT and transwell experiments were conducted to detect the capacity of cell proliferation and migration for these cells. Downregulation of E-cad expression was observed in 60 % of ovarian cancer tissues (30/50) by IHC, whereas MS-PCR result indicated that E-cad was methylated in 64 % of (32/50) ovarian cancer specimens. And E-cad expression was significantly correlated with E-cad methylation (P = 0.004). 5-Aza-dC was used to process SKOV3 and ES2 ovarian cancer cell lines. By MTT experiment, we found that the proliferation of 5-Aza-dC-treated SKOV3 and ES2 was significantly suppressed by 28.0 % (P < 0.05) and 32.3 % (P < 0.05). By transwell experiment, the motility of SKOV3 and ES2 was found to be significantly suppressed by 38.2 and 27.4 % (P < 0.05), respectively, after treated with 5-Aza-dC. E-cad methylation is one of the main reasons for the expression reduction in ovarian cancer. 5-Aza-dC treatment could significantly restore the expression of E-cad and suppress growth and invasion of SKOV3 and ES2 cells. These results suggest E-cad methylation may be a promising target for ovarian cancer therapy.

Abstract 3557: The aging suppressor hormone Klotho inhibits ovarian cancer cell proliferation by down-regulation of the estrogen receptor.

Abstract Klotho is a 1012 amino acid (aa) long transmembranal protein, which can be cleaved, shed and act as a circulating hormone. Klotho contains two regions named KL1 and KL2, each about 500aa long. Klotho deficient mice present a syndrome of early aging, while overexpression of klotho extends lifespan. Klotho is an essential co-factor for the activity of FGF23 and thus serves as a major regulator of phosphate homeostasis. Klotho is also a potent inhibitor of the insulin growth factor (IGF)-1 pathway. We have identified klotho as a tumor suppressor in breast and pancreatic cancer. Recently, klotho activity as a tumor suppressor was observed also in melanoma and in cervix, lung, colon and gastric cancers. As klotho is expressed in the normal ovary, we undertook to study the expression and activity of klotho in ovarian cancer. We first analyzed klotho expression, using quantitative RT-PCR and Western blotting, in 18 ovarian cancer cell lines and noted high levels of klotho mRNA and protein in only three lines. We next used immunohistochemistry analysis to study klotho expression in a tissue array containing 277 primary and recurrent ovarian, fallopian and primary peritoneal cancers, along with 22 normal and benign tumor samples. While high klotho expression was noted in all normal samples, reduced expression was noted in 30% of the tumors. Reduced expression was not associated with histology, tumor location or stage. Overexpression of either full length klotho or KL1 reduced clonal growth of OvCa432, SKOV3 and ES2 ovarian cancer cell lines by 80%, 60%, and 0%, respectively. Klotho inhibits the IGF-1 pathway in pancreatic and breast cancer cells. As the IGF-1 pathway plays an important role in ovarian cancer development, we studied the effect of klotho on this pathway in ovarian cancer cell lines. Klotho inhibited IGF-1-mediated AKT activation, and to a lesser degree ERK1/2 phosphorylation, in SKOV3 and OvCa432 cells. Interestingly, ES2 cells did not respond to IGF-1. Estrogen receptor (ER)α and progesterone receptor (PR) have a significant role in ovarian cancer development. ERα is expressed in a majority of ovarian cancers, while PR is less abundant, and its expression is a marker for better prognosis. We found that klotho reduces ERα expression and activity in SKOV3 and OvCa432. Interestingly, klotho elevated PR-A isoform expression in these cell lines. In conclusion, unlike in breast and pancreas cancer, where klotho was identified as a potent tumor suppressor in most cancer subtypes, and the IGF-1 pathway was inhibited in a wide array of cell lines, in the ovary, klotho plays a role in a smaller subset of tumors. The characteristics of this group have not been identified yet. However, it appears that ERα inhibition and PR-A up-regulation may play a role in mediating klotho activities in this subset. Citation Format: Irina Lojkin, Omer Schwartzman, Tami Rubinek, Ido Wolf. The aging suppressor hormone Klotho inhibits ovarian cancer cell proliferation by down-regulation of the estrogen receptor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3557. doi:10.1158/1538-7445.AM2013-3557

Epigenetic silencing of DKK2 and Wnt signal pathway components in human ovarian carcinoma

Wnt/β-Catenin signaling dysregulation is involved in tumorigenesis. Furthermore, epigenetic modification of the Dickkopf (DKK) family (DKK1-DKK4) has been shown to be important in Wnt signaling regulation. In this study, the role of DKK2, a Wnt antagonist, in epithelial ovarian cancer (EOC) was evaluated by examining the expression and methylation of DKK2 in SKOV3 and ES-2 ovarian cancer cell lines and 78 tissues collected from patients (50 ovarian carcinoma, 20 benign tumor and 8 normal ovarian tissues). DKK2 is highly downregulated in EOCs; however, DKK2 expression levels are higher in both normal tissues and benign tumors. In most cases of ovarian carcinoma, DKK2 is methylated, compared with the more common unmethylated form present in benign tumors and normal ovarian tissues. Additionally, DKK2 may be epigenetically silenced by methylation in higher grades and stages of EOC. Functional analysis revealed that overexpression of DKK2 suppressed malignant cell growth and invasion in SKOV3 and ES-2 cell lines. The expression of the downstream genes of Wnt signaling, including β-catenin, c-Myc and cyclin D1, was decreased in DKK2-transfected cells compared with mock cells. The expression of matrix metalloproteinase-2 and focal adhesion kinase were also decreased in DKK2 transfectants, supporting findings indicating inhibition of cell migration and invasion. This report provides novel indications that DKK2 is a unique hypermethylated target gene in EOC and that DKK2 may contribute to tumorigenesis in EOC through the Wnt/β-catenin signaling mechanisms.

Abstract 1687: A novel PKC inhibitor: Inhibition of cell proliferation in cancer cell lines and pharmacokinetics in Sprague-Dawley rats

Abstract Background: Cancer treatment by conventional chemotherapy is hindered by toxic side effects and the frequent development of multi-drug resistance by cancer cells. Cytotoxic peptides are a promising class of drugs that avoid the shortcomings of conventional chemotherapy because certain peptides exhibit selective cytotoxicity against a broad spectrum of human cancer cells (Mader J.S. and Hoskin D.W., 2006). Protein kinase C (PKC) is a family of kinases involved in the transduction of signals for cell proliferation, differentiation, angiogenesis, migration and apoptosis. PKC has been implicated in tumorigenesis (Griner E.M. and Kazanietz M.G., 2007). PharmaGap based its anticancer therapy strategy on the use of proprietary computer modelling for the design of novel cytotoxic peptide candidates for select PKC peptides in order to develop novel drugs targeting PKC. Objective: 1) To test novel GAP-107B8 peptide-based PKC inhibitors’ capability to inhibit cell proliferation in various cancer cell lines; and 2) To gather information about the pharmacokinetics of an inhibitor in a rodent model. Methods: Cell proliferation was measured in LS513 colon, A2780cp ovarian, and ES-2 ovarian cancer cell lines after treatment with different peptides (concentrations in the micromolar range) up to 48 hours. Proliferation was measured using CyQuant Assay and data was normalized to untreated controls. Pharmacokinetic data was obtained from a bolus study and from a 2 hour continuous infusion study performed in Sprague-Dawley rats. Single bolus doses ranged from 1.0 to 5.5 mg/kg. Blood sampling was obtained over 120 minutes following administration. Doses for the 2 hour continuous infusion study ranged from 10 to 40 mg/kg. Blood sampling was obtained prior to infusion start, and up to 120 minutes post-infusion. Plasma was used for all subsequent pharmacokinetic analyses. Results: In different screenings, a peptide dose-response inhibition on cell proliferation was observed in all cell lines tested. In one screening study, at the highest peptide concentration after a 48 hour incubation, the peptide decreased proliferation by 67%, 71%, and 28% in LS513, A2780cp, and ES-2 cells respectively. Another screening showed decreased proliferation by 92%, 92%, and 75% in LS513, A2780cp, and ES-2 cells respectively. From the bolus study it was found that the pharmacokinetic profiles of the cytotoxic peptide were similar for all doses and for both genders. Elimination appeared to be biphasic, and plasma half-life values were dose dependent. Exposure, based on AUC0-t, increased in a dose related fashion (pseudo linear) for both genders. Conclusions: Novel PKC inhibitors have been found to inhibit cell proliferation of various cancer cell lines. The first pharmacokinetic data for these novel inhibitors was obtained after a bolus and an infusion study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1687. doi:10.1158/1538-7445.AM2011-1687

Glycoproteomic Analyses of Ovarian Cancer Cell Lines and Sera from Ovarian Cancer Patients Show Distinct Glycosylation Changes in Individual Proteins

Ovarian cancer is difficult to diagnose in women because symptoms of the disease are often not noticed until the disease has progressed to an advanced untreatable stage. Although a serum test, CA125, is currently available to assist with monitoring treatment of ovarian cancer, this test lacks the necessary specificity and sensitivity for early detection. Therefore, better biomarkers of ovarian cancer are needed. A glycoprotein analysis approach was undertaken using high resolution Fourier transform ion cyclotron resonance mass spectrometry to analyze glycosylated proteins present in the conditioned media of ovarian cancer cell lines and in sera obtained from ovarian cancer patients and normal controls. In this study, glycosylated proteins were separated by gel electrophoresis, and individual glycoproteins were selected for glycosylation analysis and protein identification. The attached glycans from each protein were released and profiled by mass spectrometry. Glycosylation of a mucin protein and a large glycosylated protein isolated from the ES2 ovarian cancer cell line was determined to consist of mostly O-linked glycans. Four prominent glycoproteins of approximate 517, 370, 250, 163 kDa from serum samples were identified as two forms of apolipoprotein B-100, fibronectin, and immunoglobulin A1, respectively. Mass spectrometric analysis of glycans isolated from apolipoprotein B-100 (517 kD) showed the presence of small, specific O-linked oligosaccharides. In contrast, analysis of fibronectin (250 kD) and immunoglobulin A1 (163 kD) produced N-linked glycan fragments in forms that were sufficiently different from the glycans obtained from the corresponding protein band present in the normal serum samples. This study shows that not only a single protein but several are aberrantly glycosylated, and those abnormal glycosylation changes can be detected and may ultimately serve as glycan biomarkers for ovarian cancer.

Luteinizing hormone-releasing hormone as an autocrine growth factor in ES-2 ovarian cancer cell line.

Agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LHRH) inhibit the growth of various cancers in vivo. This effect is mainly exerted through the suppression of the pituitary-gonadal axis and the creation of a state of sex steroid deprivation. In addition, much evidence has been accumulated in the past few years that LHRH analogs can also have direct effects on tumor growth mediated by specific LHRH receptors (-R) on tumor cells. Although an involvement of LHRH in the proliferation of some cancer cells has been postulated, it is still not clear at present whether LHRH produced locally has a stimulatory or inhibitory effect. In the present study we investigated whether LHRH can function as an autocrine growth factor in ovarian cancer. ES-2 human ovarian cancer cell line expresses mRNA for LHRH, which is apparently translated into peptide LHRH and then secreted by the cells, as demonstrated for the first time by the detection of LHRH-like immunoreactivity in conditioned media from the cells cultured in vitro. ES-2 cells also express mRNA for LHRH receptors. [D-Trp6]LHRH agonist at 10 ng/ml stimulates the proliferation of ES-2 in vitro after 48 h, but is inhibitory after 72 h and at concentrations of 1000 ng/ml. LHRH antagonist Cetrorelix inhibits growth of ES-2 cell line only at 1000 ng/ml. The incubation of ES-2 ovarian cancer cells in vitro with an LHRH antibody inhibited cell proliferation in a time and concentration-dependent manner. Collectively, our results suggest that LHRH may function as an autocrine growth factor in ovarian cancer.