ES-2 Cells Research Articles

Alpinumisoflavone Disrupts Endoplasmic Reticulum and Mitochondria Leading to Apoptosis in Human Ovarian Cancer.

Alpinumisoflavone is a prenylated isoflavonoid derived from the Cudrania tricuspidate fruit and Genista pichisermolliana. Alpinumisoflavone has anticancer properties in a variety of cancer cells, including colorectal, esophageal, renal and hepatocellular carcinoma. However, its mechanisms and effects in ovarian cancer remain unexplored. Our findings indicate that alpinumisoflavone triggers anti-proliferation in 2D- and 3D-cultured human ovarian cancer (ES2 and OV90) cells, including a reduction in the proliferating cell nuclear antigen expression and sub-G1 phase arrest of the cell cycle. Both alpinumisoflavone-treated ES2 and OV90 cells exhibited an augmentation in late apoptotic cells and the depolarization of mitochondrial membrane potential (MMP). We also observed a decrease in respiratory chain activity in ovarian cancer cells, owing to lower energy output by the alpinumisoflavone. In addition, combining cisplatin (a chemotherapeutic drug used in several malignancies) with alpinumisoflavone boosted apoptosis in ES2 and OV90 cells via a reduction in cell proliferation, induction of late apoptotic cells, and depolarization of MMP. Furthermore, alpinumisoflavone also regulated the PI3K/AKT, MAPK and endoplasmic reticulum (ER) stress regulatory signaling pathways, leading to cell death in both ES2 and OV90 cells. In general, our findings verified that alpinumisoflavone inhibited ovarian cancer cell growth via mitochondrial malfunction.

Dihydrotanshinone I inhibits ovarian tumor growth by activating oxidative stress through Keap1-mediated Nrf2 ubiquitination degradation

Dihydrotanshinone I (DHT), a bioactive compound in Salvia miltiorrhiza, was reported to exhibit cytotoxicity against various malignancies. However, the underlying mechanism on ovarian cancer remains unclear. Here, DHT inhibited cell viability of ovarian cancer HO8910PM, SKOV3, A2780 and ES2 cells. It showed moderate inhibitory effect on ovarian epithelial IOSE80 cells and lower toxicity than chemotherapy drugs. DHT induced apoptosis and G2 cell cycle arrest accompanied by reduced expression of Bcl-2, Caspase-3, and increased Bax. Meanwhile, DHT increased ROS accumulation, decreased mitochondrial membrane potential and activated oxidative stress in HO8910PM and ES2 cells. Mechanistically, DHT inhibited Nrf2 and p62 expression, Nrf2 target genes and enzymes, and Nrf2 nuclear translocation, while increased the expression of Nrf2 inhibitor Keap1. NAC, a ROS scavenger, rescued DHT-induced proliferation inhibition, ROS generation and Nrf2 inhibition. DHT alleviated tBHQ-induced Nrf2 expression and increased its mRNA level. However, the proteasome inhibitor MG132 blocked DHT-induced Nrf2 inhibition, suggesting a post-translational regulation manner. DHT enhanced Nrf2 binding with Keap1, leading to potentiated Nrf2 ubiquitination degradation. Furthermore, Nrf2 and p62 overexpression blocked DHT-induced Nrf2 and p62 inhibition. Consistent with the in vitro results, DHT significantly delayed tumor growth in HO8910PM and ES2 xenograft nude mice, decreased tumor marker HE4 and CA125 levels, reversed the abnormally expressed proteins including Ki67, Nrf2, p62, Keap1, Bcl-2, CyclinB1, Cdc-2, and antioxidant enzymes SOD, CAT in vivo. Serum from DHT-treated mice also inhibited cell growth in vitro. Taken together, DHT exhibits anti-ovarian tumor effect by activating oxidative stress through ubiquitination-mediated Nrf2 degradation. Our findings implicate a potential application of DHT for ovarian cancer therapy.

OSI-906 restores the sensitivity of ovarian clear cell carcinoma to cisplatin by targeting the IGF1R/AKT pathway.

Among the various histologic subtypes of ovarian cancers (OCs), ovarian clear cell carcinoma (OCCC) represents a great challenge due to its disease aggressiveness and resistance to chemotherapy. IGF1 is overexpressed in epithelial ovarian cancer (EOC), and IGF1 pathway activation is related to the chemoresistance of various cancers. In this study, we found that the expression level of IGF1 was higher in OCCC than in the most common type of OC, high-grade serous adenocarcinoma (HGSC). Then, we investigated the role of IGF1 pathway activation in the progression of OCCC, observing that activation of the IGF1 pathway using IGF1 promoted the proliferation and migration of ES2 cells, while inactivation of the IGF1 pathway using the selective IGF1R inhibitor OSI-906 reversed the alteration mediated by IGF1. Based on the role of the IGF1 pathway in cancer chemoresistance, we proposed that OSI-906 may restore the sensitivity of OCCC to cisplatin. We first validated that IGF1 increased the IC50 value of cisplatin in ES2 cells, while OSI-906 decreased it. Then we confirmed that IGF1 decreased the apoptosis rate of ES2 cells induced by cisplatin, while OSI-906 increased it. Finally, we conducted animal experiments to investigate whether OSI-906 helps cisplatin control the growth of OCCC. As expected, OSI-906 increased the effect of cisplatin in attenuating the growth of OCCC in vivo. Therefore, we conclude that using OSI-906 may be an effective method to restore the sensitivity of OCCC to cisplatin by targeting the IGF1R/AKT pathway.

Hypoxia-induced Maspin Expression Affects the Prognosis of Ovarian Clear Cell Carcinoma.

To investigate the role of the expression of hypoxia-related genes on the prognosis of ovarian clear cell carcinoma (OCCC). Basal mRNA levels of eight hypoxia-related genes were compared. Cell viability was assayed after treating ES-2 cells under hypoxic conditions. The mRNA and protein levels were evaluated after the induction of hypoxia and administration of increased doses of N-acetylcysteine (NAC). Finally, the prognostic role of their expression levels was evaluated in 61 patients with OCCC. The mRNA and protein levels of maspin increased gradually with the induction of hypoxia. Maspin protein expression decreased after treatment with paclitaxel and NAC. High expression of maspin was related to poor progression-free and overall survival in patients with OCCC (adjusted hazard ratios, 3.97 and 7.47; 95% confidence intervals=1.34-11.81, and 1.98-28.13). High expression of maspin induced by hypoxia might be associated with poor prognosis of OCCC.

Splice-Variant Knock-Out of TGFβ Receptors Perturbates the Proteome of Ovarian Carcinoma Cells.

The aim of this study was to analyze the biological role of different transforming growth factor-β (TGFβ) receptor splice variants in ovarian carcinoma (OC). Specific receptor variant knockouts (KO) were prepared using the CRISPR/Cas9 genome editing system in two OC cell lines, TβRI variant 1 (TβRIv1) KO in ES-2 cells and TβRII variant 1 (TβRIIv1) KO in OVCAR-8 cells. Control and KO cells were compared by proteomic analysis, functional tests, analysis of epithelial–mesenchymal transition (EMT) drivers, and Western blot of signaling proteins. Proteomic analysis revealed significant changes in protein pathways in the KO cells. TβRIv1 KO resulted in a significant reduction in both cellular motility and invasion, while TβRIIv1 KO significantly reduced cellular motility and increased Reactive Oxygen Species (ROS) production. Both receptor variant KOs reduced MET protein levels. Of the EMT drivers, a significant decrease in TWIST protein expression, and increase in SNAIL protein and MALAT1 mRNA levels were observed in the TβRIIv1 KO compared to control. A significant decrease in JNK1 and JNK2 activation was found in the TβRIv1 KO compared to control cells. These findings provide new insight regarding the biological role of the TGFβ receptor variants in the biology and potentially the progression of OC.

RNA-binding protein IGF2BP2 enhances circ_0000745 abundancy and promotes aggressiveness and stemness of ovarian cancer cells via the microRNA-3187-3p/ERBB4/PI3K/AKT axis

BackgroundCircular RNAs (circRNAs) are increasingly recognized as important regulators in cancer including ovarian cancer (OC). This work focuses on the effects of circ_0000745 on the OC development of and molecules involved.MethodsExpression of circ_0000745 in collected OC tissues and the acquired OC cell lines was examined by RT-qPCR. The stability of circ_0000745 in cells was examined by RNase R treatment. The target transcripts interacted with circ_0000745 were predicted using bioinformatic systems. Gain- and loss-of-function studies of circ_0000745, microRNA (miR)-3187-3p and erb-b2 receptor tyrosine kinase 4 (ERBB4) were conducted to determine their functions on proliferation, migration, invasion and stem cell property of OC cells.ResultsCirc_0000745 and ERBB4 were abundantly expressed while miR-3187-3p was poorly expressed in OC tissues and cells. Circ_0000745 sequestered miR-3187-3p and blocked its repressive effect on ERBB4. Downregulation of circ_0000745 reduced proliferation, aggressiveness, epithelial-mesenchymal transition, and stemness of SK-OV-3 cells, but this reduction was blocked upon miR-3187-3p inhibition or ERBB4 upregulation. By contrast, artificial induction of circ_0000745 upregulation, miR-3187-3p upregulation and ERBB4 downregulation led to inverse trends in ES-2 cells. ERBB4 promoted the phosphorylation of the PI3K/AKT signaling pathway. An RNA binding protein IGF2BP2 was found to circ_0000745 bind to and promote its expression and stability.ConclusionThis study demonstrated that circ_0000745 upregulated by IGF2BP2 promotes aggressiveness and stemness of OC cells through a miR-3187-3p/ERBB4/PI3K/AKT axis. Circ_0000745 may serve as a promising target for OC treatment.

Ultrasound-stimulated microbubbles enhances radiosensitivity of ovarian cancer.

Radiation therapy is regarded as an effective treatment for early ovarian cancer (OC). However, due to radiation resistance caused by DNA double-strand breaks (DSBs) and angiogenesis, the efficacy of radiotherapy for advanced OC is limited and controversial. To explore whether ultrasound-stimulated microbubbles (USMBs) can enhance the radiosensitivity of OC. OC cells (ES-2) were respectively irradiated with 5-Gy and 10-Gy radiation doses with or without exposure to USMB. Methyl thiazolyltetrazolium (MTT) and colony-formation assays were conducted to detect the viability and proliferation of ES-2 cells after USMBs and ionizing radiation (IR) treatment. Immunofluorescence assays were conducted to examine levels of gamma-H2A histone family member X (γ-H2AX), an indicator for DSBs. Flow cytometry analyses were carried out to assess the apoptosis of ES-2 cells. The angiogenic activity of human umbilical vein endothelial cells (HUVECs) was measured by tube formation assays. USMBs enhanced IR-induced suppressive effect on the viability and proliferation of OC cells. The protein levels of phosphorylated γ-H2AX and CHK1 were significantly upregulated after IR treatment and further enhanced by USMBs. In addition, USMBs enhanced the promotion of IR-mediated OC cell apoptosis. The inhibitory effect of IR on angiogenesis was further enhanced by USMBs, and protein levels of AT1R, VEGFA, and EGFR were downregulated by IR in a dose-dependent way and then enhanced by USMB treatment in HUVECs. USMB exposure significantly enhances the radiosensitivity of OC by suppressing cell proliferation, promoting OC cell apoptosis, and inhibiting angiogenesis.

Wnt5a influences the directional displacement of ovarian cancer cells

Objectives: The noncanonical Wnt ligand Wnt5a is found in high concentrations in ovarian cancer ascites. This microenvironmental Wnt5a has recently been shown to potentiate ovarian cancer metastasis. We sought to identify whether Wnt5a modulates ovarian cancer cell motility. Methods: We engineered and validated a microfluidic device that establishes stable gradients of soluble ligands. After a Wnt5a gradient was established, ovarian cancer cells (ES2 or OVCAR5) were introduced and allowed to adhere to a 2-dimensional fibronectin substrate. Real time imaging was carried out over 7 hours and cellular migration was analyzed with respect to speed, displacement, and directionality. In a subset of experiments, ROR2, a noncanonical Wnt5a receptor involved in planar cell polarity (PCP) pathways, was depleted with siRNA to assess contribution of ROR2-dependent PCP to Wnt5a mediated behaviors. Results: In the presence of Wnt5a, ES2 and OVCAR5 ovarian cancer cells demonstrated an increase in both mean and instantaneous velocity of migration when compared to absence of ligand (P Conclusions: Wnt5a promotes the motility of ovarian cancer cells by influencing both directionality of motion as well as degree of displacement. Furthermore, the Wnt5a receptor ROR2 was shown to be critical in mediating these behaviors, suggesting a role for planar cell polarity pathways in ovarian cancer metastasis. As Wnt5a is produced at high levels by peritoneal mesothelial cells and omental adipose tissue in the metastatic microenvironment of ovarian cancer, these data suggest a potential mechanism by which ovarian cancer cells ‘home’ to their metastatic niche. ROR2 represents an attractive target for disrupting this tumor cell/microenvironment interaction and represents a possible new therapeutic target.

Increasing sensitivity to olaparib through inhibition of discoidin domain receptor 2 (DDR2) in homologous-recombination proficient ovarian cancer models

<h3>Objectives:</h3> Discoidin Domain Receptor 2 (DDR2) is a tyrosine kinase receptor which binds the most common extracellular matrix protein, fibrillar collagen type 1. DDR2 expression is critical for invasion and migration of ovarian cancer tumor cells, making DDR2 a potential target for new treatments. This study aimed to determine whether genetic inactivation of DDR2 in ovarian cancer cells would increase sensitivity to treatment with PARP inhibitor. <h3>Methods:</h3> Two ovarian cancer cells lines, ES2 and COV362, were used for <i>in vitro</i> assays. Stable hairpin control and DDR2 knockdowns (shSCRM and shDDR2) were used for ES2 and transient small interfering RNA knockdown of DDR2 (siControl and siDDR2) were used for COV362. Homologous recombination (HR) status was determined by quantification of RAD-51 foci relative to controls using immunofluorescence after irradiation. Cell viability and survival after treatment with olaparib was quantified using MTS assays and clonogenics. The DNA damage response after treatment with olaparib was analyzed using immunofluorescence. <i>In vivo</i> studies to evaluate the sensitivity to olaparib in the presence or absence of DDR2 expression were performed in nude mice after intraperitoneal injection with ES2shSCRM or shDDR2 cells. <h3>Results:</h3> ES2 and COV362 cells were found to have a 2-fold increase in RAD51 foci after irradiation indicating HR proficiency. DDR2 genetically inactivated cells (ES2shDDR2) were found to be more HR deficient than DDR2 expressing cells (ES2shSCRM) which remained HR proficient (0.80 vs 4.78 relative RAD51 foci per cell). We found that genetic inactivation of DDR2 in the ES2s and COV362s led to increased sensitivity to olaparib with an 80% (15uM vs 73uM) and 68% (62uM vs 195uM) reduction in IC50s, respectively, compared to DDR2 expressing cells. Additionally, ES2shDDR2 cells had decreased cell survival by clonogenic assay after treatment with olaparib compared to ES2shSCRM cells as measured by absorbance (0.64 vs 0.80, p=0.005). We found that DNA damage and the repair response was altered in DDR2 genetically inactivated cells treated with olaparib with an increase in gamma-H2AX (8 vs 6 foci per cell, p<0.0001) and 53BP1 foci (3 vs 1 foci per cell, p<0.0001) when compared to DDR2 expressing cells. In an intraperitoneal metastatic mouse model with the HR-proficient cell line ES2, we found that genetic inactivation of DDR2 led to decreased tumor burden when treated with olaparib compared to DDR2-expressing cells treated with olaparib (1499mm³ vs 3236mm³, p=0.04). <h3>Conclusions:</h3> DDR2 knockdown sensitizes HR proficient ovarian cancer cells to treatment with PARP inhibitor in both <i>in vitro</i> and <i>in vivo</i> models through a mechanism of induced HR deficiency and increased DNA damage. Future experiments will explore whether a DDR2 inhibitor can improve sensitivity to PARP inhibitor in HR proficient ovarian cancer models.

TRAIL produced by SAM-1-activated CD4+ and CD8+ subgroup T cells induces apoptosis in human tumor cells through upregulation of death receptors

Bacterial superantigens potently activate conventional T-cells to induce massive cytokine production and mediate tumor cell death. To engineer superantigens for immunotherapy against tumors in clinic, we previously generated SAM-1, a staphylococcal enterotoxins C2 (SEC2) mutant, that exhibited significantly reduced toxicity but maintained the superantigen activity in animal models. This present study aimed to investigate whether SAM-1 activates T cells and induces apoptosis in human tumor cells. We found that SAM-1 induced the maturation of dendritic cells (DCs) with upregulating expression of the surface markers CD80, CD86 and HLA-DR, which secreted high levels of IL-12p70 by activating TLR2-NF-κB signaling pathways. SAM-1 could activate human CD4+ subgroup T cells and CD8+ subgroup T cells in the presence of mature dendritic cells (DCs), leading to the productions of cytokines TRAIL, IL-2, IFN-γ and TNF-α. We observed that TRAIL mediated the apoptosis and S-phase and G2/M-phase arrest in HGC-27 tumor cells via binding to upregulated death receptors DR4 and DR5. Using shRNA knockdown in HGC-27 cells or constitutive overexpression in ES2 cells for DR4 and DR5, we demonstrated the vital requirement of DR4 and DR5 in apoptosis of tumor cells in response to TRAIL secreted from SAM-1-activated T cells. Collectively, our results will facilitate better understanding of SAM-1-based immunotherapies for cancer.

CCNE1 Is a Putative Therapeutic Target for ARID1A-Mutated Ovarian Clear Cell Carcinoma.

Background: Ovarian clear cell carcinoma (OCCC) is resistant to platinum chemotherapy and is characterized by poor prognosis. Today, the use of poly (ADP-ribose) polymerase (PARP) inhibitor, which is based on synthetic lethality strategy and characterized by cancer selectivity, is widely used for new types of molecular-targeted treatment of relapsed platinum-sensitive ovarian cancer. However, it is less effective against OCCC. Methods: We conducted siRNA screening to identify synthetic lethal candidates for the ARID1A mutation; as a result, we identified Cyclin-E1 (CCNE1) as a potential target that affects cell viability. To further clarify the effects of CCNE1, human OCCC cell lines, namely TOV-21G and KOC7c (ARID1A mutant lines), and RMG-I and ES2 (ARID1A wild type lines) were transfected with siRNA targeting CCNE1 or a control vector. Results: Loss of CCNE1 reduced proliferation of the TOV-21G and KOC7c cells but not of the RMG-I and ES2 cells. Furthermore, in vivo interference of CCNE1 effectively inhibited tumor cell proliferation in a xenograft mouse model. Conclusion: This study showed for the first time that CCNE1 is a synthetic lethal target gene to ARID1A-mutated OCCC. Targeting this gene may represent a putative, novel, anticancer strategy in OCCC treatment.

Potential of Pharmaceutical Intervention in Platelets and Cancer Positive Feedback Loop

Significance High platelet counts and advanced stage of ovarian cancer go hand-in-hand in promoting each other in a feed-forward loop that results in blood coagulation and chemotherapy resistance. This results in a high incidence of death due to thrombosis in ovarian cancer patients, and especially in patients with the ovarian clear cell carcinoma histologic type. Objective and Hypothesis We sought to develop an experimental model of the positive interactions between platelets and cancer cells and test the hypothesis that interference with platelet clotting will inhibit this interaction. Approach The effects of platelets on spheroid formation by cancer or healthy epithelial cells were evaluated using a magnetic 3D cancer spheroids assay. The ES2 and MESOV cell lines, which represent clear cell carcinoma and high grade serous histologies, respectively were used for the ovarian cancer cells. Primary human fallopian tube secretory epithelial cell cultures were used to represent healthy cells. The spheroids were imaged and measured using the Optronix GelCount colony counter and evaluated using metabolic viability (MTT) and protein concentration (SRB) assays. The shear-free platelet aggregation assay was performed in the presence of healthy or cancer cells or their conditioned media. Furthermore, we evaluated possible interruption of this feed-forward loop using antiplatelet agents: aspirin—a cyclooxygenase (COX)-1 and -2 inhibitor, celecoxib—a selective COX-2 inhibitor, clopidogrel—an ADP binding inhibitor, dipyridamole—an ADP uptake inhibitor, eptifibatide—a platelet's GP IIb/IIIa inhibitor, and prostacyclin—a platelet aggregation inhibitor. Results Incubation of platelets with cancer spheroids as they are forming decreased the size and density of the spheres in less than 15 minutes of exposure. The MTT assay indicated that condensed spheres were just as live and viable as the spheres that formed in the absence of platelets. Incubation of cancer cells or cancer cells’ conditioned media with platelets caused clumping of platelets in a cancer cells number-dependent manner. Healthy cells’ conditioned media did not cause platelets aggregation. Pre-treating platelets with up to a 1 mM of aspirin, clopidogrel, dipyridamole, and prostacyclin did not prevent cancer cell-induced aggregation, unlike celecoxib, which prevented aggregation at high concentrations, and eptifibatide, which was able to prevent aggregation at low concentrations as 0.1 µM. Conclusions The positive interaction between platelets and cancer cells can be mimicked in co-culture conditions. This interaction appears to involve platelets GPIIb/IIIa binding.

Propofol inhibits proliferation, migration, invasion and promotes apoptosis by regulating HOST2/JAK2/STAT3 signaling pathway in ovarian cancer cells.

We aimed to investigate the effect of propofol on the growth of human ovarian cancer cells ES2 and OVCAR-3 in vitro by regulating long non-coding RNA HOST2 (human ovarian cancer-specific transcript 2) and the inhibition of JAK2/STAT3 signaling pathway. In the present study, ES-2 and OVCAR-3 cells were firstly treated with different concentrations of propofol (0, 1, 5 and 10μg/ml). The expression of HOST2 in ES-2 and OVCAR-3 cells were detected by quantitative reverse transcription-PCR (qRT-PCR). Then, the expression of HOST2 was changed by transfection of HOST2 overexpression plasmid into ES-2 and OVCAR-3 cells. Cell proliferation, migration, invasion and apoptosis were performed using CCK-8, wound-healing, Transwell assays and Flow Cytometry. Western blot analysis was performed to detect the expressions of apoptosis-associated proteins and JAK2/STAT3 pathway-related proteins. Results showed that cell viability and intracellular HOST2 expression in ES-2 and OVCAR-3 cells were decreased gradually with the increase of propofol concentration in a dose-dependent manner. Compared with the propofol group, overexpression of HOST2 significantly promoted the cell proliferation, migration, invasion and inhibited apoptosis, and ameliorated the inhibitory effect of propofol on the growth of tumor cells. Western blot analysis showed that compared with propofol group, the expression of Bcl-2 was significantly increased whereas Bax and the ratio of Cleaved caspase3/caspase3 were significantly decreased in pcDNA-HOST2 group. In addition, overexpression of HOST2 significantly enhanced the phosphorylation level of JAK2 and STAT3, and reduced the suppressive effect of propofol on JAK2/STAT3 signaling. Our results illustrated that propofol could significantly inhibit the proliferation, migration, invasion and induce apoptosis of ES-2 and OVCAR-3 cells by downregulating HOST2. The regulation mechanism may be achieved by inhibiting the activation of JAK2/STAT3 signaling pathway.

Synthesis and evaluation of 68Ga-labeled dimeric cNGR peptide for PET imaging of CD13 expression with ovarian cancer xenograft.

Introduction: Previous studies have shown that peptides containing the asparagine-glycine-arginine (NGR) sequence can specifically bind to CD13 (aminopeptidase N) receptor, a tumor neovascular biomarker that is over-expressed on the surface of angiogenic blood vessels and various tumor cells, and it plays an important role in angiogenesis and tumor progression. In the present study, we aimed to evaluate the efficacy of a gallium-68 (68Ga)-labeled dimeric cyclic NGR (cNGR) peptide as a new molecular probe that binds to CD13 in vitro and in vivo.Materials and Methods: A dimeric cNGR peptide conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) was synthesized and labeled with 68Ga. In vitro uptake and binding analyses of the 68Ga- DOTA-c(NGR)2 were performed in two ovarian tumor cell lines, ES2 and SKOV3, which had different CD13 expression patterns. An in vivo biodistribution study was performed in normal mice, and micro positron emission tomography (PET) imaging was conducted in nude mice bearing ES2 and SKOV3 tumors.Results: 68Ga-DOTA-c(NGR)2 was prepared with high radiochemical purity (>95%), and it was stable both in saline at room temperature and in bovine serum at 37°C for 3 h. In vitro studies showed that the uptake of 68Ga-DOTA-c(NGR)2 in ES2 cells was higher compared with SKOV3 cells, and such uptake could be blocked by the cold DOTA-c(NGR)2. Biodistribution studies demonstrated that 68Ga-DOTA-c(NGR)2 was rapidly cleared from blood and mainly excreted from the kidney. MicroPET imaging of ES2 tumor xenografts showed the focal uptake of 68Ga-DOTA-c(NGR)2 in tumors from 1 to 1.5 h post-injection. The high-contrast tumor visualization occurred at 1 h, corresponding to the highest tumor/background ratio of 10.30±0.26. The CD13-specific tumor targeting of the 68Ga-DOTA-c(NGR)2 was further supported by the reduced uptake of the probe in ES2 tumors by co-injection of the unlabeled cold peptide. In SKOV3 tumor models, the tumor was not obviously visible under the same imaging conditions.Conclusions: 68Ga-DOTA-c(NGR)2 was easily synthesized, and it showed favorable CD13-specific targeting ability by in vitro data and microPET imaging with ovarian cancer xenografts. Collectively, 68Ga-DOTA-c(NGR)2 might be a potential PET imaging probe for non-invasive evaluation of the CD13 receptor expression in tumors.

GADD45B Facilitates Metastasis of Ovarian Cancer Through Epithelial-Mesenchymal Transition.

BackgroundGrowth arrest and DNA-damage-inducible 45 beta (GADD45B) is overexpressed and is associated with poor clinical outcomes in many human cancers, but the clinical implication of GADD45B in epithelial ovarian cancer (EOC) remains unclear.MethodsBioinformatics analysis of The Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) cohorts was used to illustrate the relationship between GADD45B expression and metastasis, as well as the survival time of EOC. GADD45B was downregulated by siRNAs in EOC cells, and migration ability was determined by a transwell assay and wound-healing assay. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and gene set enrichment analysis (GSEA) were conducted to discover the downstream pathway of GADD45B. The regulation of epithelial–mesenchymal transition (EMT) by GADD45B was verified by Western blotting and qRT-PCR. Finally, the correlation of GADD45B expression with EOC metastasis was investigated in EOC tissues by immunohistochemistry.ResultsOverexpression of GADD45B indicates shorter overall survival time and progression-free survival time, and it is an independent risk factor for poor survival in EOC patients. Elevated GADD45B is related to venous invasion, lymphatic invasion and peritoneal carcinomatosis. Downregulation of GADD45B decreases the migration of ES2 and SKOV3 cells. Further KEGG enrichment analysis and GSEA revealed that EMT may be the downstream pathway of GADD45B. In addition, reduced GADD45B increases the expression of E-cadherin and decreases that of N-cadherin and vimentin. Finally, immunohistochemical analysis of GADD45B expression revealed that the expression of GADD45B in omental metastatic tissues was higher than that in matched primary ovarian cancer tissues. These results suggest that elevated GADD45B promotes the motility of ovarian cancer cells through EMT and is associated with EOC metastasis.ConclusionGADD45B can promote the motility of ovarian cancer cells through EMT, is associated with EOC metastasis, and may be a new biomarker of metastasis and prognosis.

Suppression of Ovarian Cancer Cell Growth by AT-MSC Microvesicles

Transport of bioactive cargo of microvesicles (MVs) into target cells can affect their fate and behavior and change their microenvironment. We assessed the effect of MVs derived from human immortalized mesenchymal stem cells of adipose tissue-origin (HATMSC2-MVs) on the biological activity of the ovarian cancer cell lines ES-2 (clear cell carcinoma) and OAW-42 (cystadenocarcinoma). The HATMSC2-MVs were characterized using dynamic light scattering (DLS), transmission electron microscopy, and flow cytometry. The anti-tumor properties of HATMSC2-MVs were assessed using MTT for metabolic activity and flow cytometry for cell survival, cell cycle progression, and phenotype. The secretion profile of ovarian cancer cells was evaluated with a protein antibody array. Both cell lines internalized HATMSC2-MVs, which was associated with a decreased metabolic activity of cancer cells. HATMSC2-MVs exerted a pro-apoptotic and/or necrotic effect on ES-2 and OAW-42 cells and increased the expression of anti-tumor factors in both cell lines compared to control. In conclusion, we confirmed an effective transfer of HATMSC2-MVs into ovarian cancer cells that resulted in the inhibition of cell proliferation via different pathways, apoptosis and/or necrosis, which, with high likelihood, is related to the presence of different anti-tumor factors secreted by the ES-2 and OAW-42 cells.

Metalloprotease ADAMTS-1 decreases cell migration and invasion modulating the spatiotemporal dynamics of Cdc42 activity

ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn2+/Ca2+, involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.

Butorphanol Inhibits the Malignant Biological Behaviors of Ovarian Cancer Cells via Down-Regulating the Expression of TMEFF1.

PurposeAn important issue with compounds for treating ovarian cancer is the development of drug resistance and side effects. Butorphanol is a synthetic opioid. Opioids have been shown to promote or prevent tumor growth and metastasis. This research aimed to reveal the affection of Butorphanol on the malignant biological behaviors of ovarian cancer cells.MethodsDifferent concentrations of Butorphanol were used to treat ovarian cancer cell lines, ES-2 and SKOV3. Biological functions of cells were performed by CCK-8 assay, colony formation assay, apoptosis analysis, transwell assays and scratch assays. The differences in the transcriptome of the Butorphanol treated and negative control (NC) cells were analyzed by RNA-Seq.ResultsButorphanol treatment significantly inhibited the viability, colony-forming, migration and invasion of ES-2 and SKOV3 cells compared to NC. Furthermore, Butorphanol treatment obviously induced the apoptosis of ES-2 and SKOV3 cells and regulated the expression of apoptosis-related proteins. Additionally, Butorphanol treatment significantly reduced the expression of p-AKT, p-mTOR and P70S6K without affecting the expression of AKT and mTOR in ES-2 and SKOV3 cells. Forty-four genes were identified to up-regulate its expression, while 17 genes were identified to down-regulate its expression in Butorphanol-treated cells. Among them, TMEFF1 was found to be significantly down-regulated in Butorphanol-treated cells. Additionally, the restoration of TMEFF1 expression complemented the inhibitory effect of Butorphanol treatment on cell proliferation and invasion.ConclusionIn conclusion, Butorphanol is a compound with potential to treat ovarian cancer. TMEFF1 may play a key role in inhibiting the malignant proliferation and metastasis of Butorphanol treatment on ovarian cancer cells.

Therapeutic potential of α,β-thujone through metabolic reprogramming and caspase-dependent apoptosis in ovarian cancer cells.

The therapeutic potential of α,β-thujone, a functional compound found in many medicinal plants of the Cupressaceae, Asteraceae, and Lamiaceae families, has been demonstrated, including in inflammation and cancers. However, its pharmacological functions and mechanisms of action in ovarian cancer remain unclear. We investigated the anticancer properties of α,β-thujone in ES2 and OV90 human ovarian cancer cells and its effect on sensitization to cisplatin. α,β-thujone inhibited cancer cell proliferation and induced cell death through caspase-dependent intrinsic apoptotic pathways. Moreover, α,β-thujone-mediated endoplasmic reticulum stress was associated with the loss of mitochondrial functions and altered metabolic landscape of ovarian cancer cells. α,β-Thujone attenuated blood vessel formation in transgenic zebrafish, implying it has significant antiangiogenic potential. In addition, α,β-thujone sensitized ovarian cancer cells to cisplatin, causing synergistic pharmacological effects. Collectively, our results suggest that α,β-thujone has therapeutic potential in human ovarian cancer and functions via regulating multiple intracellular stress-associated metabolic reprogramming and caspase-dependent apoptotic pathways.

USP48 Sustains Chemoresistance and Metastasis in Ovarian Cancer.

Ubiquitin specific protease 48 (USP48) is a member of the deubiquitinating enzymes (DUBs) family. However, the function of USP48 in ovarian cancer remains unclear. The present study reveals that USP48 knockdown could significantly inhibit cell migration and invasion in ES2, 3AO and A2780 cells, without affecting cell proliferation. After carboplatin (CBP) treatment, the USP48 ablation increases the apoptosis rate, and the cleaved PARP and cleaved caspase 3 expression levels in ES2, 3AO and A2780 cells. The subcutaneous tumor and intraperitoneally injected experiments demonstrated that the USP48 knockdown significantly increases responsiveness to CBP, and alleviates the metastasis in vivo. Meanwhile, USP48 deficiency results in the improved survival of mice. Finally, the analysis of clinical samples and the TCGA and Kaplan-Meier Plot database revealed that the high expression of USP48 in ovarian cancer patients is associated with poor survival and resistance to CBP therapy. In summary, USP48 may be a potential therapeutic target for ovarian cancer patients.