Epithelial Ovarian Cancer Cell SKOV3 Research Articles

Differentially expression and function of circular RNAs in ovarian cancer stem cells

BackgroundCircular RNAs (circRNAs) are noncoding RNAs that regulate miRNA expression; however, their functions in cancer stem cells (CSCs) are not well known.MethodsTo determine the function of differentially expression of circRNAs associated with ovarian CSCs, circRNA profiling was conducted using a circRNA-based microarray on sphere-forming cells derived from A2780 and SKOV3 epithelial ovarian cancer cells termed A2780-SP and SKOV3-SP compared to monolayer cells such as A2780 and SKOV3 cells, respectively. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to predict the biological functions of the circRNAs expressed in CSCs.ResultsThe circRNA-based microarray data showed that 159 circRNAs were significantly upregulated (fold change > 1.5) and 55 circRNAs were downregulated in ovarian CSCs compared to monolayer cells. GO and KEGG enrichment analysis of differentially expressed circRNAs in ovarian CSCs showed that they were mainly involved in cell cycle, histone modification, cellular protein metabolic process, cell cycle, apoptotic signaling pathway, and ubiquitin-mediated proteolysis in ovarian cancer. In addition, the hsa-circRNA000963-miRNA-mRNA regulatory network was constructed based on potential target of miRNAs. These analyses involved that the biological function of the hsa-circRNA00096/miRNA/mRNA network was involved in signaling pathways regulating pluripotency of stem cells, PI3K-Akt signaling pathway, cell cycle, p53 signaling pathway, Wnt signaling pathway, calcium modulating pathway, and production of miRNAs involved in gene silencing by miRNA.ConclusionsOur data demonstrate the expression profiles of circRNAs in ovarian CSCs and suggest that circRNAs may be potential diagnostic and predictive biomarkers of ovarian cancer.

Identification of Novel Drug Candidate for Epithelial Ovarian Cancer via In Silico Investigation and In Vitro Validation.

Epithelial ovarian cancer (EOC) has a poor prognosis and high mortality rate; patients are easy to relapse with standard therapies. So, there is an urgent need to develop novel drugs. In this study, differentially expressed genes (DEGs) of EOC were identified in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Enrichment and protein–protein interaction (PPI) analyses were performed. The drug candidate which has the possibility to treat EOC was predicted by Connectivity Map (CMAP) databases. Moreover, molecular docking was selected to calculate the binding affinity between drug candidate and hub genes. The cytotoxicity of drug candidates was assessed by MTT and colony formation analysis, the proteins coded by hub genes were detected by Western blots, and apoptosis analysis was evaluated by flow cytometry. Finally, 296 overlapping DEGs (|log 2 fold change|>1; q-value <0.05), which were principally involved in the cell cycle (p < 0.05), and cyclin-dependent kinase 1 (CDK1) were screened as the significant hub gene from the PPI network. Furthermore, the 21 drugs were extracted from CMAPs; among them, piperlongumine (PL) showed a lower CMAP score (-0.80, -62.92) and was regarded as the drug candidate. Furthermore, molecular docking results between PL and CDK1 with a docking score of –8.121 kcal/mol were close to the known CDK1 inhibitor (–8.24 kcal/mol). Additionally, in vitro experiments showed that PL inhibited proliferation and induced apoptosis via targeting CDK1 in EOC SKOV3 cells. Our results reveal that PL may be a novel drug candidate for EOC by inhibiting cell cycle.

PLGA Nanoparticles for the Intraperitoneal Administration of CBD in the Treatment of Ovarian Cancer: In Vitro and In Ovo Assessment

The intraperitoneal administration of chemotherapeutics has emerged as a potential route in ovarian cancer treatment. Nanoparticles as carriers for these agents could be interesting by increasing the retention of chemotherapeutics within the peritoneal cavity. Moreover, nanoparticles could be internalised by cancer cells and let the drug release near the biological target, which could increase the anticancer efficacy. Cannabidiol (CBD), the main nonpsychotropic cannabinoid, appears as a potential anticancer drug. The aim of this work was to develop polymer nanoparticles as CBD carriers capable of being internalised by ovarian cancer cells. The drug-loaded nanoparticles (CBD-NPs) exhibited a spherical shape, a particle size around 240 nm and a negative zeta potential (−16.6 ± 1.2 mV). The encapsulation efficiency was high, with values above 95%. A controlled CBD release for 96 h was achieved. Nanoparticle internalisation in SKOV-3 epithelial ovarian cancer cells mainly occurred between 2 and 4 h of incubation. CBD antiproliferative activity in ovarian cancer cells was preserved after encapsulation. In fact, CBD-NPs showed a lower IC50 values than CBD in solution. Both CBD in solution and CBD-NPs induced the expression of PARP, indicating the onset of apoptosis. In SKOV-3-derived tumours formed in the chick embryo model, a slightly higher—although not statistically significant—tumour growth inhibition was observed with CBD-NPs compared to CBD in solution. To sum up, poly-lactic-co-glycolic acid (PLGA) nanoparticles could be a good strategy to deliver CBD intraperitoneally for ovarian cancer treatment.

MiR‐200c overexpression inhibits the invasion and tumorigenicity of epithelial ovarian cancer cells by suppressing lncRNA HOTAIR in mice

Epithelial ovarian cancer (EOC) is a common ovarian cancer in gynecological cancers today. It has been found that microRNAs and long-chain noncoding RNA (lncRNA) regulate the gene transcriptional expression in cells. However, it is not well understood that the upstream and downstream regulatory molecules of lncRNA HOX antisense intergenic RNA (HOTAIR). The effects of miR-200c overexpression on the invasion and nude mouse tumorigenicity, as well as lncRNA HOTAIR and snail expression of EOC SKOV3 cells, should be further explored. The expression of miR-200c and lncRNA HOTAIR was detected by reverse transcription PCR (RT-PCR) in EOC SKOV3 cells. The whole miR-200c gene fragment was cloned into a lentiviral plasmid vector. The miR-200c expression in transducted SKOV3 cells with reconstructed miR-200c lentivirus was significantly higher than the negative control (P < .01). The lentivirus-miR-200c-SKOV3 cells show that the invasion ability was significantly decreased compared with the negative control (P < .01). The nude mouse tumorigenicity was significantly decreased compared with that of the control group (P < .01). The snail protein expression in lentivirus-miR-200c-SKOV3 xenograft tumor was significantly decreased compared with the negative control lentivirus-SKOV3 group (P < .05). The miR-200c overexpression significantly decreased the expressions of lncRNA HOTAIR and snail, but increased E-cadherin expression in the lentivirus-miR-200c transducted SKOV3 cells of xenograft tumor, compared with the negative control (P < .05). The miR-200c overexpression in SKOV3 cells with transducted lentivirus-miR-200c can inhibit lncRNA HOTAIR expression, decrease snail, increase E-cadherin and significantly reduce the invasion and tumorigenicity of EOC SKOV3 cells. These results suggest that the miR-200c and lncRNA HOTAIR could be effective therapeutic targets for human epithelial ovarian cancer treatment.

Expression of transcription factor SOX12 in lung adenocarcinoma and its clinical significance

To explore the expression patterns of transcription factor SOX12 in lung adenocarcinoma and its significance in the diagnosis and prognosis of the malignancy. Large cancer genome databases were used to analyze SOX12 expression level in lung adenocarcinoma. Immunohistochemistry (IHC) and semiquantitative PCR were used to detect the expression of SOX12 in 36 specimens of lung adenocarcinoma tissues, 15 adjacent tissues and 21 normal lung tissues. The prognostic value of SOX12 in lung adenocarcinoma and lung squamous cell carcinoma were analyzed using Kaplan-Meier Plotter database, and the relationship between SOX12 expression and the overall survival (OS) and progression free survival (PPS) of the patients were analyzed. Analysis of TCGA database and GEO (GSE40419) database showed that SOX12 expression levels were significantly higher in in lung adenocarcinoma than in normal lung tissues (P &lt; 0.001). The results of IHC and semiquantitative PCR revealed that SOX12 was expressed at significantly higher levels in lung adenocarcinoma than in normal lung tissues (P &lt; 0.001). Kaplan-Meier survival analysis showed that patients with lung adenocarcinoma positive for SOX12 had a significantly shorter OS and PPS time than those negative for SOX12 (P &lt; 0.05), but SOX12 positivity did not significantly affect OS and PPS of patients with lung squamous cell carcinoma. High expression levels of SOX12 in lung adenocarcinoma are significantly associated with a poor OS of the patients, suggesting the value of SOX12 to assist in early diagnosis and prognostic evaluation of lung adenocarcinoma.

Peptide P7 inhibits the bFGF-stimulated proliferation and invasion of SKOV3 cells.

Peptide P7 specifically binds with basic fibroblast growth factor (bFGF) to inhibit the proliferation and invasion of numerous types of cancer cell. However, this effect has remained to be demonstrated in ovarian cancer-derived cell lines. In the present study, the protein P7 was used treat bFGF-stimulated SKOV3 epithelial ovarian cancer cells to explore the therapeutic potential of P7. An MTT and a scratch wound assay were used to respectively evaluate the proliferation and migration of bFGF-stimulated SKOV3 cells treated with P7. Reverse transcription-quantitative polymerase chain reaction analysis was used to detect the gene expression of urokinase-type plasminogen activator (uPA), as well as matrix metallopeptidase (MMP)-2 and −9, which have a role in cell migration/invasion. The morphology and proliferation of SKOV3 cells were not significantly affected by different concentrations of P7. However, P7 had an obvious inhibitory effect on the proliferation and migration of bFGF-stimulated SKOV3 cells. Treatment with P7 significantly lowered the gene expression of uPA, MMP-2 and MMP-9 compared with that in the control group. In conclusion, the present results suggested that P7, which, at least in part, acts through inhibition of bFGF, may have a potential therapeutic application in epithelial ovarian cancer.

“Regulator of G‐protein Signaling 10 (RGS10) inhibits NF‐κB signaling, COX‐2 and inflammatory cytokine production in ovarian cancer cells”

Epithelial Ovarian Cancer (EOC) is the fifth leading cause of cancer‐related deaths among females in the United States. NF‐κB signaling and production of inflammatory mediators have been strongly linked to ovarian cancer progression and chemoresistance. Regulator of G‐protein Signaling 10 (RGS10) regulates ovarian cancer survival and chemoresistance, and has been linked to NF‐κB signaling in immune cells. The goal of the current study was to determine the ability of RGS10 to inhibit NF‐κB signaling and subsequent inflammatory effects in ovarian cancer. RGS10 is a negative regulator of Gαi signaling and multiple Gi coupled GPCRs are implicated in ovarian cancer. Therefore, we predicted that RGS10 regulates NF‐κB signaling and inflammatory cytokines downstream of Gαi mediated‐pathway. We found that knock‐down of RGS10 enhanced NF‐κB phosphorylation and produced significantly higher levels of COX‐2 and TNF‐α mRNA compared to cells transfected with control siRNA in SKOV‐3 EOC cells. Surprisingly, we found that Pertussis toxin (ptx), which is a Gαi inhibitor, had no effects on upregulation of p‐NF‐κB, COX‐2 and TNF‐α expressions‐mediated by RGS10 knock‐down. These results provide insight in the mechanism that RGS10 utilizes to regulate ovarian cancer survival and chemoresistance.Support or Funding Information National Institutes of Health Rivkin Center for Ovarian Cancer. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sanguinarine inhibits epithelial ovarian cancer development via regulating long non-coding RNA CASC2-EIF4A3 axis and/or inhibiting NF-κB signaling or PI3K/AKT/mTOR pathway

ObjectiveThis study aimed to investigate the antitumor effects and possible regulatory mechanisms of sanguinarine in epithelial ovarian cancer. Material and methodsThe effects of sanguinarine on the malignant behaviors of epithelial ovarian cancer SKOV3 cells and the expression of long non-coding RNA CASC2 were investigated. The expression of CASC2 and EIF4A3 in epithelial ovarian cancer tissues and cells were detected, and the potential mechanisms of sanguinarine were explored by investigating the interactions between CASC2 and EIF4A3. Furthermore, the regulatory relationship between sanguinarine and nuclear factor-κB (NF-κB) signaling or PI3K/AKT/mTOR pathway was explored. ResultsSanguinarine exhibited antitumor effects in SKOV3 cells by significantly inhibiting cell viability, migration and invasion and promoting cell apoptosis. Moreover, sanguinarine induced CASC2 expression and silencing of CASC2 reversed the effects of sanguinarine in epithelial ovarian cancer cells. CASC2 was significantly lowly expressed in ovarian cancer tissues and cells, while EIF4A3 was highly expressed. EIF4A3 was identified as a CASC2 binding protein. Knockdown of EIF4A3 reversed the effects of sanguinarine plus CASC2 silencing. Besides, sanguinarine markedly inhibited the activation of NF-κB signaling or PI3K/AKT/mTOR pathway, which was reversed by CASC2 silencing. And the effects of sanguinarine plus CASC2 silencing on the activation of these pathways were further reversed after knockdown of EIF4A3 at the same time. ConclusionsOur findings reveal that sanguinarine exhibits antitumor effects in epithelial ovarian cancer cells possible via regulating CASC2-EIF4A3 axis and/or inhibiting NF-κB signaling or PI3K/AKT/mTOR pathway. Sanguinarine may serve as a potential therapeutic reagent for epithelial ovarian cancer.

Effect of S1PR2 inhibition on epithelial ovarian cancer SKOV3 cell proliferation in vitro and in vivo

Objective: To study the effect and mechanism of S1PR2 inhibition on epithelial ovarian cancer SKOV3 cell proliferation in vitro and in vivo. Methods: (1) A pair of S1PR2 gene small interference RNA (siRNA) , namely si-S1PR2, and a pair of negative control siRNA were designed. Western blot methods were used to detect the silence efficiency of the S1PR2 in the si-S1PR2 group, blank control group and negative control group. (2) Study in vitro: the experiment included three groups, namely si-S1PR2 group, blank control group and negative control group. Cell counting kit-8 (CCK-8) assay was used to detect the proliferation inhibition rates of the transfected cells. The cell cycles of the transfected cells were measured by flow cytometry. Western blot was used to detect the levels of phosph-extracellular regulated protein kinase 1/2 (p-ERK1/2) of the transfected cells. (3) Study in vivo:to establish intraperitoneal transplantation models, 8 mice in each group were intraperitoneally injected with 5×10(6) SKOV3 cells. Phosphate buffered saline (PBS) or JTE-013 were administered into mice twice per week starting on day 7 after the injection of the cancer cells. Twenty-eight days after nude mice intraperitoneal injection with JTE-013 or PBS, the mice were sacrificed and the number and the weight of visible tumors were calculated. Results: (1) The results of western blot showed that the relative S1PR2 protein expression levels were 0.24±0.04 in the si-S1PR2 group, which was lower than that in the blank control group (1.10±0.14, P<0.01) and negative control group (1.07±0.13, P<0.01) . (2) The results of CCK-8 assay indicated that after transfected for 24, 48 and 72 hours, the proliferation inhibition rate of si-S1PR2 group were respectively (26.6±3.3) %, (35.0±3.4) %, and (34.0±2.8) %, significantly lower than those in the blank control group (all 0; all P<0.01) and negative control group [ (1.7±0.9) %, (2.5±0.5) %,and (2.4±1.1) % respectively; all P<0.01]. The results of flow cytometry showed that the G(0)/G(1) ratio in the si-S1PR2 group [ (70.9±2.8) %] was significantly higher than those in the blank control group [ (61.7±2.4) %, P<0.01] and negative control group [ (62.1±3.3) %, P<0.01]. Western blot showed that the relative expression level of p-ERK1/2 in si-S1PR2 group (0.11±0.03) was significantly lower than those in the blank control group [ (0.62±0.09) , P<0.01] and negative control group [ (0.68±0.09) , P<0.01]. (3)Twenty-eight days after nude mice intraperitoneal injection with JTE-013 or PBS, the tumor number of the control group and JTE-013 group were respectively 15.4±4.3 and 8.2±3.7, the tumor weight were (0.45±0.12) and (0.21±0.07) g, respectively. The tumor number and weight in the JTE-013 group were significantly less than those in the control group (all P<0.01) . Conclusions: The growth of ovarian cancer cells could be decreased by S1PR2 inhibition in vitro and in vivo. One of the mechanisms of the growth inhibitory effect is probably that S1PR2 inhibition lower the phosphorylation level of extracellular regulated protein kinase 1/2 (ERK1/2) pathway, which prevent the transformation of ovarian cancer cells from phase G(1) to S.

Effect of resveratrol on cisplatin chemotherapy sensitivity of ovarian cancer cells

Objective To investigate the effect and mechanism of resveratrol on cisplatin chemotherapy sensitivity of human epithelial ovarian cancer SKOV3 cells. Methods The cultured in vitro human ovarian cancer SKOV3 cells in logarithmic growth phase were treated with resveratrol (20 μg/ml), cisplatin (40 μg/ml) and resveratrol (20 μg/ml) + cisplatin (20 μg/ml) respectively, and the blank group was set, n=10. After 48 hours, the cellular growth inhibition rate was calculated by methyl thiazolyl thiazolium (MTT), cell cycle and apoptosis rate were analyzed by flow cytometry, the expressions of Bax mRNA and Bcl-2 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of cleaved Caspase-3 protein was detected by Western blotting. Results The cellular growth inhibition rate of SKOV3 cells in resveratrol + cisplatin group was significantly increased than that in cisplatin group [(53.0±8.1)% vs. (37.5±6.5)%, P=0.017], the proportion of G0-G1 phase cells in resveratrol + cisplatin group was significantly higher than that in cisplatin group [(61.2±3.4)% vs. (54.9±2.8)%, P=0.017], the proportion of G2-M phase cells in resveratrol + cisplatin group was significantly lower than that in cisplatin group [(5.1±0.5)% vs. (8.6±0.9)%, P=0.008], the apoptosis index in resveratrol + cisplatin group was significantly increased than that in cisplatin group [(59.3±7.4)% vs. (43.6±6.0)%, P=0.015], the ratio of Bax/Bcl-2 in resveratrol + cisplatin group was significantly increased than that in cisplatin group (4.6±1.8 vs. 3.3±1.4, P=0.026), and the expression of cleaved Caspase-3 protein in resveratrol + cisplatin group was significantly increased than that in cisplatin group (0.47±0.15 vs. 0.38±0.12, P=0.011). Compared with blank group, the G0-G1 phase in cell cycle of resveratrol group was significantly increased [(46.5±2.4)% vs. (37.8±1.9)%, P=0.023], the expression of Bcl-2 mRNA was significantly decreased [(32.9±11.2) ×10-3vs. (44.8±13.0)×10-3,P=0.028], the ratio of Bax/Bcl-2 was significantly increased (2.1±0.8 vs. 1.5±0.6, P=0.019), and the expression of cleaved Caspase-3 protein was significantly increased (0.26±0.10 vs. 0.08±0.03, P<0.001). Conclusion Resveratrol can effectively enhance the effect on human epithelial ovarian cancer SKOV3 cells treated by cisplatin, which is perhaps related to its effects of blocking the cell cycle G0-G1 and altering the expression of apoptosis-related genes and proteins. Key words: Ovarian neoplasms; Cisplatin; Drug therapy; Apoptosis; Resveratrol

AEG-1 Contributes to Metastasis in Hypoxia-Related Ovarian Cancer by Modulating the HIF-1alpha/NF-kappaB/VEGF Pathway.

Objective Ovarian carcinoma represents one of the deadliest malignancies among female cancer patients. Astrocyte-elevated gene-1 (AEG-1) participates in the ontogenesis of multiple human malignant diseases. Here we evaluated AEG-1, hypoxia-inducible factor- (HIF-) 1α, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and vascular endothelial growth factor (VEGF) amounts in hypoxia induced ovarian carcinoma cells. This study aimed to explore the mechanism by which AEG-1 regulates metastasis in hypoxia induced ovarian carcinoma. Patients and Methods AEG-1, HIF-1α, and VEGF protein amounts were evaluated by immunohistochemistry in 40 and 170 normal ovary and ovarian cancer tissue specimens, respectively. In addition, AEG-1, HIF-1α, NF-κB, and VEGF mRNA and protein levels were determined by reverse quantified RT-PCR and WB, respectively, at different time periods (0–24 h) in epithelial ovarian cancer (EOC) SKOV3 cells treated in a hypoxia incubator. Furthermore, NF-κB and VEGF gene and protein expression levels in AEG-1 knockdown EOC cells were quantitated by RT-PCR and WB, respectively. Results AEG-1, HIF-1α, and VEGF amounts were significantly elevated in EOC tissue samples compared with normal ovary specimens (p < 0.001). Positive expression of HIF-1α and AEG-1 was associated with higher metastatic rate (p < 0.01), lower FIGO stage (p < 0.001), and degree of differentiation (p < 0.001). Meanwhile, EOC SKOV3 cells grew upon exposure to hypoxia for 8 h (p < 0.001); at this time point, AEG-1, HIF-1α, NF-κB, and VEGF amounts peaked (p < 0.001), at both the gene and the protein levels. After AEG-1 knockdown, HIF-1α, NF-κB, and VEGF amounts were significantly decreased in EOC SKOV3 cells, also under hypoxic conditions (p < 0.01). Conclusions As an independent prognostic factor, AEG-1 was found to be significantly associated with hypoxia in ovarian cancer by regulating the HIF-1alpha/NF-kappaB/VEGF pathway. Therefore, AEG-1 may be useful in determining disease stage and prognosis in ovarian cancer.

Increased expression of protein kinase CK2α correlates with poor patient prognosis in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is one of the deadly gynecological malignancies. The function of protein kinase CK2α (CK2α) in EOC is still unknown. Our study aimed to investigate the relationship between the protein expression of CK2α and the tumor progression, the prognosis of human EOC. In this study, we analyzed the expression levels of CK2α through Western blot, using EOC cell lines like A2780, HO8910, COV644, OVCAR3, SKOV3, and the primary normal ovarian surface epithelial (NOSE) cells. Furthermore, OVCAR3 and SKOV3 EOC cells were employed as a cellular model to study the role of CK2α on cell growth, migration, invasion, apoptosis, and cell cycle distribution. In addition, we investigated CK2α protein expression in tumor tissues from patients with EOC by immunohistochemistry and analyzed the association between CK2α expression and clinicopathologic parameters and prognosis of EOC patients. And we found that compared with NOSE cells, CK2α protein expression was increased in A2780, HO8910, OVCAR3, and SKOV3 ovarian cancer cell lines. Decreased CK2α expression suppressed OVCAR3 and SKOV3 cell growth and induced more apoptosis. CK2α knockdown using specific siRNAs inhibited migration and invasion ability of OVCAR3 and SKOV3 cells. In addition, high CK2α protein expression was found in 68.4% (80/117) of EOC patients. Increased CK2α expression of was significantly correlated with FIGO staging and peritoneal cytology. Patients with higher CK2α expression had a significantly poorer overall survival compared with those with lower CK2α expression. Multi-variate Cox regression analysis proved that increased CK2α expression was an independent prognostic marker for EOC. Taken together, our data displayed that CK2α may play a role in tumor aggressive behavior of EOC and could be used as a marker for predicting prognosis of EOC patient. High CK2α expression might predict poor patient survival.

Atractylenolide I modulates ovarian cancer cell-mediated immunosuppression by blocking MD-2/TLR4 complex-mediated MyD88/NF-κB signaling in vitro.

BackgroundTLR4/MD-2 complex-mediated MyD88-dependent activation of NF-κB and Akt promotes tumor-associated immunosuppression in epithelial ovarian cancer (EOC) via induction of immunesuppressive cytokines and indoleamine 2,3-dioxygenase (IDO). Atractylenolide I (AO-1) is a naturally occurring sesquiterpene lactone known to change the conformational ensemble of human MD-2 on EOC cells. This study examined the modulation by AO-1 of TLR4/MD-2 complex-mediated MyD88/NF-κB signaling.MethodsThe expression and activation of NF-κB, Akt and IDO1 by MyD88+ EOC SKOV3 cells was determined using western blot; the TLR4/MD-2 complex on SKOV3 cells and the phenotype of T lymphocytes were determined using flow cytometry; IDO activity was evaluated by measuring l-kynurenine; Immunesuppressive cytokines were detected using ELISA; T‐cell proliferation to mitogen stimulation was assessed by MTT assay; the cytotoxicity of lymphocytes and NK cells was measured using LDH-cytotoxicity assay.ResultsAO-1 could down-regulate expression of TLR4/MD-2 complex, resulting in downregulation of MyD88/NF-κB signaling and activation of NF-κB, Akt and IDO1 and secretion of IL-6, TGF-β1, VEGF and IL-17A by EOC SKOV3 cells, and further reduce increased levels of regulatory T cells (Treg cells) and improve decreased proliferative response and antitumor cytotoxicity of T lymphocytes exposed to EOC SKOV3 cell supernatant.ConclusionAO-1 may reverse EOC cell-mediated immunosuppression through blocking TLR4/MD-2 complex-mediated MyD88/NF-κB signaling.

Downregulation of MACC1 expression enhances cisplatin sensitivity in SKOV-3/DDP cells.

The aim of this study was to investigate the correlation between MACC1 expression and resistance to cisplatin (DDP) in DDP-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP). MACC1 mRNA and protein expression levels in SKOV-3 and SKOV-3/DDP cells were detected by reverse transcriptase polymerase chain reaction and western blot. The SKOV-3/DDP cells were divided into 5 groups: control, shVect (transfected with p-super-EGFP-1 plasmid), pshMACC1 (transfected with psuper-EGFP-shMACC1 plasmid), PD (pretreated with 20 μM PD98059), and combined (transfected with psuper-EGFP-shMACC1 plasmid and pretreated with 20 μM PD98059) groups. Cisplatin sensitivity and cell apoptosis in SKOV-3/DDP cells were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. ERK1/2 and p-ERK1/2 expression was determined by western blot. MACC1 mRNA and protein expression levels in SKOV-3/DDP cells were 2.66 ± 0.54 and 1.95 ± 0.45 times those seen in SKOV-3 cells (P < 0.05). Cisplatin sensitivity of pshMACC1 group was much higher than that in the control and shVect groups. Cisplatin-induced cell apoptosis rates increased significantly in the pshMACC1, PD, and combined groups, compared to the control and shVect groups. Moreover, the apoptosis rate was the highest in the combined group among the 5 groups (IC50 = 20.836 ± 0.629 μM). p-ERK1/2 expression decreased significantly in the pshMACC1, PD, and combined groups (this decrease was the most obvious in the combined group). In conclusion, downregulation of MACC1 expression could enhance cisplatin sensitivity and decrease drug resistance in SKOV- 3/DDP cells.

Downregulated lincRNA HOTAIR expression in ovarian cancer stem cells decreases its tumorgeniesis and metastasis by inhibiting epithelial-mesenchymal transition.

BackgroundEmerging evidence indicates that dysregulated long intervening non-coding RNA (lincRNA) HOTAIR correlates highly with tumor invasion and metastasis but a link between the high expression of HOTAIR and the metastatic cascade of cancer stem cells (CSCs) needs to be further studied. The purpose of this study was to investigate the effect of down-regulated HOTAIR expression on tumorgeniesis and metastasis of epithelial ovarian cancer (EOC) CSCs. CD117+CD44+CSCs were isolated from human EOC SKOV3 cell line by using a magnetic-activated cell sorting system, and were then transfected with the expression vector-based small hairpin RNA targeting HOTAIR; the stably transfected cells were selected for the study. Colony-forming, wound-healing, cellular metastasis and tumorigenicity assays were performed.ResultsThe results demonstrated that the HOTAIR expression in clinical EOC tissues and SKOV3 CD117+CD44+CSCs was higher than in SKOV3 tumor tissues and non-CD117+CD44+CSCs. The CD117+CD44+-shHOTAIR showed an inhibited HOTAIR expression, reduced cell migration and invasion than CD117+CD44+- scramble, suggesting the inhibition of an epithelial-mesenchymal transition. Moreover, the downregulated HOTAIR expression in CD117+CD44+ CSCs significantly decreased the tumor growth and lung metastasis in xenograft mice.ConclusionOur findings demonstrated the shHOTAIR-mediated down-regulation of the HOTAIR expression in CD117+CD44+ CSCs can be a promising new opportunity for future clinical trials.

Ubiquitin-specific protease 14 (USP14) regulates cellular proliferation and apoptosis in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Thus, there is an emergent need to invest a novel therapeutic for EOC. In this study, we defined ubiquitin-specific protease 14 (USP14) as a therapeutic target for EOC. Western blot was used to evaluate the expression of USP14 in nine fresh EOC tissues and three fresh normal ovarian tissues. The protein level of USP14 was higher in the cancer samples compared with that in the normal ovary tissues. Immunohistochemistry analysis was performed on formalin-fixed paraffin-embedded section of 116 cases of EOCs and indicated that USP14 was significantly associated with clinical pathologic variables. Kaplan-Meier curve showed that high expression of USP14 was related to poor prognosis of EOC patients. Starvation and re-feeding assay was used to imitate cell cycle, suggesting that USP14 played a critical role in SKOV3 cell proliferation. CCK-8 assay showed that SKOV3 cells treated with USP14-shRNA (shUSP14) grew more slowly than control group. Flow cytometry revealed that the reduced expression of USP14 induced the apoptosis of the SKOV3 EOC cells. In summary, our findings suggest that USP14 is involved in the progression of EOC and that it may be a useful target of therapy in EOC.

Effect of Diallyl Trisulfide on Human Ovarian Cancer SKOV-3/DDP Cell Apoptosis

To investigate the effects of diallyl trisulfide (DT) on apoptosis of cisplatin (DDP)-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP), and the role of p53 upregulated modulator of apoptosis (PUMA). SKOV-3/DDP cells were randomly divided into control, DT, DPP and DPP+DT groups, which were treated with DT or combined DT and DDP. All cells were incubated for 48 h. and apoptosis rates were assessed by flow cytometry. mRNA and protein expression of PUMA, Bax and Bcl-2 was determined by RT-PCR and Western blot assays, respectively. Compared with control group, the apoptosis rates of SKOV-3/DDP cells in DT groups were obviously increased, with dose-dependence (P < 0.05), the mRNA and protein expressions of PUMA, Bax also being up-regulated (P < 0.05), while those of Bcl-2 were down-regulated (P < 0.05). Compared with DT groups, the apoptosis rate in the DDP+DT group was significantly increased (P < 0.05). After knockdown of PUMA with specific siRNA, the apoptosis rate of SKOV-3/DDP cells was obviously decreased (P < 0.05). DT can promote the apoptosis of SKOV-3/DDP cells with PUMA playing a critical role.

Study of growth, invasion and metastasis on ovarian epithelial cancer cell line with CCL18 over-expression by mediated in vitro

To investigate the effect of growth, invasion and metastasis on ovarian cancer cell line SKOV3 with chemokine (C-C motif) ligand 18 (CCL18) over-expression by mediated in vitro. The restructuring plasmid of CCL18 expression was constructed and SKOV3 cells was transfected with plasmid DNA in vitro. The growth curve, cell cycle, cell migration, invasion and adhesion capacity in SKOV3-CCL18 cells and control cells were detected by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, transwell chamber, migration invasion and fibronectin adhesion method, respectively. (1) CCL18-pEGFP-N1 plasmid was successfully constructed and transmitted to the SKOV3 cells, the stable growth of the subculture SKOV3-CCL18 cells was screening and completed. (2) Compared with SKOV3-CCL18 cells and SKOV3 or SKOV3-pEGFP-N1 cells, there was not differences statistically significant in growth curve (all P > 0.05); but the rate of the SKOV3-CCL18 cells in a proliferative field (S+G2+M) was significantly higher than that in SKOV3 or SKOV3-pEGFP-N1 cells (32.80% versus 27.06%, 32.80% versus 26.98%, respectively; all P < 0.05). (3) The invasion, migration and adhesion capacity of SKOV3-CCL18 cells (being 0.49 ± 0.18, 1.16 ± 0.25 and 0.39 ± 0.10, respectively) in vitro were significantly higher than those in SKOV3 (being 0.23 ± 0.13, 0.36 ± 0.10 and 0.16 ± 0.03, respectively) or SKOV3-pEGFP-N1 cells (being 0.19 ± 0.05, 0.38 ± 0.23 and 0.13 ± 0.11, respectively; all P < 0.05). The CCL18 over-expression in epithelial ovarian cancer SKOV3 cells could lead to strengthen ability of invasion, migration and adhesion in vitro.

Evaluation of characteristics of CD44+CD117+ ovarian cancer stem cells in three dimensional basement membrane extract scaffold versus two dimensional monocultures

BackgroundCancer stem cells (CSCs) are thought to be capable of surviving conventional chemotherapeutic treatments because the cells have more resistant to anticancer drugs than common cancer cells. Most in vitro studies in experimental cancer cells have been done in a two-dimensional (2D) monocultures, while accumulating evidence suggests that cancer cells behave differently when they are grown within a three-dimensional (3D) culture system.ResultsThe CD44+CD117+cells isolated from human epithelial ovarian cancer SKOV-3 cell line using magnetic-activated cell sorting were found to grow faster than the SKOV-3 cells in the 3D culture and in the nude mice. Anticancer drugs 5FU, docetaxel, cisplatin, and carboplatin were seen to inhibit growth of the CD44+CD117+ cells by 50% in the 2D culture with IC50 concentration, whereas, in the 3D culture, the four drugs inhibited the cell growth by only 34.4%, 40.8%, 34.8% and 21.9% at 3D one, respectively. Effect of paclitaxel on the CD44+CD117+cell viability indicated that fewer cells underwent apoptosis in 3D culture than that in 2D one. In addition, anticancer drugs markedly increased the expression of ABCG2 and ABCB1 of CD44+CD117+cells in 3D culture.ConclusionOur assay demonstrated that human epithelial ovarian cancer CD44+CD117+cells possessed the properties of CSCs that exhibited more chemoresistance in the 3D culture than that of in 2D one. The 3D culture provides a realistic model for study of the CSC response to anticancer drugs.

Effects of Valproic Acid on Proliferation, Apoptosis, Angiogenesis and Metastasis of Ovarian Cancer in Vitro and in Vivo

Inhibitors of histone deacetylase activity are emerging as a potentially important new class of anticancer agents. In this study, we assessed the anticancer effects of valproic acid (VPA) on ovarian cancer in vitro and in vivo. Cultured SKOV3 cells were treated by VPA with different concentrations and time, then the effects on cell growth, cell cycle, apoptosis, and related events were investigated. A human ovarian cancer model transplanted subcutaneously in nude mice was established, and the efficacy of VPA used alone and in combination with diammine dichloroplatinum (DDP) to inhibit the growth of tumors was also assessed. Proliferation of SKOV3 cells was inhibited by VPA in a dose and time dependent fashion. The cell cycle distribution changed one treatment with VPA, with decrease in the number of S-phase cells and increase in G1-phase. VPA could significantly inhibit the growth of the epithelial ovarian cancer SKOV3 cells in vivo without toxic side effects. Treatment with VPA combined with DDP demonstrated enhanced anticancer effects. The result of flow cytometry (FCM) indicated that after VPA in vitro and in vivo, the expression of E-cadherin was increased whereas vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were decreased. This study suggests that VPA could be a novel attractive agent for treatment of ovarian cancer.