Ovarian Cancer Cell Line SKOV3 Research Articles

Chemotherapy induces adaptive drug resistance and metastatic potentials via phenotypic CXCR4-expressing cell state transition in ovarian cancer.

Ovarian cancer (OVC) patients who receive chemotherapy often acquire drug resistance within one year. This can lead to tumor reoccurrence and metastasis, the major causes of mortality. We report a transient increase of a small distinctive CXCR4High/CD24Low cancer stem cell population (CXCR4High) in A2780 and SKOV-3 OVC cell lines in response to cisplatin, doxorubicin, and paclitaxel, treatments. The withdrawal of the drug challenges reversed this cell-state transition. CXCR4High exhibits dormancy in drug resistance and mesenchymal-like invasion, migration, colonization, and tumor formation properties. The removal of this cell population from a doxorubicin-resistant A2780 lineage (A2780/ADR) recovered the sensitivity to drug treatments. A cytotoxic peptide (CXCR4-KLA) that can selectively target cell-surface CXCR4 receptor was further synthesized to investigate the therapeutic merits of targeting CXCR4High. This peptide was more potent than the conventional CXCR4 antagonists (AMD3100 and CTCE-9908) in eradicating the cancer stem cells. When used together with cytotoxic agents such as doxorubicin and cisplatin, the combined drug-peptide regimens exhibited a synergistic cell-killing effect on A2780, A2780/ADR, and SKOV-3. Our data suggested that chemotherapy could establish drug-resistant and tumor-initiating properties of OVC via reversible CXCR4 cell state transition. Therapeutic strategies designed to eradicate rather than antagonize CXCR4High might offer a far-reaching potential as supportive chemotherapy.

The translational blocking of α5 and α6 integrin subunits affects migration and invasion, and increases sensitivity to carboplatin of SKOV-3 ovarian cancer cell line

Epithelial ovarian cancer is the most lethal gynecologic malignancy. Integrins, overexpressed in cancer, are involved in various processes that favor the development of the disease. This study focused on determining the degree of involvement of α5, α6 and β3 integrin subunits in the establishment/development of epithelial ovarian cancer (EOC), such as proliferation, migration, invasion, and response to carboplatin. The translation of the α5, α6 and β3 integrins was blocked using morpholines, generating morphant cells for these proteins, which were corroborated by immunofluorescence assays. WST-1 proliferation assay showed that silencing of α5, α6, and β3 integrins does not affect the survival of morphants. Wound healing and transwell chamber assays showed that blocking α5 and α6 integrins decrease, in lesser and greater level respectively, the migratory and the invasive capacity of SKOV-3 cells. Finally, blocking α5 and α6 integrins partially sensitized the cells response to carboplatin, while blocking integrin β3 generated resistance to this drug. Statistical analyses were performed with the GraphPad Prism 5.0 software employing one way and two-way ANOVA tests; data are shown as average±SD. Results suggest that α5 and α6 integrins could become good candidates for chemotherapy targets in EOC.

Genome-wide DNA copy number analysis in clonally expanded human ovarian cancer cells with distinct invasive/migratory capacities.

Ovarian cancer has the worst prognosis of any gynecological malignancy, and generally presents with metastasis at advanced stages. Copy number variation (CNV) frequently contributes to the alteration of oncogenic drivers. In this study, we sought to identify genetic targets in heterogeneous clones from human ovarian cancers cells. We used array-based technology to systematically assess all the genes with CNVs in cell models clonally expanded from A2780 and SKOV3 ovarian cancer cell lines with distinct highly and minimally invasive/migratory capacities. We found that copy number alterations differed between matched highly and minimally invasive/migratory subclones, differentially affecting specific functional processes including immune response processes, DNA damage repair, cell cycle and cell proliferation. We also identified seven genes as strong candidates, including DDB1, ERCC1, ERCC2, PRPF19, BCAT1, CDKN1B and MARK4, by integrating the above data with gene expression and clinical outcome data. Thus, by determining the molecular signatures of heterogeneous invasive/migratory ovarian cancer cells, we identified genes that could be specifically targeted for the treatment and prognosis of advanced ovarian cancers.

Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

C‐Met inhibition enhances chemosensitivity of human ovarian cancer cells

In clinical practice, human ovarian cancer shows considerable resistance to chemotherapy. This study aimed to investigate the role of c-Met in the chemoresistance of ovarian cancer. Ovarian cancer cell line SKOV3 and OVCAR-3 were pretreated with c-Met inhibitor INCB28060, and then treated with paclitaxel. Cell survival, cell cycle and apoptosis were analyzed by MTT assay, flow cytometry analysis and TUNEL assay, respectively. The activation of c-Met signalling was detected by western blot analysis. INCB28060 inhibited the survival of SKOV3 and OVCAR-3 cells and enhanced the chemosensitivity of SKOV3 and OVCAR-3 cells to paclitaxel. INCB28060 inhibited c-Met signalling, caused mitochondrial membrane depolarization and DNA repair, and induced the apoptosis of SKOV3 and OVCAR-3 cells. c-Met plays an important role in mediating the chemoresistance of ovarian cancer. The combination of c-Met inhibitor and chemotherapy is a promising strategy to human ovarian cancer.

Inhibition of gamma-secretase in Notch1 signaling pathway as a novel treatment for ovarian cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death for gynecological cancer. Most patients are not diagnosed until the cancer is at an advanced stage with poor prognosis. Notch1 signaling pathway plays an oncogenic role in EOC. There have been few studies on enzymatic activity of γ-secretase and the mechanism of how γ-secretase inhibitor works on cancer cell. Here, we show that Jagged1 and NICD were highly expressed in ovarian carcinoma. The expressions of Notch1, Jagged1 and NICD in Notch1 pathway did not correlate with outcome in ovarian cancer. The enzymatic activity of γ-secretase in ovarian cancer cell lines SKOV3, CAOV3 and ES2 is significantly higher than in normal ovarian epithelial cell line T29. DAPT (a γ-secretase inhibitor) reduced the enzymatic activity of γ-secretase, inhibited the proliferation, and increased the apoptosis in ovarian cancer cell lines. Hence, γ-secretase inhibitor may become a highly promising novel therapeutic strategy against ovarian cancer in the field of precision medicine.

Human C1q Induces Apoptosis in an Ovarian Cancer Cell Line via Tumor Necrosis Factor Pathway.

Complement protein C1q is the first recognition subcomponent of the complement classical pathway that plays a vital role in the clearance of immune complexes, pathogens, and apoptotic cells. C1q also has a homeostatic role involving immune and non-immune cells; these functions not necessarily involve complement activation. Recently, C1q has been shown to be expressed locally in the microenvironment of a range of human malignant tumors, where it can promote cancer cell adhesion, migration, and proliferation, without involving complement activation. C1q has been shown to be present in the ascitic fluid formed during ovarian cancers. In this study, we have examined the effects of human C1q and its globular domain on an ovarian cancer cell line, SKOV3. We show that C1q and the recombinant globular head modules induce apoptosis in SKOV3 cells in a time-dependent manner. C1q expression was not detectable in the SKOV3 cells. Exogenous treatment with C1q and globular head modules at the concentration of 10 µg/ml induced apoptosis in approximately 55% cells, as revealed by immunofluorescence microscopy and FACS. The qPCR and caspase analysis suggested that C1q and globular head modules activated tumor necrosis factor (TNF)-α and upregulated Fas. The genes of mammalian target of rapamycin (mTOR), RICTOR, and RAPTOR survival pathways, which are often overexpressed in majority of the cancers, were significantly downregulated within few hours of the treatment of SKOV3 cells with C1q and globular head modules. In conclusion, C1q, via its globular domain, induced apoptosis in an ovarian cancer cell line SKOV3 via TNF-α induced apoptosis pathway involving upregulation of Bax and Fas. This study highlights a potentially protective role of C1q in certain cancers.

The Heat Shock Transcription Factor HSF1 Induces Ovarian Cancer Epithelial-Mesenchymal Transition in a 3D Spheroid Growth Model.

Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. The proteotoxic stress-responsive transcription factor HSF1 is frequently overexpressed in a variety of cancers and is vital to cellular proliferation and invasion in some cancers. Upon analysis of various patient data sets, we find that HSF1 is frequently overexpressed in ovarian tumor samples. In order to determine the role of HSF1 in ovarian cancer, inducible HSF1 knockdown cell lines were created. Knockdown of HSF1 in SKOV3 and HEY ovarian cancer cell lines attenuates the epithelial-to-mesenchymal transition (EMT) in cells treated with TGFβ, as determined by western blot and quantitative RT-PCR analysis of multiple EMT markers. To further explore the role of HSF1 in ovarian cancer EMT, we cultured multicellular spheroids in a non-adherent environment to simulate early avascular tumors. In the spheroid model, cells more readily undergo EMT; however, EMT inhibition by HSF1 becomes more pronounced in the spheroid model. These findings suggest that HSF1 is important in the ovarian cancer TGFβ response and in EMT.

Effect of ginsenoside Rh-2 via activation of caspase-3 and Bcl-2-insensitive pathway in ovarian cancer cells.

Ginsenoside has been reported to have therapeutic effects for some types of cancer, but its effect on ovarian cancer cells has not been evaluated. In this study, we monitored the effects of ginsenoside-Rh2 (Rh2) on the inhibition of cell proliferation and the apoptotic process in the ovarian cancer cell line SKOV3 using an MTT assay and TUNEL assay. We found that Rh2 inhibited cell proliferation and significantly induced apoptosis. We confirmed the apoptotic effects of Rh2 using western blot analysis of apoptosis-related proteins. Specifically, the levels of cleaved poly ADP ribose polymerase (PARP) and cleaved caspase-3 significantly increased in SKOV3 cells treated with Rh2. Therefore, Rh2 clearly suppressed the growth of SKOV3 cells in vitro, which was associated with induction of the apoptosis pathway. Moreover, the migration assay showed that Rh2 inhibited the invasive ability of SKOV3 cells. Taken together, our results suggest that Rh2 has anticancer effects in SKOV3 cells through inhibition of cell proliferation and induction of apoptosis. Considering the therapeutic potential of Rh2, more studies should be carried out to facilitate the future application of this natural product as a potential anti-cancer agent.

Abstract B065: Use of simultaneous detection of externalized CD107a and CD137 for evaluation of specificity and effector functions of polyclonal T-cells produced for adoptive cellular immunotherapy of prostate cancer

Abstract The ex vivo production of T-cells that can recognize tumor-associated antigens (TAAs) and through this recognition eliminate cancer cells is the goal of T-cell-based adoptive cellular immunotherapy (ACI). The technological approaches that use whole cancer cells or their pertinent lysates as a source of TAAs for production of these T-cells lead to generation of polyclonal T-cells their antigen specificity of which is often unknown. Evaluation of such polyclonal T-cells is therefore challenging because their target and off-target performance might be difficult to predict. In our study we used human dendritic cells (DCs) pulsed with inactivated prostate cancer cell line LNCaP and matured with polyinosinic:polycytidylic acid (poly(I:C)) to induce expansion of autologous lymphocytes. The expanded lymphocytes were then challenged with live LNCaP cells and analyzed by simultaneous detection of a marker of T-cell cytotoxicity and antigen-induced T-cell stimulation, the surface externalization of, respectively, CD107a and CD137. Our data showed that lymphocytes expanded with LNCaP-pulsed and poly(I:C)-matured DCs contained a notable fraction of CD137 and/or CD107a positive T-cells after their challenge with live LNCaP cells. No such notable fraction was however observed in resting lymphocytes, or when the lymphocytes were challenged with ovarian cancer cell line SKOV-3. Importantly, autologous lymphocytes that were expanded with poly(I:C)-matured DCs not previously pulsed with the inactivated LNCaP cells did not show a notable population of the marker positive cells. Collectively, our data showed that simultaneous detection of externalized CD107a and CD137 on the surface of polyclonal T-cells may allow for evaluation of polyclonal T-cell specificity and effector functions after their challenge with complex antigens. Citation Format: Pavla Taborska, Dmitry Stakheev, Katerina Vavrova, Petra Vrabcova, Jirina Bartunkova, Daniel Smrz. Use of simultaneous detection of externalized CD107a and CD137 for evaluation of specificity and effector functions of polyclonal T-cells produced for adoptive cellular immunotherapy of prostate cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B065.

Expression and mechanism of action of miR-196a in epithelial ovarian cancer.

To explore the expression, biological function and possible mechanism of action of microRNA molecular-196a (miR-196a) in epithelial ovarian cancer. RT-PCR was used to detect the expression quantities of epithelial ovarian tissue, benign ovarian tissue, normal ovary epithelial tissue, ovarian cancer cell lines and miR-196a in normal ovarian epithelial cells to analyze the relationship between the expression of miR-196a and the clinical pathologic parameters of ovarian cancer. Among those cell lines, the cell line of which miR-196a expressed the most or least was selected and transfected the ovarian cancer cell line by using negative control plasma and miR-196a inhibitor. After transfection, RT-PCR was used to test the expression quantity of miR-196a, Transwell chamber method was applied to determine the migration and invasion abilities of ovarian carcinoma cells and Western blot was employed to detect the expression of HOXA10 protein. The relative expression quantities of miR-196a in ovarian cancer tissue and benign ovarian tissue were significantly higher than that in normal ovarian epithelial tissue, and the expression quantity of miR-196a in ovarian cancer tissue was distinctively higher than that in benign ovarian tissue (P<0.05). Among 78 cases of epithelial ovarian cancer, the expression quantities of miR-196a in patients with low differentiation were all significantly higher than those in patients with high differentiation (P<0.05). The expression of miR-196a showed no significant relation with age, clinical stage and whether CA125 was positive or not in patients (P>0.05). Compared with normal ovarian epithelial cell line IOSE80, the expression quantities of miR-196a of all ovarian cancer cell lines increased obviously and differences were statistically significant (P<0.05). Among them, the expression of miR-196a of ovarian cancer cell line SKOV3 was the highest, while it decreased significantly (4.678±0.785 vs. 2.131±0.345, t=2.938, P<0.05) after the ovarian cancer cell line SKOV3 was transfected by miR-196a inhibitor. The results of Transwell chamber method showed that the migration and invasion abilities of ovarian cancer cells SKOV3 were declined significantly after the expression of miR-196a was down-regulated and the difference showed statistical significance (P<0.05). The results of Western blot revealed that the relative expression of HOXA10 decreased distinctly after the expression of miR-196a was down-regulated and also the difference showed statistical significance (P<0.05). The miR-196a might serve as a cancer-promoting gene to promote the migration and invasion of epithelial ovarian cancer by downstream target gene HOXA10.

CLC-3 Cl- channel-mediated invasion and migration of human ovarian cancer cells.

To investigate the potential role of CLC-3, a member of the voltage-gated chloride channel (CLC) superfamily, in invasion and migration of ovarian cancer cell line SKOV3. CLC-3 antisense oligonucleotides were transfected into ovarian cancer cell line SKOV3, and its effects on cell invasion and migration were analyzed by using Transwell chamber assay and wound healing assay in vitro. The efficiency of CLC-3 antisense was determined with RT-PCR and Western blotting. The protein concentrations of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) were determined using ELISA kits. Cell volume measurements were performed. Studies in vitro revealed that the CLC-3 antisense inhibited invasion and migration of ovarian cancer cell line SKOV3. CLC-3 antisense treatment decreased protein levels of MMP-2, MMP-9, and VEGF in culture medium of SKOV3 cells. In addition, the authors found that the capability for regulatory volume decrease (RVD) was much attenuated in SKOV3 cells transfected with CLC-3 antisense. These results strongly suggest that CLC-3 may get involved in proliferation, invasion, and migration of ovarian cancer cells and thus may be a useful therapeutic target.

A Combination of Two Receptor Tyrosine Kinase Inhibitors, Canertinib and PHA665752 Compromises Ovarian Cancer Cell Growth in 3D Cell Models

IntroductionAdvanced ovarian cancer is often a fatal disease as chemotherapeutic drugs have limited effectiveness. Better targeted therapy is needed to improve the survival and quality of life for these women. Receptor tyrosine kinases including EGFR, Her-2 and c-Met are associated with a poor prognosis in ovarian cancer. Therefore, the co-activation of these receptors may be crucial for growth promoting activity. In this study, we explored the effect of combining two small molecule inhibitors that target the EGFR/Her-2 and c-Met receptor tyrosine kinases in two ovarian cancer cell lines. The aim of this study was to investigate the combined inhibition activity of a dual EGFR/Her-2 inhibitor (canertinib) and a c-Met inhibitor (PHA665752) in ovarian cancer cell lines in 3D cell aggregates.MethodsOVCAR-5 and SKOV-3 ovarian cancer cell lines were cultured on a non-adherent surface to produce 3D cell clusters and aggregates. Cells were exposed to canertinib and PHA665752, both individually and in combination, for 48 h. The effect on growth, metabolism and the expression/phosphorylation of selective signaling proteins associated with EGFR, Her-2 and c-Met were investigated.ResultsThe single drug treatments significantly decreased cell growth and altered the expression of signaling proteins in OVCAR-5 and SKOV-3 cell lines. The combination treatment showed greater reduction of cell numbers for both cell lines. Total expression and phosphorylation of signaling proteins were further reduced in the combination drug treatments, compared to the single inhibitor treatments.ConclusionOur findings suggest that the concurrent targeting of more than one receptor tyrosine kinase may be useful in developing more effective targeted drug regimens for patients, who have EGFR, Her-2 and c-Met positive ovarian cancer cells.

Tumor-associated macrophages promote invasion via Toll-like receptors signaling in patients with ovarian cancer

Tumor-associated macrophages (TAMs) derived from peripheral blood monocytes recruit into tumor microenvironment and display functions associated with tumor progression. The mechanisms by which TAMs display roles that associated with the invasion ability of ovarian cancer have not been well investigated. In our research, we found abundant TAMs infiltrate in ovarian cancer compared with benign ovarian tumor tissues. Levels of matrix metalloproteinase (MMP)-2, MMP-9 and MMP-10, and Toll-like receptors (TLRs) signaling proteins were evaluated in ovarian cancer. The high level of TAMs was associated with metastasis and advance of patients with ovarian cancer. TAMs and ovarian cancer cell line SKOV3 were cocultured in vitro, MMPs level and the invasion ability of SKOV3 cells were significantly up-regulated. The coculture process was correlated with the activation of TLRs signaling and downstream nuclear factor (NF)-κB p65 and microtubule-associated proteins (MAPs) kinases pathway in SKOV3. In addition, pre-incubation with TLRs signaling inhibitors remarkably suppressed invasion ability of SKOV3. Levels of TLRs signaling pathways proteins were also down-regulated in this blocking process. These findings demonstrated that TAMs promoted up-regulation of MMP-2, MMP-9 and MMP-10 expressions and enhanced ovarian cancer cells invasion via TLRs signaling pathway. We conclude that TAMs could enhance ovarian cancer cells invasion and ultimately promote ovarian cancer progression.

WNT5A-ROR2 is induced by inflammatory mediators and is involved in the migration of human ovarian cancer cell line SKOV-3.

BackgroundWnt5A, which is a member of the non-transforming Wnt protein family, is implicated in inflammatory processes. It is also highly expressed by ovarian cancer cells. ROR2, which is a member of the Ror-family of receptor tyrosine kinases, acts as a receptor or co-receptor for Wnt5A. The Wnt5A–ROR2 signaling pathway plays essential roles in the migration and invasion of several types of tumor cell and influences their cell polarity. We investigated the modulation of Wnt5A–ROR2 by inflammatory mediators and its involvement in the migration of the human ovarian cancer cell line SKOV-3.MethodsSKOV-3 cells were treated with LPS (lipopolysaccharide), LTA (lipoteichoic acid) and recombinant human IL-6 alone or in combination with STAT3 inhibitor (S1155S31-201) or NF-kB inhibitor (BAY11-7082) for 4, 8, 12, 24 and 48 h. The Wnt5A and ROR2 expression levels were determined at the gene and protein levels. Cells were transfected with specific siRNA against Wnt5A in the absence or presence of human anti-ROR2 antibody and cell migration was assessed using transwells.ResultsThere was a strong downregulation of Wnt5A expression in the presence of STAT3 or NF-kB inhibitors. Cell stimulation with LTA or IL-6 for 8 h led to significantly increased levels of Wnt5A (5- and 3-fold higher, respectively). LPS, LTA or IL-6 treatment significantly increased ROR2 expression (2-fold after 48 h). LPS- or LTA-induced Wnt5A or ROR2 expression was abrogated in the presence of STAT3 inhibitor (p < 0.001). IL-6-induced Wnt5A expression was abrogated by both STAT3 and NF-kB inhibitors (p < 0.001). Although not significant, IL-6-induced ROR2 expression showed a modest decrease when STAT3 inhibitor was used. Moreover, cell migration was decreased by 80 % in siRNA Wnt5A-transfected cells in the presence of anti-human ROR2 antibody (p < 0.001).ConclusionsThis study revealed for the first time that inflammatory mediators modulate Wnt5A and ROR2 through NF-kB and STAT3 transcription factors and this may play a role in ovarian cancer cell migration. The results described here provide new insight into the role of the Wnt5A–ROR2 complex in ovarian cancer progression in relation to inflammation.

Abstract 1373: IHC and RT-PCR assays for detection of cancer antigen NY-ESO-1 in human tissues

Abstract Introduction: NY-ESO-1 is a cancer testis antigen frequently expressed in a broad range of tumors: lung, breast, ovarian, bladder, liver cancer, melanoma, sarcoma and myeloma. CDX-1401, a fully human monoclonal antibody linked to full length NY-ESO-1, binds to dendritic cell (DC) receptor DEC-205 to stimulate NY-ESO-1 specific CD4 and CD8 responses. Immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR) assays were developed to determine NY-ESO-1 expression in human tumors and normal adjacent tissues (NAT). A qRT-PCR assay for the detection of LAGE-1, a cancer testis antigen with significant DNA homology to NY-ESO-1, was also developed but no commercially specific antibody was available for IHC. Methods: Formalin fixed, paraffin embedded tumors and NAT were procured from a commercial source or obtained from Mosaic Laboratories; split anonymized samples were analyzed for NY-ESO-1 by IHC at Mosaic and for NY-ESO-1 and LAGE-1 by qRT-PCR at MolecularMD (OR) under IRB-approved protocols allowing in vitro analysis of remnant human samples. The study included: 18 each melanoma and ovarian cancers; 13 each colorectal (CRC), head &amp; neck (H&amp;N), and non-small cell lung cancers (NSCLC); and 38 NAT. HT1080 fibrosarcoma and SKOV3 ovarian cancer cell lines (ATCC, VA) and normal testis were used as controls. The qRT-PCR assays were validated using in vitro transcribed RNA on NY-ESO-1 and LAGE-1 kits provided by Life Technologies and the NY-ESO-1 IHC was developed using a mouse monoclonal antibody (Sigma, MO). Results: The NY-ESO-1 and LAGE-1 qRT-PCR assays had a linear range of 100-1,000,000 copies/reaction with limit of detection between 1-10 copies. An excess of LAGE-1 copies did not impact the specificity of the NY-ESO-1 assay and vice-versa. NY-ESO-1 protein was detected by IHC and NY-ESO-1 transcripts and LAGE-1 transcripts were detected in 9 (12%), 12 (16%) and 16 (21.3%), respectively, of the 75 tumor tissues while only 2 of the 38 normal samples were positive for LAGE-1. When stromal staining was considered in addition to epithelial staining, 16 (21.3%) tumor samples were positive by IHC. NY-ESO-1 protein was more prevalent in NSCLC (38%), ovarian cancer (11%) and melanoma (11%) vs. other tumor types. Positive staining by IHC was achieved in tumor tissues shown positive for NY-ESO-1 and negative for LAGE-1 transcripts. In general, tumor tissues obtained from Mosaic were of better quality, as assessed by the pathologist, and more positive than commercially procured samples. Conclusions: Two qRT-PCR assays were developed to specifically quantify NY-ESO-1 or LAGE-1 mRNA without cross reaction. The antibody used in IHC assay seemed specific to NY-ESO-1. Inadequate sample quality impacted target prevalence. Implementing these assays in clinical trials may aid in prospectively identifying patients with NY-ESO-1 positive malignancies who could derive benefit from investigational NY-ESO-1 vaccines designed to induce anti-tumor immunity, such as CDX-1401. Citation Format: Abdel Halim, Rebecca G. Bagley, Lisa Dauffenbach, Eric P. Olsen, Tibor Keler. IHC and RT-PCR assays for detection of cancer antigen NY-ESO-1 in human tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1373.

Effects of chrysin on sphere formation and CK2α expression of ovarian cancer stem-like cells derived from SKOV3 cell line

To investigate whether inhibitory effect of chrysin on sphere formation of ovarian cancer stem-like cells(spheroids derived from human ovarian cancer SKOV3 cell line ) is involved in the down-regulating of the protein expression of casein kinase CK2α. SKOV3-derived ovarian cancer stem-like cells obtained by suspension culture in stem cell-condition medium using ultra-low adhesion plate were treated with various concentrations (5.0, 10.0 and 20.0 μmol/L) of chrysin. Sphere formation assay was used to determine the sphere forming rate of SKOV3-derived ovarian cancer stem-like cells. Western blot was used to analyze the protein expressions of CK2α and cancer stem cell markers CD133 and CD44. Silence of CK2α by siRNA and ectopic expression of CK2α by transfection with pcDNA3.1-CK2α plasmid were used to explore the mechanism underlying the effect of chrysin on sphere formation of SKOV3-derived ovarian cancer stem-like cells. Chrysin (5.0, 10.0 and 20.0 μmol/L) significantly reduced the sphere forming rate of SKOV3-derived ovarian cancer stem-like cells, in a concentration-dependent manner (22.3%±2.5% vs 14.7%±2.1%, 8.6%± 1.7% and 3.8% ± 1.1% respectively; P<0.05). In addition, chrysin (5.0, 10.0 and 20.0 μmol/L) obviously down-regulated the protein expressions of CK2α, CD133 and CD44 in SKOV3-derived ovarian cancer stem-like cells. In combination with CK2α siRNA transfection and chrysin synergistically decreased sphere formation (P<0.05) and the protein expressions of CK2α, CD133 and CD44 in SKOV3-derived ovarian cancer stem-like cells. However, transfection with pcDNA3.1-CK2α plasmid attenuated inhibitory effects of chrysin on sphere formation capability and the expressions of CK2α, CD133 and CD44 of SKOV3-derived ovarian cancer stem-like cells. Down-regulation of CK2α protein expression is involved in the inhibition effect of chrysin on the sphere formation capability of SKOV3-derived ovarian cancer stem-like cells.

Anticancer role of antidiabetic drug Metformin in ovarian cancer cells

Purpose: Epithelial ovarian cancer is the most common ovarian cancer and has life threatening implications. Despite the progress in surgical and therapeutic strategies, resistance to chemotherapy is still a major concern. Chemotherapeutic agents cause cytotoxicity, primarily by the induction of apoptosis. The status of p53 is a key factor in determining the efficacy of apoptotic signaling. p53 is the most commonly mutated tumor suppressor gene in ovarian cancer. Metformin (an antidiabetic drug) has shown putative effects in many solid tumors. Hence we aimed to study the role of metformin in p53 mutated cancer cells. Methods: SKOV3 and OAW42 ovarian cancer cell line were used. The cancer cells were treated with metformin. MTT, Flow cytometry and Western blotting were used to characterize the effects of the different treatments. Results: Metformin treatment leads to cell cycle arrest in the G0/G1, S and G2/M phase of the cell cycle in SKOV3 and OAW42 respectively. Moreover, there was upregulation of Bax and downregulation of Bcl-2 protein and increased apoptosis in SKOV3 and OAW42 ovarian cancer cells. Conclusion: These findings support the potential of metformin to be used as chemoadjuvant and reflects its ability to sensitize cancer cells to apoptosis independent of p53 status.

NFATc1 regulates cell proliferation, migration, and invasion of ovarian cancer SKOV3 cells in vitro and in vivo

NFATc1 (nuclear factor of activated T‑cells c1) is associated with malignancy in several cancer models. However, the expression and function of NFATc1 in ovarian cancer remain elusive. In the present study, we investigated the role of NFATc1 in human epithelial ovarian cancer (EOC) using human ovarian adenocarcinoma SKOV3 cells and patient characteristics. NFATc1 expression was silenced by siRNA in the SKOV3 ovarian cancer cell line and in human ovarian cancer nude mouse xenografts. Real‑time PCR, western blotting, immunohistochemical staining, MTT, flow cytometry, transwell, erasion trace and mouse assays were used to detect NFATc1 expression, cell proliferation, apoptosis, cell invasion and migration, tumor growth and angiogenesis. Survival analysis was performed to assess the correlation between NFATc1 expression and survival. NFATc1 was overexpressed in the SKOV3 ovarian cancer cell line and in human serous/mucinous ovarian cancer tissues. The silencing of NFATc1 expression by siRNA reduced cell proliferation and migration and promoted apoptosis invitro and decreased the ovarian cancer cell tumorigenesis invivo in nude mice. NFATc1 overexpression in high‑grade serous ovarian carcinomas was an independent prognostic factor of poor overall survival and of early relapse (P<0.01) in a univariate analysis. Our present data provide evidence that NFATc1 is overexpressed in human serous/mucinous ovarian cancer and is associated with a poor prognosis. NFATc1 silencing regulates the cell cycle, apoptosis, invasion and migration. NFATc1 thus has the potential to be a therapeutic target and to be used in EOC diagnosis and prognosis.

Decreased expression of microRNA-148a predicts poor prognosis in ovarian cancer and associates with tumor growth and metastasis.

MicroRNA-148a (MiR-148a) had been reported to take part in some cancer progresses, but its clinical significance in ovarian cancer had been rarely reported. The purpose of this study was to evaluate the prognostic value of miR-148a as well as its roles in ovarian cancer progression. Relative expression of miR-148a in the plasma specimens of ovarian cancer patients was detected by qRT-PCR. Chi-square test was used to analyze the relationship between miR-148a expression and clinical characteristics. The overall survival was analyzed by Kaplan-Meier method and Cox regression analysis was used to evaluate the prognostic value of miR-148a. In addition, the ovarian cancer cell line SKOV-3 was separately transfected with pcDNA3-microRNA-148a over-expression vector and pcDNA3 empty vector to detect the functional roles of miR-148a in ovarian cancer progression. Decreased level of plasma miR-148a was observed in ovarian cancer patients compared with healthy controls. The expression level was associated with histopathologic grade, TNM stage and lymph node metastasis (P<0.05 for all). Besides, patients with high level of miR-148a had a longer survival time than those with low level (40.3 months vs 31.6 months, log rank test, P=0.002). Cox regression analysis indicated that miR-148a might be a potential biomarker for ovarian cancer prognosis (HR=1.699, 95%CI=1.175-2.456, P=0.005). Moreover, cell experiments confirmed that miR-148a could inhibit proliferation, migration and invasion of ovarian cancer cells. MiR-148a may be a potential prognostic factor for ovarian cancer and it can suppress tumor progression.