n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and Ovarian-Neoplasms

Background: Ovarian cancer is one of the most important gynecological malignancies, causing significant mortality.\ Recently, there has been extensive attention to the involvement of signaling cascades in its initiation/progression. In this\ study, we focused on the possible role of Notch signal transduction in proliferation and metalloproteinase 2 and 9 function\ in human ovarian cancer OVCAR-3 cells. Methods: MTT proliferation assays were used to evaluate effects of a DAPT\ inhibitor on cell proliferation. For measurement of Hes-1 mRNA levels, quantitative reverse transcription polymerase\ chain reaction (qRT-PCR) was applied following 48 h incubation with the inhibitor. In addition, metalloproteinase\ (MMPs) activity was assessed by zymography. Results: Inhibition of Notch signaling resulted in a significant reduction\ in OVCAR-3 cell proliferation. Additionally, DAPT treatment of cells significantly decreased Hes-1 mRNA levels\ (p < 0.05) as well as activity of MMP-2 and -9 (p < 0.05). Conclusion: Our results suggested that suppression of Notch\ signaling by a specific inhibitor can effectively decrease proliferation and the potential for metastasis of OVCAR-3 cells\ via a reduction in the activity of metalloproteinases 2 and 9. Thus, pharmacological targeting of the Notch signaling\ pathway could be a promising future treatment for ovarian cancer.

Topics: Cell Line, Tumor; Cell Proliferation; Dipeptides; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Molecular Targeted Therapy; Ovarian Neoplasms; Receptor, Notch1; Receptors, Notch; RNA, Messenger; Signal Transduction; Transcription Factor HES-1

Ovarian cancer is characterized by being highly metastatic, a feature that represents the main cause of failure of the treatment. This study investigated the effects of γ-secretase inhibition on the TGF-β-induced epithelial-mesenchymal transition (EMT) process in ovarian cancer cell lines. SKOV3 cells incubated in the presence of TGF-β showed morphological and biochemical changes related to EMT, which were blocked by co-stimulation with TGF-β and the γ-secretase inhibitor DAPT. In SKOV3 and IGROV1 cells, the co-stimulation blocked the cadherin switch and the increase in the transcription factors Snail, Slug, Twist and Zeb1 induced by TGF-β. DAPT impaired the translocation of phospho-β-catenin to the inner cell compartment observed in TGF-β-treated cells, but was not able to block the induction at protein level induced by TGF-β. Moreover, the inhibitor blocked the increased cell migration and invasiveness ability of both cell lines induced by TGF-β. Notch target genes (Hes1 and Hey1) were induced by TGF-β, decreased by DAPT treatment and remained low in the presence of both stimuli. However, DAPT alone caused no effects on most of the parameters analyzed. These results demonstrate that the γ-secretase inhibitor used in this study exerted a blockade on TGF-β-induced EMT in ovarian cancer cells.

Topics: Actin Cytoskeleton; Amyloid Precursor Protein Secretases; beta Catenin; Cadherins; Cell Line, Tumor; Cell Movement; Cell Shape; Dipeptides; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Ovarian Neoplasms; Phosphorylation; Receptors, Notch; RNA, Messenger; Transforming Growth Factor beta

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.

Topics: Amyloid Precursor Protein Secretases; Antineoplastic Agents; Apoptosis; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Proliferation; Dipeptides; Dose-Response Relationship, Drug; Female; Humans; Jagged-1 Protein; Kaplan-Meier Estimate; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Proportional Hazards Models; Protease Inhibitors; Protein Domains; Receptor, Notch1; Signal Transduction; Time Factors

Aldehyde dehydrogenase 1 (ALDH1) is a cancer stem-like cell (CSC) marker in human cancers; however, the specific ALDH1-regulated function and its underlying signalling pathways have not been fully demonstrated. Here, we investigated the ALDH1-regulated function and its underlying signalling and tested whether all-trans retinoic acid (ATRA) can suppress ALDH1-regulated tumour behaviour in ovarian cancer cells. By modulating ALDH1 expression using flow cytometry enrichment and exogenous overexpression or knockdown, we showed that the ALDH1 activity is positively correlated with stemness in ovarian cancer cells according to measures such as sphere formation and CSC marker expression as well as tumourigenesis in a mouse xenograft model. The findings indicate that the ALDH1 directly regulates the functions of ovarian cancer cells. We also showed that ALDH1 can regulate the expression of FoxM1 and Notch 1, which are involved in the downstream signalling of ALDH1-mediated biofunctions. Inhibition of FoxM1 by Thiostrepton and of Notch1 by DAPT downregulated the sphere formation ability of cells. ATRA reduced ALDH1 expression, suppressed tumour formation and inhibited sphere formation, cell migration and invasion in ALDH1-abundant ovarian cancer cells. We conclude that ATRA downregulates ALDH1/FoxM1/Notch1 signalling and suppresses tumour formation in ovarian cancer cells.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Antineoplastic Agents; Cell Movement; Cell Transformation, Neoplastic; Dipeptides; Down-Regulation; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; Humans; Isoenzymes; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplasm Transplantation; Neoplastic Stem Cells; Ovarian Neoplasms; Receptor, Notch1; Retinal Dehydrogenase; RNA Interference; RNA, Small Interfering; Spheroids, Cellular; Thiostrepton; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

Ovarian cancer is the most deadly gynecological malignancy. Understanding the molecular pathogenesis of ovarian cancer is critical to provide new targeted therapeutic strategies. Recent evidence supports a role for Notch in ovarian cancer progression and associates its dysregulation to poor overall survival. Similarly, CXCR4/SDF1α signalling correlates with ovarian cancer progression and metastasis. Recent findings indicate that Notch promotes CXCR4/SDF1α signalling and its effect on cell growth and migration; nonetheless, up to now, the association between Notch and CXCR4/SDFα in ovarian cancer has not been reported. Thereby, the aim of this study was to investigate if Notch and CXCR4/SDF1α cooperate in determining ovarian cancer growth, survival and migration. To address this issue, Notch signalling was inhibited by using γ-secretase inhibitors, or upregulated by forcing of Notch1 expression in ovarian cancer cell lines. Our results indicated that Notch activity influenced tumour cell growth and survival and positively regulated CXCR4 and SDF1α expression. CXCR4/SDF1α signalling mediated the effect of Notch pathway on ovarian cancer cell growth and SDF1α-driven migration. Additionally, for the first time, we demonstrated that Notch signalling activation can be detected in ovarian cancer specimens by immunohistochemistry analysis of the Notch transcriptional target, HES6 and is positively correlated with high expression levels of CXCR4 and SDF1α. Our results demonstrate that Notch affects ovarian cancer cell biology through the modulation of CXCR4/SDF1α signalling and suggest that Notch inhibition may be a rationale therapeutic approach to hamper ovarian cancer progression mediated by the CXCR4/SDF1α axis.

Topics: Amyloid Precursor Protein Secretases; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Chemokine CXCL12; Dipeptides; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ovarian Neoplasms; Receptor, Notch1; Receptors, CXCR4; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Notch signaling is implicated in ovarian cancer tumorigenesis and inhibition of Notch signaling with γ-secretase inhibitor DAPT resulted in reduction of tumor cell viability and induction of apoptosis in ovarian cancer cells. This study investigated whether DAPT has the same effect on ovarian cancer cells that are resistant to cisplatin and the underlying molecular events. Ovarian cancer cell lines resistant to cisplatin were treated with DAPT, cisplatin or combination for cell viability MTT, flow cytometric cell cycle, ELISA apoptosis and colony formation assays. qRT-PCR and western blotting were used to detect gene expressions. We found that pretreatment of ovarian cancer cisplatin-resistant cell lines with DAPT for 24 h and then with cisplatin for 72 h showed a synergistic antitumor activity in these cell lines, while cisplatin treatment and then addition of DAPT just showed an additive or antagonistic effects on these cisplatin-resistant ovarian cancer cells. Moreover, pretreatment of ovarian cancer cell lines with DAPT and then with cisplatin also inhibited tumor cell colony formation capacity, arrested tumor cells at G2 phase of the cell cycle and induced apoptosis. The cell cycle and apoptosis-related genes, such as cyclin B1, Bcl-2 and caspase-3, were also modulated by the treatment. Pretreatment of ovarian cancer cell lines with DAPT and then with cisplatin downregulated Notch1 and Hes1 expression dose- and time-dependently. The current data demonstrate that DAPT pretreatment was able to sensitize cisplatin-resistant human ovarian cancer cells to cisplatin by downregulation of Notch signaling.

Topics: Amyloid Precursor Protein Secretases; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinogenesis; Caspase 3; Cell Line, Tumor; Cell Survival; Cisplatin; Cyclin B1; Dipeptides; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Female; G2 Phase Cell Cycle Checkpoints; Homeodomain Proteins; Humans; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1

To determine the effect and mechanism of gamma-secretase inhibitor DAPT on the growth and apoptosis of human ovarian carcinoma SKOV3 cells.. The effect of gamma-secretase inhibitor DAPT was tested in vitro using SKOV3 cells. Its inhibition effect on cell proliferation was determined by CCK-8 assay. The cell apoptosis was detected by AO/EB double staining and flow cytometry. The expression of Notch1 mRNA and protein was detected by RT-PCR and Western blot.. Compared with controls, 5 micromol/L, 10 micromol/L and 20 micromol/L of DAPT showed an effect of cell growth inhibition in a dose-dependent manner, with 19.87%, 28.38%, and 46.67% of 24 h inhibitory rates, respectively. Dose-dependent effect of DAPT on cell apoptosis was also evident, with (5.80 +/- 0.98)%, (12.96 +/- 4.99)%, (30.88 +/- 7.63)%, and (42.98 +/- 1.46)% apoptosis rates for the control, 5 micromol/L, 10 micromol/I, and 20 micromol/L DAPT groups, respectively. RT-PCR analysis demonstrated that the expression of Notch1 mRNA decreased significantly in the DAPT groups, with an inhibition rate of 10.23%, 20.50%, and 38.83% for the three DAPT groups, respectively. Western blot results demonstrated that the expression of Notch1 protein decreased significantly, with an inhibition rate of 12.89%, 27.47%, and 49.84% for the three DAPT groups, respectively.. Gamma-secretase inhibitor DAPT can block Notch signaling pathway, inhibit proliferation, and induce apoptosis of SKOV3 cells through down-regulation of the expression of Notch1.

Topics: Amyloid Precursor Protein Secretases; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dipeptides; Down-Regulation; Female; Humans; Ovarian Neoplasms; Receptor, Notch1; Signal Transduction

Ovarian granulosa cell tumors (GCTs) represent 3%-5% of all ovarian malignancies. Treatments have limited proven efficacy and biologically targeted treatment is lacking. The aim of this study was to investigate the role of Notch signaling in the proliferation, steroidogenesis, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/AKT pathway in a FOXL2-mutated granulosa tumor cell line (KGN) representative of the adult form of GCTs. When Notch signaling is initiated, the receptors expose a cleavage site in the extracellular domain to the metalloproteinase TACE and, following this cleavage, Notch undergoes another cleavage mediated by the presenilin-gamma-secretase complex. To achieve our goal, DAPT, an inhibitor of the gamma-secretase complex, was used to investigate the role of the Notch system in parameters associated with cell growth and death, using a human granulosa cell tumor line (KGN) as an experimental model. We observed that JAGGED1, DLL4, NOTCH1, and NOTCH4 were highly expressed in KGN cells as compared to granulosa-lutein cells obtained from assisted reproductive techniques patients. The proliferation and viability of KGN cells, as well as progesterone and estradiol production, decreased in the presence of 20 μM DAPT. Apoptotic parameters like PARP and caspase 8 cleavages, BAX, and BCLXs increased in KGN cells cultured with DAPT, whereas others such as BCL2, BCLXl, FAS, and FAS ligand did not change. AKT phosphorylation decreased and PTEN protein increased when Notch signaling was inhibited in KGN cells. We conclude that the Notch system acts as a survival pathway in KGN cells, and might be interacting with the PI3K/AKT pathway.

Topics: Adaptor Proteins, Signal Transducing; Amyloid Precursor Protein Secretases; Apoptosis; Calcium-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Dipeptides; Female; Forkhead Box Protein L2; Forkhead Transcription Factors; Gonadal Steroid Hormones; Granulosa Cell Tumor; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Membrane Proteins; Mutation; Ovarian Neoplasms; Receptors, Notch; Serrate-Jagged Proteins

Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.

Topics: Amyloid Precursor Protein Secretases; Animals; Carcinoma, Papillary; Cell Line, Tumor; Cell Proliferation; Cystadenocarcinoma, Serous; Dipeptides; Dose-Response Relationship, Drug; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neoplasm Staging; Neoplasms, Experimental; Neoplastic Stem Cells; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Tumor Burden; Tumor Cells, Cultured

The release of Notch intracellular domain (NICD) is mediated by gamma-secretase. gamma-Secretase inhibitors have been shown to be potent inhibitors of NICD. We hypothesized that Notch1 is acting as an oncogene in ovarian cancer and that inhibition of Notch1 would lead to inhibition of cell growth and apoptotic cell death in ovarian cancer cells. In this study, expressions of Notch1 and hes1 in four human ovarian cancer (A2780, SKOV3, HO-8910, and HO-8910PM), and one ovarian surface (IOSE 144) cell lines were detected by Western blot and quantitative real-time RT-PCR. The effects of gamma-secretase inhibition (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, DAPT) were measured by MTT assay, flow cytometry, ELISA and colony-forming assay. Our results showed that Notch1 and hes1 were found in all the four human ovarian cancer and IOSE 144 cell lines, and they were significantly higher in ovarian cancer cells A2780 compared to another four ovarian cells. Down-regulation of Notch1 expression by DAPT was able to substantially inhibit cell growth, induce G1 cell cycle arrest and induce cell apoptosis in A2780 in dose- and time-dependent manner. In addition, hes1 was found to be down-regulated in dose- and time-dependent manner by DAPT in A2780. These results demonstrate that treatment with DAPT leads to growth inhibition and apoptosis of A2780 cells in dose- and time-dependent manner. These findings also support the conclusion that blocking of the Notch1 activity by gamma-secretase inhibitors represents a potentially attractive strategy of targeted therapy for ovarian cancer.

Topics: Amyloid Precursor Protein Secretases; Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Line, Tumor; Cell Proliferation; Dipeptides; Female; Homeodomain Proteins; Humans; Ovarian Neoplasms; Protease Inhibitors; Receptor, Notch1; Transcription Factor HES-1