Molecular characterization of highly tumorigenic cell lines used in a xenograph model to investigate cellular therapy for the treatment of refractory ovarian cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e16516-e16516
Author(s):  
S. B. Ingersoll ◽  
P. Yue ◽  
S. Ahmad ◽  
J. Turkson ◽  
J. R. Edwards ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Lines ◽  
Cellular Therapy ◽  
Fluorescent Protein ◽  
Tumor Incidence ◽  
Mobility Shift ◽  
Term Survival ◽  
Innovative Strategies ◽  
Mobility Shift Assay

e16516 Background: Because current therapies for ovarian cancer (OC) have little impact on the long-term survival, there is a compelling need to develop innovative strategies. Our aim was to characterize OC cell lines which can be utilized to test cellular therapy in combination with cytokines or chemotherapies to elicit a graph versus tumor response to treat refractory OC patients. Methods: SKOV-3-RFP, a red fluorescent protein (RFP) expressing line, was passaged through nude mice to create more tumorigenic lines compared to parental SKOV-3 cells. Three SKOV-3-derived lines were established and characterized for IFNα-2b sensitivity, E-cadherinand CCN1gene expression, and STAT3 activation. The cells were grown in the presence of IFNα-2b to determine proliferative effects. Tumors were harvested when mice became moribund; ascitic fluid (AF) and solid tumor (ST) tissue were snap-frozen. Expression of E-cadherinand CCN1, which have been implicated to play role(s) in OC pathobiology, were analyzed in ST harvested from the mice and from SKOV3-derived lines (SKOV3-RFP, AF1, AF2, and AF3). We investigated the activation status in the SKOV3-derived cells of STAT3, which has been associated with malignant transformation and tumor progression. Results: Parental SKOV3-RFP cells when injected at a dose of 5x106 gave a tumor incidence of 4/6 in 14 weeks. AF1 and AF2, when injected at a dose of 1x106 resulted in 100% tumor incidence in 5-weeks (n = 5). These two lines were also more resistant to IFNα-2b compared to RFP. All SKOV3-derived lines and ST expressed E-cadherin by RT-PCR. None of the SKOV3-derived lines expressed CCN1; however, one of the two AF1 ST tested expressed CCN1. Both AF2 ST evaluated showed weak expression of CCN1. In vitro DNA-binding with electrophoretic mobility shift assay (EMSA), showed AF1 and AF3 cells harbor constitutively-active STAT3, whereas the RFP line does not. Conclusions: The SKOV3-derived lines that we developed will be a better model to test novel OC treatment regiments because these lines exhibit increased resistance to IFNα-2b, are more tumorigenic in a xenograph model, show aberrant STAT3 activation, and tumors harvested from these lines express genes that make these lines more aggressive. No significant financial relationships to disclose.

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

scholarly journals Mechanism of MicroRNA-375 Promoter Methylation in Promoting Ovarian Cancer Cell Malignancy

2021 ◽  
Vol 20 ◽  
pp. 153303382098011
Author(s):  
Junjun Shu ◽  
Ling Xiao ◽  
Sanhua Yan ◽  
Boqun Fan ◽  
Xia Zou ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Cell Invasion ◽  
Promoter Methylation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Cell Malignancy

Objective: Ovarian cancer (OC) ranks one of the most prevalent fatal tumors of female genital organs. Aberrant promoter methylation triggers changes of microRNA (miR)-375 in OC. Our study aimed to evaluate the mechanism of methylated miR-375 promoter region in OC cell malignancy and to seek the possible treatment for OC. Methods: miR-375 promoter methylation level in OC tissues and cells was detected. miR-375 expression in OC tissues and cell lines was compared with that in demethylated cells. Role of miR-375 in OC progression was measured. Dual-luciferase reporter gene assay was utilized to verify the targeting relationship between miR-375 and Yes-associated protein 1 (YAP1). Then, Wnt/β-catenin pathway-related protein expression was tested. Moreover, xenograft transplantation was applied to confirm the in vitro experiments. Results: Highly methylated miR-375 was seen in OC tissues and cell lines, while its expression was decreased as the promoter methylation increased. Demethylation in OC cells brought miR-375 back to normal level, with obviously declined cell invasion, migration and viability and improved apoptosis. Additionally, miR-375 targeted YAP1 to regulate the Wnt/β-catenin pathway protein expression. Overexpressed YAP1 reversed the protein expression, promoted cell invasion, migration and viability while reduced cell apoptosis. Overexpressed miR-375 in vivo inhibited OC progression. Conclusion: Our study demonstrated that demethylated miR-375 inhibited OC growth by targeting YAP1 and downregulating the Wnt/β-catenin pathway. This investigation may offer novel insight for OC treatment.

Pre-clinical models of cellular therapy combined with cytokines demonstrate in vitro and in vivo ovarian cancer cell killing

2012 ◽  
Vol 125 ◽  
pp. S45
Author(s):  
S. Ingersoll ◽  
S. Ahmad ◽  
G. Stoltzfus ◽  
M. Merchant ◽  
A. Ahmed ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cell ◽  
Cellular Therapy ◽  
Ovarian Cancer Cell ◽  
Cell Killing ◽  
Clinical Models

scholarly journals Cytocidal Antitumor Effects against Human Ovarian Cancer Cells Induced by B-Lactam Steroid Alkylators with Targeted Activity against Poly (ADP-Ribose) Polymerase (PARP) Enzymes in a Cell-Free Assay

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 1028
Author(s):  
Nikolaos Nikoleousakos ◽  
Panagiotis Dalezis ◽  
Aikaterini Polonifi ◽  
Elena G. Geromichalou ◽  
Sofia Sagredou ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Mrna Expression ◽  
Cancer Cells ◽  
Cell Lines ◽  
Synthetic Lethality ◽  
Parp Inhibitor ◽  
Positive Control ◽  
Ovarian Cancer Cells ◽  
Antitumor Effects

We evaluated three newly synthesized B-lactam hybrid homo-aza-steroidal alkylators (ASA-A, ASA-B and ASA-C) for their PARP1/2 inhibition activity and their DNA damaging effect against human ovarian carcinoma cells. These agents are conjugated with an alkylating component (POPA), which also served as a reference molecule (positive control), and were tested against four human ovarian cell lines in vitro (UWB1.289 + BRCA1, UWB1.289, SKOV-3 and OVCAR-3). The studied compounds were thereafter compared to 3-AB, a known PARP inhibitor, as well as to Olaparib, a standard third-generation PARP inhibitor, on a PARP assay investigating their inhibitory potential. Finally, a PARP1 and PARP2 mRNA expression analysis by qRT-PCR was produced in order to measure the absolute and the relative gene expression (in mRNA transcripts) between treated and untreated cells. All the investigated hybrid steroid alkylators and POPA decreased in vitro cell growth differentially, according to the sensitivity and different gene characteristics of each cell line, while ASA-A and ASA-B presented the most significant anticancer activity. Both these compounds induced PARP1/2 enzyme inhibition, DNA damage (alkylation) and upregulation of PARP mRNA expression, for all tested cell lines. However, ASA-C underperformed on average in the above tasks, while the compound ASA-B induced synthetic lethality effects on the ovarian cancer cells. Nevertheless, the overall outcome, leading to a drug-like potential, provides strong evidence toward further evaluation.

scholarly journals Long non-coding RNA PTPRG-AS1 promotes cell tumorigenicity in epithelial ovarian cancer by decoying microRNA-545-3p and consequently enhancing HDAC4 expression

2020 ◽  
Author(s):  
Juanjuan Shi ◽  
Xijian Xu ◽  
Dan Zhang ◽  
Jiuyan Zhang ◽  
Hui Yang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Epithelial Ovarian Cancer ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Abstract Background: Long non-coding RNA PTPRG antisense RNA 1 (PTPRG-AS1) deregulation has been reported in various human malignancies and identified as an important modulator of cancer development. Few reports have focused on the detailed role of PTPRG-AS1 in epithelial ovarian cancer (EOC) and its underlying mechanism. This study aimed to determine the physiological function of PTPRG-AS1 in EOC. A series of experiments were also performed to identify the mechanisms through which PTPRG-AS1 exerts its function in EOC.Methods: Reverse transcription-quantitative polymerase chain reaction was used to determine PTPRG-AS1 expression in EOC tissues and cell lines. PTPRG-AS1 was silenced in EOC cells and studied with respect to cell proliferation, apoptosis, migration, and invasion in vitro and tumor growth in vivo. The putative miRNAs that target PTPRG-AS1 were predicted using bioinformatics analysis and further confirmed in luciferase reporter and RNA immunoprecipitation assays.Results: Our data verified the upregulation of PTPRG-AS1 in EOC tissues and cell lines. High PTPRG-AS1 expression was associated with shorter overall survival in patients with EOC. Functionally, EOC cell proliferation, migration, invasion in vitro, and tumor growth in vivo were suppressed by PTPRG-AS1 silencing. In contrast, cell apoptosis was promoted by loss of PTPRG-AS1. Regarding the mechanism, PTPRG-AS1 could serve as a competing endogenous RNA in EOC cells by decoying microRNA-545-3p (miR-545-3p), thereby elevating histone deacetylase 4 (HDAC4) expression. Furthermore, rescue experiments revealed that PTPRG-AS1 knockdown-mediated effects on EOC cells were, in part, counteracted by the inhibition of miR-545-3p or restoration of HDAC4.Conclusions: PTPRG-AS1 functioned as an oncogenic lncRNA that aggravated the malignancy of EOC through the miR-545-3p/HDAC4 ceRNA network. Thus, targeting the PTPRG-AS1/miR-545-3p/HDAC4 pathway may be a novel strategy for EOC anticancer therapy.

scholarly journals LY2157299 Monohydrate, a TGF-βR1 Inhibitor, Suppresses Tumor Growth and Ascites Development in Ovarian Cancer

Cancers ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 260 ◽  
Author(s):  
Qing Zhang ◽  
Xiaonan Hou ◽  
Bradley Evans ◽  
Jamison VanBlaricom ◽  
Saravut Weroha ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Growth ◽  
Cell Lines ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Migration And Invasion ◽  
Ascites Formation ◽  
Tgf Β1

Transforming growth factor beta (TGF-β) signaling has pleiotropic functions regulating cancer initiation, development, and metastasis, and also plays important roles in the interaction between stromal and cancer cells, making the pathway a potential therapeutic target. LY2157299 monohydrate (LY), an inhibitor of TGF-β receptor I (TGFBRI), was examined for its ability to inhibit ovarian cancer (OC) growth both in high-grade serous ovarian cancer (HGSOC) cell lines and xenograft models. Immunohistochemistry, qRT-PCR, and Western blot were performed to study the effect of LY treatment on expression of cancer- and fibroblast-derived genes. Results showed that exposure to TGF-β1 induced phosphorylation of SMAD2 and SMAD3 in all tested OC cell lines, but this induction was suppressed by pretreatment with LY. LY alone inhibited the proliferation, migration, and invasion of HGSOC cells in vitro. TGF-β1-induced fibroblast activation was blocked by LY. LY also delayed tumor growth and suppressed ascites formation in vivo. In addition, independent of tumor inhibition, LY reduces ascites formation in vivo. Using OVCAR8 xenograft specimens we confirmed the inhibitory effect of LY on TGF-β signaling and tumor stromal expression of collagen type XI chain 1 (COL11A1) and versican (VCAN). These observations suggest a role for anti-TGF-β signaling-directed therapy in ovarian cancer.

scholarly journals Transcriptional activation of the H-ferritin gene in differentiated Caco-2 cells parallels a change in the activity of the nuclear factor Bbf

1995 ◽  
Vol 311 (3) ◽  
pp. 769-773 ◽  
Author(s):  
M A Bevilacqua ◽  
M C Faniello ◽  
P D′Agostino ◽  
B Quaresima ◽  
M T Tiano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Mobility Shift ◽  
Differentiated Cells ◽  
Binding Factor ◽  
H Gene ◽  
Ferritin Gene ◽  
Mobility Shift Assay ◽  
Promoter Interaction

In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.

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