15 research outputs found
Citrate Synthase Expression Affects Tumor Phenotype and Drug Resistance in Human Ovarian Carcinoma
<div><p>Citrate synthase (CS), one of the key enzymes in the tricarboxylic acid (TCA) cycle, catalyzes the reaction between oxaloacetic acid and acetyl coenzyme A to generate citrate. Increased CS has been observed in pancreatic cancer. In this study, we found higher CS expression in malignant ovarian tumors and ovarian cancer cell lines compared to benign ovarian tumors and normal human ovarian surface epithelium, respectively. <i>CS</i> knockdown by RNAi could result in the reduction of cell proliferation, and inhibition of invasion and migration of ovarian cancer cells in vitro. The drug resistance was also inhibited possibly through an excision repair cross complementing 1 (ERCC1)-dependent mechanism. Finally, upon <i>CS</i> knockdown we observed significant increase expression of multiple genes, including <i>ISG15</i>, <i>IRF7</i>, <i>CASP7</i>, and <i>DDX58</i> in SKOV3 and A2780 cells by microarray analysis and real-time PCR. Taken together, these results suggested that CS might represent a potential therapeutic target for ovarian carcinoma.</p></div
<i>CS</i> silencing increases drug sensitivity in ovarian cancer cells.
<p>(<b>A</b>) Protein level of CS was examined in <i>CS</i>-silenced SKOV3 and A2780 cells. (<b>B</b>) SKOV3 and A2780 cells were treated with <i>CS</i> siRNA or negative siRNA for 48 h, then cells were treated with different concentrations of DDP for another 24 h. Cell viability was measured after incubation with CCK8 for 1.5 h. (<b>C</b>) 48 h after <i>CS</i> siRNA transfection, SKOV3 and A2780 cells were treated with 1 µg/ml DDP for 12 h and 1 h, respectively. Then cells were plated in 6-well plates, colonies were stained and counted after incubation for 8 days. Results shown were representative of three independent experiments. (<b>D</b>) SKOV3 and A2780 cells were treated with <i>CS</i> siRNA for 48 h, ERCC1 and γ-H2AX protein levels were compared between NC and siCS1078 group with β-tubulin used as a loading control. Mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p
Up regulated expression of citrate synthase (CS) in human ovarian tumors and human ovarian tumor cell lines.
<p>(<b>A</b>) <i>CS</i> mRNA and (<b>B</b>) protein expression was assessed in normal human ovarian surface epithelium (HOSE), ovarian cancer cell lines, benign (n = 11) and malignant ovarian tumors (n = 21) using real-time PCR and western blot, respectively (B =  ovarian benign tumor, M =  ovarian malignant tumor). Mean ± SEM. **<i>P</i><0.01 and ***<i>P</i><0.001.</p
<i>CS</i> silencing affects AMPK/P38 MAPK pathway in ovarian cancer cell lines.
<p>(<b>A</b>) mRNA and <b>(B)</b> protein expression level of CS by real-time PCR and western blot in SKOV3 and A2780 cells after <i>CS</i> siRNA (100 nM) for 24 h and 48 h after transfection, respectively. (<b>C</b>) Decreased CS activity after 48 h transfection in SKOV3 and A2780 cells. (<b>D</b>) ATP level was examined 48 h after <i>CS</i> silencing. (<b>E, F</b>) p-AMPKα and p-p38 were analyzed in <i>CS</i>-silenced cancer cells by western blot. Mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p
<i>CS</i> silencing inhibits SKOV3 and A2780 cell invasion and migration in <i>vitro</i>.
<p>48 h after transfection, cell invasiveness was evaluated using the transwell assay and the Boyden Chamber test was used to measure the extent of cell migration. The number of invaded and migrated cells was counted using a bright-field microscope (200×). Representative images and the relative cell numbers were shown in (<b>A</b>) and (<b>B</b>). (<b>C</b>) p-FAK, MMP2 and Vimentin in <i>CS</i> knockdown cancer cells were analyzed using western blot. Mean ± SEM. *<i>P</i><0.05.</p
Gene expression profile related to drug resistance and apoptosis in SKOV3 and A2780 cells after <i>CS</i> silencing.
<p>(<b>A, B</b>) Ovarian cancer cells were treated with siRNA for 48 h, total RNA was extracted for gene expression analysis. Expression of <i>CASP7 (CASPASE7), IRF7 (</i>interferon regulatory factor 7), <i>DDX58 (</i>DEAD (Asp-Glu-Ala-Asp) box polypetide 58<i>)</i>, and <i>ISG15 (IFN</i>-stimulated gene 15<i>)</i> was increased significantly whereas <i>ATG12 (</i>autophagy related 12<i>)</i> expression was decreased in SKOV3 and A2780 cells after <i>CS</i> knockdown. <i>β-actin</i> was used as an internal control gene. Mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001. (<b>C</b>) The diagram was shown to identify potential signaling pathways modulated by CS.</p
<i>CS</i> silencing results in proliferation reduction in SKOV3 and A2780 cells.
<p>(<b>A</b>) Cell proliferation was measured in <i>CS</i>-silenced cancer cells and control groups. (<b>B</b>) p-ERK and <b>(C)</b> Bcl-2 expression were measured after <i>CS</i> siRNA for 48 h in SKOV3 and A2780 cells by western blot. β-tubulin was used as a loading control. (<b>D</b>) 48 h after <i>CS</i> siRNA, cells were treated with indicated concentrations of DDP (15 µg/mL or 10 µg/mL) for 24 h. Cleaved caspase 3 was measured by western blot. Mean ± SEM. *<i>P</i><0.05 and **<i>P</i><0.01.</p
FOXM1 Modulates Cisplatin Sensitivity by Regulating EXO1 in Ovarian Cancer
<div><p>Cisplatin is commonly used in ovarian cancer chemotherapy, however, chemoresistance to cisplatin remains a great clinical challenge. Oncogenic transcriptional factor FOXM1 has been reported to be overexpressed in ovarian cancer. In this study, we aimed to investigate the potential role of FOXM1 in ovarian cancers with chemoresistance to cisplatin. Our results indicate that FOXM1 is upregulated in chemoresistant ovarian cancer samples, and defends ovarian cancer cells against cytotoxicity of cisplatin. FOXM1 facilitates DNA repair through regulating direct transcriptional target EXO1 to protect ovarian cancer cells from cisplatin-mediated apoptosis. Attenuating FOXM1 and EXO1 expression by small interfering RNA, augments the chemotherapy efficacy against ovarian cancer. Our findings indicate that targeting FOXM1 and its target gene EXO1 could improve cisplatin effect in ovarian cancer, confirming their role in modulating cisplatin sensitivity.</p></div
DNA repair regulation of FOXM1 is partially mediated by EXO1.
<p>A2780 and SKOV3 cells were transfected with EXO1 specific siRNA or NC siRNA. (A)The expression of EXO1 was determined by real-time PCR analysis 48 h after transfection. (B) Western blotting analysis was done to determine the expression level of EXO1. (C) 48 h after transfection, A2780 and SKOV3 cells were treated with increasing concentrations of cisplatin for another 48 h and their rates of viability were measured by CCK8 and compared to cells without cisplatin treatment. (D) 48 h after siRNA transfection, A2780 and SKVO3 cells were treated for 1 h with 1 µg/ml and 2 µg/ml cisplatin, respectively. Then cells were plated in 6-well plates and incubated for 8–10 d. Then colonies were stained and counted. Results shown are representative of three independent experiments. Graphs provide quantification as a percentage of the nontreated wells. Each column and bar represents mean±s.d. of triplicate determinations. (E) 48 h after transfection, A2780 and SKOV3 cells were treated for 48 h with 1 µg/ml and 2 µg/ml cisplatin, respectively. After treatment, apoptosis was determined by flow cytometric analysis of Annexin V and PI staining. The right panel shows means±s.d. of three independent experiments. (F) 48 h after transfection, A2780 and SKOV3 cells were treated for 24 h with 2 µg/ml and 4 µg/ml cisplatin, respectively. After treatment, cell lysates were prepared, resolved by SDS-PAGE and subjected to immunoblotting analysis of FOXM1, EXO1, γH2AX, cleaved caspase-3 and β-actin. (G) A2780 and SKOV3 cells with or without silencing of EXO1 expression, were treated with 1 µg/ml and 2 µg/ml cisplatin for 1 h, respectively. γH2AX foci of A2780 and SKOV3 cells were quantified at different time point: 24, 48 and 72 h after cisplatin treatment. The percentage of γH2AX positive cells were plotted. Untreated cells were used as control.</p
Expression of FOXM1 target genes in response to cisplatin in A2780 and SKVO3 cells.
<p>A2780 and SKOV3 cells were treated with the indicated concentration of cisplatin for 24-time PCR analysis (A) and western blotting (B). Results are shown from three independent experiments in triplicates. Columns, mean; bars, SD. (C) A2780 and SKOV3 cells were treated with 2 µg/ml and 4 µg/ml cisplatin for 12 h respectively, then were cultured in fresh complete medium for indicated time points (the time when complete medium was added was set as 0 h). Western blot was performed to determine the expression of EXO1 and β-actin. (D) A2780 and SKO3 cells were transfected with FOXM1 siRNA, 48 h later, EXO1 expression was determined by real-time PCR analysis. Results are shown from three independent experiments in triplicates. Columns, mean; bars, SD. (E) 24 h after FOXM1 siRNA transfection, A2780 and SKOV3 cells were treated with 2 µg/ml and 4 µg/ml cisplatin for 12 h respectively, then were cultured in fresh complete medium for indicated time points (the time when complete medium was added was set as 0 h). The arrow (↓) indicates transfection and subsequent cisplatin treatment. Western blot was performed to determine the expression of FOXM1, EXO1 and β-actin.</p