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

Fabrication of Hyperbranched Block-Statistical Copolymer-Based Prodrug with Dual Sensitivities for Controlled Release

Dendrimer with hyperbranched structure and multivalent surface is regarded as one of the most promising candidates close to the ideal drug delivery systems, but the clinical translation and scale-up production of dendrimer has been hampered significantly by the synthetic difficulties. Therefore, there is considerable scope for the development of novel hyperbranched polymer that can not only address the drawbacks of dendrimer but maintain its advantages. The reversible addition–fragmentation chain transfer self-condensing vinyl polymerization (RAFT-SCVP) technique has enabled facile preparation of segmented hyperbranched polymer (SHP) by using chain transfer monomer (CTM)-based double-head agent during the past decade. Meanwhile, the design and development of block-statistical copolymers has been proven in our recent studies to be a simple yet effective way to address the extracellular stability vs intracellular high delivery efficacy dilemma. To integrate the advantages of both hyperbranched and block-statistical structures, we herein reported the fabrication of hyperbranched block-statistical copolymer-based prodrug with pH and reduction dual sensitivities using RAFT-SCVP and post-polymerization click coupling. The external homo oligo­(ethylene glycol methyl ether methacrylate) (OEGMA) block provides sufficient extracellularly colloidal stability for the nanocarriers by steric hindrance, and the interior OEGMA units incorporated by the statistical copolymerization promote intracellular drug release by facilitating the permeation of GSH and H⁺ for the cleavage of the reduction-responsive disulfide bond and pH-liable carbonate link as well as weakening the hydrophobic encapsulation of drug molecules. The delivery efficacy of the target hyperbranched block-statistical copolymer-based prodrug was evaluated in terms of <i>in vitro</i> drug release and cytotoxicity studies, which confirms both acidic pH and reduction-triggered drug release for inhibiting proliferation of HeLa cells. Interestingly, the simultaneous application of both acidic pH and GSH triggers promoted significantly the cleavage and release of CPT compared to the exertion of single trigger. This study thus developed a facile approach toward hyperbranched polymer-based prodrugs with high therapeutic efficacy for anticancer drug delivery

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

(A) The H/ACA snoRNA locus CeN55 is located in an intron of the protein coding gene D1046

1. Tiling array probe signal intensities indicate that the UM2 sequence upstream of snoRNA CeN55 might also be expressed (red box). (B) Model of RACE amplification of the putative UM2-snoRNA dicistronic fragments. 3AD is a 3'end adaptor ligated to the ncRNAs; 3RT is the primer complementary to 3AD, primer_UM2 located in the region of UM2. The RACE analysis was performed by reverse transcription using the 3RT as primer followed by PCR with primers primer_UM2 and 3RT. (C) Gel analysis of the RACE fragments.<p><b>Copyright information:</b></p><p>Taken from "analysis of noncoding RNA promoter motifs"</p><p>http://www.biomedcentral.com/1471-2199/9/71</p><p>BMC Molecular Biology 2008;9():71-71.</p><p>Published online 5 Aug 2008</p><p>PMCID:PMC2527325.</p><p></p

Expression of the reporter gene was detected by RT-PCR using DNase I digested RNA extracted from transgenic worms as template

For the negative controls (RNA-PCR) RT-PCR was performed using the same template without adding reverse transcriptase.<p><b>Copyright information:</b></p><p>Taken from "analysis of noncoding RNA promoter motifs"</p><p>http://www.biomedcentral.com/1471-2199/9/71</p><p>BMC Molecular Biology 2008;9():71-71.</p><p>Published online 5 Aug 2008</p><p>PMCID:PMC2527325.</p><p></p

<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

<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

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 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

<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