Down-regulation of Egr-1 by siRNA inhibits growth of human prostate carcinoma cell line PC-3.
ABSTRACT The inhibitory effect of a specific small EGR-1 interfering RNA (siRNA) on cell proliferation and the expression of EGR-1 in human prostate carcinoma cell lines PC-3 and LNCaP was investigated. To knockdown Egr-1 expression, a siRNA targeting against Egr-1 was synthesized and transfected into PC-3 and LNCaP cells. The down-regulation of Egr-1 expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The transcription activity was determined by luciferase expression. Cell proliferation inhibition rates were determined by soft agar and methyl thiazolyl tetrazolium (MTT) assay. The effect of Egr-1 siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry (FCM). RNA interference efficiently suppressed the Egr-1 expression in PC-3 and LNCaP cells. At 96 h after transfection, the expression inhibition rate was 44.52% at mRNA level detected by RT-PCR and 40.17% at protein level by Western blot analysis. The cell proliferation inhibition rates at 24, 48, 96 and 120 h after Egr-1 siRNA and non-silencing siRNA transfection, were 5, 25.06, 65.61 and 78.36%, respectively for PC-3 cells and 23, 40.3, 75.9 and 67.4%, respectively for LNCaP cells. The apoptosis rate was similar for both PC-3 and LNCaP and the number of cells was increased in G0/G1 phase from 38.2 to 88.6%, and decreased in S and G2/M phase at 96 h after transfection. Down-regulation of Egr-1 results in significant inhibition of tumor growth in vitro. The inhibition of Egr-1 expression can induce apoptosis of PC-3 cells. The use of Egr-1 siRNA deserves further investigation as a novel approach to cancer therapy.
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ABSTRACT: Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme that couples with cyclooxygenase-2 (COX-2) for the production of PGE(2). Although COX-2 is known to mediate the growth and progression of several human cancers including hepatocellular carcinoma (HCC), the role of mPGES-1 in hepatocarcinogenesis is not well established. This study provides novel evidence for a key role of mPGES-1 in HCC growth and progression. Forced overexpression of mPGES-1 in two HCC cell lines (Hep3B and Huh7) increased tumor cell growth, clonogenic formation, migration and invasion, whereas knockdown of mPGES-1 inhibited these parameters, in vitro. In a mouse tumor xenograft model, mPGES-1-overexpressed cells formed palpable tumors at earlier time points and developed larger tumors when compared with the control (P<0.01); in contrast, mPGES-1 knockdown delayed tumor development and reduced tumor size (P<0.01). Mechanistically, mPGES-1-induced HCC cell proliferation, invasion and migration involve PGE(2) production and activation of early growth response 1 (EGR1) and β-catenin. Specifically, mPGES-1-derived PGE(2) induces the formation of EGR1-β-catenin complex, which interacts with T-cell factor 4/lymphoid enhancer factor 1 transcription factors and activates the expression of β-catenin downstream genes. Our findings depict a novel crosstalk between mPGES-1/PGE(2) and EGR1/β-catenin signaling that is critical for hepatocarcinogenesis.Oncogene 07/2011; 31(7):842-57. · 7.36 Impact Factor
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ABSTRACT: Combined treatment of anticancer drugs and small interfering RNAs (siRNAs) have emerged as a new modality of anticancer therapy. Here, we describe a co-delivery system of anticancer drugs and siRNA in which anticancer drug-derived lipids form cationic nanoparticles for siRNA complexation. The anticancer drug mitoxantrone (MTO) was conjugated to palmitoleic acid, generating two types of palmitoleyl MTO (Pal-MTO) lipids: monopalmitoleyl MTO (mono-Pal-MTO) and dipalmitoleyl MTO (di-Pal-MTO). Among various lipid compositions of MTO, nanoparticles containing mono-Pal-MTO and di-Pal-MTO at a molar ratio of 1:1 (md11-Pal-MTO nanoparticles) showed the most efficient cellular delivery of siRNA, higher than that of Lipofectamine 2000. Delivery of red fluorescence protein-specific siRNA into B16F10-RFP cells using md11-Pal-MTO nanoparticles reduced the expression of RFP at both mRNA and protein levels, demonstrating silencing of the siRNA target gene. Moreover, delivery of Mcl-1-specific anticancer siRNA (siMcl-1) using md11-Pal-MTO enhanced antitumor activity in vitro, reducing tumor cell viability by 81% compared to a reduction of 68% following Lipofectamine 2000-mediated transfection of siMcl-1. Intratumoral administration of siMcl-1 using md11-Pal-MTO nanoparticles significantly inhibited tumor growth, reducing tumor size by 83% compared to untreated controls. Our results suggest the potential of md11-Pal-MTO multifunctional nanoparticles for co-delivery of anticancer siRNAs for effective combination therapy.Biomaterials 09/2011; 32(36):9785-95. · 7.60 Impact Factor
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ABSTRACT: Vascular endothelial growth factor (VEGF) regulates several cell functions including; proliferation, differentiation, permeability, vascular tone, and the production of vasoactive molecules. The purpose of this study was to evaluate the potency of specific short-interfering RNA (siRNA) to suppress human VEGF expression by siRNA and investigate the effects of VEGF down-regulation on the cell proliferation and apoptosis of the human prostate cancer cell lines DU-145. Transfection was performed using X-tremeGENE siRNA transfection reagent. At different time intervals, transfected cells were harvested and total RNA was extracted for RT-PCR. The VEGF content in supernatants were measured by ELISA. Inhibition of cell growth by hVEGF-siRNA was measured by using cell proliferation ELISA BrdU assay. Apoptotic cells were evaluated by using annexin-V-FITC apoptotic detection method. Transfection of hVEGF-siRNA resulted in statistically significant inhibition of hVEGF-mRNA that in turn caused a marked reduction in the expression of hVEGF. The cell growth was assessed every 24 h for 4 days after siRNA treatment resulted in a marked inhibition of cell proliferation as compared to scramble siRNA. The results of apoptosis showed that approximately 15 % of the cells treated with control-siRNA manifested evident apoptotic changes after 24 hpt, whereas DU-145 cells treated with hVEGF-siRNA significantly were positive, that is to say, 53 % at 72 hpt 23.9 ± 2.78 % (P < 0.001) and 13 ± 1.57 % at 96 hpt. Our findings indicate that siRNA are effective in eliciting the RNAi pathway in cancerous cells and that specific siRNA efficiently down-regulate VEGF expression. They could decrease VEGF production and induce apoptosis, which may also be linked to the inhibition of cancerous cell proliferation. Therefore, it can be concluded that siRNA-mediated suppression of VEGF represents a powerful tool against prostate cancer cell proliferation. VEGF down-regulation exerts a direct anti-apoptotic function in the DU-145 cell lines and promises the development of drugs for cancer therapy.Clinical and Translational Oncology 07/2012; 14(10):791-9. · 1.28 Impact Factor