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 downregulation 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 G 0 /G 1 phase from 38.2 to 88.6%, and decreased in S and G 2 /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.

Abstract. To address elements that might uniquely characterize EGR-1 mediated signaling, the expression of two transcription factors, namely, nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) were studied. PC-3 and LNCaP prostate carcinoma cell lines were transiently transfected with wildtype Egr-1 expression plasmid (pCMV-Egr-1) and treated with cisplatin and TPA. Overexpression of EGR-1 was found to induce nuclear expression of both, NF-κB and AP1. However, the intensity of the induced AP-1 and NF-κB was diminished after cisplatin treatment, but not after TPA. Our findings confirm that the overexpression of wild-type Egr-1 caused a marked increase in cell proliferation in PC-3 and LNCaP proliferation in a 14-day soft agar colony forming assay. In addition, luciferase reporter gene assay showed that the transcriptional activity of AP-1 and NF-κB in PC-3 and LNCaP prostate carcinoma cell lines was also modulated by the overexpression of EGR-1 in these cells using tandem repeated Luc-AP-1 and Luc-NF-κB. The activation of both NF-κB and AP-1 are key steps in the cascade of events following the activation of the EGR-1 gene. It was revealed that overexpression of EGR-1 selectively increased AP-1 and NF-κB activation, and that the activation of these nuclear factors appears to be essential for the induction of proliferation and anchorage independence in activated PC-3 and LNCaP cells. However, the mechanism underlying the modulation of AP-1 and NF-κB by the overexpression of EGR-1 is still unknown.