Abstract
IL-8 produced by prostate cancer cells may be responsible for the androgen-independent growth of advanced prostate cancers. Accumulating evidence from microarray analyses and animal genetic models highlights the central involvement of the transcription factor early growth response-1 (EGR-1) in prostate carcinoma progression. It is unknown, however, whether knockdown of EGR-1 inhibits IL-8 production and IL-8-mediated tumor metastasis. Here we show that EGR-1 knockdown by a specific shRNA-Egr1 inhibited gene transcription and production of IL-8 by the human prostate cancer cell line DU145. Conversely, enforced expression of EGR-1 in EGR-1-lacking PC3 prostate cancer cells markedly enhanced IL-8 transcription and secretion. By using wild type and a series of mutant IL-8 promoter luciferase constructs, we found that the NF-kappaB binding site is important for EGR-1 regulation of IL-8. Furthermore, silencing EGR-1 suppressed a synergistically functional interaction between EGR-1 and NF-kappaB. Consequently, knockdown of EGR-1 inhibited IL-8-mediated tumor colony formation and invasion. Thus, targeted knockdown of EGR-1 could be an effective therapeutic approach against prostate cancer.
Highlights
Markedly different distribution patterns for interleukin 8 (IL-8) and its receptors
Knockdown of early growth response-1 (EGR-1) Decreases Transcription and Secretion of IL-8 by Prostate Cancer Cells—To determine whether EGR-1 regulates transcription and secretion of IL-8 by prostate cancer cells, DU145 cells were separated on a 2% agarose gel and visualized by ethidium were transfected with shRNA-EGR-1 plasmids or control plasbromide staining
The GAPDH promoter fragment acted as the mids to generate the DU145/sh-EGR-1 and DU145/sh-Con stainternal negative control for this assay
Summary
QCMTM 24-Well Cell Invasion Assay kit was purchased from Chemicon Corp. ShRNA Expression, siRNA Duplex, and Reporter Gene Constructs—The empty pU6 ϩ 27, shRNA-control and shRNA-p65 vectors were purchased from Panomics Corp. Equivalent amount of total protein was incubated with 2 g of specific antibody or control IgG at 4 °C overnight. The immune complexes were captured by incubation with protein A-agarose at 4 °C for 2 h, and the immunoprecipitated proteins were subjected to Western blotting with specific antibodies. DNA from these samples was subjected to PCR analyses with NF-B-BSpf: 5Ј-CAT CAG TTG CAA ATC GTG GA-3Ј and NF-B-BSpr: 5Ј-GAA GCT TGT GTG CTC TGC TG-3Ј; GAPDH-pf: 5Ј-GTA TTC CCC CAG GTT TAC AT-3Ј and GAPDH-pr: 5Ј-TTC TGT CTT CCA CTC ACT CCT-3Ј.
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