Regulation of TGF‐β1 gene transcription in human prostate cancer cells by nitric oxide

Abstract

Overexpression of transforming growth factor (TGF)-beta1 is associated with advanced prostate cancer. Our previous studies showed an inverse correlation between the expressions of TGF-beta1 and inducible nitric oxide synthase (iNOS) in prostatic tumors in mice. The purpose of this study was to investigate regulation of TGF-beta1 expression in human prostate cancer cells by nitric oxide (NO). Expression of TGF-beta1 in the three well-characterized lines of human prostate cancer cells (PC-3MM2, LNCaP, and DU145) was determined by using the enzyme-linked immunoabsorbance assay (ELISA), real-time reverse-transcriptase PCR (RT-PCR), nuclear run-on, and promoter activity analyses. Expression of both TGF-beta1 protein and mRNA was inhibited in both dose- and time-dependent manners by NO donors sodium nitroprusside (SNP), S-nitroso-N-acetylpenicilamine (SNAP), S-nitrosoglutathione (GSNO), and (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR-1) and by transfection of iNOS. The inhibitory effects of SNP and iNOS on TGF-beta1 expression were reduced in cells treated with NO scavengers N-dithiocarboxysarcosine (DTCS), 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), and hemoglobin, or with the iNOS inhibitor N-methyl-arginine (NMA). SNP downregulated the in vitro transcription of TGF-beta1 mRNA, inhibited TGF-beta1 promoter activity, but had no significant effects on TGF-beta1 mRNA stability. These results show that NO downregulates TGF-beta1 expression in prostate cancer cells at transcription level by suppressing the de novo synthesis of TGF-beta1 mRNA.