Regulation of TGF-beta1 gene transcription in human prostate cancer cells by nitric oxide.
BACKGROUND
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).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wang
Daren
D
Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA. dongzu@ucmail.uc.edu
Lu
Shan
S
Dong
Zhongyun
Z
D045744:Cell Line, Tumor; D016166:Free Radical Scavengers / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis* / Q000235:genetics
C477506:(+-)-E-4-methyl-2-((E)-hydroxylamino)-5-nitro-6-methoxy-3-hexanamide; D016166:Free Radical Scavengers; D006898:Hydroxylamines; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D009599:Nitroprusside; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D026423:S-Nitroso-N-Acetylpenicillamine; D052247:Nitric Oxide Synthase Type II