Involvement of FLIP in 2-methoxyestradiol-induced tumor regression in transgenic adenocarcinoma of mouse prostate model.
OBJECTIVE
The purpose of this study is to investigate whether Fas-associated death domain interleukin-1 converting enzyme like inhibitory protein (FLIP) inhibition is a therapeutic target associated with 2-methoxyestradiol (2-ME2)-mediated tumor regression.


METHODS
Expression and levels of FLIP were analyzed using (a) real-time PCR and immunoblot analysis in androgen-independent PC-3 cells treated with the newly formulated 2-ME2 and (b) immunohistochemistry in different Gleason pattern human prostate tumors. Transient transfections and chromatin immunoprecipitation (ChIP) assays were used to identify the transcription factors that regulate FLIP. Involvement of FLIP in 2-ME2-induced tumor regression was evaluated in transgenic adenocarcinoma mouse prostate (TRAMP) mice.


RESULTS
High Gleason pattern (5+5) human prostate tumors exhibit significant increase in FLIP compared with low Gleason pattern 3+3 (P=or<0.04). 2-ME2 reduced the levels and promoter activity of FLIP (P=0.001) in PC-3 cells. Transient expression assays show sequences between -503/+242 being sufficient for 2-ME2-induced inhibition of FLIP promoter activity. Cotransfection experiments show that overexpression of Sp1 activated, whereas Sp3 inhibited, Sp1 transactivation of FLIP promoter activity (P=0.0001). 2-ME2 treatment reduced binding of Sp1 to the FLIP promoter as evidenced by ChIP. Further, levels of FLIP associated with Fas or FADD decreased, whereas cleavage of caspase-8, levels of Bid, and apoptosis increased in response to 2-ME2 treatment in PC-3 cells. Administration of 2-ME2 regressed established prostate tumors in TRAMP mice that were associated with reduced expression of FLIP and Sp1.


CONCLUSIONS
Targeting Sp1-mediated FLIP signaling pathway may provide a novel approach for prostate cancer management.
Ganapathy
Manonmani
M
Department of Urology, School of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Ghosh
Rita
R
Jianping
Xie
X
Zhang
Xiaoping
X
Bedolla
Roble
R
Schoolfield
John
J
Yeh
I-Tien
IT
Troyer
Dean A
DA
Olumi
Aria F
AF
Kumar
Addanki P
AP
D000077584:2-Methoxyestradiol; D000230:Adenocarcinoma / Q000188:drug therapy* / Q000473:pathology; D000818:Animals; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004195:Disease Models, Animal; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic / Q000235:genetics; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000378:metabolism; D014162:Transfection; D050257:Tubulin Modulators / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053200:Fas-Associated Death Domain Protein; D016329:Sp1 Transcription Factor; D050257:Tubulin Modulators; D051705:Sp3 Transcription Factor; D004958:Estradiol; D000077584:2-Methoxyestradiol