Tumor necrosis factor-alpha-induced apoptosis in prostate cancer cells through inhibition of nuclear factor-kappaB by an IkappaBalpha "super-repressor".
Prostate cancer patients experiencing a relapse in disease often express high serum tumor necrosis factor-alpha (TNF-alpha) levels. Many androgen-insensitive prostate cancer cells are TNF-alpha insensitive because of the expression of antiapoptotic genes as part of the nuclear factor-kappaB (NF-kappaB) family of transcription factors. NF-kappaB stimulates gene transcription when expressed in the nucleus; however, in resting cells, this nuclear import is prevented by association with the cytoplasmic inhibitor IkappaBalpha. This cytoplasmic retention of NF-kappaB is uncoupled by many extracellular signals including low levels of TNF-alpha. During normal cell activation, nuclear translocation of NF-kappaB is preceded by phosphorylation and degradation of IkappaBalpha. When phosphorylation is blocked, IkappaBalpha remains intact, thereby blocking NF-kappaB translocation to the nucleus and subsequent activation of antiapoptotic genes that cause TNF-alpha insensitivity. We tested whether a "super-repressor" of NF-kappaB activity could be transfected into prostate cancer cells and make them TNF-alpha sensitive. PC-3 and LNCaP cells were stimulated with TNF-alpha (10 ng/ml) for 24 h in the presence or absence of the IkappaBalpha "super-repressor" (p6R-IkappaB(S32A + S36A)). NF-kappaB activity was measured by electrophoretic mobility shift assay and the steady state levels of the cytoplasmic IkappaBalpha protein were measured by Western blot. Secretory IL-6 and IL-6 mRNA were measured by ELISA. p6R-IkappaB(S32A + S36A) blocked the stimulation of NF-kappaB activity by TNF-alpha in prostate cancer cells. It also subsequently decreased IL-6 production by TNF-alpha. We conclude that these data demonstrate that inhibition of NF-kappaB selectively sensitizes previously insensitive prostate cancer cells to TNF-alpha.
Muenchen
H J
HJ
Department of Internal Medicine, University of Michigan, Ann Arbor 48109-0946, USA. muenchen@umich.edu
Lin
D L
DL
Walsh
M A
MA
Keller
E T
ET
Pienta
K J
KJ
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D001692:Biological Transport; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020672:I-kappa B Proteins; D015850:Interleukin-6 / Q000235:genetics; D008297:Male; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D015850:Interleukin-6; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha