Characterizing the involvement of the nuclear factor-kappa B (NF kappa B) transcription factor in uveal melanoma.
Purpose. To examine the involvement of nuclear factor-kappa B (NFkappaB) pathways in uveal melanoma (UM) and to assess their potential as a therapeutic target for metastatic UM. Methods. Samples from primary (n = 7) and metastatic (n = 7) UM were evaluated for NFkappaB transcription factor family expression by quantitative PCR (QPCR), immunofluorescent staining, and Western blot analysis. The effect of two NFkappaB inhibitors, DHMEQ and BMS-345541, on two cell lines derived from UM liver metastases was assessed. Cell proliferation was examined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, methylene blue assay, and immunostaining for Ki-67. Apoptosis was assessed by immunostaining for activated caspase 3. Results. NFkappaB1, NFkappaB2, RelA, RelB, and NIK were expressed in primary UM and in its liver metastases. NFkappaB2, RelB, and NIK showed significantly higher mRNA levels in metastases from UM compared with primary tumors (3.4-fold, P = 0.03; 3.6-fold, P = 0.05; 3.5-fold, P = 0.03; respectively). NFkappaB2 protein activation was 3.9-fold higher in metastases (P = 0.03). NFkappaB inhibition reduced metastatic cell proliferation by 9.2-fold and 1.9-fold according to Ki67 staining (P = 0.04) and methylene blue assay (P = 6 x 10(-7)), respectively. Both NFkappaB inhibitors achieved dose-dependent reductions of UM cell proliferation in both cell lines (P < 0.001). NFkappaB inhibition resulted in a 6.3-fold increase of apoptosis (P = 7 x 10(-7)). Conclusions. These data indicate that the NFkappaB1 and NFkappaB2 pathways are active in both primary and metastatic UM and that these pathways regulate metastatic cell proliferation and apoptosis. The role of NFkappaB as a therapeutic target for UM should be further evaluated.
Dror
Rinat
R
Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Lederman
Michal
M
Umezawa
Kazuo
K
Barak
Vivian
V
Pe'er
Jacob
J
Chowers
Itay
I
D017209:Apoptosis; D001549:Benzamides / Q000494:pharmacology; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation; D003512:Cyclohexanones / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D019394:Ki-67 Antigen / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism* / Q000556:secondary; D008545:Melanoma / Q000378:metabolism* / Q000556:secondary; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000502:physiology*; D011810:Quinoxalines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014604:Uveal Neoplasms / Q000378:metabolism* / Q000473:pathology
C471109:4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; D001549:Benzamides; D003512:Cyclohexanones; D007093:Imidazoles; D019394:Ki-67 Antigen; D016328:NF-kappa B; D011810:Quinoxalines; D012333:RNA, Messenger; C464444:dehydroxymethylepoxyquinomicin; D053148:Caspase 3