Intracellular inhibitory effects of Velcade correlate with morphoproteomic expression of phosphorylated-nuclear factor-kappaB and p53 in breast cancer cell lines.
Velcade, a proteasome inhibitor, has been shown to inhibit DNA binding activity of nuclear factor-kappaB (NF-kappaB) and to stabilize p53 in vitro. But its impact, in the context of activated (phosphorylated and translocated) NF-kappaB and the expression of p53, has not been studied in breast cancer. It would be desirable to determine whether or not the immunohistochemical (IHC) expressions of activated NF-kappaB and of p53 can predict the effects of Velcade in viable tumor cells. To answer these questions, we selected 3 breast cancer cell lines (SKBR-3, MDA-175, and MDA-231), which are negative for hormonal receptors, but differ in HER-2/neu expression (strong, mild, and minimal, respectively). The 3 cell lines showed different expressions of phosphorylated (p)- NF-kappaB and p53, as evaluated using immunohistochemistry with visual quantification by brightfield microscopy. After being treated with Velcade for 2 days, MDA-231 cells showed markedly reduced proliferation, followed by SKBR-3 cells, and then by MDA-175 cells. There was strong correlation between the nuclear expression of either p-NF-kappaB or p53 and the inhibitory rate of Velcade in the 3 cell lines (r = 0.987 and 0.807, respectively). Western blotting showed an increase in inhibitor-kappaB (I-kappaB) expression in nuclei of MDA-231 and SKBR-3 cells, but not in MDA-175 cells, following exposure to Velcade. Velcade treatment resulted in cleaved caspase-3 expression in MDA-231 cells and in the overexpression of p53 and p21WAF1 in all 3 cell lines, as evaluated using Western blotting. In summary, morphoproteomic analysis of p-NF-kappaB and p53 can be correlated with the inhibitory effect of Velcade in vitro. We propose that this proliferative inhibition is variably associated with blocking p-NF-kappaB function by upregulation of nuclear I-kappaB, stabilization of p53, and induction of p21WAF1.
Lun
Mingyue
M
Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania 17822, USA.
Zhang
Ping L
PL
Siegelmann-Danieli
Nava
N
Blasick
Thomas M
TM
Brown
Robert E
RE
D000970:Antineoplastic Agents / Q000494:pharmacology; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D001943:Breast Neoplasms; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D020543:Proteome; D011719:Pyrazines / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D000970:Antineoplastic Agents; D001897:Boronic Acids; D016328:NF-kappa B; D011480:Protease Inhibitors; D020543:Proteome; D011719:Pyrazines; D016159:Tumor Suppressor Protein p53; D000069286:Bortezomib