While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-kappaB (NF-kappaB) in the suppression of apoptosis are known, their role in survival of prostate cancer cells is not well understood. We investigated the role of NF-kappaB and AP-1 in the survival of human androgen-independent (DU145) and -dependent (LNCaP) prostate cancer cell lines. Our results show that the faster rate of proliferation of DU145 cells when compared to LNCaP cells correlated with the constitutive expression of activated NF-kappaB and AP-1 in DU-145 cells. The lack of constitutive expression of NF-kappaB and AP-1 in LNCaP cells also correlated with their sensitivity to the antiproliferative effects of tumor necrosis factor (TNF). TNF induced NF-kappaB activation but not AP-1 activation in LNCaP cells. In DU145 cells both c-Fos and c-Jun were expressed and treatment with TNF activated c-Jun NH2-terminal kinase (JNK), needed for AP-1 activation. In LNCaP cells, however, only low levels of c-Jun was expressed and treatment with TNF minimally activated JNK. Treatment of cells with curcumin, a chemopreventive agent, suppressed both constitutive (DU145) and inducible (LNCaP) NF-kappaB activation, and potentiated TNF-induced apoptosis. Curcumin alone induced apoptosis in both cell types, which correlated with the downregulation of the expression of Bcl-2 and Bcl-xL and the activation of procaspase-3 and procaspase-8. Overall, our results suggest that NF-kappaB and AP-1 may play a role in the survival of prostate cancer cells, and curcumin abrogates their survival mechanisms.
The very process of deregulated oncogene expression during cancer development also sensitizes cancer cells to apoptotic signals (1-3). Deregulated oncoproteins such as E1a and c-Myc promote apoptosis by activating multiple downstream proapoptotic effector pathways (4, 5). Additional mechanisms of sensitizing cancer cells to apoptosis by an activated oncoprotein have been described (6, 7). For example, E2F sensitized cells to apoptosis through down-regulation of anti-apoptotic signals (7). Here we show that cancer cells can also be sensitized to apoptosis by up-regulating the expression levels of RKIP (Raf kinase inhibitor protein). RKIP was originally identified as an interacting partner of Raf-1 and a negative regulator of the mitogen-activated protein kinase cascade initiated by Raf-1 (8). RKIP also inhibits nuclear factor B (NF-B)1 signaling by negatively modulating the activating phosphorylation of IKK␣ and IKK via upstream kinases (9). Although the molecular mechanism by which RKIP inhibits the Raf and NF-B signaling pathways has been partially delineated, little is known about the biological relevance of the inhibition of these pathways by RKIP. In addition to these functions, we presently demonstrate the rapid up-regulation of RKIP during induction of chemotherapy-triggered apoptosis in human prostate and breast cancer cells. However, in DNA-damaging agent-resistant cancer cells, treatment with the drugs does not up-regulate RKIP expression. Ectopic expression of RKIP sensitizes DNA damage agentresistant cells to undergo apoptosis. Down-regulation of RKIP expression confers resistance to 9-nitrocamptothecin (9NC) by releasing its inhibitory constraint on two major survival pathways in cancer cells. Our studies suggest that RKIP represents a novel apoptotic marker in human cancer cells. MATERIALS AND METHODSCell Lines, Plasmid Constructs, and Chemicals-The human breast cell lines 578T and 578Bst were purchased from American Type Culture Collection (Manassas, VA). A human breast cancer MCF7 cell subline resistant to 9NC treatment was a gift from Dr. Ray Frackelton (Brown University). The human prostate cell lines LNCaP, DU145, and PC3 were purchased from American Type Culture Collection. Early (Ͻ30)-or late (Ͼ100)-passage DU145 cells were not used for this study. The 9NC-resistant DU145 cell subline, RC1, was established by continuous exposure of DU145 cells to 9NC (10). All cell lines were grown in conditions suggested by American Type Culture Collection. MCF7 and
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