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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Purpose: Purpose of this study has been the assessment of nuclear factor-nB (NF-nB) as a survival factor in human mesothelial cells (HMC), transformed HMC and malignant mesothelioma (MMe) cells.We aimed at verifying whether the proteasome inhibitor Bortezomib could abrogate NF-nB activity in MMe cells, leading to tumor cell death and may be established as a novel treatment for this aggressive neoplasm. Experimental Design: In HMC and MMe cells, NF-nB nuclear translocation and DNA binding were studied by electrophoretic mobility shift assay, following treatment with tumor necrosis factor-a (TNF-a). The IKK inhibitor Bay11-7082 was also tested to evaluate its effects on HMC, transformed HMC, and MMe cell viability upon exposure to asbestos fibers. Following Bortezomib treatment, cytotoxicity of MMe cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, whereas apoptosis and cell-cycle blockade were investigated by high-content analysis. Bortezomib was also given to mice bearing i.p. xenografts of MMe cells, and its effects on tumor growth were evaluated. Results: Here, we show that NF-nB activity is a constitutive survival factor in transformed HMC, MMe cells, and acts as a survival factor in HMC exposed to asbestos fibers. Bortezomib inhibits NF-nB activity in MMe cells and induces cell cycle blockade and apoptosis in vitro as well as tumor growth inhibition in vivo. Conclusions: Inhibition of NF-nB constitutive activation in MMe cells by Bortezomib resulted in in vitro cytotoxicity along with apoptosis and in vivo tumor regression. Our results support the use of Bortezomib in the treatment of MMe and has led to a phase II clinical trial currently enrolling in Europe.

Taurolidine (TRD) was designed in the 1970s as a broad-spectrum antibiotic and is used clinically at high doses without systemic toxicity. We have found that this agent possesses cytotoxic activity in human tumor cell lines and antineoplastic activity in mice bearing i.p. human tumor xenografts. We now report the mechanism by which TRD induces cell death in DU145 human prostate tumor cells. The IC50 (3 days) of TRD in this model was 16.8+/-1.1 microM. Cytotoxicity was associated with DNA debris and increased membrane phosphatidylserine externalization, both suggesting the induction of apoptosis. This was confirmed by the ability of TRD to induce PARP cleavage in these cells, an effect prevented by coexposure to the pan-caspase inhibitor zVAD-FMK. TRD exposure also resulted in the appearance of cytochrome c in the cytoplasm, procaspase 9 activation within 2 h of drug exposure and procaspase 8 activation 4 h after exposure. Parallel experiments revealed that cytochrome c appearance in the cytoplasm was not blocked by preexposure to zVAD-FMK, while activation of both procaspase 9 and procaspase 8 was prevented. Finally, antineoplastic activity was assessed in mice bearing subcutaneous xenografts of DU145 cells. Initial studies quantitated the toxicity of three i.p. injections of TRD, administered as one injection on three alternate days per week, at doses ranging from 500 to 700 mg/kg per injection. The 500 mg/kg dose produced about 7% mortality after three cycles and effectively inhibited tumor growth. Thus, TRD induced mitochondrial-mediated apoptosis in DU145 human prostate tumor cells and this effect could be exploited for therapeutic advantage.