The blockade of Akt activation through the inhibition of 3-phosphoinositide-dependent kinase-1 (PDK-1) represents a major signaling mechanism whereby celecoxib mediates apoptosis. Celecoxib, however, is a weak PDK-1 inhibitor (IC 50 , 48 M), requiring at least 30 M to exhibit discernable effects on the growth of tumor cells in vitro. Here, we report the structure-based optimization of celecoxib to develop PDK-1 inhibitors with greater potency in enzyme inhibition and growth inhibition. Kinetics of PDK-1 inhibition by celecoxib with respect to ATP suggest that celecoxib derivatives inhibit PDK-1 by competing with ATP for binding, a mechanism reminiscent to that of many kinase inhibitors. Structureactivity analysis together with molecular modeling was used to generate compounds that were tested for their potency in inhibiting PDK-1 kinase activity and in inducing apoptosis in PC-3 prostate cancer cells. Docking of potent compounds into the ATP-binding site of PDK-1 was performed for lead optimization, leading to two compounds, OSU-03012 and OSU-03013, with IC 50 values in PDK-1 inhibition and apoptosis induction in the low M range. Exposure of PC-3 cells to these agents led to Akt dephosphorylation and inhibition of p70 S6 kinase activity. Moreover, overexpression of constitutively active forms of PDK-1 and Akt partially protected OSU-03012-induced apoptosis. Screening in a panel of 60 cell lines and more extensive testing in PC-3 cells indicated that the mean concentration for total growth inhibition was ϳ3 M for both agents. Considering the conserved role of PDK-1/Akt signaling in promoting tumorigenesis, these celecoxib analogs are of translational relevance for cancer prevention and therapy.

Certain members of the thiazolidenedione family

No abstract

In light of the clinical relevance of targeting cyclin D1 in breast cancer, we have investigated the mechanism underlying the effect of the peroxisome proliferator-activated receptor-␥ (PPAR␥) agonists troglitazone and ciglitazone on cyclin D1 repression. We obtain evidence that the ability of high doses of troglitazone and ciglitazone to repress cyclin D1 is independent of PPAR␥ activation. PPAR␥-inactive troglitazone and ciglitazone analogs 5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl-methoxy)-benzylidene]-2,4-thiazolidinedione (⌬2-TG) and 5-[4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-dione are able to facilitate cyclin D1 ablation with potency similar to that of troglitazone and ciglitazone in MCF-7 cells. Reverse transcription-polymerase chain reaction shows that the mRNA level of cyclin D1 remains unaltered in drug-treated cells, indicating the repression is mediated at the posttranscriptional level. Moreover, the ablative effect of these agents is specific to cyclin D1, in that the expression levels of many other cyclins and cyclin-dependent kinases examined remain unchanged after drug treatment. Our data indicate that troglitazone-and ⌬2-TG-induced cyclin D1 repression is mediated via proteasome-facilitated proteolysis because it is inhibited by different proteasome inhibitors, including N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norleucinal (MG132), lactacystin, and epoxomicin, and is preceded by increased ubiquitination. The dissociation of these two pharmacological activities (i.e., PPAR␥ activation and cyclin D1 ablation) provides a molecular basis to use ⌬2-TG as a scaffold to develop a novel class of cyclin D1-ablative agents. Therefore, a series of ⌬2-TG derivatives have been synthesized. Among them,5,7,-benzylidene]-2,4-thiazolidinedione represents a structurally optimized agent with potency that is an order of magnitude higher than that of ⌬2-TG in cyclin D1 repression and MCF-7 cell growth inhibition.Cyclin D1 represents an important downstream effector of diverse proliferative and transforming signaling pathways, including those mediated by ␤-catenin (Shtutman et al., 1999), estrogen receptor ␣ (ER␣) (Lukas et al

Although the antitumor effect of ␣-tocopheryl succinate (vitamin E succinate) has been well demonstrated, its underlying mechanism remains elusive. This study provides evidence that inhibition of BclxL/Bcl-2 function represents a major pathway whereby ␣-tocopheryl succinate mediates apoptosis induction in prostate cancer cells. In vitro data indicate that ␣-tocopheryl succinate was able to disrupt the binding of Bak BH3 peptide to Bcl-xL and Bcl-2 with IC 50 of 26 M, in line with its potency in antiproliferation. Treatment of PC-3 cells with this agent led to reduced association of Bcl-2 and Bcl-xL with Bak, leading to caspase-dependent apoptosis. Moreover, overexpression of Bcl-xL protected LNCaP cells from the apoptosis induction. This mechanistic finding provided a basis to develop potent Bcl-xL/Bcl-2 inhibitors. Docking of ␣-tocopheryl succinate into the Bak peptide-binding site indicates that it adopted a unique hairpin-shaped conformation for protein interactions. We rationalized that the hemisuccinate and the two proximal isopranyl units of the side chain played a crucial role in ligand anchoring and protein-ligand complex stabilization, respectively. However, exposure of the distal isopranyl unit to a polar environment might diminish the binding affinity of ␣-tocopheryl succinate. This premise was corroborated by a structure-activity analysis of a series of derivatives with truncated side chains and/or altered carboxyl terminus. This computer model predicted that the removal of the distal isopranyl unit from the side chain would improve binding affinity, leading to two agents with significantly higher potency in inhibiting Bak peptide binding and in suppressing prostate cancer cell proliferation.Although numerous epidemiological and intervention studies have failed to establish a correlation between vitamin E intake and the incidence of cancer, recent investigations have suggested the potential use of ␣-tocopheryl succinate (vitamin E succinate) as a therapeutic agent for cancer (for reviews see Refs. 1 and 2). Evidence indicates that ␣-tocopheryl succinate induces apoptosis in cells with a malignant or transformed phenotype without incurring significant toxicity to normal cells (3-6). Moreover, its in vivo efficacy has been demonstrated in a number of animal model experiments, including suppression of breast and melanoma tumor growth (7-9), inhibition of colon cancer liver metastases (10), and sensitization of colon tumor cells to the tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) (11). Despite these advances, the mechanism underlying the effect of this redox-inactive vitamin E derivative on apoptosis remains elusive. Putative signaling mechanisms include inhibition of cyclin A binding to the transcription factor E2F (12), mitochondrial destabilization through sphingomyelinase activation (13), suppression of NFB activation (14), activation of protein kinase C ␣ (15), and up-regulation of mitogen-activated protein kinase signaling (16,17). Another school of thought is that ␣-tocopheryl su...