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Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 µM 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. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The phosphatidylinositol 3'-kinase/PDK-1/Akt signaling cascade represents a convergence point for a plethora of receptor tyrosine kinase and cytokine-mediated pathways that regulate cell proliferation and survival and offers a framework to account for the ability of many extracellular trophic factors to maintain cell survival (9, 10, 11, 12, 13, 14, 15)
. Dysregulation of this signaling cascade due to constitutive growth factor-receptor activation and/or PTEN mutations results in Akt up-regulation, which subsequently promotes tumor invasiveness, angiogenesis, and progression (16, 17, 18, 19)
. Thus, PDK-1/Akt signaling inhibitors are of translational relevance for development into useful chemotherapeutic or chemopreventive agents. In this study, we carried out structure-based optimization of celecoxib using an integrated approach combining structure-activity analysis and molecular modeling, leading to two potent PDK-1 inhibitors, OSU- 03012 and OSU-03013, with IC50 values at the low µM range. These two PDK-1 inhibitors could achieve total growth inhibition in 60 different human tumor cell lines at a mean concentration of 3 µM.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) are reported in parts per million relative to tetramethylsilane peak with CDCl3 as solvent unless otherwise mentioned. High-resolution electrospray ionization mass spectrometry analyses were performed with a 3-Tesla Finnigan FTMS-2000 Fourier Transform mass spectrometer.
Synthesis of Chemicals.
In this article, we discussed 36 compounds, of which the chemical names, proton nuclear magnetic resonance (1H NMR), and high-resolution mass spectrometry data are summarized in Table 1
. The procedures used to synthesize compounds 1–36 are described in the Supplementary Data section.
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Cell Viability Analysis.
The effect of celecoxib and its derivatives on PC-3 cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay in six replicates. Cells were grown in 10% FBS- supplemented RPMI 1640 in 96-well, flat-bottomed plates for 24 h, and were exposed to various concentrations of celecoxib derivatives dissolved in DMSO (final concentration 
0.1%) in 1% serum-containing RPMI 1640 for different time intervals. Controls received DMSO vehicle at a concentration equal to that in drug-treated cells. The medium was removed, replaced by 200 µl of 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide in 10% FBS-containing RPMI 1640, and cells were incubated in the CO2 incubator at 37°C for 2 h. Supernatants were removed from the wells, and the reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye was solubilized in 200 µl/well DMSO. Absorbance at 570 nm was determined on a plate reader.
Cell Proliferation.
PC-3 cells were seeded into six-well plates at 75,000 cells/well in 10% FBS-containing RPMI 1640. After a 24-h attachment period, cells were treated in triplicate with the indicated concentration of celecoxib derivatives or DMSO vehicle in 10% FBS-containing RPMI 1640. At different time intervals, cells were harvested by trypsinization and numerated using a Coulter counter model Z1 D/T (Beckman Coulter, Fullerton, CA).
Apoptosis Analysis.
Two methods were used to assess drug-induced apoptotic cell death: detection of DNA fragmentation by the Cell Death Detection ELISA kit (Roche Diagnostics) and Western blot analysis of PARP cleavage. The ELISA was performed according to the manufacturer’s instructions and is based on the quantitative determination of cytoplasmic histone-associated DNA fragments in the form of mononucleosomes or oligonucleosomes generated after induced apoptotic death. In brief, 4 x 105 PC-3 cells were cultured in a T-25 flask for 24 h before treatment. Cells were treated with the DMSO vehicle or the test agent at the indicated concentrations for 6–24 h, collected, and cell lysates equivalent to 2 x 103 PC-3 cells were used in the ELISA. For the PARP cleavage assay, drug-treated cells were collected 4–8 h after treatment, washed with ice-cold PBS, and resuspended in lysis buffer containing 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1 mM CaCl2, 10% glycerol, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 100 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, leupeptin at 10 µg/ml, and aprotinin at 10 µg/ml. Soluble cell lysates were collected after centrifugation at 10,000 x g for 5 min. Equivalent amounts of proteins (60–100 µg) from each lysate were resolved in 8% SDS- polyacrylamide gels. Bands were transferred to nitrocellulose membranes and analyzed by immunoblotting with anti-PARP antibody.
Immunoblotting.
The general procedure for the Western blot analysis of Akt and phospho-Akt is described as follows. Cells were washed in PBS, resuspended in SDS sample buffer, sonicated by an ultrasonic sonicator for 5 s, and boiled for 5 min. After brief centrifugation, equivalent protein concentrations from the soluble fractions were resolved in 10% SDS-polyacrylamide gels on a Minigel apparatus, and transferred to a nitrocellulose membrane using a semi-dry transfer cell. The transblotted membrane was washed three times with TBS containing 0.05% Tween 20 (TBST). After blocking with TBST containing 5% nonfat milk for 60 min, the membrane was incubated with the primary antibody at 1:1000 dilution in TBST-5% low fat milk at 4°C for 12 h, and was then washed three times with TBST. The membrane was probed with goat antirabbit IgG-horseradish peroxidase conjugates (1:1000) for 1 h at room temperature and was washed with TBST three times. The immunoblots were visualized by enhanced chemiluminescence.
PDK-1 Kinase Assay.
This in vitro assay was performed using a PDK-1 kinase assay kit (Upstate) according to the vendor’s instructions. This cell-free assay is based on the ability of recombinant PDK-1, in the presence of DMSO vehicle or the test agent, to activate its downstream kinase serum- and glucocorticoid-regulated kinase which, in turn, phosphorylates the Akt/serum- and glucocorticoid-regulated kinase-specific peptide substrate RPRAATF with [
-32P]ATP. The 32P-phosphorylated peptide substrate was then separated from the residual [-32P]-ATP using P81 phosphocellulose paper and quantitated by a scintillation counter after three washes with 0.75% phosphoric acid. The reported values represent the means of two independent determinations.
Immunoprecipitated Akt Kinase Assay.
Akt immunoprecipitation was carried out according to a modified, published procedure (7)
. PC-3 cells were treated with DMSO vehicle or the test agents at the indicated concentrations for 2 h and then lysed at 4°C for 1 h in buffer A containing 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 10 mM sodium ß-glycerophosphate, 0.1% 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of aprotinin, pepstatin, and leupeptin. Cell lysates were centrifuged at 10,000 x g for 5 min, and the supernatant was treated with anti-Akt at 4°C for 60 min, followed by protein G-agarose beads for additional 60 min. The immunoprecipitate was used to analyze Akt kinase activity by using the Akt/serum- and glucocorticoid-regulated kinase-specific peptide substrate RPRAATF as described above. Values represent the means of two independent determinations.
Immunoprecipitated p70 S6 kinase (p70S6K) Assay.
Immunoprecipitation of p70S6K was carried out according to a modification of a published procedure (20)
. In brief, PC-3 cells were cultured in T-75 flasks (1.5 x 106/flask), and treated with OSU-03012 at the indicated concentrations in 1% FBS-containing RPMI 1640 for 6 h. Both floating and adherent cells were collected and lysed in 1 ml of lysis buffer [50 mM Tris (pH7.4), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM EGTA, and 10% protease inhibitor mixture (Calbiochem)] for 30 min on ice. Lysates were centrifuged at 10,000 x g at 4°C for 20 min. Equal amounts of total protein were subject to immunoprecipitation with anti-p70S6K antibody (sc-8418; Santa Cruz Biotechnology, Santa Cruz, CA). The mixture was incubated on ice with rocking for 1 h, followed by incubation with protein A-Sepharose bead for 2 h. The immunocomplex was washed with lysis buffer twice, followed by assay buffer [20 mM 4-morpholinepropanesulfonic acid (pH 7.2), 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM DTT], and resuspended in assay buffer. The assay was carried out using a p70S6K assay kit (Upstate) according to the manufacturer’s instruction.
Transient Transfection.
The constitutively active Akt construct HA-PKBT308D/S473D and the constitutively active PDK-1 construct pcDNA-PDK1-A280V were kindly provided by Dr. Brain Hemmings (Friedrich Miescher Institute, Basel, Switzerland) and Dr. Feng Liu (University of Texas Health Science Center, San Antonio, TX), respectively. PC-3 cells were seeded into T-75 flasks (1.5 x 106/flask). Aliquots containing 3 µg of each plasmid or a control pcDNA3.1(+) vector in 750 µl of Opti-MEM (Invitrogen-Life Technologies, Inc.) were incubated with 9 µl of the FuGene 6 reagent (Roche Diagnostics Corp., Indianapolis, IN) for 15 min. Each flask was washed with Opti-MEM and then received the plasmid-FuGene 6 mixture and 4 ml of Opti-MEM. The flask was placed in a CO2 incubator for 4 h, and the transfection medium was replaced with 10% FBS-supplemented RPMI 1640. After 24 h, Mock-, Akt-, and PDK-1-transfected PC-3 cells were seeded into 96-well plates at 5000 cells/well in 10% FBS-supplemented RPMI 1640. On the next day, cells were treated in four replicates with the indicated concentrations of OSU-03012 in 1% FBS- containing medium for 24 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to determine the cell viability.
Molecular Modeling.
The crystal structure of PDK-1 in complex with ATP, obtained from the Brookhaven Protein Data Bank (entry code 1H1W), was subject to the deletion of heteroatoms and the addition of polar hydrogens, partial atomic charges, and atomic solvation parameters using the molecular modeling software AutoDock Tools.1
The three-dimensional structures of small molecules were generated using the software SYBYL 6.9 (Tripos Associate, St. Louis, MO; 2002). Gasteiger charges were computed, and energy minimization was carried out with default parameters. AutoDock Tools software was used to designate the rotable bonds and generate grid parameter file and docking parameter file using default parameters. Docking was performed with AutoDock 3.05,2
which predicts the bound conformations of a small, flexible ligand to a nonflexible macromolecular target of known structure (21)
. This software is an automated docking package that combines a rapid grid-based method for energy evaluation with a Lamarckian genetic algorithm method of conformation search. All of the molecular modeling calculations and manipulations were performed on Silicon Graphics O2 (Silicon Graphics Inc., Mountain View, CA).
Statistical Analysis.
Each experiment was performed in triplicate unless otherwise mentioned. All of the experiments were carried out at least twice on different occasions. Where appropriate, the data are presented as the mean + SD.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These IC50 values corresponded to a 5–6-fold improvement over the activities of celecoxib (48 µM and 30 µM, respectively). However, the OSU-02067 isomer 24 exhibited a precipitous decrease in PDK-1 inhibitory activity, which might be attributable to steric hindrance imposed by an unfavorable orientation of the tricyclic aromatic ring.
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, top panel) and Akt phosphorylation status (Fig. 2B
, bottom panel). As shown, both assays gave consistent results.
According to the kinase assay, the IC50 of OSU-02067 for inhibiting intracellular Akt activation was 5 µM vis-à-vis 28 µM for celecoxib. Neither OSU-02067 nor other celecoxib derivatives displayed a direct inhibitory effect on immunoprecipitated Akt activity. Meanwhile, Western blot analysis shows that treatment of PC-3 cells with OSU- 02067 at 
5 µM led to significant Akt dephosphorylation.
The inhibition of PDK-1/Akt signaling led to apoptotic death in PC-3 cells in 1% of FBS-containing RPMI 1640 in a dose-dependent manner (Fig. 2C)
, as evidenced by DNA fragmentation (Fig. 2D
, top panel) and PARP cleavage (Fig. 2D
, bottom panel). The dose of OSU-02067 required to induce 50% PC-3 cell death at 24 h was 5 µM, as compared with that of 30 µM for celecoxib (data not shown). The IC50 values for both OSU-02067 and celecoxib to induce PC-3 cell death was consistent with that of inhibiting Akt activation in drug-treated cells. Furthermore, the effect of OSU-02067 vis-à-vis celecoxib on PC-3 cell proliferation was examined in 10% FBS-supplemented RPMI 1640 (Fig. 2E)
. As shown, OSU-02067 at 1 µM showed substantial antiproliferative activity, exceeding that of 30 µM celecoxib. Together, these data clearly indicated the in vitro efficacy of OSU-02067 in PC-3 growth inhibition and prompted us to undertake additional lead optimization via structure-based design.
Molecular Modeling and Structure-Based Optimization.
Kinetics of PDK-1 inhibition by celecoxib with respect to ATP was examined to shed light onto the mechanistic basis by which these celecoxib derivatives mediated enzyme inhibition. The inhibitory effect of celecoxib on PDK-1 kinase activity was determined in the presence of various concentrations of ATP. Kinetic data revealed an inverse relationship between the degree of celecoxib-exerted PDK-1 inhibition and ATP concentrations. The resulting Lineweaver-Burke plot indicated that celecoxib inhibited PDK-1 through competition with ATP (Fig. 3)
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Together, these findings provided a molecular rationale for structure-based optimization on the basis of the framework generated by the crystal structure of PDK-1-ATP complex (31)
. OSU-02067 was docked into the ATP-binding domain that is located within a deep cleft between the two lobes of PDK-1 (Fig. 4A)
. Although OSU-02067 competed with ATP for binding, the mode of binding for OSU-02067 was found to be somewhat different from that of ATP (Fig. 4B)
.
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Å2(32)
. Considering that the binding of the phenanthrene ring of OSU-02067 covers 32 Å2 more apolar surface than the methylphenyl moiety does, the corresponding free energy change was estimated to be –4.35 kJ/mol, which is consistent with the experimentally measured free energy difference.
The sulfonamide group (either oxygen or nitrogen) formed hydrogen bonds with the backbone amide of Ala162 in the hinge region. This bond lattice mimicked one of the two hydrogen bonds formed by the adenine of ATP in the same microdomain, in which 6-NH donates a hydrogen bond to carbonyl of Ser160 and 1-N accepts a hydrogen bond from NH of Ala162 (31)
. No hydrogen bond was formed between the pyrazole ring and Glu166, but the ribose of ATP accepts a hydrogen bond from Glu166. In light of the importance of hydrogen bonding with Ala 162 and Ser160 in ligand anchoring, we carried out structure optimization of OSU-02067 by replacing its sulfonamide moiety with a series of heteroatom-rich functional groups that could potentially form hydrogen bonding with the backbone amide and/or carbonyl of these two residues. Structures of 13 representative derivatives, their potency against PDK-1, and their ability to cause apoptotic death in PC-3 cells are summarized in Fig. 5
.
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. The guanidino group of OSU-03013 resembled the partial structure of the purine ring of ATP, which allowed the formation of hydrogen bonds with Ser160 and Ala162 as depicted by the docking model.
Like OSU-02067, they exhibited no appreciable direct inhibition on immunoprecipitated Akt kinase activity (data not shown) nor was any measurable COX-2 inhibitory activity detected at concentrations up to 50 µM. As shown in Fig. 6B
, exposure of PC-3 cells to either agent, even at 1 µM, resulted in a substantial decrease in the phospho-Akt level. Because PDK-1 also phosphorylates other members of the AGS protein kinase family such as p70S6K (20
, 33, 34, 35)
, we assessed the kinase activity of immunoprecipitated p70S6K in drug-treated PC-3 cells. As indicated in Fig. 6C
, the activity of immunoprecipitated p70S6K was reduced in a dose-dependent manner after exposing PC-3 cells to OSU-03012 at the indicated concentrations for 6 h.
Cellular Effects of PDK-1/Akt Signaling Inhibitors.
Both OSU-03012 and OSU- 03013 induced apoptotic death in PC-3 cells in 1% FBS-containing medium in a dose-dependent manner, as demonstrated by DNA fragmentation and PARP cleavage (Fig. 7, A and B)
. These agents exhibited higher potency than OSU-02067 in apoptosis induction at concentrations >2.5 µM.
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. High levels of serum might affect the efficacy of these PDK-1 inhibitors due to several factors. First, these celecoxib derivatives might display high serum-binding affinity, thereby sequestering them from entering cells. Second, continuous stimulation of phosphatidylinositol 3'-kinase/Akt signaling through various growth factor receptors counters the inhibitory effect of these agents on Akt. Third, serum could up-regulate Bcl-xL in PC-3 cells, which enhances the threshold to apoptotic signals emanating from phosphatidylinositol 3'-kinase/Akt inhibition (36)
. As shown, OSU-03012 was effective in suppressing PC-3 cell proliferation at sub-µM, consistent with that noted in 1% serum. In contrast, OSU-03013 required higher concentrations than OSU-03012 to achieve the same degree of antiproliferative effects although it had higher PDK-1 inhibitory potency.
To confirm that inhibition of PDK-1/Akt signaling represented the underlying antitumor mechanism for these agents, we assessed the protective effect of the transient expression of the constitutively active forms of PDK-1 and Akt [PDK-1A280V (37)
and AktT308D/S473D (38)
, respectively] on the drug-induced PC-3 cell death. Western blot analysis using antibodies against PDK-1, Akt, and the respective myc and hemagglutinin tags confirmed that transient transfection of PDK-1A280V and AktT308D/S473D into PC-3 cells led to increase in the expression levels of the respective kinases (Fig. 8A)
. These transient transfectants were exposed to 1–10 µM OSU-03012 in 1% FBS-containing medium for 24 h to examine the susceptibility to OSU-03012-induced cell death vis-à-vis transfectants with an empty pcDNA vector. As shown, both PDK-1A280V and AktT308D/S473D expression gave partial, yet significant, protection against OSU-03012-induced apoptotic death (Fig. 8B)
, which underscores the involvement of both kinases in the antitumor effects of these celecoxib derivatives.
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, which include: (a) RPMI- 8226 leukemia cells; (b) NCI-H322M non-small cell lung cancer cells; (c) HT29 colon cancer cells; (d) U251 glioblastoma cancer cells; (e) SK-MEL-28 melanoma cancer cells; (f) SK-OV-3 ovarian cancer cells; (g) RXF 393 renal cancer cells; (h) PC-3 prostate cancer cells; and (i) MDA-MB-231 breast cancer cells. Many of these cell lines were responsive to the growth inhibitory effect of both agents at concentrations as low as 0.1 µM.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Accordingly, replacement of the sulfonamide function with 2-aminoacetamide and guanidine led to OSU-03012 and OSU-03013, respectively, both of which exhibited improved PDK-1 inhibition with IC50 values of 5 and 2 µM, respectively (Fig. 6A)
. Docking of OSU-03013 into the ATP binding site revealed increased hydrogen bonding between the guanidine moiety and the backbone oxygen of Ser160. The effect of these substituents on ligand binding, however, was subtle, as illustrated by the structure-activity relationship summarized in Fig. 5
.
The high potency of OSU-03012 and OSU-03013 in PDK-1 inhibition was reflected in their abilities to effectively block the activation of Akt and p70S6K (Fig. 6)
and to induce apoptotic cell death in PC-3 cells at low µM concentrations (Fig. 7)
. More importantly, due to the conserved role of PDK-1/Akt signaling in cell proliferation and survival, these agents were potent in inhibiting cell growth in serum-containing medium in all 60 of the human tumor cell lines examined, with mean concentration resulting in 50% growth inhibition values of 1.2 µM and 1.3 µM, respectively, and concentration resulting in total growth inhibition (total growth inhibition) values of 3.2 and 2.9 µM, respectively.
In summary, the present study has clearly demonstrated the successful development of a new class of PDK-1/Akt signaling inhibitors via structure-based optimization of celecoxib. In light of the prominent role of PDK-1/Akt signaling in different stages of tumorigenesis, these molecules have translational potential to be developed into antitumor agents for the prevention and/or therapy of cancers alone or in combination with other treatments. Testing of in vivo efficacy against different tumor xenografts in nude mice is currently under way in this laboratory.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article can be obtained from the corresponding author upon request.
Requests for reprints:Ching-Shih Chen, College of Pharmacy, The Ohio State University, 336 Parks Hall, 500 West 12th Avenue, Columbus, OH 43210-1291. Phone: (614) 688-4008; Fax: (614) 688-8556; E-mail: chen.844{at}osu.edu
1 Internet address: http://www.scripps.edu/pub/olson-web/doc/autodock/tools.html. 
2 Internet address: http://www.scripps.edu/pub/olson-web/people/gmm/autodock/obtaining.html.
3 Internet address: http://dtp.nci.nih.gov/index.html.
Received 12/29/03. Revised 3/18/04. Accepted 4/ 2/04.
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 J. Lou, N. Fatima, Z. Xiao, S. Stauffer, G. Smythers, P. Greenwald, and I. U. Ali Proteomic profiling identifies cyclooxygenase-2-independent global proteomic changes by celecoxib in colorectal cancer cells. Cancer Epidemiol. Biomarkers Prev., September 1, 2006; 15(9): 1598 - 1606. [Abstract] [Full Text] [PDF]
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K. Liang, Y. Lu, X. Li, X. Zeng, R. I. Glazer, G. B. Mills, and Z. Fan Differential Roles of Phosphoinositide-Dependent Protein Kinase-1 and Akt1 Expression and Phosphorylation in Breast Cancer Cell Resistance to Paclitaxel, Doxorubicin, and Gemcitabine Mol. Pharmacol., September 1, 2006; 70(3): 1045 - 1052. [Abstract] [Full Text] [PDF]
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 A. J. Johnson, D. M. Lucas, N. Muthusamy, L. L. Smith, R. B. Edwards, M. D. De Lay, C. M. Croce, M. R. Grever, and J. C. Byrd Characterization of the TCL-1 transgenic mouse as a preclinical drug development tool for human chronic lymphocytic leukemia Blood, August 15, 2006; 108(4): 1334 - 1338. [Abstract] [Full Text] [PDF]
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A. Yacoub, M. A. Park, D. Hanna, Y. Hong, C. Mitchell, A. P. Pandya, H. Harada, G. Powis, C.-S. Chen, C. Koumenis, et al. OSU-03012 Promotes Caspase-Independent but PERK-, Cathepsin B-, BID-, and AIF-Dependent Killing of Transformed Cells Mol. Pharmacol., August 1, 2006; 70(2): 589 - 603. [Abstract] [Full Text] [PDF]
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J. A. McCubrey, M. M. LaHair, and R. A. Franklin OSU-03012 in the Treatment of Glioblastoma Mol. Pharmacol., August 1, 2006; 70(2): 437 - 439. [Abstract] [Full Text] [PDF]
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 S. Grosch, T. J. Maier, S. Schiffmann, and G. Geisslinger Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst, June 7, 2006; 98(11): 736 - 747. [Abstract] [Full Text] [PDF]
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C. A. Granville, R. M. Memmott, J. J. Gills, and P. A. Dennis Handicapping the Race to Develop Inhibitors of the Phosphoinositide 3-Kinase/Akt/Mammalian Target of Rapamycin Pathway Clin. Cancer Res., February 1, 2006; 12(3): 679 - 689. [Abstract] [Full Text] [PDF]
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A. Kardosh, N. Soriano, Y.-T. Liu, J. Uddin, N. A. Petasis, F. M. Hofman, T. C. Chen, and A. H. Schonthal Multitarget inhibition of drug-resistant multiple myeloma cell lines by dimethyl-celecoxib (DMC), a non-COX-2 inhibitory analog of celecoxib Blood, December 15, 2005; 106(13): 4330 - 4338. [Abstract] [Full Text] [PDF]
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S. Ali, B. F. El-Rayes, F. H. Sarkar, and P. A. Philip Simultaneous targeting of the epidermal growth factor receptor and cyclooxygenase-2 pathways for pancreatic cancer therapy Mol. Cancer Ther., December 1, 2005; 4(12): 1943 - 1951. [Abstract] [Full Text] [PDF]
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 N. Kusunoki, T. Ito, N. Sakurai, T. Suguro, H. Handa, and S. Kawai A Novel Celecoxib Derivative Potently Induces Apoptosis of Human Synovial Fibroblasts J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 796 - 803. [Abstract] [Full Text] [PDF]
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P.-H. Tseng, H.-P. Lin, J. Zhu, K.-F. Chen, E. M. Hade, D. C. Young, J. C. Byrd, M. Grever, K. Johnson, B. J. Druker, et al. Synergistic interactions between imatinib mesylate and the novel phosphoinositide-dependent kinase-1 inhibitor OSU-03012 in overcoming imatinib mesylate resistance Blood, May 15, 2005; 105(10): 4021 - 4027. [Abstract] [Full Text] [PDF]
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A. J. Johnson, L. L. Smith, J. Zhu, N. A. Heerema, S. Jefferson, A. Mone, M. Grever, C.-S. Chen, and J. C. Byrd A novel celecoxib derivative, OSU03012, induces cytotoxicity in primary CLL cells and transformed B-cell lymphoma cell line via a caspase- and Bcl-2-independent mechanism Blood, March 15, 2005; 105(6): 2504 - 2509. [Abstract] [Full Text] [PDF]
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R. W. Caron, A. Yacoub, M. Li, X. Zhu, C. Mitchell, Y. Hong, W. Hawkins, T. Sasazuki, S. Shirasawa, A. P. Kozikowski, et al. Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the effect of therapeutic kinase inhibitors on cell survival Mol. Cancer Ther., February 1, 2005; 4(2): 257 - 270. [Abstract] [Full Text] [PDF]
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X. Liu, P. Yue, Z. Zhou, F. R. Khuri, and S.-Y. Sun Death Receptor Regulation and Celecoxib-Induced Apoptosis in Human Lung Cancer Cells J Natl Cancer Inst, December 1, 2004; 96(23): 1769 - 1780. [Abstract] [Full Text] [PDF]
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H.-P. Lin, S. K. Kulp, P.-H. Tseng, Y.-T. Yang, C.-C. Yang, C.-S. Chen, and C.-S. Chen Growth inhibitory effects of celecoxib in human umbilical vein endothelial cells are mediated through G1 arrest via multiple signaling mechanisms Mol. Cancer Ther., December 1, 2004; 3(12): 1671 - 1680. [Abstract] [Full Text] [PDF]
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