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1.
Mol Ther ; 26(11): 2567-2579, 2018 11 07.
Article in English | MEDLINE | ID: mdl-30274786

ABSTRACT

Wnt/ß-catenin signaling mediates cancer immune evasion and resistance to immune checkpoint therapy, in part by blocking cytokines that trigger immune cell recruitment. Inhibition of ß-catenin may be an effective strategy for increasing the low response rate to these effective medicines in numerous cancer populations. DCR-BCAT is a nanoparticle drug product containing a chemically optimized RNAi trigger targeting CTNNB1, the gene that encodes ß-catenin. In syngeneic mouse tumor models, ß-catenin inhibition with DCR-BCAT significantly increased T cell infiltration and potentiated the sensitivity of the tumors to checkpoint inhibition. The combination of DCR-BCAT and immunotherapy yielded significantly greater tumor growth inhibition (TGI) compared to monotherapy in B16F10 melanoma, 4T1 mammary carcinoma, Neuro2A neuroblastoma, and Renca renal adenocarcinoma. Response to the RNAi-containing combination therapy was not dependent on Wnt activation status of the tumor. Importantly, this drug combination was associated with elevated levels of biomarkers of T cell-mediated cytotoxicity. Finally, when CTLA-4 and PD-1 antibodies were combined with DCR-BCAT in MMTV-Wnt1 transgenic mice, a genetic model of spontaneous Wnt-driven tumors, complete regressions were achieved in the majority of treated subjects. These data support RNAi-mediated ß-catenin inhibition as an effective strategy to increase response rates to cancer immunotherapy.


Subject(s)
CTLA-4 Antigen/antagonists & inhibitors , Carcinoma, Renal Cell/drug therapy , Melanoma, Experimental/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , beta Catenin/genetics , Animals , CTLA-4 Antigen/genetics , CTLA-4 Antigen/immunology , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/immunology , Carcinoma, Renal Cell/pathology , Combined Modality Therapy , Female , Humans , Immunotherapy/methods , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Mice , Mice, Transgenic , Programmed Cell Death 1 Receptor/immunology , RNA Interference , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , T-Lymphocytes/immunology , Wnt Signaling Pathway/genetics , Wnt1 Protein/genetics , beta Catenin/antagonists & inhibitors
2.
Mol Cancer Ther ; 17(2): 544-553, 2018 02.
Article in English | MEDLINE | ID: mdl-29282298

ABSTRACT

Colorectal carcinomas harbor well-defined genetic abnormalities, including aberrant activation of Wnt/ß-catenin and MAPK pathways, often simultaneously. Although the MAPK pathway can be targeted using potent small-molecule drugs, including BRAF and MEK inhibitors, ß-catenin inhibition has been historically challenging. RNAi approaches have advanced to the stage of clinical viability and are especially well suited for transcriptional modulators, such as ß-catenin. In this study, we report therapeutic effects of combined targeting of these pathways with pharmacologic agents. Using a recently described tumor-selective nanoparticle containing a ß-catenin-targeting RNAi trigger, in combination with the FDA-approved MEK inhibitor (MEKi) trametinib, we demonstrate synergistic tumor growth inhibition in in vivo models of colorectal cancer, melanoma, and hepatocellular carcinoma. At dose levels that were insufficient to significantly impact tumor growth as monotherapies, combination regimens resulted in synergistic efficacy and complete tumor growth inhibition. Importantly, dual MEKi/RNAi therapy dramatically improved survival of mice bearing colorectal cancer liver metastases. In addition, pharmacologic silencing of ß-catenin mRNA was effective against tumors that are inherently resistant or that acquire drug-induced resistance to trametinib. These results provide a strong rationale for clinical evaluation of this dual-targeting approach for cancers harboring Wnt/ß-catenin and MAPK pathway mutations. Mol Cancer Ther; 17(2); 544-53. ©2017 AACR.


Subject(s)
Colorectal Neoplasms/therapy , MAP Kinase Kinase Kinases/antagonists & inhibitors , Pyridones/pharmacology , Pyrimidinones/pharmacology , RNA, Messenger/genetics , RNA, Small Interfering/administration & dosage , beta Catenin/genetics , Animals , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Drug Synergism , Gene Silencing , Heterografts , Humans , Liver Neoplasms, Experimental/secondary , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System/drug effects , Mice , Mice, Nude , Nanoparticles/administration & dosage , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Wnt Signaling Pathway/drug effects , beta Catenin/metabolism
3.
Mol Cancer Ther ; 15(9): 2143-54, 2016 09.
Article in English | MEDLINE | ID: mdl-27390343

ABSTRACT

The Wnt/ß-catenin pathway is among the most frequently altered signaling networks in human cancers. Despite decades of preclinical and clinical research, efficient therapeutic targeting of Wnt/ß-catenin has been elusive. RNA interference (RNAi) technology silences genes at the mRNA level and therefore can be applied to previously undruggable targets. Lipid nanoparticles (LNP) represent an elegant solution for the delivery of RNAi-triggering oligonucleotides to disease-relevant tissues, but have been mostly restricted to applications in the liver. In this study, we systematically tuned the composition of a prototype LNP to enable tumor-selective delivery of a Dicer-substrate siRNA (DsiRNA) targeting CTNNB1, the gene encoding ß-catenin. This formulation, termed EnCore-R, demonstrated pharmacodynamic activity in subcutaneous human tumor xenografts, orthotopic patient-derived xenograft (PDX) tumors, disseminated hematopoietic tumors, genetically induced primary liver tumors, metastatic colorectal tumors, and murine metastatic melanoma. DsiRNA delivery was homogeneous in tumor sections, selective over normal liver and independent of apolipoprotein-E binding. Significant tumor growth inhibition was achieved in Wnt-dependent colorectal and hepatocellular carcinoma models, but not in Wnt-independent tumors. Finally, no evidence of accelerated blood clearance or sustained liver transaminase elevation was observed after repeated dosing in nonhuman primates. These data support further investigation to gain mechanistic insight, optimize dose regimens, and identify efficacious combinations with standard-of-care therapeutics. Mol Cancer Ther; 15(9); 2143-54. ©2016 AACR.


Subject(s)
Neoplasms/genetics , RNA Interference , RNA, Small Interfering/genetics , beta Catenin/genetics , Animals , Apolipoproteins E/chemistry , Apolipoproteins E/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Lipids/chemistry , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Male , Melanoma, Experimental , Mice , Nanoparticles/chemistry , Neoplasm Metastasis , Neoplasms/metabolism , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/chemistry , Structure-Activity Relationship , Wnt Signaling Pathway , Xenograft Model Antitumor Assays , beta Catenin/metabolism , ras Proteins/genetics , ras Proteins/metabolism
4.
Nucleic Acid Ther ; 24(6): 405-12, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25272050

ABSTRACT

Decreased production of erythropoietin (EPO) causes anemia in patients with chronic kidney disease, and recombinant human EPO is used to treat renal failure associated anemia. The liver, the main EPO-producing organ in utero, maintains the capacity to produce EPO in the adult but in insufficient quantities to restore hemoglobin levels to normal in patients with impaired renal function. Inhibition of prolyl-4-hydroxylase domain (PHD) proteins is known to cause an increase in EPO production through its effects on hypoxia inducible factor. Here, we utilized small interfering RNA (siRNA) targeting EGLN1, the gene encoding the PHD2 protein, to investigate the phenotypic consequences in nonhuman primates. A single, well-tolerated intravenous dose of an optimized EGLN1 siRNA encapsulated in a lipid nanoparticle formulation caused robust mRNA silencing in the liver, leading to increases in serum EPO and hemoglobin. The siRNA-induced erythropoiesis was dose-dependent and was sustained for at least 2 months. These data point to the potential for an RNA interference-based, liver-targeted therapeutic approach for the treatment of anemia.


Subject(s)
Erythropoiesis/drug effects , Hypoxia-Inducible Factor-Proline Dioxygenases/antagonists & inhibitors , RNA Interference , RNA, Small Interfering/administration & dosage , Animals , Erythropoietin/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Liver/drug effects , Macaca mulatta
5.
Int J Pharm ; 466(1-2): 58-67, 2014 May 15.
Article in English | MEDLINE | ID: mdl-24607208

ABSTRACT

A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.


Subject(s)
Apolipoproteins B/genetics , Ornithine/chemistry , Peptides/administration & dosage , Phenylalanine/chemistry , Polymers/administration & dosage , RNA, Small Interfering/administration & dosage , Animals , Female , Liver/metabolism , Molecular Weight , Peptides/chemistry , Polymers/chemistry , RNA, Messenger/genetics , RNA, Small Interfering/chemistry , Rats, Sprague-Dawley
6.
J Control Release ; 183: 124-37, 2014 Jun 10.
Article in English | MEDLINE | ID: mdl-24657948

ABSTRACT

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.


Subject(s)
Biocompatible Materials/chemical synthesis , Drug Carriers/chemical synthesis , Liver/metabolism , Nylons/chemical synthesis , Peptides/chemical synthesis , RNA, Small Interfering/administration & dosage , Animals , Autoradiography , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacokinetics , Biocompatible Materials/toxicity , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Drug Carriers/toxicity , Drug Design , Drug Stability , Female , Hep G2 Cells , Hepatocytes/metabolism , Humans , Liver/diagnostic imaging , Macaca mulatta , Nylons/chemistry , Nylons/pharmacokinetics , Nylons/toxicity , Peptides/chemistry , Peptides/pharmacokinetics , Peptides/toxicity , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacokinetics , RNA, Small Interfering/toxicity , Radionuclide Imaging , Rats, Sprague-Dawley , Species Specificity , Structure-Activity Relationship , Tissue Distribution
7.
RNA ; 16(12): 2553-63, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20940339

ABSTRACT

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.


Subject(s)
Gene Transfer Techniques , RNA, Small Interfering/genetics , Animals , Animals, Genetically Modified , Antibodies/isolation & purification , Antibodies/metabolism , Antibodies/pharmacology , Antibody Specificity , Argonaute Proteins , Cells, Cultured , Eukaryotic Initiation Factor-2/immunology , Eukaryotic Initiation Factor-2/metabolism , Evaluation Studies as Topic , Female , Gene Silencing/physiology , Gene Targeting/methods , Gene Transfer Techniques/standards , Humans , Immunoprecipitation/methods , Immunoprecipitation/standards , Macaca mulatta , Mice , Mice, Inbred ICR , Protein Binding , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Rodentia
8.
Mol Ther ; 18(1): 171-80, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19738601

ABSTRACT

Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.


Subject(s)
Dexamethasone/therapeutic use , Nanoparticles/adverse effects , RNA, Small Interfering/immunology , RNA, Small Interfering/metabolism , Animals , Female , Gene Silencing , Inflammation/chemically induced , Inflammation/drug therapy , Mice , Nanoparticles/administration & dosage , RNA, Small Interfering/administration & dosage , Receptors, Glucocorticoid/agonists
9.
Bioorg Med Chem Lett ; 19(4): 1240-4, 2009 Feb 15.
Article in English | MEDLINE | ID: mdl-19155174

ABSTRACT

A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site approximately 13A from the ATP binding site. Preliminary data is presented for several of these compounds.


Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , Quinazolines/chemical synthesis , Quinazolines/pharmacology , Binding Sites , Checkpoint Kinase 1 , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Humans , Molecular Conformation , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Kinases/chemistry , Protein Kinases/metabolism , Quinazolines/chemistry
10.
J Med Chem ; 51(14): 4239-52, 2008 Jul 24.
Article in English | MEDLINE | ID: mdl-18578472

ABSTRACT

Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Kinesins/antagonists & inhibitors , Neoplasms/enzymology , Piperidines/pharmacology , Pyrroles/pharmacology , Taxoids/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Humans , Neoplasms/drug therapy , Taxoids/therapeutic use
11.
Bioorg Med Chem Lett ; 17(22): 6280-5, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17900896

ABSTRACT

From HTS lead 1, a novel benzoisoquinolinone class of ATP-competitive Chk1 inhibitors was devised and synthesized via a photochemical route. Using X-ray crystallography as a guide, potency was rapidly enhanced through the installation of a tethered basic amine designed to interact with an acidic residue (Glu91) in the enzyme pocket. Further SAR was explored at the solvent front and near to the H1 pocket and resulted in the discovery of low MW, sub-nanomolar inhibitors of Chk1.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Protein Kinases/drug effects , Quinolones/chemical synthesis , Quinolones/pharmacology , Apoptosis/drug effects , Binding Sites , Cell Line, Tumor , Checkpoint Kinase 1 , Crystallography, X-Ray , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Photochemistry , Protein Kinases/chemistry , Quinolones/chemistry , Structure-Activity Relationship
12.
Bioorg Med Chem Lett ; 17(10): 2697-702, 2007 May 15.
Article in English | MEDLINE | ID: mdl-17395460

ABSTRACT

Installation of a C2-aminopropyl side chain to the 2,4-diaryl-2,5-dihydropyrrole series of kinesin spindle protein (KSP) inhibitors results in potent, water soluble compounds, but the aminopropyl group induces susceptibility to cellular efflux by P-glycoprotein (Pgp). We show that by carefully modulating the basicity of the amino group by beta-fluorination, this series of inhibitors maintains potency against KSP and has greatly improved efficacy in a Pgp-overexpressing cell line. The discovery that cellular efflux by Pgp can be overcome by carefully modulating the basicity of an amine may be of general use to medicinal chemists attempting to transform leading compounds into cancer cell- or CNS-penetrant drugs.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Fluorine/metabolism , Kinesins/antagonists & inhibitors , Propylamines/pharmacology , Pyrroles/pharmacology , Biological Transport , Cytoskeleton , Hydrogen-Ion Concentration , Kinesins/metabolism , Solubility , Water
13.
Cancer Biol Ther ; 5(5): 498-504, 2006 May.
Article in English | MEDLINE | ID: mdl-16582597

ABSTRACT

Activation of the epidermal growth factor receptor (EGFR) provides a measure of protection to immortalized epidermal keratinocytes (HaCaT cells) against apoptosis induced by diverse cellular stressors. This effect is due, in part, to sustained MAPK-dependent Bcl-xL expression. Here, we report a second EGFR/MAPK-dependent signaling event that protects HaCaT cells against apoptosis incurred during forced suspension culture (anoikis). This pathway targets Bim, a pro-apoptotic BH3-only Bcl-2 family member. Bim expression was functionally relevant to HaCaT cell survival as demonstrated by partial protection against anoikis provided by siRNA-induced Bim downregulation. Growth factor starvation of attached and suspended cells was associated with enhanced Bim expression whereas EGFR activation reduced Bim expression by inducing Bim phosphorylation and proteasomal degradation. EGFR-dependent Bim phosphorylation required MAPK activation. Furthermore, PKC-delta activity contributed to both MEK/MAPK phosphorylation and Bim phosphorylation as demonstrated using both pharmacological inhibitors of PKC-delta and siRNA-mediated PKC-delta knockdown. In addition to HaCaT cells, EGFR activation supported survival and induced Bim phosphorylation in several squamous carcinoma cell lines in a strictly MAPK-dependent fashion. These results establish that EGFR activation attenuates susceptibility of immortalized and malignant keratinocytes to apoptosis by post-translational control of Bim-EL expression through a pathway requiring PKC-delta and MEK/MAPK activation.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Epithelial Cells/metabolism , ErbB Receptors/metabolism , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Protein Kinase C-delta/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction , Anoikis , Apoptosis , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cells, Cultured , Down-Regulation , Humans , Keratinocytes/metabolism , MAP Kinase Kinase 1 , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Phosphorylation , Protein Kinase C-delta/antagonists & inhibitors , Protein Kinase C-delta/genetics , Protein Processing, Post-Translational , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , RNA, Small Interfering/pharmacology
14.
J Cell Biochem ; 98(2): 440-50, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16440325

ABSTRACT

Insulin receptor substrate 1 (IRS-1) is a major downstream signaling protein for insulin and insulin-like growth factor I (IGF-I) receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In breast cancer, IRS-1 overexpression has been associated with tumor development, hormone-independence and antiestrogen-resistance. In part, these effects are related to potentiation of IRS-1/PI-3K/Akt signaling. In estrogen sensitive breast cancer cell lines, tamoxifen treatment reduces IRS-1 expression and function; consequently, inhibiting IRS-1/PI-3K signaling. We tested whether anti-IRS1 siRNA could inhibit growth and survival of estrogen-sensitive MCF-7 breast cancer cells, when used alone or in combination with TAM. Our results indicated: (a) out of four tested anti-IRS1 siRNAs, two siRNAs reduced IRS-1 protein by approximately three-fold in both growing and IGF-I-stimulated cells without affecting a closely related protein, IRS-2; (b) these effects paralleled IRS1 mRNA downregulation by approximately three-fold, measured by quantitative real time-polymerase chain reaction; (c) action of anti-IRS1 siRNAs induced the apoptotic response, observed by altered mitochondrial membrane potential coupled with downregulation of NF-kappaB target Bcl-xL and reduced cell viability; (d) anti-IRS1 siRNA treatment enhanced the cytotoxic effects of TAM by approximately 20%. In summary, anti-IRS1 RNAi strategy could become a potent tool to induce breast cancer cell death, especially if combined with standard TAM therapy.


Subject(s)
Breast Neoplasms/metabolism , Cell Death/drug effects , Phosphoproteins/pharmacology , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Signal Transduction/drug effects , Tamoxifen/pharmacology , Breast Neoplasms/drug therapy , Female , Humans , Insulin Receptor Substrate Proteins , Phosphoproteins/chemistry , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Receptors, Estrogen/antagonists & inhibitors , Tumor Cells, Cultured
15.
J Cancer Res Clin Oncol ; 131(6): 347-54, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15856297

ABSTRACT

PURPOSE: To determine the effect of modulating MAP kinase phosphatase-1 (MKP-1) expression levels on cell death induced by glucocorticoid (GC) or hydroxyurea (HU) treatment in the human pre-B acute lymphoblastic leukemia cell line 697. METHODS: Stable MKP-1 overexpressing transformants of the 697 pre-B acute lymphoblastic leukemia cell line were created and tested for sensitivity to the GC triamcinolone acetonide (TA) and HU, and compared to a control 697 cell line containing normal MKP-1 expression levels. Small interfering RNAs (siRNAs) were designed to inhibit MKP-1 expression and evaluated for their effect on GC-mediated cell death. RESULTS: MKP-1 overexpression caused a phenotype of partial resistance to HU-induced apoptosis but not to GC-induced apoptosis. Electroporation of siRNAs effectively silenced MKP-1 expression, and increased sensitivity to TA by 9.6+/-1.9%. CONCLUSIONS: Because MKP-1 protects certain tumor cells from chemotherapy-induced apoptosis, its inhibition is being considered as a possible strategy for combination cancer therapy. However, this study suggests that while MKP-1 inhibition may improve the efficacy of DNA damaging agents, it may have only limited utility in combination with glucocorticoids.


Subject(s)
Cell Cycle Proteins/metabolism , Gene Silencing , Glucocorticoids/pharmacology , Immediate-Early Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Protein Tyrosine Phosphatases/metabolism , Triamcinolone Acetonide/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Base Sequence , Caspase 3 , Caspases/metabolism , Cell Cycle Proteins/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm , Dual Specificity Phosphatase 1 , Humans , Hydroxyurea/pharmacology , Immediate-Early Proteins/genetics , Immunoblotting , Molecular Sequence Data , Phosphoprotein Phosphatases/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Protein Phosphatase 1 , Protein Tyrosine Phosphatases/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Transfection
16.
J Biol Chem ; 279(53): 55809-17, 2004 Dec 31.
Article in English | MEDLINE | ID: mdl-15509554

ABSTRACT

Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells.


Subject(s)
Alternative Splicing , Apoptosis , Glucocorticoids/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Acetylcysteine/metabolism , Blotting, Northern , Caspase 3 , Caspases/metabolism , Cell Line , Cell Line, Tumor , Cell Survival , Dose-Response Relationship, Drug , Electroporation , Humans , Immunoblotting , Lentivirus/genetics , Lentivirus/metabolism , Lymphocytes/metabolism , Protein Isoforms , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Interference , Receptors, Glucocorticoid/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Time Factors , Transfection
18.
Cancer Res ; 63(1): 172-8, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12517795

ABSTRACT

Glucocorticoid (GC) sensitivity in hematopoietic cells requires the activation and nuclear translocation of the glucocorticoid receptor (GR) and the subsequent activation of caspases. To gain insight into the caspase cascade responsible for the execution phase of GC-induced apoptosis, 697 pre-B leukemic cells were stably transfected with dominant negative forms of caspase-8, caspase-9, or caspase-10 and the caspase-8 inhibitor CrmA. We observed that inhibition of caspase-9 or caspase-10 activity, but not caspase-8, caused partial resistance of 697 cells to GC-induced apoptosis. Inhibition of multiple caspases through the use of specific peptide inhibitors had an additive effect and caused complete resistance. To identify GR-regulated genes upstream of caspase activation in 697 cells, we performed DNA microarray analysis. 113 genes were identified, which were induced or repressed at least 3-fold by GC. Surprisingly, mitogen-activated protein kinase phosphatase-1 (MKP-1), a GR-induced gene in other cell types, was repressed 3-fold and correlated with an induction of JNK activity. These results suggest the involvement of mitogen activated protein kinases and apical caspase-9 and caspase-10 in the GC-induced apoptosis of pre-B lymphocytes.


Subject(s)
Apoptosis/drug effects , Caspases/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Glucocorticoids/pharmacology , Leukemia, B-Cell/pathology , Preleukemia/pathology , Cell Survival , Clone Cells , DNA Primers , Humans , JNK Mitogen-Activated Protein Kinases , Kinetics , Leukemia, B-Cell/genetics , Mitogen-Activated Protein Kinases/metabolism , Oligonucleotide Array Sequence Analysis , Preleukemia/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tumor Cells, Cultured
19.
Bioorg Med Chem Lett ; 12(15): 2027-30, 2002 Aug 05.
Article in English | MEDLINE | ID: mdl-12113834

ABSTRACT

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Binding Sites , Binding, Competitive , Inhibitory Concentration 50 , Protein Binding , Structure-Activity Relationship , Tumor Cells, Cultured , rap GTP-Binding Proteins/drug effects , rap GTP-Binding Proteins/metabolism
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