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1.
Mol Cancer Ther ; 12(11): 2308-18, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23943805

ABSTRACT

Successful siRNA therapeutics requires the optimal integration of multiple components, including an efficient delivery system, a disease indication that is appropriate for siRNA-based therapy, and a potent and nontoxic siRNA against a robust therapeutic target. Although all currently available delivery systems have limitations, it is important to recognize that a careful selection of the disease indication, therapeutic target, and siRNA molecule could partially compensate for deficiencies associated with the delivery system and makes it possible to advance a therapeutic siRNA regimen. In this study, we present the development of siRNA therapeutics for hepatocellular carcinoma using an integrated approach, including the development of an efficient lipid nanoparticle delivery system, the identification of a robust therapeutic target that does not trigger liver toxicity upon target knockdown, and the selection of potent and nonimmunogenic siRNA molecules against the target. The resulting siRNA-containing lipid nanoparticles produced significant antitumor efficacy in orthotopic hepatocellular carcinoma models, and, thus, represent a promising starting point for the development of siRNA therapeutics for hepatocellular carcinoma.


Subject(s)
Carcinoma, Hepatocellular/drug therapy , Drug Delivery Systems/methods , Genetic Therapy/methods , Liver Neoplasms/drug therapy , Nanoparticles , RNA, Small Interfering/administration & dosage , Animals , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Female , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms, Experimental , Mice , Mice, SCID , RNA, Small Interfering/genetics , RNA, Small Interfering/toxicity
2.
Cancer Res ; 67(9): 4390-8, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17483353

ABSTRACT

To identify new candidate cancer drug targets, we used RNAi as a tool to functionally evaluate genes that play a role in maintaining human tumor cell survival. We screened a small interfering RNA (siRNA) library directed against approximately 3,700 individual genes to assess the ability of siRNAs to induce cell death in an in vitro cell cytotoxicity assay. We found that siRNAs specifically targeting ras-related nuclear protein (Ran), targeting protein for Xenopus kinesin-like protein 2 (TPX2), and stearoyl-CoA desaturase 1 (SCD1), significantly reduced the survival of multiple human tumor cell lines. Further target validation studies revealed that treatment with Ran and TPX2 siRNAs differentially reduced the survival of activated K-Ras-transformed cells compared with their normal isogenic counterparts in which the mutant K-Ras gene had been disrupted (DKS-8). Knockdown of Ran and TPX2 in activated mutant K-Ras cells selectively induced S-phase arrest or transient G(2)-M arrest phenotypes, respectively, that preceded apoptotic cell death. Given our observations that Ran and TPX2 depletion preferentially reduces the survival of activated K-Ras-transformed cells, these two proteins may serve as useful anticancer targets in tumors expressing the activated K-Ras oncogene.


Subject(s)
Cell Cycle Proteins/genetics , Microtubule-Associated Proteins/genetics , Neoplasm Proteins/genetics , Neoplasms/genetics , Nuclear Proteins/genetics , Phosphoproteins/genetics , RNA, Small Interfering/genetics , Stearoyl-CoA Desaturase/genetics , Xenopus Proteins/genetics , ran GTP-Binding Protein/genetics , Cell Cycle/genetics , Cell Death/genetics , Cell Line, Tumor , Cell Survival/genetics , Gene Library , Genes, ras , Humans , Neoplasms/pathology , RNA Interference
3.
Cancer Res ; 67(3): 1176-83, 2007 Feb 01.
Article in English | MEDLINE | ID: mdl-17283153

ABSTRACT

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.


Subject(s)
Biphenyl Compounds/pharmacology , Carcinoma, Small Cell/drug therapy , Carcinoma, Small Cell/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Nitrophenols/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfonamides/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/biosynthesis , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Biphenyl Compounds/administration & dosage , Carboplatin/administration & dosage , Carcinoma, Small Cell/pathology , Cell Growth Processes/drug effects , Cell Line, Tumor , Down-Regulation , Drug Synergism , Etoposide/administration & dosage , Humans , Lung Neoplasms/pathology , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Nitrophenols/administration & dosage , Piperazines/administration & dosage , Piperazines/pharmacology , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Small Interfering/genetics , Sulfonamides/administration & dosage , Transfection , Up-Regulation
4.
Mol Cancer Ther ; 6(1): 269-76, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17237286

ABSTRACT

To identify cancer-specific targets, we have conducted a synthetic lethal screen using a small interfering RNA (siRNA) library targeting approximately 4,000 individual genes for enhanced killing in the DLD-1 colon carcinoma cell line that expresses an activated copy of the K-Ras oncogene. We found that siRNAs targeting baculoviral inhibitor of apoptosis repeat-containing 5 (survivin) significantly reduced the survival of activated K-Ras-transformed cells compared with its normal isogenic counterpart in which the mutant K-Ras gene had been disrupted (DKS-8). In addition, survivin siRNA induced a transient G(2)-M arrest and marked polyploidy that was associated with increased caspase-3 activation in the activated K-Ras cells. These results indicate that tumors expressing the activated K-Ras oncogene may be particularly sensitive to inhibitors of the survivin protein.


Subject(s)
Cell Transformation, Neoplastic/pathology , Genes, ras , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/deficiency , Neoplasm Proteins/metabolism , Alleles , Apoptosis , Cell Death , Cell Survival , Clone Cells , G2 Phase , Genes, Neoplasm , Humans , Inhibitor of Apoptosis Proteins , Mitosis , Mutant Proteins/metabolism , Polyploidy , RNA, Small Interfering/metabolism , Survivin
5.
Proc Natl Acad Sci U S A ; 100(11): 6347-52, 2003 May 27.
Article in English | MEDLINE | ID: mdl-12746500

ABSTRACT

Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.


Subject(s)
Gene Expression , RNA, Small Interfering/metabolism , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction
6.
J Biol Chem ; 278(1): 486-90, 2003 Jan 03.
Article in English | MEDLINE | ID: mdl-12419797

ABSTRACT

Survivin, one of the most tumor-specific gene products, has been implicated in both anti-apoptosis and cytokinesis. However, the mechanism by which survivin regulates these two different processes is still elusive. Here, we show that survivin binds to the catalytic domain of Aurora-B. We demonstrate that in the presence of survivin, Aurora-B phosphorylates histone H3 much more efficiently than in the absence of survivin in a cell-free system. Furthermore, we confirm that cells lacking survivin due to survivin antisense oligonucleotide-treatment have lower Aurora-B kinase activity, whereas cells overexpressing survivin have higher Aurora-B kinase activity. We also provide evidence that depletion of survivin by survivin antisense oligonucleotide treatment causes significant reduction of endogenous phosphorylated histone H3 and mislocalization of Aurora-B. These results indicate that survivin stimulates Aurora-B kinase activity and helps correctly target Aurora-B to its substrates during the cell cycle, thus providing a mechanism as to how survivin exerts its function in human cells.


Subject(s)
Cysteine Proteinase Inhibitors/metabolism , Microtubule-Associated Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Aurora Kinase B , Aurora Kinases , Catalytic Domain , Cell Cycle/physiology , Cell Line , Down-Regulation/physiology , Histones/metabolism , Humans , Immunohistochemistry , Inhibitor of Apoptosis Proteins , Macromolecular Substances , Microtubule-Associated Proteins/genetics , Neoplasm Proteins , Oligonucleotides, Antisense/metabolism , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/genetics , Recombinant Fusion Proteins/metabolism , Survivin
7.
Microbiology (Reading) ; 143 ( Pt 2): 367-376, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9043114

ABSTRACT

The BGL2 gene encodes a unique 1,3-beta-glucosyltransferase (Bgl2p) present in the cell wall of Candida albicans and other fungi. Although believed to be involved in cell wall assembly, disruption of the gene in saccharomyces cerevisiae showed no apparent phenotype. We performed sequential disruptions of the BGL2 loci in a homozygous ura3 clinical isolate of C. albicans using the URA3 blaster method, in order to investigate the role of Bgl2p in this dimorphic, pathogenic fungus. Strain CACW-1 contained disruptions of both homologues of the BGL2 gene and lacked Bgl2p, as assessed by protein extraction, SDS-PAGE and Western blot analysis, and enzyme assay; however, residual non-Bgl2p transferase activity was detected. CACW-1 was attenuated in virulence for mice when compared to an isogenic parent strain, and fewer organisms were recovered from the kidneys of infected animals. Additional phenotypic changes included: (1) a dramatic increase in the sensitivity to the chitin synthesis inhibitor nikkomycin Z when CACW-1 cells were incubated at 37 or 42 degrees C; (2) an 8.7 +/- 1.6% slower growth rate at 37 degrees C for CACW-1 when compared to its isogenic parent; and (3) aggregation of CACW-1 cells during stationary phase and/or incubation of stationary phase cells in phosphate buffer. Characterization of SDS-extracted cell walls did not reveal any significant differences in the levels of 1,3-beta- or 1,6-beta-glucan. These data reveal that loss of Bgl2p does have a phenotype in C. albicans, and indicate that (1) loss of Bgl2p function renders cells more dependent on chitin for wall integrity, and attenuates virulence (probably due to subtle changes in wall structure), and (2) that additional 1,3-beta-glucosyltransferases are present in the C. albicans BGL2 disruptant.


Subject(s)
Candida albicans/genetics , Fungal Proteins/genetics , Genes, Fungal , Glucan Endo-1,3-beta-D-Glucosidase/genetics , Glucosyltransferases/genetics , Animals , Antifungal Agents/pharmacology , Candida albicans/enzymology , Candida albicans/pathogenicity , Candidiasis , Cell Wall/chemistry , Cell Wall/metabolism , Cell Wall/ultrastructure , Glucans/chemistry , Glycosylation , Kidney/microbiology , Mice , Microbial Sensitivity Tests , Mutagenesis , Phenotype , Virulence/genetics
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