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1.
Nucleic Acid Ther ; 25(4): 198-208, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26042602

ABSTRACT

We studied the ability of polypurine reverse Hoogsteen hairpins (PPRHs) to silence a variety of relevant cancer-related genes in several human cell lines. PPRHs are hairpins formed by two antiparallel polypurine strands bound by intramolecular Hoogsteen bonds linked by a pentathymidine loop. These hairpins are able to bind to their target DNA sequence through Watson-Crick bonds producing specific silencing of gene expression. We designed PPRHs against the following genes: BCL2, TOP1, mTOR, MDM2, and MYC and tested them for mRNA levels, cytotoxicity, and apoptosis in prostate, pancreas, colon, and breast cancer cell lines. Even though all PPRHs were effective, the most remarkable results were obtained with those against BCL2 and mammalian target of rapamycin (mTOR) in decreasing cell survival and mRNA levels and increasing apoptosis in prostate, colon, and pancreatic cancer cells. In the case of TOP1, MDM2, and MYC, their corresponding PPRHs produced a strong effect in decreasing cell viability and mRNA levels and increasing apoptosis in breast cancer cells. Thus, we confirm that the PPRH technology is broadly useful to silence the expression of cancer-related genes as demonstrated using target genes involved in metabolism (DHFR), proliferation (mTOR), DNA topology (TOP1), lifespan and senescence (telomerase), apoptosis (survivin, BCL2), transcription factors (MYC), and proto-oncogenes (MDM2).


Subject(s)
Gene Silencing , Genes, Neoplasm , Inverted Repeat Sequences , Poly T/genetics , RNA, Messenger/antagonists & inhibitors , Apoptosis/genetics , Base Pairing , Cell Line, Tumor , Cell Survival , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , Female , Humans , Male , Poly T/metabolism , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
2.
BMC Res Notes ; 6: 454, 2013 Nov 12.
Article in English | MEDLINE | ID: mdl-24215842

ABSTRACT

BACKGROUND: MicroRNAs are small non-coding RNAs involved in gene expression regulation by targeting specific regions in the 3'-UTR of the mRNA of their target genes. This binding leads to a decrease in the protein levels of such genes either by mRNA degradation or mRNA destabilization and translation inhibition. The interaction between a miRNA and its target mRNAs is usually studied by co-transfection of a reporter expression vector containing the 3'-UTR region of the mRNA and an inhibitory or precursor molecule for the miRNA. This approach, however, does not measure the direct and physical interaction between a miRNA and a specific mRNA. FINDINGS: RNA molecules corresponding to miR-224 and to the 3'-UTR of SLC4A4 were incubated together and their interaction studied under different binding conditions using electrophoretic mobility shift assays. A direct and specific interaction between miR-224 and SLC4A4 mRNA was observed. This interaction was abolished in the presence of competitors. CONCLUSIONS: In this study, we explored a new application for the electrophoretic mobility shift assay and we demonstrated that it is a useful alternative method to assess, in a direct and specific manner, whether a miRNA binds to a specific predicted target mRNA.


Subject(s)
3' Untranslated Regions/genetics , Electrophoretic Mobility Shift Assay , MicroRNAs/chemistry , Ribonucleotides/chemistry , Sodium-Bicarbonate Symporters/chemistry , Binding Sites , Gene Expression Regulation , Humans , MicroRNAs/genetics , Phosphorus Radioisotopes , RNA Stability , Ribonucleotides/genetics , Sodium-Bicarbonate Symporters/genetics
3.
PLoS One ; 8(5): e63276, 2013.
Article in English | MEDLINE | ID: mdl-23675469

ABSTRACT

BACKGROUND: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. RESULTS: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexate-induced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. CONCLUSIONS: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Cytochromes c/genetics , Gene Expression Regulation, Neoplastic/drug effects , Methotrexate/pharmacology , Apoptosis/drug effects , Ascorbic Acid/pharmacology , Cytochromes c/metabolism , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Glutathione/pharmacology , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , MCF-7 Cells , Oxidation-Reduction/drug effects , Reducing Agents/pharmacology , Signal Transduction , Tetramethylphenylenediamine/pharmacology
4.
Biochem Pharmacol ; 84(12): 1581-91, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-23018034

ABSTRACT

Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the Match™ software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.


Subject(s)
Cell Proliferation , Genomics , Neoplasms/pathology , Sp1 Transcription Factor/genetics , Base Sequence , Blotting, Western , Chromatin Immunoprecipitation , DNA Primers , HeLa Cells , Humans , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Real-Time Polymerase Chain Reaction , Sp1 Transcription Factor/metabolism
5.
Biochem Pharmacol ; 82(11): 1572-82, 2011 Dec 01.
Article in English | MEDLINE | ID: mdl-21864507

ABSTRACT

MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA silencing that play a role in many biological processes. They are involved in the development of many diseases, including cancer. Extensive experimental data show that they play a role in the pathogenesis of cancer as well as the development of drug resistance during treatments. The aim of this work was to detect differentially expressed miRNAs in MTX-resistant cells. Thus, miRNA microarrays of sensitive and MTX-resistant HT29 colon cancer cells were performed. The results were analyzed using the GeneSpring GX11.5 software. Differentially expressed miRNAs in resistant cells were identified and miR-224, which was one of the most differentially expressed miRNAs and with high raw signal values, was selected for further studies. The underexpression of miR-224 was also observed in CaCo-2 and K562 cells resistant to MTX. Putative targets were predicted using TargetScan 5.1 software and integrated with the data from expression microarrays previously performed. This approach allowed us to identify miR-224 targets that were differentially expressed more than 2-fold in resistant cells. Among them CDS2, DCP2, HSPC159, MYST3 and SLC4A4 were validated at the mRNA level by qRT-PCR. Functional assays using an anti-miR against miR-224 desensitized the cells towards MTX, mimicking the resistant phenotype. On the other hand, siRNA treatment against SLC4A4 or incubation of Poly Purine Reverse Hoogsteen (PPRH) hairpins against CDS2 or HSPC159 increased sensitivity to MTX. These results revealed a role for miR-224 and its targets in MTX resistance in HT29 colon cancer cells.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Drug Resistance, Neoplasm , Methotrexate/pharmacology , MicroRNAs/metabolism , 3' Untranslated Regions , Caco-2 Cells , Cell Line, Tumor , Colonic Neoplasms , Humans , K562 Cells , Lectins/metabolism , MicroRNAs/antagonists & inhibitors , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sodium-Bicarbonate Symporters/genetics , Sodium-Bicarbonate Symporters/metabolism
6.
Hum Gene Ther ; 22(4): 451-63, 2011 Apr.
Article in English | MEDLINE | ID: mdl-20942657

ABSTRACT

Polypurine reverse-Hoogsteen hairpins (PPRHs) are double-stranded DNA molecules formed by two polypurine stretches linked by a pentathymidine loop, with intramolecular reverse-Hoogsteen bonds that allow a hairpin structure. PPRHs bind to polypyrimidine targets by Watson-Crick bonds maintaining simultaneously a hairpin structure due to intramolecular Hoogsteen bonds. Previously, we described the ability of Template-PPRHs to decrease mRNA levels because these PPRHs target the template DNA strand interfering with the transcription process. Now, we designed Coding-PPRHs, a new type of PPRHs that directly target the pre-mRNA. The dihydrofolate reductase (dhfr) gene was selected as a target in breast cancer therapy. These PPRHs caused a high degree of cytotoxicity and a decrease in DHFR mRNA and protein levels, but by a different mechanism of action than Template-PPRHs. Coding-PPRHs interfere with the splicing process by competing with U2 auxiliary factor 65 for binding to the polypyrimidine target sequence, leading to a lower amount of mature mRNA. These new PPRHs showed high specificity as no off-target effects were found. The application of these molecules as therapeutic tools was tested in breast cancer cells resistant to methotrexate, obtaining a noticeable cytotoxicity even though the dhfr locus was amplified. Coding-PPRHs can be considered as new molecules to decrease gene expression at the mRNA level and an alternative to other antisense molecules.


Subject(s)
Breast Neoplasms/metabolism , DNA/genetics , DNA/metabolism , Inverted Repeat Sequences/genetics , Breast Neoplasms/genetics , Cell Death/genetics , Cell Line, Tumor , DNA/pharmacology , DNA/toxicity , DNA, Single-Stranded/metabolism , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Enzyme Activation/drug effects , Female , Humans , Methotrexate/pharmacology , Nuclear Proteins/metabolism , Protein Binding/drug effects , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism , Splicing Factor U2AF , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolism
7.
BMC Cancer ; 10: 250, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20515499

ABSTRACT

BACKGROUND: Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance. METHODS: The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for beta-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway. RESULTS: S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. beta-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells. CONCLUSIONS: S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistance.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Colonic Neoplasms/metabolism , Drug Resistance, Neoplasm , Methotrexate/pharmacology , S100 Proteins/metabolism , Blotting, Western , Caco-2 Cells , Cell Survival/drug effects , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Drug Resistance, Neoplasm/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , HT29 Cells , Humans , Oligonucleotide Array Sequence Analysis , RNA Interference , RNA, Messenger/metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , S100 Calcium-Binding Protein A4 , S100 Proteins/genetics , Transcription, Genetic , Transfection , Up-Regulation , beta Catenin/genetics , beta Catenin/metabolism
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