ABSTRACT
Wnt-5a is a secreted glycoprotein which has been shown to be involved in the regulation of cell adhesion and motility, processes which are of importance in metastasis formation by cancer cells. We here present an initial study aiming at evaluating whether small interfering RNA (siRNA) in combination with cisplatin can be used to modulate protein expression levels under in vitro conditions. For this purpose, an AU-rich region corresponding to the initial 260 bases of the Wnt-5a 3' untranslated region was chosen as the target. The effect of four different siRNAs was evaluated by analysis of protein suppression levels in rabbit reticulocyte lysate (RRL) and an immortalized noncancerous mammary epithelial (HB2) cell line by monitoring the activity of transiently expressed luciferase. The specificity and kinetics for hybridization of the siRNA with the messenger RNA target were followed by digestion techniques and analysis by polyacrylamide gel electrophoresis. Specific and temperature-dependent hybridization was observed, with a half-life of approximately 0.5 h at 4 degrees C. Significant downregulation of luciferase activity was obtained in the micromolar and nanomolar range, for RRL and HB2, respectively. In addition, the downregulation of protein production caused by addition of cisplatin could be further potentiated by addition of siRNA in a selective manner. The latter observation suggests that combined use of cisplatin and siRNA could be a method to decrease therapeutically used cisplatin concentrations. Thus, toxic side effects could be minimized while key proteins are targeted in a highly specific manner.
Subject(s)
3' Untranslated Regions , Cisplatin/pharmacology , Proto-Oncogene Proteins/genetics , RNA, Messenger/chemistry , RNA, Small Interfering , Wnt Proteins/genetics , Base Sequence , Cell Line, Transformed , DNA, Complementary , Drug Evaluation, Preclinical , Humans , In Vitro Techniques , Molecular Sequence Data , Phosphorylation , Wnt-5a ProteinABSTRACT
We here report the synthesis of the two polyamine-based nucleoside derivatives 5-{[bis-(3-aminopropyl)amino]acetamido-1-propynyl}uridine and 2-{[bis-(3-aminopropyl)amino]-acetamido-1-propynyl}adenosine. The various polyamine derivatives have been used in thermal melting analysis using DNA from herring testes, and in cellular studies using four different cell lines. The compounds were all found to be non-toxic, thus holding good promise for future use as siRNA building blocks.
Subject(s)
Adenosine/analogs & derivatives , Adenosine/chemical synthesis , Adenosine/pharmacology , Breast Neoplasms/drug therapy , DNA/drug effects , Polyamines/chemistry , Uridine/analogs & derivatives , Uridine/chemical synthesis , Uridine/pharmacology , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Circular Dichroism/methods , DNA/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , In Vitro Techniques , Molecular Structure , Structure-Activity Relationship , Temperature , Tumor Cells, CulturedABSTRACT
The use of short interfering RNAs (siRNA) for selective suppression of protein production has rapidly become a commonly used technique for transient modulation of protein levels. In the present paper, we investigate whether introduction of platinated bases in the sense strand can be used to modulate the efficacy of siRNAs. Four different siRNAs were studied, all targeting the initial AU-rich 3' UTR of Wnt-5a mRNA. The siRNAs were characterized with respect to melting properties and translational inhibitory effect in vitro using luciferase as a reporter gene. The translation inhibition studies reveal that all platinated siRNA remain efficient. For an siRNA with partial complementarity to the luciferase gene, platination was shown to reduce the off-target effects. All siRNAs were found to be active in cellular in vitro translation systems, reaching suppression levels well above 80% for the majority of siRNAs investigated.
Subject(s)
RNA Interference , RNA, Small Interfering/chemistry , 3' Untranslated Regions/chemistry , Cell Line , Cisplatin/chemistry , Humans , Protein Biosynthesis/drug effects , RNA, Small Interfering/pharmacology , Temperature , Wnt Proteins/geneticsABSTRACT
The DNA interference pathways exhibited by cisplatin and related anticancer active metal complexes have been extensively studied. Much less is known to what extent RNA interaction pathways may operate in parallel, and perhaps contribute to both antineoplastic activity and toxicity. The present study was designed with the aim of comparing the reactivity of two model systems comprising RNA and DNA hairpins, r(CGCGUUGUUCGCG) and d(CGCGTTGTTCGCG), towards a series of platinum(II) complexes. Three platinum complexes were used as metallation reagents; cis-[PtCl(NH3)2(OH2)]+ (1), cis-[PtCl(NH3)(c-C6H11NH2)(OH2)]+ (2), and trans-[PtCl(NH3)(quinoline)(OH2)]+ (3). The reaction kinetics were studied at pH 6.0, 25 degrees C, and 1.0 mM < or = I < or = 500 mM. For both types of nucleic acid targets, compound 3 was found to react about 1 order of magnitude more rapidly than compounds 1 and 2. Further, all platinum compounds exhibited a more pronounced salt dependence for the interaction with r(CGCGUUGUUCGCG). Chemical and enzymatic cleavage studies revealed similar interaction patterns with r(CGCGUUGUUCGCG) after long exposure times to 1 and 2. A substantial decrease of cleavage intensity was found at residues G4 and G7, indicative of bifunctional adduct formation. Circular dichroism studies showed that platinum adduct formation leads to a structural change of the ribonucleic acid. Thermal denaturation studies revealed platination to cause a decrease of the RNA melting temperatures by 5-10 degrees C. Our observations therefore suggest that RNA is a kinetically competitive target to DNA. Furthermore, platination causes destabilization of RNA structural elements, which may lead to deleterious intracellular effects on biologically relevant RNA targets.
