The constitutively active serine/threonine kinase Pim-1 is upregulated in different cancer types, mainly based on the action of several interleukines and growth factors at the transcriptional level. So far, a regulation of oncogenic Pim-1 by microRNAs (miRNAs) has not been reported. Here, we newly establish miR-33a as a miRNA with potential tumor suppressor activity, acting through inhibition of Pim-1. A screen for miRNA expression in K562 lymphoma, LS174T colon carcinoma and several other cell lines revealed generally low endogenous miR33a levels relative to other miRNAs. Transfection of K562 and LS174T cells with a miR-33a mimic reduced Pim-1 levels substantially. In contrast, the cell-cycle regulator cyclin-dependent kinase 6 predicted to be a conserved miR-33a target, was not downregulated by the miR-33a mimic. Seed mutagenesis of the Pim-1 3 0 -untranslated region in a luciferase reporter construct and in a Pim-1 cDNA expressed in Pim-1-deficient Skov-3 cells demonstrated specific and direct downregulation of Pim-1 by the miR-33a mimic. The persistence of this effect was comparable to that of a small interfering RNA-mediated knockdown of Pim-1, resulting in decelerated cell proliferation. In conclusion, we demonstrate the potential of miR-33a to act as a tumor suppressor miRNA, which suggests miR-33a replacement therapy through delivery of miR mimics as a novel therapeutic strategy.
PurposeWhile the benefits of ascorbic acid (vitamin C, ascorbate) as an essential nutrient are well established, its effects on tumor cells and in tumor treatment are controversial. In particular, conflicting data exist whether ascorbate may increase the cytotoxic effects of antineoplastic drugs or may rather exert adverse effects on drug sensitivity during cancer treatment. Findings are further obscured regarding the distinction between ascorbate and dehydroascorbate (DHA). Thus, the purpose of this study was to evaluate and directly compare the cytotoxic efficacy of ascorbate compared to DHA, and to analyse if ascorbate at pharmacological concentrations affects the efficacy of antineoplastic agents in prostate carcinoma cells.MethodsWe directly compare the effects of ascorbate (supplied as ‘Pascorbin® solution for injection’) and DHA on tumor cell viability, and determine IC50 values for various cell lines. At concentrations well below the IC50, ascorbate effects on cell proliferation and cell cycle are analysed. We furthermore determine changes in cellular sensitivity towards various cytostatic drugs upon pre-treatment of cells with ascorbate.ResultsWe demonstrate higher therapeutic efficacy of ascorbate over DHA in various cell lines, independent of cell line-specific differences in ascorbate sensitivity, and identify the extracellular generation of H2O2 as critical mechanism of ascorbate action. We furthermore show that, in addition to pro-apoptotic effects described previously, ascorbate treatment already at concentrations well below the IC50 exerts anti-proliferative effects on tumor cells. Those are based on interference with the cell cycle, namely by inducing a G0/G1 arrest. Pre-treatment of tumor cells with ascorbate leads to increased cellular sensitivity towards Docetaxel, Epirubicin, Irinotecan and 5-FU, but not towards Oxaliplatin and Vinorelbin. For Docetaxel and 5-FU, a linear correlation between this sensitizing effect and the ascorbate dosage is observed.ConclusionsThe redox-active form of vitamin C, ascorbate, shows therapeutic efficacy in tumor cells. These antitumor effects of ascorbate are mainly based on its extracellular action and, in addition to the induction of apoptosis, also include an anti-proliferative effect by inducing cell cycle arrest. Furthermore, ascorbate treatment specifically enhances the cytostatic potency of certain chemotherapeutics, which implicates therapeutic benefit during tumor treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s00280-010-1418-6) contains supplementary material, which is available to authorized users.
Polycationic polymers like poly(ethylene imine)s (PEIs) are extensively explored for the nonviral transfer of DNA or small RNAs (siRNAs). To enhance biocompatibility and alter pharmacokinetic properties, hyperbranched PEI was recently grafted with the nonligand oligosaccharides maltose or maltotriose at various degrees in a systematic study to yield (oligo-)maltose PEIs (OM-PEIs). In this paper, we investigate the in vivo biocompatibility and efficacy of a whole set of (OM-)PEIs and the corresponding (OM-)PEI-based DNA or siRNA complexes upon systemic (intravenous, i.v.) administration in mice. We determine the overall survival and animal welfare, hepatotoxicity, immune stimulation, erythrocyte aggregation, and the efficacy of DNA delivery in vivo. Higher-degree oligomaltose-grafting of PEI substantially decreases weight loss, abolishes lethality upon repeated treatment with the free polymers or with complexes, and abrogates hepatotoxicity, as determined by serum levels of liver enzymes. Immunostimulatory effects (TNF-α, IFN-γ) and erythrocyte aggregation are mainly observed upon treatment with partially maltotriose-grafted PEI or PEI-based complexes and are largely abolished upon higher-degree grafting. In vivo transfection experiments in mice bearing subcutaneous (s.c.) tumor xenografts reveal a strong dependence of reporter gene expression in a given organ on the mode of complex administration (i.v. vs intraperitoneal injection) and the OM-PEI architecture, with high-level maltose-grafted PEI (PEI-(2-Mal)) being most efficient for DNA delivery. We conclude that distinct differences between different patterns of maltose- or maltotriose-grafting are observed with regard to both biocompatibility and in vivo efficacy and identify optimal oligomaltose-PEIs for therapeutic applications.
RNA interference (RNAi) is a naturally occurring, powerful mechanism for gene silencing, based on the cleavage of a given target mRNA. It relies on small interfering RNAs (siRNAs) in the cell. Being similar in structure, microRNAs (miRNAs) are important regulators of gene expression which mainly act by blocking mRNA translation. In cancer, certain miRNAs have been found to be pathologically downregulated. The therapeutic application of siRNAs or miRNAs for the induction of RNAi or miRNA replacement, respectively, relies on their efficient delivery through a non-viral formulation. Complexation of siRNAs/miRNAs in polymeric nanoparticles based on polyethylenimines (PEIs) offers protection against degradation, delivery to the target site, cellular uptake, and intracellular release. This chapter provides protocols for therapeutic gene silencing and miRNA replacement therapy, based on PEI complexes for in vitro and in vivo use.
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