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1.
J Mater Chem B ; 8(10): 2020-2031, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32091038

RESUMO

Intracellular delivery has been critical for the success of siRNA and related therapeutic nucleic acids. Improvement of delivery carriers will positively influence the efficacy of future nanomedicines. Our strategy for optimizing siRNA nanocarriers focuses on a bioinspired sequence-defined process including (i) identification of artificial amino acids active in specific delivery steps, (ii) assembly into defined sequences by solid phase-assisted synthesis (SPS), and (iii) screening for siRNA delivery, selection of top candidates and understanding structure-activity relations, followed by (iv) sequence variation for the next round of carrier selection. In the current review, our experience with this artificial peptide evolution in tumor-directed siRNA delivery is addressed. The medium-sized oligoaminoamides show better biological compatibility and can be functionalized to meet the requirements of siRNA delivery, such as formation of stable nanoparticles, shielding against proteins in the bloodstream, targeting into tumor tissue, and intracellular siRNA release in bioactive form.

2.
Bioconjug Chem ; 31(3): 729-742, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-31967454

RESUMO

The programmable endonuclease activity and simple usage of CRISPR/Cas9 have revolutionized the field of genome editing. The binding of single guide RNA (sgRNA) by the Cas9 protein results in the formation of negatively charged ribonucleoprotein (RNP) complexes. The presence of this functional complex inside cells is imperative for the intended specific genome modifications. The direct intracellular delivery of Cas9/sgRNA RNP complexes is of great advantage. In this work, a compound library of sequence-defined oligo(ethylenamino) amides containing structural motifs for stable nanoparticle formation, cellular uptake, and endosomal release was used for the screening and development of suitable Cas9 RNP delivery vehicles. Lipid-containing oligoaminoamides (lipo-OAAs) were identified as the most efficient carriers for intracellular Cas9/sgRNA delivery and gene disruption. Fluorescence correlation spectroscopy measurements indicated that the lipo-OAAs only interact with sgRNA-loaded Cas9 protein, which suggests exclusive ionic interaction with the negatively charged RNPs. The type of contained fatty acid turned out to have a critical impact on the knock out efficiency: the presence of one hydroxy group in the fatty acid dramatically changes the properties and performance of the resulting Cas9/sgRNA lipo-OAA complexes. The lipo-OAA-containing hydroxy-stearic acid (OHSteA) was superior to the analogues with saturated or unsaturated fatty acids without hydroxylation; it formed smaller and more defined nanoparticles with Cas9/sgRNA and improved the cellular uptake and endosomal release, which altogether resulted in an increased nuclear association and the highest gene knock out levels. The efficient and adaptable delivery platform has high potential for the future development of therapeutics based on precise genome modifications.

3.
PLoS One ; 14(11): e0224314, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31747409

RESUMO

Cancer progression and metastases are frequently related to changes of cell motility. Amongst others, the microRNA-200c (miR-200c) was shown to maintain the epithelial state of cells and to hamper migration. Here, we describe two miR-200c inducible breast cancer cell lines, derived from miR-200c knock-out MCF7 cells as well as from the miR-200c-negative MDA-MB-231 cells and report on the emerging phenotypic effects after miR-200s induction. The induction of miR-200c expression seems to effect a rapid reduction of cell motility, as determined by 1D microlane migration assays. Sustained expression of miR200c leads to a changed morphology and reveals a novel mechanism by which miR-200c interferes with cytoskeletal components. We find that filamin A expression is attenuated by miRNA-200c induced downregulation of the transcription factors c-Jun and MRTF/SRF. This potentially novel pathway that is independent of the prominent ZEB axis could lead to a broader understanding of the role that miR200c plays in cancer metastasis.

4.
Sci Rep ; 9(1): 15548, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664083

RESUMO

Sunitinib is a multispecific kinase inhibitor and one of its targets is the kinase GRK5, which is regulating a multitude of G protein-coupled receptors (GPCRs). In this study we demonstrate that a decreased GRK5 expression induced by knock-down experiments or sunitinib treatment hampers the migration of cancer cell lines. A proteomic analysis revealed many pathways related to cell migration which were down regulated upon the GRK5 knock-down. Furthermore, we found in MDA-MB-231 breast cancer cells that the inhibition of migration is mediated by the GPCR gastrin releasing peptide receptor (GRPR) leading to a reduced expression of migration regulating downstream targets like CDC42 and ROCK1. An in silico Kaplan Meier analysis revealed that GRK5 and GRPR overexpression reduces the distant metastasis free survival in triple-negative breast cancer (TNBC) patients. Thus, we suggest a novel anti-migratory effect of impaired GRK5 expression which induces a negative feedback loop on GRPR signalling.

5.
Biomacromolecules ; 20(10): 3613-3626, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31497946

RESUMO

Within the last two decades, a series of novel therapeutic nucleic acids entered research and clinical evaluation. Their differences both in biophysical properties as well as in mode and site of biological action provide polymer-based carriers with new delivery challenges. Recent tailor-made designs of polymeric carriers are reviewed that were optimized for nucleic acid cargos such as plasmid DNA, siRNA, and micro RNA, mRNA, or genome-modifying nucleic acids. The specific requirements for the various therapeutic cargos are discussed. Future directions include dynamic bioresponsive polymers as components of nanomachines, multifunctional sequence-defined carriers for evolution-based selective optimization, and organic-inorganic multicomponent nanoassemblies.

6.
Methods Mol Biol ; 2036: 141-164, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31410795

RESUMO

Lipopolyplexes present well-established nucleic acid carriers assembled from sequence-defined cationic lipo-oligomers and DNA or RNA. They can be equipped with additional surface functionality, like shielding and targeting, in a stepwise assembly method using click chemistry. Here, we describe the synthesis of the required compounds, an azide-bearing lipo-oligomer structure and dibenzocyclooctyne (DBCO) click agents as well as the assembly of the compounds with siRNA into a surface-functionalized formulation. Both the lipo-oligomer and the DBCO-equipped shielding and targeting agents are produced by solid-phase synthesis (SPS). This enables for precise variation of all functional units, like variation in the amount of DBCO attachment sites or polyethylene glycol (PEG) length. Special cleavage conditions with only 5% trifluoroacetic acid (TFA) must be applied for the synthesis of the shielding and targeting agents due to acid lability of the DBCO unit. The two-step lipopolyplex assembly technique allows for separate optimization of the core and the shell of the formulation.

7.
Int J Pharm ; 569: 118570, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31352048

RESUMO

Small interfering RNA (siRNA) represents a new class of therapeutic agents. Its successful intracellular delivery is a major challenge. Lipo-oligomeric carriers can complex siRNA into lipopolyplexes and thus mediate its cellular uptake. In this study, siRNA against the kinesin related mRNA EG5 gene (siEG5) and the microtubule inhibitor pretubulysin (PT) were co-formulated into polyplexes using azide-containing lipo-oligomer 1198. Nanoparticles were further modified by click reaction using shielding agent DBCO-PEG or EGFR targeting peptide GE11 (DBCO-PEG-GE11). Polyplexes displayed efficient payload incorporation and homogenous particle sizes of 200 nm. The biological effects of the unmodified and surface-functionalized polyplexes were investigated. The successful GE11-mediated intracellular delivery of siRNA into the EGFR overexpressing KB and Huh7 cell lines facilitated potent silencing of an EGFP-luciferase reporter gene by GFP siRNA. Specific downregulation of EG5 mRNA by siEG5 resulted in the expected antitumoral activity. The combination formulation 1198 siEG5 + PT provided superior antitumoral activity over free PT and 1198 siEG5.


Assuntos
Cinesina/genética , Oligopeptídeos/administração & dosagem , Peptídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Moduladores de Tubulina/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/genética , Proteínas de Fluorescência Verde/genética , Humanos , Luciferases/genética , Polietilenoglicóis/administração & dosagem
8.
Clin Cancer Res ; 25(19): 5997-6008, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31196853

RESUMO

PURPOSE: The innate tumor homing potential of mesenchymal stem cells (MSCs) has been used for a targeted delivery of the theranostic sodium iodide symporter (NIS) transgene into solid tumors. We have previously shown that external beam radiotherapy (EBRT) results in the enhanced recruitment of NIS-expressing MSCs into human hepatocellular carcinoma (HuH7). In parallel, the tumor-associated cytokine TGFB1 becomes strongly upregulated in HuH7 tumors in response to EBRT. EXPERIMENTAL DESIGN: We therefore evaluated the effects of combining focused EBRT (5 Gy) with MSC-mediated systemic delivery of the theranostic NIS transgene under control of a synthetic TGFB1-inducible SMAD-responsive promoter (SMAD-NIS-MSCs) using 123I-scintigraphy followed by 131I therapy in CD1 nu/nu mice harboring subcutaneous human hepatocellular carcinoma (HuH7). RESULTS: Following tumor irradiation and SMAD-NIS-MSC application, tumoral iodide uptake monitored in vivo by 123I-scintigraphy was enhanced as compared with nonirradiated tumors. Combination of EBRT and SMAD-NIS-MSC-mediated 131I therapy resulted in a significantly improved delay in tumor growth and prolonged survival in therapy mice as compared with the combined therapy using CMV-NIS-MSCs or to control groups receiving EBRT or saline only, or EBRT together with SMAD-NIS-MSCs and saline applications. CONCLUSIONS: MSC-based NIS-mediated 131I therapy after EBRT treatment dramatically enhanced therapeutic efficacy when a TGFB1-inducible SMAD-responsive promoter was used to drive NIS expression in adoptively applied MSCs. The remarkable therapeutic effect seen is thought to be linked in large part to the enhanced TGFB1 produced in this context, which leads to a highly selective and focused amplification of MSC-based NIS expression within the tumor milieu.

9.
Methods Mol Biol ; 1974: 83-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31098997

RESUMO

As synthetic small interfering RNA (siRNA) against antitumoral gene targets show promise for cancer treatment, different siRNA delivery systems have sparkled intense investigations. To develop tumor-specific carriers for cytosolic and systemic siRNA delivery, our laboratory has recently generated folate-conjugated targeted combinatorial siRNA polyplexes based on sequence-defined oligomer platform compatible with solid-phase-supported synthesis. These polyplexes presented efficient siRNA-mediated gene silencing in folate receptor-expressing tumors in vitro and in vivo. In this chapter, we provide a brief background on the formulation design and detailed protocols to evaluate polyplex formation, gene silencing efficiency, and receptor-directed cell killing in cancer cells using targeted combinatorial siRNA polyplexes.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Receptor 1 de Folato/genética , Neoplasias/genética , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Receptor 1 de Folato/antagonistas & inibidores , Receptor 1 de Folato/química , Ácido Fólico/genética , Inativação Gênica , Humanos , Neoplasias/terapia , Polietilenoglicóis/química , Polímeros/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/uso terapêutico
10.
Cell Death Dis ; 10(4): 302, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944311

RESUMO

Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin.

11.
Mol Pharm ; 16(6): 2405-2417, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31025870

RESUMO

In the current study, nanoparticles containing the antimetabolite drug methotrexate (MTX) and the novel tubulin-binding drug pretubulysin (PT) were developed for combination chemotherapy. Polyelectrolyte complexes were formed based on ∼20 nm cationic nanomicelles of lipo-oligomer 454 with the anionic MTX at the molar ratio of 3:1, resulting in spherical nanoparticles with sizes of 150 nm (454 MTX). Particle formation in the presence of PT, which also interacts with 454, resulted in coloaded micelle complexes (454 PT+MTX) of 170 nm as demonstrated by transmission electron microscopy and dynamic light scattering measurements. Both drugs were incorporated to a high extent (∼85% for MTX, ∼70% for PT). Nanoparticles were stable in up to 20% serum and physiological NaCl solution. Cellular internalization of 454 PT+MTX into L1210 leukemia and KB cervix carcinoma cells was determined by confocal light scattering microscopy. The antitumor activity of the drug combination PT+MTX in both cell lines was strongly increased by drug formulation with 454 with IC50 values of PT+MTX decreasing 11-fold from 0.22 nM to 19 pM on L1210 cells and 6-fold from 2.8 to 0.48 nM on KB cervix carcinoma cells. Systemic treatment of NMRI nu/nu mice bearing subcutaneous L1210 tumors with 454 PT+MTX nanoparticles resulted in a more effective delay of tumor growth in comparison to the free drug combination of PT+MTX without 454. Importantly, nanoparticle formulation of PT+MTX with 454 increased the survival of mice by more than 100% compared to that of the buffer treated group and more than 40% compared to that of the free drug group.

12.
Methods Mol Biol ; 1943: 1-25, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838606

RESUMO

Sequence-defined cationic lipo-oligomers containing unsaturated fatty acids are potent nucleic acid carriers that are produced by solid-phase supported synthesis. However, the trifluoroacetic acid (TFA)-mediated removal of acid-labile protecting groups and cleavage from the resin can be accompanied by side products caused by an addition of TFA to the double bonds of unsaturated fatty acids. These TFA adducts are converted into hydroxylated derivatives under aqueous conditions. Here we describe an optimized cleavage protocol (precooling cleavage solution to 4 °C, 20 min cleavage at 22 °C), which minimizes TFA adduct formation, retains the unsaturated hydrocarbon chain character, and ensures high yields of the synthesis.


Assuntos
Ácidos Graxos Insaturados/química , Nanopartículas/química , RNA Interferente Pequeno/genética , Transfecção/métodos , Sequência de Aminoácidos , Aminoácidos/química , Animais , Cátions/química , Linhagem Celular Tumoral , Terapia Genética/métodos , Humanos , Camundongos , Estrutura Molecular , Polimerização , Interferência de RNA , Temperatura Ambiente , Ácido Trifluoracético/química
13.
Methods Mol Biol ; 1943: 83-99, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838611

RESUMO

Nucleic acid-based therapies offer the option to treat tumors in a highly selective way, while toxicity towards healthy tissue can be avoided when proper delivery vehicles are used. We have recently developed carrier systems based on linear polyethylenimine, which after chemical coupling of protein- or peptide-based ligands can form nanosized polyplexes with plasmid DNA (pDNA) or RNA and deliver their payload into target cells by receptor-mediated endocytosis. This chapter describes the synthesis of LPEI from a precursor polymer and the current coupling techniques and purification procedure for peptide conjugates with linear polyethylenimine. A protocol is also given for the formation and characterization of polyplexes formed with LPEI conjugate and pDNA.


Assuntos
Técnicas de Química Sintética/métodos , Nanoconjugados/química , Polietilenoimina/síntese química , Terapia Genética/métodos , Humanos , Neoplasias/genética , Neoplasias/terapia , Ácidos Nucleicos/administração & dosagem , Ácidos Nucleicos/genética , Transfecção/métodos
14.
ACS Nano ; 13(4): 3884-3895, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30844241

RESUMO

Metal-organic framework nanoparticles (MOF NPs) are of growing interest in diagnostic and therapeutic applications, and due to their hybrid nature, they display enhanced properties compared to more established nanomaterials. The effective application of MOF NPs, however, is often hampered by limited control of their surface chemistry and understanding of their interactions at the biointerface. Using a surface coating approach, we found that coordinative polymer binding to Zr- fum NPs is a convenient way for peripheral surface functionalization. Different polymers with biomedical relevance were assessed for the ability to bind to the MOF surface. Carboxylic acid and amine containing polymers turned out to be potent surface coatings and a modulator replacement reaction was identified as the underlying mechanism. The strong binding of polycarboxylates was then used to shield the MOF surface with a double amphiphilic polyglutamate-polysarcosine block copolymer, which resulted in an exceptional high colloidal stability of the nanoparticles. The effect of polymer coating on interactions at the biointerface was tested with regard to cellular association and protein binding, which has, to the best of our knowledge, never been discussed in literature for functionalized MOF NPs. We conclude that the applied approach enables a high degree of chemical surface confinement, which could be used as a universal strategy for MOF NP functionalization. In this way, the physicochemical properties of MOF NPs could be tuned, which allows for control over their behavior in biological systems.

15.
Cancer Res ; 79(9): 2298-2313, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30718358

RESUMO

Antiangiogenic therapy of glioblastoma (GBM) with bevacizumab, a VEGFA-blocking antibody, may accelerate tumor cell invasion and induce alternative angiogenic pathways. Here we investigate the roles of the proangiogenic apelin receptor APLNR and its cognate ligand apelin in VEGFA/VEGFR2 antiangiogenic therapy against distinct subtypes of GBM. In proneural GBM, apelin levels were downregulated by VEGFA or VEGFR2 blockade. A central role for apelin/APLNR in controlling GBM vascularization was corroborated in a serial implantation model of the angiogenic switch that occurs in human GBM. Apelin and APLNR are broadly expressed in human GBM, and knockdown or knockout of APLN in orthotopic models of proneural or classical GBM subtypes significantly reduced GBM vascularization compared with controls. However, reduction in apelin expression led to accelerated GBM cell invasion. Analysis of stereotactic GBM biopsies from patients as well as from in vitro and in vivo experiments revealed increased dissemination of APLNR-positive tumor cells when apelin levels were reduced. Application of apelin-F13A, a mutant APLNR ligand, blocked tumor angiogenesis and GBM cell invasion. Furthermore, cotargeting VEGFR2 and APLNR synergistically improved survival of mice bearing proneural GBM. In summary, we show that apelin/APLNR signaling controls GBM angiogenesis and invasion and that both pathologic features are blunted by apelin-F13A. We suggest that apelin-F13A can improve the efficiency and reduce the side effects of established antiangiogenic treatments for distinct GBM subtypes. SIGNIFICANCE: Pharmacologic targeting of the APLNR acts synergistically with established antiangiogenic treatments in glioblastoma and blunts therapy resistance to current strategies for antiangiogenesis.See related commentary by Amoozgar et al., p. 2104.


Assuntos
Glioblastoma , Adulto , Inibidores da Angiogênese , Animais , Apelina , Receptores de Apelina , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular
16.
Gene Ther ; 26(3-4): 93-108, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30683895

RESUMO

Tumor heterogeneity, within and between tumors, may have severe implications for tumor therapy, especially for targeted gene therapy, where single-targeted approaches often result in limited efficacy and therapy resistance. Polymer-formulated nonviral vectors provide a potent delivery platform for cancer therapy. To improve applicability for future clinical use in a broad range of patients and cancer types, a dual-targeting approach was performed. Synthetic LPEI-PEG2kDa-based polymer backbones were coupled to two tumor-specific peptide ligands GE11 (EGFR-targeting) and cMBP (cMET-targeting). The dual-targeting approach was used to deliver the theranostic sodium iodide symporter (NIS) gene to hepatocellular cancer. NIS as auspicious theranostic gene allows noninvasive imaging of functional NIS gene expression and effective anticancer radioiodide therapy. Enhanced tumor-specific transduction efficiency of dual-targeted polyplexes compared to single-targeted polyplexes was demonstrated in vitro using tumor cell lines with different EGFR and cMET expression and in vivo by 124I-PET-imaging. Therapeutic efficacy of the bispecific concept was mirrored by significantly reduced tumor growth and perfusion, which was associated with prolonged animal survival. In conclusion, the dual-targeting approach highlights the benefits of a bifunctional strategy for a future clinical translation of the bioimaging-based NIS-mediated radiotherapy allowing efficient targeting of heterogeneic tumors with variable receptor expression levels.


Assuntos
Carcinoma Hepatocelular/genética , Terapia Genética/métodos , Nanomedicina Teranóstica/métodos , Animais , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Receptores ErbB/análise , Receptores ErbB/genética , Feminino , Expressão Gênica/genética , Técnicas de Transferência de Genes , Heterogeneidade Genética , Xenoenxertos , Humanos , Ligantes , Neoplasias Hepáticas/genética , Camundongos , Camundongos Nus , Peptídeos/síntese química , Peptídeos/genética , Polímeros , Proteínas Proto-Oncogênicas c-met/análise , Proteínas Proto-Oncogênicas c-met/genética
17.
Pharmacol Res Perspect ; 7(1): e00460, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30693087

RESUMO

Pretubulysin (PT), a potent tubulin-binding antitumoral drug, and the well-established antimetabolite methotrexate (MTX) were tested separately or in combination (PT+MTX) for antitumoral activity in L1210 leukemia cells or KB cervix carcinoma cells in vitro and in vivo in NMRI-nu/nu tumor mouse models. In cultured L1210 cells, treatment with PT or MTX displays strong antitumoral effects in vitro, and the combination PT+MTX exceeds the effect of single drugs. PT also potently kills the MTX resistant KB cell line, without significant MTX combination effect. Cell cycle analysis reveals the expected arrest in G1/S by MTX and in G2/M by PT. In both cell lines, the PT+MTX combination induces a G2/M arrest which is stronger than the PT-triggered G2/M arrest. PT+MTX does not change rates of apoptotic L1210 or KB cells as compared to single drug applications. Confocal laser scanning microscopy images show the microtubule disruption and nuclear fragmentation induced by PT treatment of L1210 and KB cells. MTX changes the architecture of the F-actin skeleton. PT+MTX combines the toxic effects of both drugs. In the in vivo setting, the antitumoral activity of drugs differs from their in vitro cytotoxicity, but their combination effects are more pronounced. MTX on its own does not display significant antitumoral activity, whereas PT reduces tumor growth in both L1210 and KB in vivo models. Consistent with the cell cycle effects, MTX combined at moderate dose boosts the antitumoral effect of PT in both in vivo tumor models. Therefore, the PT+MTX combination may present a promising therapeutic approach for different types of cancer.


Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Metotrexato/uso terapêutico , Oligopeptídeos/uso terapêutico , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia L1210/tratamento farmacológico , Metotrexato/farmacologia , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Oligopeptídeos/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico
18.
Endocr Relat Cancer ; 26(1): 89-101, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30121623

RESUMO

Based on their excellent tumor-homing capacity, genetically engineered mesenchymal stem cells (MSCs) are under investigation as tumor-selective gene delivery vehicles. Transgenic expression of the sodium iodide symporter (NIS) in genetically engineered MSCs allows noninvasive tracking of MSC homing by imaging of functional NIS expression as well as therapeutic application of 131I. The use of tumor stroma-activated promoters can improve tumor-specific MSC-mediated transgene delivery. The essential role of transforming growth factor B1 (TGFB1) and the SMAD downstream target in the signaling between tumor and the surrounding stroma makes the biology of this pathway a potential option to better control NIS expression within the tumor milieu. Bone marrow-derived MSCs were stably transfected with a NIS-expressing plasmid driven by a synthetic SMAD-responsive promoter (SMAD-NIS-MSCs). Radioiodide uptake assays revealed a 4.9-fold increase in NIS-mediated perchlorate-sensitive iodide uptake in SMAD-NIS-MSCs after TGFB1 stimulation compared to unstimulated cells demonstrating the successful establishment of MSCs, which induce NIS expression in response to activation of TGFB1 signaling using a SMAD-responsive promoter. 123I-scintigraphy revealed significant tumor-specific radioiodide accumulation and thus NIS expression after systemic application of SMAD-NIS-MSCs into mice harboring subcutaneous tumors derived from the human hepatocellular carcinoma (HCC) cell line HuH7, which express TGFB1. 131I therapy in SMAD-NIS-MSCs-treated mice demonstrated a significant delay in tumor growth and prolonged survival. Making use of the tumoral TGFB1 signaling network in the context of MSC-mediated NIS gene delivery is a promising approach to foster tumor stroma-selectivity of NIS transgene expression and tailor NIS-based gene therapy to TGFB1-rich tumor environments.

19.
Breast ; 43: 31-38, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30415143

RESUMO

BACKGROUND: MicroRNA-27a (miR-27a) is a small non-coding RNA, shown to play a role in multiple cancers, including the regulation of ERα expression in breast cancer. Most ERα positive tumors are treated with Selective Estrogen Receptor Modulators (SERMs) and thus the role of miR-27a expression in response to SERM treatment is of interest. METHODS: Tamoxifen resistant cells were generated by molecular evolution with six cycles of tamoxifen treatment. MCF7 and T47D luminal A breast cancer cell lines were either treated with miR-27a mimics, or ER-signaling was modulated ectopically. The changes were analyzed with RT-qPCR, western blotting and transcriptional activity ERE-reporter assays. Moreover, the response to SERM treatments (tamoxifen, endoxifen and toremifen) was investigated by cell viability and apoptosis measurements. An in silico analysis of survival data from the METABRIC study was performed in order to assess the prognostic value of miR-27a for response to SERM treatment. RESULTS: Tamoxifen-resistant cells showed decreased expression of ERα and miR-27a. The overexpression of miR-27a increased the levels of ERα, while modulation of ERα decreased miR-27a expression. High miR-27a expression increased the sensitivity of MCF7 and T47D cells to SERM treatments and re-sensitized the cells to tamoxifen. Patient survival of luminal A breast cancer patients that underwent endocrine therapies was better in groups with high miR-27a expression. CONCLUSION: MiR-27a sensitizes luminal A breast cancer cells to SERM treatments based on a positive feedback loop with ERα. An increased overall-survival of ER-positive breast cancer patients that underwent endocrine treatments and displayed high miR-27a levels was found.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , MicroRNAs/genética , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Simulação por Computador , Retroalimentação Fisiológica , Feminino , Humanos , Células MCF-7 , Prognóstico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Análise de Sobrevida , Tamoxifeno/uso terapêutico
20.
Bioconjug Chem ; 29(11): 3649-3657, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30235924

RESUMO

Sequence-defined cationic lipo-oligomers are potent siRNA carriers, forming stable lipo-polyplexes based on both electrostatic and hydrophobic interactions and, after endocytosis and endosomal protonation, facilitating the delivery of siRNA into the cytosol. After completion of the nucleic acid delivery process, carriers should be readily biodegradable to ensure minimum accumulation of amphiphilic molecules that are harmful to lysosomes and other intracellular organelles. Endolysosomal enzymes may degrade a surplus of carrier molecules left over in lysosomes and thereby facilitate the generation and rapid excretion of cleavage products. By solid-phase supported synthesis, a library of sequence-defined lipo-oligomers was generated containing artificial and natural amino acids comprising precise enzymatic cleavage sites. Incorporating either short cleavable l-arginine sequences (RR), noncleavable d-arginine linkers (rr), or varieties of both tailored the degradability of lipo-oligomers, as demonstrated upon incubation with the endolysosomal protease cathepsin B. Cleavage products were identified by MALDI-TOF mass spectrometry. The effect of improved intracellular degradation on cell tolerability was studied by transfecting Huh7-eGFPLuc and DU145-eGFPLuc cells. Positioning of enzymatic cleavage sites between a lipophilic diacyl domain and an ionizable oligocationic siRNA binding unit enabled efficient enzymatic degradation of the carrier and reduced the lytic potential under lysosomal conditions. Highly degradable carriers containing at least one l-arginine dipeptide linker significantly improved the viability of transfected cells without hampering gene silencing activity. Therefore, the precise integration of enzymatic cleavage sites in lipo-oligomers is a promising strategy toward biocompatible nucleic acid carriers.


Assuntos
Catepsina B/metabolismo , Metabolismo dos Lipídeos , RNA Interferente Pequeno/metabolismo , Aminoácidos/metabolismo , Linhagem Celular , Inativação Gênica , Humanos , Hidrólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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