1,720,982 research outputs found
Potent Apoptotic Response Induced by Chloroacetamidine Anthrathiophenediones in Bladder Cancer Cells.
Full text linkWe previously found that two neighboring G-quadruplexes behave as a molecular switch controlling the expression of HRAS (Cogoi, S.; Schekotikhin, A. E.; Xodo, L. E. Nucl. Acids Res. 2014, DOI: 10.1093/nar/gku574). In this study we have designed anthrathiophenediones with two hloroacetamidine-containing side chains (CATDs) as G-quadruplex binders and have examined their anticancer activity in T24 bladder cancer cells bearing mutant HRAS and in T24 xenografts. The designed CATDs (3a−e), bearing alkyl side chains of different length, penetrate T24 cancer cells more than their analogues with guanidine-containing side chains. The lead compounds 3a and 3c inhibit HRAS expression, metabolic activity, and colony formation in T24 cancer cells. They also activate a strong apoptotic response, as indicated by PARP-1, caspases 3/7, and annexin V/propidium iodide assays.
Apoptosis occurs under conditions where cyclin D1 is down-regulated and the cell cycle arrested in G2 phase. Finally, compound 3a inhibits the growth of T24 xenografts and increases the median survival time of nude mice
Antisense locked nucleic acids efficiently suppress BCR/ABL and induce cell growth decline and apoptosis in leukemic cells
No full textChronic myeloid leukemia (CML) develops when a hematopoietic stem cell acquires the Philadelphia chromosome carrying the BCR/ABL fusion gene. This gives the transformed cells a proliferative advantage over normal hematopoietic cells. Silencing the BCR/ABL oncogene by treatment with specific drugs remains an important therapeutic goal. In this work, we used locked nucleic acid (LNA)-modified oligonucleotides to silence BCR/ ABL and reduce CML cell proliferation, as these oligonucleotides are resistant to nucleases and exhibit an exceptional affinity for cognate RNA. The anti-BCR/ABL oligonucleotides were designed as LNA-DNA gapmers, consisting of end blocks of 3/4 LNA monomers and a central DNA stretch of 13/14 deoxyribonucleotides. The gapmers were complementary to the b2a2 and b3a2 mRNA junctions with which they form hybrid duplexes that have melting temperatures of 79°C and 75°C, respectively, in a 20 mmol/L NaCl-buffered (pH 7.4) solution. Like DNA, the designed LNA-DNA gapmers were capable of activating RNase H and promote cleavage of the target b2a2 and b3a2 BCR/ABL mRNAs. The treatment of CML cells with junction-specific antisense gapmers resulted in a strong and specific reduction of the levels of BCR/ABL transcripts (∼ 20% of control) and protein p210BCR/ABL (∼ 30% of control). Moreover, the antisense oligonucleotides suppressed cell growth up to 40% of control and induced apoptosis, as indicated by the increase of caspase-3/7 activity in the treated cells. Finally, the b2a2-specific antisense gapmer used in combination with STI571 (imatinib mesylate), a tyrosine kinase inhibitor of p210BCR/ABL, produced an enhanced antiproliferative effect in KYO-1 cells, which compared with K562 cells are refractory to STI571. The data of this study support the application of BCR/ABL antisense LNA-DNA gapmers, used either alone or in combination with STI571, as potential antileukemic agents
Guanidino anthrathiophenediones as G-quadruplex binders: uptake, intracellular localization, and anti-Harvey-Ras gene activity in bladder cancer cells.
No full textWe prepared a series of anthrathiophenediones
(ATPDs) with guanidino-alkyl side chains of different length
(compounds 1, 10−13). The aim was to investigate their interaction
with DNA and RNA G-quadruplexes, their uptake in malignant and
nonmalignant cells, and their capacity to modulate gene expression and
inhibit cell growth. Flow cytometry showed that the ATPDs enter
more efficiently in malignant T24 bladder cells than in nonmalignant
embryonic kidney 293 or fibroblast NIH 3T3 cells. In T24 malignant
cells, compound 1, with two ethyl side chains, is taken up by
endocytosis, while 12 and 13, with respectively propyl and butyl side chains, are transported by passive diffusion. The designed ATPDs localize in the cytoplasm and nucleus and tightly bind to DNA and RNA G-quadruplexes. They also decrease HRAS expression, increase the cell population in G0/G1, and strongly inhibit proliferation in malignant T24 bladder cells, but not in nonmalignant 293 or NIH 3T3 cells
Postsynthetic conjugation of biopolymers with high molecular mass poly(ethylene glycol): Optimization of a solution process tested on synthetic oligonucleotides
No full textThe reaction of oligonucleotides with high molecular weight monomethoxy poly(ethylene glycol)s (MPEGs) has been tested to set up a convenient procedure for the postsynthetic conjugation in solution of biopolymers. A first oligonucleotide was previously modified in 5', using a liquid-phase procedure, with a linker carrying a terminal primary amino group to enhance its nucleophilic reactivity. Two procedures commonly utilized for the activation of the terminal OH groups of the MPEG were evaluated, that is, the reaction with pNO(2)-phenyl chloroformate and with N,N-disuccinimidyl carbonate. Both water as well as organic solution conditions were employed and compared. In a second test, a X-amino modified, commercial 20-mer was also conjugated in a microscale condition to verify the effect of size and concentration of MPEG on the postsynthetic conjugation of these biopolymers under troublesome synthetic conditions
Small Interfering RNA-Mediated Silencing of Glutathione-S-transferase A1 Sensitizes Hepatic Carcinoma Cells to Photodynamic Therapy with Pentaphyrins
No full textPhotodynamic therapy (PDT) uses nontoxic photosensitizers and visible light to produce reactive oxygen species that kill malignant cells by apoptosis or necrosis. Silencing the antioxidant GSTA1-1 gene by siRNA sensitizes hepatic HepG2 cells to PDT with pentaphyrins. The study is a proof-of-concept for combining PDT with antigene molecules that decrease cellular response to oxidative stress
Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins
No full textThe human KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) upstream of the major transcription initiation site. In this article, we demonstrate by primer-extension experiments, PAGE, chemical footprinting, CD, UV and FRET experiments that the G-rich strand of NHE (32R) folds into intra-molecular G-quadruplex structures. Fluorescence data show that 32R in 100 mM KCl melts with a biphasic profile, showing the formation of two distinct G-quadruplexes with T(m) of approximately 55 degrees C (Q(1)) and approximately 72 degrees C (Q(2)). DMS-footprinting and CD suggest that Q(1) can be a parallel and Q(2) a mixed parallel/antiparallel G-quadruplex. When dsNHE (32R hybridized to its complementary) is incubated with a nuclear extract from Panc-1 cells, three DNA-protein complexes are observed by EMSA. The complex of slower mobility is competed by quadruplex 32R, but not by mutant oligonucleotides, which cannot form a quadruplex structure. Using paramagnetic beads coupled with 32R, we pulled down from the Panc-1 extract proteins with affinity for quadruplex 32R. One of these is the heterogeneous nuclear ribonucleoprotein A1, which was previously reported to unfold quadruplex DNA. Our study suggests a role of quadruplex DNA in KRAS transcription and provides the basis for the rationale design of molecular strategies to inhibit the expression of KRAS
Antiproliferative activity of atripelx forming oligonucleotide recognizing a Ki-ras polypurine/polypyrimidine motif correlates with protein binding
No full textThe Ki-ras gene is frequently mutated and/or overexpressed in human cancer. Since it is suspected to play a key role in the pathogenesis of many tumors, there is interest to search for strategies aiming at the specific inhibition of this oncogene. In this paper, we investigated the capacity of a 20 mer G-rich oligonucleotide (ODN20) conjugated to high molecular weight monomethoxy polyethylene glycol (MPEG) to inhibit the expression of the Ki-ras gene and the proliferation of pancreatic cancer cells. The conjugate, MPEG ODN20, was designed to form a triplex with a critical pur/pyr sequence located in the promoter of the Ki-ras gene. To make the conjugate resistant to endogenous and exogenous nucleases, five phosphorothioate linkages were introduced in its backbone. Confocal microscopy and FACS experiments showed that MPEG ODN20 had a higher capacity to penetrate the cell membranes and accumulate in the nucleus of Panc-1 cells than ODN20. Incubation of Panc-1 cells with MPEG ODN20 reduced specifically the levels of Ki-ras mRNA and RAS protein p21RAS. A single-dose administration of MPEG ODN20 was sufficient to inhibit cell proliferation by about 50% compared with control. By contrast, the antiproliferative activity of the unconjugated ODN20 analog was found to be not significant. Band-shift and footprinting experiments showed that MPEG ODN20 formed a weak triplex (Kd ∼ 1.5 μM at 37°C, 50 mM Tris-acetate, pH 7.4, 10 mM NaCl, 10 mM MgCl2, 5 mM spermidine) with the Ki-ras pyr/pur motif, suggesting that its bioactivity can hardly be mediated by a triplex-based mechanism. Here, we provide evidence that, in vitro, ODN20 and MPEG ODN20 competitively inhibit the binding to the Ki-ras pur/pyr motif of a nuclear protein, suggesting that the activity of MPEG ODN20 occurs with an aptameric mechanism. The biological implications of this study are discussed
Critical role of hnRNP A1 in activating KRAS transcription in pancreatic cancer cells: A molecular mechanism involving G4 DNA
No full textKRAS is one of the most mutated genes in human cancer. Its crucial role in the tumourigenesis of pancreatic ductal adenocarcinoma (PDAC) has been widely demonstrated. As this deadly cancer does not sufficiently respond to conventional chemotherapies, it is important to increase our knowledge of pancreatic cancer biology, in particular how oncogenic KRAS is regulated. The promoter of KRAS contains a GA-element composed of runs of guanines that fold into a G4 structure. This unusual DNA conformation is recognized by several nuclear proteins, including MAZ and hnRNP A1. Recent data have revealed that KRAS is interconnected to ILK and hnRNPA1 in a circuitry that enables pancreatic cancer cells to maintain an aggressive phenotype. The present review illustrates recent advances on how KRAS is regulated in pancreatic cancer cells, focusing on the formation of G4 structures in the KRAS promoter and their interaction with hnRNP A1. The newly discovered KRAS-ILK-hnRNP A1 regulatory loop is discussed, emphasizing its potential as a therapeutic target for PDAC-specific molecules
Postsynthetic conjugation of biopolymers with high-molecular mass poly(ethylene glycol): optimization of a solution process tested on synthetic oligonucleotides
No full textLipid-modified G4-decoy oligonucleotide anchored to nanoparticles: delivery and bioactivity in pancreatic cancer cells.
Full text linkKRAS is mutated in >90% of pancreatic ductal adenocarcinomas. As its inactivation leads to tumour regression, mutant KRAS is considered an attractive target for anticancer drugs. In this study we report a new delivery strategy for a G4-decoy oligonucleotide that sequesters MAZ, a transcription factor essential for KRAS transcription. It is based on the use of palmitoyl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with lipid-modified G4-decoy oligonucleotides and a lipid-modified cell penetrating TAT peptide. The potency of the strategy in pancreatic cancer cells is demonstrated by cell cytometry, confocal microscopy, clonogenic and qRT-PCR assays
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