A ruthenium-oligonucleotide bioconjugated photosensitizing aptamer for cancer cell specific photodynamic therapy
Luke K. McKenzieMarie FlammePatrick FelderJohannes KargesFrédéric BonhommeAlbert GandiosoChristian MalosseGilles GasserMarcel Hollenstein
0
Citation
0
Reference
10
Related Paper
Abstract:
Ruthenium complexes have emerged as a promising class of compounds for use as photosensitizers (PSs) in photodynamic therapy (PDT) due to their attractive photophysical properties and relative ease of chemical alteration. While promising, they generally are not inherently targeting to disease sites and may therefore be prone to side effects and require higher doses. Aptamers are short oligonucleotides that bind specific targets with high affinity. One such aptamer is AS1411, a nucleolin targeting, G-quadruplex forming, DNA aptamer. Here we present the first example of direct conjugation of a Ru(II) polypyridyl complex-based PS to an aptamer and an assessment of its in vitro cancer cell specific photosensitization including discussion of the challenges faced.Keywords:
Aptamer
Nucleolin
Background Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in tumorigenesis and angiogenesis. Emerging evidence suggests that the cell-surface expressed nucleolin is a strategic target for an effective and nontoxic cancer therapy. Methodology/Principal Findings By monitoring the expression of nucleolin mRNA, and by measuring the level of nucleolin protein recovered from the surface and nucleus of cells, here we show that the presence of nucleolin at the cell surface is dependent on the constant induction of nucleolin mRNA. Indeed, inhibitors of RNA transcription or translation block expression of surface nucleolin while no apparent effect is observed on the level of nucleolin in the nucleus. The estimated half-life of surface nucleolin is less than one hour, whereas that of nuclear nucleolin is more than 8 hours. Nucleolin mRNA induction is reduced markedly in normal fibroblasts that reach confluence, while it occurs continuously even in post-confluent epithelial tumor cells consistent with their capacity to proliferate without contact inhibition. Interestingly, cold and heat shock induce nucleolin mRNA concomitantly to enhanced mRNA expression of the heat shock protein 70, thus suggesting that surface nucleolin induction also occurs in response to an environmental insult. At the cell surface, one of the main functions of nucleolin is to shuttle specific extracellular ligands by an active transport mechanism, which we show here to be calcium dependent. Conclusion/Significance Our results demonstrate that the expression of surface nucleolin is an early metabolic event coupled with tumor cell proliferation and stress response. The fact that surface nucleolin is constantly and abundantly expressed on the surface of tumor cells, makes them a preferential target for the inhibitory action of anticancer agents that target surface nucleolin.
Nucleolin
Cite
Citations (188)
Abstract Nucleolin is a multifunctional abundant nucleolar protein whose transcriptional expression is trans-activated by the proto-oncogene c-myc during exponential cellular growth. This non-ribosomal protein has defined roles in ribosomal RNA processing and transcriptional regulation of various genes including rRNA. Following cellular stress, nucleolin has been shown to inhibit chromosomal replication and to relay nucleolar stress signals mediated by its sub-nuclear re-localization. Thus, nucleolin has been demonstrated to have both positive and negative effects on cell proliferation. We have previously demonstrated that nucleolin binds to the p53-antagonist Hdm2 and inhibits Hdm2-mediated degradation of p53 albeit independent of the ARF (Alternate Reading Frame) tumor suppressor protein. Nucleolin also reduces Hdm2 protein levels by an unknown mechanism when over-expressed in cells to levels found in a variety of cancer cells. Here we have delineated that multiple domains of both nucleolin and Hdm2 are involved in distinct nucleolin-Hdm2 interactions in unstressed cells. Our data indicates that the nucleolin N-terminus as well as the central RNA-binding domain (RBD) are predominantly involved in binding to Hdm2, as determined by Far-Western as well as GST-binding assays in vitro. While the N-terminus of nucleolin was found associated with the Hdm2 RING domain, the nucleolin RBDs robustly bound to the NLS/NES (nuclear localization and export signals) domain of Hdm2. The C-terminal GAR (Glycine-Arginine Rich) domain of nucleolin, shown previously to be essential for association with p53, did not bind Hdm2 yet was found to stimulate Hdm2-mediated p53-ubiquitination. Conversely, the nucleolin RBD that strongly interacted with Hdm2 was found to inhibit the Hdm2 E3 ligase activity against p53, leading to p53 stabilization. There was no significant effect of over-expression of nucleolin or its various domains on Hdm2-autoubiquitination in cells. Similarly, the phosphorylation status of nucleolin did not significantly influence its binding to Hdm2. As might be expected from a key rRNA processing factor, our findings suggest that the activity of nucleolin towards Hdm2 can be modulated to yield either p53 up- or down-regulation. Our studies can aid in the future design of small molecule-based cancer therapeutics that can stimulate the wild-type p53 checkpoint in variety of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4532.
Nucleolin
ribosome biogenesis
Ribosomal protein
Cite
Citations (0)
Nucleolin is an abundant non-ribosomal protein found in nucleolus and a major component of silver-stained nucleolar organizer region (AgNOR), a histopathological marker of cancer which is highly elevated in cancer cells. We recently reported that nucleolin on the cell surface of mouse gastric cancer cells acts as a receptor for tumor necrosis factor-α-inducing protein (Tipα), a new carcinogenic factor of Helicobacter pylori. In this study, we first examined the localization of nucleolin on cell surface of five gastric cancer cell lines by cell fractionation and flow cytometry: We found that large amounts of nucleolin were present on surface of MKN-45, KATOIII, MKN-74, and AGS cells, with smaller amounts on surface of MKN-1 cells. The membrane fraction of normal epithelial cells of mouse glandular stomach did not contain much nucleolin, suggesting that translocation of nucleolin to the cell surface occurs during carcinogenesis, making for easier binding with Tipα. AS1411, a nucleolin targeted DNA aptamer, inhibited growth of gastric cancer cell lines in this order of potency: MKN-45>KATOIII>AGS>MKN-74=MKN-1, associated with induction of S-phase cell cycle arrest. Fluorescein isothiocyanate (FITC)-AS1411 was more rapidly incorporated into MKN-45 and AGS than into MKN-1 cells, based on varying amounts of cell surface nucleolin. We think that AS1411 first binds to nucleolin on the cell surface and that the binding complex is then incorporated into the cells. All results indicate that nucleolin on the cell surface is a new and promising therapeutic target for treatment of gastric cancer.
Nucleolin
Cite
Citations (88)
: Nucleolin is a protein abundantly present in the nucleolus, but its expression on the surface of cells is potentially associated with various types of malignancies. So far, several nucleolin-targeting strategies, including the nucleolin-targeting peptide, anti-nucleolin pseudopeptides, anti-nucleolin antibodies, and the anti-nucleolin aptamer, AS1411, have been developed and investigated in different types of studies. The AS1411 aptamer has been known as the outstanding approach for targeting nucleolin with superior specificity and therapeutic potential in comparison with other targeting strategies. In this review, we highlight different nucleolin-targeting strategies for the targeted delivery of chemotherapeutic drugs, proteins with therapeutic potential, and toxins.
Nucleolin
Aptamer
Targeted Therapy
Cite
Citations (1)
AS1411 is an antiproliferative DNA aptamer, which binds the ubiquitous protein, nucleolin. In this study, we show that constitutive overexpression of nucleolin confers increased sensitivity to the growth inhibitory effects of AS1411. HeLa cells overexpressing nucleolin have an increased growth rate and invasiveness relative to control cells. Nucleolin overexpressing cells demonstrate increased growth inhibition in response to the AS1411 treatment, which correlates with increased apoptosis and cell cycle arrest, when compared to non-transfected cells. AS1411 induces nucleolin expression at the RNA and protein level in HeLa cells, suggesting a feedback loop with important implications for the clinical use of AS1411.
Nucleolin
HeLa
Aptamer
Cite
Citations (30)
Nucleolin
Ribosomal protein
Cite
Citations (22)
AGRO100, a G-rich oligonucleotide is an extremely stable molecule which is stabilized by G-quartet formation. It has growth inhibitory activity for malignant cells which correlates with binding to a target protein, nucleolin. Nucleolin is a multifunctional protein which plays an important role in regulating ribosomal RNA synthesis and processing as well as DNA replication. We have shown that nucleolin is present on the surface of transformed, but not normal cells. Nucleolin binding results in translocation of the oligonucleotide to the cell nucleus. AGRO100 is growth inhibitory for a wide variety of tumor cell T types, causing S-phase arrest and inducing apoptosis. Its growth inhibitory activity is synergistic with a number of chemotherapeutic agents. A phase I clinical trial was performed to determine the clinical toxicity of AGRO100. Seventeen patients were treated with a four-day infusion of AGRO100. Five patients with evidence of clinical response or stable disease were given an additional monthly course of AGRO100. No clinical or laboratory toxicity was noted. Significant clinical activity was seen in these patients. In summary, the G-rich oligonucleotide AGRO100 inhibits cancer cell growth by binding to nucleolin. There is evidence of clinical activity without significant toxicity.
Nucleolin
Aptamer
Cite
Citations (0)
Aptamer
Nucleolin
Linker
Cite
Citations (1)
Nucleolin
Cite
Citations (83)
Nucleolin, a multifunctional protein distributed in the nucleolus, participates in many modulations including rDNA transcription, RNA metabolism, and ribosome assembly. Nucleolin is also found in the cytoplasm and on the cell membrane, and surface nucleolin can bind to various ligands to affect many physiological functions. The expression and localization of nucleolin is often abnormal in cancers, as the differential distribution of nucleolin in cancer can influence the carcinogenesis, proliferation, survival, and metastasis of cancer cells, leading to the cancer progression. Thus, nucleolin may be a novel and promising target for anti-cancer treatment. Here, we describe how nucleolin act functions in cancer development and describe nucleolin-dependent anti-cancer therapies.
Nucleolin
ribosome biogenesis
Cite
Citations (72)