Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.

The iron chelator deferriferrichrysin induces paraptosis via extracellular signal-related kinase activation in cancer cells.

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.

Significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.

We selected the G-quadruplex motif located in the nuclease-hypersensitive elements (NHE) III1 region of the c-Myc promoter and for the first time performed its interaction studies with a designed peptide (QW10). Our CD results showed that the peptide bound to the c-Myc G-quadruplex and induced a significant blue shift in the positive peak of 20 nm in KCl alone or with 40wt% PEG200 or 20wt% PEG8000 in comparison to NaCl. Our Native Gel results confirmed that peptide binding destabilized the duplex and stabilized the unimolecular G-quadruplex and not binding to i-motif. UV thermal results confirmed destabilization of bimolecular structure and stabilization of unimolecular G-quadruplex. QW10 showed preferential binding towards c-MYC promoter G4 with binding constant (K b) values of the order of 0.05 ± 0.2 µM, 0.12 ± 0.1 µM and 0.05 ± 0.3 µM for complexes in K+ alone or 40wt% PEG 200 or 20wt% PEG 8000 respectively. QW10 showed preferential cytotoxicity with IC50 values of 11.10 µM and 6.44 µM after 72 and 96 hours' incubation on Human Breast Carcinoma MDA-MB 231 cells and was found to be non-toxic with Human Embryonic Kidney (HEK-1) cells. Interestingly, we observed reduction of c-Myc gene expression by 2.5 fold due to QW10 binding and stabilizing c-MYC G4. Our study for the first time provides an expanded overview of significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.

Detection of Intracellular Reactive Oxidative Species Using the Fluorescent Probe Hydroxyphenyl Fluorescein.

Various fluorescent probes for the detection of intracellular reactive oxidative species (ROS) have been developed because ROS levels are closely associated with cellular states. Here, we describe a method for detection of intracellular ROS in living cells using the fluorescent probe, hydroxyphenyl fluorescein (HPF), which detects hydroxyl radicals and peroxynitrite. NIH3T3 cells and p53 knockout (p53-/-) mouse embryonic fibroblasts (MEFs) were transformed by expressing oncogenic RAS using a retrovirus system. The cells were treated with HPF at 37 °C for 30 min, and subsequently, images were acquired using a confocal fluorescence microscope at an excitation wavelength of 488 nm after washing with PBS.

Photosensitizers Based on G-Quadruplex Ligand for Cancer Photodynamic Therapy.

G-quadruplex (G4) is the non-canonical secondary structure of DNA and RNA formed by guanine-rich sequences. G4-forming sequences are abundantly located in telomeric regions and in the promoter and untranslated regions (UTR) of cancer-related genes, such as RAS and MYC. Extensive research has suggested that G4 is a potential molecular target for cancer therapy. Here, we reviewed G4 ligands as photosensitizers for cancer photodynamic therapy (PDT), which is a minimally invasive therapeutic approach. The photosensitizers, such as porphyrins, were found to be highly toxic against cancer cells via the generation of reactive oxidative species (ROS) upon photo-irradiation. Several porphyrin derivatives and analogs, such as phthalocyanines, which can generate ROS upon photo-irradiation, have been reported to act as G4 ligands. Therefore, they have been implicated as promising photosensitizers that can selectively break down cancer-related DNA and RNA forming G4. In this review, we majorly focused on the potential application of G4 ligands as photosensitizers, which would provide a novel strategy for PDT, especially molecularly targeted PDT (mtPDT).

Osmolyte-Enhanced Protein Synthesis Activity of a Reconstituted Translation System.

Molecular crowding is receiving great attention in cell-free synthetic biology because molecular crowding is a critical feature of natural cell discrimination from artificial cells. Further, it has significant and generic influences on biomolecular functions. Although there are reports on how the macromolecular crowder reagents affect cell-free systems such as transcription and translation, the second class of molecular crowder reagents with low molecular weight, osmolyte, was much less studied in cell-free systems. In the present study, we focused on trimethylamine- N-oxide (TMAO) and betaine, methylamine osmolytes, and investigated the effectiveness of these osmolytes on gene expression activity of reconstituted cell-free protein synthesis. The gene expression activity of the fluorescent proteins Venus and tdTomato and the enzymes ß-galactosidase and dihydrofolate reductase were tested. At 37 °C, 0.4 M TMAO showed the highest enhancement of translational activity by a factor of 1.6-3.8, regardless of protein type. In contrast, betaine showed only a moderate effect that was limited to fluorescent proteins. Excess amounts of osmolytes suppressed gene expression activity. An mRNA-start assay and SDS-PAGE quantitative analysis provided firm evidence that TMAO enhances the translation process, instead of transcription, folding, or the maturation of fluorescent proteins. Interestingly, at 26 °C, TMAO and betaine showed the highest enhancement of protein synthesis activity at lower concentrations than at 37 °C. These findings provide implications on how osmolytes assist translation in natural cells. Further, they provide guidelines for modulation of protein synthesis activity in artificial cells through osmolyte addition.

Metal sensitive and DNA concentration dependent structural rearrangement of short oligonucleotide into large suprastructures.

Formation of higher order structures, such as G-quadruplexes and G-quadruplex based large suprastructures into long G-wires and liquid crystals is promising elements for use in healthcare for drug delivery as they are mechanically and thermally stable. In this study, we studied the structures of short 11-mer oligonucleotide 5'-G2AG5AG2-3'(11Pu) which is observed in 3'-UTR region of c-jun protooncogene. We used circular dichroism, UV-thermal melting, native gel electrophoresis and atomic force microscopy to determine the structure of 11Pu. CD results showed that 11Pu formed a mixed G-quadruplex in the presence of Na+ with and without Mg2+, while it formed a parallel G-quadruplex in the presence of 100 mM K+ with or without Mg2+. Cation selectivity in inducing the formation of large superstructures was observed in the presence of 100 mM K+ with 10 mM Mg2+. On the contrary, 10 mM Ca2+ did not induce the suprastructures. It was further demonstrated that Mg2+ at low concentration induced a parallel G-quadruplex of 11Pu, whereas at 10 mM Mg2+ induced a large suprastructure. AFM Images showed that 11Pu formed a G-wire, a liquid crystals and a crystalline lattice depending on the concentration of 11Pu and Mg2+. These insights may be employed to design G quadruplex-based nanowires for targeted drug delivery as well as interesting candidates for molecular nanowires. Communicated by Ramaswamy H. Sarma.

Selective recognition of human telomeric G-quadruplex with designed peptide via hydrogen bonding followed by base stacking interactions.

We described a novel synthetic peptide in which a glutamine residue binds through hydrogen bonding to a guanine-base and a trytophan residue intercalates with K+ resulting in stabilization of a human telomeric G-quadruplex with high selectivity over its complementary c-rich strand and a double-stranded DNA and its complementary C-rich strand. This peptide offers great potential for cancer treatment by inhibiting the telomere extension by telomerase.

An anionic phthalocyanine decreases NRAS expression by breaking down its RNA G-quadruplex.

Aberrant activation of RAS signalling pathways contributes to aggressive phenotypes of cancer cells. The RAS-targeted therapies for cancer, therefore, have been recognised to be effective; however, current developments on targeting RAS have not advanced due to structural features of the RAS protein. Here, we show that expression of NRAS, a major isoform of RAS, can be controlled by photo-irradiation with an anionic phthalocyanine, ZnAPC, targeting NRAS mRNA. In vitro experiments reveal that ZnAPC binds to a G-quadruplex-forming oligonucleotide derived from the 5'-untranslated region of NRAS mRNA even in the presence of excess double-stranded RNA, which is abundant in cells, resulting in selective cleavage of the target RNA's G-quadruplex upon photo-irradiation. In line with these results, upon photo-irradiation, ZnAPC decreases NRAS mRNA and NRAS expression and thus viability of cancer cells. These results indicate that ZnAPC may be a prominent photosensitiser for a molecularly targeted photodynamic therapy for cancer.

DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity.

The development of a switching system for guanine nanowire (G-wire) formation by external signals is important for nanobiotechnological applications. Here, we demonstrate a DNA nanostructural switch (G-wire <--> particles) using a designed peptide and a protease. The peptide consists of a PNA sequence for inducing DNA to form DNA-PNA hybrid G-quadruplex structures, and a protease substrate sequence acting as a switching module that is dependent on the activity of a particular protease. Micro-scale analyses via TEM and AFM showed that G-rich DNA alone forms G-wires in the presence of Ca2+, and that the peptide disrupted this formation, resulting in the formation of particles. The addition of the protease and digestion of the peptide regenerated the G-wires. Macro-scale analyses by DLS, zeta potential, CD, and gel filtration were in agreement with the microscopic observations. These results imply that the secondary structure change (DNA G-quadruplex <--> DNA/PNA hybrid structure) induces a change in the well-formed nanostructure (G-wire <--> particles). Our findings demonstrate a control system for forming DNA G-wire structures dependent on protease activity using designed peptides. Such systems hold promise for regulating the formation of nanowire for various applications, including electronic circuits for use in nanobiotechnologies.

Highly Sensitive Telomerase Assay Insusceptible to Telomerase and Polymerase Chain Reaction Inhibitors for Cervical Cancer Screening Using Scraped Cells.

A sensitive telomerase assay based on asymmetric-polymerase chain reaction (A-PCR) on magnetic beads and subsequent application of cycling probe technology, STAMC, which is insusceptible to DNase and PCR inhibitors, was for the first time applied to clinical specimens in addition to a conventional telomeric repetitive amplification protocol (TRAP). The electrophoresis results showed that an increase in scraped cervical cancer cells not only reduced TRAP products but also increased smaller products, suggesting the unreliability of TRAP for clinical samples. To achieve the required sensitivity of STAMC for clinical application, the sequence and concentration conditions were explored for the forward and reverse primers for A-PCR, which resulted in a detection limit of only two HeLa cells with 1 µM TS primer (5'-AATCCGTCGAGCAGAGTT-3') and 0.04 µM ACX primer (5'-GCGCGGCTTACCCTTACCCTTACCCTAACC-3'). Under the same primer conditions, the fluorescence signal of STAMC increased as scraped cervical cancer cells increased despite showing a negligible intensity for benign tumors. Furthermore, STAMC showed no signal for a cervical cancer patient treated with irradiation therapy. These results indicate that STAMC is useful for not only cervical cancer screening but also investigating the effect of cancer treatments such as radiation therapy and drug administration.

Effects of background anionic compounds on the activity of the hammerhead ribozyme in Mg(2+)-unsaturated solutions.

Cellular ribozymes exhibit catalytic activity in media containing large numbers of anionic compounds and macromolecules. In this study, the RNA cleavage activity of the hammerhead ribozyme induced by Mg(2+) was investigated using the solutions containing background nucleic acids, small phosphate and carboxylic acid compounds, and neutral polymers. Analysis of the substrate cleavage kinetics showed that the anionic compounds do not affect the ribozyme activity in Mg(2+)-saturated solutions and there is almost no effect of the anion-Mg(2+) complexes formed. On the other hand, the rate of substrate cleavage in Mg(2+)-unsaturated solutions was reduced under conditions of a high background of anionic compounds found in cells. The extent of the reduction was more with a greater net negative charge, caused by decreased amounts of Mg(2+) that could be used for the ribozyme reaction. It was remarkable that background DNA and RNA molecules having phosphodiester bonds reduced the cleavage rate as much as adenosine monophosphates having a charge of -2 when the effects of the same amount of phosphate groups were compared. Greater reductions in rates than those expected from the molecular charge were also observed in the background of fatty acids that form micelles. An addition of poly(ethylene glycol) to the solutions partially restored the ribozyme activity, suggesting a possible role of macromolecular crowding in counteracting the inhibitory effects of background anions on the ribozyme reaction. The results have biological and practical implications with respect to the effects of molecular environment on the efficiency of ion binding to RNA.

Stabilization of DNA Structures with Poly(ethylene sodium phosphate).

The structure and stability of biomolecules under molecular crowding conditions are of interest because such information clarifies how biomolecules behave under cell-mimicking conditions. The anionic surfaces of chromatin, which is composed of DNA strands and histone complexes, are concentrated in cell nuclei and thus generate a polyanionic crowding environment. In this study, we designed and synthesized an anionic polymer, poly(ethylene sodium phosphate) (PEP·Na), which has a nucleic acid phosphate backbone and created a cell nucleus-like environment. The effects of molecular crowding with PEP·Na on the thermodynamics of DNA duplexes, triplexes, and G-quadruplexes were systematically studied. Thermodynamic analysis demonstrated that PEP·Na significantly stabilized the DNA structures; e.g., a free energy change at 25 °C for duplex formation decreased from -6.6 to -12.8 kcal/mol with 20 wt % PEP·Na. Thermodynamic parameters further indicated that the factors for the stabilization of the DNA structures were dependent on sodium ion concentration. At lower polymer concentrations, the stabilization was attributed to a shielding of the electrostatic repulsion between DNA strands by the sodium ions of PEP·Na. In contrast, at higher polymer concentrations, the DNA structures were entropically stabilized by volume exclusion, which could be enhanced by electrostatic repulsion between phosphate groups in DNA strands and in PEP·Na. Additionally, increasing PEP·Na concentration resulted in increasing enthalpy of the DNA duplex but decreasing enthalpy of DNA G-quadruplex, indicating that the polymers also promoted dehydration of the DNA strands. Thus, polyanionic crowding affects the thermodynamics of DNA structures via the sodium ions, volume exclusion, and hydration. The stabilization of DNA by the cell nucleus-like polyanionic crowding provides new information regarding DNA structures and allows for modeling reactions in cell nuclei.

A mRNA-Responsive G-Quadruplex-Based Drug Release System.

G-quadruplex-based drug delivery carriers (GDDCs) were designed to capture and release a telomerase inhibitor in response to a target mRNA. Hybridization between a loop on the GDDC structure and the mRNA should cause the G-quadruplex structure of the GDDC to unfold and release the bound inhibitor, anionic copper(II) phthalocyanine (CuAPC). As a proof of concept, GDDCs were designed with a 10-30-mer loop, which can hybridize with a target sequence in epidermal growth factor receptor (EGFR) mRNA. Structural analysis using circular dichroism (CD) spectroscopy showed that the GDDCs form a (3 + 1) type G-quadruplex structure in 100 mM KCl and 10 mM MgCl2 in the absence of the target RNA. Visible absorbance titration experiments showed that the GDDCs bind to CuAPC with Ka values of 1.5 × 10(5) to 5.9 × 105 M(-1) (Kd values of 6.7 to 1.7 µM) at 25 °C, depending on the loop length. Fluorescence titration further showed that the G-quadruplex structure unfolds upon binding to the target RNA with Ka values above 1.0 × 10(8) M(-1) (Kd values below 0.01 µM) at 25 °C. These results suggest the carrier can sense and bind to the target RNA, which should result in release of the bound drug. Finally, visible absorbance titration experiments demonstrated that the GDDC release CuAPC in response to the target RNA.

A fluorescent probe for detection of an intracellular prognostic indicator in early-stage cancer.

Cyclin A2 is a promising cancer prognostic indicator, but its intracellular in situ imaging is still a challenging task. This work designs an "off-on" fluorescent probe, which can fluorescently detect intracellular cyclin A2 and distinguish cancer cells. In addition, this work sheds light on the development of future protein biosensors.

Reduced graphene oxide upconversion nanoparticle hybrid for electrochemiluminescent sensing of a prognostic indicator in early-stage cancer.

Upconversion nanoparticles (UCNPs) have been proposed as a promising new class of biological luminescent labels because of their weak auto-fluorescence background, strong penetration ability under near-infrared (NIR) radiation, resistance to photobleaching, and low toxicity. Although UCNPs hold great promise in nanotechnology and nanomedicine, their applications in ECL fields still remain unexplored. Herein, a label-free, ultra-sensitive and selective electrochemiluminescence (ECL) assay is developed for detection of cyclin A2 by using highly efficient ECL graphene-upconversion hybrid. Being an important member of the cyclin family, cyclin A2 is involved in the initiation of DNA replication, transcription and cell cycle reg-ulation through the association of cyclin-dependent kinases (CDK). Cyclin A2 is a prognostic indicator in early-stage cancers and a target for treatment of different types of cancers. However, the expression level of cyclin A2 is quite low, direct detection of cyclin A2 in crude cancer cell extracts is challenging and important for both clinical diagnosis of cancer in the early stage and the treatment. By chemically grafting cyclin A2 detection specific probe, a PEGlyted hexapeptide, to graphene-upconversion hybrid, the constructed ECL biosensor displays a superior performance for cyclin A2 , which can not only detect cyclin A2 directly in cancer cell extracts, but also discriminate between normal cells and cancer cells. More importantly, the ECL biosensor has different responses between clinical used anticancer drug-treated and non-treated cancer cells, which demonstrates that the sensor can be potentially used for drug screening, and for evaluation of therapeutic treatments in early-stage cancers.

A simple "add and measure" FRET-based telomeric tandem repeat sequence detection and telomerase assay method.

A simple and sensitive method for measuring telomeric tandem repeat DNA and telomerase activity based on fluorescence resonance energy transfer (FRET) with a FAM-modified 12-mer ODN probe as a donor (fluorophore) and ethidium bromide (EB) as an acceptor (quencher) is proposed. When telomeric DNA and the FAM-modified probe form a duplex, EB intercalates between base-pairs, resulting in fluorescence quenching of FAM through FRET from FAM to EB. This method can be used to estimate the amount of telomeric DNAs in a sample solution as the molar concentration of the telomeric DNA unit [5'-(TTA GGG TTA GGG)-3']. A linear fluorescence quenching ratio was obtained in 5-1000 pM of telomeric DNA units by adjusting the amount of FAM-modified probe. A PCR-free telomerase activity assay using this FRET-based method could be applied to ≥400 HeLa cells per µL. This assay represents a novel technique for initial screenings of cancer diagnosis and is a facile method for quantifying telomeric DNA or other tandem repeat sequences.

Study on effects of molecular crowding on G-quadruplex-ligand binding and ligand-mediated telomerase inhibition.

The telomere G-quadruplex-binding and telomerase-inhibiting capacity of two cationic (TMPyP4 and PIPER) and two anionic (phthalocyanine and Hemin) G-quadruplex-ligands were examined under conditions of molecular crowding (MC). Osmotic experiments showed that binding of the anionic ligands, which bind to G-quadruplex DNA via π-π stacking interactions, caused some water molecules to be released from the G-quadruplex/ligand complex; in contrast, a substantial number of water molecules were taken up upon electrostatic binding of the cationic ligands to G-quadruplex DNA. These behaviors of water molecules maintained or reduced the binding affinity of the anionic and the cationic ligands, respectively, under MC conditions. Consequently, the anionic ligands (phthalocyanine and Hemin) robustly inhibited telomerase activity even with MC; in contrast, the inhibition of telomerase caused by cationic TMPyP4 was drastically reduced by MC. These results allow us to conclude that the binding of G-quadruplex-ligands to G-quadruplex via non-electrostatic interactions is preferable for telomerase inhibition under physiological conditions.

Specific binding of anionic porphyrin and phthalocyanine to the G-quadruplex with a variety of in vitro and in vivo applications.

The G-quadruplex, a four-stranded DNA structure with stacked guanine tetrads (G-quartets), has recently been attracting attention because of its critical roles in vitro and in vivo. In particular, the G-quadruplex functions as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplex can show peroxidase-like activity with an anionic porphyrin, iron (III) protoporphyrin IX (hemin). Importantly, hemin binds to G-quadruplexes with high selectivity over single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is attributable to an electrostatic repulsion of phosphate groups in ssDNA and dsDNA. The G-quadruplex and hemin-G-quadruplex complex allow development of sensing techniques to detect DNA, metal ions and proteins. In addition to hemin, anionic phthalocyanines also bind to the G-quadruplex formed by human telomere DNA, specifically over ssDNA and dsDNA. Since the binding of anionic phthalocyanines to the G-quadruplex causes an inhibition of telomerase activity, which plays a role in the immortal growth of cancer cells, anionic phthalocyanines are promising as novel anticancer drug candidates. This review focuses on the specific binding of hemin and anionic phthalocyanines to G-quadruplexes and the applications in vitro and in vivo of this binding property.