Direct Determination of miR‐21 in Total RNA Extracted from Breast Cancer Samples Using Magnetosensing Platforms and the p19 Viral Protein as Detector Bioreceptor

A novel magnetobiosensing approach for the rapid, sensitive and selective miR‐21 detection is reported involving the use of a specific RNA probe (antimiR‐21), streptavidin‐magnetic beads (Strep‐MBs), the siRNA Binding Protein p19 as detector bioreceptor, and amperometric detection with the H₂O₂/hydroquinone (HQ) system at disposable screen‐printed carbon electrodes. The magnetosensor exhibited a dynamic range from 1.4 to 10 nM and a detection limit of 4.2 fmol of synthetic target miR‐21 without any amplification step in 75 min. The usefulness of the approach was evaluated by analyzing total RNA (RNA_t) extracted from metastatic breast cancer cell lines, human tissues and breast cytologies.     

Multiplexed Immunosensing Platform Coupled to Hybridization Chain Reaction for Electrochemical Determination of MicroRNAs in Clinical Samples

There is an urgent need for development of rapid and inexpensive techniques for detection of microRNAs (miRNAs), which are potential biomarkers of various types of cancer. In this paper, we describe a multiplexed electrochemical platform for determination of three cancer‐relevant miRNAs: miR‐21, let‐7a and miR‐31. The strategy combines the use of magnetic beads (MBs) modified with a commercial antibody for the efficient capture of the heteroduplexes formed by hybridization of the target miRNA with DNA probe. Free non‐hybridized region of the DNA probe was thereafter hybridized with two biotin‐labeled auxiliary DNA probes in a process of hybridization chain reaction (HCR), resulting in a long hybrid bearing a large number of biotin molecules. Labeling of these multiple biotin units with streptavidin‐peroxidase conjugates allowed an amplification of the amperometric signal measured after capturing the modified MBs at a screen‐printed carbon electrode array of eight electrodes. The combined strategy demonstrated in a similar assay time significantly higher sensitivity than those previously described using modified MBs with the same capture antibody (without amplification by HCR) or a HCR strategy implemented on the surface of MBs, respectively. The methodology exhibits a good selectivity for discriminating single mismatches and was applied to the determination of the three target miRNAs in total RNA (RNA_t) extracted from various cancer cell lines and from cervical precancerous lesions.     

Exploratory Study on the RNA‐Binding Structural Motifs by Library Screening Targeting pre‐miRNA‐29 a

The metabolic stream of microRNA (miRNA) production, the so‐called maturation process of miRNAs, became one of important metabolic paths for drug‐targeting to modulate the expression of genes related to a number of diseases. We carried out discovery studies on small molecules binding to the precursor of miR‐29a (pre‐miR‐29a) from a chemical library containing 41 119 compounds (AQ library) by the fluorescent indicator displacement (FID) assay using the xanthone derivative X2SdiMe as a fluorescent indicator. The FID assay provided 1075 compounds, which showed an increase of fluorescence. These compounds were subsequently submitted to a binding analysis in a surface plasmon resonance (SPR) assay on a pre‐miR‐29a immobilized surface. 21 hit compounds were identified with a good reproducibility in the binding. These compounds have not been reported to bind to RNA until now and can be classified into two groups on the basis of the kinetics in the binding. To gain more information on the motif structures that could be necessary for the binding to pre‐miR‐29a, 19 substructures were selected from the hit compounds. The substructure library (SS library) which consisted of 362 compounds was prepared from the AQ library. An SPR assay of the SS library on pre‐miR‐29a‐immobilized surface suggested that five substructures could potentially be important structural motifs to bind to pre‐miR‐29a. These studies demonstrate that the combination of FID‐based screening of chemical library and subsequent SPR assay would be one way for obtaining practical solutions for the discovery of molecules which bind to the target pre‐miRNAs.     

Single‐Vehicular Delivery of Antagomir and Small Molecules to Inhibit miR‐122 Function in Hepatocellular Carcinoma Cells by using “Smart” Mesoporous Silica Nanoparticles

MicroRNAs (miRNAs) regulate a variety of biological processes. The liver‐specific, highly abundant miR‐122 is implicated in many human diseases including cancer. Its inhibition has been found to result in a dramatic loss in the ability of Hepatitis C virus (HCV) to infect host cells. Both antisense technology and small molecules have been used to independently inhibit endogenous miR‐122 function, but not in combination. Intracellular stability, efficient delivery, hydrophobicity, and controlled release are some of the current challenges associated with these novel therapeutic methods. Reported herein is the first single‐vehicular system, based on mesoporous silica nanoparticles (MSNs), for simultaneous cellular delivery of miR‐122 antagomir and small molecule inhibitors. The controlled release of both types of inhibitors depends on the expression levels of endogenous miR‐122, thus enabling these drug‐loaded MSNs to achieve combination inhibition of its targeted mRNAs in Huh7 cells.     

Polymersome‐assisted delivery of curcumin: A suitable approach to decrease cancer stemness markers and regulate miRNAs expression in HT29 colorectal cancer cells

Curcumin as a safe traditional compound has various benefits such as anticancer activities. However, low solubility in water is a problem. Herein, curcumin encapsulated in polymersome nanoparticles (CPNs) have been developed, the physicochemical properties have been evaluated, and cytotoxicity effects on HT29 cells were evaluated by MTT assay and annexin V/PI staining. The expression of stemness markers including CD44, CD133, and CD24, as well as miRNAs (miR‐126, miR‐34a, miR‐21, miR‐155, miR‐221, and miR‐222) and some potential targets, was evaluated in CPNs‐treated and untreated cells. Physicochemical analysis confirmed the encapsulation of curcumin in polymersomes and showed a spherical shape, an appropriate mean size of 259.5±1.5 nm, the acceptable polydispersity index of ~ 0.465, and the zeta potential of (‐8.74±0.2), as well as long‐term storage of CPNs at 4°C. According to the result, CPNs with the IC50 of 14 μg/ml increased apoptosis and induced S arrest in treated cells. Flow cytometry analysis showed the decrease in cancer stemness markers. RT‐qPCR analysis identified the downregulation of miR‐21, miR‐155, and miR‐221/222, as well as upregulation of miR‐34a, miR‐126, and deregulation of some apoptotic targets such as P53, CASP9, CASP8, CASP3, BAX, and BCl‐2 in CPNs‐treated cells. As a result, CPNs can be a safe and effective complementary agent to diminish cancer stem cells and tumor recurrence in colorectal cancer therapy.     

MicroRNA Delivery with Bioreducible Polyethylenimine as a Non‐Viral Vector for Breast Cancer Gene Therapy

Polyethylenimines (PEIs) are outstanding macromolecules belonging to the polycations used in gene transfection. The transfection efficiency and cytotoxicity of PEIs increase with the increase in their molecular weight. To break up the correlation between transfection efficiency and cytotoxicity for non‐viral gene delivery, disulfide cross‐linked polyethylenimine (PEI‐SS) has been widely employed as highly efficient gene vectors for DNA/siRNA delivery in numerous efforts. In this work, PEI‐SS is described as a non‐viral vector for miRNA delivery for the first time. PEI‐SS is synthesized via cross‐linking using disulfide bonds as the cross‐linker from low molecular weight PEI. PEI‐SS can efficiently bind anti‐miR‐155 to form the polyplex with nano‐sized spherical structures in the size range of 10–100 nm. The polyplex is degraded by glutathione (GSH, a reducing agent) in cancer cells. Anti‐miR‐155 is then released to efficiently inhibit tumor growth.     

Curcumin loaded PEG400‐OA nanoparticles: A suitable system to increase apoptosis,decrease migration,and deregulate miR‐125b/miR182 in MDA‐MB‐231 human breast cancer cells

Curcumin is an anti‐cancerous agent, but its low‐solubility limits its clinical use. The relationship between deregulation of miRNAs and their targets suggested that miRNAs can be interest targets of curcumin in treatment of different cancers. In this study, to overcome essential defects of the clinical usage of this golden drug, curcumin‐encapsulated polymersome nanoparticles (CPNs) have been developed, and the cytotoxicity effects were studied on MDA‐MB‐231 breast cancer cells. The expression level of miR‐182/125b and the expression pattern of some potential targets in apoptotic pathway, predicted by in silico approaches, were analyzed by RT‐qPCR in CPNs‐treated and untreated cells. Moreover, the amount of CASP9 and CASP8 proteins were determined by Western blotting. The effect of CPNs on cell migration were studied by scratch test and the level of EGFR, E‐cadherin, and beta‐catenin proteins were monitored in CPNs‐treated and untreated cells by western blotting. RT‐qPCR analysis identified the downregulation of miR‐125b and miR‐182 in CPNs‐treated cells and the upregulation of some predicted apoptotic target genes such as P53, CASP9 and BAX after 24 hours. Western blotting confirmed the effects of curcumin on the increase of cleaved CASP9 protein. Based on data from the current experiment, the migration of MDA‐MB‐231 cells was decreased after CPNs treatment. According to the results, CPNs, as suitable and compatible nanocarriers, can deliver curcumin into cancerous cells more effectively and can increase the therapeutic effects of curcumin on MDA‐MB‐231 cells partly by suppression of miR‐125b and miR‐182 as well as induction of apoptosis and inhibition of metastatic progression.     

Cold atmospheric plasma modification of curcumin loaded in tri‐phosphate chitosan nanoparticles enhanced breast cancer cells apoptosis

The anti‐cancer mechanisms of curcumin have been reported to include suppressions of angiogenesis and tumor proliferation. The main goal of this research is to increase the solubility of curcumin by cold atmospheric plasma (CAP) and assess the effects of modified curcumin by charging with tri‐polyphosphate chitosan nanoparticles for MCF‐7, MDA‐MB‐231 breast cancer cells, and human fibroblast cells. Curcumin modification was done by CAP and its solubility was evaluated by spectrophotometry. After loading modified curcumin into nano‐chitosan‐TPP, nanocurcumin was characterized by scanning electron microscopy. Cellular viability and apoptosis of treated cells were assessed by MTT and Annexin V. The changes of messenger RNA expression of TP5353 and VEGF genes were analyzed by real‐time PCR. CAP was able to transform the curcumin to possess hydrophilic characteristics after 90 seconds. The mean diameter of Curcumin loaded chitosannanoparticles (NPs) were determined as 48 nm. MTT results showed that the IC₅₀ of nano Cur‐chitosan‐TPP was effectively decreased compared to free curcumin in MCF‐7 (15 μg/mL at 72 hours vs 20 μg/mL at 48 hours). Additionally, nano Cur‐chitosan‐TPP had no significant effect on normal cells (Human dermal fibroblas: HDF), whereas it also decreased the viability of triple negative breast cancer cell line (MDA‐MB‐231). Real‐time PCR results showed that expression level of TP53 gene was upregulated (P = .000), whereas VEGF gene downregulated (P = .000) in treated MCF‐7 cells. Curcumin loaded chitosan nanoparticles have led to an induction of apoptosis (79.93%) and cell cycle arrest (at S and G2M). Modified‐curcumin‐tri‐polyphosphate chitosan nanoparticles using CAP can be considered as a proper candidate for breast cancer treatment.     

Prebiotic Oligosaccharides: Special Focus on Fructooligosaccharides,Its Biosynthesis and Bioactivity

MicroRNAs (miRNAs) are important nonprotein-coding genes involved in almost all biological processes during biotic and abiotic stresses in plants. To investigate the miRNA-mediated plant response to drought stress, two drought-tolerant (C-306 and NI-5439) and two drought-sensitive (HUW-468 and WL-711) wheat genotypes were exposed to 25 % PEG 6000 for 1, 12 and 24 h. Temporal expression patterns of 12 drought-responsive miRNAs and their corresponding nine targets were monitored by quantitative real-time PCR (qRT-PCR). The results showed differential expression of miRNAs and their targets with varying degree of upregulation and downregulation in drought-sensitive genotypes. Likewise, in drought-tolerant wheat genotypes, maximum accumulation of miR393a and miR397a was observed at 1 h of stress. In addition, nearly perfect negative correlation was observed in four miRNA and target pairs (miR164-NAC, miR168a-AGO, miR398-SOD and miR159a-MYB) across all the temporal period studied which could be a major player during drought response in wheat. We, for the first time, validated the presence of miR529a and miR1029 in wheat. These findings gives a clue for temporal and variety-specific differential regulation of miRNAs and their targets in wheat in response to osmotic shock and could help in defining the potential roles of miRNAs in plant adaptation to osmotic stress in future.     

Expression of Luteinizing Hormone-Releasing Hormone (LHRH) and Type-I LHRH Receptor in Transitional Cell Carcinoma Type of Human Bladder Cancer

Bladder cancer (BC) is the tenth most frequently detected cancer in both sexes. Type-I luteinizing hormone-releasing hormone (LHRH) receptor (LHRH-R-I) is expressed not only in the pituitary, but also in several types of cancer disease. There are few data about LHRH-R-I expression in human BC. This study aimed to investigate the expression of LHRH and LHRH-R-I in the transitional cell carcinoma (TCC) type of human BC. RNA was extracted from 24 human bladder tumor specimens and three BC cell lines. RT-PCR was performed to detect mRNA for LHRH and LHRH-R-I. The protein of LHRH-R-I was further studied by immunohistochemistry (IHC), ligand competition assay, and Western Blot. PCR products of LHRH were found in 19 of 24 (79%) specimens and mRNA of LHRH-R-I was detected in 20 of 24 specimens (83%). Positive immunostaining for LHRH-R-I with different expression intensity was found in all samples examined, showing negative correlation with TCC grade. Radioligand binding studies also showed the presence of specific LHRH-R-I and high affinity binding of LHRH analogs. The high incidence of LHRH-R in BC suggests that it could serve as a molecular target for therapy of human BC with cytotoxic LHRH analogs or modern powerful antagonistic analogs of LHRH.     

Chitosan/Nitrogen Doped Reduced Graphene Oxide Modified Biosensor for Impedimetric Detection of microRNA

The development of a low‐cost and disposable biosensor platform for the sensitive and rapid detection of microRNAs (miRNAs) is of great interest for healthcare, pharmaceuticals, and medical science. We designed an impedimetric biosensing platform using Chitosan (CHIT)/nitrogen doped reduced graphene oxide (NRGO) conductive composite to modify the surface of pencil graphite electrodes (PGE) for the sensitive detection of miRNAs. An initial optimisation protocol involved investigation of the effect of NRGO concentration and miR 660 DNA probe concentration on the response of the modified electrode. After the optimization protocol, the sequence‐selective hybridization between miR 660 DNA probe and its RNA target was evaluated by measuring changes on charge transfer resistance, R_ct values. Moreover, the selectivity of impedimetric biosensor was tested in the presence of non‐complementary miRNA (NC) sequences, such as miR 34a and miR 16. The hybridization process was examined both in phosphate buffer (PBS) and in PBS diluted fetal bovine serum (FBS:PBS) solutions. The biosensor demonstrated a detection limit of 1.72 μg/mL in PBS and 1.65 μg/mL in FBS:PBS diluted solution. Given the easy, quick and disposable attributes, the proposed conductive nanocomposite biosensor platform shows great promise as a low‐cost sensor kit for healthcare monitoring, clinical diagnostics, and biomedical devices.     

Synthesis and characterization of chroman‐containing compounds and their preliminary assessment of cytotoxicity toward two human cancer cell lines

A series of chroman derivatives were synthesized by employing o‐methyl phenol as a precursor. These compounds were fully characterized using IR, NMR spectroscopic techniques, and elemental analysis. Mechanisms for the formation of the chroman moiety of these compounds were also proposed. MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay was used to estimate their cytotoxicity toward two cancer cell lines: human ovarian cancer (A2780) and human cervical cancer (Hela). © 2010 Wiley Periodicals, Inc. Heteroatom Chem 21:423–429, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/hc.20621     

Network-Based Identification of Novel Connections Among Apoptotic Signaling Pathways in Cancer

MicroRNAs (miRNAs), highly conserved, non-coding endogenous RNA and nearly ~22 nucleotides (nt) in length, are well-known to regulate several apoptotic pathways in cancer. In this study, we computationally constructed the initial human apoptotic PPI network by several online databases, and further integrated these high-throughput datasets into a Na?ve Bayesian model to predict protein functional connections. Based on the modified apoptotic network, we identified several apoptotic hub proteins such as TP53, SRC, M3K3/5/8, cyclin-dependent kinase2/6, TNFR16/19, and TGF-?? receptor 1/2. Subsequently, we identified some microRNAs that could target the aforementioned apoptotic hub proteins by using TargetScan, PicTar, and Diana-MicroH. In conclusion, these results demonstrate the PPI network-based identification of new connections amongst apoptotic pathways in cancer, which may shed new light on the intricate relationships between core apoptotic pathways and some targeted miRNAs in human cancers.     

Investigation of volatile biomarkers in liver cancer blood using solid-phase microextraction and gas chromatography/mass spectrometry

Solid-phase microextraction (SPME) followed by gas chromatography/mass spectrometry (GC/MS) was used for the detection of liver cancer volatile biomarkers. Headspace SPME conditions (fiber coating, extraction temperature and extraction time) and desorption conditions were optimized and applied to the determination of volatiles in human blood. Between the liver cancer group (n = 19) and the normal group (n = 18), positive rates of 19 volatile compounds among the total of 47 detected were found to be different with statistical significance (p < 0.05, chi-squared test). We suggested hexanal, 1-octen-3-ol and octane, of the 19 compounds, as biomarkers of liver cancer with clinical diagnostic value. The sensitivity and specificity of 94.7% and 100% for hexanal, 84.2% and 100% for 1-octen-3-ol, and 89.5% and 100% for octane were obtained, respectively, after the cutoff values had been properly established. These results show that SPME-GC/MS is a simple, rapid and sensitive method for the investigation of volatile disease markers in human blood.     

Cyclometalated Gold(III) Complexes Containing N‐Heterocyclic Carbene Ligands Engage Multiple Anti‐Cancer Molecular Targets

Metal N‐heterocyclic carbene (NHC) complexes are a promising class of anti‐cancer agents displaying potent in vitro and in vivo activities. Taking a multi‐faceted approach employing two clickable photoaffinity probes, herein we report the identification of multiple molecular targets for anti‐cancer active pincer gold(III) NHC complexes. These complexes display potent and selective cytotoxicity against cultured cancer cells and in vivo anti‐tumor activities in mice bearing xenografts of human cervical and lung cancers. Our experiments revealed the specific engagement of the gold(III) complexes with multiple cellular targets, including HSP60, vimentin, nucleophosmin, and YB‐1, accompanied by expected downstream mechanisms of action. Additionally, Pt^II and Pd^II analogues can also bind the cellular proteins targeted by the gold(III) complexes, uncovering a distinct pincer cyclometalated metal–NHC scaffold in the design of anti‐cancer metal medicines with multiple molecular targets.     

Beyond the BET Family: Targeting CBP/p300 with 4‐Acyl Pyrroles

Bromodomain and extra‐terminal domain (BET) inhibitors are widely used both as chemical tools to study the biological role of their targets in living organisms and as candidates for drug development against several cancer variants and human disorders. However, non‐BET bromodomains such as those in p300 and CBP are less studied. XDM‐CBP is a highly potent and selective inhibitor for the bromodomains of CBP and p300 derived from a pan‐selective BET BRD‐binding fragment. Along with X‐ray crystal‐structure analysis and thermodynamic profiling, XDM‐CBP was used in screenings of several cancer cell lines in vitro to study its inhibitory potential on cancer cell proliferation. XDM‐CBP is demonstrated to be a potent and selective CBP/p300 inhibitor that acts on specific cancer cell lines, in particular malignant melanoma, breast cancer, and leukemia.     

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” through the conventional small‐molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure–activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras‐binding and cell‐penetrating properties. These cell‐permeable cyclic peptides inhibit Ras signaling by binding to Ras‐GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein–protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.     

Nuclear annexin II negatively regulates growth of LNCaP cells and substitution of ser 11 and 25 to glu prevents nucleo-cytoplasmic shuttling of annexin II

Background  

Annexin II heavy chain (also called p36, calpactin I) is lost in prostate cancers and in a majority of prostate intraepithelial neoplasia (PIN). Loss of annexin II heavy chain appears to be specific for prostate cancer since overexpression of annexin II is observed in a majority of human cancers, including pancreatic cancer, breast cancer and brain tumors. Annexin II exists as a heterotetramer in complex with a protein ligand p11 (S100A10), and as a monomer. Diverse cellular functions are proposed for the two forms of annexin II. The monomer is involved in DNA synthesis. A leucine-rich nuclear export signal (NES) in the N-terminus of annexin II regulates its nuclear export by the CRM1-mediated nuclear export pathway. Mutation of the NES sequence results in nuclear retention of annexin II.