Bringing to Light the Importance of the miRNA Methylome in Colorectal Cancer Prognosis Through Electrochemical Bioplatforms.

This work reports the first electrochemical bioplatforms developed for the determination of the total contents of either target miRNA or methylated target miRNA. The bioplatforms are based on the hybridization of the target miRNA with a synthetic biotinylated DNA probe, the capture of the formed DNA/miRNA heterohybrids on the surface of magnetic microcarriers, and their recognition with an antibody selective to these heterohybrids or to the N6-methyladenosine (m6A) epimark. The determination of the total or methylated target miRNA was accomplished by labeling such secondary antibodies with the horseradish peroxidase (HRP) enzyme. In both cases, amperometric transduction was performed on the surface of disposable electrodes after capturing the resulting HRP-tagged magnetic bioconjugates. Because of their increasing relevance in colorectal cancer (CRC) diagnosis and prognosis, miRNA let-7a and m6A methylation were selected. The proposed electrochemical bioplatforms showed attractive analytical and operational characteristics for the determination of the total and m6A-methylated target miRNA in less than 75 min. These bioplatforms, innovative in design and application, were applied to the analysis of total RNA samples extracted from cultured cancer cells with different metastatic profiles and from paired healthy and tumor tissues of patients diagnosed with CRC at different stages. The obtained results demonstrated, for the first time using electrochemical platforms, the potential of interrogating the target miRNA methylation level to discriminate the metastatic capacities of cancer cells and to identify tumor tissues and, in a pioneering way, the potential of the m6A methylation in miRNA let-7a to serve as a prognostic biomarker for CRC.

Advanced technologies towards improved HPV diagnostics.

Persistent infection with high-risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low-income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time-consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR-Cas-based systems, as well as microfluidics, paperfluidics and lab-on-a-chip devices, ideal for point-of-care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.

Electrochemical biosensors for analysis of DNA point mutations in cancer research.

Cancer is a genetic disease induced by mutations in DNA, in particular point mutations in important driver genes that lead to protein malfunctioning and ultimately to tumorigenesis. Screening for the most common DNA point mutations, especially in such genes as TP53, BRCA1 and BRCA2, EGFR, KRAS, or BRAF, is crucial to determine predisposition risk for cancer or to predict response to therapy. In this review, we briefly depict how these genes are involved in cancer, followed by a description of the most common techniques routinely applied for their analysis, including high-throughput next-generation sequencing technology and less expensive low-throughput options, such as real-time PCR, restriction fragment length polymorphism, or high resolution melting analysis. We then introduce benefits of electrochemical biosensors as interesting alternatives to the standard methods in terms of cost, speed, and simplicity. We describe most common strategies involved in electrochemical biosensing of point mutations, relying mostly on PCR or isothermal amplification techniques, and critically discuss major challenges and obstacles that, until now, prevented their more widespread application in clinical settings.

Electrochemical LAMP-based assay for detection of RNA biomarkers in prostate cancer.

Current molecular diagnostics of prostate cancer relies on detection of elevated levels of PSA protein in serum, but its specificity has been questioned due to its higher levels also in non-malignant prostate diseases. A long non-coding RNA biomarker, PCA3, demonstrated excellent specificity for prostate cancer, and thus has become an interesting alternative to PSA monitoring. Its detection utilizes mostly reverse transcription PCR with optical detection, making the protocol longer and more expensive. To avoid PCR, we have developed an electrochemical assay coupled with LAMP, an isothermal amplification technique showing high sensitivities at constant temperatures and shorter reaction times. We amplified PCA3 RNA as well as PSA mRNA (serving as a control), hybridized LAMP products on magnetic beads and measured them with chronoamperometry at carbon electrode chips. We show good sensitivity and specificity for both biomarkers in prostate cancer cell lines, and successful detection of PCA3 in clinical samples, i.e., urine samples from 11 prostate cancer patients and 7 healthy controls, where we obtained excellent correlation with clinical data. This is to our knowledge a first such attempt to apply electrochemistry to determine two RNA biomarkers directly in urine samples of prostate cancer patients in a minimally invasive diagnostics format.

New Trends in the Detection of Gynecological Precancerous Lesions and Early-Stage Cancers.

The prevention and early diagnostics of precancerous stages are key aspects of contemporary oncology. In cervical cancer, well-organized screening and vaccination programs, especially in developed countries, are responsible for the dramatic decline of invasive cancer incidence and mortality. Cytological screening has a long and successful history, and the ongoing implementation of HPV triage with increased sensitivity can further decrease mortality. On the other hand, endometrial and ovarian cancers are characterized by a poor accessibility to specimen collection, which represents a major complication for early diagnostics. Therefore, despite relatively promising data from evaluating the combined effects of genetic variants, population screening does not exist, and the implementation of new biomarkers is, thus, necessary. The introduction of various circulating biomarkers is of potential interest due to the considerable heterogeneity of cancer, as highlighted in this review, which focuses exclusively on the most common tumors of the genital tract, namely, cervical, endometrial, and ovarian cancers. However, it is clearly shown that these malignancies represent different entities that evolve in different ways, and it is therefore necessary to use different methods for their diagnosis and treatment.

Electrochemical bioassay coupled to LAMP reaction for determination of high-risk HPV infection in crude lysates.

Electrochemical (EC) detection of DNA biomarkers represents an interesting tool in molecular oncology due to its sensitivity, simplicity, low cost or rapid times of measurement. However, majority of EC assays, same as most optical-based techniques, require preceding DNA extraction step to remove other cellular components, making these assays more laborious and time-consuming. One option to circumvent this is to use LAMP (loop-mediated amplification), an isothermal amplification technique that can amplify DNA directly in crude lysates in a short time at a constant temperature. Here, we coupled the LAMP reaction with EC readout to detect DNA from the two most common oncogenic human papillomavirus (HPV) types that cause cervical cancer in women, i.e. HPV 16 and HPV 18, directly in crude lysates without a need for DNA extraction step. We show that in crude lysates, the LAMP reaction was superior to PCR, with very good selectivity on a panel of cancer cell lines and with high sensitivity, enabling detection of HPV DNA from as few as 10 cells. As a proof of principle, we applied the assay to nineteen clinical samples both from uninfected women and from women suffering from cervical precancerous lesions caused by HPV 16 or HPV 18 genotypes. Clinical samples were simply boiled for 5 min in homogenization buffer without DNA extraction step, and amplified with LAMP. We obtained excellent concordance of our assay with PCR, reaching 100% sensitivity for both genotypes, 81.82% specificity for HPV 16 and 94.12% specificity for HPV 18. Proposed assay could be a straightforward, simple, rapid and sensitive alternative for early diagnostics of precancerous cervical lesions.

DNA Methylation in Solid Tumors: Functions and Methods of Detection.

DNA methylation, i.e., addition of methyl group to 5'-carbon of cytosine residues in CpG dinucleotides, is an important epigenetic modification regulating gene expression, and thus implied in many cellular processes. Deregulation of DNA methylation is strongly associated with onset of various diseases, including cancer. Here, we review how DNA methylation affects carcinogenesis process and give examples of solid tumors where aberrant DNA methylation is often present. We explain principles of methods developed for DNA methylation analysis at both single gene and whole genome level, based on (i) sodium bisulfite conversion, (ii) methylation-sensitive restriction enzymes, and (iii) interactions of 5-methylcytosine (5mC) with methyl-binding proteins or antibodies against 5mC. In addition to standard methods, we describe recent advances in next generation sequencing technologies applied to DNA methylation analysis, as well as in development of biosensors that represent their cheaper and faster alternatives. Most importantly, we highlight not only advantages, but also disadvantages and challenges of each method.

A novel zinc finger protein-based amperometric biosensor for miRNA determination.

This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (- 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA-RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP's non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.

Ferrocenes as new anticancer drug candidates: Determination of the mechanism of action.

Chemotherapy plays an essential role in the management of cancer worldwide. However, it is a non-specific treatment limited by major drawbacks, thus identification and testing of new promising molecular structures representing potential drug candidates are urgently needed. In this work, ferrocene complexes as potential antitumor drugs that display cytotoxicity in low micromolar concentrations against ovarian cancer cells A2780 and SK-OV-3 were investigated to identify their mode of action. Their mechanism of cellular accumulation was studied using differential pulse voltammetry and inductively coupled plasma - mass spectrometry. Their mode of cell death induction was determined by changes in the mitochondrial membrane potential, production of reactive oxygen species and by Annexin V staining. Transferrin receptors were identified as key mediators of intracellular accumulation of ferrocenes and the extent of cellular uptake reflected the anticancer activity of individual compounds. Functional analysis revealed activation of intrinsic apoptosis as a dominant mechanism leading to regulated cell death induced in ovarian cancer cells by ferrocenes. Ferrocenes represent a group of promising sandwich organometallic complexes exerting cytotoxic activity. We suggest their application not only as standalone chemotherapeutics but also as modifying substituents of known drugs to improve their antitumor effects.

The role of miR-409-3p in regulation of HPV16/18-E6 mRNA in human cervical high-grade squamous intraepithelial lesions.

Cervical cancer is one of the most common malignancies in women. MicroRNAs (miRNAs) are involved in a variety of fundamental cellular processes, including carcinogenesis. The potential utilization of aberrantly expressed miRNAs as novel biomarkers in cervical cancer diagnostics is growing. We investigated miRNA expression profiles during the progression of dysplasia in cervical epithelium to identify aberrantly expressed miRNAs. High-throughput miRNA profiling of high-grade precancerous lesions identified 79 miRNAs showing significant difference in expression values compared to normal cervical epithelium. Ten selected miRNAs were subsequently measured in an independent group of samples to validate them as promising biomarkers of cervical carcinogenesis. MicroRNAs miR-10b-5p, miR-34c-5p, miR-409-3p and miR-411-5p were confirmed as downregulated, while miR-10a-5p, miR-132-3p, miR-141-5p were significantly upregulated in dysplastic cervical tissues. Further investigation revealed an inverse correlation of miR-409-3p with E6 mRNA levels in precancerous cervical lesions. Subsequent in vitro analyses showed a direct involvement of this miRNA in the regulation of E6 oncogene levels, thus confirming a potential tumor suppressor function of miR-409-3p in cervical malignancies. Hence, miR-409-3p may represent a useful early marker and a potential therapeutic target for cervical cancer.

Genomagnetic LAMP-based electrochemical test for determination of high-risk HPV16 and HPV18 in clinical samples.

Major cause of cervical cancer is a persistent infection with high-risk types of human papillomaviruses (HPV). For that reason, HPV testing is now becoming an important addition to standard cytological screening of cervical malignancies in women (known as Pap test). New methods are sought which could offer rapid and inexpensive detection schemes, such as those based on electrochemical (EC) readout. Here, we developed an assay for parallel detection of two most oncogenic high-risk HPV types, HPV16 and HPV18, by combining loop-mediated amplification (LAMP) of template DNA, its separation using magnetic beads and detection with amperometry at carbon-based electrode chips. Our EC-LAMP test enabled us to successfully discriminate both HPV types not only in cancer cell lines, but also using clinical material obtained from HPV-positive patient samples.

[Human Papillomavirus - Role in Cervical Carcinogenesis and Methods of Detection]. / Lidský papilomavirus - role v karcinogenezi cervixu a moznosti jeho detekce.

BACKGROUND: Persistent infection with high-risk human papillomavirus (HPV) strains, especially HPV 16 and HPV 18, is associated with the onset of various malignant diseases, including cervical carcinoma in women. HPV DNA testing is thus being implemented as a complementary method to standard cytological examination, mainly due to its increased sensitivity. AIM: This review outlines the role of HPV in cervical carcinogenesis, with a focus on the formation of cervical intraepithelial neoplasias (CIN1-3) and the molecular mechanism underlying cellular transformation. Current biomarkers used to screen premalignant lesions are described, including mRNA transcripts of the E6 and E7 genes, protein p16 (a cyclin-dependent kinase inhibitor that regulates cell cycle progression from G1 to S phase), altered DNA methylation patterns, and actions of specific microRNAs (short (18-22 bp), non-coding, single-stranded RNA molecules that regulate gene expression at the post-transcriptional level). This review also describes the advantages and drawbacks of commercial HPV tests, and depicts novel methods for more cost-effective and faster HPV diagnostics based on optical or electrochemical detection. CONCLUSION: Although great progress has been made, the incidence and mortality rates of cervical malignancies remain relatively high, especially in developing countries. Incorporation of HPV testing into routine screening programs could help to decrease mortality rates; however, the cost of such testing must be reduced if it is to compete with current cytology-based examinations.Key words: HPV - cervical carcinoma - HPV testing - nucleic acid hybridization - mRNA - DNA methylation - microRNA This work was supported by MEYS-NPS I-LO1413 and GACR 17-08971S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 25. 9. 2017Accepted: 26. 1. 2018.

Current Methods of microRNA Analysis.

BACKGROUND: MicroRNA (miRNA) are a class of short non-coding RNA molecules that regulate gene expression at the post-transcription level by binding to mRNA. By affecting many physiological processes, including cellular proliferation, differentiation, and apoptosis, they have a major impact on the development of cancer as well as other diseases. Hence, miRNAs could serve as potential tumor biomarkers in e.g. early diagnostics, predicting responses to therapy, monitoring relapse, and molecular classification of tumors. AIM: miRNA detection requires various sophisticated strategies due to the small size, sequence similarity among family members, and often very low levels of miRNAs in analyzed samples. This review describes standard techniques of miRNA detection, such as the reverse transcriptase polymerase chain reaction, microarrays, and next-generation sequencing, and compares several commercially available detection kits. Major emphasis is given to newly developed technologies and methods, which could make the analysis cheaper and quicker. We present, for instance, alternative amplification techniques (isothermal amplification and the hybridization chain reaction), different types of nanomaterials, special proteins used in miRNA analysis, and a number of biosensors utilizing optical or electrochemical detection. CONCLUSION: The importance of miRNA has led to a huge increase in the number of new methods. Most of them, however, have not been tested on clinical material, and thus it is difficult to assess their potential usefulness in routine practice. Their commercial application strongly depends on strict validation with standard techniques using not only model systems, but also clinical samples. Key words: microRNA - gene expression regulation - tumour biomarkers - reverse transcription PCR - biosensors This work was supported by MEYS - NPS I - LO1413 and GAČR 17-08971S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 9. 7. 2018.

Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs.

Cytochrome c (cyt c) is one of the most studied conjugated proteins due to its electron-transfer properties and ability to regulate the processes involved in homeostasis or apoptosis. Here we report an electrochemical strategy for investigating the electroactivity of cyt c and its analogs with a disrupted heme moiety, i.e. apocytochrome c (acyt c) and porphyrin cytochrome c (pcyt c). The electrochemical data are supplemented with low-temperature and spin-probe electron paramagnetic resonance (EPR) spectroscopy. The main contribution of this report is a complex evaluation of cyt c reduction and oxidation at the level of surface-localized amino acid residues and the heme moiety in a single electrochemical scan. The electrochemical pattern of cyt c is substantially different to both analogs acyt c and pcyt c, which could be applicable in further studies on the redox properties and structural stability of cytochromes and other hemeproteins.

Portable Lock-in Amplifier-Based Electrochemical Method to Measure an Array of 64 Sensors for Point-of-Care Applications.

We present a portable lock-in amplifier-based electrochemical sensing system. The basic unit (cluster) consists of four electrochemical cells (EC), each containing one pseudoreference electrode (PRE) and one working electrode (WE). All four ECs are simultaneously interrogated, each at different frequencies, with square wave pulses superposed on a sawtooth signal for cyclic voltammetry (CV). Lock-in amplification provides independent read-out of four signals, with excellent noise suppression. We expanded a single cluster system into an array of 16 clusters by using electronic switches. The chip with an array of ECs was fabricated using planar technology with a gap between a WE and a PRE of ≈2 µm, which results in partial microelectrode-type behavior. The basic electrode characterization was performed with the model case using a ferricyanide-ferrocyanide redox couple (Fe2+/Fe3+) reaction, performing CV and differential pulse voltammetry (DPV). We then used this system to perform cyclic lock-in voltammetry (CLV) to measure concurrently responses of the four ECs. We repeated this method with all 64 ECs on the chip. The standard deviation of a peak oxidation and reduction current in a single channel consisting of 13 ECs was ≈7.46% and ≈5.6%, respectively. The four-EC configuration in each measured spot allows determination of nonperforming ECs and, thus, to eliminate potential false results. This system is built in a portable palm-size format suitable for point-of-care applications. It can perform either individual or multiple measurements of active compounds, such as biomarkers.

Titanocenes as anticancer agents: recent insights.

BACKGROUND: Enormous success of antitumor agent cisplatin initiated interest in other organometallic complexes. A subclass of organometallic compounds termed metallocenes, characterized by a transition metal central atom (M) bound to cyclopentadienide (Cp-/[C5H5]-) ligands with the basic formula Cp2M, has gained increasing interest as promising anticancer agents. OBJECTIVES: This review is aimed at a progress in the development of organometallic titanium-based compounds focusing primarily on the evaluation of their cytotoxic activity and mechanism of action in relation to potential utilization as anticancer drugs. RESULTS: Metallocenes bearing titanium as central atom were sorted according to their structure and modifications and their anticancer activity is further discussed. CONCLUSION: Titanocenes represent family of promising compounds exerting cytostatic activity. We suggest that their application not only as separate agents, but also in combination with newly developed carriers, e.g. nanomaterials, may lead to improvement of their delivery into tumor cells and following utilization in cancer treatment.

Electrochemical chip-based genomagnetic assay for detection of high-risk human papillomavirus DNA.

Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice.

Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder.

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein-ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.

Magnetic bead-based hybridization assay for electrochemical detection of microRNA.

Aberrant expression of microRNAs (miRNAs), short non-coding RNA molecules regulating gene expression, is often found in tumor cells, making the miRNAs suitable candidates as cancer biomarkers. Electrochemistry is an interesting alternative to current standard methods of miRNA detection by offering cheaper instrumentation and faster assays times. In this paper, we labeled miRNA in a quick, simple, two-step procedure with electroactive complex of osmium(VI) and 2,2'-bipyridine, Os(VI)bipy, which specifically binds to the ribose at the 3'-end of the miRNA, and hybridized such labeled miRNA with biotinylated capture probe attached to the streptavidin magnetic beads. Labeled miRNA was then detected at hanging mercury drop electrode at femtomole level due to an electrocatalytic nature of the peak from the Os(VI)bipy label. We obtained good selectivity of the assay using elevated hybridization temperatures for better discrimination of perfect duplex from single and double mismatches. After optimization of the protocol, we demonstrated feasibility of our assay by detecting target miRNA in real total RNA samples isolated from human cancer cells.