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Ferrocenes represent an interesting group of drugs with potential antitumor properties. Moreover, their electronic properties make them suitable for electrochemical studies. We determined an uptake of a novel ferrocene derivative in low μM concentrations by selected cancer cell lines and showed its localization predominantly in cytoplasm, using glassy carbon electrodes. [ABSTRACT FROM AUTHOR]

Electrochemical methods hold promise for fast and inexpensive determination of cancer biomarkers, including microRNAs playing an important role in regulation of gene expression. We developed a simple assay utilizing (i) microRNA labeling with osmium(VI) complexes, (ii) hybridization of microRNA with magnetic bead-immobilized capture probe, and (iii) adsorptive transfer stripping (AdTS) technique in which working electrode is dipped into a microliter-sized drop for direct adsorption of microRNA. Increased sensitivity and decreased sample consumption was achieved, and nanogram quantities of microRNA present in 500-fold excess of total RNA were determined. Our results indicate only negligible interference during hybridization from other RNA molecules. [ABSTRACT FROM AUTHOR]

Dysregulation of gene expression mechanisms has been observed in many tumors, making their analysis of utmost importance. These mechanisms include DNA methylation, an epigenetic mechanism in which 5-carbon of cytosine becomes methylated, leading to gene silencing, and action of short RNA molecules called microRNAs, which regulate protein synthesis at post-transcriptional level by binding to mRNAs. In this review, we describe major roles of both mechanisms in carcinogenesis, offer an overview of currently used methods for their analysis, and summarize most recent advances in electrochemical-based assays and strategies. Advantages of electrochemistry, including favorable cost, time of experiment, or simple instrumentation, are highlighted, along with current challenges that need to be addressed prior to successful application into clinical routine.

Abstract Background ΔNp63α is an epithelial progenitor cell marker that maintains epidermal stem cell self-renewal capacity. Previous studies revealed that UV-damage induced p53 phosphorylation is confined to ΔNp63α-positive cells in the basal layer of human epithelium. Results We now report that phosphorylation of the p53 tumour suppressor is positively regulated by ΔNp63α in immortalised human keratinocytes. ΔNp63α depletion by RNAi reduces steady-state ATM mRNA and protein levels, and attenuates p53 Serine-15 phosphorylation. Conversely, ectopic expression of ΔNp63α in p63-null tumour cells stimulates ATM transcription and p53 Serine-15 phosphorylation. We show that ATM is a direct ΔNp63α transcriptional target and that the ΔNp63α response element localizes to the ATM promoter CCAAT sequence. Structure-function analysis revealed that the ΔNp63-specific TA2 transactivation domain mediates ATM transcription in coordination with the DNA binding and SAM domains. Conclusions Germline p63 point mutations are associated with a range of ectodermal developmental disorders, and targeted p63 deletion in the skin causes premature ageing. The ΔNp63α-ATM-p53 damage-response pathway may therefore function in epithelial development, carcinogenesis and the ageing processes.

Abstract Background Cisplatin and its derivatives are commonly used anti-cancer drugs. However, cisplatin has clinical limitations including serious side effects and frequent emergence of intrinsic or acquired resistance. Thus, the novel platinum(IV) complex LA-12 represents a promising treatment modality, which shows increased intracellular penetration resulting in improved cytotoxicity in various cancer cell lines, including cisplatin resistant cells. Results LA-12 disrupts cellular proliferation regardless of the p53 status in the cells, however the potency of the drug is greatly enhanced by the presence of a functional p53, indicating several mechanisms of action. Similarly to cisplatin, an interaction of LA-12 with molecular chaperone Hsp90 was proposed. Binding of LA-12 to Hsp90 was demonstrated by Hsp90 immunoprecipitation followed by platinum measurement using atomic absorption spectrometry (AAS). An inhibitory effect of LA-12 on Hsp90 chaperoning function was shown by decrease of Hsp90-assisted wild-type p53 binding to p21WAF1 promoter sequence in vitro and by accelerated ubiqutination and degradation of primarily unfolded mutant p53 proteins in cells exposed to LA-12. Conclusions To generalize our findings, LA-12 induced degradation of other Hsp90 client proteins such as Cyclin D1 and estrogen receptor was shown and proved as more efficient in comparison with cisplatin. This newly characterised molecular mechanism of action opens opportunities to design new cancer treatment strategy profitable from unique LA-12 properties, which combine DNA damaging and Hsp90 inhibitory effects.

Brain metastases (BMs) are the most common intracranial tumors in adults and occur 3-10 times more frequently than primary brain tumors. Despite intensive multimodal therapies, including resection, radiotherapy, and chemotherapy, BMs are associated with poor prognosis and remain challenging to treat. BMs predominantly originate from primary lung (20-56%), breast (5-20%), and melanoma (7-16%) tumors, although they can arise from other cancer types less frequently. The metastatic cascade is a multistep process involving local invasion, intravasation into the bloodstream or lymphatic system, extravasation into normal tissue, and colonization of the distal site. After reaching the brain, circulating tumor cells (CTCs) breach the blood-brain barrier (BBB).The selective permeability of the BBB poses a significant challenge for therapeutic compounds, limiting the treatment efficacy of BMs. Understanding the mechanisms of tumor cell interactions with the BBB is crucial for the development of effective treatments. This review provides an in-depth analysis of the brain barriers, including the BBB, blood-spinal cord barrier, blood-meningeal barrier, blood-arachnoid barrier, and blood-cerebrospinal fluid barrier. It explores the molecular and cellular components of these barriers and their roles in brain metastasis, highlighting the importance of this knowledge for identifying druggable targets to prevent or limit BM formation., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Galectin-1 (Gal-1) displays unique sensitivity to oxidative inactivation which appears critical in regulating its spatial and temporal activity. The two physicochemical states, i.e. monomer-dimer equilibrium and redox states, are related to Gal-1 varying functionality. In this work, we used chronopotentiometric stripping analysis, intrinsic fluorescence spectroscopy, and mobility shift assay to follow changes in the structure and lectin activity of reduced and oxidized Gal-1 forms. Our results show that monomers and dimers are similarly distributed under mild reduction and oxidation conditions. Gal-1 after its oxidation consists of at least three different monomeric forms while reduced Gal-1 only one. Lectin activity, affinity to N-acetyllactosamine, is relatively similar for low Gal-1 concentrations for both, reduced and oxidized Gal-1. However, at higher Gal-1 concentrations, we observed a ten times higher affinity for reduced than oxidized form. Further, our data indicate that the monoclonal antibodies bind preferentially to Gal-1 dimers and specifically to only some forms of its oxidized form., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Veronika Ostatna reports financial support was provided by Czech Science Foundation. Roman Hrstka reports financial support was provided by Ministry of Health of the Czech Republic. Roman Hrstka reports financial support was provided by European Commission. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

Despite extensive research, the molecular role of AGR2 in the progression and metastasis of colorectal cancer (CRC) has not been fully characterized. We used quantitative mass spectrometry (SWATH MS) to identify differentially expressed proteins in paired CRC cell models of the SW480 and SW620 cell lines in response to AGR2 protein level manipulation. Relying on the results from SWATH MS and subsequent immunochemical validation, we selected NMP3 as the top candidate protein associated with AGR2 in CRC tumour cells in our screen. RT‒qPCR and immunochemical analysis confirmed the involvement of AGR2-mediated regulation of NPM3 at the transcriptional and posttranscriptional levels. Since PD-L1 is a constituent of the NPM3 regulatory axis, we aimed to correlate the changes in PD-L1 to the differential expression of AGR2 in our cell models. We found that AGR2 positively regulates PD-L1 levels in both SW480 and SW620 cell lines; additionally, several different CRC patient transcriptome cohorts confirmed the association of AGR2 with PD-L1. Our work reveals a new AGR2-NPM3 regulatory axis and the involvement of AGR2 in the regulation of PD-L1, which paves the way for the association of AGR2 with immune evasion in CRC cells., (© 2024. The Author(s).)

Aim: Patients with multiple brain metastases (BM) benefit from hippocampal-avoiding whole brain radiotherapy (HA-WBRT), the challenging and less available form of WBRT. This study explores potential of pre-radiotherapy (pre-RT) hippocampal magnetic resonance spectroscopy (MRS) measuring hippocampal neuronal density as an imaging surrogate and predictive tool for assessing neurocognitive functions (NCF)., Methods: 43 BM patients underwent pre-RT hippocampal MRS. N-acetyl aspartate (NAA) concentration, a marker for neuronal density (weighted by creatine (Cr) and choline (Cho) concentrations), and neurocognitive function (NCF) tests (HVLT and BVMT) performed by certified psychologists were evaluated. Clinical variables and NAA concentrations were correlated with pre-RT NCFs., Results: HVLT and BVMT subtests showed pre-RT deterioration except for BVMT recognition. Significantly better NCFs were observed in women in HVLT subsets. Significantly higher NAA/Cr + Cho was measured in women (median 0.63 vs. 0.55; P=0.048) in the left hippocampus (no difference in the right hippocampus). In men, a positive correlation (0.51, P=0.018) between total brain volume and HVLT-TR, between left hippocampal NAA/Cr + Cho and HVLT-R (0.45, P=0.063), and between right hippocampal NAA/Cr + Cho and BVMT-recognition (0.49, P=0.054) was observed. In women, a borderline significant negative correlation was observed between left hippocampal NAA/Cr + Cho and BVMT-TR (-0.43, P=0.076) and between right NAA/Cr + Cho and HVLT-DR (-0.42, P=0.051)., Conclusion: Borderline statistically significant correlations were observed with speculative interpretation underlying the challenges of hippocampal MRS as a surrogate for neurocognitive impairment. Further studies need to be done to ascertain the opportunities for imaging predictors of benefit from memory sparing radiotherapy., Competing Interests: The authors report no conflicts of interest in this work.

Background: The aim of this retrospective study is to analyze a consecutive cohort of brain metastasis (BM) patients treated off clinical trials through combination of surgery and radiotherapy over the last 15 years in a tertiary neurooncology center., Materials and Methods: All BM patients operated between 2007-2019 received adjuvant linac-based radiotherapy categorized to whole brain radiotherapy (WBRT) and tumor bed stereotactic radiotherapy. Survival outcomes and local control was analyzed., Results: In total, 118 patients were enrolled, those with stereotactic radiotherapy (41%) had better baseline characteristics mirrored in longer overall survival (OS) [18 vs . 7.1 months, p < 0.001; hazard ratio (HR) 0.47, p = 0.004] with median follow-up of 58 months. Cumulative incidence for local, distant, and extracranial control was not significantly different between groups, with 12-month cumulative control of 22% vs . 18%, 44% vs . 29%, and 35% vs . 32% for stereotactic and WBRT group, respectively. WBRT was an independent factor for better distal brain control., Conclusions: Real world data demonstrating significantly better overall survival in patients treated with postoperative targeted radiotherapy compared with postoperative WBRT is presented, with no significant difference in cumulative incidence for local or distant brain control. The majority of patients with targeted radiotherapy had a fractionated dose schedule with outcomes comparable to single-dose radiation trials of postoperative targeted radiotherapy., Competing Interests: Conflicts of interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (© 2024 Greater Poland Cancer Centre.)

A new hydrophilic interaction liquid chromatography - mass spectrometry method is developed for low-abundant phospholipids and sphingolipids in human plasma and serum. The optimized method involves the Cogent Silica type C hydride column, the simple sample preparation by protein precipitation, and the removal of highly abundant lipid classes using the postcolumn valve directed to waste during two elution windows. The method allows a highly confident and sensitive identification of low-abundant lipid classes in human plasma (246 lipid species from 24 lipid subclasses) based on mass accuracy and retention dependencies in both polarity modes. The method is validated for quantitation using two internal standards (if available) for each lipid class and applied to human plasma and serum samples obtained from patients with pancreatic ductal adenocarcinoma (PDAC), healthy controls, and NIST SRM 1950. Multivariate data analysis followed by various statistical projection methods is used to determine the most dysregulated lipids. Significant downregulation is observed for lysophospholipids with fatty acyl composition 16:0, 18:0, 18:1, and 18:2. Distinct trends are observed for phosphatidylethanolamines (PE) in relation to the bonding type of fatty acyls, where most PE with acyl bonds are upregulated, while ether/plasmenyl PE are downregulated. For the sphingolipid category, sphingolipids with very long N-acyl chains are downregulated, while sphingolipids with shorter N-acyl chains were upregulated in PDAC. These changes are consistently observed for various classes of sphingolipids, ranging from ceramides to glycosphingolipids, indicating a possible metabolic disorder in ceramide biosynthesis caused by PDAC., Competing Interests: Declaration of competing interest M.H., R.J., and D.W. are listed as inventors on the patent EP 3514545 related to this work. All other authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© 2024 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Estrogen receptor alpha (ER) is a key biomarker for breast cancer, and the presence or absence of ER in breast and other hormone-dependent cancers decides treatment regimens and patient prognosis. ER is activated after ligand binding - typically by steroid. 2682 steroid compounds were used in a molecular docking study to identify novel ligands for ER and to predict compounds that may show anticancer activity. The effect of the most promising compounds was determined by a novel luciferase reporter assay. Two compounds, 7 and 12, showing ER inhibitory activity comparable to clinical inhibitors such as tamoxifen or fulvestrant were selected. We propose that the inhibitory effect of compounds 7 and 12 on ER is related to the presence of a double bond in their D-ring, which may protect against ER activation by reducing the electron density of the keto group, or may undergo metabolism leading to an active compound. Western blotting revealed that compound 12 decreased the level of ER in the breast cancer cell line MCF7, which was associated with reduced expression of both isoforms of the progesterone receptor, a well-known downstream target of ER. However, compound 12 has a different mechanism of action from fulvestrant. Furthermore, we found that compound 12 interferes with mitochondrial functions, probably by disrupting the electron transport chain, leading to induction of the intrinsic apoptotic pathway even in ER-negative breast cancer cells. In conclusion, the combination of computational and experimental methods shown here represents a rapid approach to determine the activity of compounds towards ER. Our data will not only contribute to research focused on the regulation of ER activity but may also be useful for the further development of novel steroid receptor-targeted drugs applicable in clinical practice., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Direct infusion of lipid extracts into the ion source of a mass spectrometer is a well-established method for lipid analysis. In most cases, nanofluidic devices are used for sample introduction. However, flow injection analysis (FIA) based on sample infusion from a chromatographic pump can offer a simple alternative to shotgun-based approaches. Here, we describe important modification of a method based on FIA and tandem mass spectrometry (MS/MS). We focus on minimizing contamination of the FIA/MS both to render the lipidomic platform more robust and to increase its capacity and applicability for long-sequence measurements required in clinical applications. Robust validation of the developed method confirms its suitability for lipid quantitation in human plasma analysis. Measurements of standard human plasma reference material (NIST SRM 1950) and a set of plasma samples collected from kidney cancer patients and from healthy volunteers yielded highly similar results between FIA-MS/MS and ultra-high-performance supercritical fluid chromatography (UHPSFC)/MS, thereby demonstrating that all modifications have practically no effect on the statistical output. Newly modified FIA-MS/MS allows for the quantitation of 141 lipid species in plasma (11 major lipid classes) within 5.7 min. Finally, we tested the method in a clinical laboratory of the General University Hospital in Prague. In the clinical setting, the method capacity reached 257 samples/day. We also show similar performance of the classification models trained based on the results obtained in clinical settings and the analytical laboratory at the University of Pardubice. Together, these findings demonstrate the high potential of the modified FIA-MS/MS for application in clinical laboratories to measure plasma and serum lipid profiles., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

Catechol-O-methyl transferase (COMT) is involved in detoxification of catechol estrogens, playing cancer-protective role in cells producing or utilizing estrogen. Moreover, COMT suppressed migration potential of breast cancer (BC) cells. To delineate COMT role in metastasis of estrogen receptor (ER) dependent BC, we investigated the effect of COMT overexpression on invasion, transcriptome, proteome and interactome of MCF7 cells, a luminal A BC model, stably transduced with lentiviral vector carrying COMT gene (MCF7-COMT). 2D and 3D assays revealed that COMT overexpression associates with decreased cell invasion (p < 0.0001 for Transwell assay, p < 0.05 for spheroid formation). RNA-Seq and LC-DIA-MS/MS proteomics identified genes associated with invasion (FTO, PIR, TACSTD2, ANXA3, KRT80, S100P, PREX1, CLEC3A, LCP1) being downregulated in MCF7-COMT cells, while genes associated with less aggressive phenotype (RBPMS, ROBO2, SELENBP, EPB41L2) were upregulated both at transcript (|log2FC|> 1, adj. p < 0.05) and protein (|log2FC|> 0.58, q < 0.05) levels. Importantly, proteins driving MET signaling were less abundant in COMT overexpressing cells, and pull-down confirmed interaction between COMT and Kunitz-type protease inhibitor 2 (SPINT2), a negative regulator of MET (log2FC = 5.10, q = 1.04 -7 ). In conclusion, COMT may act as tumor suppressor in ER dependent BC not only by detoxification of catechol estrogens but also by suppressing cell invasion and interplay with MET pathway., (© 2023. The Author(s).)

Most of the organs of the digestive tract comprise secretory epithelia that require specialized molecular machines to achieve their functions. As such anterior gradient (AGR) proteins, which comprise AGR1, AGR2, and AGR3, belong to the protein disulfide isomerase family, and are involved in secretory and transmembrane protein biogenesis in the endoplasmic reticulum. They are generally expressed in epithelial cells with high levels in most of the digestive tract epithelia. To date, the vast majority of the reports concern AGR2, which has been shown to exhibit various subcellular localizations and exert pro-oncogenic functions. AGR2 overexpression has recently been associated with a poor prognosis in digestive cancers. AGR2 is also involved in epithelial homeostasis. Its deletion in mice results in severe diffuse gut inflammation, whereas in inflammatory bowel diseases, the secretion of AGR2 in the extracellular milieu participates in the reshaping of the cellular microenvironment. AGR2 thus plays a key role in inflammation and oncogenesis and may represent a therapeutic target of interest. In this review, we summarize the already known roles and mechanisms of action of the AGR family proteins in digestive diseases, their expression in the healthy digestive tract, and in digestive oncology. At last, we discuss the potential diagnostic and therapeutic implications underlying the biology of AGR proteins., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1 , COL4A2 , FLNA , VAV3 , VEGFA , and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-β treatment and were validated by RT-qPCR of GPX2 , PTGS2 , and PLA2G4A . Since PGE 2 is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2 -knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway., Competing Interests: The authors declare no conflict of interest.

A residual cancer burden after neoadjuvant therapy (NAT) for breast cancer (BC) is associated with worse treatment outcomes compared to patients who achieved pathologic complete remission. This single-institutional retrospective study of 767 consecutive patients, including 468 patients with assessable residual cancer burden (aRCB) after NAT, with a median follow-up of 36 months, evaluated the biomarkers assessed before NAT from a biopsy and after NAT from a surgical specimen, their dynamics, and effect on long-term outcomes in specific breast cancer subtypes. The leading focus was on proliferation index Ki-67, which was significantly altered by NAT in all BC subtypes (p < 0.001 for HER2 positive and luminal A/B HER2 negative and p = 0.001 for TNBC). Multivariable analysis showed pre-NAT and post-NAT Ki-67 as independent predictors of survival outcomes for luminal A/B HER2 negative subtype. For TNBC, post-NAT Ki-67 was significant alone, and, for HER2 positive, the only borderline association of pre-NAT Ki-67 was observed in relation to the overall survival. Steroid and HER2 receptors were re-assessed just in a portion of the patients with aRCB. The concordance of both assessments was 92.9% for ER status, 80.1% for PR, and 92.2% for HER2. In conclusion, these real-world data of a consecutive cohort confirmed the importance of biomarkers assessment in patients with aRCB, and the need to consider specific BC subtypes when interpreting their influence on prognosis.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Anterior gradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) known to be overexpressed in many human epithelial cancers and is involved in cell migration, cellular transformation, angiogenesis, and metastasis. This protein inhibits the activity of the tumor suppressor p53, and its expression levels can be used to predict cancer patient outcome. However, the precise network of AGR2-interacting partners and clients remains to be fully characterized. Herein, we used label-free quantification and also stable isotope labeling with amino acids in cell culture-based LC-MS/MS analyses to identify proteins interacting with AGR2. Functional annotation confirmed that AGR2 and its interaction partners are associated with processes in the ER that maintain intracellular metabolic homeostasis and participate in the unfolded protein response, including those associated with changes in cellular metabolism, energy, and redox states in response to ER stress. As a proof of concept, the interaction between AGR2 and PDIA3, another ER-resident PDI, was studied in more detail. Pathway analysis revealed that AGR2 and PDIA3 play roles in protein folding in ER, including post-translational modification and in cellular response to stress. We confirmed the AGR2-PDIA3 complex formation in cancer cells, which was enhanced in response to ER stress. Accordingly, molecular docking characterized potential quaternary structure of this complex; however, it remains to be elucidated whether AGR2 rather contributes to PDIA3 maturation in ER, the complex directly acts in cellular signaling, or mediates AGR2 secretion. Our study provides a comprehensive insight into the protein-protein interaction network of AGR2 by identifying functionally relevant proteins and related cellular and biochemical pathways associated with the role of AGR2 in cancer cells., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Pancreatic cancer has the worst prognosis among all cancers. Cancer screening of body fluids may improve the survival time prognosis of patients, who are often diagnosed too late at an incurable stage. Several studies report the dysregulation of lipid metabolism in tumor cells, suggesting that changes in the blood lipidome may accompany tumor growth. Here we show that the comprehensive mass spectrometric determination of a wide range of serum lipids reveals statistically significant differences between pancreatic cancer patients and healthy controls, as visualized by multivariate data analysis. Three phases of biomarker discovery research (discovery, qualification, and verification) are applied for 830 samples in total, which shows the dysregulation of some very long chain sphingomyelins, ceramides, and (lyso)phosphatidylcholines. The sensitivity and specificity to diagnose pancreatic cancer are over 90%, which outperforms CA 19-9, especially at an early stage, and is comparable to established diagnostic imaging methods. Furthermore, selected lipid species indicate a potential as prognostic biomarkers., (© 2022. The Author(s).)

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 (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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Long-term dysbiosis of the gut microbiome has a significant impact on colorectal cancer (CRC) progression and explains part of the observed heterogeneity of the disease. Even though the shifts in gut microbiome in the normal-adenoma-carcinoma sequence were described, the landscape of the microbiome within CRC and its associations with clinical variables remain under-explored. We performed 16S rRNA gene sequencing of paired tumour tissue, adjacent visually normal mucosa and stool swabs of 178 patients with stage 0-IV CRC to describe the tumour microbiome and its association with clinical variables. We identified new genera associated either with CRC tumour mucosa or CRC in general. The tumour mucosa was dominated by genera belonging to oral pathogens. Based on the tumour microbiome, we stratified CRC patients into three subtypes, significantly associated with prognostic factors such as tumour grade, sidedness and TNM staging, BRAF mutation and MSI status. We found that the CRC microbiome is strongly correlated with the grade, location and stage, but these associations are dependent on the microbial environment. Our study opens new research avenues in the microbiome CRC biomarker detection of disease progression while identifying its limitations, suggesting the need for combining several sampling sites (e.g., stool and tumour swabs).

In this paper we compare electrochemical behavior of two homolog proteins, namely anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), playing an important role in cancer cell biology. The slight variation in their protein structures has an impact on protein adsorption and orientation at charged surface and also enables AGR2 and AGR3 to form heterocomplexes. We confirm interaction between AGR2 and AGR3 (i) in vitro by immunochemical and constant current chronopotentiometric stripping (CPS) analysis and (ii) in vivo by bioluminescence resonance energy transfer (BRET) assay. Mutation of AGR2 in dimerization domain (E60A) prevents development of wild type AGR2 dimers and also negatively affects interaction with wild type AGR3 as shown by CPS analysis. Beside new information about AGR2 and AGR3 protein including their joint interaction, our work introduces possible applications of CPS in bioanalysis of protein complexes, including those relatively unstable, but important in the cancer research., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fully acetylated deoxyfluorinated hexosamine analogues and non-fluorinated 3,4,6-tri-O-acylated N-acetyl-hexosamine hemiacetals have previously been shown to display moderate anti-proliferative activity. We prepared a set of deoxyfluorinated GlcNAc and GalNAc hemiacetals that comprised both features: O-acylation at the non-anomeric positions with an acetyl, propionyl and butanoyl group, and deoxyfluorination at selected positions. Determination of the in vitro cytotoxicity towards the MDA-MB-231 breast cancer and HEK-293 cell lines showed that deoxyfluorination enhanced cytotoxicity in most analogues. Increasing the ester alkyl chain length had a variable effect on the cytotoxicity of fluoro analogues, which contrasted with non-fluorinated hemiacetals where butanoyl derivatives had always higher cytotoxicity than acetates. Reaction with 2-phenylethanethiol indicated that the recently described S-glyco-modification is an unlikely cause of cytotoxicity.

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.

Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1-14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.

A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.

Epithelial-mesenchymal transition (EMT) is a process involved not only in morphogenesis and embryonic development, but also in cancer progression, whereby tumor cells obtain a more aggressive metastatic phenotype. Anterior gradient protein 2 (AGR2) maintains the epithelial phenotype and blocks the induction of EMT, thus playing an undeniable role in tumor progression. However, the mechanism through which AGR2 expression is regulated, not only during EMT, but also in the early stages of cancer development, remains to be elucidated. In the present study, we show an inverse correlation of AGR2 with ZEB1 (zinc finger enhancer binding protein, δEF1) that was verified by analysis of several independent clinical data sets of lung adenocarcinomas. We also identified the ZEB1 binding site within the AGR2 promoter region and confirmed AGR2 as a novel molecular target of ZEB1. The overexpression of ZEB1 decreased the promoter activity of the AGR2 gene, which resulted in reduced AGR2 protein level and the acquisition of a more invasive phenotype of these lung cancer cells. Conversely, silencing of ZEB1 led not only to increased levels of AGR2 protein, but also attenuated the invasiveness of tumor cells. The AGR2 knockout, vice versa, increased ZEB1 expression, indicating that the ZEB1/AGR2 regulatory axis may function in a double negative feedback loop. In conclusion, we revealed for the first time that ZEB1 regulates AGR2 at the transcriptional level, while AGR2 presence contributes to ZEB1 mRNA degradation. Thus, our data identify a new regulatory mechanism between AGR2 and ZEB1, two rivals in the EMT process, tightly associated with the development of metastasis.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

There is growing epidemiological evidence indicating an association between diabetes mellitus and the increased incidence of colorectal cancer (CRC). The preferred initial and most widely used pharmacological agent for the treatment of type 2 diabetes is metformin, which in parallel reduces the risk of CRC and improves patient prognosis. AMP-activated protein kinase (AMPK) appears to be tightly associated with the beneficial metabolic effects of metformin, serving as a cellular energy sensor activated in response to a variety of conditions that deplete cellular energy levels. Such conditions include nutrient starvation (particularly glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex. The aim of the present study was to determine the effect of metformin on CRC cell lines, with different levels of anterior gradient 2 (AGR2) expression, exposed to 5-fluorouracil (5-FU) and oxaliplatin, alone or in combination with metformin. AGR2 has recently emerged as a factor involved in colon carcinogenesis. In AGR2 -knockout cells, markedly higher levels of phosphorylated-AMPK were observed in comparison with control cells transfected with GFP -scrambled guide RNA, which indicated that the presence of AGR2 may interfere with the metformin-dependent activation of AMPK. In addition, metformin in combination with 5-FU and oxaliplatin induced ROS production and attenuated autophagy. This effect was enhanced in AGR2 -knockout cells., (Copyright © 2019, Spandidos Publications.)

Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas and are often secreted in cancer patients' specimens, which suggests a role for AGR proteins in intra and extracellular compartments. Although these proteins exhibit high sequence homology, AGR2 is predominantly described as a pro-oncogene and a potential prognostic biomarker. However, little is known about the function of AGR3. Therefore, the aim of the present study was to investigate the role of AGR3 in breast cancer. The results demonstrated that breast cancer cells secrete AGR3. Furthermore, it was revealed that extracellular AGR3 (eAGR3) regulates tumor cell adhesion and migration. The current study indicated that the pharmacological and genetic perturbation of Src kinase signaling, through treatment with Dasatinib (protein kinase inhibitor) or investigating cells that express a dominant-negative form of Src, significantly abrogated eAGR3-mediated breast cancer cell migration. Therefore, the results indicated that eAGR3 may control tumor cell migration via activation of Src kinases. The results of the present study indicated that eAGR3 may serve as a microenvironmental signaling molecule in tumor-associated processes., (Copyright: © Obacz et al.)

Background: Organometallic compounds are chemical substances containing a carbon-metal bond. From a biological point of view, these compounds are generally considered to be toxic for living organisms. They may exert therapeutic potential, especially as anticancer or antimicrobial drugs. Their structural variability and usually uncharged and mostly lipophilic character are particularly advantageous properties. Platinum derivatives (predominately cisplatin) are the most proven advantageous agents in the medical field. The success of cisplatin has led the scientific community to focus on the synthesis of other organometallic compounds with improved anti-tumour effects and lower cytotoxicity towards healthy tissues. Close attention is focused on compounds bearing atoms of iron, titanium or ruthenium., Purpose: Here, we focus on summarising a description of the most important compounds containing iron, titanium or ruthenium atoms in their structure, showing potential application in cancer treatment including the mechanism of action for some of the most commonly studied compounds. The reported structures were used successfully in preclinical studies including animal models and progressed to various stages of human clinical trials. Despite the fail-ure of some of these compounds, there are still several candidates which are expected to progress to the late stages of the clinical trials either alone or as part of combined chemotherapy. Ruthenium-containing substances in particular show high potential for utilisation in cancer treatment due to low cytotoxicity associated with the ability to block neoangiogenesis and metastasis development.

Sensory neurons of the peripheral nervous system are critical in health and disease. Sensory neurons derived from induced pluripotent stem (iPS) cells are now being used increasingly for in vitro models of neuropathy, pain, and neurotoxicity. DNA methylation is critical for neurodevelopment and has been implicated in many neuronal diseases, but has not been examined in iPS-derived sensory neurons. In order to better characterize the iPS-derived sensory neuron model, we have undertaken a genome-wide DNA methylation study on the cells from human iPS to iPS-derived sensory neurons during differentiation through reduced representation and bisulfite sequencing. We report decreasing DNA methylation with iPS-derived sensory neuronal differentiation that is reflected in increasing numbers and proportions of hypomethylated individual CpGs and regions, as well as lowered DNMT3b expression. Furthermore, genes with changes in DNA methylation near their TSS suggest key pathways that may be involved in iPS-derived sensory neuronal differentiation. These findings provide insights into sensory neuronal differentiation and can be used for further in vitro modelling of disease states.

Accurate classification of breast tumors is vital for patient management decisions and enables more precise cancer treatment. Here, we present a quantitative proteotyping approach based on sequential windowed acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry and establish key proteins for breast tumor classification. The study is based on 96 tissue samples representing five conventional breast cancer subtypes. SWATH proteotype patterns largely recapitulate these subtypes; however, they also reveal varying heterogeneity within the conventional subtypes, with triple negative tumors being the most heterogeneous. Proteins that contribute most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2 and are associated with estrogen receptor (ER) status, tumor grade status, and HER2 status. Although these three key proteins exhibit high levels of correlation with transcript levels (R > 0.67), general correlation did not exceed R = 0.29, indicating the value of protein-level measurements of disease-regulated genes. Overall, this study highlights how cancer tissue proteotyping can lead to more accurate patient stratification., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)