Direct observation of DNA alterations induced by a DNA disruptor.

DNA alterations, such as base modifications and mutations, are closely related to the activity of transcription factors and the corresponding cell functions; therefore, detection of DNA alterations is important for understanding their relationships. Particularly, DNA alterations caused by exposure to exogenous molecules, such as nucleic acid analogues for cancer therapy and the corresponding changes in cell functions, are of interest in medicine for drug development and diagnosis purposes. However, detection of comprehensive direct evidence for the relationship of DNA modifications/mutations in genes, their effect on transcription factors, and the corresponding cell functions have been limited. In this study, we utilized a single-molecule electrical detection method for the direct observation of DNA alterations on transcription factor binding motifs upon exposure to a nucleic acid analogue, trifluridine (FTD), and evaluated the effects of the DNA alteration on transcriptional activity in cancer cell line cells. We found ~ 10% FTD incorporation at the transcription factor p53 binding regions in cancer cells exposed to FTD for 5 months. Additionally, through single-molecule analysis of p53-enriched DNA, we found that the FTD incorporation at the p53 DNA binding regions led to less binding, likely due to weaken the binding of p53. This work suggests that single-molecule detection of DNA sequence alterations is a useful methodology for understanding DNA sequence alterations.

Clinicopathological significance of MYL9 expression in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, and often involves invasion and distant metastasis from the early tumor stages. Myosin II reportedly plays a key role in regulating tumor progression and metastasis. AIMS: We examined whether myosin regulatory light polypeptide 9 (MYL9) regulates cancer cell proliferation. METHODS AND RESULTS: To investigate the expression pattern and clinical significance of MYL9 in pancreatic ductal adenocarcinoma, we performed immunohistochemical analysis of samples collected from 101 patients with pancreatic ductal adenocarcinoma. The expression of MYL9 was investigated to evaluate its functional role and contribution to proliferation and apoptosis in pancreatic ductal adenocarcinoma cells in vitro. The results showed that MYL9 was predominantly expressed in the cytoplasm and membrane of pancreatic ductal adenocarcinoma cells. Multivariate analysis indicated that MYL9 acted as an independent prognostic factor for overall survival and distant metastasis-free survival. MYL9 expression was strongly associated with malignancy in in vitro analyses, including proliferation and anti-apoptotic activities. CONCLUSIONS: Our findings suggest that MYL9 is an independent prognostic factor of pancreatic ductal adenocarcinoma. MYL9 is a crucial biomarker and potential therapeutic target for pancreatic ductal adenocarcinoma.

Computational healthcare: Present and future perspectives (Review).

Artificial intelligence (AI) has been developed through repeated new discoveries since around 1960. The use of AI is now becoming widespread within society and our daily lives. AI is also being introduced into healthcare, such as medicine and drug development; however, it is currently biased towards specific domains. The present review traces the history of the development of various AI-based applications in healthcare and compares AI-based healthcare with conventional healthcare to show the future prospects for this type of care. Knowledge of the past and present development of AI-based applications would be useful for the future utilization of novel AI approaches in healthcare.

Single-molecule RNA sequencing for simultaneous detection of m6A and 5mC.

Epitranscriptomics is the study of RNA base modifications involving functionally relevant changes to the transcriptome. In recent years, epitranscriptomics has been an active area of research. However, a major issue has been the development of sequencing methods to map transcriptome-wide RNA base modifications. We have proposed a single-molecule quantum sequencer for mapping RNA base modifications in microRNAs (miRNAs), such as N6-methyladenosine (m6A) or 5-methylcytidine (5mC), which are related to cancer cell propagation and suppression. Here, we investigated 5mC and m6A in hsa-miR-200c-5p extracted from colorectal cancer cells and determined their methylation sites and rates; the data were comparable to those determined by mass spectrometry. Furthermore, we evaluated the methylation ratio of cytidine and adenosine at each site in the sequences and its relationship. These results suggest that the methylation ratio of cytidine and adenosine is facilitated by the presence of vicinal methylation. Our work provides a robust new tool for sequencing various types of RNA base modifications in their RNA context.

Scent test using Caenorhabditis elegans to screen for early-stage pancreatic cancer.

Although early detection and diagnosis are indispensable for improving the prognosis of patients with pancreatic cancer, both have yet to be achieved. Except for pancreatic cancer, other cancers have already been screened through scent tests using animals or microorganisms, including Caenorhabditis elegans. While such a method may greatly improve the prognosis of pancreatic cancer, no studies have investigated the same, mainly given the difficulty of collecting suitable samples from patients with early-stage pancreatic cancer. In this study, we organized a nationwide study group comprising high-volume centers throughout Japan to collect patients with very-early-stage pancreatic cancer (stage 0 or IA). We initially performed an open-label study involving 83 cases (stage 0-IV), with subsequent results showing significant differences after surgical removal in stage 0-IA (×10 dilution: p < 0.001; ×100 dilution: p < 0.001). Thereafter, a blinded study on 28 cases (11 patients with stage 0 or IA disease and 17 healthy volunteers) was conducted by comparing very-early-stage pancreatic cancer patients with healthy volunteers to determine whether C. elegans could detect the scent of cancer for the diagnosis of early-stage pancreatic cancer. Preoperative urine samples had a significantly higher chemotaxis index compared to postoperative samples in patients with pancreatic cancer [×10 dilution: p < 0.001, area under the receiver operating characteristic curve (AUC) = 0.845; ×100 dilution: p < 0.001, AUC = 0.820] and healthy volunteers (×10 dilution: p = 0.034; ×100 dilution: p = 0.088). Moreover, using the changes in preoperative and postoperative chemotaxis index, this method had a higher sensitivity for detecting early pancreatic cancer compared to existing diagnostic markers. The clinical application C. elegans for the early diagnosis of cancer can certainly be expected in the near future.

Immuno-Surgical Management of Pancreatic Cancer with Analysis of Cancer Exosomes.

Exosomes (EXs), a type of extracellular vesicles secreted from various cells and especially cancer cells, mesenchymal cells, macrophages and other cells in the tumor microenvironment (TME), are involved in biologically malignant behaviors of cancers. Recent studies have revealed that EXs contain microRNAs on their inside and express proteins and glycolipids on their outsides, every component of which plays a role in the transmission of genetic and/or epigenetic information in cell-to-cell communications. It is also known that miRNAs are involved in the signal transduction. Thus, EXs may be useful for monitoring the TME of tumor tissues and the invasion and metastasis, processes that are associated with patient survival. Because several solid tumors secrete immune checkpoint proteins, including programmed cell death-ligand 1, the EX-mediated mechanisms are suggested to be potent targets for monitoring patients. Therefore, a companion therapeutic approach against cancer metastasis to distant organs is proposed when surgical removal of the primary tumor is performed. However, EXs and immune checkpoint mechanisms in pancreatic cancer are not fully understood, we provide an update on the recent advances in this field and evidence that EXs will be useful for maximizing patient benefit in precision medicine.

Hereditary pancreatitis model by blastocyst complementation in mouse.

The application of pluripotent stem cells is expected to contribute to the elucidation of unknown mechanism of human diseases. However, in vitro induction of organ-specific cells, such as pancreas and liver, is still difficult and the reproduction of their disorders in a model has been unfeasible. To study the mechanism of human hereditary pancreatitis (HP), we here performed the blastocyst complementation (BC) method. In the BC method, mouse embryonic stem (ES) cells harboring CRISPR/CAS9-mediated mutations in the Prss1 gene were injected into blastocysts with deficient Pdx1 gene, which is a critical transcription factor in the development of pancreas. The results showed that trypsin was activated extremely in Prss1-mutant mice. This implied that the mouse phenotype mimics that of human HP and that the BC method was useful for the reproduction and study of pancreatic disorders. The present study opens the possibility of investigating uncharacterized human diseases by utilizing the BC method.

State-of-the-Art Technology of Model Organisms for Current Human Medicine.

Since the 1980s, molecular biology has been used to investigate medical field mechanisms that still require the use of crude biological materials in order to achieve their necessary goals. Transcription factor-induced pluripotent stem cells are used in regenerative medicine to screen drugs and to support lost tissues. However, these cells insufficiently reconstruct whole organs and require various intact cells, such as damaged livers and diabetic pancreases. For efficient gene transfer in medical use, virally mediated gene transfers are used, although immunogenic issues are investigated. To obtain efficient detective and diagnostic power in intractable diseases, biological tools such as roundworms and zebrafish have been found to be useful for high-throughput screening (HST) and diagnosis. Taken together, this biological approach will help to fill the gaps between medical needs and novel innovations in the field of medicine.

Convolutional Neural Network Can Recognize Drug Resistance of Single Cancer Cells.

It is known that single or isolated tumor cells enter cancer patients' circulatory systems. These circulating tumor cells (CTCs) are thought to be an effective tool for diagnosing cancer malignancy. However, handling CTC samples and evaluating CTC sequence analysis results are challenging. Recently, the convolutional neural network (CNN) model, a type of deep learning model, has been increasingly adopted for medical image analyses. However, it is controversial whether cell characteristics can be identified at the single-cell level by using machine learning methods. This study intends to verify whether an AI system could classify the sensitivity of anticancer drugs, based on cell morphology during culture. We constructed a CNN based on the VGG16 model that could predict the efficiency of antitumor drugs at the single-cell level. The machine learning revealed that our model could identify the effects of antitumor drugs with ~0.80 accuracies. Our results show that, in the future, realizing precision medicine to identify effective antitumor drugs for individual patients may be possible by extracting CTCs from blood and performing classification by using an AI system.

COVID-19 Drug Discovery Using Intensive Approaches.

Since the infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China during December 2019, the coronavirus disease 2019 (COVID-19) has spread on a global scale, causing the World Health Organization (WHO) to issue a warning. While novel vaccines and drugs that target SARS-CoV-2 are under development, this review provides information on therapeutics which are under clinical trials or are proposed to antagonize SARS-CoV-2. Based on the information gained from the responses to other RNA coronaviruses, including the strains that cause severe acute respiratory syndrome (SARS)-coronaviruses and Middle East respiratory syndrome (MERS), drug repurposing might be a viable strategy. Since several antiviral therapies can inhibit viral replication cycles or relieve symptoms, mechanisms unique to RNA viruses will be important for the clinical development of antivirals against SARS-CoV-2. Given that several currently marketed drugs may be efficient therapeutic agents for severe COVID-19 cases, they may be beneficial for future viral pandemics and other infections caused by RNA viruses when standard treatments are unavailable.

Theoretical analyses and experimental validation of the effects caused by the fluorinated substituent modification of DNA.

Halogen-modified nucleic acid molecules, such as trifluorothymidine (FTD) and 5-fluorouracil, are widely used in medical science and clinical site. These compounds have a very similar nucleobase structure. It is reported that both of these compounds could be incorporated into DNA. The incorporation of FTD produces highly anti-tumor effect. However, it is not known whether to occur a significant effect by the incorporation of 5-fluorouracil. Nobody knows why such a difference will occur. To understand the reason why there is large differences between trifluorothymidine and 5-fluorouracil, we have performed the molecular dynamics simulations and molecular orbital calculations. Although the active interaction energy between Halogen-modified nucleic acids or and complementary adenine was increased, in only FTD incorporated DNA, more strongly dispersion force interactions with an adjacent base were detected in many thermodynamic DNA conformations. As the results, the conformational changes occur even if it is in internal body temperature. Then the break of hydrogen bonding between FTD and complementary adenine base occur more frequently. The double helix structural destabilization of DNA with FTD is resulted from autoagglutination caused by the bonding via halogen orbitals such as halogen bonding and the general van der Waals interactions such as CH-[Formula: see text], lone pair (LP)-[Formula: see text], and [Formula: see text]-[Formula: see text] interactions. Therefore, it is strongly speculated that such structural changes caused by trifluoromethyl group is important for the anti-tumor effect of FTD alone.

One-carbon metabolism for cancer diagnostic and therapeutic approaches.

Altered metabolism is critical for the rapid and unregulated proliferation of cancer cells; hence the requirement for an abundant source of nucleotides. One characteristic of this metabolic reprogramming is in one-carbon (1C) metabolism, which is particularly noteworthy for its role in DNA synthesis. Various forms of methylation are also noteworthy as they relate to cancer cell survival and proliferation. In recent years, 1C metabolism has received substantial attention for its role in cancer malignancy via these functions. Therefore, therapeutic inhibitors targeting 1C metabolism have been utilized as anticancer drugs. This review outlines the importance of 1C metabolism and its clinical application in cancer. Understanding 1C metabolism could aid the development of novel cancer diagnostic and therapeutic methods.

CD44/CD133-Positive Colorectal Cancer Stem Cells are Sensitive to Trifluridine Exposure.

Cancer stem cells (CSCs) are involved in metastatic colorectal cancer recurrence, but no effective therapy targeting these cells is currently available. Because trifluridine (FTD)/tipiracil therapy is used for refractory colorectal cancer, we sought to determine whether FTD is effective against CSC-like cells. CD44+CD133+ high-expressing and other populations of human DLD-1 colon cancer cells were separately isolated through fluorescence-activated cell sorting. The sphere-forming activity of each population and the anti-sphere-forming effects of FTD and fluorouracil (5-FU) on CD44+CD133+ cells were then measured. CD44+CD133+ DLD-1 cells formed substantially more spheres than other cells. Moreover, treating CD44+CD133+ DLD-1 cells with subtoxic concentrations of FTD (1 µM) inhibited sphere formation, and this was superior to the effect of subtoxic concentrations (1 µM) of 5-FU. The associated inhibition rates for FTD and 5-FU were 58.2% and 26.1%, respectively. Further, CD44+CD133+ DLD-1 cells expressed higher levels of thymidine kinase 1, which is responsible for FTD phosphorylation, than DLD-1 cells, and FTD was incorporated into the DNA of CD44+CD133+ DLD-1 cells. Thus, our data show that FTD treatment is effective against CSC-like cells and might be applied as CSC-targeting chemotherapy for tumor subtypes with high CD44 and CD133 expression.

Distinct methylation levels of mature microRNAs in gastrointestinal cancers.

The biological significance of micro (mi)RNAs has traditionally been evaluated according to their RNA expression levels based on the assumption that miRNAs recognize and regulate their targets in an unvarying fashion. Here we show that a fraction of mature miRNAs including miR-17-5p, -21-5p, and -200c-3p and let-7a-5p harbor methyl marks that potentially alter their stability and target recognition. Importantly, methylation of these miRNAs was significantly increased in cancer tissues as compared to paired normal tissues. Furthermore, miR-17-5p methylation level in serum samples distinguished early pancreatic cancer patients from healthy controls with extremely high sensitivity and specificity. These findings provide a basis for diagnostic strategies for early-stage cancer and add a dimension to our understanding of miRNA biology.

A Hydrogen Peroxide Activatable Gemcitabine Prodrug for the Selective Treatment of Pancreatic Ductal Adenocarcinoma.

The main concern in the use of anticancer chemotherapeutic drugs is host toxicity. Patients need to interrupt or change chemotherapy due to adverse effects. In this study, we aimed to decrease adverse events with gemcitabine (GEM) in the treatment of pancreatic ductal adenocarcinoma and focused on the difference of hydrogen peroxide levels in normal versus cancer cells. We designed and synthesized a novel boronate-ester-caged prodrug that is activated by the high H2 O2 concentrations found in cancer cells to release GEM. An H2 O2 -activatable GEM (A-GEM) has higher selectivity for H2 O2 over other reactive oxygen species (ROS) and cytotoxic effects corresponding to the H2 O2 concentration in vitro. A xenograft model of immunodeficient mice indicated that the effect of A-GEM was not inferior to that of GEM when administered in vivo. In particular, myelosuppression was significantly decreased following A-GEM treatment compared with that following GEM treatment.

Direct Analysis of Incorporation of an Anticancer Drug into DNA at Single-Molecule Resolution.

Identifying positions at which anticancer drug molecules incorporate into DNA is essential to define mechanisms underlying their activity, but current methodologies cannot yet achieve this. The thymidine fluorine substitution product trifluridine (FTD) is a DNA-damaging anticancer agent thought to incorporate into thymine positions in DNA. This mechanism, however, has not been directly confirmed. Here, we report a means to detect FTD in a single-stranded oligonucleotide using a method to distinguish single molecules by differences in electrical conductance. Entire sequences of 21-base single-stranded DNAs with and without incorporated drug were determined based on single-molecule conductances of the drug and four deoxynucleosides, the first direct observation of its kind. This methodology may foster rapid development of more effective anticancer drugs.

Mitochondrial pyruvate carrier 1 expression controls cancer epithelial-mesenchymal transition and radioresistance.

Mitochondrial pyruvate carrier (MPC) is known to cause different expressions in normal and cancer cells. We observed a change in phenotype with the suppression of MPC expression. We knocked down MPC1 and/or MPC2 using siRNA or shRNA. We observed its cell morphology and accompanying molecular marker. Furthermore, the radioresistance of the MPC knockdown cell line was examined using a colony formation assay. MPC1-suppressed cells changed their morphology to a spindle shape. Epithelial-mesenchymal transition (EMT) was suspected, and examination of the EMT marker by PCR showed a decrease in E-cadherin and an increase in fibronectin. Focusing on glutamine metabolism as the mechanism of this phenomenon, we knocked down the glutamine-metabolizing enzyme glutaminase (GLS). EMT was also observed in GLS-suppressed cells. Furthermore, when MPC1-suppressed cells were cultured in a glutamine-deficient medium, changes in EMT markers were suppressed. In addition, MPC1-suppressed cells also increased with a significant difference in radioresistance. Decreased MPC1 expression favorably affects EMT and radioresistance of cancer.

Poly(ethylene glycol)-poly(lysine) block copolymer-ubenimex conjugate targets aminopeptidase N and exerts an antitumor effect in hepatocellular carcinoma stem cells.

Previous studies highlighted that aminopeptidase N (APN)/CD13 acts as a scavenger in the survival of hepatocellular carcinoma (HCC) stem cells by reducing reactive oxygen species (ROS) levels. Hence, it has been proposed that APN/CD13 inhibition can increase cellular ROS levels and sensitize cells to chemotherapeutic agents. Although ubenimex, also known as bestatin, competitively inhibits proteases such as APN/CD13 on the cellular membrane and it is clinically used for patients with acute myeloid leukemia and lymphedema, research has demonstrated that higher concentrations of the agent induce the death of APN/CD13+ HCC stem cells. In this study, we developed a poly(ethylene glycol)-poly(lysine) block copolymer-ubenimex conjugate (PEG-b-PLys(Ube)) to increase the efficacy of reagents in APN/CD13+ cancer stem cells. Exposure to PEG-b-PLys(Ube) increased the intracellular ROS concentration by inhibiting APN enzyme activity, permitting the induction of apoptosis and attenuation of HCC cell proliferation. In addition, PEG-b-PLys(Ube) exhibited a relatively stronger antitumor effect in mice than PEG-b-PLys alone or phosphate-buffered saline. Moreover, an isobologram analysis revealed that combinations of fluorouracil, cisplatin, or doxorubicin with PEG-b-PLys(Ube) exhibited synergistic effects. This study demonstrated that PEG-b-PLys(Ube) does not impair the properties of ubenimex and exerts a potent antitumor effect.

Drug discovery of anticancer drugs targeting methylenetetrahydrofolate dehydrogenase 2.

Many anticancer drugs have serious adverse effects; therefore, it is necessary to target features specific to cancer cells to minimize the effects on healthy cells. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was reported to be specifically enhanced in cancer. We confirmed the validity of MTHFD2 as a drug discovery target using clinical data. In addition, we performed in silico screening to design an anticancer drug specifically targeting MTHFD2. Analysis of the clinical data indicated that MTHFD2 was enhanced in most cancers compared with normal tissues, and affected the prognosis in cancer patients. Candidate compounds for MTHFD2 inhibitors were identified using in silico drug discovery techniques, and the important interactions for MTHFD2 binding were determined. In addition, these candidate compounds decreased levels of MTHFD2 metabolites in cancer cells. The findings of the present study may help to develop anticancer drugs targeting MTHFD2, with a view to minimizing the adverse effects of anticancer drugs.

Polyamine flux suppresses histone lysine demethylases and enhances ID1 expression in cancer stem cells.

Cancer stem cells (CSCs) exhibit tumorigenic potential and can generate resistance to chemotherapy and radiotherapy. A labeled ornithine decarboxylase (ODC, a rate-limiting enzyme involved in polyamine [PA] biosynthesis) degradation motif (degron) system allows visualization of a fraction of CSC-like cells in heterogeneous tumor populations. A labeled ODC degradation motif system allowed visualization of a fraction of CSC-like cells in heterogeneous tumor populations. Using this system, analysis of polyamine flux indicated that polyamine metabolism is active in CSCs. The results showed that intracellular polyamines inhibited the activity of histone lysine 4 demethylase enzymes, including lysine-specific demethylase-1 (LSD1). Chromatin immunoprecipitation with Pol II antibody followed by massively parallel DNA sequencing, revealed the global enrichment of Pol II in transcription start sites in CSCs. Increase of polyamines within cells resulted in an enhancement of ID1 gene expression. The results of this study reveal details of metabolic pathways that drive epigenetic control of cancer cell stemness and determine effective therapeutic targets in CSCs.