Downregulation of miR-122-5p Activates Glycolysis via PKM2 in Kupffer Cells of Rat and Mouse Models of Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) has pathological characteristics similar to those of alcoholic hepatitis, despite the absence of a drinking history. The greatest threat associated with NASH is its progression to cirrhosis and hepatocellular carcinoma. The pathophysiology of NASH is not fully understood to date. In this study, we investigated the pathophysiology of NASH from the perspective of glycolysis and the Warburg effect, with a particular focus on microRNA regulation in liver-specific macrophages, also known as Kupffer cells. We established NASH rat and mouse models and evaluated various parameters including the liver-to-body weight ratio, blood indexes, and histopathology. A quantitative phosphoproteomic analysis of the NASH rat model livers revealed the activation of glycolysis. Western blotting and immunohistochemistry results indicated that the expression of pyruvate kinase muscle 2 (PKM2), a rate-limiting enzyme of glycolysis, was upregulated in the liver tissues of both NASH models. Moreover, increases in PKM2 and p-PKM2 were observed in the early phase of NASH. These observations were partially induced by the downregulation of microRNA122-5p (miR-122-5p) and occurred particularly in the Kupffer cells. Our results suggest that the activation of glycolysis in Kupffer cells during NASH was partially induced by the upregulation of PKM2 via miR-122-5p suppression.

Delta-like canonical Notch ligand 3 as a potential therapeutic target in malignancies: A brief overview.

Delta-like canonical Notch ligand 3 (DLL3) is a member of the Delta/Serrate/Lag2 (DSL) Notch receptor ligand family and plays a crucial role in Notch signaling, which influences various cellular processes including differentiation, proliferation, survival, and apoptosis. DLL3 is expressed throughout the presomitic mesoderm and is localized to the rostral somatic compartments; mutations in DLL3 induce skeletal abnormalities such as spondylocostal dysostosis. Recently, DLL3 has attracted interest as a novel molecular target due to its high expression in neuroendocrine carcinoma of the lung. Moreover, a DLL3-targeting Ab-drug conjugate, rovalpituzumab tesirine (ROVA-T), has been developed as a new treatment with proven antitumor activity. However, the development of ROVA-T was suspended because of shorter overall survival compared to topotecan, the second-line standard treatment. Thus, several studies on the mechanism and function of DLL3 in several malignancies are underway to find a new strategy for targeting DLL3. In this review, we discuss the roles of DLL3 in various malignancies and the future perspectives of DLL3-related research, especially as a therapeutic target.

Uptake of MicroRNAs from Exosome-Like Nanovesicles of Edible Plant Juice by Rat Enterocytes.

MicroRNAs (miRNAs) are small RNAs present in extracellular vesicles (EVs) that, when transferred to a target cell, affect its biological functions. Plant miRNAs regulate the expression of certain mammalian genes. Here, we characterized EVs in fruit and vegetable juice, and their miRNA cargo, and investigated whether such miRNA-containing EVs could be taken up by mammalian enterocytes in vitro. Using filtration and ultra-centrifugation methods, EVs were purified from commercially available and manually squeezed plant juice. EV morphological features and subcellular localization were analyzed using the NanoSight tracking system and electron microscopy. Plant EV miRNA levels were evaluated using quantitative reverse transcription PCR. For the in vitro EV uptake experiments, rat intestinal epithelial cells (IEC6) were used. Plant EVs shared morphological features with mammalian EVs and contained miR156a-5p, miR166a-3p, and miR168a-5p. EVs were present in the cell sap-filled central vacuoles and were taken up by IEC6 cells. Edible plant cells produce EVs that contain various miRNAs and release them into the central vacuole. The exogenous plant EVs are taken up by mammalian enterocytes in vitro. These findings suggest the possibility that exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract.

Delta-like 3 localizes to neuroendocrine cells and plays a pivotal role in gastrointestinal neuroendocrine malignancy.

Delta-like 3 (DLL3) is a member of the Delta/Serrate/Lag2 (DSL) group of Notch receptor ligands. Five DSL ligands are known in mammals, among which DLL3 has a unique structure. In the last few years, DLL3 has attracted attention as a novel molecular targeting gene in neuroendocrine carcinoma of the lung due to its high expression. However, the expression pattern and functions of DLL3 in the gastrointestinal tract and gastrointestinal neuroendocrine carcinoma remain unclear. In this study, we examined the expression and role of DLL3 in the gastrointestinal tract, as well as in gastrointestinal neuroendocrine carcinoma. Immunohistochemical staining of the human normal gastrointestinal tract revealed that DLL3 localized in neuroendocrine cells. DLL3 showed intense staining in chromogranin A-positive gastric cancer specimens. Real-time quantitative RT-PCR and western blotting analyses showed considerable upregulation of DLL3 in gastrointestinal neuroendocrine carcinoma cell lines. Immuno-electron microscopy demonstrated abundant expression of DLL3 in neurosecretory granules in these cells. Furthermore, gene silencing of DLL3 caused significant growth inhibition through the induction of intrinsic apoptosis. Our findings suggest that DLL3 is expressed in neuroendocrine cells of the gastrointestinal tract and that it has a pivotal role in gastrointestinal neuroendocrine carcinoma cells. Based on these findings, further investigations are required to achieve a breakthrough in developing therapeutic strategies for gastrointestinal neuroendocrine carcinoma.

α-Aminoisobutyric Acid-Containing Amphipathic Helical Peptide-Cyclic RGD Conjugation as a Potential Drug Delivery System for MicroRNA Replacement Therapy in Vitro.

Replacement therapy with tumor suppressive microRNA (TS-miRNA) might be the next-generation oligonucleotide therapy; however, a novel drug delivery system (DDS) is required. Recently, we developed the cell-penetrating peptide, model amphipathic peptide with α-aminoisobutyric acid (MAP(Aib)), as a carrier for oligonucleotide delivery to cells. In this study, we examined whether a modified MAP(Aib) analogue, MAP(Aib)-cRGD, could be a DDS for TS-miRNA replacement therapy. MIR145-5p, a representative TS-miRNA especially in colorectal cancer, was selected. The MAP(Aib)-cRGD dose was adjusted for MIR145-5p delivery to cells using peripheral blood mononuclear cells and degradation analysis. AlexaFluor488-labeled MIR145-5p incorporation into cells and negative regulation of MIR145-5p-targeting genes demonstrated MAP(Aib)-cRGD's functionality as a miRNA DDS. Treating MIR145-5p with MAP(Aib)-cRGD also revealed various anticancer effects, such as cell viability, invasion inhibition, and apoptosis induction in WiDr cells. Altogether, these findings suggest that MAP(Aib)-cRGD could be a DDS for TS-miRNA replacement therapy, but in vivo investigations are required.

Analysis of Extracellular Vesicles in Gastric Juice from Gastric Cancer Patients.

Extracellular vesicles (EVs) are secretory membrane vesicles containing lipids, proteins, and nucleic acids; they function in intercellular transport by delivering their components to recipient cells. EVs are observed in various body fluids, i.e., blood, saliva, urine, amniotic fluid, and ascites. EVs secreted from cancer cells play important roles in the formation of their environment, including fibrosis, angiogenesis, evasion of immune surveillance, and even metastasis. However, EVs in gastric juice (GJ-EVs) have been largely unexplored. In this study, we sought to clarify the existence of GJ-EVs derived from gastric cancer patients. GJ-EVs were isolated by the ultracentrifuge method combined with our own preprocessing from gastric cancer (GC) patients. We verified GJ-EVs by morphological experiments, i.e., nanoparticle tracking system analysis and electron microscopy. In addition, protein and microRNA markers of EVs were examined by Western blotting analysis, Bioanalyzer, or quantitative reverse transcription polymerase chain reaction. GJ-EVs were found to promote the proliferation of normal fibroblast cells. Our findings suggest that isolates from the GJ of GC patients contain EVs and imply that GJ-EVs partially affect their microenvironments and that analysis using GJ-EVs from GC patients will help to clarify the pathophysiology of GC.

Organ-Specific MicroRNAs (MIR122, 137, and 206) Contribute to Tissue Characteristics and Carcinogenesis by Regulating Pyruvate Kinase M1/2 (PKM) Expression.

Pyruvate kinase is known as the glycolytic enzyme catalyzing the final step in glycolysis. In mammals, two different forms of it exist, i.e., pyruvate kinase M1/2 (PKM) and pyruvate kinase L/R (PKLR). Also, PKM has two isoforms, i.e., PKM1 and PKM2. These genes have tissue-specific distribution. Namely, PKM1 is distributed in high-energy-demanding organs, such as brain and muscle. Also, PKM2 is distributed in various other organs, such as the colon. On the other hand, PKLR is distributed in liver and red blood cells (RBCs). Interestingly, PKM2 has been recognized as one of the essential genes for the cancer-specific energy metabolism termed the “Warburg effect”. However, the mechanism(s) underlying this fact have remained largely unclear. Recently, we found that some organ-specific microRNAs (miRNAs, MIR) regulate PKM isoform expression through direct targeting of polypyrimidine tract binding protein 1 (PTBP1), which is the splicer responsible for PKM2-dominant expression. In this study, we examined whether this machinery was conserved in the case of other PTBP1- and PKM-targeting miRNAs. We focused on the MIRs 122, 137, and 206, and investigated the expression profiles of each of these miRNAs in tissues from mouse and human organs. Also, we examined the regulatory mechanisms of PKM isoform expression by testing each of these miRNAs in human cancer cell lines. Presently, we found that brain-specific MIR137 and muscle-specific MIR206 predominantly induced PKM1 expression through direct targeting of PTBP1. Also, liver-specific MIR122 suppressed the expression of both PKM1 and PKM2, which action occurred through direct targeting of PKM to enable the expression of PKLR. Moreover, the expression levels of these miRNAs were downregulated in cancer cells that had originated from these tissues, resulting in PKM2 dominance. Our results suggest that the organ-specific distribution of miRNAs is one of the principal means by which miRNA establishes characteristics of a tissue and that dysregulation of these miRNAs results in cancer development through a change in the ratio of PKM isoform expression. Also, our results contribute to cancer diagnosis and will be useful for cancer-specific therapy for the Warburg effect in the near future.

Glycolysis in gastrointestinal stromal tumor: a brief overview.

Gastrointestinal stromal tumor (GIST) is the most prevalent mesenchymal tumor of the digestive tract. Its growth is primarily influenced by mutations in KIT or PDGFRA. Surgery is the primary treatment option for GIST; however, KIT inhibitors, such as imatinib, are used for inoperable cases. Resistance to imatinib is an upcoming challenge, especially because the effectiveness of alternative drugs is limited. Enhancement of the glycolysis pathway in cancer cells has been identified as a key feature in cancer. This unique metabolic activity has implications on tumor growth, prognosis, and resistance to therapy, even in GIST. Members of the glucose transporter (GLUT) family (particularly GLUT-1) play a significant role in GIST progression and response to treatment. Diagnostic imaging using 18F-fluorodeoxyglucose positron emission tomography/computed tomography, which enables visualization of glucose metabolism, can aid in GIST diagnosis and risk assessment. The interplay between glycolysis and GIST can lead to the development of various therapeutic strategies, especially those involving glycolysis-related molecules, such as hexokinase and lactate dehydrogenase. However, further research is required to understand the full spectrum of glycolysis in GIST and its therapeutic potential. Herein, we present an exhaustive overview and analysis of the role of glycolysis in GIST, especially as a therapeutic target.

Antitumor effects of chemically modified miR-143 lipoplexes in a mouse model of pelvic colorectal cancer via myristoylated alanine-rich C kinase substrate downregulation.

Replenishing tumor-suppressor miRNAs (TS-miRNAs) is a potential next-generation nucleic acid-based therapeutic approach. Establishing an effective miRNA delivery system is essential to successful TS-miRNA therapy. To overcome vulnerability to RNA nucleases, we previously developed a chemically modified miRNA143-3p (CM-miR-143). In clinical practice, colorectal cancer (CRC) pelvic recurrence is an occasional challenge following curative resection, requiring a novel therapy because reoperative surgery poses a significant burden to the patient. Hence, we considered the use of CM-miR-143 as an alternative treatment. In this study, we used a mouse model bearing pelvic CRC adjacent to the rectum and investigated the anticancer effects of CM-miR-143 lipoplexes formulated from miRNA and a cationic liposome. Compared with commercial synthetic miR-143, CM-miR-143 lipoplexes accumulated heavily in regions of the pelvic CRC tumor where the blood flow was high. As a result, systemic administration of CM-miR-143 lipoplexes improved animal survival by significantly suppressing pelvic CRC tumors and relieving a lethal bowel obstruction caused by rectal compression. Detailed protein analysis revealed that the myristoylated alanine-rich C kinase is a novel target for CM-miR-143 lipoplexes. Our results suggest that CM-miR-143 is a potential next-generation drug candidate in the treatment of CRC pelvic recurrence.

Keloid Nodule Metabolic Activity for Continuous Expansion.

Background: We previously reported that keloid nodules had such specific structures that higher expression of autophagy proteins and glycolytic markers was observed in the central zone fibroblasts than in marginal zone fibroblasts. The purpose of this study is to investigate how keloid nodules play a role in metabolic activity for continuous expansion. Methods: A total of 57 nodules were randomly chosen from seven keloid samples and divided into four groups of disease duration (2, 4, 6, and 17 years). Immunohistochemical and immunofluorescent analyses were performed. Results: Immunohistochemical analysis with anti-CD-31 confirmed that the nodules had a structure with a greater number of vessels in the marginal zone than in the central zone. The density of fibroblasts in nodules (190.29 ± 64.45) was significantly higher than that of surrounding connective tissue (140.18 ± 63.94) (P < 0.05).The area of each nodule increased for 2 to 4 years, then decreased, graphically represented by an approximately horizontal line, to 17 years. The ratio of total nodule area/dermis area increased as disease duration lengthened. The maximum ratio was the 17-year group at 48.01%. The nodule number/dermis area ratio rose approximately with advancing disease duration. Conclusions: Instead of increasing the size of the nodules, their number and total area increased within the keloid lesions. We believe that the keloid nodules must play an important role in energy metabolic activity for continuous growth by increasing in number and total area.

Glucose transporter­1 inhibition overcomes imatinib resistance in gastrointestinal stromal tumor cells.

Imatinib mesylate (imatinib) is the primary agent of choice used to treat gastrointestinal stromal tumors (GIST). However, drug resistance to imatinib poses a major obstacle to treatment efficacy. In addition, the relationship between imatinib resistance and glycolysis is poorly understood. Glucose transporter (GLUT)­1 is a key component of glycolysis. The present study aimed to assess the potential relationship between components in the glycolytic pathway and the acquisition of imatinib resistance by GIST cells, with particular focus on GLUT­1. An imatinib­resistant GIST cell line was established through the gradual and continuous imatinib treatment of the parental human GIST cell line GIST­T1. The expression of glycolysis­related molecules (GLUT­1, hexokinase 2, pyruvate kinase M2 and lactate dehydrogenase) was assessed in parental and imatinib­resistant cells by western blotting, reverse transcription­quantitative PCR and glucose and lactate measurement kits. In addition, clinical information and transcriptomic data obtained from the gene expression omnibus database (GSE15966) were used to confirm the in vitro results. The potential effects of GLUT­1 inhibition on the expression of proteins in the glycolysis (GLUT­1, hexokinase 2, pyruvate kinase M2 and lactate dehydrogenase) and apoptosis pathways (Bcl­2, cleaved PARP, caspase-3 and caspase-9) in imatinib­resistant cells were then investigated following gene silencing and treatment using the GLUT­1 inhibitor WZB117 by western blotting. For gene silencing, the mature siRNAs for SLC2A1 were used for cell transfection. Annexin V­FITC/PI double­staining followed by flow cytometry was used to measure apoptosis whereas three­dimensional culture experiments were used to create three­dimensional spheroid cells where cell viability and spheroid diameter were measured. Although imatinib treatment downregulated GLUT­1 expression and other glycolysis pathway components hexokinase 2, pyruvate kinase M2, and lactate dehydrogenase in parental GIST­T1 cells even at low concentrations. By contrast, expression of these glycolysis pathway components in imatinib­resistant cells were increased by imatinib treatment. WZB117 administration significantly downregulated AKT phosphorylation and Bcl­2 expression in imatinib­resistant cells, whereas the combined administration of imatinib and WZB117 conferred synergistic growth inhibition effects in apoptosis assay. WZB117 was found to exert additional inhibitory effects by inducing apoptosis in imatinib­resistant cells. Therefore, the present study suggests that GLUT­1 is involved in the acquisition of imatinib resistance by GIST cells, which can be overcome by combined treatment with WZB117 and imatinib.

Anti-tumor effect of boron neutron capture therapy in pelvic human colorectal cancer in a mouse model.

Local recurrence of colorectal cancer (CRC) can occur in patients after curative resection, and additional surgical resection may therefore be required; however, this is a significant burden for patients, because additional surgical resection may necessitate the resection of other organs such as the bladder, prostate, uterus, or sacral bone. Therefore, there is a need for alternative therapeutic strategies. We focused on boron neutron capture therapy (BNCT) as a treatment modality that can selectively target tumor cells without excessive damage to normal tissues. The usefulness of BNCT to pelvic CRC remains unknown. This study investigated the anti-cancer effect of boronophenylalanine (BPA)-mediated BNCT in a previously established mouse model of pelvic recurrence of CRC. Uptake of BPA in CRC was observed both in vitro and in vivo, and the concentrations were sufficient for BNCT. Our results are the first to show that BPA-mediated BNCT prolonged the survival of experimental mice with pelvic tumors; moreover, it did not cause any obvious severe side effects in the treated animals. In conclusion, BPA-mediated BNCT could contribute to treating local recurrence of pelvic CRC.

γ-H2AX as a potential indicator of radiosensitivity in colorectal cancer cells.

Preoperative radiotherapy improves local disease control and disease-free survival in patients with advanced rectal cancer; however, a reliable predictive biomarker for the effectiveness of irradiation has yet to be elucidated. Phosphorylation of H2A histone family member X (H2AX) to γ-H2AX is induced by DNA double-strand breaks and is associated with the development of colorectal cancer (CRC). The current study aimed to clarify the relationship between γ-H2AX expression and CRC radiosensitivity in vitro and in vivo. H2AX levels were analyzed in datasets obtained from cohort studies and γ-H2AX expression was investigated by performing immunohistochemistry and western blotting using clinical CRC samples from patients without any preoperative therapy. In addition, the CRC cell lines WiDr and DLD-1 were subjected to irradiation and/or small interfering RNA-H2AX, after which the protein levels of γ-H2AX were examined in samples obtained from patients undergoing preoperative chemoradiotherapy. To quantify the observable effect of treatment on cancer cells, outcomes were graded as follows: 1, mild; 2, moderate; and 3, marked, with defined signatures of cellular response. Datasets obtained from cohort studies demonstrated that H2AX mRNA levels were significantly upregulated and associated with distal metastasis and microsatellite instability in CRC tissues, in contrast to that of normal tissues. In addition, γ-H2AX was overexpressed in clinical samples. In vitro, following irradiation, γ-H2AX expression levels increased and cell viability decreased in a time-dependent manner. Combined irradiation and γ-H2AX knockdown reduced the viability of each cell line when compared with irradiation or γ-H2AX knockdown alone. Furthermore, among clinical CRC samples from patients undergoing preoperative chemoradiotherapy, levels of γ-H2AX in the grade 1 group were significantly higher than those in grade 2 or grade 3. In conclusion, γ-H2AX may serve as a novel predictive marker and target for preoperative radiotherapy effectiveness in patients with CRC.

An In Vivo Mouse Model of Pelvic Recurrence of Human Colorectal Cancer.

Pelvic recurrence of colorectal cancer is a crucial problem because radical surgery can lead to excessive invasion. Novel therapeutic strategies are required instead of surgery. However, there are few suitable models because of the difficulty in transplanting and observing tumors in the pelvis. We have established an appropriate injection site suitable for the establishment of colorectal cancer pelvic recurrence that allows for the observation of tumor growth. DLD-1 cells stably expressing luciferase (DLD-1 clone#1-Luc) were inoculated into various points of female BALB/c nude mice and the engrafted cells were analyzed with an imaging system employing bioluminescent signals and computed tomography. Weekly analysis with the imaging system showed that a triangular area defined by the vagina, the anus, and the ischial spine was suitable for the engraftment of pelvic tumors. The imaging system was able to detect the engrafted tumor 7 days after the inoculation of cells. Weight loss was observed in our model, and overall survival was 21-42 days. Tumor involvement of adjacent organs was detected histopathologically, as is the case in the clinical situation. These findings suggest that this model is valid for evaluations of the therapeutic effects of novel treatments under development. It is hoped that this model will be used in preclinical research.