Peptide mimetic NC114 induces growth arrest by preventing PKCδ activation and FOXM1 nuclear translocation in colorectal cancer cells.

The peptide mimetic, NC114, is a promising anticancer compound that specifically kills colorectal cancer cells without affecting normal colon epithelial cells. In our previous study, we observed that NC114 inhibited the Wnt/ß-catenin pathway, with significant downregulation of both Ser 675-phosphorylated ß-catenin and its target genes, cyclin D1 and survivin. However, the molecular mechanism responsible for its cytotoxic effect has not yet been fully characterized. In the present study, we demonstrated that NC114 prevented cell cycle progression from S to G2/M phase by downregulating cell cycle-related gene expression, and also induced growth arrest in SW480 and HCT-116 colorectal cancer cells. A novel covariation network analysis combined with transcriptome analysis revealed a series of signaling cascades affected by NC114 treatment, and identified protein kinase C-δ (PKCδ) and forkhead box protein M1 (FOXM1) as important regulatory factors for NC114-induced growth arrest. NC114 treatment inhibits the activation of PKCδ and its kinase activity, which suppresses MEK/ERK signaling. Attenuated MEK/ERK signaling then results in a reduction in FOXM1 phosphorylation and subsequent nuclear translocation of FOXM1 and ß-catenin. Consequently, formation of a T-cell factor-4 (TCF4)/ß-catenin transcription complex in the nucleus is inhibited and transcription of its target genes, such as cell cycle-related genes, is downregulated. The efficacy of NC114 on tumor growth was confirmed in a xenograft model. Collectively, elucidation of the mechanism by which NC114 induces growth arrest in colorectal cancer cells should provide a novel therapeutic strategy for colorectal cancer treatment.

Microscopic image-based classification of adipocyte differentiation by machine learning.

Adipocyte differentiation is a sequential process involving increased expression of peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte-specific gene expression, and accumulation of lipid droplets in the cytoplasm. Expression of the transcription factors involved is usually detected using canonical biochemical or biomolecular procedures such as Western blotting or qPCR of pooled cell lysates. While this provides a useful average index for adipogenesis for some populations, the precise stage of adipogenesis cannot be distinguished at the single-cell level, because the heterogenous nature of differentiation among cells limits the utility of averaged data. We have created a classifier to sort cells, and used it to determine the stage of adipocyte differentiation at the single-cell level. We used a machine learning method with microscopic images of cell stained for PPARγ and lipid droplets as input data. Our results show that the classifier can successfully determine the precise stage of differentiation. Stage classification and subsequent model fitting using the sequential reaction model revealed the action of pioglitazone and rosiglitazone to be promotion of transition from the stage of increased PPARγ expression to the next stage. This indicates that these drugs are PPARγ agonists, and that our classifier and model can accurately estimate drug action points and would be suitable for evaluating the stage/state of individual cells during differentiation or disease progression. The incorporation of both biochemical and morphological information derived from immunofluorescence image of cells and so overcomes limitations of current models.

Microscopic image-based covariation network analysis for actin scaffold-modified insulin signaling.

To infer a "live" protein network in single cells, we developed a novel Protein Localization and Modification-based Covariation Network (PLOM-CON) analysis method using a large set of quantitative data on the abundance (quantity), post-translational modification state (quality), and localization/morphological information of target proteins from microscope immunostained images. The generated network exhibited synchronized time-dependent behaviors of the target proteins to visualize how a live protein network develops or changes in cells under specific experimental conditions. As a proof of concept for PLOM-CON analysis, we applied this method to elucidate the role of actin scaffolds, in which actin fibers and signaling molecules accumulate and form membrane-associated protein condensates, in insulin signaling in rat hepatoma cells. We found that the actin scaffold in cells may function as a platform for glycogenesis and protein synthesis upon insulin stimulation.

Establishment and phenotyping of disease model cells created by cell-resealing technique.

Cell-based assays are growing in importance for screening drugs and investigating their mechanisms of action. Most of the assays use so-called "normal" cell strain because it is difficult to produce cell lines in which the disease conditions are reproduced. In this study, we used a cell-resealing technique, which reversibly permeabilizes the plasma membrane, to develop diabetic (Db) model hepatocytes into which cytosol from diabetic mouse liver had been introduced. Db model hepatocytes showed several disease-specific phenotypes, namely disturbance of insulin-induced repression of gluconeogenic gene expression and glucose secretion. Quantitative image analysis and principal component analysis revealed that the ratio of phosphorylated Akt (pAkt) to Akt was the best index to describe the difference between wild-type and Db model hepatocytes. By performing image-based drug screening, we found pioglitazone, a PPARγ agonist, increased the pAkt/Akt ratio, which in turn ameliorated the insulin-induced transcriptional repression of the gluconeogenic gene phosphoenolpyruvate carboxykinase 1. The disease-specific model cells coupled with image-based quantitative analysis should be useful for drug development, enabling the reconstitution of disease conditions at the cellular level and the discovery of disease-specific markers.

L-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2.

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic ß-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.

[Ulcerative colitis in remission with cerebral abscess and septic pulmonary emboli: a case report].

A 69-year-old man with a 4-year history of ulcerative colitis (UC) presented at our hospital with high fever, dysarthria, and right hemiparesis. Computed tomography (CT) of the head revealed a low-density area in the left temporal lobe. Chest CT exposed multiple pulmonary nodules in his right lung. Gadolinium-enhanced magnetic resonance imaging (MRI) indicated a 3-cm tumor with ring enhancement located in the left temporal lobe. The patient was diagnosed with a brain abscess and septic pulmonary emboli (SPE); antibiotic therapy was initiated. Shrinkage of the brain abscess was not observed in a follow-up MRI;thus, he underwent aspiration and drainage of the abscess 11 days after his hospitalization. Intravenous antibiotic therapy was continued for 6 weeks after the operation. Follow-up chest CT performed 48 days after his hospitalization revealed disappearance of the SPE. Follow-up head MRI conducted 63 days after his hospitalization indicated that the cyst had almost disappeared. Occurrence of a brain abscess in patients with UC has been very rarely reported in Japan. To the best of our knowledge, this is the first report of a case of a brain abscess in conjunction with UC and SPE. It is believed that patients with UC have compromised immunity and exhibit activation of the blood coagulation system. Our report suggests that medical practitioners should consider the possibility of a brain abscess and SPE for patients with UC.

A resealed-cell system for analyzing pathogenic intracellular events: perturbation of endocytic pathways under diabetic conditions.

Cell-based assay systems that can serve as cellular models of aberrant function in pathogenic organs would be novel and useful tools for screening drugs and clarifying the molecular mechanisms of various diseases. We constructed model cells that replicated the conditions in diabetic hepatocytes by using the cell resealing technique, which enables the exchange of cytosol. The plasma membrane of HeLa cells was permeabilized with the streptococcal toxin streptolysin O, and cytosol that had been prepared from wild-type or db/db diabetic mice was introduced into the resulting semi-intact cells. By resealing the plasma membrane by exposure to Ca(2+), we created WT or Db model cells, in which the cytosolic conditions replicated those of healthy or diabetic liver. Interestingly, phosphorylation of p38 MAPK was promoted, whereas the level of endosomal phosphatidylinositol-3-phosphate was decreased, in Db cells. We investigated several endocytic pathways in WT and Db cells, and found that retrograde endosome-to-Golgi transport was delayed in a p38 MAPK-dependent manner in Db cells. Furthermore, the degradation pathway of the EGF receptor from endosomes to lysosomes was enhanced in Db cells, and this did not depend on the activation of p38 MAPK. The disease model cell system should become a powerful tool for the detection of aberrant processes in cells under pathogenic conditions and for therapeutic applications.

Molecular size as a limiting characteristic for bioconcentration in fish.

The relationships between the bioconcentration factor (BCF) of chemicals in fish and their size, as characterized by molecular weight (MW), effective cross sectional diameter (Deff), and maximum diameter (Dmax) have been investigated using an experimental data set of 737 new and 441 existing chemicals monitored by the Japanese Chemical Substances Control Law (CSCL). Substances with BCF > or = 5000 (very high bioconcentration potential) typically have MW < 550, Deff < 1.1 nm and Dmax < 2.0 nm, respectively and the substances with BCF > or = 1000 (high bioconcentration potential) have MW < 550, Deff < 1.4 nm and Dmax < 2.9 nm, respectively Therefore, the previously suggested threshold values for Deff (0.95 nm) and Dmax (1.5 nm) used for discriminating between bioconcentrative and non-bioconcentrative substances were found to be somewhat small. We found that many substances with BCF > or = 1000 and Dmax > or = 1.5 nm have Deff < 0.95 nm.