The cooperative induction of macrophage activation by fucoidan derived from Cladosiphon okamuranus and ß-glucan derived from Saccharomyces cerevisiae.

The present study investigated immune stimulatory effects of Cladosiphon okamuranus-derived fucoidan to activate murine macrophage-like cell line RAW264, and the functional relationship with zymosan, a Saccharomyces cerevisiae-derived ß-glucan. The production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in RAW264 cells were remarkably enhanced in the presence of 10 µg/mL fucoidan, and the stimulatory effects of fucoidan were maximally augmented in combinational treatment with 500 ng/mL zymosan, whereas any TLR ligands had no those effects. Confocal microscopic analyses suggested that fucoidan bound on plasma membrane, and it was estimated that some cell surface molecules acted as receptor for fucoidan because cytochalasin D, an inhibitor of phagocytosis, did not affect the immune enhancing activities, whereas methyl-ß-cyclodextrin (MßCD), a general agent for disruption of lipid rafts, diminished that. Furthermore, it was revealed that the additive effects of zymosan on the immune activation with fucoidan was thought to be mediated by dectin-1 based on the results with dectin-1-knockdown RAW264 cells. All of results suggested that fucoidan and some kinds of ß-glucan would cooperatively reinforce the activity of innate immune cells via interactive receptor crosstalk.

The Nuclear Zinc Finger Protein Zfat Maintains FoxO1 Protein Levels in Peripheral T Cells by Regulating the Activities of Autophagy and the Akt Signaling Pathway.

Forkhead box O1 (FoxO1) is a key molecule for the development and functions of peripheral T cells. However, the precise mechanisms regulating FoxO1 expression in peripheral T cells remain elusive. We previously reported that Zfat(f/f)-CD4Cre mice showed a marked decline in FoxO1 protein levels in peripheral T cells, partially through proteasomal degradation. Here we have identified the precise mechanisms, apart from proteasome-mediated degradation, of the decreased FoxO1 levels in Zfat-deficient T cells. First, we confirmed that tamoxifen-inducible deletion of Zfat in Zfat(f/f)-CreERT2 mice coincidently decreases FoxO1 protein levels in peripheral T cells, indicating that Zfat is essential for maintaining FoxO1 levels in these cells. Although the proteasome-specific inhibitors lactacystin and epoxomicin only moderately increase FoxO1 protein levels, the inhibitors of lysosomal proteolysis bafilomycin A1 and chloroquine restore the decreased FoxO1 levels in Zfat-deficient T cells to levels comparable with those in control cells. Furthermore, Zfat-deficient T cells show increased numbers of autophagosomes and decreased levels of p62 protein, together indicating that Zfat deficiency promotes lysosomal FoxO1 degradation through autophagy. In addition, Zfat deficiency increases the phosphorylation levels of Thr-308 and Ser-473 of Akt and the relative amounts of cytoplasmic to nuclear FoxO1 protein levels, indicating that Zfat deficiency causes Akt activation, leading to nuclear exclusion of FoxO1. Our findings have demonstrated a novel role of Zfat in maintaining FoxO1 protein levels in peripheral T cells by regulating the activities of autophagy and the Akt signaling pathway.

Molecular mechanisms of transcriptional regulation by the nuclear zinc-finger protein Zfat in T cells.

Zfat is a nuclear protein with AT-hook and zinc-finger domains. We previously reported that Zfat plays crucial roles in T-cell survival and development in mice. However, the molecular mechanisms whereby Zfat regulates gene expression in T cells remain unexplored. In this study, we analyzed the genome-wide occupancy of Zfat by chromatin immunoprecipitation with sequencing (ChIP-seq), which showed that Zfat bound predominantly to a region around a transcription start site (TSS), and that an 8-bp nucleotide sequence GAA(T/A)(C/G)TGC was identified as a consensus sequence for Zfat-binding sites. Furthermore, about half of the Zfat-binding sites were characterized by histone H3 acetylations at lysine 9 and lysine 27 (H3K9ac/K27ac). Notably, Zfat gene deletion decreased the H3K9ac/K27ac levels at the Zfat-binding sites, suggesting that Zfat may be related to the regulation of H3K9ac/K27ac. Integrated analysis of ChIP-seq and transcriptional profiling in thymocytes identified Zfat-target genes with transcription to be regulated directly by Zfat. We then focused on the chromatin regulator Brpf1, a Zfat-target gene, revealing that Zfat bound directly to a 9-bp nucleotide sequence, CGAANGTGC, which is conserved among mammalian Brpf1 promoters. Furthermore, retrovirus-mediated re-expression of Zfat in Zfat-deficient peripheral T cells restored Brpf1 expression to normal levels, and shRNA-mediated Brpf1 knockdown in peripheral T cells increased the proportion of apoptotic cells, suggesting that Zfat-regulated Brpf1 expression was important for T-cell survival. Our findings demonstrated that Zfat regulates the transcription of target genes by binding directly to the TSS proximal region, and that Zfat-target genes play important roles in T-cell homeostasis.

Zfat-deficient CD4⁺ CD8⁺ double-positive thymocytes are susceptible to apoptosis with deregulated activation of p38 and JNK.

Zfat, which is a nuclear protein harboring an AT-hook domain and 18-repeats of C2H2 zinc-finger motif, is highly expressed in immune-related tissues, including the thymus and spleen. T cell specific deletion of the Zfat gene by crossing Zfat(f/f) mice with LckCre mice yields a significant reduction in the number of CD4(+) CD8(+) double-positive (DP) thymocytes. However, physiological role for Zfat in T cell development in the thymus remains unknown. Here, we found that Zfat-deficient DP thymocytes in Zfat(f/f)-LckCre mice were susceptible to apoptosis both at an unstimulated state and in response to T cell receptor (TCR)-stimulation. The phosphorylation levels of p38 and JNK were elevated in Zfat-deficient thymocytes at an unstimulated state with an enhanced phosphorylation of ATF2 and with an over-expression of Gadd45α⋅ On the other hand, the activation of JNK in the Zfat-deficient thymocytes, but not p38, was strengthened and prolonged in response to TCR-stimulation. All these results demonstrate that Zfat critically participates in the development of DP thymocytes through regulating the activities of p38 and JNK.

Highly-sensitive detection of free advanced glycation end-products by liquid chromatography-electrospray ionization-tandem mass spectrometry with 2,4,6-trinitrobenzene sulfonate derivatization.

The common derivatization method of primary amino groups by 2,4,6-trinitrobenzene sulfonate (TNBS) was applied to the detection of free adducts of advanced glycation end-products (AGEs), in combination with liquid chromatography-tandem mass spectrometry-multiple reaction monitoring (LC-MS/MS-MRM). The proposed TNBS-MS method provided a surprisingly significant improvement of the detection of AGE-free adducts (e.g., by a factor of >1000 for methylglyoxal-derived hydroimidazolone) with a detection limit of 1.0 nM (10 fmol/injection volume), which was due to the high ionization efficiency of the derived trinitrophenol moiety and its hydrophobicity. With the aid of stable-isotope-labeled internal standard (MG-H1-d3), the convenient TNBS method that allowed the derivation of AGE-free adducts bearing amino groups under mild reaction conditions (30 mM, pH 8.5, 30 min, 30 °C) also permitted successive detection and quantification of five typical AGE-free adducts at nanomolar levels in rat plasma (50 µL) with high reproducibility (2-9% of RSD) and recovery (93-113%), using LC-MS/MS-MRM.

Application of 13C stable isotope labeling liquid chromatography-multiple reaction monitoring-tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats.

The liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) method using (13)C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting (13)C-labeled peptides in blood sample with a C(18) cartridge, the extract was applied to a (13)C monoisotopic transition LC-MRM-MS/MS system with D-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5-100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The (13)C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three (13)C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each (13)C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC-MRM-MS/MS method.

ROS-induced cleavage of NHLRC2 by caspase-8 leads to apoptotic cell death in the HCT116 human colon cancer cell line.

Excess production of reactive oxygen species (ROS) is known to cause apoptotic cell death. However, the molecular mechanisms whereby ROS induce apoptosis remain elusive. Here we show that the NHL-repeat-containing protein 2 (NHLRC2) thioredoxin-like domain protein is cleaved by caspase-8 in ROS-induced apoptosis in the HCT116 human colon cancer cell line. Treatment of HCT116 cells with the oxidant tert-butyl hydroperoxide (tBHP) induced apoptosis and reduced NHLRC2 protein levels, whereas pretreatment with the antioxidant N-acetyl-L-cysteine prevented apoptosis and the decrease in NHLRC2 protein levels seen in tBHP-treated cells. Furthermore, the ROS-induced decrease in NHLRC2 protein levels was relieved by the caspase inhibitor z-VAD-fmk. We found that the thioredoxin-like domain of NHLRC2 interacted with a proenzyme form of caspase-8, and that caspase-8 cleaved NHLRC2 protein at Asp580 in vitro. Furthermore, siRNA-mediated knockdown of caspase-8 blocked the ROS-induced decrease in NHLRC2 protein levels. Both shRNA and CRISPR-Cas9-mediated loss of NHLRC2 resulted in an increased susceptibility of HCT116 cells to ROS-induced apoptosis. These results suggest that excess ROS production causes a caspase-8-mediated decrease in NHLRC2 protein levels, leading to apoptotic cell death in colon cancer cells, and indicate an important role of NHLRC2 in the regulation of ROS-induced apoptosis.

Apremilast Induces Apoptosis of Human Colorectal Cancer Cells with Mutant KRAS.

BACKGROUND/AIM: We previously reported the crucial roles of oncogenic Kirsten rat sarcoma viral oncogene homologue (KRAS) in inhibiting apoptosis and disrupting cell polarity via the regulation of phosphodiesterase type 4B2 (PDE4B2) expression in human colorectal cancer (CRC) HCT116 cells in a three-dimensional culture (3DC). Here, we evaluated the effects of apremilast, a selective PDE4 inhibitor, on luminal apoptosis in 3DC and nude mice assay using HKe3 human CRC cells stably expressing wild-type (wt)PDE4B2 (HKe3-wtPDE4B2), mutant (mt)PDE4B2 (kinase dead) (HKe3-wtKRAS), wtKRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS). MATERIALS AND METHODS: Apoptosis was detected by immunofluorescence using confocal laser scanning microscopy or western blot in HKe3-wtPDE4B2, HKe3-mtPDE4B2, HKe3-wtKRAS and mtKRAS cells treated with or without apremilast in 3DC. Tumourigenicity was assessed in nude mice assay using these cells. RESULTS: Apremilast did not inhibit the proliferation of HKe3-wtPDE4B2 cells or HKe3-mtKRAS in two-dimensional cultures, whereas the number of apoptotic HKe3-wtPDE4B2 cells and HKe3-mtKRAS cells increased after apremilast treatment in 3DC, leading to formation of a luminal cavity. Tumour growth in nude mice was dramatically reduced by intraperitoneal injection of apremilast. Notably, a decreased level of caspase-1 expression was observed in HKe3-wtPDE4B2 and HKe3-mtKRAS cells. CONCLUSION: Apremilast induces tumour regression in nude mice, possibly by inducing caspase-1 expression.

An Alpha-kinase 2 Gene Variant Disrupts Filamentous Actin Localization in the Surface Cells of Colorectal Cancer Spheroids.

BACKGROUND/AIM: Alpha-kinase 2 (ALPK2), suggested to be a novel tumour-suppressor gene down-regulated by oncogenic KRAS, plays a pivotal role in luminal apoptosis in normal colonic crypts. The aim of this study was to determine the association between ALPK2 germline variants and colorectal cancer. MATERIALS AND METHODS: Missense single nucleotide variants in the exons of the ALPK2 gene in 2,343 consecutive autopsy cases (1,446 cases with cancer and 897 cases without cancer) were screened using HumanExome BeadChip arrays. To address the functional effect of a missense ALPK2 variant, a 3D floating cell culture was performed using HCT116-derived human colorectal cancer cells stably expressing wild-type (wt) ALPK2 (HCT116-wtALPK2) or amino acid-substituted (sub) ALPK2 (HCT116-subALPK2). RESULTS: We identified that one of the ALPK2 germline variants, rs55674018 (p.Q1853E), was significantly associated with the presence of cancer (adjusted odds ratio(OR)=4.39; 95% confidence interval(CI)=1.31-14.78, p=0.001). The p.Q1853E variant was present in the East Asian population and located in the immunoglobulin-like domain. Notably, the basolateral polarity of actin in the surface of HCT116-wtALPK2 spheroids was more attenuated compared to that of HCT116-subALPK2 spheroids. Furthermore, luminal apoptosis and cell aggregation were promoted by wtALPK2, but not by subALPK2 in 3D culture. CONCLUSION: The p.Q1853E variant of ALPK2, which had been accumulating in the Japanese population, induced a metastatic phenotype by disrupting ALPK2 function.

Marked Reduction in FoxO1 Protein by its Enhanced Proteasomal Degradation in Zfat-deficient Peripheral T-Cells.

BACKGROUND: Zfat is a nuclear protein that harbours putative DNA-binding domains. T-cell specific deletion of Zfat in Zfat(f/f)-CD4Cre mice yields a significant decrease in the number of peripheral T-cells with a lower surface expression of interleukin-7 receptor-α (IL-7Rα). However, the molecular mechanism by which Zfat controls IL-7Rα expression remains unknown. MATERIALS AND METHODS: Expression levels of the molecules involved in IL-7Rα expression were determined by immunoblotting. RESULTS: Zfat-deficient peripheral T-cells showed a marked reduction in the FoxO1 protein that regulates IL-7Rα expression; however, the FoxO1 mRNA expression level was not affected by Zfat-deficiency. Furthermore, treatment of Zfat-deficient T-cells with a proteasome inhibitor, epoxomicin, restored FoxO1 expression levels, indicating that the loss of Zfat enhanced the proteasomal degradation of the FoxO1 protein. CONCLUSION: These results suggest that Zfat is required for peripheral T-cell homeostasis through IL-7Rα expression by controlling the FoxO1 protein.

Establishment of a Three-dimensional Floating Cell Culture System for Screening Drugs Targeting KRAS-mediated Signaling Molecules.

BACKGROUND/AIM: Oncogenic mutations in the KRAS gene are critically involved in many human tumors but drugs targeting oncogenic KRAS have not yet been clinically developed. Herein, we established a three-dimensional floating (3DF) culture system for screening drugs that target KRAS-mediated signaling molecules. MATERIALS AND METHODS: HKe3 cells, derived from colorectal cancer HCT116 cells and disrupted at mutated (mt) KRAS gene, were infected with a retrovirus expressing wild-type (wt) KRAS or mtKRAS to establish HKe3-derived cells expressing wtKRAS or mtKRAS. Established cells were cultured in 96-well plates with an ultra-low attachment surface and round bottom for 3DF culture. RESULTS: HKe3-wtKRAS and HKe3-mtKRAS cells in 3DF culture rapidly assembled into respective single spherical structures (spheroids). Furthermore, mtKRAS but not wtKRAS expression inhibited luminal apoptosis in spheroids indicating that the 3DF culture was compatible with the 3D matrigel culture. CONCLUSION: This 3DF culture system could be useful for screening drugs that target KRAS-mediated signaling molecules.

Resveratrol induces luminal apoptosis of human colorectal cancer HCT116 cells in three-dimensional culture.

BACKGROUND: We have previously reported the crucial roles of oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) in inhibiting apoptosis and disrupting cell polarity via the regulation of phosphodiesterase 4 (PDE4) expression in human colorectal cancer HCT116 cells in three-dimensional cultures (3DC). Herein we evaluated the effects of resveratrol, a PDE4 inhibitor, on the luminal cavity formation and the induction of apoptosis in HCT116 cells. MATERIALS AND METHODS: Apoptosis was detected by immunofluorescence using confocal laser scanning microscopy with an antibody against cleaved caspase-3 in HCT116 cells treated with or without resveratrol in a two-dimensional culture (2DC) or 3DC. RESULTS: Resveratrol did not induce apoptosis of HCT116 cells in 2DC, whereas the number of apoptotic HCT116 cells increased after resveratrol treatment in 3DC, leading to formation of a luminal cavity. CONCLUSION: Resveratrol induced apoptosis of HCT116 cells in 3DC, resulting in the formation of a luminal cavity, probably by inhibiting PDE4 activity.