Study of chondrogenesis of umbilical cord mesenchymal stem cells in curdlan- poly(vinyl alcohol) composite hydrogels and its mechanical properties of freezing-thawing treatments.

The curdlan gel is a natural material produced by bacteria. It utilizes chemical cross-linking reactions to form a 3D porous composite hydrogel, increasing its porosity and water content, and improving its mechanical properties. It can be used in tissue repair and regenerative medicine. Curdlan-Poly(vinyl alcohol) (PVA) composite hydrogel can rapidly swell within 1 min due to its porous structure. Compression tests confirmed that it still maintains its original mechanical strength, even after five repeated freeze-thaw (FT) processes, making it suitable for long-term cryopreservation. The purpose of this study is to transplant umbilical cord mesenchymal stem cells (UC-MSCs) on Curdlan-PVA composite hydrogel and observe the chondrocytes on the material. The results of using 4',6-diamidino-2-phenylindole (DAPI), hematoxylin and eosin (H&E), calcein-acetoxymethyl ester (calcein AM), and Collagen type II-Fluorescein isothiocyanate (FITC) staining, confirmed that UC-MSCs can attach and differentiate into chondrocytes on 3D Curdlan-PVA composite hydrogel.

Induction of MYCN-amplified neuroblastoma differentiation through NMYC suppression using PPAR-γ antagonist.

Neuroblastomas are the most common extracranial solid tumors in children and have a unique feature of neuronal differentiation. Peroxisome proliferator-activated receptor (PPAR)-γ is reported to have neuroprotective effects in addition to having antitumor effects in various cancers. Thus, we aimed to clarify the role of PPAR-γ agonist and antagonist in malignant neuroblastomas, which also possess neuronal features. In MYCN-amplified neuroblastoma CHP212 cells, treatment with the PPAR-γ antagonist GW9662 induced growth inhibition in a dose-dependent manner. In addition, the PPAR-γ antagonist treatment changed cell morphology with increasing expression of the neuronal differentiation marker tubulin beta 3 (TUBB3) and induced G1 phase arrest and apoptosis in MYCN-amplified neuroblastoma. Notably, the PPAR-γ antagonist treatment significantly decreased expression of NMYC, B-cell lymphoma 2 (BCL2) and bromodomain-containing protein 4 (BRD4). It is implied that BRD4, NMYC, BCL2 suppression by the PPAR-γ antagonist resulted in cell growth inhibition, differentiation, and apoptosis induction. In our in vivo study, the PPAR-γ antagonist treatment induced CHP212 cells differentiation and resultant tumor growth inhibition. Our results provide a deeper understanding of the mechanisms of tumor cell differentiation and suggest that PPAR-γ antagonist is a new therapeutic and prevention option for neuroblastomas.

Citrus limonL.-Derived Nanovesicles Show an Inhibitory Effect on Cell Growth in p53-Inactivated Colorectal Cancer Cells via the Macropinocytosis Pathway.

Edible plant-derived nanovesicles have been explored as effective materials for preventing colorectal cancer (CRC) incidence, dependent on gene status, as a K-Ras-activating mutation via the macropinocytosis pathway. Approximately 70% of CRC harbors the p53 mutation, which is strongly associated with a poor prognosis for CRC. However, it has not been revealed whether p53 inactivation activates the macropinocytosis pathway or not. In this study, we investigated parental cells, wild-type or null for p53 treated with Citrus limon L.-derived nanovesicles, as potential materials for CRC prevention. Using ultracentrifugation, we obtained C. limon L.-derived nanovesicles, the diameters of which were approximately 100 nm, similar to that of the exosomes derived from mammalian cells. C. limon L.-derived nanovesicles showed inhibitory effects on cell growth in not p53-wild, but also in p53-inactivated CRC cells. Furthermore, we revealed that the macropinocytosis pathway is activated by p53 inactivation and C. limon L.-derived nanovesicles were up taken via the macropinocytosis pathway. Notably, although C. limon L.-derived nanovesicles contained citrate, the inhibitory effects of citrate were not dependent on the p53 status. We thus provide a novel mechanism for the growth inhibition of C. limon L.-derived nanovesicles via macropinocytosis and expect to develop a functional food product containing them for preventing p53-inactivation CRC incidence.

Sodium salicylate and 5-aminosalicylic acid synergistically inhibit the growth of human colon cancer cells and mouse intestinal polyp-derived cells.

As colon cancer is one of the most common cancers in the world, practical prevention strategies for colon cancer are needed. Recently, treatment with aspirin and/or 5-aminosalicylic acid-related agents was reported to reduce the number of intestinal polyps in patients with familial adenomatous polyposis. To evaluate the mechanism of aspirin and 5-aminosalicylic acid for suppressing the colon polyp growth, single and combined effects of 5-aminosalicylic acid and sodium salicylate (metabolite of aspirin) were tested in the two human colon cancer cells with different cyclooxygenase-2 expression levels and intestinal polyp-derived cells from familial adenomatous polyposis model mouse. The combination induced cell-cycle arrest at the G1 phase along with inhibition of cell growth and colony-forming ability in these cells. The combination reduced cyclin D1 via proteasomal degradation and activated retinoblastoma protein. The combination inhibited the colony-forming ability of mouse colonic mucosa cells by about 50% and the colony-forming ability of mouse intestinal polyp-derived cells by about 90%. The expression level of cyclin D1 in colon mucosa cells was lower than that in intestinal polyp-derived cells. These results suggest that this combination may be more effective in inhibiting cell growth of intestinal polyps through cyclin D1 down-regulation.

Induction of DNA Damage in Mouse Colorectum by Administration of Colibactin-producing Escherichia coli, Isolated from a Patient With Colorectal Cancer.

BACKGROUND/AIM: Among colorectal cancer-associated intestinal microbiota, colibactin-producing (clb+) bacteria are attracting attention. We aimed to clarify the interaction between clb+ Escherichia coli and normal colorectal epithelial cells in vivo and in vitro. MATERIALS AND METHODS: Five-week-old female Balb/c mice were divided in an untreated group, a group treated with clb+ E. coli isolated from a Japanese patient with colorectal cancer (E. coli-50), and a group treated with non colibactin-producing E. coli (E. coli-50/ΔclbP). Mice were sacrificed at 18 weeks of treatment. RESULTS: Treatment with clb+ E. coli increased positivity for H2A histone family member X phosphorylated at Ser-139 (γH2AX) in epithelial cells of the luminal surface of the mouse rectum but this did not occur in the E. coli-50/ΔclbP and untreated groups. In an in vitro setting, the ratio of apoptotic cells was increased and cell counts were reduced by treatment with clb+ E. coli more than in untreated cells and normal rat colorectal epithelial cells. CONCLUSION: E. coli-50 induced DNA damage in the mouse rectum, possibly by direct interaction between clb+ E. coli and normal colorectal epithelial cells. Our findings imply that regulation of clb+ E. coli infection may be a useful strategy for colorectal cancer control.

Complex Modulating Effects of Dietary Calcium Intake on Obese Mice.

BACKGROUND/AIM: Οverweight and obesity are risk factors for chronic diseases. Dietary calcium has been reported to exert anti-obesity effects. However, the complex modulating effects of calcium intake on obese mice have not been clarified. MATERIALS AND METHODS: The effects of calcium intake on body weight/visceral fat mass were examined in the obese mouse model, KK-Ay Results: Body weight gain decreased in mice fed a diet containing 0.4 to 3.2% calcium at the age of 11 and 13 weeks, but not at 12 weeks after normalization for food intake. Calcium intake also decreased serum insulin levels and increased the amount of feces excreted. Fecal deoxycholate levels were lower in the high-calcium group than in the normal diet control group. Furthermore, the ratio of the deoxycholate-producing microbiome in feces decreased. CONCLUSION: Dietary calcium has anti-obesity effects in obese KK-Ay mice. Inhibition of insulin production and an increased amount of feces excreted with calcium intake may affect body weight.

Artesunate inhibits intestinal tumorigenesis through inhibiting wnt signaling.

Artesunate (ART) is a clinically approved antimalarial drug and was revealed as a candidate of colorectal cancer chemopreventive agents in our drug screening system. Here, we aimed to understand the suppressive effects of ART on intestinal tumorigenesis. In vitro, ART reduced T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter transcriptional activity. In vivo, ART inhibited intestinal polyp development. We found that ART reduces TCF1/TCF7 nuclear translocation by binding the Ras-related nuclear protein (RAN), suggesting that ART inhibits TCF/LEF transcriptional factor nuclear translocation by binding to RAN, thereby inhibiting Wnt signaling. Our results provide a novel mechanism through which artesunate inhibits intestinal tumorigenesis.

The Radical Scavenger NZ-419 Suppresses Intestinal Polyp Development in Apc-Mutant Mice.

Colorectal cancer is the fourth leading cause of cancer death worldwide, and it is important to establish effective methods for preventing colorectal cancer. One effective prevention strategy could be the use of antioxidants. However, the role of the direct antioxidative function of antioxidants against carcinogenesis has not been clarified. Thus, we aimed to determine whether the direct removal of reactive oxygen species by a hydroxyl radical scavenger, NZ-419, could inhibit colorectal carcinogenesis. NZ-419 is a creatinine metabolite that has been shown to be safe and to inhibit the progression of chronic kidney disease in rats, and it is now under clinical development. In the present study, we demonstrated that NZ-419 eliminated reactive oxygen species production in HCT116 cells after H2O2 stimulation and suppressed H2O2-induced Nrf2 promoter transcriptional activity. The administration of 500 ppm NZ-419 to Apc-mutant Min mice for 8 weeks resulted in a decrease in the number of polyps in the middle segment of the small intestine to 62.4% of the value in the untreated control (p < 0.05 vs. control group). As expected, NZ-419 treatment affected the levels of reactive carbonyl species, which are oxidative stress markers in the serum of Min mice. Suppression of the mRNA levels of the proliferation-associated factor c-Myc was observed in intestinal polyps of Min mice after NZ-419 treatment, with a weak suppression of epithelial cell proliferation assessed by proliferation cell nuclear antigen (PCNA) staining in the intestinal polyps. This study demonstrated that NZ-419 suppress the development of intestinal polyps in Min mice, suggesting the utility of radical scavenger/antioxidants as a cancer chemopreventive agent.

Theracurmin inhibits intestinal polyp development in Apc-mutant mice by inhibiting inflammation-related factors.

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Therefore, it is important to establish useful methods for preventing CRC. One prevention strategy involves the use of cancer chemopreventive agents, including functional foods. We focused on the well-known cancer chemopreventive agent curcumin, which is derived from turmeric. However, curcumin has the disadvantage of being poorly soluble in water due to its high hydrophobicity. To overcome this problem, the formation of submicron particles with surface controlled technology has been applied to curcumin to give it remarkably improved water solubility, and this derived compound is named Theracurmin. To date, the preventive effects of Theracurmin on hereditary intestinal carcinogenesis have not been elucidated. Thus, we used Apc-mutant mice, a model of familial adenomatous polyposis, to evaluate the effects of Theracurmin. First, we showed that treatment with 10-20 µM Theracurmin for 24 hours reduced nuclear factor-κB (NF-κB) transcriptional activity in human colon cancer DLD-1 and HCT116 cells. However, treatment with curcumin mixed in water did not change the NF-κB promoter transcriptional activity. As NF-κB is a regulator of inflammation-related factors, we next investigated the downstream targets of NF-κB: monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-6. We found that treatment with 500 ppm Theracurmin for 8 weeks inhibited intestinal polyp development and suppressed MCP-1 and IL-6 mRNA expression levels in the parts of the intestine with polyps. This report provides a proof of concept for the ongoing Theracurmin human trial (J-CAP-C study).

Activation of NF-E2 p45-related factor-2 transcription and inhibition of intestinal tumor development by AHCC, a standardized extract of cultured Lentinula edodes mycelia.

It has been reported that activation of NF-E2 p45-related factor-2 (NRF2), a transcription factor, induces a variety of antioxidant enzymes, and plays an important role in preventing carcinogenesis. AHCC is a standardized extract of cultured Lentinula edodes mycelia and it has been demonstrated to improve cancer. However, the effects of AHCC on NRF2 have not been examined, and the effects on intestinal adenoma development are not yet fully understood. We first investigated the effects of AHCC (1-5 mg/ml) on NRF2 activity in human colon cancer cell lines by a luciferase reporter gene assay, and found NRF2 transcriptional activities were increased ~12.6-fold. In addition, AHCC dose-dependently increased HO-1 and NQO-1 mRNA levels, and decreased interleukine-6 mRNA levels. Next, we administered 1,000 ppm AHCC for 8 weeks in the diet of Apc mutant Min mice, and found that AHCC significantly reduced the total number of intestinal polyps to 57.7% and to 67.6% of the control value in male and female Min mice, respectively, with suppression of interleukine-6 in the polyp part. These data suggest that AHCC possesses an ability to suppress cellular oxidative stress through activation of NRF2, thereby lowering intestinal polyp development in Min mice.

Characterization of unique metabolites in γ-aminobutyric acid-rich cheese by metabolome analysis using liquid chromatography-mass spectrometry.

Fermented dairy products comprise many functional components. Our previous study using fermented milk showed that the γ-aminobutyric acid (GABA)-producing Lactococcus lactis 01-7 strain can produce unique metabolites such as antihypertensive peptides, whereas this study was designed to find the unique metabolites in GABA-rich cheese using the 01-7 strain. Metabolites between cheese ripening with the non-GABA-producing L. lactis 01-1 strain (control) and GABA-rich cheese ripening with a mixture of 01-1 and 01-7 strains were compared. GABA and ornithine were detected in GABA-rich cheese using an amino acid analyzer and citrate was detected in the control cheese using HPLC. Metabolome analysis using LC-MS showed that peptides with unknown function and those with antihypertensive activity were higher in the GABA-rich cheese than in the control cheese. Further analysis of the amount of the YLGY derivatives showed that the amount of YL in the GABA-rich cheese was lower than that in the control. PRACTICAL APPLICATIONS: Clarification of metabolites in cheese contributes to the improvement of cheese ripening, thereby providing consumers with unique cheese with good nutritional and functional characteristics. The use of the 01-7 strain as a cheese starter might provide a functional cheese with antihypertensive-, antioxidative-, and anxiolytic-like activities.

Novel screening system revealed that intracellular cholesterol trafficking can be a good target for colon cancer prevention.

In conventional research methods for cancer prevention, cell proliferation and apoptosis have been intensively targeted rather than the protection of normal or benign tumor cells from malignant transformation. In this study, we aimed to identify candidate colon cancer chemopreventive drugs based on the transcriptional activities of TCF/LEF, NF-κB and NRF2, that play important roles in the process of malignant transformation. We screened a "validated library" consisting of 1280 approved drugs to identify hit compounds that decreased TCF/LEF and NF-κB transcriptional activity and increased NRF2 transcriptional activity. Based on the evaluation of these 3 transcriptional activities, 8 compounds were identified as candidate chemopreventive drugs for colorectal cancer. One of those, itraconazole, is a clinically used anti-fungal drug and was examined in the Min mouse model of familial adenomatous polyposis. Treatment with itraconazole significantly suppressed intestinal polyp formation and the effects of itraconazole on transcriptional activities may be exerted partly through inhibition of intracellular cholesterol trafficking. This screen represents one of the first attempts to identify chemopreventive agents using integrated criteria consisting of the inhibition of TCF/LEF, NF-κB and induction of NRF2 transcriptional activity.

Inhibition of NF-kappaB transcriptional activity enhances fucoxanthinol-induced apoptosis in colorectal cancer cells.

BACKGROUND: Evidence from epidemiological and experimental studies has shown that the etiology of colorectal cancer (CRC) is related to lifestyle, mainly diet. At the same time, there are many foods and beverages that have been shown to provide protection against CRC. We turned our attention to a traditional Japanese food, brown algae, that contains carotenoids and various functional polyphenols, especially fucoxanthin (FX) and fucoxanthinol (FxOH). RESULTS: Both FX and FxOH treatments induced apoptosis in a dose-dependent and time-dependent manner as detected by annexin V / propidium iodide and the presence of a subG1 population in human colon cancer HCT116 cells. This apoptotic effect of FxOH was stronger than that of FX. We also found that nuclear factor-kappa B (NF-κB) transcriptional activity was significantly increased by treatment with ≥5 µM FxOH. Thus, we cotreated the cells with FxOH plus NF-κB inhibitor, and the results demonstrated that this cotreatment strongly enhanced the induction of apoptosis compared with the effects of FxOH or NF-κB inhibitor treatment alone and resulted in X-linked inhibitor of apoptosis (IAP) downregulation. CONCLUSIONS: This study suggested that FxOH is a more potent apoptosis-inducing agent than FX and that its induction of apoptosis is enhanced by inhibiting NF-κB transcriptional activity via suppression of IAP family genes.

Descriptive Sensory Traits of Cooked Eggs Laid from Hens Fed Rice Grain.

Descriptive sensory characteristics of eggs produced by conventional corn-based feeding and unhulled whole rice grain-feeding were compared in two cooking procedures using a trained panel. Rice-feeding significantly decreased brothy and roasted odor in eggs cooked into half-cooked egg yolks, and decreased the creamy odor, smoothness and moisture of eggs cooked into custard puddings. However, a statistical interaction between rice-feeding and production farm was not observed in every sensory attribute. These findings indicated that replacing corn with unhulled whole rice grain in diets for laying hens alters the sensory attributes of eggs.

SREBP-1c-Dependent Metabolic Remodeling of White Adipose Tissue by Caloric Restriction.

Caloric restriction (CR) delays the onset of many age-related pathophysiological changes and extends lifespan. White adipose tissue (WAT) is not only a major tissue for energy storage, but also an endocrine tissue that secretes various adipokines. Recent reports have demonstrated that alterations in the characteristics of WAT can impact whole-body metabolism and lifespan. Hence, we hypothesized that functional alterations in WAT may play important roles in the beneficial effects of CR. Previously, using microarray analysis of WAT from CR rats, we found that CR enhances fatty acid (FA) biosynthesis, and identified sterol regulatory element-binding protein 1c (SREBP-1c), a master regulator of FA synthesis, as a mediator of CR. These findings were validated by showing that CR failed to upregulate factors involved in FA biosynthesis and to extend longevity in SREBP-1c knockout mice. Furthermore, we revealed that SREBP-1c is implicated in CR-associated mitochondrial activation through the upregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. Notably, these CR-associated phenotypes were observed only in WAT. We conclude that CR induces SREBP-1c-dependent metabolic remodeling, including the enhancement of FA biosynthesis and mitochondrial activation, via PGC-1α in WAT, resulting in beneficial effects.

Differential response to caloric restriction of retroperitoneal, epididymal, and subcutaneous adipose tissue depots in rats.

The beneficial actions of caloric restriction (CR) are partially mediated by metabolic remodeling of white adipose tissue (WAT). Recently, we showed that CR enhances de novo fatty acid (FA) biosynthesis and mitochondrial biogenesis, particularly in WAT. Here, to better understand the response of WAT to CR, we compare the effects of CR on three WAT depots in rats: retroperitoneal (rWAT), epididymal (eWAT) and subcutaneous (sWAT). Computed tomography and histological analysis showed that CR reduced the volume and average size of rWAT adipocytes. In all WAT depots, CR markedly upregulated the expression of proteins involved in FA biosynthesis in fed rats. In visceral WAT (rWAT and eWAT), hormone-sensitive lipase (lipolytic form) phosphorylation was increased by CR under fed conditions, and decreased by CR under fasted conditions. Conversely, in sWAT, hormone-sensitive lipase phosphorylation was increased by CR under fasted conditions. CR enhanced the effect of feeding on AKT activity in sWAT (indicative of a positive effect on insulin sensitivity) but not in rWAT or eWAT. These data suggest that CR improves lipid metabolism in an insulin signaling-dependent manner in sWAT only. The effects of CR on adipokine (adiponectin and leptin) expression were also different among rWAT, eWAT and sWAT, and CR reduced the gene expression of M2 macrophage markers in rWAT and sWAT, but not in eWAT. We conclude that CR differentially affects the characteristics of WAT depots in rats, including adipocyte size, lipid metabolism, insulin signaling, adipocytokine profile and macrophage infiltration.

Mitochondrial intermediate peptidase is a novel regulator of sirtuin-3 activation by caloric restriction.

Sirtuin-3 (SIRT3) regulates mitochondrial quality and is involved in the anti-ageing and pro-longevity actions of caloric restriction (CR). Here, we show that CR upregulates the mature form of SIRT3 and mitochondrial intermediate peptidase (MIPEP), a mitochondrial signal peptidase (MtSPase), in white adipose tissue. We also demonstrate that upregulation of mature SIRT3 is dependent on MIPEP in 3T3-L1 cells, suggesting that MIPEP may contribute to the maintenance of mitochondrial quality during CRvia activation of SIRT3. This novel mechanism of SIRT3 activation through MIPEP facilitates the elucidation of additional molecular pathways of CR.

Sterol regulatory element-binding protein-1c orchestrates metabolic remodeling of white adipose tissue by caloric restriction.

Caloric restriction (CR) can delay onset of several age-related pathophysiologies and extend lifespan in various species, including rodents. CR also induces metabolic remodeling involved in activation of lipid metabolism, enhancement of mitochondrial biogenesis, and reduction of oxidative stress in white adipose tissue (WAT). In studies using genetically modified mice with extended lifespans, WAT characteristics influenced mammalian lifespans. However, molecular mechanisms underlying CR-associated metabolic remodeling of WAT remain unclear. Sterol regulatory element-binding protein-1c (Srebp-1c), a master transcription factor of fatty acid (FA) biosynthesis, is responsible for the pathogenesis of fatty liver (steatosis). Our study showed that, under CR conditions, Srebp-1c enhanced mitochondrial biogenesis via increased expression of peroxisome proliferator-activated receptor gamma coactivator-1α (Pgc-1α) and upregulated expression of proteins involved in FA biosynthesis within WAT. However, via Srebp-1c, most of these CR-associated metabolic alterations were not observed in other tissues, including the liver. Moreover, our data indicated that Srebp-1c may be an important factor both for CR-associated suppression of oxidative stress, through increased synthesis of glutathione in WAT, and for the prolongevity action of CR. Our results strongly suggested that Srebp-1c, the primary FA biosynthesis-promoting transcriptional factor implicated in fatty liver disease, is also the food shortage-responsive factor in WAT. This indicated that Srebp-1c is a key regulator of metabolic remodeling leading to the beneficial effects of CR.

Classification and characterization of Japanese consumers' beef preferences by external preference mapping.

BACKGROUND: Over the past few decades, beef producers in Japan have improved marbling in their beef products. It was recently reported that marbling is not well correlated with palatability as rated by Japanese consumers. This study sought to identify the consumer segments in Japan that prefer sensory characteristics of beef other than high marbling. RESULTS: Three Wagyu beef, one Holstein beef and two lean imported beef longissimus samples were subjected to a descriptive sensory test, physicochemical analysis and a consumer (n = 307) preference test. According to consumer classification and external preference mapping, four consumer segments were identified as 'gradual high-fat likers', 'moderate-fat and distinctive taste likers', 'Wagyu likers' and 'distinctive texture likers'. Although the major trend of Japanese consumers' beef preference was 'marbling liking', 16.9% of the consumers preferred beef samples that had moderate marbling and distinctive taste. The consumers' attitudes expressed in a questionnaire survey were in good agreement with the preference for marbling among the 'moderate-fat and distinctive taste likers'. CONCLUSION: These results indicate that moderately marbled beef is a potent category in the Japanese beef market. © 2017 Society of Chemical Industry.