Intestinal aberrant sphingolipid metabolism shaped-gut microbiome and bile acids metabolome in the development of hepatic steatosis.

Conjugated bile acids (CBAs) play major roles in hepatic gene regulation via nuclear S1P-inhibited histone deacetylase (HDACs). Gut microbiota modifies bile acid pool to generate CBAs and then CBAs returned to liver to regulate hepatic genes, fatty liver, and non-alcoholic fatty liver disease (NAFLD). However, it is not yet known how the gut microbiota was modified under the environment of inflammatory bowel disease (IBD). Here, we revealed that aberrant intestinal sphingosine kinases (SphKs), a major risk factor of IBD, modified gut microbiota by increasing the proportions of Firmicutes and Verrucomicrobia, which were associated with the increase in CBAs. When exposed to a high-fat diet (HFD), sphingosine kinases 2 knockout (SphK2KO) mice developed more severity of intestinal inflammation and hepatic steatosis than their wild-type (WT) littermates. Due to knockdown of nuclear SphK2, Sphk2KO mice exhibited an increase in sphingosine kinases 1 (SphK1) and sphingosine-1-phosphate (S1P) in intestinal epithelial cells. Therefore, the microbiota was modified in the environment of the SphK1/S1P-induced IBD. 16S rDNA amplicon sequencing of cecal contents indicated an increase of Firmicutes and Verrucomicrobia. Ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) measured an increase in CBAs, including taurocholic acid (TCA), taurodeoxycholic acid (TDCA), and glycocholic acid (GCA), in cecal contents and liver tissues of Sphk2KO mice. These CBAs accumulated in the liver promoted hepatic steatosis through downregulating the acetylation of H3K9, H3K14, H3K18 and H3K27 due to the CBAs-S1PR2-nuclear SphK2-S1P signaling pathway was blocked in HFD-SphK2KO mice. In summary, intestinal aberrant sphingolipid metabolism developed hepatic steatosis through the increase in CBAs associated with an increase in Firmicutes and Verrucomicrobia.

Atypical chemokine receptor 3 induces colorectal tumorigenesis in mice by promoting ß-arrestin-NOLC1-fibrillarin-dependent rRNA biogenesis.

Atypical chemokine receptor 3 (ACKR3) has emerged as a key player in various biological processes. Its atypical "intercepting receptor" properties have established ACKR3 as the major regulator in the pathophysiological processes in many diseases. In this study, we investigated the role of ACKR3 activation in promoting colorectal tumorigenesis. We showed that ACKR3 expression levels were significantly increased in human colon cancer tissues, and high levels of ACKR3 predicted the increased severity of cancer. In Villin-ACKR3 transgenic mice with a high expression level of CKR3 in their intestinal epithelial cells, administration of AOM/DSS induced more severe colorectal tumorigenesis than their WT littermates. Cancer cells of Villin-ACKR3 transgenic mice were characterised by the nuclear ß-arrestin-1 (ß-arr1)-activated perturbation of rRNA biogenesis. In HCT116 cells, cotreatment with CXCL12 and AMD3100 selectively activated ACKR3 and induced nuclear translocation of ß-arr1, leading to an interaction of ß-arr1 with nucleolar and coiled-body phosphoprotein 1 (NOLC1). NOLC1, as the phosphorylated protein, further interacted with fibrillarin, a conserved nucleolar methyltransferase responsible for ribosomal RNA methylation in the nucleolus, thereby increasing the methylation in histone H2A and promoting rRNA transcription in ribosome biogenesis. In conclusion, ACKR3 promotes colorectal tumorigenesis through the perturbation of rRNA biogenesis by the ß-arr1-induced interaction of NOLC1 with fibrillarin.

Increased S1P induces S1PR2 internalization to blunt the sensitivity of colorectal cancer to 5-fluorouracil via promoting intracellular uracil generation.

Sphingosine-1-phosphate (S1P), the backbone of most sphingolipids, activating S1P receptors (S1PRs) and the downstream G protein signaling has been implicated in chemoresistance. In this study we investigated the role of S1PR2 internalization in 5-fluorouracil (5-FU) resistance in human colorectal cancer (CRC). Clinical data of randomly selected 60 CRC specimens showed the correlation between S1PR2 internalization and increased intracellular uracil (P < 0.001). Then we explored the regulatory mechanisms in CRC model of villin-S1PR2-/- mice and CRC cell lines. We showed that co-administration of S1P promoted S1PR2 internalization from plasma membrane (PM) to endoplasmic reticulum (ER), thus blunted 5-FU efficacy against colorectal tumors in WT mice, compared to that in S1PR2-/- mice. In HCT116 and HT-29 cells, application of S1P (10 µM) empowered S1PR2 to internalize from PM to ER, thus inducing 5-FU resistance, whereas the specific S1PR2 inhibitor JTE-013 (10 µM) effectively inhibited S1P-induced S1PR2 internalization. Using Mag-Fluo-AM-labeling [Ca2+]ER and LC-ESI-MS/MS, we revealed that internalized S1PR2 triggered elevating [Ca2+]ER levels to activate PERK-eLF2α-ATF4 signaling in HCT116 cells. The activated ATF4 upregulated RNASET2-mediated uracil generation, which impaired exogenous 5-FU uptake to blunt 5-FU therapy. Overall, this study reveals a previously unrecognized mechanism of 5-FU resistance resulted from S1PR2 internalization-upregulated uracil generation in colorectal cancer, and provides the novel insight into the significance of S1PR2 localization in predicting the benefit of CRC patients from 5-FU-based chemotherapy.

M10, a novel derivative of Myricetin, prevents ulcerative colitis and colorectal tumor through attenuating robust endoplasmic reticulum stress.

Chronic gut inflammation disposes to an increased risk of colitis-associated cancer. Chemoprevention is an attractive complementary strategy. We aimed to evaluate the chemopreventive effects of M10, a novel derivative of Myricetin, in the murine azoxymethane/dextran sodium sulfate model. Oral administration of M10 at 50-100 mg/kg once a day for consecutive 12 weeks significantly prevented ulcerative colitis (UC) and colorectal tumor. Pathological analysis of intestines showed that M10 reduced the degree of chronic inflammation and prevented the progression of colorectal tumorigenesis. Flow cytometry analysis of the immunocytes isolated from intraepithelial and lamina propria showed that M10 prevented the infiltration of myeloid-derived suppressor cells and increased CD8+T and CD4+T cells in colorectal tissues. Enzyme-linked immunosorbent analysis revealed the reduction of pro-inflammatory mediators granulocyte-macrophage colony-stimulating factor/macrophage colony-stimulating factor, IL-6 and TNF-α in colonic mucosa. Western blot assay also showed M10 prevention of the NF-κB/IL-6/STAT3 pathways and the biomarkers of inflammation and colorectal tumorigenesis. Electron microscopy analysis revealed that M10 prevent robust endoplasmic reticulum (ER) stress-induced autophagy in inflamed colonic mucosal cells. In conclusion, oral administration of Myricetin derivative M10 exerts chemoprevention of UC and colorectal tumor in mice. The mechanism of chemoprevention is associated with the reduction of biomarkers of chronic inflammation and proliferation through attenuating robust ER stress in inflamed colonic mucosal cells. M10 exerts chemoprevention activity without evidence of toxicity in mice. These results justify further evaluation of M10 in clinical trials. M10 could develop a promising regimen in the chemoprevention of colitis and colorectal cancer.

Knockdown of CXCR4 Inhibits CXCL12-Induced Angiogenesis in HUVECs through Downregulation of the MAPK/ERK and PI3K/AKT and the Wnt/ß-Catenin Pathways.

CXCL12 is an extracellular chemokine binding to cell surface receptor CXCR4. We found that activation of CXCL12/CXCR4 axis stimulated angiogenesis in endothelial cells. Knockdown of CXCR4 in endothelial cells prevented the branch points of angiogenesis. Endothelial cells exposed to CXCL12 presented high level of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and matrix metalloproteinase MMP-2, but not in CXCR4 knockdown cells. Further studies revealed that activation of CXCL12/CXCR4 axis in vascular endothelial cells stimulates the angiogenesis through upregulation of the MAPK/ERK and PI3K/AKT and Wnt/ß-catenin pathways. Conclusion, downregulation of CXCR4 could inhibit angiogenesis in cancer tissues.

Roles and Signaling Pathways of Des-γ-Carboxyprothrombin in the Progression of Hepatocellular Carcinoma.

Des-γ-carboxyprothrombin (DCP), an abnormal prothrombin produced in human hepatocellular carcinoma (HCC), plays crucial roles in the progression of HCC. DCP binding to cellular mesenchymal-epithelial transition factor (c-Met) is an initial event and consequently stimulates HCC through the increase of c-Met-Janus kinase 1- signal transducers and activators of transcription pathways. DCP stimulates HCC invasion through activation of matrix metalloproteinase via upregulation of extracellular signal-regulated kinase-mitogen-activated protein kinase (MAPK) pathway. DCP stimulates HCC angiogenesis through activation of the DCP-kinase insert domain receptor-phospholipaseC-γ-MAPK pathway. Identification of these pathways is important for designing the therapeutic strategy for HCC.

Des-gamma-carboxy prothrombin (DCP) antagonizes the effects of gefitinib on human hepatocellular carcinoma cells.

BACKGROUND/AIMS: Des-gamma-carboxy prothrombin (DCP), an aberrant prothrombin produced by hepatocellular carcinoma (HCC) cells, is known as a marker for HCC. Recent studies indicated that high levels of DCP are associated with the malignant potential of HCC. In this study, we aimed to investigate the association of DCP with gefitinib treatment failure in HCC and whether DCP counteracts gefitinib-induced growth inhibition and apoptosis of HCC. METHODS: The experiments were performed in HCC cell lines HepG2 and PLC/PRF/5. The effects of gefitinib on HCC in the presence or absence of DCP were evaluated by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Apoptotic cells were identified by Annexin V-FITC/PI staining. Western blotting was performed to analyze the expressions of molecules related to the apoptotic caspase-dependent pathway and epidermal growth factor receptor (EGFR) pathway. RESULTS: Gefitinib inhibited HCC cell proliferation and induced apoptosis in HCC cells. The effects of gefitinib on HCC cells were antagonized by DCP. In the presence of DCP, HCC cells were resistant to the gefitinib-induced inhibition of proliferation and stimulation of apoptosis. DCP prevented the activation of the apoptotic caspase-dependent pathway induced by gefitinib. These antagonistic effects of DCP also arose from its ability to up-regulate EGFR, c-Met and hepatocyte growth factor (HGF) in HCC cells. CONCLUSION: DCP antagonized gefitinib-induced HCC cell growth inhibition by counteracting apoptosis and up-regulating the EGFR pathway. High levels of DCP might thus lead to low response rates or possibly no response to gefitinib in patients with HCC.

Acetyl-11-keto-beta-boswellic acid (AKBA) prevents human colonic adenocarcinoma growth through modulation of multiple signaling pathways.

BACKGROUND: Acetyl-11-keto-beta-boswellic acid (AKBA) is a derivative of boswellic acid. We have previously reported that AKBA can reduce the number and size of colonic adenomatous polyps in the APC(Min/+) mouse model. In this study, we evaluated the effect of AKBA on human colonic adenocarcinoma growth. Its efficacy and toxicity were compared with those of the non-steroidal anti-inflammatory drug aspirin. METHODS: The inhibition of cancer cell growth was estimated by colorimetric and clonogenic assay. Cell cycle distribution was analyzed by the flow cytometry assay. Annexin V-FITC/PI staining and JC-1 fluorescence probe assays were performed to determine the apoptotic cells. Further experiment was carried out in mice with HT-29 xenografts. AKBA was orally administered for 24days. The HT-29 xenografts were removed for TUNEL staining and western blotting analysis. Blood was obtained for clinical chemical analysis, and samples of organs were sectioned for microscopic assessment. RESULTS: AKBA significantly inhibited human colon adenocarcinoma growth, showing arrest of the cell cycle in G1-phase and induction of apoptosis. AKBA administration in mice effectively delayed the growth of HT-29 xenografts without signs of toxicity. The activity of AKBA was more potent than that of aspirin. Western blotting suggested that this activity may arise from its multiple effects on the activation of apoptotic proteins, suppression of inflammatory cytokines and modulation of EGFR and ATM/P53 signaling pathways in the HT-29 xenografts. CONCLUSIONS: AKBA prevents the growth of colonic adenocarcinoma through modulation of multiple signaling pathways. GENERAL SIGNIFICANCE: AKBA could be a promising agent in the prevention of colonic adenocarcinomas.

Des-γ-carboxy prothrombin (DCP) as a potential autologous growth factor for the development of hepatocellular carcinoma.

Des-γ-carboxy prothrombin (DCP) is a prothrombin precursor produced in hepatocellular carcinoma (HCC). Because of deficiency of vitamin K or γ-glutamyl carboxylase in HCC cells, the 10 glutamic acid (Glu) residues in prothrombin precursor did not completely carboxylate to γ-carboxylated glutamic acid (Gla) residues, leaving some Glu residues remained in N-terminal domain. These prothrombin precursors with Glu residues are called DCPs. DCP displays insufficient coagulation activity. Since Liebman reported an elevated plasma DCP in patients with HCC, DCP has been used in the diagnosis of HCC. Recently, its biological malignant potential has been specified to describe DCP as an autologous growth factor to stimulate HCC growth and a paracrine factor to integrate HCC with vascular endothelial cells. DCP was found to stimulate HCC growth through activation of the DCP-Met-JAK1-STAT3 signaling pathway. DCP might increase HCC invasion and metastasis through activation of matrix metalloproteinase (MMPs) and the ERK1/2 MAPK signaling pathway. DCP has also been found to play a crucial role in the formation of angiogenesis. DCP could increase the angiogenic factors released from HCC and vascular endothelial cells. These effects of DCP in angiogenesis might be related to activation of the DCP-KDR-PLC-γ-MAPK signaling pathway. In this article, we summarized recent studies on DCP in biological roles related to cancer progression and angiogenesis in HCC.

Chemoprevention of intestinal adenomatous polyposis by acetyl-11-keto-beta-boswellic acid in APC(Min/+) mice.

Acetyl-11-keto-beta-boswellic acid (AKBA) is a derivative of boswellic acid, which is an active component of the gum resin of Boswellia serrata. AKBA has been used as an adjuvant medication for treatment of inflammatory diseases. In this study, we aimed to evaluate the efficacy of AKBA as a chemopreventive agent against intestinal adenomatous polyposis in the adenomatous polyposis coli multiple intestinal neoplasia (APC(Min/+) ) mouse model. APC(Min/+) mice were administered AKBA by p.o. gavage for 8 consecutive weeks. The mice were sacrificed and the number, size and histopathology of intestinal polyps were examined by light microscopy. AKBA decreased polyp numbers by 48.9% in the small intestine and 60.4% in the colon. An even greater AKBA effect was observed in preventing the malignant progression of these polyps. The number of large (>3 cm) colonic polyposis was reduced by 77.8%. Histopathologic analysis demonstrated a significant reduction in the number of dysplastic cells and in the degree of dysplasia in each polyp after AKBA treatment. There was no evidence of high grade dysplasia or intramucosal carcinoma in any of the polyps examined within the treated group. More interestingly, interdigitated normal appearing intestinal villi were observed in the polyps of the treated group. During the course of the study, AKBA was well tolerated by the mice with no obvious signs of toxicity. Results from immunohistochemical staining, Western blotting and enzyme-linked immunosorbent assay indicated that the chemopreventive effect of AKBA was attributed to a collection of activities including antiproliferation, apoptosis induction, antiangiogenesis and anti-inflammation. AKBA was found to exert its chemopreventive action through the inhibition of the Wnt/ß-catenin and NF-κB/cyclooxygenase-2 signaling pathways. Our findings suggest that AKBA could be a promising regimen in chemoprevention against intestinal tumorigenesis.

A new chromone from Angelica polymorpha with cytotoxic activity.

A new chromone polymorchromone B was isolated and characterized from the roots of Angelica polymorpha Maxim, a commonly used traditional Chinese medicine. On the basis of spectroscopic analysis, including IR, HR-ESI-MS, 1D and 2D NMR spectral data, and hydrolysis followed by chromatographic analysis, the structure of the new chromone was elucidated as 5-hydroxy-2-[(angeloyloxy)methyl]-2'-(dimethyl)-3'-(2S,3S-epoxy-2-methylbutanoate)-dihydropyran [3',2':6,7]chromone. Moreover, it was found that polymorchromone B showed remarkable cytotoxic activity against A-549 cell lines.

LYP, a novel bestatin derivative, inhibits cell growth and suppresses APN/CD13 activity in human ovarian carcinoma cells more potently than bestatin.

LYP is a bestatin dimethylaminoethyl ester which inhibits aminopeptidase N (APN/CD13). Our goal in this study was to evaluate LYP as a candidate compound for cancer treatment, beginning by studying its inhibitory effects on tumors and then comparing it to bestatin. Experiments were performed on human ovarian carcinoma (OVCA) ES-2 and SKOV-3 cell lines, which have high and low levels of APN/CD13 respectively. LYP effectively inhibited ES-2 cell growth as estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the trypan blue dye-exclusion test. LYP significantly suppressed APN/CD13 activity on the surface of ES-2 cells as measured by quantifying the enzymatic cleavage of the substrate L-leucine-p-nitroanilide. The inhibitory effects of LYP were greater than those of bestatin at the same concentrations. In contrast, LYP was a weak inhibitor of SKOV-3 cell growth, suggesting that LYP may inhibit ES-2 cell growth via suppression of APN/CD13. Inhibition of APN/CD13 expression was also demonstrated with immunofluorescent flow cytometry and Western blot analysis. Inhibitory effects of LYP were confirmed by using a mouse model in which LYP delayed the growth of ES-2 xenografts in mice after 2 weeks of LYP injections. Inhibition of APN/CD13 expression was demonstrated in the ES-2 xenografts using Western blot analysis. The inhibitory effects of LYP on the ES-2 xenografts were stronger than those of bestatin. These results suggest that LYP has a powerful inhibitory effect on the growth of OVCA cells and that the mechanism may be via a decrease in the expression of APN/CD13.

LYP, a bestatin dimethylaminoethyl ester, inhibited cancer angiogenesis both in vitro and in vivo.

Our previous study revealed that LYP, a bestatin dimethylaminoethyl ester, inhibited the growth of human ovarian carcinoma ES-2 xenografts in mice and suppressed aminopeptidase N (APN/CD13) activity more potently than bestatin. In this study, we examined the inhibitory effect of LYP on migration and formation of capillary tube of human umbilical vascular endothelial cells (HUVECs) in vitro and anti-angiogenesis in ES-2 xenografts in mice. LYP did not possess cytotoxicity to HUVEC proliferation according to the MTT assay and trypan blue exclusion assay. However, APN/CD13 activity on cell surface of HUVECs was suppressed in the presence of LYP as measured by quantifying the enzymatic cleavage of the substrate l-leucine-p-nitroanilide. The assays of scratch and transwell chamber showed that LYP significantly inhibited HUVEC migration and invasion through Matrigel coated polycarbonate filters. Capillary tube formation assay revealed that the number of branch points formed by HUVECs on 3-D Matrigel was reduced after incubation with LYP. The anti-angiogenesis of LYP was verified in ES-2 xenografts in mice. The mean vascular density (MVD) and mean vascular luminal diameter (MVLD) were markedly reduced by LYP after two weeks of intravenous injection as evaluated by CD34 immunohistochemical staining. LYP suppression of cancer angiogenesis was greater than that of bestatin. The inhibition of angiogenic molecules may involve in anti-angiogenesis of LYP. The levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF-α) were decreased in HUVECs and ES-2 xenografts after treatment with LYP as determined by Western blot analysis. These results indicated that the high efficacy of LYP may partially relate to the inhibition of angiogenesis.

Myricetin and M10, a myricetin-3-O-ß-d-lactose sodium salt, modify composition of gut microbiota in mice with ulcerative colitis.

Our previous studies found that M10, a myricetin-3-O-ß-d-lactose sodium salt, possessed higher effects of ameliorating ulcerative colitis (UC) than Myricetin in mice. Here, we aim to investigate whether the inhibition of UC is the consequence of the effects of M10 that leads to the changed microbiota. Mice model of UC was induced by dextran sulfate sodium (DSS) treatment. M10 and Myricetin were orally administrated for 12 weeks. We performed 16S rDNA sequencing assay to analyze the composition of gut microbiota isolated from ileocecum. Both M10 and Myricetin normalized the composition of Firmicutes and Actinobacteria as healthy mice had. At genus level, the effects of M10 and Myricetin on colitis were associated to the increase of probiotics, such as Akkermansia, and the inhibition of pathogenic microorganisms, such as Ruminococcus and Parabacteroides. M10 had stronger activity than Myricetin in the improvement of biosynthesis and degradation activities, resulting to increasing metabolism of sulfur, pyruvate, steroid biosynthesis and unsaturated fatty acid biosynthesis in gut. Furthermore, M10 normalized the proportion of Firmicutes and Actinobacteria in gut microbiota. It suggests that the improvements in UC are the consequence of the effect of M10 that leads to the changed intestinal microbiota. Conclusion: M10 contributed the pharmacological effects on UC by modification of the intestinal microbiota.

Nuclear translocation of ATG5 induces DNA mismatch repair deficiency (MMR-D)/microsatellite instability (MSI) via interacting with Mis18α in colorectal cancer.

BACKGROUND AND PURPOSE: It is well known that microsatellite instability-high (MSI-H) is associated with 5-fluorouracil (5-FU) resistance in colorectal cancer. MSI-H is the phenotype of DNA mismatch repair deficiency (MMR-D), mainly occurring due to hypermethylation of MLH1 promoter CpG island. However, the mechanisms of MMR-D/MSI-H are unclear. We aim to investigate the pathway of MMR-D/MSI-H involved in 5-FU resistance. EXPERIMENTAL APPROACH: Human colorectal cancer specimens were diagnosed for MSI-H by immunohistochemistry and western blotting. Proteome microarray interactome assay was performed to screen nuclear proteins interacting with ATG5. Nuclear ATG5 and ATG5-Mis18α overexpression were analysed in ATG5high colorectal cancer bearing mice. The methylation assay determined the hypermethylation of hMLH1 promoter CpG island in freshly isolated human colorectal cancer tissue samples and HT29atg5 and SW480atg5 cancer cells. KEY RESULTS: In ATG5high colorectal cancer patients, 5-FU-based therapy resulted in nuclear translocation of ATG5, leading to MSI-H. Colorectal cancer in Atg5 Tg mice demonstrated 5-FU resistance, compared to Atg5+/- and WT mice. Proteome microarray assay identified Mis18α, a protein localized on the centromere and a source for methylation of the underlying chromatin, which responded to the translocated nuclear ATG5 leading to ATG5-Mis18α conjugate overexpression. This resulted in MLH1 deficiency due to hypermethylation of hMLH1 promoter CpG island, while the deletion of nuclear Mis18α failed to induce ATG5-Mis18α complex and MMR-D/MSI-H. CONCLUSIONS AND IMPLICATIONS: Nuclear ATG5 resulted in MMR-D/MSI-H through its interaction with Mis18α in ATG5high colorectal cancer cells. We suggest that ATG5-Mis18α or Mis18α may be a therapeutic target for treating colorectal cancer.

M10, a Myricetin-3-O-b-D-Lactose Sodium Salt, Prevents Ulcerative Colitis Through Inhibiting Necroptosis in Mice.

BACKGROUND: M10 is a derivative of Myricetin by adding a hydrophilic glycosylation group. Our previous study revealed that M10 by oral administration prevented colitis-associated colonic cancer (CAC) through attenuating endoplasmic reticulum stress in mice. In current study, we evaluated the inhibitory effects of M10 on ulcerative colitis in mice model, the mechanism of M10 in preventing colitis was further investigated. METHODS: Mice model of ulcerative colitis was induced by continuous oral dextran sodium sulfate (DSS). M10 was given gavage once a day for 12 consecutive weeks. Disease activity index (DAI) was recorded by analyzing the symptoms of colitis. Intestinal barrier was analyzed by the Immunofluorescence staining assay. The structure of microvilli of intestinal epithelial cells was analyzed under Transmission electron microscopy (TEM). TEM assay was also performed to determine the formation of necroptosis in the colonic epithelium with ulcerative colitis. We performed Western blotting assay to analyze the IL-6 and NF-κB pathways, as well as the cytokine cascades related to TNF-α signaling pathway during necroptosis. RESULTS: M10 by oral administration demonstrated a prevention of ulcerative colitis, showing a significant decrease of DAI as compared to the model mice. Pathological analysis indicated that M10 attenuated the degree of colonic inflammation in colonic tissues. M10 restored the structures of intestinal barrier damaged by DSS. M10 prevented the activation of the IL-6 and NF-κB signaling pathways in the inflamed colonic epithelium. Further, M10 prevented necroptosis in the inflamed colonic mucosal cells through down-regulating the TNF-α pathway. Importantly, M10 demonstrated higher activities in preventing ulcerative colitis than Myricetin and control drug Mesalazine. CONCLUSIONS: Myricetin derivative M10 prevents chronic ulcerative colitis through inhibiting necroptosis. M10 could be developed as a promising drug for the treatment of chronic ulcerative colitis.

SphK2 confers 5-fluorouracil resistance to colorectal cancer via upregulating H3K56ac-mediated DPD expression.

Aberrant sphingolipid metabolism has been implicated in chemoresistance, but the underlying mechanisms are still poorly understood. Herein we revealed a previously unrecognized mechanism of 5-fluorouracil (5-FU) resistance contributed by high SphK2-upregulated dihydropyrimidine dehydrogenase (DPD) in colorectal cancer (CRC), which is evidenced from human CRC specimens, animal models, and cancer cell lines. TMA samples from randomly selected 60 CRC specimens firstly identified the clinical correlation between high SphK2 and increased DPD (p < 0.001). Then the regulatory mechanism was explored in CRC models of villin-SphK2 Tg mice, SphK2-/-mice, and human CRC cells xenografted nude mice. Assays of ChIP-Seq and luciferase reporter gene demonstrated that high SphK2 upregulated DPD through promoting the HDAC1-mediated H3K56ac, leading to the degradation of intracellular 5-FU into inactive α-fluoro-ß-alanine (FBAL). Lastly, inhibition of SphK2 by SLR080811 exhibited excellent inhibition on DPD expression and potently reversed 5-FU resistance in colorectal tumors of villin-SphK2 Tg mice. Overall, this study manifests that SphK2high conferred 5-FU resistance through upregulating tumoral DPD, which highlights the strategies of blocking SphK2 to overcome 5-FU resistance in CRC.

Vitamin K2 inhibits the growth of hepatocellular carcinoma via decrease of des-gamma-carboxy prothrombin.

BACKGROUND: Des-gamma-carboxy prothrombin (DCP) is a serum protein produced by hepatocellular carcinoma (HCC) cells in the absence of vitamin K. Serum and tissue DCP expressions are thought to reflect the biological malignant potential of HCC. Hence, we aimed to examine the efficacy of vitamin K(2) on the production of DCP as well as tumor cell growth and invasion. METHODS: Cell growth and viability were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The in vivo efficacy of vitamin K(2) was examined in nude mice bearing HCC cells. A 24-well transwell chamber was used to evaluate the motility and invasive ability of HCC cells. Levels of DCP in supernatant of cultures and in serum of mice were measured using an electrochemiluminescence immunoassay method. Western blot and immunohistochemical analysis were employed to evaluate the expression of DCP in HCC. RESULTS: Vitamin K(2) (2-40 muM) significantly decreased the levels of DCP production in supernatant of PLC/PRF/5 and HepG2 cells and in serum of nude mice bearing HCC xenografts. The inhibition of DCP was also observed using the assays of Western blot analysis in HCC cultures and immunohistochemical analysis in HCC xenografts in mice. As a result of administration of vitamin K(2), the capacity of HCC growth was inhibited and the invasion and migration of tumor cells were decreased. Furthermore, the inhibitory effects of HCC growth were also observed in vivo and the sensitivity was well correlated with the decrease of DCP in the serum of mice. CONCLUSION: Vitamin K(2) might suppress the growth and invasion of HCC cells via decrease of DCP.

Metformin inhibited colitis and colitis-associated cancer (CAC) through protecting mitochondrial structures of colorectal epithelial cells in mice.

Although a mountain of papers have showed that metformin plays a role in inhibiting cancers, but the mechanism underpinning this has not yet fully elucidated. Herein, we used AOM/DSS model, the clinicopathological features are similar to those found in humans, to investigate the effects of metformin as well as combination with 5-FU in the prevention of colitis and colitis associated cancer (CAC). Oral metformin significantly inhibited DSS-induced ulcerative colitis and AOM/DSS-induced CAC. Metformin also ameliorated 5-FU-induced colorectal gastrointestinal symptoms in mice. Metformin combination with 5-FU strongly inhibited colorectal cancer. Metformin reduced levels of the NFκB signaling components p-IKKα/ß, p-NFκB, p-IκBα in colorectal mucosal cells. Transmission electron microscopy analysis suggested that the inhibition of metformin on colitis and CAC might associate with its biological activity of protecting mitochondrial structures of colorectal epithelial cells. Further analysis by Mito Tracker Red staining assay indicated that metformin prevented H2O2-induced mitochondrial fission correlated with a decrease of mitochondrial perimeter. In addition, metformin increased the level of NDUFA9, a Q-module subunit required for complex I assembly, in colorectal epithelial cells. These observations of metformin in the inhibition of colitis and CAC might associate with its activity of activating the LKB1/AMPK pathway in colorectal epithelial cells. In conclusion, metformin inhibited colitis and CAC through protecting the mitochondrial structures of colorectal epithelial cells.

CXCR7/CXCR4 heterodimer-induced histone demethylation: a new mechanism of colorectal tumorigenesis.

Both chemokine receptors (CXCRs) 7 and 4 can facilitate immune cell migration and mediate a vast array of physiological and pathological events. Herein we report, in both human and animal studies, that these two CXCRs can form heterodimers in vivo and promote colorectal tumorigenesis through histone demethylation. Compared with adjacent non-neoplastic tissue, human colorectal cancer (CRC) tissue showed a significant higher expression of CXCR4 and CXCR7, which was colocalized in the cancer cell epithelium. The CXCR/CXCR4 heterodimerization was associated with increased histone demethylase JMJD2A. Villin-CXCR7-CXCR4 transgenic mice demonstrated a greater degree of exacerbated colitis and tumorigenesis than villin-CXCR7 and villin-CXCR4 mice. The CXCR7/CXCR4 heterodimerization also promoted APC mutation-driven colorectal tumorigenesis in APCMin/+/villin-CXCR7-CXCR4 mice. Further analysis showed that the CXCR7/CXCR4 heterodimer induced nuclear ßarr1 recruitment and histone demethylase JMJD2A, leading to histone demethylation and resulting in transcription of inflammatory factors and oncogenes. This study uncovered a novel mechanism of colorectal tumorigenesis through the CXCR7/CXCR4 heterodimer-induced histone demethylation. Inhibition of CXCR7/CXCR4 heterodimer-induced histone demethylation could be an effective strategy for the prevention and treatment of colorectal cancer.