Bovine Lactoferrin Protects Dextran Sulfate Sodium Salt Mice Against Inflammation and Impairment of Colonic Epithelial Barrier by Regulating Gut Microbial Structure and Metabolites.

Background: Ulcerative colitis is characterized by relapsing and remitting mucosal inflammation. Bovine lactoferrin (BL) is a multifunctional protein that could regulate the intestinal flora and has anti-inflammatory effects. The aim of this study was to investigate the therapeutic effect of BL on colitis. Methods: Dextran sulfate sodium salt (DSS) was utilized to establish a mouse model of colitis. BL was administered to treat DSS mice. The weight, the activity, and fecal status of the mice were recorded every day. Disease activity index was calculated. After the mice were euthanized, the colon length was measured. Hematoxylin and eosin staining was used to observe the pathological changes of the colon, and histological activity index was calculated. The myeloperoxidase (MPO) activity of colon tissue was measured. Western blot and immunohistochemistry were used to detect the expressions of Claudin-1, Occludin, and ZO-1. The expressions of IL-1ß, IL-6, IL-10, TNF-α, and TGF-ß in colon tissue were detected by ELISA. The protein expressions of MUC2, Reg3γ, ß-defensin (HBD-2), and cAMP were detected by immunofluorescence (IF). 16S rDNA sequencing determined the type and structure of intestinal flora. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) measured the metabolites of the intestinal flora. Results: Compared with the DSS group, the mice's weight in the BL group was higher and the length of the colon was longer. At the 14th day, MPO activity was higher in the BL group. The expressions of Claudin-1, Occludin, and ZO-1 in the colon were up-regulated in the BL group compared with the DSS group. The expressions of IL-1ß, IL-6, and TNF-α were lower. The expressions of IL-10 and TGF-ß were higher. IF showed that the expressions of MUC2 and ß-defensin (HBD-2) were down-regulated, and the expressions of Reg3γ and cAMP were up-regulated. The 16S rDNA sequencing results showed that the alpha diversity and beta diversity were notably changed in the DSS mice treated with BL. Metabolomics results showed that BL changed purine metabolism in the DSS mice. Conclusion: BL alleviated colitis in mice by improving the inflammatory response and the structure of the colon barrier in the colon. BL changed the composition and metabolites of the intestinal flora. Thus, BL might be an effective nutritional supplement for colitis treatment.

MicroRNA-520b Functions as a Tumor Suppressor in Colorectal Cancer by Inhibiting Defective in Cullin Neddylation 1 Domain Containing 1 (DCUN1D1).

MicroRNAs (miRs), a class of small noncoding RNAs, are important regulators for gene expression through directly binding to the 3'-untranslated region (3'-UTR) of their target mRNA. Recently, downregulation of miR-520b has been observed in several common human cancers. However, the exact role of miR-520b in colorectal cancer (CRC) has not previously been studied. In this study, our data showed that miR-520b was significantly downregulated in CRC and cell lines when compared with adjacent normal tissues and a normal intestinal epithelial cell line. Low expression of miR-520b was notably associated with the malignant progress and a shorter survival time for CRC patients. Restoration of miR-520b inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in CRC cells. Defective in cullin neddylation 1 domain containing 1 (DCUN1D1) was then identified as a novel target gene of miR-520b in CRC cells. The expression of DCUN1D1 was significantly increased in CRC, with a negative correlation to miR-520b expression in CRC tissues. Moreover, a high expression of DCUN1D1 was significantly associated with the malignant progress and a poor prognosis for CRC patients. Furthermore, overexpression of DCUN1D1 rescued the miR-520b-mediated malignant phenotypes and EMT in CRC cells. The data demonstrate that miR-520b functions as a tumor suppressor in CRC through targeting DCUN1D1, suggesting that miR-520b may become a potential therapeutic target for the treatment of CRC.

Cancer stem cell vaccine inhibits metastases of primary tumors and induces humoral immune responses against cancer stem cells.

The inability to target cancer stem cells (CSC) may be a significant factor contributing to treatment failure. We have developed a strategy to target the CSC populations in melanoma and squamous cell carcinoma using CSC lysate-pulsed dendritic cells (DCs). The CSC-DC vaccine was administered in the adjuvant setting after localized radiation therapy of established tumors. Using mouse models we demonstrated that DCs pulsed with CSCs enriched by virtue of their expression of the CSC marker ALDH (termed CSC-DC) significantly inhibited tumor growth, reduced development of pulmonary metastases and prolonged survival. The effect was associated with downregulation of chemokine (C-C motif) receptors CCR7 and CCR10 in tumor cells and decreased expression of the chemokine (C-C motif) ligands CCL21, CCL27 and CCL28 in lung tissue. The CSC-DC vaccine significantly reduced ALDHhigh CSC frequency in primary tumors. Direct targeting of CSCs was demonstrated by the specific binding of IgG produced by ALDHhigh CSC-DC vaccine-primed B cells to ALDHhigh CSCs, resulting in lysis of these target CSCs in the presence of complement. These data suggest that the CSC-DC vaccine approach may be useful in the adjuvant setting where local and systemic relapse are high after conventional treatment of cancers.

Antitumor effector B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10.

We have previously reported that adoptive transfer of tumor-draining lymph node (TDLN) B cells confers tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer. In this study, we identified IL-10-producing cells within these B cells, and found that IL-10 removal, either by using IL-10(-/-) TDLN B cells or by systemic neutralization of IL-10, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. Depletion of IL-10 in B-cell adoptive transfers significantly increased CTLs and B-cell activity of PBMCs and splenic cells in the recipient. Activated TDLN B cells express Fas ligand, which was further enhanced by coculture of these TDLN B cells with 4T1 tumor cells. Effector B cells killed tumor cells directly in vitro in an antigen specific and Fas ligand-dependent manner. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. These findings further define the biological function of antitumor effector B cells, which may offer alternative cellular therapies to cancer.

The expression of p66Shc protein in benign, premalignant, and malignant gastrointestinal lesions.

ROS produced from Oxidative stress have long been recognized to be involved in carcinogenesis. p66Shc generates H(2)O(2) by oxidizing cytochrome c, and its expression has been reported to be elevated in several tumors. However, the expression of p66Shc in gastric cancer has not been reported, and its role in colorectal cancer has not been well elucidated. This study investigated p66Shc expression in benign, premalignant, and malignant gastric and colorectal lesions. p66Shc expression in 146 gastric tumors, 136 colorectal tumors, 45 gastric hyperplastic polyps, 33 gastric low-grade intraepithelial neoplasias, 41 gastric high-grade intraepithelial neoplasias, 42 colorectal hyperplastic polyps, 21 colorectal low-grade intraepithelial neoplasias, 38 colorectal high-grade intraepithelial neoplasias, and 30 normal gastric and colorectal tissues was measured by immunohistochemistry. Most normal gastric and colorectal tissues exhibited low or no p66Shc expression (93.4 %), while most gastric and colorectal tumors exhibited moderate to high p66Shc expression (78.1 %-80.9 %). The p66Shc expression in normal gastric and colorectal tissues were significantly lower than that in the low-grade intraepithelial neoplasias (p < 0.05), high-grade intraepithelial neoplasias (p < 0.01), and gastric adenocarcinomas (p < 0.01 or <0.001). No differences in p66Shc expression were observed in gastric and colorectal hyperplastic polyps compared to the normal tissues. No statistically significant differences in p66Shc expression were observed between patients with different disease stages, different tumor grades, and with or without lymph node metastasis in gastric and colorectal cancers. In conclusion, p66Shc may be involved in the carcinogenesis of gastric and colorectal cancers and could be a marker for the diagnosis of gastric and colorectal cancers.

Knockdown of p62/sequestosome 1 attenuates autophagy and inhibits colorectal cancer cell growth.

p62/sequestosome-1 is a multifunctional adapter protein implicated in selective autophagy, cell signaling pathways, and tumorigenesis, and plays an important role at the crossroad between autophagy and cancer. But, the connection between autophagy and cancer is complex and in some cases contradictory. Human colorectal cancer tissues from patients were analyzed for expression of p62 and Microtubule-associated protein light chain 3 (LC3, an autophagosome marker) using immunostaining, western blotting, real-time PCR, and confocal microscopy. To study the effects of p62 on autophagy and cell growth, shRNA for p62 was applied and cell growth curve was monitored in human colorectal cancer cell. In vivo experiments were done using the mouse xenograft model. We showed that up-regulated expression of p62 and LC3 in colorectal cancer tissues. We also demonstrated that specifically knockdown the expression of p62 showed significantly inhibitory effects not only on autophagy activation, but also on tumor growth both in vitro and xenograft tumors model. The ectopic overexpression of p62 and autophagy activation contributes to colorectal tumorigenesis. p62 and autophagy will be therapy targets for the treatment of colorectal cancer.

Restoration of klotho gene expression induces apoptosis and autophagy in gastric cancer cells: tumor suppressive role of klotho in gastric cancer.

BACKGROUND: The loss of tumor suppressor gene expression is involved in the carcinogenesis of gastric cancer (GC). Klotho is a recently identified tumor suppressor gene that epigenetically inactivated in gastric cancer. However, the signaling pathways involved in the suppressive role of klotho have rarely been reported in gastric cancer. In this study, we investigated the involvement of klotho in gastric cancer cell proliferation, apoptosis, and autophagy as well as the associated signaling. METHODS: Methylation of klotho gene promoter in GC-7901, MNK-45 and AGS gastric cancer cells as well as GES-1 normal gastric epithelial cells was detected by bisulfate-based PCR. Restoration of klotho gene expression was established by applying a demethylating agent and delivering aklotho gene expression vector into GC-7901 cells. Cell viability was measured by CCK-8 assay. Cell apoptosis and cycling were analyzed by flow cytometry. Autophagy was measured by detecting LC3-I and LC3-II expression. Protein levels and phosphorylation were measured by Western blot assay. RESULTS: Methylation of klotho gene promoter and expression of the klotho gene were detected in GC cells. Restoration of klotho gene expression significantly inhibited cell proliferation, induced cell apoptosis, and increased LC3-I/LC3-II expression in GC cells. Restoration of klotho gene expression downregulated the phosphorylation levels of IGF-1 receptor, IRS-1, PI3K, Akt, and mTOR proteins. Both apoptosis and autophagy inhibitors blocked klotho-induced apoptosis and autophagy. CONCLUSION: Klotho is a tumor suppressor in gastric cancer, which regulates IGF-1R phosphorylation and the subsequent activation of IRS-1/PI3K/Akt/mTOR signaling, tumor cell proliferation, apoptosis, and autophagy.

Immunohistochemical study of PUMA, c-Myb and p53 expression in the benign and malignant lesions of gallbladder and their clinicopathological significances.

BACKGROUND: Gallbladder cancers have a very poor prognosis without specific molecular marker being identified. In this study we studied PUMA, c-Myb and p53 expression in benign and malignant lesions of gallbladder and analyzed their clinicopathological significance. METHOD: Immunohistochemical staining of PUMA, c-Myb and p53 protein was performed in 108 gallbladder adenocarcinomas, 46 peritumoral tissues, 15 polyps, and 35 chronic cholecystitis. RESULTS: We demonstrated that the percent of positive PUMA, c-Myb and p53 expression was significantly higher in gallbladder adenocarcinomas than in peritumoral tissues, polyps and chronic cholecystitis (p < 0.05 or 0.01). Benign gallbladder epithelium with positive PUMA, c-Myb or p53 expression showed moderately or severely atypical hyperplasia. The percent of positive PUMA, c-Myb and p53 expression was significantly higher in the cases having poorly differentiated adenocarcinoma with large tumor mass, lymph node metastasis and high invasiveness than cases with well-differentiated adenocarcinoma with small tumor mass and without metastasis and invasiveness (p < 0.05 or p < 0.01). The percent of positive PUMA, c-Myb and p53 expression was significantly higher in cases with radical resection than without resection (p < 0.05). Univariate Kaplan-Meier analysis showed that PUMA, c-Myb and p53 expression was associated with decreased overall survival (p < 0.05 or p < 0.01). Multivariate Cox regression analysis showed that PUMA, c-Myb or p53 expression was a poor-prognostic predictor in gallbladder adenocarcinoma. CONCLUSION: PUMA, c-Myb and p53 expression closely relates to the carcinogenesis, fast-progression, easy-metastasis, high-invasion, and poor-prognosis in gallbladder adenocarcinoma.

Epigenetic silencing of Klotho expression correlates with poor prognosis of human hepatocellular carcinoma.

Klotho is identified as a tumor suppressor in several tumors, but the expression of the Klotho gene (KL) and its regulative mechanism are not reported in hepatocellular carcinoma (HCC). The messenger RNA and protein levels of Klotho were measured in 64 HCC tumor tissues by real-time polymerase chain reaction and immunohistochemistry, respectively. The methylation of KL promoter DNA was examined by bisulfite-based polymerase chain reaction. The correlation of Klotho protein expression and methylation with survival of HCC was analyzed using Kaplan-Meier analysis. The interference of KL gene expression was conducted in HCC cells by DNA demethylating agent and/or histone deacetylase inhibitor. In HCC tissues, a significant loss of Klotho messenger RNA and protein expression was observed, which parallels the increased methylation in KL promoter DNA. Both Klotho expression and methylation correlated with the poor prognosis of HCC. Experiments with HCC cell lines showed that a combination of DNA demethylating agent and histone deacetylase inhibitor fully recovered Klotho expression and subsequently induced cell apoptosis. In conclusion, Klotho is a tumor suppressor in HCC. Both hypermethylation and acetylation are involved in the loss of Klotho expression in HCC cells. Both KL gene expression and its promoter DNA methylation are predictive factors for the poor prognosis of HCC. Our study also suggests that the Klotho gene could be a target for therapy of HCC.

Restoration of klotho expression induces apoptosis and autophagy in hepatocellular carcinoma cells.

PURPOSE: Klotho has been identified as a tumor suppressor in several human malignancies including hepatocellular carcinoma (HCC). However, the signaling pathways involved in the tumor suppressive role of klotho in HCC have not been reported. Here, we investigated the role of klotho in HCC cell proliferation, apoptosis, autophagy, and invasion, as well as its associated signal transduction pathways. METHODS: Restoration of klotho gene expression was established by delivering a klotho gene expression vector into the human HCC cell lines HepG2 and MHCC-97-H. Cell viability was measured using a cell counting (CCK-8) assay and apoptosis was analyzed through flow cytometry. Autophagy was measured via LC3-I and LC3-II protein expression levels and tumor cell invasion was assessed using a Matrigel invasion chamber assay. Expression and phosphorylation of several apoptosis and survival related proteins were assessed using Western blot assays. RESULTS: Exogenous klotho gene expression significantly inhibited HCC cell proliferation, induced HCC cell apoptosis, increased LC3-I and LC3-II protein expression in HCC cells, and decreased migration of HCC cells in a Matrigel invasion chamber assay. Exogenous klotho gene expression also down-regulated the phosphorylation levels of the IGF-1 receptor, and the downstream Akt, ERK, and p70S6K proteins. Both apoptosis and autophagy inhibitors decreased klotho-induced apoptosis and autophagy. CONCLUSION: Klotho is a tumor suppressor that, through the regulation of IGF-1R phosphorylation and subsequent activation of downstream Akt-p70S6K and ERK signaling, regulates HCC tumor cell proliferation, apoptosis, autophagy and invasion.

Expression of CDC6 and GDF-9 and their clinicopathological significances in benign and malignant lesions of the gallbladder.

PURPOSE: Gallbladder cancers are cancers with high disease-specific mortality rates due to the lack of early diagnosis and effective therapy. In this study, we evaluated whether CDC6 and GDF-9 could be a marker for early diagnosis and target therapy. METHODS: CDC6 and GDF-9 expressions in 108 gallbladder adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues were detected using immunohistochemistry (IHC). RESULTS: We demonstrated that positive CDC6 and GDF-9 expressions were significantly higher in adenocarcinomas than that in peritumoral tissues, polyps, and chronic cholecystitis (p < 0.01). Benign lesions with positive CDC6 and negative GDF-9 expression showed moderately- or severely-atypical hyperplasia. The positive rates of CDC6 were significantly lower in cases with well-differentiated adenocarcinoma, small tumor mass, no metastasis of the lymph node, and no invasion of regional tissues (p < 0.05 or p < 0.01). In contrast, GDF-9 expression was significantly lower in the cases with poorly-differentiated adenocaarcinoma, lymph node metastasis, and invasion (p < 0.05 or p < 0.01). Univariate Kaplan-Meier analysis showed that CDC6 (p=0.046) or GDF-9 (p=0.032) expression was associated with decreased overall survival. Multivariate Cox regression analysis showed that increased expression of CDC6 (p=0.042) or decreased expression of GDF-9 (p=0.031) was an independent poor-prognostic predictor in gallbladder adenocarcinoma. CONCLUSION: CDC6 and GDF-9 might be closely related to the carcinogenesis, clinical biological behaviors, and prognosis of gallbladder adenocarcinoma. The positive expression of CDC6 and negative expression of GDF-9 have poor-prognosis in gallbladder carcinoma.

Immunohistochemical study of Dicer and Drosha expression in the benign and malignant lesions of gallbladder and their clinicopathological significances.

Dicer and Drosha are two key enzymes that are involved in the processing of miRNA production. Their expressions in gallbladder cancer have not been investigated. In this study, we studied Dicer and Drosha expression in 21 non-dysplastic gallbladder epithelia and 108 gallbladder adenocarcinomas by immunohistochemical staining. The clinicopathological significance of Dicer and Drosha expressions was analyzed. We demonstrated that the percent of positive Dicer or Drosha expression was significantly lower in gallbladder adenocarcinoma than that in non-dysplastic gallbladder epithelia (p<0.01). The percent of positive Dicer and Drosha expression was significantly lower in poorly differentiated adenocarcinoma with lymph node metastasis, invasiveness, and no resection than in well-differentiated adenocarcinoma without metastasis, invasiveness, and with radical resection (p<0.05 or p<0.01). Univariate Kaplan-Meier analysis showed that loss of Dicer and Drosha expression was associated with decreased overall survival (p<0.05 or p<0.01). Multivariate Cox regression analysis showed that loss of Dicer and Drosha expression was an independent poor-prognostic predictor in patients with gallbladder adenocarcinoma. In conclusion, loss of Dicer and Drosha expression is closely related to the metastasis, invasion, and poor-prognosis in gallbladder adenocarcinoma.

Pedicled ileal flap for duodenal defect after right hemicolectomy.

BACKGROUND/AIMS: To describe a convenient approach for repairing a large duodenal defect after right hemicolectomy for right side colon neoplasm. METHODOLOGY: Six patients with a large duodenal defect after right hemicolectomy with partial duodenectomy for right side colon neoplasm were treated with a pedicled ileal flap to cover the large duodenal defect. RESULTS: Six cases recovered smoothly. There was no perioperative morbidity and mortality in this series, except for one patient who had a duodenal leakage but who got well with medical treatment. Postoperative radiography showed normal duodenal peristalsis. CONCLUSIONS: The pedicled ileal flap procedure for repairing a large duodenal defect after right hemicolectomy is safe and convenient, and is especially indicated for elderly patients or patients with poor general condition.