[Palmitic acid induces hepatocellular oxidative stress and activation of inflammasomes].

OBJECTIVE: To evaluate the effect of palmitic acid (PA) on oxidative stress and activation of inflammasomes in hepatocytes. METHODS: To test the dose-dependent effect of PA on normal murine hepatocytes AML12, the cells were treated with 0, 0.15, 0.25 and 0.4 mmol/L of palmitic acid (PA). The cells were also divided into blank control group, 0.25 mmol/L PA group and 0.25 mmol/L PA+N-acetylcysteine (NAC) group to examine the effect of reactive oxygen species (ROS) on the activation of inflammasomes. After 24 h of treatment, lipid accumulation, total ROS, mitochondrial ROS, expression and localization of NOX4, and expressions of inflammasomes and IL-1ß were detected in the hepatocytes. RESULTS: Compared with the control cells, PA treatment of the cells significantly increased cytoplasmic lipid accumulation, concentrations of total ROS (12 463.09±2.72 vs 6691.23±2.45, P=0.00) and mitochondrial ROS (64.98±0.94 vs 45.04±0.92, P=0.00), and the expressions of NOX4, NLRP3, ASC, caspase-1, and IL-1ß (1603.52±1.32 vs 2629.33±2.57, P=0.00). The mitochondria and NOX4 were found to be co-localized in the cytoplasm. NAC obviously reduced cellular ROS level stimulated by PA (7782.15±2.87 vs 5445.6±1.17, P=0.00) and suppressed the expressions of NLRP3, ASC and caspase-1. CONCLUSION: PA treatment can stimulate lipid accumulation in hepatocytes and induce oxidative stress through NOX4 and mitochondria pathway to activate inflammasomes and stimulate the secretion of IL-1ß.

[Value of urgent colonoscopy in diagnosis of severe acute lower gastrointestinal bleeding in patients with different bowel cleanliness].

OBJECTIVE: To investigate the value of urgent colonoscopy in the diagnosis of severe acute lower gastrointestinal bleeding and the optimal bowel preparation before examination. METHODS: The clinical data were collected from 188 patients undergoing wither urgent or elective colonoscopy for severe acute lower gastrointestinal bleeding in Nanfang Hospital. Univariate analysis was used to assess the effect of the timing of colonoscopy on the diagnostic rate of hemorrhage, and a multivariate model which stratified bowel cleanliness was used to analyze the impact of bowel cleanliness on the diagnostic rate of urgent colonoscopy. RESULTS: Of the 188 patients, 118 underwent urgent colonoscopy and 70 underwent elective colonoscopy examinations. The diagnostic rates were comparable between the two groups (44.1% vs 41.4%, P=0.724), but urgent colonoscopy resulted in a significantly higher diagnostic rate for identifying the bleeding source (32.2% vs 18.6%, P=0.041). The proportion of the patients taking oral laxatives was significantly lower in urgent colonoscopy group (P<0.001). Oral laxatives versus enema resulted in good, moderate, and poor bowel cleanliness in 63.6% vs 13.5%, 28.6% vs 24.3%, and 7.8% vs 62.2% of the patients (P<0.001). Univariate analysis indicated that good bowel cleanliness was associated with a significantly higher diagnostic rate of colonoscopy than poor bowel cleanliness (P=0.012). Multivariate analysis showed that with good bowel cleanliness, urgent colonoscopy yielded a significantly higher diagnostic rate than elective colonoscopy (P=0.030); subgroup analyses suggested that good bowel cleanliness improved the diagnostic rate of urgent colonoscopy as compared with poor bowel cleanliness (P=0.015). CONCLUSION: In patients with good bowel cleanliness, urgent colonoscopy yields a higher diagnostic rate than elective colonoscopy for severe acute lower gastrointestinal bleeding. Poor bowel cleanliness resulting from bowel preparation by enema significantly lowers the diagnostic performance of urgent colonoscopy. Oral laxatives are recommended over enemas for bowel preparation before urgent colonoscopy when the patients have stable hemodynamics.

TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways.

BACKGROUND: Transducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/ß-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce. METHODS: Gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR. RESULTS: TLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1. CONCLUSION: This study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis.

Gastrointestinal hormone abnormalities and G and D cells in functional dyspepsia patients with gastric dysmotility.

AIM: To investigate the relationship between gastric dysmotility, gastrointestinal hormone abnormalities, and neuroendocrine cells in gastrointestinal mucosa in patients with functional dyspepsia (FD). METHODS: Gastric emptying was assessed with solid radiopaque markers in 54 FD patients, and the patients were divided into two groups according to the results, one with delayed gastric emptying and the other with normal gastric emptying. Seventeen healthy volunteers acted as normal controls. Fasting and postprandial plasma levels and gastroduodenal mucosal levels of gastrointestinal hormones gastrin, somatostatin (SS) and neurotensin (NT) were measured by radioimmunoassay in all the subjects. G cells (gastrin-producing cells) and D cells (SS-producing cells) in gastric antral mucosa were immunostained with rabbit anti-gastrin polyclonal antibody and rabbit anti-SS polyclonal antibody, respectively, and analyzed quantitatively by computerized image analysis. RESULTS: The postprandial plasma gastrin levels, the fasting and postprandial plasma levels and the gastric and duodenal mucosal levels of NT were significantly higher in the FD patients with delayed gastric emptying than in those with normal gastric emptying and normal controls. The number and gray value of G and D cells and the G cell/D cell number ratio did not differ significantly between normal controls and the FD patients with or without delayed gastric emptying. CONCLUSION: Our findings suggest that the abnormalities of gastrin and NT may play a role in the pathophysiology of gastric dysmotility in FD patients, and the abnormality of postprandial plasma gastrin levels in FD patients with delayed gastric emptying is not related to the changes both in the number and gray value of G cells and in the G cell/D cell number ratio in gastric antral mucosa.

[Effects of combined use of curcumin and catechin on cyclooxygenase-2 mRNA expression in dimethylhydrazine-induced rat colon carcinogenesis].

OBJECTIVE: To examine the effect of combined use of curcumin and catechin on the number of aberrant crypt foci (ACF) and expression levels of cyclooxygenase-2 (COX-2) mRNA in rat colon carcinogenesis. Methods Dimethylhydrazine (DMH)-induced rats colon carcinogenesis model was used for evaluation of the synergistic inhibitory effect between curcumin and catechin in light of ACF formation and tumor incidence. COX-2 mRNA expression was also detected in rat colon carcinogenesis. RESULTS: Curcumin, catechin and their co-treatment caused significant inhibition of DMH-induced ACF and colon carcinogenesis as compared with untreated DMH-induced rat models (P<0.01). Co-treatment with curcumin and catechins caused greater inhibition of DMH-induced ACF and colon carcinogenesis than the single use of curcumin or catechin (P<0.05). A synergistic inhibitory effect between curcumin and catechin on the expression of COX-2 mRNA was observed in the early stage of rat colon carcinogenesis but not in colon tumor tissues. CONCLUSION: Curcumin and catechin have synergistic effect on ACF and COX-2 mRNA expression in rat colon carcinogenesis, suggesting their potential value in the prevention of human colon cancers.

Identification of the sAPRIL binding peptide and its growth inhibition effects in the colorectal cancer cells.

BACKGROUND: A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) super family. It binds to its specific receptors and is involved in multiple processes during tumorigenesis and tumor cells proliferation. High levels of APRIL expression are closely correlated to the growth, metastasis, and 5-FU drug resistance of colorectal cancer. The aim of this study was to identify a specific APRIL binding peptide (BP) able to block APRIL activity that could be used as a potential treatment for colorectal cancer. METHODS: A phage display library was used to identify peptides that bound selectively to soluble recombinant human APRIL (sAPRIL). The peptides with the highest binding affinity for sAPRIL were identified using ELISA. The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively. An in vivo mouse model of colorectal cancer was used to determine the anti-tumor efficacy of the sAPRIL-BP. RESULTS: Three candidate peptides were characterized from eight phage clones with high binding affinity for sAPRIL. The peptide with the highest affinity was selected for further characterization. The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner. In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally. Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells. CONCLUSIONS: sAPRIL-BP significantly suppressed tumor growth in vitro and in vivo and might be a candidate for treating colorectal cancers that express high levels of APRIL.

The tumor-suppressor gene LZTS1 suppresses colorectal cancer proliferation through inhibition of the AKT-mTOR signaling pathway.

The Leucine zipper tumor suppressor gene 1 (LZTS1/FEZ1) gene was originally identified as a potential tumor suppressor. However, the expression pattern and the role of LZTS1 in the progression of colorectal cancer (CRC) have not been well characterized. Herein, we reported that LZTS1 was markedly reduced in CRC tissues compared with matched adjacent normal intestine epithelial tissues. In analysis of 160 CRC specimens, we revealed that decreased expression of LZTS1 was correlated to aggressive characteristics and poor survival of patients with CRC. Moreover, we found that expression of LZTS1 in CRC cells significantly inhibited cell proliferation in vitro and prohibited tumor growth in vitro. On the contrary, silence of LZTS1 promoted cell proliferation and tumor growth in CRC cells. Furthermore, we demonstrated that LZTS1 inhibited cell proliferation and tumor growth in CRC in part via suppression of AMT-mTOR, subsequently down-regulating p27Kip and up-regulating cyclin D1. These findings suggest that LZTS1 plays a potential tumor suppressor role in CRC progression and represents a valuable clinical prognostic marker of this disease.

[Effects of the antibacterial peptide cecropins from Chinese oak silkworm, Antheraea pernyi on 1, 2-dimethylhydrazine-induced colon carcinogenesis in rats].

OBJECTIVE: To gain insight into the putative anticancer effect of the antibacterial peptides, cecropins, from Chinese oak silkworm, Antheraea pernyi, on the cancer cells and 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats. METHODS: Growth inhibitory effect of the cecropins on normal human gastric epithelial cell line (GES-1) and human colon adenocarcinoma cell line (LS-174T) was observed using a microculture tetrazolium (MTT) colorimetric methods. Male Wistar rats were divided into 4 groups. Group1 was given on a weekly basis cecropins from Antheraea pernyi (3,000 Ua/ml) by gavage at 2 doses of 10 ml/kg body weight and exposed to subcutaneous injection of DMH at the dose of 20 mg/kg body weight. Group 2 was received weekly DMH only. Group 3 was given the cecropins by gavage at the same dose as in group 1. Group 4 was weekly exposed to subcutaneous injection of EDTA (1 mmol/L). All treatments were completed in a course of 18 weeks and the experiment was finished at week 33. RESULTS: MTT assay showed selective cytotoxic activity of the cecropins against the human colon adenocarcinoma cells line. The viability of the cancer cells was about 54% and 100% for the normal cells. There was a significantly lower incidence of large intestinal tumors in rats gavaged with cecropins (65%, P<0.01), but the tumor burden (tumors/tumor-bearing animal) and tumor mass index were comparable between the groups (P>0.05). CONCLUSION: The cecropins possess effective anti-tumor activity with no cytotoxicity against normal eukaryotic cells, and impede the neoplastic process in murine large intestines.

[Electron microscopic observation of endocrine cells in the antrum in response to Helicobacter pylori infection].

OBJECTIVE: To observe the ultrastructural changes of the endocrine cells in the antrum after the onset of Helicobacter pylori (Hp) infection. METHODS: Seven patients with chronic gastritis were included in this study, and toluidine blue staining and rapid urease test were performed to determine the Hp status of the gastric antral mucosa biopsies. Five patients identified as being positive for Hp constituted the test group, leaving the 2 Hp-negative patients serving as control. The endocrine cells in the antrum of the patients were sampled to observe the ultrastructural changes by transmission electron microscopy (TEM). RESULTS: TEM showed increased electron density of the secretion granules in the endocrine cells, and secretions in the parietal cells were active. CONCLUSION: The ultrastructural changes of the endocrine cells in the antrum might explain high gastrin levels after the onset of Hp infection.

Akt promotes irradiation-induced regulatory T-cell survival in hepatocellular carcinoma.

BACKGROUND: Radiotherapy is an efficient remedy in the treatment of hepatocellular carcinoma (Hca); however, some cancer cells can still survive from the radiation and the therapeutic effect is to be improved. Regulatory T cell (Tregs)-induced tumor tolerance and Akt expression play important roles in the tumor survival. This study aims to elucidate the role of radiation induces Akt expression in Tregs. METHODS: A rat Hca model was developed. Hca tissue was collected from the rats with or without radiotherapy. The frequency of Treg and apoptotic Treg in Hca tissue was assessed by flow cytometry. A cell culture model was used to investigate the mechanism by which the tumor-infiltrating Tregs survive from irradiation. RESULTS: After radiotherapy, the frequency of Treg was increased in Hca, the frequency of apoptotic Tregs was decreased and the expression of Akt was increased in the remaining Tregs. The results were reproduced in vitro with a cell culture model. The addition of Akt inhibitor blocked the irradiation-induced Treg survival. Tregs with high levels of Akt still preserve the immune suppressor function. CONCLUSIONS: Akt plays an important role in the radiation-induced tumor-infiltrating Treg survival in Hca.

microRNA-224 promotes cell proliferation and tumor growth in human colorectal cancer by repressing PHLPP1 and PHLPP2.

PURPOSE: To investigate the clinicopathologic significance, role, and mechanism of action of microRNA-224 (miR-224) in colorectal cancer. EXPERIMENTAL DESIGN: Real-time PCR was used to quantify miR-224 expression. The association of miR-224 with the clinicopathologic features and survival was evaluated in 110 colorectal cancer patients. The role of miR-224 in colorectal cancer was investigated using in vitro and in vivo assays. Luciferase reporter assays were conducted to confirm target gene associations. RESULTS: miR-224 was overexpressed in colorectal cancer. High-level expression of miR-224 was significantly associated with an aggressive phenotype and poor prognosis. Overexpression of miR-224 promoted colorectal cancer cell proliferation in vitro and tumor growth in vivo. Specifically, miR-224 accelerated the G1-S phase transition through activation of AKT/FOXO3a signaling, downregulation of p21Cip1 and p27Kip1, and upregulation of cyclin D1. Moreover, both PH domain leucine-rich-repeats protein phosphatase 1 (PHLPP1) and PHLPP2, antagonists of PI3K/AKT signaling, were confirmed as bona fide targets of miR-224. miR-224 directly targeted the 3'-untranslated regions of the PHLPP1 and PHLPP2 mRNAs and repressed their expression. CONCLUSION: This study reveals functional and mechanistic links between miRNA-224 and the tumor suppressors PHLPP1 and PHLPP2 in the pathogenesis of colorectal cancer. miR-224 not only plays important roles in the regulation of cell proliferation and tumor growth in colorectal cancer, but also has potential as a prognostic marker or therapeutic target for colorectal cancer.

[Lentivirus-mediated RNA interference suppresses APRIL expression and enhances chemosensitivity in colorectal cancer cells].

OBJECTIVE: To investigate the effects of lentivirus-mediated RNA interference (RNAi) targeting a proliferation-inducing ligand (APRIL) on the chemosensitivity to 5-FU of colorectal cancer cell line LoVo. METHODS: The lentiviral vector siRNA-APRIL was constructed and verified by PCR and DNA sequencing. LoVo cells were transfected with siRNA-APRIL plasmid, non-targeting siRNA plasmid, or empty plasmid. Forty-eight hours after the transfection, the cells were examined for APRIL expression using Western blot. Seventy-two hours after treatment with 10 µg/ml 5-FU, flow cytometry was used to detect the cell apoptosis and cell cycle changes. The cell growth inhibition rate following 5-FU exposure was detected by MTT assay. RESULTS: PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting APRIL gene was successfully inserted into the lentiviral vector. siRNA-APRIL transfection resulted in obviously reduced expression of APRIL in LoVo cells. After 5-FU exposure, the apoptosis rate of siRNA-APRIL-transfected cells were increased to (21.12∓3.35)%, significantly higher than that in cells transfected with the non-targeting plasmid or the empty plasmid [(13.06∓1.92)% and (12.28∓1.79)%, respectively, P<0.01]; the cell number in G0/G1 phase increased while that in G2/M phase decreased in siRNA-APRIL-transfected cells. The growth inhibition rate in siRNA-APRIL group was (59.67∓5.03)%, significantly higher than that in the other two groups [(42.33∓4.16)% and (39.67∓4.73)%, respectively, P<0.01]. CONCLUSION: Lentivirus-mediated RNAi targeting APRIL can effectively suppress the expression of APRIL in LoVo cells and enhance the chemosensitivity of the cells to 5-FU.

[Selective COX-2 inhibitor delays experimental gastric ulcer healing by stimulating gastric acid secretion in rats].

OBJECTIVE: To observe the effect of selective cyclooxygenase-2 (COX-2) inhibitor on the healing of experimental gastric ulcer in rats and explore its mechanisms in light of gastric acid secretion. METHODS: Gastric ulcers were induced in rats by an application of acetic acid to the serosal surface of the anterior gastric body. The effects of selective COX-2 inhibitor, celecoxib, on the healing of gastric ulcer, the total acidity of gastric juice, the expressions of H+, K+-ATPase mRNA and protein, and the ultrastructure of the parietal cell were observed in comparison with the effects of normal saline. RESULTS: Nine days after ulcer induction, the ulcer area was 11.9-/+3.1 mm square and 19.7-/+3.8 mm square in rats with normal saline and celecoxib treatments, respectively (P<0.01). The total acidity of gastric juice and the expressions of H+, K+-ATPase mRNA and protein in celecoxib group were significantly higher than that in normal saline group at both 6 and 9 days after ulcer induction, but no significant difference was found between the two groups in the amount of secretary canaliculus and microvillus. CONCLUSION: Selective COX-2 inhibitor can significantly delay the healing of experimental gastric ulcer in rats, the mechanism of which might be associated with enhanced digestive action of gastric acid on the new granulation tissue at the ulcer base as a result of celecoxib-stimulated gastric acid secretion of the parietal cells.