PIAS1 upregulation confers protection against Cerulein-induced acute pancreatitis via FTO downregulation by enhancing sumoylation of Foxa2.

OBJECTIVE: This research discussed the specific mechanism by which PIAS1 affects acute pancreatitis (AP). METHODS: PIAS1, Foxa2, and FTO expression was assessed in Cerulein-induced AR42J cells and mice. Loss- and gain-of-function assays and Cerulein induction were conducted in AR42J cells and mice for analysis. The relationship among PIAS1, Foxa2, and FTO was tested. Cell experiments run in triplicate, and eight mice for each animal group. RESULTS: Cerulein-induced AP cells and mice had low PIAS1 and Foxa2 and high FTO. Cerulein induced pancreatic injury in mice and inflammation and oxidative stress in pancreatic tissues, which could be reversed by PIAS1 or Foxa2 upregulation or FTO downregulation. PIAS1 elevated SUMO modification of Foxa2 to repress FTO transcription. FTO upregulation neutralized the ameliorative effects of PIAS1 or Foxa2 upregulation on Cerulein-induced AR42J cell injury, inflammation, and oxidative stress. CONCLUSION: PIAS1 upregulation diminished FTO transcription by increasing Foxa2 SUMO modification, thereby ameliorating Cerulein-induced AP.

Blockade of the Arid5a/IL-6/STAT3 axis underlies the anti-inflammatory effect of Rbpjl in acute pancreatitis.

BACKGROUND: The microarray data analysis predicted that Rbpjl is poorly expressed in acute pancreatitis (AP). Activated IL-6/STAT3 signaling is further known to contribute to the progression of AP through immune regulation, and both IL-6 and STAT3 were bioinformatically predicted to interact with Arid5a. Accordingly, we aimed to investigate the potential involvement of the Arid5a/IL-6/STAT3 axis in the regulatory role of Rbpjl in the inflammation of AP. METHODS: Pancreatic acinar cells were exposed to lipopolysaccharide (LPS) to induce the pancreatic cell damage, and mice were subjected to supramaximal cerulein stimulation to induce AP. Expression patterns of Rbpjl and the Arid5a/IL-6/STAT3 axis were measured in mouse and cell models. Their expression was further manipulated to explore their effects on pancreatic cell injury and inflammation, as reflected by cell viability and apoptosis as well as reactive oxygen species (ROS) accumulation and proinflammatory cytokine secretion. Moreover, ChIP, EMSA, and dual-luciferase reporter assays were carried out to identify the interactions between Rbpjl and Arid5a. RESULTS: Rbpjl was found to be down-regulated in pancreatic tissues of AP mice and LPS-induced pancreatic acinar cells, while re-expression of Rbpjl led to enhanced cell viability, suppressed LPS-induced inflammation and ROS accumulation, and alleviation of AP-induced damage. Mechanistically, Rbpjl could bind to the promoter region of Arid5a and down-regulated its expression, thus repressing the activation of the IL-6/STAT3 signal axis. Furthermore, Rbpjl impaired Arid5a-dependent IL-6/STAT3 activation, hence alleviating pancreatic acinar cell inflammation. Furthermore, these effects were validated with in vivo experiments. CONCLUSION: Collectively, our findings highlight that Rbpjl attenuates AP by down-regulating Arid5a and inactivating the IL-6/STAT3 pathway.

The anti-inflammatory mechanism of SAHA in acute pancreatitis through HDAC5/SLIT2/Akt/ß-catenin axis.

Acute pancreatitis (AP) is widely recognized to be an inflammation-related disease, in which HDAC was upregulated. The anti-inflammatory role of suberoylanilide hydroxamic acid (SAHA), a HDAC inhibitor, has been documented. In this context, this research was implemented to figure out whether SAHA manipulated inflammation in AP. Subsequent to induction of AP mouse model, HDAC5 expression was detected. The binding of HDAC5 and SLIT2 was detected by Co-Immunoprecipitation and Chromatin immunoprecipitation assays. SAHA treatment and gain- and loss-of-function approaches were used in AP mice and lipopolysaccharide (LPS)-induced pancreatic acinar cells. In mice, biochemical methods were implemented to measure activities of pancreatic lipase, trypsin, myeloperoxidase (MPO) and pancreatic edema, TUNEL staining to determine pancreatic cell apoptosis, and flow cytometry to assess the total number of leukocytes and neutrophils in pancreas. In pancreatic acinar cells, CCK-8 was performed to evaluate cell viability. HDAC5 exhibited overexpression in AP mice. Mechanical analysis showed that HDAC5 facilitated SLIT2 deacetylation to downregulate SLIT2, thus activating Akt/ß-catenin pathway in pancreatic acinar cells. SAHA treatment, HDAC5 silencing or SLIT2 overexpression diminished inflammation in AP in vivo and in vitro. SAHA treatment, HDAC5 silencing or SLIT2 overexpression reduced activities of pancreatic lipase, trypsin, MPO, pancreatic edema and cell apoptosis in AP mice as well as elevated viability of LPS-induced pancreatic acinar cells. SAHA might exert anti-inflammatory effects in AP mice via HDAC5/SLIT2/Akt/ß-catenin axis.

Alcohol Aggravates Acute Pancreatitis by Impairing Autophagic Flux Through Activation of AMPK Signaling Pathway.

OBJECTIVE: Alcohol consumption is always the main cause of acute pancreatitis (AP). It has been reported that alcohol exerts direct damage to the pancreas. However, the specific role of alcohol during AP needs to be investigated. This study aims to examine the effects of alcohol in cerulein-induced AP and the role of the AMPK pathway. METHODS: Human subjects from operations, cerulein-induced AP rat, and cerulein-stimulated AR42J cell line were enrolled in this study. Electron microscopy was employed for observation of cell morphology, immunohistochemistry for identification of cells, ELISA for detection of inflammation factors, Annexin V/PI double staining for evaluation of cell apoptosis, immunofluorescence for assessment of autophagic flux, oil red O staining for examination of lipid droplet accumulation, and Western blot for measurement of expressions of proteins related to autophagy, apoptosis, and AMPK signal pathway. PI3K inhibitor 3-MA and AMPK inhibitor BML-275 were utilized for investigation of the relationship between impaired autophagic flux and the AMPK pathway by inhibiting or stimulating the formation of autophagosome. RESULTS: Alcohol consumption caused lipid droplet accumulation in the pancreas, and it also activated AMPK signaling pathway, thus aggravating the autophagic flux during AP. Alcohol up-regulated the expressions of anti-apoptotic proteins during the induction of AP to inhibit cell apoptosis and enhance cell necrosis. Inhibition of autophagosome formation by AMPK inhibitor BML-275 ameliorated the decreased cell viability caused by alcohol and cerulein in vitro. CONCLUSION: Alcohol aggravates AP progression by impairing autophagic flux and enhancing cell autophagy through the AMPK signaling pathway.

ZRANB1 enhances stem-cell-like features and accelerates tumor progression by regulating Sox9-mediated USP22/Wnt/ß-catenin pathway in colorectal cancer.

The pathogenesis of colorectal cancer (CRC) is a multistep process characterized by the accumulation of gene mutations and epigenetic alterations. Tumor necrosis factor receptor-associated factor-binding protein domain (ZRANB1) is a deubiquitinase that mediates tumor growth and metastasis by deubiquitinating target proteins. In this study, we examined the regulatory effects of ZRANB1 on the maintenance of cancer stem cell (CSC) properties and tumor growth in CRC. Human CRC tissue samples and matched normal tissues were collected for the analysis of ZRANB1 expression. ZRANB1 was upregulated in CRC human tissues and cell lines, and its expression was positively correlated with advanced tumor stage and poor survival of CRC patients. The overexpression of ZRANB1 also induced the expression of CSC markers in CRC cells. Then, a xenograft model was established by inoculating BALB/c mice with CRC cells. The upregulation of ZRANB1 promoted tumorigenesis in vivo. Sox9 is a transcription factor that acts as an oncogene in human cancers. ZRANB1 increased the stability of Sox9 in CRC cells by decelerating its ubiquitination. Further analysis revealed that Sox9 regulated the transcription activity of USP22 by binding to its promoter. Moreover, ZRANB1 enhances stem-cell-like features of CRC cells and activated the Wnt/ß-catenin pathway through USP22. Our results highlighted the role of ZRANB1 as a molecular target for CRC treatment, which may contribute to the development of novel therapies with better efficacy.

Determining the effects of Ephrin Type B Receptor 6 and Type A Receptor 3 on facilitating colorectal epithelial cell malignant transformation.

Ephrin Type-A Receptor 3 (EphA3) and Ephrin Type-B Receptor 6 (EphB6) belong to the ephrin receptor group consisting of the largest subset of receptor tyrosine kinases (RTKs) and are essential for neurogenesis and embryogenesis. The current study aimed to evaluate their functional roles in transforming colorectal epithelial cells and dissect the underlying molecular mechanisms. We observed altered EphA3 and EphB6 expression in tumor tissues as compared to normal tissues in a tissue microarray study. Enforced EphB6 expression promoted IMCE cell proliferation, migration, and invasion in vitro and tumor formation in nude mice, with a stronger oncogenic activity than EphA3. Pathway analysis of differentially expressed genes from a gene microarray study provided important insight into potential mechanisms through which EphB6 may regulate the malignant transformation of colorectal epithelial cells. This study represents the first demonstration of EphB6 in enhancing colorectal epithelial cell transformation, suggesting its stipulative role in the early stage of colorectal tumorigenesis. Our findings primarily uncover novel biomarkers and therapeutic targets of colorectal cancer.

MiR-30-5p suppresses cell chemoresistance and stemness in colorectal cancer through USP22/Wnt/ß-catenin signaling axis.

Colorectal cancer (CRC) remains both common and fatal, and its successful treatment is greatly limited by the development of stem cell-like characteristics (stemness) and chemoresistance. MiR-30-5p has been shown to function as a tumor suppressor by targeting the Wnt/ß-catenin signaling pathway, but its activity in CRC has never been assessed. We hypothesized that miR-30-5p exerts anti-oncogenic effects in CRC by regulating the USP22/Wnt/ß-catenin signaling axis. In the present study, we demonstrate that tissues from CRC patients and human CRC cell lines show significantly decreased miR-30-5p family expression. After identifying the 3'UTR of USP22 as a potential binding site of miR-30-5p, we constructed a luciferase reporter containing the potential miR-30-5p binding site and measured the effects on USP22 expression. Western blot assays showed that miR-30-5p decreased USP22 protein expression in HEK293 and Caco2 CRC cells. To evaluate the effects of miR-30-5p on CRC cell stemness, we isolated CD133 + CRC cells (Caco2 and HCT15). We then determined that, while miR-30-5p is normally decreased in CD133 + CRC cells, miR-30-5p overexpression significantly reduces expression of stem cell markers CD133 and Sox2, sphere formation, and cell proliferation. Similarly, we found that miR-30-5p expression is normally reduced in 5-fluorouracil (5-FU) resistant CRC cells, whereas miR-30-5p overexpression in 5-FU resistant cells reduces sphere formation and cell viability. Inhibition of miR-30-5p reversed the process. Finally, we determined that miR-30-5p attenuates the expression of Wnt/ß-catenin signaling target genes (Axin2 and MYC), Wnt luciferase activity, and ß-catenin protein levels in CRC stem cells.

Prognostic significance of glutathione peroxidase 2 in gastric carcinoma.

Increasing evidence suggests that the glutathione peroxidase 2 may actually play important roles in tumorigenesis and progression in various human cancers such as colorectal carcinomas and lung adenocarcinomas. However, the role of glutathione peroxidase 2 in gastric carcinoma remains to be determined. In this study, the expression and prognostic significance of glutathione peroxidase 2 in gastric carcinoma were investigated and the well-known prognostic factor Ki-67 labeling index was also assessed as positive control. Glutathione peroxidase 2 expression levels in the tumor tissue specimens, the matched adjacent normal tissue specimens, and the lymph node metastases of 176 patients with gastric carcinoma were evaluated by quantitative polymerase chain reaction, western blotting, and immunohistochemical staining. The associations between glutathione peroxidase 2 expression levels, as determined by immunohistochemical staining, and multiple clinicopathological characteristics were determined by Pearson's chi-square test and Spearman's correlation analysis. The relationships between glutathione peroxidase 2 expression and other clinicopathological variables and patient prognoses were analyzed further by the Kaplan-Meier method, the log-rank test, and Cox multivariate regression. The quantitative polymerase chain reaction, western blotting, and immunohistochemical staining results showed that glutathione peroxidase 2 expression levels were upregulated in both the primary tumor foci and the lymph node metastases of patients with gastric carcinoma (all p values < 0.05). Furthermore, Pearson's chi-square tests, as well as Spearman's correlation analysis, revealed that glutathione peroxidase 2 expression levels were strongly correlated with the Ki-67 labeling index, differentiation, histological patterns, Lauren classifications, lymph node metastasis, vascular invasion, tumor-node-metastasis stages, Helicobacter pylori infection, and overall survival (all p values < 0.05). Kaplan-Meier analysis, as well as the log-rank test and multivariate Cox regression analysis, showed that multiple clinicopathological risk factors and glutathione peroxidase 2 expression were novel independent prognostic factors for gastric carcinoma (all p values < 0.05). Glutathione peroxidase 2 expression is a novel independent prognostic biomarker for gastric carcinoma that may be used to devise personalized therapeutic regimens and precision treatments for this disease.

EphA3 promotes malignant transformation of colorectal epithelial cells by upregulating oncogenic pathways.

Ephrin Type-A Receptor 3 (EphA3) belongs to the ephrin receptor subfamily of the protein tyrosine kinase family, and plays an important role in embryogenesis and neurogenesis. This study aimed to investigate the role of EphA3 in promoting malignant transformation of colorectal epithelial cells, and explore underlying molecular mechanisms. Colorectal cancer tissue specimens from 68 patients were analyzed for EphA3 expression. EphA3 expression levels were manipulated in rat colon epithelial cell lines. We found that EphA3 expression level in tumor tissues was associated with patient age (P = 0.015), tumor differentiation (P = 0.001), and lymph node metastasis (P = 0.039). Overexpression of EphA3 and its constitutively active mutants promoted colony formation, migration and invasion, and tumorigenicity of colon epithelial cells in nude mice. The cDNA and lncRNA microarray profiling data revealed that differentially expressed genes and lncRNAs in EphA3 or mutant-transfected cells were associated with cell proliferation, invasion and angiogenesis. Our findings reveal the mechanisms underlying the oncogenic activities of EphA3 in colorectal cells, which could provide novel targets for the prevention, early diagnosis, and treatment of colorectal cancer.

High extracellular pressure promotes gastric cancer cell adhesion, invasion, migration and suppresses gastric cancer cell differentiation.

Slightly increased pressure stimulates tumor cell adhesion and proliferation. In the present study, we aimed to evaluate the effects of high pressure on gene expression and the biological behavior of gastric cancer cells. After incubation for 30 min at 37˚C under ambient and increased pressure, one portion of SGC7901 cells was used for cell proliferation and apoptosis assays, cell cycle analysis, adhesion invasion or migration assays. The other portion of cells was harvested for detection of matrix metalloproteinase-2 (MMP-2), inhibitor of DNA binding-1 (ID1), sonic Hedgehog (SHH) and E-cadherin expression by western blotting or RT-PCR. In addition, we investigated the effects of high pressure on SGC7901 cell ultrastructure by transmission electron microscopy. We found that the adhesion fold under increased pressure of 760 and 1,520 mmHg was 2.39±1.05 (P<0.05) and 2.47±0.85 (P<0.01) as compared with the control, respectively. The invasion fold was 3.42±2.06 (P<0.05) and 5.13±2.49 (P<0.01) as compared with the control, respectively. The migration was 1.65±0.20 (P<0.001) and 2.53±0.50 (P<0.001) as compared with the control, respectively. At increased pressure, MMP-2 and ID1 expression increased significantly, while the expression of SHH decreased significantly. However, we did not find significant change in proliferation, apoptosis, cell cycle or ultrastructure of the SGC7901 cells under high pressure. In conclusion, high pressure promoted the adhesion, invasion and migration of SGC7901 cells. Moreover, the present study suggests that the pressure-augmented invasion and migration may be related to the increase in MMP-2 expression. Moreover, high pressure may suppress SGC7901 cell differentiation, which may result from the change in SHH and ID1 expression.

EphB6 overexpression and Apc mutation together promote colorectal cancer.

The erythropoietin-producing hepatocyte (Eph) family tyrosine kinases play important roles in tumorigenesis and cancer aggression. In this study, we investigated the role of EphB6 in oncogenic transformation of colorectal epithelial cells in vitro and in vivo. EphB6 is upregulated in human colorectal cancer (CRC) tissues as compared to normal tissues, and its overexpression promotes proliferation, migration and invasion by IMCE colorectal adenoma cells, in which one Apc allele is mutated. EphB6 overexpression together with Apc mutation leads to the development of colorectal tumors in vivo. Expression microarrays using mRNAs and lncRNAs isolated from EphB6-overexpresssing IMCE and control cells revealed a large number of dysregulated genes involved in cancer-related functions and pathways. The present study is the first to demonstrate that EphB6 overexpression together with Apc gene mutations may enhance proliferation, invasion and metastasis by colorectal epithelial cells. Microarray data and pathway analysis of differentially expressed genes provided insight into possible EphB6-regulated mechanisms promoting tumorigenesis and cancer progression. EphB6 overexpression may represent a novel, effective biomarker predictive of cell proliferation, invasion and metastasis patterns in CRC tumors.

MALDI-TOF Mass Array Analysis of Nell-1 Promoter Methylation Patterns in Human Gastric Cancer.

Mass spectrometry (MS) enables rapid and sensitive qualitative and quantitative analyses of biomolecules (proteins, peptides, oligosaccharides, lipids, DNA, and RNA), drugs, and metabolites. MS has become an essential tool in modern biomedical research, including the analysis of DNA methylation. DNA methylation has been reported in many cancers, suggesting that it can be utilized as an early biomarker to improve the early diagnosis rate. Using matrix-assisted laser desorption/ionization time-of-flight MS and MassCLEAVE reagent, we compared Nell-1 hypermethylation levels among tumor tissues, paracarcinoma tissues, and normal tissues from gastric cancer patients. Almost 80% of the CpG sites in the amplicons produced were covered by the analysis. Our results indicate a significant difference in methylation status between gastric cancer tissue (a higher level) and normal tissue. The same trend was identified in gastric cancer tissue versus paracarcinoma tissue. We also detected lower relative expression of Nell-1 by real-time PCR. Furthermore, immunohistochemical analyses revealed that Nell-1 staining was less intense in cancer tissue relative to normal tissue and that the tumor cells had spread to the muscle layer. These findings may serve as a guide for the early diagnosis of gastric cancer.

S-1 combined with cisplatin versus cisplatin alone for the treatment of advanced gastric cancer: a pilot randomized-controlled trial.

We aimed to assess the efficacy and safety of S-1 combined with cisplatin (SC) over cisplatin alone (C) for the treatment of advanced gastric cancer in China. Between July 2009 and June 2011, 72 eligible patients with advanced gastric cancer were selected and divided randomly into two groups. Thirty-six patients received SC, with S-1 on days 1 through 14 of a 21-day cycle and cisplatin (60 mg/m on day 1) every 4 weeks for two cycles. The other 36 patients were administered only cisplatin (in the same manner as SC). The primary outcome was overall survival. The secondary outcomes were progression-free survival and adverse events. The 2-year overall response rate was 51.5 and 42.3% for the SC and C groups, respectively, and the difference was statistically significant, whereas the median overall survival was 9.4 months (range, 1.9-24.4 months) and 7.6 months (range, 1.7-21.4 months), respectively (P=0.039). The median progression-free survival was 7.7 months for SC (range, 1.8-19.4 months), whereas it was 6.5 months (range, 1.5-16.4 months) for C (P=0.047). The toxicity profile was similar in both groups. In summary, we have shown that S-1 combined with cisplatin is more effective, with acceptable toxicity in comparison with cisplatin alone in Chinese patients with advanced gastric cancer. Chinese Clinical Trials Register: ChiCTR-TRC-13003993.

Overexpression of the HIF hydroxylase PHD3 is a favorable prognosticator for gastric cancer.

Hypoxia-induced factors (HIFs) play a central role in the adaptive mechanisms of cancer cells to survive under conditions of hypoxia. HIFs are regulated by prolyl hydroxylases (PHDs) among which PHD3 is implicated as a tumor suppressor. We aimed to correlate PHD3 expression with clinicopathologic parameters and to evaluate its prognostic significance in gastric cancer. The 101 tissue samples were collected from 83 resected stages I­IV gastric cancer patients, which were grouped as non-cancerous mucosa (n=18) and primary carcinoma (n=83). PHD3 expression was evaluated by immunohistochemistry. We adopted Pearson chi-square test, univariate analysis, multivariate analysis and Kaplan­Meier method. The positive frequency of PHD3 in cancer cells was 42.2%, whereas non-cancerous mucosa had no detectable PHD3. The expression of PHD3 increased significantly from non-cancerous mucosa to cancer. A significant difference was observed between PHD3 expression and tumor differentiation (P=0.007). The overexpression of PHD3 was associated with well differentiation. In univariate analyses, American Joint Committee on Cancer (AJCC) stage (P<0.0001), pT classification (P<0.0001), pN classification (P<0.0001), differentiation (P=0.0121), peritoneal metastasis (P=0.0006) and gross features (P=0.0104) were significantly associated with survival except PHD3 (P=0.2228) (Table 3). In multivariate analysis, AJCC stage was prognostically independent [hazard ratio (HR), 3.078; 95% confidence interval (CI), 2.228­4.252; P<0.0001]. Overexpression of PHD3 is a favorable prognosticator for gastric cancer. AJCC stage is an independent prognostic factor of gastric cancer.

The co-expression of USP22 and BMI-1 may promote cancer progression and predict therapy failure in gastric carcinoma.

Recent experimental evidence support the model in which the simultaneous induction of BMI-1 and USP22 is critical during cancer progression. Whether this model may affect gastric cancer (GC) progression is worthy of additional study. In this study, we examined the significance of the USP22 and BMI-1 expression in GC (n = 219), non-cancerous mucosa (n = 37), and lymph node metastasis (n = 37). The protein expression level of USP22 and BMI-1 were concomitantly up-regulated from non-cancerous mucosa to primary carcinoma and from carcinomas to lymph node metastasis (P < 0.001). A statistical correlation was observed between USP22 and BMI-1 expression in GC tissues (n = 219, r = 0.634, P < 0.001) and in lymph node metastasis (n = 37, r = 0.689, P < 0.001). The incidence of positive expression was 57.08% for USP22, 49.32% for BMI-1, and 45.21% for USP22/BMI-1 in 219 GC tissues, respectively. Co-positive of USP22/BMI-1 was significantly correlated with gross features (x(2) = 14.256, P < 0.001), differentiation (x(2) = 5.872, P = 0.015), pT classification (x(2) = 18.486, P < 0.001), pN classification (x(2) = 9.604, P = 0.002), pM classification (x(2) = 32.766, P < 0.001), and AJCC stage (x(2) = 58.278, P < 0.001). Notably, high USP22/BMI-1 expression was significantly associated with shorter disease-specific survival (P < 0.001). By Cox regression analysis, co-positive of USP22/BMI-1 was found to be an independent prognostic factor (P = 0.002). Our results indicated the simultaneous activation of USP22 and BMI-1 may associate with GC progression and therapy failure.

Is gastrointestinal dysfunction induced by gastric cancer peritoneal metastasis relevant to impairment of interstitial cells of Cajal?

Although impaired gastrointestinal motility from gastric cancer peritoneal metastasis (GCPM) causes extraordinary pain, its cause is unclear. Interstitial cells of Cajal (ICC) are apparently pacemaker cells, and their loss could cause motor dysfunction. In this study, we developed a mouse model for GCPM, and investigated electrophysiological changes in the small intestine and attendant changes in ICC. We found decreased ICC and disrupted electrical rhythm in the model. Pathologic ICC changes were well described. Cancer peritoneal metastasis may impair intestinal myoelectrical activity by damaging ICC and ICC networks. Interstitial cells of Cajal will be a target of palliative treatment and merit further study.